Clarke's Analysis of Drugs and Poisons, 4th Edition

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Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 20:30:51

Clarke’s Analysis of Drugs and Poisons

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 20:30:51

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 20:30:51

Clarke’s Analysis of Drugs and Poisons in pharmaceuticals, body fluids and postmortem material FOURTH EDITION

Consulting Editors

Anthony C Moffat M David Osselton Brian Widdop Executive Development Editor

Jo Watts

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 20:30:51

Published by Pharmaceutical Press 1 Lambeth High Street, London SE1 7JN, UK 1559 St Paul Avenue, Gurnee, IL 60031, USA # Pharmaceutical Press 2011 Chapter 13: Figures 13.1–13.21, 13.25–13.32 # TICTAC Communications is a trade mark of Pharmaceutical Press Pharmaceutical Press is the publishing division of the Royal Pharmaceutical Society First edition, edited by EGC Clarke, published 1969 (Vol. 1) and 1975 (Vol. 2) Second edition (in one volume) published 1986 Third edition published 2004 Fourth edition published 2011 Typeset by Thomson Digital, Noida, India Printed in Italy by LEGO S.p.A. ISBN 978 0 85369 711 4 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without the prior written permission of the copyright holder. The publisher makes no representation, express or implied, with regard to the accuracy of the information contained in this book and cannot accept any legal responsibility or liability for any errors or omissions that may be made. A catalogue record for this book is available from the British Library.

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 20:30:51

Contents

VOLUME 1 Editorial Advisory Board vii Editorial and Production Staff Contributors ix About the Editors xi Foreword xii Preface xiii General Notices xv Abbreviations xix

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Part One: Chapters 1 Hospital Toxicology 3 DRA Uges 2 Therapeutic Drug Monitoring 59 M Hallworth 3 Workplace Drug Testing 73 A Verstraete M Peat 4 Driving Under the Influence of Alcohol 87 AW Jones 5 Driving Under the Influence of Drugs 115 BK Logan MD Osselton 6 Drug Testing in Human Sport 127 DA Cowan 7 Drug Testing in Animal Sport 138 P Teale 8 Drug-facilitated Sexual Assault 147 MD Osselton 9 Forensic Toxicology 160 MD Osselton AC Moffat B Widdop

15 Natural Toxins 243 JF de Wolff FA de Wolff 16 Pesticides 258 M Kała 17 Metals and Anions 288 R Braithwaite 18 Drugs in Saliva 308 V Spiehler 19 Hair Analysis 323 P Kintz 20 Method Development and Validation 334 FT Peters 21 Quality Control in the Pharmaceutical Industry 350 P Graham 22 Quality Control and Accreditation in the Toxicology Laboratory 361 AC Moffat 23 Measuring and Reporting Uncertainty 371 MA LeBeau 24 Pharmacokinetics and Metabolism 388 OH Drummer 25 Pharmacogenomics 401 SHY Wong 26 Interpretation of Toxicological Data 417 OH Drummer S Karch 27 Paediatric Toxicology 429 D Reith 28 Sampling, Storage and Stability 445 S Kerrigan

10 Postmortem Toxicology 176 G Jones

29 Extraction 458 T Stimpfl

11 Drugs of Abuse 190 SD McDermott

30 Colour Tests 471 B Widdop

12 Medicinal Products 208 AC Moffat AG Davidson

31 Immunoassays 496 RS Niedbala JM Gonzalez

13 Solid Dosage Form Identification 219 J Ramsey

32 Ultraviolet, Visible and Fluorescence Spectrophotometry 507 J Cordonnier J Schaep

14 Volatile Substances 230 RJ Flanagan

33 Infrared Spectroscopy 521 RD Jee

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Contents

34 Near-infrared Spectroscopy 538 RD Jee

44 Emerging Techniques 787 D Rudd

35 Raman Spectroscopy 553 DE Bugay PA Martoglio Smith FC Thorley

Subject Index Ii

36 Nuclear Magnetic Resonance Spectroscopy 564 JC Lindon JK Nicholson 37 Mass Spectrometry 577 D Watson 38 Liquid Chromatography–Mass Spectrometry 594 HH Maurer 39 Thin-layer Chromatography 600 CF Poole 40 Gas Chromatography 636 S Dawling 41 High Performance Liquid Chromatography 718 T Kupiec P Kemp 42 Capillary Electrophoresis 758 F Tagliaro A Fanigliulo J Pascali F Bortolotti 43 Atomic Absorption Spectroscopy, Inductively Coupled Plasma–Mass Spectrometry and Other Techniques for Measuring the Concentrations of Metals 773 A Taylor

VOLUME 2 Part Two: Monographs 807 Part Three: Indexes of Analytical Data CAS Numbers 2263 Molecular Formulae 2278 Functional Classes: Therapeutic 2305 Functional Classes: Pesticides 2315 Functional Classes: Other Substances 2317 Molecular Weights 2328 Melting Points 2342 Colour Tests 2353 Thin-layer Chromatographic Data 2358 Gas Chromatographic Data 2392 High Performance Liquid Chromatographic Data 2410 Ultraviolet Absorption Data 2427 Infrared Peaks 2442 Mass Spectral Data of Drugs 2451 Mass Spectral Data of Pesticides 2460 Reagents 2461 Pharmacological Terms 2463

Subject Index Ii

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 10:1:25

Editorial Advisory Board

Dr Craig Chatterton

Dr Jan-Piet Franke

Dr Christine Moore

CNC Forensic Toxicology Services, c/o 7 Sawley Close, Darwen, Lancashire BB3 3QY, UK

Department of Pharmaceutical Analysis, University of Groningen, A Deusinglaan 1, 9713 AV Groningen, The Netherlands

Immunalysis Corporation, 829 Towne Centre Drive, Pomona, CA 91767, USA

Dr Hee-Sun Chung

National Forensic Service (formally NISI), 331-1 Sinwol 7-Dong, Yang Chun-Ku, Seoul 158-097, Korea Dr Gail Cooper

Forensic Medicine and Science, University of Glasgow, Scotland G12 8QQ, UK Mr Simon Cosbey

5A Carnalea Aveue, Bangor BT19 1HF, Northern Ireland Dr Simon Elliott

(ROAR) Forensics, Ltd, Malvern Hills Science Park, Malvern, Worcestershire, WR14 3SZ, UK

Dr Sue Paterson Professor Bruce Goldberger

University of Florida – College of Medicine, Department of Pathology and Laboratory Medicine, 4800 SW 35th Drive, Gainesville, FL 32608, USA

Toxicology Unit, Imperial College, St Dunstan’s Road, London W6 8RP, UK Mr Chip H Walls

Forensic Toxicology Laboratory, 12500 SW 152Nd Street, Bldg B, Miami, FL 33177, USA

Dr Rodney G Gullberg

Washington State Toxicology Laboratory, 2203 Airport Way S, Suite 360, Seattle, WA 98134, USA Dr Dan S Isenschmid

Wayne County Medical Examiner’s Office, 1300 E Warren Avenue, Detroit, MI 48207, USA

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Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 10:1:25

Editorial and Production Staff

Emma Burrows Helen Carter Tamsin Cousins Amy Cruse Simon Dunton Marian Fenton Kelly Davey Rebecca Garner Austin Gibbons David Granger Jo Humm

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Jean Macpherson Julie McGlashan Louise McIndoe Ithar Malik Jason Norman Karl Parsons The Prescribers, The School of Pharmacy, London, UK Jo Watts Lucy White John Wilson

Freelance Staff Irene Chiwele Millie Davis Laurent Y Galichet Poppy McLaughlin Eva Reichardt A team of dedicated copyeditors, proofreaders and indexers also helped in the preparation of this publication.

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 21:0:29

Contributors

Dr Federica Bortolotti

Mr Mike Hallworth

Dr Sean D McDermott

Department of Public Health and Community Medicine, Section of Forensic Medicine, University of Verona, Verona, Italy

Department of Clinical Biochemistry, Royal Shrewsbury Hospital, Shrewsbury, UK

Drugs Intelligence Unit, Forensic Science Laboratory, Dublin, Ireland

Dr Roger D Jee

Professor Anthony C Moffat

Dr Robin A Braithwaite

Regional Toxicology Laboratory, City Hospital NHS Trust, Birmingham, UK Dr David E Bugay

SSCI Inc., West Lafayette, USA Dr Jan Cordonnier

The School of Pharmacy, University of London, UK Dr Graham Jones

Office of Chief Medical Examiner, Edmonton, Canada Dr A Wayne Jones

Formerly, Royal Pharmaceutical Society of Great Britain and, The School of Pharmacy, University of London, London, UK Professor Jeremy K Nicholson

Department of Surgery and Cancer, Imperial College of Science, Technology and Medicine, London, UK

Dorpsstaat 106, B-8340 Sysele-Damme, Belgium

National Board of Forensic Medicine, Department of Forensic Chemistry, Linkoping, Sweden

Professor David A Cowan

Dr Maria Kała

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania, USA

Department of Forensic Toxicology, Institute of Forensic Research, Cracow, Poland

Professor M David Osselton

Drug Control Centre, King’s College London, UK

Dr R Sam Niedbala

University of Bournemouth, Dorset, UK Dr Alastair G Davidson

Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK Dr Sheila Dawling

Vanderbilt University Medical Centre, Diagnostic Labs – TVC, Nashville, Tennessee, USA Professor Frederik A de Wolff

Toxicology Laboratory, Leiden University Medical Center, Leiden, The Netherlands

Dr Steven Karch

PO Box 5139, Berkeley, CA 94705, USA

Dr Jennifer Pascali

Dr Phil Kemp

Department of Public Health and Community Medicine, Section of Forensic Medicine, University of Verona, Verona, Italy

Analytical Research Laboratories, Oklahoma City, USA Dr Sarah Kerrigan

SHSU Regional Crime Laboratory, The Woodlands, Texas, USA

Dr Jacob F de Wolff

University College London Hospitals NHS Foundation Trust, London, UK

Dr Thomas Kupiec

Victorian Inst. of Forensic Medicine, Southbank, Victoria, Australia Dr Ameriga Fanigliulo

Department of Public Health and Community Medicine, Section of Forensic Medicine, University of Verona, Verona, Italy Dr Robert J Flanagan

Analytical Research Laboratories, Oklahoma City, USA

Institut f€ ur Rechtsmedizin, Universit€atsklinikum Jena, Jena, Germany Professor Colin F Poole

Department of Chemistry, Wayne State University, Detroit, Michigan, USA

Dr Marc LeBeau

Dr John Ramsey

Chemistry Unit, FBI Laboratory, Quantico, Virginia, USA

Division of Cardiological Sciences, St George’s Hospital Medical School, London, UK

Professor John C Lindon

Division of Biomedical Sciences, Imperial College of Science, Technology and Medicine, London, UK

Medical Toxicology Unit, Guy’s and St Thomas’ Hospital Trust, London, UK

Dr Barry K Logan

Dr Jesus M Gonzalez

Dr Pamela A Martoglio Smith

Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania, USA

Quest Diagnostics, Houston, Texas, USA Dr Frank T Peters

Dr Pascal Kintz

Laboratoire Chemtox, Illkirch-Graffenstaden, France

Dr Olaf H Drummer

Dr Michael A Peat

NMS Labs, Willow Grove, Pennsylvania, USA SSCI Inc., West Lafayette, USA

Dr David Reith

Dunedin School of Medicine, University of Otago, New Zealand Dr Dave Rudd

GlaxoSmithKline Manufacturing, Ware, Hertfordshire, UK Dr Johan Schaep

Chemiphar n.v., Brugge, Belgium Professor Hans H Maurer

Dr Paul Graham

Walker Graham Pharma Consulting Ltd, Ashington, Northumberland, UK

Department of Experimental and Clinical Toxicology, University of Saarland, Homburg/ Saar, Germany

Dr Vina Spiehler

422 Tustin Avenue, Newport Beach, CA 92663, USA ix

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 21:0:29

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Contributors

Dr Thomas Stimpfl

Dr Fiona C Thorley

Dr Jo Watts

Department of Forensic Medicine, Wien, Austria

SSCI, A Division of Aptuit, Abingdon, Oxfordshire, UK

Pharmaceutical Press, Royal Pharmaceutical Society of Great Britain, London, UK

Professor Franco Tagliaro

Professor Donald RA Uges

Dr Brian Widdop

Department of Medicine and Public Health, University of Verona, University Hospital, Verona, Italy

Laboratory for Clinical and Forensic Toxicology and Drug Analysis, University Hospital Groningen and University Centre of Pharmacy, Groningen, The Netherlands

Formerly, Medical Toxicology Unit, Guy’s and St Thomas’ Hospital Trust, London, UK

Dr Andrew Taylor

Dr Alain Verstraete

Royal Surrey County Hospital, Guildford, Surrey, UK

Klinische Biologie, Universitair Ziekenhuis, Ghent, Belgium

Dr Phil Teale

Dr David Watson

Medication and Doping Control, HFL Sport Science Quotient Bioresearch, UK

Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK

Dr Steve HY Wong

Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 10:24:0

About the Editors

Professor Anthony C Moffat BPharm, PhD, DSc, CChem, FRSC, FRPharmS, FFIP, FFSSoc

Professor Anthony C Moffat is Emeritus Professor of Pharmaceutical Analysis at The School of Pharmacy, University of London, where he was previously Head of the Centre for Pharmaceutical Analysis. He was also Chief Scientist at the Royal Pharmaceutical Society. He has over 350 publications as well as the co-authorship of eight books. Previously he worked for the Forensic Science Service for 23 years as Research Co-ordinator (Birmingham Laboratory), Resources Manager (Huntingdon Laboratory), Head of Quality Management (HQ, London), Assistant Director (Huntingdon Laboratory), and Head of the Drugs and Toxicology Division at the Home Office Central Research Establishment, Aldermaston. He has also been a Superintendent Pharmacist in a community pharmacy, Assistant Professor of Biochemistry, Baylor College of Medicine, Houston, Texas, and Chief Pharmacist, St Leonard’s Hospital, London. An active member of many professional and learned societies, his fellowships include the Royal Pharmaceutical Society, Royal Society of Chemistry, Forensic Science Society, International Pharmaceutical Federation and the American Association of Pharmaceutical Scientists as well as the membership of the International Association of Forensic Toxicologists. Professor M David Osselton BSc, PhD, CSci, CChem, FRSC, MEWI

Professor M David Osselton started his forensic toxicology career in 1974 when he went to work with Dr Alan Curry at the Home Office Central Research Establishment, Aldermaston. He gained casework experience as Senior Toxicologist working at the Home Office Forensic Science laboratories in Nottingham and Huntingdon before returning to Aldermaston in 1984 to succeed Dr Anthony Moffat as Head of Research in Alcohol, Drugs and Toxicology. In 1991, he was appointed Head of Toxicology for the Forensic Science Service. In 2007, Professor Osselton went to Bournemouth University as Head of the Centre for Forensic Sciences. He has wide experience in toxicology casework and has been involved in numerous high profile cases working for the defence and prosecution both in the UK and overseas. He is

internationally known for his research interests in toxicology and lectures widely at conferences as a plenary and keynote speaker. Between 2003 and 2009 he was Lead Assessor (Toxicology) for the UK Council for the Registration of Forensic Practitioners (CRFP) and was Visiting Professor to the Department of Forensic Science and Drug Monitoring at Kings College, University of London (2004–2007). He is a Fellow/Member of a number of professional and learned bodies including the Royal Society of Chemistry, Royal Society of Medicine, Expert Witness Institute, International Association of Forensic Toxicologists (TIAFT), Society of Forensic Toxicologists (SOFT), LTG (formally the London Toxicology Group), UK Workplace Drug Testing Forum and is chair of the United Kingdom and Ireland Association of Forensic Toxicologists. Dr Brian Widdop BSc, PhD, SRCS, CChem, FRCS, FRCPath

Dr Brian Widdop was Director of the Medical Toxicology Unit Laboratory at Guy’s Hospital, London, from 1970 until 2002. Dr Widdop has been a Speciality Assessor for the Council for the Registration of Forensic Practitioners, a member of the WHO IPCS Working Group on Analytical Toxicology, Chief Advisor to the South East Asia Regional Office of the WHO on analytical toxicology and was a Director of the Board of the UK Horse-racing Forensic Laboratory from 1991 to 2002. From 1997 to 2001, he was joint co-ordinator of the European proficiency Testing Scheme for Drugs of Abuse. Dr Widdop has published over 80 papers on various aspects of clinical and analytical toxicology and has spoken at many international meetings. He is also a member of the editorial board of the Journal of Analytical Toxicology. Dr Widdop belongs to several international scientific societies and was a founder member of the London Toxicology Group. He has been a member of The International Association of Forensic Toxicologists for 42 years and was the recipient of the Alan Curry Award in 2002. Dr Jo Watts BSc, PhD

Dr Jo Watts attained her degree in pharmacology and toxicology followed by a PhD in neuropharmacology, both at The School of Pharmacy, University of London. She is a member of TIAFT and the LTG.

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Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 10:27:22

Foreword

As one of the past presidents of the International Association of Forensic Toxicologists (TIAFT) it is an honour as well as a pleasure for me to write a foreword for the fourth edition of this prestigious publication. Indeed, when a publication is prepared by an impressive number of leading toxicologists working in world-famous institutions, as editors or as former or new authors – all outstanding specialists in their respective fields of activity – we as toxicologists can only expect to have another great database in our hands with which to do research or our daily work. In addition to the monographs revised from the previous editions, dealing with physicochemical and pharmacotoxicological properties of drugs and poisons, and the indexes of analytical data, review chapters have been included on various basic subjects of toxicology, such as hospital and forensic toxicology; immunoassays; analysis of alternative matrices; doping; driving under the influence of drugs and alcohol; therapeutic drug monitoring; workplace testing; quality assurance; pharmacokinetics; pesticides; volatile organic substances; natural toxins; different separation technologies; and spectrometric methods. New chapters by renowned experts have been added that deal with method development and validation; sampling, storage and stability; extraction; more recently developed analytical technologies such as liquid chromatography coupled to mass spectrometry; atomic absorption spectrometry, or inductively coupled mass spectrometry for the determination of inorganic poisons; pharmacogenomics; measuring and reporting uncertainty; drug facilitated sexual assaults; and paediatric forensic toxicology. These topics enable our younger and less young colleagues alike to familiarise themselves with these developments or to improve their knowledge. Especially at a time when shortcuts are made for budgetary reasons in healthcare and forensic systems, we need to document our performances of reliable analytical results followed by correct interpretations of these results to proof our usefulness to decision makers. Therefore, this fourth edition is published at the perfect moment. I remember very well at the beginning of my career in Luxembourg the moment I held the first edition from 1969, which was recommended to me by a French colleague. It was always a bible for me and is still an important part of my personal library. Several similar publications have been produced in the past, but they have never had the same impact on

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toxicology as Clarke. My professional work was also influenced by the renowned British scientists who I met during a TIAFT conference in Ghent back in the early ’70s. What a lot has changed since those days! It is an important advance that a chapter on interpretation has been added, as this is the major difference between analytical toxicology and analytical chemistry. Toxicological analysis is not analytical toxicology. From my work in forensic toxicology, I know that in court we are questioned more about interpretation of our results than about the performance of the analytical methods. Our customers should be aware that we are not ‘only making measurements’. Since toxicology is a multidisciplinary science, toxicologists need not only to have comprehensive knowledge of analytical methodology, but also to be proficient in the basics of medicine, physiology, clinical chemistry, biochemistry, pharmacodynamics and pharmacokinetics in order to provide the best possible contributions to clinicians and to forensic authorities. Toxicology is a difficult and a complex issue requiring collective information exchange among toxicology specialists from all related fields. Even the publishing of routine cases that may not be routine for other colleagues should be encouraged. In spite of the fantastic efforts in recent years to establish correlations between toxicant concentrations in body tissues and pharmacodynamic action, behaviour impairment, interindividual variability, pharmacogenetics, postmortem changes or concurrent existing pathologies, there is still a lot of research required to improve our knowledge. So, I can only recommend to my colleagues: let us do it! Even now that a staggering amount of information is available via the internet, Clarke remains a reference for old and young toxicologists. It is an easily accessible tool which can be consulted either by reading the book or by browsing and searching the online version, to give us useful structured, and peer-reviewed information written by well-known experts. I wish Clarke the best success that it deserves, to equal that of the previous three editions of this publication. Robert Wennig, PhD Past President of TIAFT

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 16:42:57

Preface

Clarke’s Analysis of Drugs and Poisons aims to be the world’s leading text on the analysis of drugs and poisons. Not only does it contain chapters on the methodology and techniques of modern analytical toxicology, but the monographs include analytical data on therapeutic drugs, drugs of abuse, drugs misused in sport as well as pesticides, metals and other poisons. This fourth edition builds on the previous editions with significant updating and improvements in scope and electronic form. Clarke, as it is affectionately known, has gained a world-wide reputation as a reliable source of toxicological information. Its presence on the benches of many different types of pharmaceutical and toxicological laboratories is a testament to its usefulness. Since the third edition was published in 2004, there have been about 120 new chemical entities brought onto the market. Some of these come from completely new chemical or pharmacological groups, but most are ‘me too’ drugs. In addition, there has been a growth of drugs misused in sport and those subject to abuse; eg legal cannabinoids and Mephedrone, and their derivatives. Nearly 400 monographs have been added to the fourth edition, with priority based on the importance of the substances covered in one of the following areas: drugs of abuse, forensic toxicology, hospital emergency toxicology, doping in sport, drugs subject to therapeutic drug monitoring and environmental toxicology. It has been impossible to include all the new drugs and poisons available, but work is continuing to add further data in the future. The information in Clarke has been designed to provide methods and data to enable analysts to detect, identify, quantify and profile drugs and poisons in a wide variety of situations. In addition, information on how to interpret the analytical data is included, since this is often the most difficult part. The book has been designed for use not only in hospital and toxicology laboratories, but also in numerous other analytical establishments. This includes quality control laboratories, and clinical laboratories engaged in drug investigations for purposes such as therapeutic drug monitoring or research into pharmacokinetics and patterns of drug metabolism. In addition, there is much information that will be of use in environmental toxicology, particularly the analysis of toxic metals and pesticides. The needs of students studying analytical and forensic toxicology have not been forgotten and the chapters form an excellent basis for study. The spin-off book Clarke’s Analytical Forensic Toxicology from the third edition of Clarke is a testament to meeting the requirements of the university teaching sector. The book is in two volumes to make it easier to use. Volume 1 contains chapters comprising methodology and analytical techniques, and the subject index to both volumes; Volume 2 contains the analytical and toxicological data, indexes to the analytical data, a list of reagents and a repeat of the subject index to both volumes. Those who regularly use Clarke will be pleased to see that the original style and form of presentation of the information has been retained from the previous edition. This tried and tested format is clear, making it easy to find relevant information. Clarke is now an established publication on MedicinesComplete, which provides online access to some of the world’s leading drug and healthcare references. This includes such reference sources as Martindale: The Complete Drug Reference, British National Formulary, The Merck Index and Stockley’s Drug Interactions. The online version of Clarke has the advantage that text searches can be performed thus aiding the reader to access relevant information more rapidly, either in Clarke alone or across multiple reference sources. Another advantage of the

online version is that it can be updated online far more frequently and easily than the conventional book form.

Volume 1 Part 1: methodology and analytical chapters This part now contains 44 chapters describing methodology and analytical techniques, which is an increase of 13 chapters from the previous edition. Three of the previous chapters have each been split into two because of the increased complexity of the topics covered. Thus there are now chapters on Driving Under the Influence of Alcohol as well as Driving Under the Influence of Drugs; Drugs in Human Sport as well as Drugs in Animal Sport; and Quality Control and Assessment in the Pharmaceutical Industry as well as Quality Control and Assessment in the Toxicology Laboratory. This latter chapter recognises the increase role of accreditation in the forensic toxicology laboratory and gives guidance on how to achieve this. A new chapter on methodology in Drug-facilitated Sexual Assault has been included to recognise the rise in this type of crime and the need for good forensic toxicological analyses. In terms of the use of particular analytical techniques, Method Development and Validation is a new chapter to assist those who need to develop their own methods and demonstrate that they are fit for purpose. Also included in this area are two new chapters on Sampling, Storage and Stability, as well as Extraction, since many toxicologists have asked for information on these topics. The increased use of liquid chromatography–mass spectrometry to replace gas chromatography–mass spectrometry in the analysis of organic compounds has been covered by a new chapter on this topic. Similarly, a new chapter on Atomic Absorption Spectroscopy, Inductively Coupled Plasma–Mass Spectrometry has been added to recognise the increased use of this combination of techniques in inorganic analysis. Four new chapters have been included to assist the toxicologist to interpret analytical data and report the results in a meaningful and clear manner. The chapter on Measuring and Reporting Uncertainty is a clear exposition that all measurements are subject to error and gives guidance on how to measure and report the uncertainty. A chapter on Paediatric Forensic Toxicology recognises that children are not just small adults and need to be treated as a separate population. Similarly, the chapter on Pharmacogenomics clearly shows how we as individuals differ in our genetic makeup and how that might affect our response to drugs. Often one of the most difficult tasks a toxicologist has is to do is to interpret the results of the analyses; a new chapter on Interpretation of Results, together with the updated chapter on Pharmacokinetics, aims to assist toxicologists in this area. This backs up the information on interpretation given in each of the methodology chapters. All the other chapters have been revised to bring them fully up to date. The structure of the spectroscopic and chromatographic chapters has been retained from the previous edition to ensure that all the relevant information is given in an easy-to-read form. The chapter on emerging techniques has been completely rewritten to acknowledge the regulatory aspects of introducing new techniques and what new instrumentation might be available in the future. The chromatographic and capillary electrophoresis systems have been extensively expanded and revised to include general screening systems as well as specialised systems for particular classes of drugs and poisons. The general systems for use have all been proven as robust xiii

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Preface

and reproducible over the years, and give excellent results for use in systematic toxicological analysis. Subject index The subject index covering both volumes can be found at the end of Volume 1.

The section entitled Disposition in the Body gives data on therapeutic concentration, toxicity, bioavailability, half-life, volume of distribution, clearance, distribution in blood, plasma : saliva ratio, protein binding and dose to enable analytical data to be interpreted in the context of a given case. In addition, abstracts from published clinical studies and case histories are included. Part three: indexes to analytical data

Volume 2 Part 2: analytical and toxicological data This part contains monographs for 2111 drugs and poisons, which is an increase of around 370 from the last edition. Not only have totally new monographs been introduced, but monographs from previous editions that were excluded from the third edition have been reinstated because the drugs concerned are still used in some parts of the world. The new additions have been chosen for drugs and poisons that are new and widely used prescription drugs, novel drugs of abuse or common poisons not previously included. For example, there are now 15 new monographs on metal salts. All the other monographs have been updated from the third edition. The use of the Recommended International Nonproprietary Name (rINN) for the drug name has been continued as this is now the international standard method of nomenclature. The orientation of the chemical structures has been normalised so that the structures of similar compounds may be compared more easily. In addition, new chemical and analytical data have been added to aid the toxicologist and pharmaceutical analyst. This includes information on stability of drugs in solution and biological fluids at different temperatures, 1-chlorobutane extraction data, and infrared spectra of drug salts. Analytical data for compounds on colour tests, thin-layer chromatography, gas chromatography and high performance liquid chromatography are given from which to choose systems that will separate and identify drugs, poisons and their metabolites. This is followed by full ultraviolet, infrared and mass spectral data together with listings of the major peaks to assist further in identifying compounds. A major change has been made to the Quantification section of each monograph: it has been rearranged to give details of the analysis of each biological fluid or tissue separately instead of being ordered by technique. This makes finding an analytical method to use for a particular tissue very much easier. Additional data such as a method’s limit of detection and limit of quantification have been added when available. This has meant a considerable increase in the size of each monograph and the list of the references at the end of the monograph, but it has improved the usability of the information. All the monographs have been brought up to date by the inclusion of new references and the deletion of old ones whenever possible. The references cited give further information on published methods for separating, identifying and quantifying drugs, poisons and their metabolites. Review articles are given whenever relevant to act as a further source of concise information.

This part contains indexes of analytical, chemical and therapeutic data, arranged in a similar order to how they appear in the monographs: CAS numbers, molecular formulae, therapeutic classes, molecular weights, melting points, colour tests, thin-layer chromatographic data, gas chromatographic data, high performance liquid chromatographic data, ultraviolet absorption maxima, infrared peaks, mass spectral data of drugs, and mass spectral data of pesticides. A list of reagents and proprietary test materials mentioned in the analytical procedures in Parts One and Two is also provided, as is a list of pharmacological terms. Subject index The subject index covering both volumes is repeated at the end of Volume 2.

Preparation of this edition We are grateful to the editorial and production staff at Pharmaceutical Press who have helped in this project: Emma Burrows, Helen Carter, Tamsin Cousins, Amy Cruse, Simon Dunton, Marian Fenton, Rebecca Garner, Austin Gibbons, David Granger, Jo Humm, Jean Macpherson, Julie McGlashan, Louise McIndoe, Ithar Malik, Jason Norman, Karl Parsons, The Prescribers at The School of Pharmacy (London), Lucy White and John Wilson. There were also the freelance staff who wrote and updated the monographs, to whom we owe thanks: Irene Chiwele, Mildred Davies, Laurent Y Galichet, Poppy McLaughlin, and Eva Reichardt. A team of copyeditors, proofreaders and indexers also contributed to the production of this publication. Without the enthusiasm and dedication of these people this work would not have been published. The Editorial Board members have also assisted in many ways: they authored, refereed manuscripts and monographs, and provided analytical data from their own laboratories. They and the authors have done a great job in providing up-to-date information in an easily accessible and readable manner. A C Moffat M D Osselton B Widdop J Watts January 2011

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General Notices

Health and Safety This work is intended to be used by appropriately qualified and experienced scientists. Processes and tests described should be performed in suitable premises by personnel with adequate training and equipment. Care should be taken to ensure the safe handling of all chemical or biological materials, and particular attention should be given to the possible occurrence of allergy, infection, fire, explosion or poisoning (including inhalation of toxic vapours). Cautionary notes have been included in a number of monograph entries, but the possibility of danger should always be kept in mind when handling biological samples, and medicinal or other chemical substances.

Classification At the head of each monograph, an indication is given of the classification of the compound according to its therapeutic or commercial use, its pharmacological action and/or its chemical group. The substance may, of course, have other uses or actions in addition to that stated.

Nomenclature

lauril laurilsulfate meglumine mesilate metilsulfate mofetil napadisilate napsilate octil pivalate steaglate tebutate teoclate tosilate xinafoate

n-dodecyl n-dodecylsulfate N-methylglucamine methanesulfonate methylsulfate 2-morpholinoethyl 1,5-naphthalenedisulfonate 2-naphthalenesulfonate octyl trimethylacetate steroyl-glycolate tert-butylacetate 8-chlorotheophyllinate p-toluenesulfonate 1-hydroxy-2-naphthoate

IUPAC Names The nomenclature generally follows the definitive rules issued by IUPAC, 1993.

Monograph Titles

Proprietary Names and Synonyms

The main titles of the monographs are the Recommended International Non-Proprietary Names (rINNs), this includes both drugs and pesticides. For drugs of abuse, the most common chemical names or abbreviations have been used. It is worth noting that for rINNs and chemical nomenclature, it is now general policy to use ‘f’ for ‘ph’ (e.g. in sulpha), ‘t’ for ‘th’ and ‘i’ for ‘y’. For this reason, entries in alphabetical lists and indexes should be sought in alternative spellings if the expected spellings are not found. The main title of a monograph is generally that of the free acid or base as this is the form in which the compound will usually be isolated in an analysis; details of the commonly available salts are included in subsidiary paragraphs within the monograph. The following abbreviated names for radicals and groups are used in the titles.

A selection of proprietary names have been included in the monographs. These can generally be applied to the UK, USA, Japan and a selection of African, Asian and European countries. Comprehensive lists of proprietary names worldwide, can be found in Martindale: The Complete Drug Reference, 37th edn, London, Pharmaceutical Press, 2011. Only singlesubstance preparations have been included except in the case of certain major classes of drugs for which the names of some compound preparations have been added. Some proprietary names that are not in current use have been retained. Names under the heading ‘Synonyms’ include alternative names, common titles, abbreviations and drug trial numbers.

Recommended name acetonide aceturate amsonate besilate camsilate caproate cipionate closilate edetate edisilate eglumine embonate enantate erbumine esilate gluceptate hibenzate isetionate

Chemical name (isopropylidenedioxy) N-acetylglycinate 4,40 -diaminostilbene-2,20 -disulfonate benzenesulfonate camphorsulfonate hexanoate cyclopentanepropionate p-chlorbenzenesulfonate ethylenediaminetetraacetate 1,2-ethanedisulfonate N-ethylglucamine 4,40 -methylenebis (3-hydroxy-2-naphthoate) (=pamoate) heptanoate tert-butylamine ethane sulfonate glucoheptonate o-(4-hydroxybenzoyl)benzoate 2-hydroxyethanesulfonate

CAS Registry Numbers Chemical Abstract Service (CAS) registry numbers are provided, where available, in the monographs to assist readers to refer to other information databases.

Molecular Weights Molecular weights have been calculated using the table of Atomic Weights as revised in 2001 by the Commission on Atomic Weights, IUPAC General Assembly, and based on the 12C scale. Molecular weights have been corrected to one decimal place and are listed in ascending order in the index of Molecular Weights.

Physical Characteristics Dissociation Constants Numerous methods can be used for the determination of dissociation constants, and there are often differences in the various values reported in the scientific literature. The pKa values given in the monographs have been taken from published data and should be regarded only as approximate. The temperature at which the determination was made is given where known. xv

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General Notices

Information on the theory, measurement and evaluation of dissociation constants is given in The Pharmaceutical Codex, 12th edn, London, Pharmaceutical Press, 1994.

of the phase volume ratios needed for a successful extraction from 1chlorobutane. Colour Tests

Melting Points The melting points recorded in the individual monographs are listed in ascending order in the index of Melting Points. Partition Coefficients

Where colour tests are given in the monographs, these names refer to the tests described in the Colour Tests chapter, where complete tables of colours are provided. Reference should be made to this chapter for an explanation of the system used for describing the colours. The reagents used for the colour tests are also listed within the list of reagents and additional colour reaction data for approximately 250 compounds is also presented. Colour tests applicable to biological fluids are described under the Hospital Toxicology chapter.

Values for log P are given in a number of monographs. Where the pH of the aqueous phase is stated, the values given are apparent coefficients at that pH (not ion-corrected). Where no pH is stated for the aqueous phase, it can be assumed that log P is for the neutral form of the substance even though it is potentially ionisable. The values given are approximates only but they serve to indicate the characteristics of the substance when it is submitted to an extraction process. For a comprehensive collection of partition coefficients for drugs see C. Hansch et al., Exploring QSAR: Hydrophobic, Electronic and Steric Constants, Washington, American Chemical Society, 1995. Information on the theory of partition coefficients can also be found in J. Sangster, Octanol-Water Partition Coefficients: Fundamentals and Physical Chemistry, New York, John Wiley, 1997.

The thin-layer chromatographic systems referred to in the monographs are described in the TLC chapter on together with lists of data for drugs in important chemical and pharmacological classifications. General screening systems (systems TA to TF and systems TL, TAD, TAE, TAF, TAJ, TAK and TAL), which include over 1500 drugs and metabolites, are provided (see Chapter 39 for system details and references). In order to clarify the presentation of values, the data are expressed in terms of Rf100 (hRf ). Complete lists of data, in ascending order, are given in the index of Thin-layer Chromatographic Data.

Solubility

Gas Chromatography

The solubilities given in the monographs, unless otherwise stated, apply at ordinary room temperature. They have been obtained from various sources and should not be regarded as precise because of variations depending on the method and condition of determination. In general, approximate values are given when a substance is soluble in less than1000 parts of solvent. Where no figure is given, the usual solubility terms have been adopted:

The gas chromatographic systems referred to in the monographs are described in the GC chapter, together with lists of retention data for drugs in important chemical and pharmacological classifications. A general screening system (system GA), which includes over 1500 drugs and metabolites, is provided. An alternative screening system (system GB) is also included (see Chapter 40 for system details and references). For most of the systems, the data are given in terms of Retention Index. Retention times or relative retention times are used in a few systems. Complete lists of retention data, in ascending order, are given in the indexes of Gas Chromatographic Data.

Very soluble Freely soluble Soluble Sparingly soluble Slightly soluble Very slightly soluble Practically insoluble or insoluble

1 part in less than 1 1 part in 1–10 1 part in 10–30 1 part in 30–100 1 part in 100–1000 1 part in 1000–10000 1 part in more than 10000

Thin-layer Chromatography

High Performance Liquid Chromatography

Temperatures are expressed throughout the text in degrees Celsius (centigrade).

The HPLC systems referred to in the monographs are described in the HPLC chapter, together with lists of retention data for drugs in important chemical and pharmacological classifications. Six general screening systems (systems HA, HX, HY, HZ, HAA and HBK) covering between400 and 1600 drugs are provided (please note that values for system HBK have not been included within monographs and can only be found in the index) (see Chapter 41 for system details and references). The data are given in terms of Retention Index, retention time, relative retention time and column capacity ratio k (see Chapter 41). Complete lists of retention data, in ascending order, are given in the indexes of High Performance Liquid Chromatographic Data.

Analytical Data

Ultraviolet Absorption

All analytical data in the monographs apply to the form of the substance described in the main title of the monograph, unless otherwise specified. In all lists or indexes of chromatographic data, a dash indicates that the value is not known, not that the substance does not elute.

The wavelengths of principal and subsidiary peaks are recorded in each monograph for acid, alkaline and neutral solution, where available. These are generally listed from 230 nm. Values in neutral solution are given for compounds for which values in acid or alkaline solution are not available or when the values in neutral solution differ significantly from those in acid or alkaline solution. In many monographs, the ultraviolet spectrum is reproduced. In these spectra, the following notation is used:

In the solubility statements, the word ‘water’ refers to purified water, the word ‘ether’ refers to diethyl ether and the word ‘ethanol’, without qualification, refers to ethanol (95%). Temperature

Extraction It has not been possible to give direct information on the best method for extracting individual substances from various biological samples. However, useful information can be gained from the data on solubility, dissociation constant and partition coefficient. The best solvent can be chosen by reference to solubility, the pH for extraction is indicated by the pKa value, and the partition coefficient gives a quantitative measure

————————— ...................................... -----------------

acid solution alkaline solution neutral solution

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General Notices Where more than one curve is shown, they do not necessarily relate to the same concentration and, consequently, points where the curves cross cannot be taken as true isosbestic points. The wavelengths of peaks in a few of the spectra may differ very slightly from those stated in the text. Where there is doubt, the values given in the text should be used. In monographs where the spectrum is reproduced, the A11 value for each peak is stated, if available. The A values apply to the form of the substance described in the main title of the monograph, unless otherwise stated. The A11 values are divided into 3 categories in order to provide an indication of reliability: n

n n

The letter ‘a’ after a figure indicates that the value is a mean value based on several reported figures, all of which lie within a range of 10% of the mean. The letter ‘b’ after a figure indicates that the value is a single reported value of unknown reliability. The letter ‘c’ after a figure indicates that the value is a mean value based on several reported figures, some of which lie outside 10% of the mean.

The phrase ‘no significant absorption’ indicates that no peaks are found at the concentrations normally used. The A11 values quoted in the monographs may be useful in identification, and may help in determining the strength of a solution which is required to obtain a curve within the instrumental range of absorption. They may also be useful to give an approximate indication of the amount of drug in a solution. However, because of instrumental differences and the possible effect of solvent and pH, A11 values are subject to considerable variation and the values quoted should not be used when an accurate assay is required. In this case, a reference specimen should be examined at the same time as the sample. The wavelengths of main peaks are listed for acid, alkaline and neutral solution from 230 nm in the index of Ultraviolet Absorption Data.

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Unless otherwise stated, solutions of solids in liquids are expressed as percentage w/v, and solutions of liquids in liquids as percentage v/v. When acids of various strengths are specified, e.g. 50% sulfuric acid, this implies the appropriate dilution by volume of the strong acid in water.

Disposition in the Body Many of the monographs contain a section with the heading ‘Disposition in the Body’. The information in these statements has been obtained from a detailed survey of published papers and other reference sources. Certain monographs have a single reference at the end of the statement, and this indicates that all the disposition information has been obtained from that source. Wherever possible, information is included on absorption, distribution, metabolism, excretion, therapeutic concentration, toxicity and pharmacokinetic parameters. Entry to the literature is provided by the inclusion of abstracts of published papers on clinical studies or case histories. These abstracts include details of drug concentrations in plasma or other body fluids or tissues; in these data a dash means that the particular value was not determined, and ND or 0 means that the substance was not detected. Concentrations in body fluids or tissues are expressed in mg/L or mg/g. In some monographs, the information is incomplete, the amount of detail being dependent upon that available in the literature searched. It should not be assumed that the statements presented reflect the only significant factors in the disposition of the drug concerned. Therapeutic Concentration This is the concentration range usually observed after therapeutic doses, as reported in clinical studies and other research projects. It should not be interpreted as the concentration range required for optimum therapeutic effects.

Infrared Absorption The wavenumbers of the 6 major peaks in the range 2000–650 cm1 (5– 15 mm), in descending order or amplitude, are recorded in the monographs. In many cases, the infrared spectrum is also reproduced. When selecting the 6 principal peaks, those which are in the region where Nujol absorbs (1490–1320 cm1, 6.7–7.6 mm) have been omitted. Corrections for calibration errors have been applied where these are known. The 6 principal peaks, in ascending order of the main peak, are listed in the index of Infrared Peaks. Mass Spectrum The m/z values of the 8 most abundant ions, in descending order of intensity, are included in many monographs. Where dashes occur in the listing, this indicates that less than eight ions have been observed. The 8 principal ions, in ascending order of the main peak, are listed in the index of Mass Spectral Data of Drugs. A separate index for pesticides can also be found. The full mass spectra for the majority of the listed compounds are displayed within the monographs.

Toxicity This statement may include drug concentrations in blood or other body fluids or tissues, which have been reported to be associated with toxic or lethal effects. Because of inter-subject variations or other variable factors, the reported toxic or lethal concentrations may occasionally lie close to or within the therapeutic range. In some monographs, the toxic or lethal blood concentrations are stated in the form 60–89–150 mg/L. These figures have been obtained from a survey of a number of reported cases and represent the maximum concentrations found in 10, 50 and 90% of the subjects, respectively. Maximum permitted concentrations in air (8–h exposure limit) are those recommended by the Health and Safety Executive in Occupational Exposure Limits 2002, Guidance Note EH40/2002 Supplement, London, HMSO, 2003. Volume of Distribution This relates to plasma concentrations after IV administration, unless otherwise stated. Values are based in a body-weight of 70 kg.

Quantification The methods referred to in the references quoted under the heading ‘Quantification’ in the monographs are not intended to be recommended methods. These references are intended to be used as a guide to the literature on the particular subject. Reagents Reagents required for specific tests or methods are generally described fully in the appropriate place in the text. However, certain common reagents that are used throughout the book are described in the list of Reagents and Proprietary Test Materials. Reagent solutions are made in purified water unless otherwise specified. When ethanol, without qualification, is stated to be used, this refers to ethanol (95%).

Clearance This usually refers to the total plasma clearance (or total whole blood clearance) after IV administration. In some instances, the total clearance after an oral dose has been included if the drug is known to be well absorbed and is not subject to significant first-pass metabolism. Numerous factors and inter-subject variations may affect the absorption, distribution, metabolism and excretion of drugs. These include age, sex and disease states such as renal impairment. In addition, results of analyses may be subject to unavoidable analytical inaccuracies. Consequently, there may be considerable variations in the observed drug concentrations and in values for pharmacokinetic parameters in individual cases. Hence, the values given in the monographs should be used only as a guide and should not be taken as absolute values.

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Dose The dose recorded under this heading in the monographs indicates the usual daily dose (oral unless otherwise stated) that may be administered for therapeutic purposes. It is intended solely as a guide in deciding whether the amount taken by an individual falls within the normal dosage range and should not be taken as a recommendation for treatment. More detailed information on doses in different conditions and age groups may be found in Martindale: The Complete Drug Reference, 37th edn, London, Pharmaceutical Press, 2011; the British National Formulary, latest edition; or in the manufacturers’ data sheets for the products.

Comments This edition of Clarke could not have been completed without the comments on the second and the first editions, and the contribution of analytical data from many scientists involved in the analysis of drugs. In order to assist in the preparation of the next edition, the reader is invited to send any constructive comments and relevant new data concerning the analysis of drugs in biological materials to the Editor, Clarke’s Analysis of Drugs and Poisons, Royal Pharmaceutical Society of Great Britain, 1 Lambeth High Street, London SE1 7JN, UK. In this way, future editions will be improved to the benefit of all of those who use it.

Deletions The following substances which were included in Volumes 1 and 2* of the 1st and 2nd editions are now included in this edition: Acetyldihydrocodeine Adrenalone Allantoin Allylprodine Alphameprodine Alphamethadol Aminometradine Aminopentamide Amisometradine Amolanone Amopyroquine *Amotriphene Amprotropine Amydricaine Amylocaine Apoatropine Azacosterol Azamethonium Bromide Benzalkonium Bromide Benzamine Benzathine Penicillin Benzethidine Betameprodine Betaprodine *Brocresine

Butallylonal Butethamine Butoxamine Cetoxime Chlorisondamine Chloride Citronella Oil Clamoxyquin Clonitazene *Cloponone Codeine N-Oxide Cyclamic Acid Cyprenorphine Demecolcine Demeton-O Desomorphine Diampromide Dibutoline Sulphate *Diethylaminoethyl Diphenylpropionate Dimenoxadole *Dimethocaine Dimethylthiambutene *Dimophebumine Dioxaphetyl Butyrate Dioxathion

*Dioxyamidopyrine *Diphenazoline Dithiazanine Iodide Embramine Erythrityl Tetranitrate *Ethylisobutrazine Ethylmethylthiambutene *Ethylpiperidyl Benzilate Etonitazene Etoxeridine Etymide Fenimide *Fenmetramide Furethidine Hydromorphinol Hydroxypethidine *Imidocarb *Iminodimethylphenylthiazolidine *Iopydol *Iopydone Isobutyl Aminobenzoate *Isometamidium Isomethadone Laudexium Methylsulphate Leucinocaine Levomethorphan Levomoramide Levophenacylmorphan Lucanthone Metabutethamine Metabutoxycaine Metazocine *Methadone Intermediate Methaphenilene Methoxypromazine Methylaminoheptane Methyldesorphine Methyldihydromorphine Methylhexaneamine Methyridine Metofoline Metopon *Moramide Intermediate Morpheridine Morphine N-Oxide Mustine Myrophine Naepaine *Naftazone Narcobarbital Nicocodine Nicomorphine *Nifuroxime Noracymethadol Norbutrine *Nordefrin

*Norgestrel Norlevorphanol *Octacaine Octaverine Orthocaine Pamaquin *Panidazole *Paromomycin Pentaquin *Pethidine Intermediate A Phenadoxone Phenamidine Phenampromide *Phenatine Phenisonone Phenomorphan Phenoxypropazine Phenylpropylmethylamine *Phthivazid *Picloxydine Pipamazine Piperoxan Pipethanate Plasmocide *Proadifen Probarbital Proheptazine Properidine Pulegium Oil *Pyrrocaine Quinapyramine Chloride Racemethorphan Racemoramide *Resorantel *Rifamide *Rolicypram Stilbamidine Sulphasomizole Sulphonal *Taurolin Teclothiazide *Terodiline *Tetracosactrin *Tetraethylammonium Bromide *Thozalinone Thurfyl Nicotinate *Tiletamine Tolonium Chloride Tolycaine Triclobisonium Chloridc Tropacocaine Tropine Tymazoline Viomycin Xenysalate

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Abbreviations

A11 AAFS AAS 4-ABA ABFT ABP ABV AC 2-ACB 2-ACDP ACDP ACE ACFP ACh AChE ACNB ACPO AD ADC ADCB ADH ADHD ADI AED AEME AES AFID AFM AFMAB AFNB AFS agg. AGP AIDS ALDH ALL ALS ALT 6-AM AMPA AMPK AMT amu ANB AO AOAC AORC APB APC APCI APDC APEI

1% Specific absorbance (abbreviation of A1cm ) American Academy of Forensic Sciences Anabolic/androgenic steroids; atomic absorption spectrometry 4-Aminobenzoyl-b-alanine American Board of Forensic Toxicology 2-(2-Amino-5-bromobenzoyl)pyridine Alcohol percentage by volume Acetylated 2-Amino-5-nitrobenzophenone 2-Amino-5-chlorodiphenylamine 2-Amino-20 -chloro-5-nitrobenzophenone; 2Amino-5,20 -dichlorobenzophenone Angiotensin-converting enzyme 2-Amino-5-chloro-20 -fluorobenzophenone Acetylcholine Acetylcholinesterase 2-Amino-20 -chloro-5-nitrobenzophenone Association of Chief Police Officers Alzheimer’s disease Analogue-to-digital converter 2-Amino-5,20 -dichlorobenzophenone Alcohol dehydrogenase Attention deficit hyperactivity disorder Acceptable daily intake Atomic emission detector Anhydroecgonine methylester Atomic emission spectrometry Alkali flame ionisation detection Atomic force microscopy/microscope 5-Amino-20 -fluoro-2methylaminobenzophenone 2-Amino-20 -fluoro-5-nitrobenzophenone Atomic fluorescence spectrometry aggregate (in botanical names), including two or more species which resemble each other closely a1-Acid glycoprotein acquired immunodeficiency syndrome Aldehyde dehydrogenase Acute lymphoblastic leukemia Amyloid lateral sclerosis Alanine transaminase (alanine aminotransferase) 6-Acetylmorphine a-Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid AMP-activated protein kinase a-Methyltryptamine Atomic mass units 2-Amino-5-nitrobenzophenone Aldehyde oxidase Association of Analytical Chemists Association of Official Racing Chemists 3-Amino-1-phenylbutane 7-Ethyl-10-[4[N-(5aminopentanoic acid)-1piperidino]-carbonyloxycamphothecin Atmospheric Pressure Chemical Ionisation Ammonium pyrrolidine dithiocarbamate Atmospheric pressure electrospray ionisation

API APL APT AR Art 5-ASA ASL ASP AsPEX AST ASTM ASV ATD ATR AUC AUFS AV BAC BBA BBR BC BCRP 1,4-BD BDB BDMPEA BE BEN BGE bid BMAA BMC BMI BNCT BOAA BP Bp B.P. BPH BrAC BRP BSA BSH BSTFA BuChE BUN BZP CA CAM CAP CAS 2-CB CBD CBN CBQCA

Atmospheric pressure ionisation; active pharmaceutical ingredients Acute promyelocytic leukaemia Attached proton test Analytical reagent artefact 5-Aminosalicyclic acid Average signal level Amnestic Shellfish Poisoning Allele-specific primer extension Aspartate transminase (aspartate aminotransferase) American Society for Testing and Materials Anodic stripping voltametry Automated thermal desorption Attenuated total reflectance Area under the curve Absorbance units full scale Atrioventricular Blood alcohol concentration Butyl boronic acid Blood-to-breath ratio Background correction Breast cancer resistance protein 1,4-Butanediol 3,4-Benzodioxazol butanamine 4-Bromo-2,5-dimethoxyphenethylamine Benzoylecgonine Balkan endemic neuropathy Background electrolyte Twice daily b-N-Methylamino-L-alanine 4-Bromomethyl-7-methoxycoumarin Body mass index Boron neutron capture therapy b-I-Oxalylamino-L-alanine Blood pressure; Bristish Pharmacopoeia; butyrylated; benzophenone Boiling point British Pharmacopoeia Benign prostatic hyperplasia Breath alcohol concentration Biological reference preparation Bovine serum albumin; body surface area Mercaptoundecahydrododecaborate Bis(trimethylsilyl)trifluoroacetamide Butyrylcholinesterase Blood urea nitrogen N-Benzylpiperazine Carbonic anyhdrase Base-modified PEG College of American Pathologists Chemical Abstracts Service 4-Bromo-2,5-dimethoxyphenethylamine Cannabidiol Cannabinol 3-(4-Carboxy-benzoyl)-2-quinoline carboxaldehyde xix

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Abbreviations

CCD CD 2C-D CDT CE CEC 2C-E CEDIA CFP CFTB CG CGE cGMP CHE ChE CHF -CHNO CI 2C-I CIA CID CIEF CIn CIRMS CITP CL Cl CLCR Cmax CMC CNS -CO2 COHb COMT COPD COSY COX CPMACB CRA CRS CSEI CSF CSP CT 2C-T-2 2C-T-7 CTAB CTFEAB CTX CV CVAA CVAO CVVHDF CYP CZE 2,4-D DA DAB DACB DAD DAFB DART DBD DBQ

Charge-coupled device Circular dichroism 2,5-Dimethoxy-4-methyl-b-phenethylamine Carbohydrate-deficient transferrin Capilary electrophoresis Capillary electrophoresis; collision energy 2,5-Dimethoxy-4-ethyl-b-phenethylamine Cloned enzyme donor immunoassay Ciguatera fish poisoning 5-Chloro-20 -fluoro-2-(2,2,2-trifluroethylamino)benzophenone Chorionic gonadotrophin Capillary gel electrophoresis Cyclic GMP; current good (pharmaceutical) manufacturing practice Cholinesterase Cholinesterase Congestive heart failure Descarbamoyl artefact Chemical ionisation 2,5-Dimethoxy-4-iodo-b-phenethylamine Chemiluminescent immunoassay, capillary ion analysis Collision induced dissociation Capillary isoelectric focusing Colour index Combustion isotope ratio MS Capillary isotachophoresis Clearance Clearance Creatinine clearance Mean maximum plasma concentration Critical micelle concentration Central nervous system Artefact formed by decarboxylation Carboxyhaemoglobin Catechol-O-methyltransferase Chronic obstructive pulmonary disease Correlation spectroscopy Cyclooxygenase 2-Cyclopropylmethylamino-5chlorobenzophenone Controlled Substances Act Chemical reference substance Cation selective exhaustive injection Cerebrospinal fluid Chiral stationary phase Computed tomography 2,5-Dimethoxy-4-ethylthio-b-phenethylamine 2,5-Dimethoxy-4-propylthio b-phenethylamine Cetyl trimethyl ammonium bromide 5-Chloro-2-(2,2,2-trifluoro)ethylaminobenzophenone Ciguatoxin Coefficient of variation 2-Chlorovinylarsenous acid 2-Chlorovinyl arsenous oxide Continuous veno-venous haemodiafiltration Cytochrome P450 Capillary zone electrophoresis 2,4-Dichlorophenoxyacetic acid Dialkylated 2,5-Diaminobenzophenone 2,5-Diamino-20 -chlorobenzophenone Diode array detection/detector 2,5-Diamino-20 -fluorobenzophenone Direct analysis in real time 3,4-Benzodioxazol butanamine 2,6-Dibromoquinone-4-chlorimide

DBZ DC DCCA DCMAB DDD DDE DDS DEA DEACFB DECP DEG dEPO DEPT DESI DFA DFSA DHEA DHHS dH2O DHPLC DIPT DLLME DLS DMA p-DMAB DME DMES DMF DMS DMSA DMSO DNOC DNS-Cl DOB DOD DOM DON DOT DPA DPASV DPI DPV DQ DRESS DRIFT DSHEA DSP DTAB DUI DUIA DUID DVT DWI EA EAAS EC ECD ECG ECM ECT ED EDDP EDT

Dibenzosuberamine Direct current (3(2,2-Dichlorovinyl)-2,2dimethylcyclopropane-carboxylic acid 20 ,5-Dichloro-2-(methylamino) benzophenone Dichlorodiphenyldichloroethane Dichlorodiphenyldichloroethylene Drug detection system Drug Enforcement Agency 2-Diethylaminoethylamino-5-chloro-20 fluorobenzophenone Drug Evaluation and Classification Program Diethylene glycol Darbepoietin Distortionless enhancement by polarisation transfer Desorption electrospray ionisation Drug-facilitated assault Drug-facilitated sexual assault Dehydroepiandrosterone Department of Health and Human Services Distilled water Denaturing HPLC Diisopropyltryptamine Dispersive liquid–liquid microextraction Dynamic light scattering 2,5-Dimethoxyamfetamine p-Dimethylaminobenzaldehyde Dimethyl ether Dimethylethylsilyl Dimethylformamide Differential mobility spectrometry Dimercaptosuccinic acid Dimethylsulfoxide Dinitro-o-cresol Dansyl chloride 4-Bromo-2,5-dimethoxyamfetamine (US) Department of Defense 2,5-Dimethoxy-4-methylamfetamine Deoxynivalenol (US) Department of Transport Diphenylamine Differential pulse anodic stripping voltametry Dry powder inhalation/inhaler Differential pulse voltametry Design qualification Drug rash with eosinophilia and systemic symptoms Diffuse reflectance IR Fourier transform spectroscopy Dietary Supplement and Health Education Act Diarrhetic shellfish poisoning Dodecyl trimethyl ammonium bromide Driving under the influence Driving under the influence of alcohol Driving under the influence of drugs Deep vein thrombosis Driving while intoxicated/impaired Enzyme acceptor Electrothermal AAS Electrochemical Electron capture detection Electrocardiogram Enteric coated microcapsules Electrical capacitance tomography Erectile dysfunction; enzyme donor 2-Ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine 1,2-Ethanedithiol

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Abbreviations EDTA EDXRF EEG EI EIA ELCD ELF ELISA ELS EMC EMCDDA EME EMEA EMIT EMPA ENFSI EOF EPBRP EPI EPO ESA EQA ESI ET ETAAS EtG EtS ETV EU eV EWDTS FAAS FAB FAEE FAIMS FAME FASS FDA FEI FFAP FFT fg FIA FID FISH Fp FPBA FPD FPIA FPLC FPN ft FSH FT FTD FTIR FTIRD GABA GBL GC GC-HRMS GC-MS(-MS) GCS G-CSF

Ethylene diamine tetra-acetate Energy-dispersive XRF Electroencephalogram Electron Impact Enzyme immunoassay Electrolytic conductivity detection Epithelial lining fluid Enzyme-linked immunosorbent assay Evaporative light-scattering Erythromycylamine European Monitoring Centre for Drugs and Drug Addiction Ecgonine methyl ester European Agency for the Evaluation of Medicinal Products Enzyme-multiplied immunoassay technique Ethyl methylphosphonic acid European Network of Forensic Science Institutes Electroosmotic flow European Pharmacopoeia biological reference preparations Enhanced product ion Erythropoietin Electrostatic analyser External quality assurance/assessment Electrospray ionisation Ethylated Electrothermal atomic absorption spectrometry Ethyl glucuronide Ethyl sulfate Electrothermal vaporisation European Union Electron volts European Workplace Drug Testing Society Flame atomic absorption spectrometry Fast atom bombardment Fatty acid ethyl esters Field asymmetric waveform ion mobility spectrometry Fatty acid methyl ester Field-amplification sample stacking Food and Drug Administration Federation Equestre Internationale Acid-modified PEG Fast Fourier transform Femtograms Flow injection analysis; fluorescent immunoassay Flame ionisation detection; free-induction decay (NMR) Fluorescence in-situ hybridisation Freezing point 4-Fluoro-3-phenoxybenzoic acid Flame photometric detector Fluorescence polarisation immunoassay Fast protein liquid chromatography Ferric(III) chloride-perchloric acid-nitric acid Foot (feet) Follicle stimulating hormone Fourier transform Flame thermionic detection Fourier transform infrared Fourier transform infrared detector g-Aminobutyric acid g-Butyrolactone Gas chromatography High resolution mass spectrometry Tandem GC-MS Glasgow Coma Scale Granulocyte colony-stimulating factor

GFAAS GFR GH GHB GI GLC GLP GMND GMP G6PDH GPS GRM GSR GTX h HBV HCC hCG HCL -HCl -HCN HCV HD HDO HDO2 HEACFB HEPES HERG HFB HFBA HGN HHD HIV HLA HMBC HMMC HMQC HMT -H2O HOM HPLC HR HR-MS HS HS-GC HSQC -HY I IA IBS ICADTS ICH ICP ICR ICRAV ID i.d. IDA IDLH IEC

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Graphite furnace atomic absorption spectrometry; electrothermal atomic absorption spectrometry Glomerular filtration rate Growth hormone g-Hydroxybutyric acid Gastrointestinal Gas-liquid chromatography Good laboratory practice Guamanian motor neuron disease Good manufacturing practice Glucose-6-phosphate dehydrogenase Genomic prescribing system Gastric release microcapsules Gunshot residue Gonyautoxins Hour(s) Hepatitis B virus Hepatocellular carcinoma Human chorionic gonadotrophin Hollow cathode lamp Artefact formed by the elimination of hydrochloric acid Artefact formed by the elimination of hydrogen cyanide Hepatitis C virus 3b-Hydrosteroid dehydrogenase Mustard sulfoxide Mustard sulfone 2-Hydroxyethylamino-5-chloro-20 fluorobenzophenone N-(2-Hydroxyethyl)-piperazine-N0 -2ethanesulfonic acid Human ether-a-go-go-related gene Heptaflurobutyrate Heptaflurobutyric anhydride Horizontal gaze nystagmus 2-Chloro-2-hydroxyethyl sulfoxide Human immunodeficiency virus Human leukocyte antigen Heteronuclear multiple bond correlation 4-Hydroxy-3-methoxymethcathinone Heteronuclear multiple quantum coherence Hexamethylenetetramine Artefact formed by dehydration of an alcohol or by rearrangement of an amino oxo compound Humic organic matter High performance liquid chromatography Heart rate High resolution mass spectrometry Headspace Headspace gas chromatography Heteronuclear single quantum coherence Acid-hydrolysed/acid hydrolysis Spin quantum number Immunoassay Irritable bowel syndrome International Council on Alcohol, Drugs and Traffic Safety International Conference on Harmonisation Inductively coupled plasma Ion cyclotron resonance International Conference of Racing Analysts and Veterinarians Isotope dilution Internal diameter Information dependent acquisition Immediately dangerous to life Ion exchange chromatography

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Abbreviations

IFHA Ig IGF-1 ILAC IM IMPA IMS INAA INR IOC IP IQC IR IRMA IRMS IS ISE IT IU IUPAC IV J JRES k K-EDTA KIMS LA LAAM LAL l LAMPA LC LC-MS(-MS) LCTF LD50 LFA LH LIF LLE LLOQ ln LOCI LOD log LOQ LPG LSD LTFS M M (COOH-) M (nor-) M (OH-) M (ring) MACB MACDP MALDI 6-MAM MANFB MAO MAOI MAS mAU MBA MBDB 2,3-MBDB

International Federation of Horseracing Authorities Immunoglobulin Insulin-like growth factor-1 International Laboratory Accreditation Co-operation Intramuscular Isopropylmethylphosphonic acid Ion mobility spectrometry Instrumental neutron activation analysis International normalised ratio International Olympic Committee Identification points/intraperitoneal Internal quality control Infrared Immunoradiometric assay Isotope ratio mass spectrometry Internal standard Ion selective electrode Ion trap International unit International Union of Pure and Applied Chemistry Intravenous Indirect spin coupling J resolved experiment Column capacity ratio Potassium ethylenediamine tetraacetic acid Kinetic interaction of microparticles in solution Laser ablation Levomethadyl acetate Limulus amoebocyte lysate test Wavelength Lysergic acid N-(methylpropyl) amide Liquid chromatography Tandem LC-MS Liquid crystal tuneable filter Lethal dose to 50% of a population Lateral flow assay Luteinising hormone Laser or light induced fluorescence Liquid–liquid extraction Lower limit of quantification Logarithm to the base e (natural logarithm) Luminescent oxygen channeling immunoassay Limit of detection Logarithm to the base 10 Limit of quantification Liquified petroleum gas Lysergic acid diethylamide; lysergide Low temperature fluorescence spectroscopy Molar (moles per L) Carboxy metabolite N-Desmethyl metabolite Hydroxy metabolite Ring compound as metabolite 2-Methylamino-5-chlorobenzophenone 2-Methylamino-5-chlorodiphenylamine Matrix assisted laser desorption and ionisation 6-Monoacetyl morphine 2-Methylamino-5-nitro-20 -fluorobenzophenone Monoamine oxidase Monoamine oxidase inhibitor Magic-angle-spinning Milli-absorbance units Methyl boronic acid N-Methyl-1-(1,3-benzodioxol-5-yl)-2butanamine Methyl-2,3-benzodioxazol butanamine

MBTFA MCF MCPA MCPA-CoA MCPP mCPP MDA MDE MDEA MDI MDMA MDP2P MDPPP Me MECC (or MEKC) MECK MEKC (or MECC) MEL mEq mg mm MFD MGF mM MHRA MIBK MID Min MLR MLS MMA MMDA MMDBB 6-MNA MND mol 8-MOP MOPPP MO/TMS Mp MPA MPHP MPPP MQL Mr MR MRI MRL MRM MRO MRPL MRS MS MSC MSTFA MTA MTSS m/z NA NAA NACE NAD NAPA NAPQI

N-Methylbis(trifluoroacetamide) (1R,2S,5R)-(–)-Menthylchloroformate Methylchlorophenoxy acetic acid Methylenecyclopropylacetyl-coenzyme-A 2-(2-Methyl-4-chlorophenoxy)propionic acid 1-(-3-Chlorophenyl)piperazine Methylenedioxyamfetamine Methylenedioxyethamfetamine Methylenedioxyethylamfetamine Metered-dose inhalers 3,4-Methylenedioxymetamfetamine 1-(3,4-Methylenedioxyphenyl)-2-propanone 3,4-Methylenedioxya-pyrrolidinopropiophenone Methyl Micellar electrokinetic capillary chromatography Micellar electrokinetic chromatography Micellar electrokinetic capillary chromatography Maximum exposure limit Milliequivalent(s) Microgram(s) Micrometer(s) Mass fragmentographic detection Mechano growth factor Micrometre(s) Medicines and Healthcare Products Regulatory Agency Methyl isobutyl ketone Multiple ion detector Minute Multiwavelength linear regression Multi-angle light scattering Multi-angle light scattering; 2-methoxymetamfetamine 3,4-Methylenedioxy-5-methoxyamfetamine 2,3-Dimethylbenzodioxazolbutanamine 6-Methoxy-2-naphthyl acetic acid Motor neurone disease, mono-Ndealkyldisopyramide Mole 8-Methoxypsoralen 4-Methoxy-a-pyrrolidinopropiophenone Methoxime/trimethylsilyl Melting point Methylphosphonic acid; N,N-dimethyl-pphenylenediamine hydrochloride 40 Methyl-a-pyrrolidinohexanophenone 4-Methyl-a-pyrrolidinopropiophenone Minimal quantifiable limit Relative molecular mass Metabolic ratio Magnetic resonance imaging Maximum residue limits Multiple reaction monitoring Medical Review Officer Minimum required performance level Magnetic resonance spectroscopy Mass spectrometry Multiplicative scatter correction N-Methyltrimethylsilyltrifluoroacetamide 4-Methylthioamfetamine Merck tox screening system Mass to charge ratio Numerical aperture Neutron activation analysis Non-aqueous capillary electrophoresis Nicotinamide–adenine dinucleotide Acecainide; N-acetylprocainamide N-Acetyl-p-benzoquinoneimine

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 21:7:16

Abbreviations NAT2 NBD-F NBP NCE NCI NC-SPE ND NDPX ng -NH3 NHTSA NIAPCI NICI NIDA NIR NIST NLCP nm NMDA NMR NNRTI NOE NOESY NPC NPD NRC NRG-1 NSAI NSAID NSD NSP OAB OATPT2 OC ODS OECD OES OF OOS OTA P PAD PAGE PBMS PC PCA PCB PCC PCEEA PCEPA PCMEA PCP PCPR PCR PD PDA PDHID PDT PEEK PEG PEL PFB PFK PFP

N-Acetyltransferase 2 4-Fluoro-7-nitro-2,1,3-benzoxadiazole 4-(4-Nitrobenzyl)pyridine New chemical entity Negative chemical ionisation Non-conditioned SPE Nordiazepam Norpropoxyphene Nanogram(s) Artefact formed by elimination of ammonia (US) National Highway Traffic Safety Administration Negative ion atmospheric pressure chemical ionisation Negative ion chemical ionisation National Institute for Drug Abuse Near-infrared imaging National Institute of Standards and Technology National Laboratory Certification Program Nanometer(s) N-Methyl-D-aspartate Nuclear magnetic resonance Non-nucleoside reverse transcriptase inhibitor Nuclear Overhauser enhancement Nuclear Overhauser enhancement spectroscopy Normal phase chromatography Nitrogen phosphorus detection Nuclear Regulatory Commission Naphthylpyrovalerone (naphyrone) Non-steroidal anti-inflammatory Nonsteroidal antiinflammatory drug Nitrogen specific detector Neurotoxic shellfish poisoning Overactive bladder Organic anion transporting polypeptide 2 Oesophageal cancer Octadecylsilane Organization for Economic Development and Cooperation Occupational exposure standard Oral fluid Out-of-specification Ochratoxin-A Apparent partition coefficient Peripheral arterial disease Polyacrylamide gel electrophoresis Particle beam MS Precipitation chromatography; principle component Principal component analysis Polychlorinated biphenyl Pyridinium chlorochromate N-(1-Phenylcyclohexyl)-2-ethoxyethenamine N-(1-Phenylcyclohexyl)-3-ethoxypropanamine N-(1-Phenylcyclohexyl)-2-methoxyethenamine Phencyclidine N-(1-Phenylcyclohexyl)-propanamine Polymerase chain reaction; principal component regression Pulsed discharge Photodiode array Pulsed discharge helium ionisation detector 1,3-Propanedithiol Polyether etherketone Polyethylene glycol Permissible exposure limit Pentafluorobenzoyl Perfluorokerosene Pentafluoropropionate; puffer fish poisoning

-PFP PFPA PFTBA pg pGp PGRN PGx PH PHA PhAsO Ph. Eur. Ph. Int PI PIAPCI PICI PID PIFAB PIS PJ pKa PLA PLOT PLS PLSR PM PMA PMEA PMMA PMN PMPA PN PO p.o. P-III-P PPARd ppb PPC PPD ppm PPP PQ PRP PSI PSP PSX PtE PTFE PTV PVP QA QC qPCR QQQ QTOF RCI r.d. rDNA rf RFLP rhEPO rhGH RI RIA RMTC RNA RPC RRT

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pentafluoropropionylated Pentafluropropionic anhydride Perfluorotributylamine Picogram(s) p-Glycoprotein Pharmacogenetics Research Network Pharmacogenomics Permethylated hydroxypropyl 4-Hydroxyamfetamine Phenylarsine oxide European Pharmacopoeia International Pharmacopoeia PH of the isoelectric point of a protein Positive ion atmospheric pressure chemical ionisation Positive ion chemical ionisation Photoionisation detection Positive ion fast atom bombardment Product ion spectrum Personalised justice Negative logarithm of the dissociation constant Phospholipase A Porous layer open tubular Partial least-squares Partial least-squares regression Permethylated; personalised medicine 4-Methoxyamfetamine 4-Methoxyethylamfetamine 4-Methoxy-methamfetamine Polymorphonuclear leukocytes Pinacoylmethylphosphonic acid Propionylated per os (oral) Per oral Procollagen type III Peroxisome proliferator activated receptor d Part(s) per billion 4-Phenyl-4 piperidinocyclohexanol p-Phenylenediamine Part(s) per million a-Pyrrolidinopropiophenone Performance qualification Polyribosylribitol phosphate Pre-column separating inlet Paralytic shellfish poisoning Polysiloxane Phosphatidylethanol Polytetrafluoroethylene Temperature-programmed sample inlet, programmable temperature vaporising Poly(vinylpyrrolidone) Quality assurance Quality control Quantitative PCR Triple quadrupoles Quadrupole TOF Racing Commissioners International Relative density Recombinant DNA Radio frequency Restriction fragment length polymorphism Recombinant human erythropoietin Recombinant human growth hormone Retention index Radioimmunoassay Racing Medication and Testing Consortium Ribonucleic acid Reversed-phase chromatography Relative retention time

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Abbreviations

RSD RSS RT s SAMHSA SARM SBW SC SCF SCFC SCOT SDS SDS-PAGE SEC SEP SERM SFE SFST SHGB SI SID SIM SIMCA SIR SMAP SNAP-25 SNARE SNP SNPA SNR SNV SOFT -SO2NH SOP SORS sp. sp.gr. SPE SPME SPR SRM SSI SSNMR SSRI STA STIP STOCSY STP STR STX SVT 2,4,5-T t1/2 T1 T2 TBA TBAF TBAH TBDM TBPE TBSA

Relative standard deviation Root sum square Retention time second(s) Substance Abuse and Mental Health Services Administration Selective androgen receptor modulator Spectral band width Subcutaneous Supercritical fluid Supercritical fluid chromatography Support-coated open tubular Sodium dodecyl sulfate; standard deviation Sodium dodecyl sulfate polyacrylamide gel electrophoresis Standard error of calibration; size exclusion chromatography Standard error of prediction Selective estrogen receptor modulator Supercritical fluid extraction Standardised field sobriety test Sex hormone binding globulin Systeme international d’unites Surface ionisation detection Selected ion monitoring Soft independent modelling of class analogies Selected ion recording 2-Sulfamoylacetylphenol Synaptosome-associated protein of 25,000 daltons Acronym derived from "soluble NSF attachment receptor" Single nucleotide polymorphism N-Succinimidyl-p-nitrophenylacetate Signal-to-noise ratio Standard normal variate Society of Forensic Toxicologists Artefact formed by elimination of the sulfonamide group Standard operating procedure Spatially offset Raman spectroscopy Species (plural spp.) Specific gravity Solid-phase extraction Solid phase microextraction Surface plasmon resonance Selected reaction monitoring; standard reference materials Sonic spray ionisation Solid-state NMR Selective serotonin reuptake inhibitor Systematic toxicological analysis Systematic toxicological identification procedure Statistical TOCSY 2,5-Dimethoxy-4-methylamfetamine; short tandem repeat Short tandem repeat Saxitoxin Supraventricular tachycardia 2,4,5-Trichlorophenoxyacetic acid Half-life Spin-lattice or longitudinal relaxation time Spin–spin or transverse relaxation time Tetrabutyl ammonium hydrogen sulfate Tetrabutyl ammonium fluoride Tetrabutylammonium hydroxide Tert-butyldimethylsilyl Tetrabromophenolphthalein ethyl ester Total body surface area

TBW TCA TCD TCM TCP TCRC TDGO TDGO2 TDI TDM TdP TEA Tf -TFA TFAA TFMPP TFPI TGS THA THC THCA THC-COOH THEED THF TIAFT TIC TID TIS TLC 2,3,5-TMA 3,4,5-TMA TMAH tmax TMCS TMMA TMS TMSI TMSTFA TNF TOC TOCSY TOF TPAH TPI TPMT TRXRF TSD TSP TTX TVAC UAC UDP UGT UHPLC UK ULOQ UN UPLC USA USP UV V VAMP var. VD Vet. VGDS

Total body water Tricyclic antidepressant Thermal conductivity detector Traditional Chinese medicine 3,5,6-Trichloro-2-pyridinol Time-coupled time-resolved chromatography Thiodiglycol sulfoxide Thiodyglycol sulfone Tolerable daily intake Therapeutic drug monitoring torsades des pointes Triethylamine Transferrin Trifluoroacetylated Trifluroacetic anhydride 1-(3-Trifluoromethylphenyl)piperazine Tissue factor pathway inhibitor Triglycine sulfate Tetrahexylammonium hydrogensulfate Tetrahydrocannabinol 11-Carboxytetrahydrocannabinol Tetrahydrocannabinol-11-oic-acid Tetrahydroxyethylene diamine Tetrahydrofuran The International Association of Forensic Toxicologists Total ion current Thermionic detection Turbo ion spray Thin-layer chromatography 2,3,5-Trimethoxyamfetamine 3,4,5-Trimethoxyamfetamine Tetramethylammonium hydroxide Time to maximum plasma concentration Trimethylchlorosilane 2,3,5-Trimethoxymethamfetamine Trimethylsilyl Iodotrimethylsilane Trimethylsilyltrifluoroacetyl Tumour necrosis factor Total organic carbon Total correlation spectroscopy Time of flight Tetrapentylammonium hydroxide Terahertz pulsed imaging Thiopurine methyltransferase Total reflection XRF Thermionic specific detection Trimethylsilyl [2,2,3,3-2H4]-proprionic acid sodium salt Tetrodotoxin Total viable aerobic count Urine alcohol concentration Uridine diphosphate UDP-glucuronosyltransferase Ultra-high pressure LC United Kingdom Upper limit of quantification United Nations Ultra performance liquid chromatography United States of America United States Pharmacopeia Ultraviolet Volt(s) Vesicle associated membrane protein Variety Volume of distribution Veterinary Voluntary genomics data submission

Clarke's Analysis of Drugs and Poisons Chapter No. Dated: 18/3/2011 At Time: 21:7:16

Abbreviations Vol VSA v/v WADA WCOT WDXRF WHO

Volume(s) Volatile substance abuse Volume in volume World Anti-Doping Agency Wall-coated open tubular Wavelength dispersive XRF World Health Organization

Wt w/v w/w XRD XRF XRPD

Weight Weight in volume Weight in weight X-ray diffraction X-ray fluorescence X-ray powder diffraction

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Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 17/3/2011 At Time: 19:13:48

PART ONE

Chapters Methodology and analytical techniques

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 17/3/2011 At Time: 19:13:48

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:39:58

CHAPTER

1

Hospital Toxicology DRA Uges

Hospital toxicology is concerned with individuals admitted to the hospital with suspected poisoning and its prime aim is to assist in the treatment of the patient. The range of substances that may be encountered is huge and ideally the hospital laboratory will have the capability to identify and, if required, quantify pharmaceutical agents, illicit drugs, gases, solvents, pesticides, toxic metals and a host of other industrial and environmental poisons in biological fluids. In practice, few laboratories can offer such a comprehensive menu and resources are concentrated on those compounds most often involved in poisoning and for which toxicological investigations are particularly useful to the clinical services. In developed countries, hospital clinical chemistry laboratories are geared to provide these basic services and rely on support from central specialised toxicology laboratories for the rarer cases. Fortunately, in the vast majority of cases the diagnosis can be made on circumstantial and clinical evidence; there is no need for urgent analyses and these can be carried out as a routine exercise. However, when the patient’s condition is severe and the diagnosis is not clear, toxicological tests may be crucial and the analytical results must be furnished quickly (usually within 1–2 h of the patient’s arrival) if they are to have any bearing on diagnosis and treatment. Ideally, the toxic substance can be both identified and quantified within this time frame. When this is not possible, a qualitative result still has considerable value if the symptoms are consistent with the identified toxin and should be communicated to the clinician without delay. These time constraints entail an inevitable compromise between speed and analytical accuracy and precision. Consequently, the quantitative methods used may fall short of the standards required, for example, for pharmacokinetic investigations. However, they must be of sufficient quality to allow an appropriate clinical decision to be made (Peters, Maurer 2002). In this area, close liaison between the laboratory personnel and the clinician who manages the patient is essential and can save hours of fruitless effort. An attempt must be made to obtain as much information about the patient as possible. This should include not only the clinical picture, but also any previous medical history of poisoning, details of drugs or other substances to which the patient may have had access and, in cases of accidental poisoning, substances to which the patient may have been exposed. This sort of dialogue between the clinician and an experienced analytical toxicologist can often yield clues as to what the cause of toxicity might be and therefore suggest which tests should be performed as a priority. Close communication must continue if the initial tests prove negative, so that the search can be widened, or if the clinician requires advice on the interpretation of positive results. Laboratories that provide analytical toxicology analyses to assist with cases of acute and chronic poisoning often offer additional services in the area of drug abuse. An increasing number of central laboratories started with providing blood spot services. Dried whole spots on printed paper are sent to these laboratories for analysis, e.g. tacrolimus, anticonvulsants, antibiotics (tuberculosis, cystic fibrosis). This can range from diagnostic tests to uncover the covert misuse of laxatives and diuretics through to routine screening of urine samples from patients assigned to treatment and rehabilitation programmes. For the latter, the requirement is to establish the drug-taking patterns of new patients and to monitor their subsequent compliance with the prescribed treatment regime. Details of techniques suitable for these services are given in separate sections.

Causes of hospital admissions for poisoning Social and economic stresses or mental disorders often result in suicide attempts, particularly through drug overdose, one of the most common causes of emergency hospital admissions. Homicidal poisoning is relatively rare, but surviving victims of this practice are often investigated initially in the hospital environment. Individuals who have been administered substances without their knowledge to facilitate robbery or sexual abuse may also be admitted to hospital. Although in the latter scenario the victims tend to contact the medical services several days after the incident, if teenagers or young adults arrive in hospital semiconscious or disorientated, the administration of so-called date-rape drugs such as alcohol, gamma-hydroxybutyric acid (GHB), flunitrazepam or ketamine must always be considered. Poisoning in children is mainly accidental, but deliberate poisoning by parents, guardians or siblings does occur. Accidental poisoning usually takes place in the domestic environment, with young children and the elderly particularly at risk. Children may gain access to pharmaceutical products, cleaning agents (bleach, disinfectants), pesticides, alcoholic drinks and cosmetics. The confused elderly may misjudge their intake of medications or be poisoned by inappropriate handling of toxic household products. Both are susceptible to acute or chronic poisoning with carbon monoxide emitted by faulty domestic heating appliances. The workplace is another environment in which accidental poisoning occurs and the analytical results from the hospital laboratory can be important not only in medical diagnosis but also in any subsequent legal investigations that involve insurance claims. Iatrogenic intoxications occur through inappropriate medical or paramedical treatment. Neonates require intravenous dosing and the need to work out doses per kilogram of body mass or per square metre (m2) of body area introduces the risk that the total amount and volume of medicine to be administered may be miscalculated. Other causes of iatrogenic poisoning include drug interactions, use of the wrong route of administration and failure to take note of impaired liver or renal function, which reduces the patient’s ability to eliminate the drug. A common example is the accumulation of digoxin in elderly patients with reduced renal function.

Qualitative screening or quantitative analysis? Laboratories adopt different approaches to hospital toxicology. To a large extent, the range of equipment available and the skills and knowledge of the staff govern the policy adopted. Where resources are scarce, only a limited screen for common drugs and poisons may be carried out, with the main effort directed towards quantitative analyses for toxins indicated by circumstantial evidence and the patient’s clinical signs. Specialised toxicology laboratories may pursue a systematic and comprehensive toxicological screen in every case, on the grounds that the clinical and circumstantial indicators are seldom reliable, and then proceed to quantify any substances detected. While the latter approach is more likely to yield useful information, it is expensive and timeconsuming. As stated above, close liaison with the clinicians to obtain a comprehensive case history and a full clinical picture can often help to focus the resources on the qualitative and quantitative tests that are most relevant. The guidelines given in Table 1.1 are useful in this context. 3

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4

Hospital Toxicology

Table 1.1 Guidelines to help focus resources on the most relevant qualitative and quantitative tests Indications for qualitative screening

Indications for quantitative analyses

To distinguish between apparent intoxication and poisoning

When the type and duration of treatment depends on the concentration (e.g. antidotes for paracetamol and thallium)

When information about the patient is lacking (no medical history)

When the prognosis is gauged by the plasma concentration (e.g. paraquat)

When the clinical picture is ambiguous (e.g. seizures)

To distinguish between therapeutic and toxic ingestion of drugs

Where the clinical picture may be caused by a pharmacological group of drugs rather than one particular substance (e.g. laxatives, diuretics)

Mixed intoxications (e.g. methanol and ethanol)

Cases of mixed intoxication (drugs of abuse, alcohol)

Toxicological monitoring (e.g. aluminium, Munchausen's syndrome)

Poisoning with no immediately evident clinical picture (e.g. paracetamol)

Toxicokinetic calculations

Where no reliable or selective quantitative method is available (e.g. herbal preparations)

Research (e.g. efficacy of treatment), education, prevention, etc.

Diagnosis of brain death A patient with brain death may be a potential donor of organs. In such cases, the patient should have a deep coma of known origin with no indication of a central infection and normal metabolic parameters. When the primary cause of coma is drug overdose, it is important to ensure that the drug has been eliminated prior to confirming the diagnosis of brain death. This also applies to drugs that may have been given in therapy. For example, thiopental is often given in the treatment of brain oedema and during neurosurgery. The half-lives of thiopental and its metabolite, pentobarbital, increase if cardiac function is diminished or the patient is hypothermic, and therefore plasma concentrations of both compounds must always be measured. Midazolam and diazepam are also administered frequently in treating cases of brain damage, and the continued presence of active concentrations of these drugs and their metabolites should also be excluded using specific and sensitive procedures, such as high performance liquid chromatography (HPLC) and LC-MS. Even if benzodiazepines or their metabolites cannot be detected, there remains the possibility that some may still be present; for instance, active concentrations of hydroxymidazolam glucuronide may be present, since the half-life may be increased considerably with end-stage organ failure. This may suggest a provocation test with the specific benzodiazepine antagonist flumazenil. Similarly, the presence of active levels of anticonvulsants (phenobarbital, carbamazepine, phenytoin and valproate), which are also given in the treatment of brain damage, must be excluded. Again, the use of sensitive and specific chromatographic methods is essential.

For forensic reasons At the special request of the clinician For purposes of statistics, research, education, prevention, etc.

In larger clinical laboratories, the use of various liquid chromatography–mass spectrometry (LC-MS) techniques for therapeutic drug monitoring and toxicological assays has increased considerably and the introduction of liquid chromatography linked to triple quadrupole mass spectrometry (LC-MS(-MS)) has brought about an enormous increase in reliability and sensitivity both in this application (Boermans et al. 2006) and in forensic toxicology (Roman et al. 2008). Although LC-MS(-MS) is not a comprehensive screening method, if sufficient information on the likely cause of poisoning in a drugs overdose case is available, it is possible to obtain both qualitative and quantitative data for a selection of up to 15 drugs and their metabolites within 40 minutes.

Applications Confirmation of diagnosis Most patients who reach hospital in time respond well to measures designed to support the vital processes of respiratory and cardiovascular function and, as mentioned above, toxicological investigations are of only historical value. However, it is still useful to have objective evidence of self-poisoning as this usually instigates psychiatric treatment and follow-up. Differential diagnosis of coma When circumstantial evidence is lacking, a diagnosis of poisoning may be difficult to sustain simply on the basis of clinical examination, since coma induced by drugs is not readily differentiated from that caused by disease processes. Apparent poisonings can be caused by hypoglycaemic coma, a cerebrovascular accident, exhaustion (after seizures), brain damage, meningitis, withdrawal symptoms, idiosyncratic reactions (e.g. to theophylline and caffeine), allergic reactions (shock), viral infections or unexpected symptoms of a disease (e.g. Lyme disease). In these situations, toxicological analyses serve either to confirm poisoning as the cause of coma or to rule it out in favour of an organic disorder that requires alternative medical and pathological investigations.

Influence on active therapy Although supportive therapy remains the cornerstone of the management of acute poisoning, specific antidotes are available for metals (chelation agents), anticholinesterase inhibitors (atropine), methanol and ethylene glycol (ethyl alcohol, formepizole, 4-methylpyrazole), paracetamol/acetaminophen (N-acetylcysteine), digoxin (antibody fragments), calcium blockers (calcium salt), cumarines (phytonadione) and opioids (naloxone). Given a clear diagnosis, a clinician usually administers the antidote without waiting for laboratory confirmation, but subsequent analyses may help to decide whether to continue with the therapy. For example, both parenteral and oral therapy with desferrioxamine in cases of iron poisoning is indicated if patients deteriorate and the serum iron concentration is extremely high. Measurements of cholinesterase activity in serum or red cells are useful in a situation of high-dose infusions of atropine into patients exposed to organophosphate insecticides or thiocarbamates. Measures designed to reduce the absorption of poisons from the gut, such as the use of emetics, purgatives, gastric lavage and irrigation, are now considered to be of limited value and unwarranted in most cases of poisoning. The efficacy of whole-bowel irrigation is also questionable, although some advocate its use to remove sustained-release or enteric-coated preparations of, for example, iron salts and other potentially lethal poisons that have passed into the small bowel, and in the decontamination of body packers. A single oral dose of activated charcoal has largely replaced other means of reducing absorption, although it is generally useful only when given within 1 h of ingestion and fails to absorb inorganic ions, alcohols, strong acids or alkalis, or organic solvents. Techniques to increase the rate of elimination of poisons, such as diuresis, adjusting the urinary pH, haemodialysis and peritoneal dialysis, venous–venous haemofiltration and charcoal haemoperfusion, are now rarely used. Forced diuresis is now frowned upon; it is probably beneficial only in cases of poisoning with thallium and, when coupled with alkalisation of urine, chlorophenoxy herbicides. Alkalisation of urine effectively increases the elimination of salicylates, phenobarbital and chlorophenoxy herbicides. Acidification of the urine has little merit in increasing the elimination of weakly basic substances, such as amfetamines and phencyclidine. New insights provide the indication of highdose Intralipid after a severe overdose of a wide variety of drugs, e.g. lidocaine, antidepressants (see www.lipidrescue.org). Haemofiltration also has a role in this context. ‘Gut dialysis’, or the use of multiple oral doses of activated charcoal, is thought to operate by creating a drug

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Quality management concentration gradient across the gut wall that leads to movement of the drug from the blood in the superficial vessels of the gut mucosa into the lumen. So far, its efficacy has been demonstrated for carbamazepine, dapsone, phenobarbital, quinine and theophylline, and there is evidence for its application in poisoning with calcium antagonists. Most of these procedures carry inherent risks to the patient and, as pointed out already, their applications are limited to only a handful of poisons. Toxicological analyses to identify and quantify the poison should be used to ensure that they are used appropriately and at the same time to prevent overtreatment of patients who would recover without such interventions.

Table 1.2 Disturbance of clinical features and indications of possible causes Clinical feature

Disturbances and poisons indicated

General appearance

Restlessness or agitation (amfetamines, cocaine, lysergide (LSD), opiate withdrawal), apathy, drowsiness, coma (hypnotics, organic solvents, lithium)

Neurological disturbances

Electroencephalogram (EEG) (central depressants), motor functions (alcohol, benzodiazepines, GHB), speech (alcohol, drugs of abuse), movement disorders (hallucinogens, amfetamines, butyrophenones, carbamazepine, lithium, cocaine, ethylene glycol), reflexes, seizures (most centrally active substances in overdose or withdrawal), ataxia

Medicolegal aspects of hospital toxicology The primary role of the hospital toxicologist is to assist clinicians in the treatment of poisoned patients, irrespective of any other aspects that surround the case. However, some cases may have a criminal element. These can range from iatrogenic poisoning, in which a patient or relative sues a health authority and its staff for neglect, through to the malicious administration of drugs or poisons by a third party. The latter category includes victims of drug-facilitated sexual assault who have been administered drugs such as flunitrazepam or GHB to induce confusion and amnesia, and non-accidental poisoning in children. Mothers are the most frequent perpetrators of child poisoning and do so to attract sympathy and attention as a consequence of the child’s illness (Munchausen’s syndrome by proxy). When these situations arise, the hospital toxicologist is obliged to take special precautions to conserve all residual samples (human matrices as well as medicines) and documentation that may feature subsequently as part of a forensic investigation (see Chapter 9).

Vital signs Mental status

Psychosis (illicit drugs), disorientation, stupor

Blood pressure

Hypotension (phenothiazines, beta-blockers, nifedipine, nitroprusside and other vasodilators) Hypertension (corticosteroids, cocaine, phenylpropanolamines, anticholinergics)

Heart

Pulse, electrocardiogram (ECG) elevation of QT-time (tricyclic antidepressants, orphenadrine, calcium blockers, class III antiarrhythmics, fluoroquinolones, macrolide antibiotics, antipsychotics, antimycotics, lithium and many drug–drug interactions) Irregularities, torsades de pointes (phenothiazines, procainamide, amiodarone, lidocaine), heart block (calcium blockers, beta-blockers, digoxin, cocaine, tricyclic antidepressants)

Temperature

Clinical manifestations and biomedical tests Specific acute clinical manifestations and vital signs of the patient that can be important in suggesting the cause of poisoning are set out in Table 1.2. Biochemical tests that gauge the physiological status of the patient are more important in terms of the immediate management of the condition and some of the abnormalities found can also be diagnostic of the type of agent involved (see Table 1.6). These, together with the clinical manifestations and history, provide the basis for the order in which the toxicological tests are carried out.

Hyperthermia (LSD, cocaine, methylenedioxymetamfetamine (MDMA), selective serotonin reuptake inhibitors (SSRIs), dinitro-o-cresol (DNOC)) Hypothermia (alcohol, benzodiazepines)

Respiration

Depressed (opiates, barbiturates, benzodiazepines) Hypoventilation (salicylates)

Muscles

Spasm and cramp (strychnine, crimidine, botulism)

Skin

Dry (parasympatholytics, tricyclic antidepressants) Perspiration (parasympathomimetics, cocaine) Gooseflesh (strychnine, LSD, opiate withdrawal)

Other indicative features

Needle marks (parenteral injections: drugs of abuse, insulin),

Some poisons have characteristic odours that may be discerned on the patient’s body or on clothes, or in breath and samples of vomit, as listed in Table 1.3. Colours of the skin and of urine samples can also be useful indicators (Tables 1.4 and 1.5). However, these clues should be interpreted with caution and are not a substitute for proper clinical and toxicological evaluation. The results of biomedical tests are usually available before any toxicological tests have been completed; Table 1.6 highlights their potential diagnostic value.

Colour (red, carboxyhaemoglobin; blue, cyanosis, e.g. with ergotamine; yellow, DNOC)

Assays required on an emergency basis Table 1.7 lists the toxicological assays (mainly in serum, plasma or blood) that should be performed as soon as possible after admission and highlights those that should preferably be provided by all acute hospital laboratories. Emergency requests for the analysis of rarer poisons may be referred to a specialised centre. Such lists vary according to the pattern of poisoning prevalent in different countries or regions, and Table 1.7 is therefore presented only as a guideline. Notes that indicate the relevance of the assays are also included.

Quality management It is essential that the whole laboratory process be controlled strictly and subjected to regular internal and external assessments. All administrative and analytical activities should be described in detailed standard

5

Blisters (paraquat, barbiturates) Eyes

Pinpoint (opiates, cholinesterase inhibitors, quetiapine) Dilated pupils (atropine, amfetamines, cocaine) Reddish (cannabis) Reflex, movements, lacrimation, nystagmus (phenytoin, alcohol)

Nose

Nasal septum complications (cocaine)

Chest

Radiography (bronchoconstriction, metals, aspiration)

Abdomen

Diarrhoea (laxatives, organophosphates) Obstruction (opiates, sympatholytics such as atropine) Radiography (lead, thallium, condoms packed with illicit drugs)

Smell

Sweat, mouth, clothes, vomit (see Table 1.3)

operating procedures (SOPs), which should be reviewed and, if necessary, updated at regular intervals. The laboratory should have in place a system of internal quality controls and also participate in external proficiency-testing schemes. Particular attention should be given to the storage of raw analytical data, results and residual samples, and no unauthorised person should have access to patient information.

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:39:59

6

Hospital Toxicology

Table 1.3 Odours associated with poisoned patients

Table 1.5 Urine colours associated with various poisons

Odour

Potential agents or situation

Colour of urine

Poison or drug

Acetone/nail polish remover

Acetone, propan-2-ol, metabolic acidosis

Red/pink

(Aeroplane) Glue

Toluene, aromatic hydrocarbon sniffing

Ampicillin, aniline, blackberries, desferrioxamine, ibuprofen, lead, mercury, phenytoin, quinine, rifampicin

Alcohol

Ethanol (not with vodka), cleaners

Orange

Warfarin, rifampicin, paprika

Ammonia

Ammonia, uraemia

Brown/rust

Chloroquine, nitrofurantoin

Bitter almonds, silver polish

Cyanide

Bleach, chlorine

Hypochlorite, chlorine

Disinfectant

Creosote, phenol, tar

Formaldehyde

Formaldehyde, methanol

Foul

Bromides, lithium

Hemp, burnt rope

Cannabis

Garlic

Arsenic, dimethyl sulfoxide (DMSO), malathion, parathion, yellow phosphorus, selenium, zinc phosphide

Table 1.6 Biochemical and haematological abnormalities in poisoning Abnormality

Indication

Acid–base disturbances Metabolic acidosis

Ethylene glycol, salicylate, methanol, cyanide, iron, amfetamines, MDMA

Metabolic alkalosis

Chronic use of diuretics or laxatives

Camphor, naphthalene, p-dichlorobenzene

Respiratory acidosis

Opiates

Nicotine, carbon monoxide

Respiratory alkalosis

Salicylates, amfetamines, theophylline

Organic solvents

Diethyl ether, chloroform, dichloromethane

Increased anion gap

Ethylene glycol

Peanuts

Rodenticide

Increased osmolar gap

Alcohols, glycols, valproate

Pears

Chloral hydrate, paraldehyde

Electrolyte disturbances

Plants with special odours

For example Taxus, Convallaria

Hypocalcaemia

Rotten eggs

Disulfiram, hydrogen sulfide, hepatic failure, mercaptans (additive to natural gas), acetylcysteine

Ethylene glycol, oxalates, phosphates, diuretics, laxatives

Hyperkalaemia

Digoxin, potassium salts

Hypokalaemia

Theophyllline, insulin, oral antidiabetic drugs, diuretics, chloroquine

Hypernatraemia

Sodium chloride, sodium bicarbonate

Hyponatraemia

MDMA, diuretics

Mothballs Smoke

Shoe polish

Nitrobenzene

Turpentine

Turpentine, wax, solvent of parathion, polish

Glucose Table 1.4 Typical colours of the skin with poisoning

Hypoglycaemia

Colour of skin

Poison or situation

Blue, cyanosis

Hypoxia, methaemoglobinaemia, sulfhaemoglobin

Liver enzymes

Blue, pigment

Dye (amitriptyline or chloral hydrate tablets), paint

Raised transaminases

Yellow (jaundice)

Liver damage (alcohol, borate, nitrites, scombroid fish, rifampicin, mushrooms, metals, paracetamol, phosphorus, solvents)

Haematological

Insulin, oral antidiabetic drugs, ethanol (children), paracetamol (with liver failure) Paracetamol, amfetamines, MDMA, iron, Amanita phalloides, strychnine

DNOC

Anaemia, raised zinc protoporphyrin, basophilic stippling

Reddish

Carbon monoxide

Carboxyhaemoglobin

Carbon monoxide

Black, necrosis

Sodium or potassium hydroxide, sulfuric acid, burning, intra-arterial injection

Methaemoglobinaemia

Chlorates, nitrites

Raised prothrombin time

Paracetamol, coumarin anticoagulants

Yellow

Where possible, the laboratory should seek accreditation by an external authority (see Chapter 22). Request forms A specially designed request form for toxicological analyses is a useful way not only to obtain essential demographic information on the patients and the analyses required but also to gather details of symptoms, drugs prescribed, biochemical abnormalities and previous medical history. This supplements the oral information provided by the clinician. On completion of the analyses, a copy of the form with the results and interpretation entered can be returned to the clinician. An example of a request form is shown in Fig. 1.1. Collection and choice of samples Blood, serum or plasma

Blood is usually easy to obtain and the analytical results can be related to the patient’s condition and also be used in pharmacokinetic or toxicokinetic calculations. A 10 mL sample of anticoagulated blood (sodium

Lead

edetate) and 10 mL of clotted blood should be collected from adults on admission (proportionately smaller volumes from young children). Most quantitative assays are carried out on the plasma, but anticoagulated whole blood is essential if the poison is associated mainly with the red cells (e.g. carbon monoxide, cyanide, lead, mercury). Serum from coagulated blood can also be used, although the levels are almost always the same as those in plasma. Serum has the advantage that there is no potential interference from any additive. The disadvantage is that clotting takes time and occurs only at room temperature, which creates problems with the analysis of unstable analytes that require samples to be cooled immediately by immersion in ice. It is advisable in addition to collect a 2 mL blood sample into a fluoride/oxalate tube if ethanol ingestion is suspected. However, since most of the fluoride tubes used in hospitals do not contain enough sodium fluoride to completely inhibit microbial production of alcohol (the minimum fluoride concentration required in blood is 1.5% w/v), these samples are not acceptable for forensic purposes. There are conflicting reports of the dangers of contamination of samples collected after the use of disinfectant swabs containing ethanol or 2-propanol and then analysed for ethanol content. Volunteer studies (Malingre et al. 2005) have suggested that this is not a

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:39:59

Quality management

7

Table 1.7 Emergency toxicological assays Assay(s)

Intervention

Comments

Anticholinesterase inhibitors(a)

Atropine (since 2008 the use of pralidoxime or obidoxime is contraindicated)

Measure serum (or preferably red cell) cholinesterase activity

Antiepileptics (carbamazepine, phenytoin)

Multiple-dose activated charcoal

—

Benzodiazepines

Flumazenil antidote only in severe cases

Consider presence of active metabolites; withdrawal seizures

Beta-blockers

Glucagon, isoprenaline

—

Calcium antagonists

Calcium salt infusions, Intralipid

Verapamil: severe prognosis

Carboxyhaemoglobin(a)

Hyperbaric oxygen

No value after administration of oxygen

Chloroquine

High doses of diazepam

Monitor serum K+

Cocaine

Diazepam, haloperidol

—

Digoxin(a)

Potassium salts, Fab antidote

Monitor serum K+, measure serum digoxin prior to giving Fab fragments

Ecstasy group (methylenedioxyamfetamine (MDA), MDMA)

Single-dose activated charcoal, diazepam, dantrolene

Check for metabolic acidosis and hyponatraemia, hyperthermia

Ethanol(a)

Haemodialysis, vitamin B

Monitor blood glucose in children

Iron(a)

Desferrioxamine, IV þ PO

Measure unbound iron; colorimetric assays for serum iron unreliable in presence of desferrioxamine

Isoniazid

Pyridoxine

—

Lithium(a)

Haemodialysis, vitamin B

Measure serum level 6 h after ingestion

Methaemoglobin(a)

Methylene blue

Methaemoglobinaemia caused by nitrites, chlorates, dapsone, aniline

Methanol, ethylene glycol plus other alcohols

Methylpyrazole or ethanol and haemodialysis

Monitor serum ethanol levels to ensure optimum antidote administration

Methotrexate

Folinate, venous–venous haemofiltration

Measure plasma methotrexate level 4–6 h after ingestion

Nifedipine: acidosis

Opiates

Naloxone

—

Osmolality

—

Increased by alcohol, glycols, severe valproate overdose

Paracetamol(a)

N-Acetylcysteine, methionine

Measure serum level at least 4 h after ingestion; prothrombin time and international normalised ratio (INR) are useful prognostic indicators

Paraquat (qualitative urine test)(a)

Activated charcoal

Urine test diagnostic; plasma levels useful in predicting outcome

Salicylate(a)

HCO3
infusion, haemodialysis

Repeat serum salicylate assays may be needed because of continued absorption of the drug

Strychnine

Diazepam

—

Thallium

Prussian (Berlin) blue orally

Treatment continued until urine thallium levels 9 L 13–96

sum 3 L sum 5

P 5–15 0.2 L 0.5–1(d) (for addicts 1–10) Amphotericin B

924.1

S

T 0.025–1

T (3–) 5–10

P 1.5–3.5 Ampicillin

349.4

S

T 0.02–1

Amprenavir

505.6

Pl S

P (2 h) 4-6; (4–6 h) 1.5–4.5

Amrinone

187.2

Pl

1–2 (4)

Amsacrine

393.5

Pl add 1 drop lactic acid then 48 h at
20  add

T 0.03

352.5

S

0.15

0.14 (d)

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:42:8

Interpretation and advice

35

Table 1.28 continued Compound

Atenolol

Relative molecular mass 266.3

Material(a)

S

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

0.1–0.6 (1)

2 L 27(d)

Atovaquone

366.8

Pl

(10) 15–30 (50)

Atracurium

929.2

S

0.1–1 (5)

Atropine

289.4

S

0.002–0.025

0.03–0 L 0.2

Azapropazone

336.4

Pl

30–90

Azathioprine

277.3

S Pl

P 0.05–0.3

152.2

S

0.04–0.3

Azelastine

6–Mercaptopurine

383

Pl

0.002–0.003 (0.01)

1–2

Azithromycin

749.0

Pl

0.04–1

Aztreonam

435.4

S

T 1–10

Baclofen

213.7

S

0.08–0.6

1.1–3.5

Barbital

184.2

Pl

5–30

20

Tissue >2 mg/g P 50–250 L 6–9.6 L 90 Barbiturates Intermediate acting

S

1–5

10–30

Long acting

S

10–40

40–60

Short acting

S

1–5

7–10

Pl

0.001

L >30 L >80 L >10–15 Barium

137.3

Bendroflumethiazide

421.4

Pl

0.05–0.1

Benoxaprofen

301.7

Pl

Peak 0.5) 0.8–1.3

Child 0.2–0.4

L3

312.8

S

0.1–0.6

0.7

58.9

B

0.0001–0.0022

Cocaine

303.4

S

0.05–0.3

0.25–5

Codeine

299.4

S

T 0.01–0.05

0.3–1

P 0.05–0.250

L 1.6

285.4

S

399.4

S

Desmethylclozapine Cobalt

L 1–20

Morphine Colchicine Colistin

1170

S

0.15 0.0003–0.0024

0.005

P 0.003

L 0.024(d)

1–5 -(10) Cystic fibrosis 10–350 (d)

Copper

63.6

Pl

106 units

0.6–1.5

2 L5 50

Cresol

108.1

Pl

Cromoglicate sodium

512.3

Pl

0.01

26.0

B

0.001–0.006

L 120 Cyanide Cyclizine

0.5

Smoker 0.005–0.012 (–0.15)

L (1) 4–5 0.75–1

266.4

S

0.1–0.25 (0.03–0.3)

252.5

S

0.004–0.025

Cyclobarbital

236.3

S

2–10

10–15

Cyclobenzaprine

275.4

S

0.003–0.036

0.4

Cyclophosphamide

279.1

Pl

10–25

42.1

Pl

80–180

L 15 Norcyclizine

L 20

Cyclopropane Cyproheptadine

287.4

Pl

0.05

Cytarabine (Ara C)

243.2

Pl add 1 drop 1 mol/L tetrahydrouridine for stabilisation

0.05–0.5

Danazol

337.5

Pl

0.2 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:43:17

40

Hospital Toxicology

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Dantrolene

314.3

S

Toxic(c)

0.4–1.5 T 0.3–1.4 P 1–3

Dapsone

248.3

S

0.5–5

10–20 L 18(d)

Deptropine Desferrioxamine Ferrioxamine Dexamethasone

333.5

S

406.8

S

1082.5

S

0.5–3

Pl

0.05–0.265

392.5

0.015 3–15

Dexfenfluramine

231.2

S

0.03–0.06

0.15–0.25

Dextromethorphan

271.4

S

0.01–0.04

0.1

Dextromoramide

392.5

S

0.075–0.15

0.2

Dextropropoxyphene

339.5

Pl

0.05–0.75

Pl

sum 0.75–3

sum 3

Pl

Anxiolytic 0.125–0.25

1.5

Antiepileptic 0.25–0.5

L5

L3 L 0.9 1 L2 Nordextropropoxyphen Diazepam

284.7

Eclampsia, tetanus, strychnine poisoning 1–1.5 Nordazepam Diazinon

270.7

S

Approximately the same as diazepam 0.2–1.8

304.3

S

0.05–0.1 (0.5)

Diazoxide

230.7

S

10–50

Dibenzepin

295.4

S

T 0.025–0.15

50–100

P 0.1–0.5 Desmethyldibenzepin

S

sum 0.2–0.4

354.5

S

0–0.013

sum 3 L 18

Dichlorodiphenyltrichloroethane Dichloromethane

84.9

S

L 280

2,4–Dichlorophenoxyacetic acid

221.0

S

100

Dichlorvos

221.0

B

Diclofenac

296.2

S

Dicoumarol

336.3

S

8–30 (50)

50–70

Dicycloverine

309.5

Pl

0–0.1

0.2

Didanosine

236.2

Pl

0.5–2.9

Dieldrin

380.9

S

0–0.0015

L 200 L 29(d) T 0.05–0.5

50–60(d)

P 0.1–2.2

L 0.5

Diethylcarbamazine

199.3

Pl

0–0.2

Diethylpropion

205.3

Pl

0.007–0.2

0.15–0.3 2 L 5.4(d)

Difenacoum

444.0

Pl

Diflunisal

250.2

S

0.5 (9) 40–100 (200)

300–500 L 600

Digitoxin

764.9

S

0.01–0.03

0.03

Digoxin

780.9

S

T 0.0005–0.001 (was 0.002)

T (0.0014) 0.0025–0.007

L 0.04–0.1 L (0.0015) 0.01–0.03 depending on potassium level

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:43:35

Interpretation and advice

41

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c) 0.5–1

Dihydrocodeine

301.4

S

0.03–0.25

Dihydroergotamine

583.7

Pl

0.001–0.01

Diltiazem

414.5

S

0.05–0.4

0.8

Dimethadione

129.1

S

500–1000

1000

L2

L 2–6 Dimethyltryptamine

188.3

S

0.001–0.1

Dimetindene

292.4

S

P 0.01–0.05

Dinitro–o–cresol (DNOC)

198.1

S

1–5

30–60

Diphenhydramine

225.4

Pl

0.1–1

1

Diphenoxylate

452.6

S

0.01

Dipipanone

349.5

Pl

0.05

0.2

Dipyridamole

504.6

S

1–2

4

Diquat

184.2

SU

Disopyramide

339.5

Pl

2–7

8

297.5

S

20% active stronger anticholinergic

sum 8–10

296.5

S

0.05–0.4

0.5–5

171.3

S

0.3–1.4

70.1

B

427.6

B

L75–100 L5

T 0.1–1

Nordisopyramide Disulfiram

0.1–0.4

L8 Diethyldithiocarbamate Divinyl oxide Dixyrazine

L 700 0.3

L 5.5(d), 9.4(d)

Domperidone

425.9

S

0.005–0.025 (0.04)

Donepezil

379.5

Pl

0.03–0.075

(d)

Dosulepin (dothiepin)

295.4

S

0.02–0.15 (0.4)

0.8

Desmethyldosulepin

S

0.1–0.2

0.75

Dosulepin S–oxide

S

0.04–0.4

0.65–2.2

Active metabolite 6-Odesmethyldonepezil L (1) 5–19

Doxacurium chloride Doxapram

1106.1 378.5

Pl

0.01–0.3

S

(1.5) 2–5.2

9 (doxapram and keto– doxapram

Doxazosin

451.5

S

0.01–0.05

Doxepin

279.4

Pl

sum 0.05–0.35

265.4

S

sum 0.2–0.35

Doxorubicin

543.5

S

0.006–0.02

Doxycycline

444.5

S

(1-) 5–10

30

Doxylamine

270.4

S

0.05–0.2

1–2

Dronabinol (D9–tetrahydrocannabinol, THC)

314.5

Pl

0.01–0.2

0.1 L 1–18

Nordoxepin

0.5–1 L 2–4

L5

Droperidol

379.4

Pl

0.05

Dyphylline

254.3

S

6.5–14 (–20)

Edrophonium

165.2

S

0.15–0.2

0.15

Efavirenz

315.7

Pl

1.0–4.0

4–6

Emetine

480.6

Pl

-0.1

0.5

Enalapril

376.5 S

0.01–0.05 (0.1)

Desethylenalapril

40

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:43:44

42

Hospital Toxicology

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Encainide

352.2

S

3-Methoxy-O-desmethylencainide (MODE)

368.5

S

O–Desmethylencainide (ODE)

Toxic(c)

0.06–0.28

338.5

S

0.1–0.3

0.3

Endrin

380.9

S

0–0.003

0.01–0.03

Enoximone

248.3

Pl

0.2

Entacapone

305.3

Pl

0.4–1.0 (–7.0)

Ephedrine

165.2

S

0.02–0.2

1 L 5 (d)

Epirubicin

543.5

S

0.01–0.05

Eprosartan

520.6

S

0.01–0.04

Erythromycin

733.9

S

0.5–6

12–15

T 0.5–1 P 4–12 Esmolol

295.4

Pl

0.15–2

Estazolam

294.8

S

0.055–0.2

Etacrynic acid

303.1

S

0.05–0.1

Ethambutol

204.3

S

0.5–6.5

6–10

46.1

B

0–25

1000–4500 L (2250) 4000–6000

Ethchlorvynol

144.6

S

0.5–8

20 L 50

Ethinamate

167.2

S

5–10

50–100

Ethosuximide

141.2

S

40–100

Ethyl ether

74.1

S

500–1500

Ethylene glycol

62.1

S

276.4

S

Ethanol

L 100 (200 (d)) (100) 150–200 L 250 L (1400) –1900 200–500 L 2000 Etidocaine

0.5–1.5

Etilefrine

181.2

S

P 5–15

Etodolac

287.4

S

20–50

Etomidate

244.3

Pl

0.1–0.5 (–1)

Etoposide

588.6

S

T 2–6

Everolimus

958.2

B

T 0.002–0.006 (0.01)

1.6–2

P 8–14 0.006–0.009

At 2 h 0.003–0.015 Immunoassay 30–35% higher due to metabolites Famotidine

337.4

S

0.02–0.06 (0.2)

0.42 (d)

Felbamate

238.2

Pl

30–60

70–120

Felodipine

384.3

S

0.001–0.008 (0.012)

0.01–0.015

Fenbufen

254.3

S

-60

Fenfluramine

231.3

Pl

0.05–0.15

Fenitrothion

277.3

Fenofibrate

360.8

0.5–0.7 L6 1

Pl

5–30

Fenoldopam

305.8

Pl

0.003–0.06

Fenoprofen

242.3

S

15–65

Fenoterol

303.4

Pl

(0.001
) 0.01–0.04

Fentanyl

336.5

S

0.001–0.002

Fexofenadine

501.7

Pl

0.3–0.6

Finasteride

372.6

Pl

0.008–0.01

0.002–0.02

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:43:58

Interpretation and advice

43

Table 1.28 continued Compound

Flecainide

Relative molecular mass 414.4

Material(a)

S

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

T 0.45–0.9

1.5–3

P 0.75–1.25

L 2.6(d),13(d)

Flucloxacillin

453.9

Pl

3–30

Fluconazole

306.3

S

5–15 (40)

50–75

Flucytosine

129.1

S

T 25–50

100

P 50–100 Flumazenil

303.3

S

0.01–0.05

0.5

P 0.2–0.3 Flunarizine

404.5

S

0.025–0.2

0.3

Flunitrazepam

313.3

S may be reduced by microorganisms

0.005–0.015

0.05

S U not in glass container

After 24 h stop medication (equilibrium) 0.08–0.15

Fluoride

19

T 0.5–2 L3

5–Fluorouracil

130.1

S

0.05–0.3

0.4–0.6 1 neurotoxic

Fluoxetine

309.3

S

0.1–0.45

1.5–2

295.3

S

sum 0.15–0.5 (0.9)

0.4

434.5

S

0.001–0.015

Norfluoxetine

L 0.9–5.0 Flupentixol Fluphenazine

437.5

S

(0.0002–) 0.001–0.017

0.05–0.1

Flupirtine

304.3

Pl

0.5–1.5

3–4

Flurazepam

387.9

S

0.0005–0.03

0.15–0.2

Sedation 0.007

L 0.5–17

0.04–0.15

sum 0.2–0.5

N–Desalkylflurazepam

288.9

S

Flurbiprofen

244.3

5–15

Fluvoxamine

318.3

S

0.05–0.25

Furosemide

330.8

S

2–5 (10)

25–30

Gabapentin

171.2

Pl

2–20

25

0.65

Galantamine

287.4

Pl

0.03–0.14

Gallopamil

484.6

Pl

0.02–0.1

L 8(d)

255.2

S

0.5–5

T 3–5

T 0.2–1

P 20

Gamma-hydroxybutyric acid (GHB) see Hydroxybutyrate Ganciclovir

P 5–12.5 Gemcitabine

263.2

Pl

3–6

Gemfibrozil

250.3

Pl

-25

Gentamicin

449–477

S

P 4–15

T2

T 0.05–2 Glibenclamide

494.0

S

0.03–0.35

0.6

Glipizide

445.5

Pl

0.1–1.0 (2.5)

2

Glutethimide

217.3

S

2–12

12–20

Gold

197.0

S

3–8

10–15

L 30 Granisetron

312.4

Pl

0.009–0.017

Griseofulvin

352.8

Pl

0.3–1.3 (2.5)

Guaifenesin

198.2

B

0.3–1.4

Guanethidine

198.3

S

0.01

Haloperidol

375.9

S

0.005–0.015 (0.04)

0.05–0.1

Halothane

197.4

B

22–84

L 200

Heptabarb(ital)

250.3

Pl

1–4

8–15

L 0.5

L 20 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:44:6

44

Hospital Toxicology

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

0.2–1(–1.5)

Heptaminol

145.2

Pl

Heptobarbital

218.2

S

50–100

125–150

Hexachlorophene

406.9

S

0.003–0.65 (1)

L 35

0.01

n–Hexane

86.2

Pl

Hexapropymate

181.2

S

2–5

10–20

Hexobarbital

236.3

S

1–5

8 (10–20)

Hydralazine

160.2

S

(0.05) 0.2–0.9

L 50 Hydrochlorothiazide

297.7

S

0.07–0.45

Hydrocodone

299.4

S

0.002–0.024 (0.05)

Hydrogen sulfide

34.1

S

Hydromorphone

285.3

S

0.008–0.032

L >0.1

4–Hydroxybutyrate (GHB)

104.1

Pl

50–120

80 (abuse)

0.1 L 0.1 (0.2) L 0.92

L 250–280 (abuse) Hydroxychloroquine Hydroxyzine

335.9 374.9

S S

T 0.1–0.4

0.5–0.8

P 0.5–2.0

L4

P 0.05–0.09

0.1 L 39(d)

Ibuprofen

206.3

S

15–30 (5–50)

Idebenone

338.4

S

0.05–0.2

Imipenem

317.4

S

T 0.5–5

100

P 0.65–0.85 P 20–75 Imipramine Desipramine

280.4

S

0.045–0.15

266.4

S

0.075–0.25 sum 0.15–0.3

sum 0.5 L sum (0.8–) 4.5–13

Indinavir

613.8

Pl S

P (1–3 h) 7–12; (4–7 h) 3–7

10

T (10–12 h) Pl neg 0.1–0.5; resistant >0.75 Indometacin

357.8

S

0.5–3

Indoramin

347.5

Pl

0.025–0.1

Iproniazid

179.2

Pl

5

Iron

35.8

S P non-haemolytic

0.5–2

Iron

35.8

B

380–625

137.1

S

T 0.2–1

4–6

6 child 2–8 L 17

Isoniazid

P 3–10 Isopropanol

60.1

B

Acetone

58.1

B

20 L (30–) 100 200–400 L 1000

5–20

200–400 L 550

Isosorbide dinitrate

236.1

S

0.003–0.018

isosorbide mononitrate

191.1

S

0.2–0.5

Isotretinoin

300.4

Pl

T 0.4–1.8; oral 0.1–0.5

Isoxicam

335.3

S

5–25

Isradipine

371.4

Pl

0.0005–0.002 (–0.01)

Itraconazole

705.6

S

T >0.25

Hydroxyitraconazole Kanamycin

484.5

S

sum 1–4

sum 6

S

T 1–4

T 5–10

P 15–25

P 25–30

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:44:19

Interpretation and advice

45

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c) 7 (abuse)

Ketamine

237.7

S

0.5–6.5

Ketanserin

395.4

S

0.015–0.2

Ketazolam

368.8

S

0.001–0.02

P 0.08–1 Nordazepam

270.7

S

0.2–0.6

Ketobemidone

247.3

Pl

0.025–0.030

1–2

Ketoconazole

531.4

S

T 0.3–0.5

Ketoprofen

254.3

S

1–5

Ketorolac

255.3

S

0.22–0.35

5 (plasma)

Ketotifen

309.4

Pl

0.001–0.004

0.02

P 3–10 (20) P 5–15 (–20)

L 1.2 (d) Labetalol

328.4

S

0.025–0.2

Lacidipine

455.5

Pl

0.003–0.006

Lamotrigine

256.1

S

2–15

0.5–1 15 L 50(d)

Lead

207.2

B Heparinised

30 (4)

L >900 Methapyrilene

L 2–380(d) Methaqualone

250.3

Pl

0.4–5.0

>2 L >8

Methazolamide

236.3

S

Methimazole (thiamazole)

114.2

Pl

40 0.5–2 (–3)

Methocarbamol

241.2

S

25–40 (–50)

Methohexital

262.3

Pl

(0.5–) 1–6

Methotrexate

454.4

S

Active > 0.005

T >4.5 (24 h after dose); >0.45 (48 h after dose)

Methoxsalen (8-methoxypsoralene)

216.2

S

0.1–0.2

1

250

T 0.025–0.1 P 0.1–0.4 Methoxyflurane

165.0

B

30–200

2-Methyl-4-chlorophenoxyacetic acid (MCPA)

200.6

Pl

100 (500)

2-Methylchlorophenoxypropionic acid (MCPP)

214.6

Methyldopa

211.2

S

1–5

Methylenedioxyamfetamine (MDA, love drug))

179.2

S

0.4

Methylenedioxyethylamfetamine (MDEA, Eve)

207.3

Pl

0.2

L1

3,4Methylenedioxymethylamfetamine (MDMA, XTC)

193.2

S

0.1–0.35

0.35–0.5

Methylenedioxyamfetamine

L 180 100 (500) L 669 (d) 7–10 9(d) 1 (1.5) L2

179.2

S

Methylfentanyl

350.5

S

L 0.4–0.8

Methylphenidate

233.3

S

Methylthioamphetamine (4–MTA, pMTA)

181.3

Pl

Methyprylon

183.2

Pl

10–20

S

0.01–0.06

L 0.002–0.011 0.005–0.06

(0.5) 0.8 L 2.3 1 2(d),4.2(d) 12–75 (–128) L 50 (–100)

Metiamide Metildigoxin

795.0

Pl

0.0005–0.0008 (–0.003)

Metipranolol as desacetylmetipranolol

267.3

Pl

0.02–0.08g

Metoclopramide

299.8

S

0.04–0.15

0.0025–0.003 L 0.005

0.1–0.2 L 4.4(d)

Metoprolol

267.4

S

0.1–0.6

(0.65(d)) 1

T 0.02–0.34

L (4.7(d)) 12–18

Metronidazole

171.2

S

(3–)10–30

150 (200(d))

Mexiletine

179.3

S

0.5–2

2–4

Mianserin

264.4

S

0.015–0.07 (0.14–)

0.5–5 sum 0.3–0.5 L

L 35(d) 250.3

S

sum 0.04–0.125

Miconazole

Desmethylmianserin

416.1

Pl

1 (2–9)

Midazolam

325.8

S

0.08–0.25 (postoperative awake 0.1–0.04)

1–1.5 (glucuronide) -metabolites also active table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:44:39

48

Hospital Toxicology

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

Mifepristone

429.6

Pl

1–2

Milrinone

211.2

S

0.15–0.25

0.3

Minoxidil

209.3

Pl

0.04–0.25 (oral)

1.4(d),3.1(d) L 2.7(d)

Mirtazepine

265.4

S

0.02–0.1 (–0.3)

251.4

S

sum 0.05–0.3

45

L >1 (3) L (8–) 15–25

Desmethylperazine

0.5

Pericyazine (periciazine)

365.5

S

0.005–0.03

Perindopril

368.5

Pl

(0.05–) 0.08–0.15

0.1

Perphenazine

404.0

S

0.0004–0.03

0.05

Pethidine (meperidine)

247.3

S

0.1–0.8

(1–) 2

233.3

S

0.3

0.5

Phenacetin

179.2

S

5–20

50

Phenazone (antipyrine)

188.2

S

5–25

50–100

L >5 Norpethidine

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:44:58

Interpretation and advice

51

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

Phencyclidine (PCP)

243.4

S

0.007–0.24

Phendimetrazine

191.3

S

0.02–0.24 (–0.3)

Phenelzine

136.2

S

0.001–0.002 (–0.2)

0.5

Phenformin

205.3

Pl

0.03–0.1 (–0.3)

0.6

Pheniramine

240.3

Pl

0.01–0.27

L 1.9 (d)

Phenmetrazine

177.2

S

0.02–0.25

0.5 L 4

Phenobarbital

232.2

S

2–30 (–40)

30–40

94.1

S

Phenprocoumon

280.3

S

1–3

5

Phensuximide

189.2

S

4–10

80

Phentermine

149.2

S

0.03–0.1

Phenylbutazone

308.4

S

50–100

L (0.3–) 1–5

L >1.5 L3

L 45–120 Phenol

50 L 90

P 10–20 L 7.6(d) 120–200 L 400–500 Phenylephrine

167.2

S

0.03–0.1 (–0.3)

Phenylpropanolamine (norephedrine)

151.2

S

0.05–0.5

2

Phenytoin

252.3

S

8–20; baby 6–14

25; baby 15

L 48 L 70

Free fraction Physostigmine

275.3

S

0.2–2

S

8

Selenium

79.0

Pl

0.045–0.13

0.4

Sertraline

306.2

S

0.05–0.25 (–0.5)

0.29(d);1.6(d)

Sildenafil

474.6

Pl

0.025–0.25 (0.5)

Depending on cardiac function

Silver

107.9

B

0–0.005

T (10–12 h) 0.1–0.4 10 L 30 L (4–) 10–50

with silver sulfadiazine ointment for burns 0.06–0.6 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:45:6

54

Hospital Toxicology

Table 1.28 continued Compound

Sirolimus

Relative molecular mass 914.2

Material(a)

Pl

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

0.004–0.015

0.020

single therapy 0.012–0.020 Sotalol

272.4

S

0.5–3 (5)

5–10 L 40(d)

Sparteine

234.4

Pl

0.5–1

Spironolactone

416.6

S

0.1–0.5

340.5

S

0.05–0.25 (–0.5)

Stiripentol

Canrenone

234.3

Pl

4–20

20

Streptomycin

581.6

S

T 1–5

T5

P 15–40

P 40–50

Strontium

87.6

S

0.03

Strychnine

334.4

S

0.075–0.1

Sufentanil

386.6

S

L 0.2–2 0.0005–0.005

L 0.001–0.007(d)

P 0.01–0.02 Sulfaguanidine

214.2

S

30–50

Sulfamethoxazole

253.3

S

30–60 (100–200)

Sulfanilamide

172.2

S

100–150

Sulfasalazine

398.4

Pl

5–30 (–70)

Sulfinpyrazone

404.5

Pl

6–17 (–21)

Sulfonamides

SU

T 35–75

200–400

200

P 80–150 Sulindac

356.4

S

0.5–5

340.4

S

sum (including sulfone) 1–5

Sulpiride

341.4

S

0.04–0.6

Sultiam (sulthiame)

290.4

S

Sulindac sulfide

L 3.8 (d)

P 0.15–0.75 0.5–12.5

12–15 L 20–25

Sumatriptan Suramin Tacrine

295.4

Pl

0.018–0.06

1407.2

Pl

150–250

300

198.3

Pl

0.007–0.03

0.02

Tacrolimus

804.0

B

T 0.003–0.01

T 0.003–0.01

Talinolol

363.5

Pl

0.04–0.15

L 5(d)

Talipexole

282.2

Pl

Continuous infusion 0.003–0.01

Tamoxifen

371.5

S

0.05–0.5

Teicoplanin

189.4

S

10–40

200

Temazepam

300.7

S

0.3–0.9

1 L 8.2(d)

Tenoxicam

337.4

Pl

5–10

Terazosin

387.4

Pl

0.02–0.08 (–0.1)

Terbinafine

291.4

S

T 0.01–0.03

277.4

S

P 0.4–0.8

Terbutaline

225.3

S

0.001–0.006 (–0.01)

L 0.04

Terfenadine

471.7

Pl

0.0015–0.0045

0.06

T 0.02–0.15

P 0.5–3 Norterbinafine

L 0.4(d) Tetrachloroethylene

165.8

S

Tetracycline

444.4

S

5–10

L 4–5

Tetrahydrocannabinol

314.5

Pl (unstable)

P 0.05–0.125

Tetrazepam

288.8

Pl

0.05–0.6 (–1)

30

T 1–5 0.001–0.01

0.004–0.0015

Clarke's Analysis of Drugs and Poisons Chapter No. 1 Dated: 15/3/2011 At Time: 17:45:8

Interpretation and advice

55

Table 1.28 continued Compound

Relative molecular mass

Material(a)

Reference concentration (mg/L) Therapeutic(b)

Toxic(c)

Thalidomide

258.2

Pl

0.5–1.5 (–8)

Thallium

204.4

B

20

P 5–15 Thiazinamium Thiocyanate

299.5

S

0.05–0.15

58.0

S

Non-smokers 1–4

0.3 35–50

Smokers 3–12

L 200

As metabolite of nitroprusside 6–30 Thiopental

242.3

S

1–5 (flat EEG: 25–40)

10 (40–50)

Pentobarbital

226.3

S

5–10

10–15

Thioproperazine

466.6

S

0.001–0.02

0.1

Thioridazine

370.6

S

0.2–1

2 (5)

386.6

S

0.3 (0.2–1.6)

L 10–100

L 3–10 Mesoridazine

402.6

S

15 mg/L

Cathine(b)

>5 mg/L

Ephedrine

>10 mg/L

Epitestosterone(c)

>200 mg/L

Methylephedrine

>10 mg/L

Morphine(d)(e)

>1 mg/L

19-Norandrosterone(c)

>2 mg/L

Salbutamol(d)(f)

>1 mg/L

testosterone/epitestosterone (T/E) ratio(g)

>4

Table 6.4 Substances prohibited by the World Anti-Doping Agency only in-competition Stimulants

(a) Non-specified stimulants: Adrafinil; amfepramone; amiphenazole; amfetamine; amfetaminil; benzphetamine; benzylpiperazine; bromantan; clobenzorex; cocaine; cropropamide; crotetamide; dimethylamfetamine; etilamfetamine; famprofazone; fencamine; fenetylline; fenfluramine; fenproporex; furfenorex; mefenorex; mephentermine; mesocarb; methamfetamine (D-); methylenedioxyamfetamine; methylenedioxymethamfetamine; pmethylamfetamine; modafinil; norfenfluramine; phendimetrazine; phenmetrazine; phentermine; 4phenylpiracetam (carphedon); prolintane

11-Nor-delta 9-tetrahydrocannabinol-9-carboxylic acid. Unless it may be as a metabolite of a permitted substance such as pseudoephedrine. Threshold adjusted if specific gravity >1.020. (d) Sum of glucuronide conjugate and free drug concentrations. (e) Unless it may be as a metabolite of a permitted substance such as codeine. (f) Concentrations greater than 500 mg/L and less than 1 mg/L should be reported as consistent with the use of a b2-agonist. (g) Testosterone/epitestosterone ratio. Although a report must be issued for samples with a T/E ratio greater than 4, samples with lower ratios must also be reported if there is evidence of an exogenous origin of testosterone. (b) (c)

A stimulant not expressly listed in this section is a Specified Substance (b) Specified stimulants (examples): Adrenaline(b); cathine(c); ephedrine(d); etamivan; etilefrine; fenbutrazate; fencamfamin; heptaminol; isometheptene; levmetamfetamine; meclofenoxate; methylephedrine(d); methylphenidate; nikethamide; norfenefrine; octopamine; oxilofrine; p-hydroxyamfetamine; pemoline; pentetrazol; phenpromethamine; propylhexedrine; selegiline; sibutramine; strychnine; tuaminoheptane and other substances with a similar chemical structure or similar biological effect(s)

Table 6.3 Methods prohibited by the World Anti-Doping Agency at all times Enhancement of oxygen transfer The following are prohibited:

Narcotics

Chemical and physical manipulation 1. Tampering, or attempting to tamper, in order to alter the integrity and validity of Samples collected during Doping Controls is prohibited. These include but are not limited to catheterisation, urine substitution and/or alteration 2. Intravenous infusions are prohibited except in the management of surgical procedures, medical emergencies or clinical investigations Gene doping The transfer of cells or genetic elements or the use of cells, genetic elements or pharmacological agents to modulate expression of endogenous genes having the capacity to enhance athletic performance is prohibited Peroxisome proliferator activated receptor d (PPARd) agonists (e.g. GW 1516) and PPARd-AMP-activated protein kinase (AMPK) axis agonists (e.g. AICAR) are prohibited

n

The rights of the individual or team must be safeguarded against error by the analyst.

Samples in human sport are now usually collected by agencies certified in accordance with the ISO 9001 standard (www.iso.org/iso/ iso_catalogue/management_standards/iso_9000_iso_14000.htm) and using doping control officers who have been appropriately trained to collect the samples. Apart from its Code (WADA 2009h), WADA publishes an International Standard for Testing (WADA 2009c), which incorporates elements from ISO/PAS 18873 (which was withdrawn in 2005) and the ISO 9000 series of quality management system standards. WADA and most international federations always provide a second portion of the sample for defence use. This is to be opened only after the first sample has been found to contain a banned drug, and after the competitor has been notified and invited to attend the second analysis, with his or her own expert if the competitor so wishes.

All stimulants (including both their D- and Loptical isomers where relevant) are prohibited, except imidazole derivatives for topical use and those stimulants included in the 2009 Monitoring Program(a) Stimulants include:

(a)

1. Blood doping, including the use of autologous, homologous or heterologous blood or red blood cell products of any origin 2. Artificially enhancing the uptake, transport or delivery of oxygen, including but not limited to perfluorochemicals, efaproxiral (RSR13) and modified haemoglobin products (e.g. haemoglobin-based blood substitutes, microencapsulated haemoglobin products)

129

The following narcotics are prohibited: Buprenorphine, dextromoramide, diamorphine (heroin), fentanyl and its derivatives, hydromorphone, methadone, morphine, oxycodone, oxymorphone, pentazocine, pethidine

Cannabinoids

Cannabinoids (e.g. hashish, marijuana) are prohibited

Glucocorticosteroids*

All glucocorticosteroids are prohibited when administered by oral, intravenous, intramuscular or rectal routes In accordance with the International Standard for Therapeutic Use Exemptions, a declaration of use must be completed by the Athlete for glucocorticosteroids administered by intra-articular, periarticular, peritendinous, epidural, intradermal and inhalation routes, except as noted below Topical preparations when used for auricular, buccal, dermatological (including iontophoresis/phonophoresis), gingival, nasal, ophthalmic and perianal disorders are not prohibited and require neither a Therapeutic Use Exemption nor a declaration of use

(a) The following substances included in the 2009 Monitoring Program (bupropion, caffeine, phenylephrine, phenylpropanolamine, pipradol, pseudoephedrine, synephrine) are not considered as Prohibited Substances. (b) Adrenaline associated with local anaesthetic agents or by local administration (e.g. nasal, ophthalmological) is not prohibited. (c) Cathine is prohibited when its concentration in urine is greater than 5 mg/L. (d) Each of ephedrine and methylephedrine is prohibited when its concentration in urine is greater than 10 mg/L. (e) WADA use the term 'glucocorticosteroid' for what is more conventionally known as a corticosteroid.

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130

Drug Testing in Human Sport

Table 6.5 Substances prohibited by particular sports and only incompetition Alcohol Alcohol (ethanol) is prohibited in-competition only, in the following sports. Detection will be conducted by analysis of breath and/or blood. The doping violation threshold (haematological values) is 0.10 g/L. n Aeronautic (FAI) n Archery (FITA, IPC) n Automobile (FIA) n Boules (IPC bowls) n Karate (WKF) n Modern Pentathlon (UIPM) for disciplines involving shooting n Motorcycling (FIM) n Ninepin and Tenpin Bowling (FIQ) n Powerboating (UIM) Beta-blockers

Unless otherwise specified, beta-blockers are prohibited in-competition only, in the following sports. n Aeronautic (FAI) n Archery (FITA, IPC) (also prohibited Out-ofCompetition) n Automobile (FIA) n Billiards and Snooker (WCBS) n Bobsleigh (FIBT) n Boules (CMSB, IPC bowls) n Bridge (FMB) n Curling (WCF) n Golf (IGF) n Gymnastics (FIG) n Motorcycling (FIM) n Modern Pentathlon (UIPM) for disciplines involving shooting n Ninepin and Tenpin Bowling (FIQ) n Powerboating (UIM) n Sailing (ISAF) for match race helms only n Shooting (ISSF, IPC) (also prohibited Out-ofCompetition) n Skiing/Snowboarding (FIS) in ski jumping, freestyle aerials/halfpipe and snowboard halfpipe/big air n Wrestling (FILA)

Beta-blockers include, but are not limited to, the following: acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bunolol, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, metipranolol, metoprolol, nadolol, oxprenolol, pindolol, propranolol, sotalol, timolol

Urine Urine is the preferred body fluid. Its collection is non-invasive, it is generally available in sufficient quantity, and the drugs or their metabolites tend to be present in relatively high concentrations. The disadvantages are that a drug may be present as its metabolites or in a conjugated form, and the parent drug may be present only in a relatively low concentration. Furthermore, the relationship with the concentration in blood is very imprecise. Substitution of samples is clearly a possibility that must be avoided and particular care is required during the period of waiting before a sample is obtained to balance this risk against the desire for privacy on the part of a person. It has been reported that racing cyclists have carried a rubber bladder of (negative) urine under their arm, connected by a rubber tube to the appropriate discharge point. Blood The principal advantage of a blood sample is that its integrity is easier to safeguard because it is usually collected by a doctor or phlebotomist experienced in the procedure. In addition, drug concentrations in blood are interpreted more easily than those in urine and certain drugs that are not excreted in urine in significant quantities (e.g. human growth hormone) can be detected in blood. Since the 2000 Olympic Games in Sydney, blood has been collected routinely by some federations (e.g. the International Cycling Union) as a ‘health check’. Any competitor whose haematocrit is above 50% is not permitted to compete. This test is intended to limit the use of erythropoietin (EPO) to stimulate red cell production. However, this haematocrit test is readily circumvented and depends on too many factors; the use of haemoglobin concentration is preferred. Furthermore, blood samples may also be collected for more sophisticated tests to indicate the use of EPO. In addition, the administration of small doses (so-called microdosing) of EPO has been shown (Ashenden et al. 2006) to reduce the chance of detection of EPO use and yet still raise haemoglobin concentrations, hence the need to collect blood samples. Recently, tests for recombinant human growth hormone (rhGH) administration and for blood transfusion have been developed (see later) that rely on the use of blood samples.

Other matrices At the present time, WADA does not permit the use of alternative biological matrices such as oral fluid or hair to counter an analytical finding obtained from either a urine or blood sample. Nevertheless, Kintz has reviewed hair testing and doping control in human sport (Kintz 1998) and an interesting case report of its use in detecting a variety of prohibited substances in body builders has been published (Dumestre-Toulet et al. 2002) (see Chapter 19).

Table 6.6 Prohibited substances most commonly reported by WADA-accredited laboratories, in order of frequency(a) Number of reports Substance

1987

Substance

1990

Substance

1995

Substance

2000

Substance

2005

Nandrolone

262

Nandrolone

192

Testosterone

293

Salbutamol

367

Testosterone

1132

Pseudoephedrine

100

Testosterone

83

Testosterone

171

Cannabis

224

Nandrolone

325

Cannabis

503

Pseudoephedrine

123

Nandrolone

212

Testosterone

306

Salbutamol

357

Ephedrine

58

Stanozolol

79

Methandienone

132

Cannabis

295

Nandrolone

298

Phenylpropanolamine

57

Phenylpropanolamine

64

Salbutamol

132

Pseudoephedrine

136

Stanozolol

233

Methenolone

42

Ephedrine

43

Pseudoephedrine

102

Ephedrine

129

Amfetamine

194

Stanozolol

37

Codeine

32

Ephedrine

78

Stanozolol

116

Terbutaline

171

110

Methandienone

27

Methenolone

25

Stanozolol

78

Terbutaline

hCG

143

Codeine

26

Amfetamine

24

Methenolone

39

Methandienone

75

Budesonide

116

Amfetamine

24

Methandienone

23

Clenbuterol

31

Lidocaine

64

Ephedrine

(a)

1987 was first year of available data.

93

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Analytical approach

131

0.90 0.80 0.70

%

0.60 0.50 0.40

Nandrolone

Testosterone

08 20

06 20

04 20

02 20

00 20

98 19

96 19

94 19

92 19

90 19

19

88

0.30 0.20 0.10 0.00

Salbutamol

Figure 6.1 The proportion of human sports samples analysed by WADA-accredited laboratories in the years 1988 to 2008 reported for the three prohibited substances most commonly found.

Analytical approach

Table 6.7 Minimum required performance levels

With the exception of anabolic steroids, prohibited substances are generally administered at or near the therapeutic dose, which results in relatively low concentrations in biological fluids. The laboratory is provided with a coded sample to preserve the anonymity of the athlete. They may also be given a declaration of any drug that has recently been taken by the athlete but, apart from that, there is usually no evidence whether or not a drug has been administered, or what sort of drug it might be. As with equine testing, any drug used in human treatment or in veterinary practice may be found. Thus, screening procedures are designed to be both sensitive and of wide coverage. The material analysed is usually in a fairly fresh condition. The analyst thus has a clearer picture of a normal sample than does the forensic or hospital chemist, who may be required to examine a wide variety of materials in various states of decomposition. Any sample that fails a screening test is invariably submitted to rigorous confirmatory testing (see below) before an adverse report is issued. The WADA-accredited laboratory must reliably be able to detect and confirm the presence of prohibited substances or their metabolites at least down to a minimum required performance level (MRPL; see Table 6.7) (WADA 2009f). However, WADA states that ‘for non-threshold substances prohibited in-competition only, it is not recommended that laboratories report below 10% of the MRPL’. This refers to stimulants, narcotics and betablockers only (the latter being prohibited only by particular sports) since, for glucocorticoids, WADA states that ‘laboratories are not to report below the MRPL’. Although, with some exceptions, the parent drug is the entity that appears in the Prohibited List, screening procedures rely upon the detection of either the unchanged drug or its metabolites. The identification of the corresponding metabolites is often useful supplementary evidence to support the identification of the parent drug, and indeed WADA expects the laboratory to identify as many of the presumptive analytical findings from the screening procedures as possible. In addition, the presence of metabolites in the appropriate concentrations relative to the parent drug helps to support the conclusion that a drug has been administered. Conversely, the absence of any expected metabolites is possible evidence that a sample has been contaminated; this should be refutable by a proper chain of custody. Occasionally, the parent drug is not excreted in urine at a detectable concentration and a knowledge of the metabolic pathways is thus essential. An example of this is the identification of 19-norandrosterone and 19-noretiocholanolone (Fig. 6.2) in the urine of humans as evidence of the administration of the anabolic steroid nandrolone or a 19norsteroid precursor. Drugs can be used either to improve or to impair athletic performance, though in human sport the latter category of drug is

Prohibited class

Specific examples/ exceptions

Concentration

Stimulants

0.5 mg/L 0.2 mg/L

Strychnine Narcotics

0.2 mg/L 10 mg/L

Buprenorphine

10 mg/L

Anabolic agents

Hormone antagonists and modulators

Clenbuterol

2 mg/L

Methandienone

2 mg/L

Methyltestosterone

2 mg/L

Stanozolol

2 mg/L

Epitestosterone

2 mg/L

Aromatase inhibitors, SERMs and other anti-oestrogenic substances

50 mg/L

Beta2-Agonists

100 mg/L

Beta-blockers

0.5 mg/L

Diuretics

0.25 mg/L 30 mg/L

Glucocorticosteroids Peptide hormones

hCG

5 IU/L

OH H H

H

O

O

O H H

H HO

HO 19-Norandrosterone

H 19-Etiocholanolone

Figure 6.2 Main metabolites of nandrolone (19-norandrosterone).

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unlikely to be used. No single analytical scheme will suffice to cover so many different types of compound; various approaches have evolved in sports drug testing laboratories to address this challenge. Some drugs are notable for being excreted in urine almost entirely in conjugated form as, for instance, most anabolic steroids. When the presence of these drugs is suspected, hydrolysis before extraction is essential, although liquid chromatography–tandem mass spectrometry (LC-MS(-MS)) of intact conjugates is starting to be used. Liquid–liquid extraction may be used, for example with alkalinisation of the urine, to prepare samples for the detection of basic drugs by gas chromatography (GC) with nitrogen-selective detection. Alternatively, drugs have been extracted on the styrene–divinylbenzene copolymer XAD-2 resin. The development of solid-phase extraction (SPE) in the cartridge format in the late 1970s (Shackleton, Whitney 1980) and the rapid advances made in the technology associated with the technique have provided an attractive alternative to liquid–liquid extraction in many drug-screening programmes. Immunochemical methods that covered anabolic steroids were first introduced into human drug screening programmes in the 1970s (Brooks et al. 1979). However, in the late 1980s, these were largely replaced by GC-MS methods. Unlike many horseracing drug testing laboratories, human sports drug testing laboratories have not employed thin-layer chromatography (TLC) and do not use enzyme-linked immunosorbent assay (ELISA) extensively, but have used XAD-2 resin in columns for sample extraction and now, more commonly C8 and C18 SPE. Instrumental methods based on GC-MS, GC–high resolution mass spectrometry (GC-HRMS), GC-MS(-MS) and LC-MS(-MS) are preferred by most laboratories.

some polar compounds, such as ephedrine, are readily extracted in this procedure, it is best suited to less polar compounds. Anabolic steroids and corticosteroids

1. Condition C18 or C8 SPE cartridges with methanol and then water. Add 4 mL of the urine sample. 2. Wash with an equal volume of water and then elute the adsorbed steroids with an equal volume of methanol. 3. Remove the methanol under nitrogen, dissolve the residue in 1 mL of 0.1 mol/L phosphate buffer (pH 6.2) containing b-glucuronidase from Escherichia coli (approximately 1.4 units using 4-nitrophenylb-D-glucuronide as the substrate at 37 C). 4. Incubate for either a minimum of 2 h at 50 C or overnight at 37 C. 5. Cool and add approximately 100 mg potassium carbonate and extract the steroids with 5 mL distilled diethyl ether. 6. Divide the ether into two portions. 7. Evaporate each portion under nitrogen, dry the residue in a vacuum desiccator over phosphorus pentoxide–potassium hydroxide. 8. To one portion, add 200 mL of LC mobile phase, transfer to an autosampler vial and immediately cap the vial. 9. Examine the solution by LC-MS(-MS) for corticosteroids (and anabolic steroids such as tetrahydrogestrinone that are not amenable to trimethylsilylation for GC-MS analysis). 10. Derivatise the other portion by the addition of 100 mL N-methyl-Ntrimethylsilyl-trifluoroacetamide–ammonium iodide–ethanethiol (1000 : 2:6) and heating at 60 C for at least 15 min. 11. Examine the solution by capillary column GC-MS. Mass spectral and chromatographic data for many anabolic steroids and their metabolites have been published by Ayotte et al. (1996) and are given as system GAI (see Chapter 40): n

Solvent extraction In general, the choice of solvent is dictated by the wide range of drugs to be covered, or the need to extract a specific drug as in confirmatory analysis procedures. In many sports drug testing laboratories throughout the world, SPE has replaced liquid–liquid extraction for the isolation of drugs from both urine and plasma. Based upon the studies of Shackleton and Whitney (1980), the use of C18 or C8 bonded-phase cartridges for the isolation of anabolic steroids and their metabolites is the most common approach for sample extraction.

Sample preparation Extraction method for drugs and metabolites Note the initial pH and specific gravity of each sample. Basic drugs

1. Take an aliquot (4.0 mL) of each urine sample in a 10 mL centrifuge tube. 2. Add 0.5 mL of 5 mol/L potassium hydroxide solution, approximately 3 g of sodium chloride, 100 mL of diphenylamine as a reference standard solution (80 mg/L, final concentration 2 mg/L) and 1.6 mL of tbutyl methyl ether. 3. Vortex thoroughly and then mix for at least 10 min on a rotary mixer. 4. Centrifuge to separate the phases at 800g for 5 min. 5. Remove about 1 mL of the ethereal layer and transfer to an autosampler vial containing approximately 20 mg of anhydrous sodium sulfate to dry the extract (take care that none of the lower aqueous layer is transferred). 6. Examine 2 mL of this extract by GC using a cross-linked 5% phenylmethylsilicone-fused silica column (12.5 m  0.32 mm i.d., 0.52 mm) using nitrogen–phosphorus detection. The use of sodium chloride to increase the ionic strength of the aqueous phase increases the extraction of many of the compounds into the ether, which obviates the need for solvent evaporation. Although

n n

Column: methylsilicone fused silica (25 m  0.2 mm i.d., 0.11 mm), connected to a mass spectrometer. Temperature programme: 180 C for 1 min to 280 C at 8 /min. Carrier gas: He.

Diuretics

1. Take an aliquot (2.0 mL) of each urine sample in a 10 mL centrifuge tube. 2. Add 100 mL mefruside internal standard (10 mg/mL) and 2 mL 0.1 mol/L acetate buffer (pH 5.2). 3. Vortex thoroughly for 30 s. 4. Decant the urine onto a cross-linked polymeric sorbent cartridge with hydrophilic and lipophilic moieties (such as Abselut Nexus) and allow it to pass through the column. Do not let the bed dry out. 5. Add 1 mL of purified water onto each cartridge and allow it to pass through the column. Do not let the bed dry out. 6. Add 1 mL of methanol–water (20 : 80) onto each cartridge and allow it to pass through the column. Apply vacuum to the column to dry the bed. 7. Release the vacuum and place a labelled 10 mL glass centrifuge tube under the cartridge to collect the sample eluent. 8. Elute the adsorbed compounds with 3 mL methanol under gravity and then with the aid of a vacuum for a minimum of 30 s to dry the bed and achieve maximum recovery. 9. Evaporate the methanolic solution to dryness using oxygen-free nitrogen at 60 C. 10. Reconstitute the samples by adding 200 mL of LC mobile phase. 11. Vortex; transfer the reconstituted extract to a 0.2 mL tapered autosampler vial and cap the vial securely. 12. Examine 10 mL of the solution by LC-MS(-MS) for diuretics.

Gas chromatography and gas chromatography–mass spectrometry The GC procedures referred to above detect a wide range of compounds in urine samples, at concentrations in the order of 0.1 mg/L. They depend on the fact that all the compounds of interest contain at least one nitrogen atom and produce a signal in an alkali flame-ionisation

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Methods detector. The methods of extraction and the selectivity of the detector ensure minimal interference from other compounds that do not contain nitrogen, although certain plasticisers that contain phosphorus, such as tributyl phosphate, may produce signals. In addition, other nitrogencontaining compounds that are not prohibited in human sport (e.g. antihistamines) produce interfering peaks. Identification is based on the retention index; alternatively, retention time (relative to a standard) may be used. Details of retention indices or relative retention times of compounds in the systems described below are given in Chapter 40 and in the index of Gas Chromatographic Data. The identity of a substance should be confirmed using derivative formation and GC-MS, comparing the data obtained with reference material analysed contemporaneously (see Criteria for identification below).

Liquid chromatography–mass spectrometry With the development of atmospheric-pressure ionisation (API) techniques, LC-MS has found increasing application in doping control for both the qualitative and quantitative analysis of drugs. Screening methods have been developed for corticosteroids (Deventer, Delbeke 2003; Mazzarino, Botr
e 2006) and diuretics (Deventer et al. 2002; Ventura et al. 2008). In human sports drug testing, LC-MS(-MS) has been used more frequently over the last 10 years. For example, Barro´n et al. (1996) developed a direct method to determine anabolic steroids in human urine by on-line SPE LC-MS with a particle beam interface. Bean and Henion showed that it was possible to determine intact, i.e. conjugated, anabolic steroids using LC-electrospray ionisation (ESI)-MS(-MS) (Bean, Henion 1997). Thevis and colleagues used LC-MS(-MS) for the rapid screening of samples for beta-blockers (Thevis et al. 2001). Diuretics have been screened by LC-MS techniques (Ventura et al. 1991) and by LC-MS(-MS) (Thieme et al. 2001). Recently, the use of sub-2 mm porous particles to enable faster separations and greater separation power has become possible (Mazzeo et al. 2005). High-pressure pumping systems (greater than the conventional 400 bar [40 MPa] limit) needed to obtain the linear velocities desired are now readily available. Although the frictional heating caused by the velocity of the mobile phase through the column can limit the benefit that can be obtained using small particles, the use of 2.1 mm and narrower internal diameter columns has minimised this potential difficulty. Th€ orngren and colleagues have used ultra-performance liquid chromatography (UPLC)-MS(-MS) to screen 130 different substances (diuretics, masking agents, central nervous system stimulants and opiates) in urine (Th€ orngren et al. 2008) in approximately 6 min.

Isotope ratio mass spectrometry Combustion isotope ratio MS (CIRMS) is now used routinely by several WADA-accredited laboratories as an additional tool to help distinguish an individual whose testosterone : epitestosterone ratio may be naturally beyond the normal range from one who was administered testosterone. This technique relies on the fact that synthetic testosterone has a different proportion of 13C to the more abundant 12 C than the normal endogenous steroid (de la Torre et al. 2001). The extracted steroids are separated by GC and then converted into CO2 and the relative amounts of 12C to 13C as CO2 is determined for each eluting steroid in turn. Typically, the testosterone metabolites androsterone and etiocholanolone or androstanediols are monitored (Aguilera et al. 2000), or the metabolites 5a-androstanediol and 5bandrostanediol (Aguilera et al. 2001; Shackleton et al. 1997b), often comparing the results with pregnanediol or pregnanetriol as endogenous internal standards (Aguilera et al. 1999; Shackleton et al. 1997a). Flenker and colleagues and Cawley and colleagues have published reference isotope ratios for endogenous steroids (Cawley et al. 2009; Flenker et al. 2008) and Cawley has also published a method for the direct analysis of testosterone rather than its metabolites (Cawley

133

et al. 2009). Grosse and colleagues have shown that, in some urine samples, 19-norsteroids may be produced in small quantities from endogenous steroids (Grosse et al. 2005). Hebestreit and co-workers have published a method to distinguish this or the minute amounts that may be produced, especially in females, from the administration of nandrolone (Hebestreit et al. 2006). Buisson and colleagues (2009) have also used GC-CIRMS to detect exogenous hydrocortisone administration. Cawley and Flenker have published a tutorial article that reviews the use of CIRMS in doping control (Cawley, Flenker 2008).

Methods In human sports drug testing, MS is essential for the definite identification of a prohibited substance, with the exception of peptide hormones and glycoproteins, such as human chorionic gonadotrophin (hCG) for which a validated immunoassay is required for detection and quantification. For confirmation of hCG, a second different immunoassay is required. Specific techniques and methodologies for other peptide hormones and glycoproteins such as EPO have been described by WADA (WADA 2009a) and one for recombinant hCG (rhGH) is currently being considered. Criteria for identification WADA requires laboratories to be accredited to ISO 17025 to be eligible for WADA accreditation. ISO/IEC 17025 : 2005 requires traceability of measurements and for sports drug testing this is considered to be met by WADA, when identifying a prohibited substance (WADA 2003), by the direct comparison with a reference material or reference collection analysed in parallel or in series with the test sample. A reference material is generally accepted as a homogeneous, stable chemical with a wellestablished structure. The material may be characterised structurally within the laboratory using appropriate techniques or validated against a certified reference material or by comparison with uncontroversial published data. WADA permits the use of a reference collection ‘obtained from a verified administration study in which scientific documentation of the identity of metabolite(s) can be demonstrated’ (WADA 2009b). Certified reference materials issued by organisations accredited for compliance with ISO Guide 34 : 2000 are often not available and hence there is the need to use authenticated administrations for comparison purposes. Many of the most common substances are now available with certificates of analysis, and thereby fully meet the generic traceability requirements of ISO/IEC 17025 : 2005. This is particularly important for quantitative analysis, where it is usually extremely difficult for an individual laboratory to determine the purity of a non-certified material to a sufficient standard to be able to establish the measurement uncertainty. WADA generally expects a chromatographic retention time match between the analyte and a reference collection sample (see above) or reference material analysed using the same procedure in the same assay. This retention time difference must be not more than 1% or 0.2 min, whichever is the smaller, and three diagnostic ions in the electron impact (EI) and chemical ionisation (CI) mass spectra must also match to within 20% of the relative abundance of each ion (Table 6.8). Figure 6.3 illustrates the relative abundance criteria that, paradoxically, show a step change for the data obtained using CI, LC or tandem MS. These standards generally meet or exceed those required by the Substance Abuse and Mental Health Services Administration (SAMHSA) for the US federal employment drug testing programmes (see also Chapter 3). Although library data may be useful in the early phase of substance identification, especially in generic screening procedures, they are not considered sufficiently reliable for the final identification. Similarly, published data are used more to assure reliability than directly for substance identification. The required documentation for the analytical certificate is clearly set out in ISO/IEC 17025 : 2005 but merely requires a statement as to the substance found. However, WADA also sets out a

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Drug Testing in Human Sport

Table 6.8 Maximum relative ion intensity tolerances for substance identification using MS Relative abundance (% of base peak)

EI-GC-MS

CI-GC-MS; GC-MSn; LC-MS; LC-MSn 15% (absolute)

>50%

10% (absolute)

25–50%

20% (relative)

25% (relative)

1600

29

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194

Drugs of Abuse

Screening tests Colour/spot tests provide a valuable indication of the content of any particular item tested, but it must be stressed that positive results to colour tests are only presumptive indications of the possible presence of the drug. Colour tests have the advantage that they can be used as field tests by unskilled operators, with the obvious need for follow-on analysis in the laboratory. One of the most important and widely used colour tests is the Marquis reagent test. Another important screen is thin-layer chromatography (TLC). This has many advantages as an analysis/screening tool. It is quick, easy to use and has a low cost; it is relatively sensitive and can give a good degree of discrimination. Solvent systems TA and TB are suitable for many drugs (see Table 11.4 and Chapter 39). Visualisation of many of the drugs may be achieved by a variety of methods. However, spraying with acidified potassium iodoplatinate reagent is suitable for many drugs. Qualitative analysis Using a capillary column gas chromatograph and a suitable temperature programme coupled to a mass spectrometer (GC-MS; see Chapter 37), the drug components of most samples can be separated and identified. Because of the reduced capital outlay of such instruments in recent times, it is not uncommon for laboratories to have several such instruments working with automatic samplers (possibly on a 24-hour basis). The use of GC-MS has become the routine method of identification of most drugs. A general GC-MS screen method (GAK, Table 11.4) can be used to separate and/or identify most of the drugs encountered in exhibits. Figure 11.2 shows the separation achieved, by this method, of a mixture of the main drugs described in this chapter. Identification of the various components of a suspect mixture can be made by library searching against commercial libraries but it is important to run a standard of the specific drug being tested, e.g. pure heroin. This obviously will need to give a retention time and mass-spectral match. High performance liquid chromatography (HPLC) is a simple and reliable method of analysis of most drugs. It is both accurate and precise and thus lends itself to quantitative analysis. It is especially useful for compounds that are thermally labile. HPLC has some advantages over GC because of the variety and combinations of mobile phases that can be chosen. There is also a choice of detectors available for specific applications. HPLC can, however, involve significantly more method development than GC and the resolution of GC is greater. No one system is suitable for optimum separation of all the different drug types, so different systems are used to give optimum separations for specific analysis. The system that is best for the separation of heroin/ acetylcodeine/noscapine/papaverine (Huizer 1983) will not be the same as that which separates cocaine from its impurities and processing byproducts (Moore, Casale 1994). A general screening method such as HBC (Table 11.4) can be used for the separation of heroin (diamorphine), cocaine, amfetamine and metamfetamine (Fig. 11.3). Using this system, metamfetamine and MDMA co-elute. However, by changing the method to HBD, amfetamine, metamfetamine, MDMA and methylenedioxyethamfetamine (MDEA) may be separated (Fig. 11.4). This illustrates the versatility of HPLC. Separations can occur between compounds that co-elute by altering the elution system. A system such as the above could be used as a screen, but identification would necessitate some spectroscopic method such as MS or infrared (IR) spectroscopy. Most modern laboratories are now equipped with Fourier transform IR (FTIR) spectrometers (see Chapter 33). These have many advantages over traditional IR instruments. They are faster and can work with smaller samples. When they are coupled with a microscope, tiny samples can be analysed. The difficulty with IR analysis of drug samples is the presence of other material that will interfere with the spectrum. These interfering compounds could be other drugs that occur naturally in the

samples (or from the synthetic process) or could be adulterants such as caffeine and paracetamol. IR analysis can, however, give valuable information on chemicals that are not suitable for GC-MS analysis. Another technique that is popular is GC-FTIR. Because of the speed of scanning of the FTIR it can be used to obtain a spectrum of the compounds that have been separated by GC. In practice, neither spectra nor pure reference samples may be available for comparison for the more unusual substances. In this situation, nuclear magnetic resonance (NMR) spectroscopy (see Chapter 36) is the method of choice. Quantitative analysis For most controlled drugs there are no minimum quantities below which an offence does not occur. The quantitative analysis of drugs is therefore not carried out routinely on all exhibits. The main reason for determining purity/drug content of powders and tablets is to enable a court to establish a monetary value of the seizure or when sentencing structures are based on equivalent pure drug content. In some countries the death sentence can apply if one is convicted of possession/supply of greater than a specified quantity of a substance. In some situations, information on drug purity will be used for intelligence purposes such as to assess trends in the illicit drug market or for use in drug comparison and profiling. Having already identified the powder as, for example, heroin, quantitative analysis may be carried out by GC using flame ionisation detection (FID) or by HPLC. In performing quantitative analysis it is always desirable to include an internal standard in the analysis. This has the advantages of ease of use, increased accuracy, no need for volumetric glassware, no need to measure injection volume and easy determination of reproducibility, and can be used as a monitor for GC or HPLC systems. An internal standard must meet the following criteria: it must be absent from the sample; it must be readily available (and not too costly); and it must be pure, show good chromatographic behaviour, be reproducible and be soluble in the solvent used. Straight-chain hydrocarbons (for GC) fulfil all of these requirements and they elute as a homologous series, so they are a popular choice as internal standard. It has been suggested that the internal standard that one chooses should be chemically related to the compound being analysed. However, provided that it fulfils the above criteria, any compound can be chosen. A general approach to quantification by GC could be as follows. The GC conditions for the qualitative analysis can again be used. A standard curve is established by preparing up to five standard solutions of the drug being quantified. A range from 1 mg/mL to 5 mg/mL is prepared using solvent containing an internal standard. A concentration of internal standard of 0.5 or 1 mg/mL will normally be adequate. A test sample is prepared that will have a concentration between 1 and 5 mg/mL, i.e. within the range of the standard curve. If the test sample is outside the range, a second sample is prepared based on the information from the first sample. In general it is suggested that at least two samples of the powder being tested be taken for quantitative analysis and an average of these be taken as the true result. The amount of the drug in the test sample can now generally be calculated by the data-analysis function of the instrument. Both GC and HPLC are used extensively for quantitative analysis and it is useful to compare the results obtained by one method with the other, for a given drug. Profiling and comparison A more detailed analysis of drug samples can be used to provide ‘collective’ information. This is generally called profiling when it involves the chemical analysis of powders, or is known as characterisation when the physical properties of tablets and other dosage forms are measured. Chemical profiling has been the technique most widely used and is often based on the chromatographic separation of impurities and precursors (as in the case of amfetamine and metamfetamine) or other naturally occurring components and adulterants (e.g. heroin, cocaine,

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Analysis of seized drugs

195

Table 11.4 GC, HPLC and TLC conditions Condition code

Source

Details

SD McDermott (unpublished)

Column: HP Ultra-1 cross-linked methylsiloxane (12.5 m
0.2 mm i.d., 0.33 mm)

Gas chromatography GAK

Carrier gas: He, 1 mL/min, 50 : 1 split ratio Temperature programme: 60 C for 2 min to 180 C at 15 C/min to 290 C at 25 C/min for 3 min MS conditions: low mass 40, high mass 550 with a solvent delay of 1.5 min GAL

Clarke (1989)

Column: OV-1 (12 m
0.32 mm i.d., 0.52 mm) Carrier gas: He, 1.5 mL/min, 25 : 1 split ratio Temperature: 260 C

GAM

McDermott (unpublished)

Column: HP Ultra-1 (12 m
0.2 mm i.d., 0.33 mm), split ratio 50 : 1 Temperature programme: 150 C (no hold) to 295 C at 15 C/min

GAN

Lee (1995)

Column: HP-1 (12 m
0.22 mm i.d., 0.33 mm) Temperature programme: 100 C for 2 min to 300 C at 15 C/min for 5 min

GAO

Blackledge, Miller (1991)

Column: HP-1 (25 m
0.2 mm i.d., 0.33 mm) Temperature programme (lactone): 70 C for 1 min to 300 C at 20 C/min for 3 min Temperature programme (BSTFA derivative): 100 C for 1 min to 300 C at 20 C/min for 7 min

GAP

SD McDermott (unpublished)

Column: HP Ultra-1 cross-linked methyl siloxane (12.5 m
0.2 mm i.d., 0.33 mm) Carrier gas: He, 1 mL/min, 50 : 1 split ratio Temperature programme: 70 C for 2 min to 210 C at 20 C/min for 1 min MS conditions: low mass 40, high mass 550 with a solvent delay of 1.5 min

GAQ

SD McDermott (unpublished)

Column: HP 101 cross-linked methylsiloxane (12 m
0.2 mm i.d., 0.33 mm) Carrier gas: He, 1 mL/min, 50 : 1 split ratio Temperature programme: 200 C for 1 min to 270 C at 5 C/min to 295 C at 20 C/min for 3 min

High performance liquid chromatography HBC

McDermott (unpublished)

Column: Spherisorb 5 ODS-1 (150
4.6 mm i.d.) at 30 C Mobile phase: acetonitrile: TEAP(a) (50 : 50), flow rate 1.5 mL/min Detector: DAD (l = 254 nm)

HBD

McDermott (unpublished)

Column: Spherisorb 5 ODS-1 (150
4.6 mm i.d.) at 30 C Mobile phase: acetonitrile: TEAP(a) (20 : 80), flow rate 1.0 mL/min Detector: DAD (l = 254 nm)

HBE

Clarke (1989)

Column: Supelcosil 5 LC-18 (250
4.6 mm i.d.) Mobile phase: phosphate buffer(b) : methanol (60 : 40) Detector: UV (l = 309 nm)

HBF

Borner, Brenneisen (1987)

Column: Spherisorb ODS-1 (250
4.6 mm i.d., 10 mm) Mobile phase: 0.3 mol/L ammonium acetate in water (buffered to pH 8 with ammonia): 0.3 mol/L ammonium acetate in methanol (100 : 0 for 2 min to 5 : 95 at 14 min) Detector: UV (l = 269 nm)

HBG

Mesmer, Satzger (1998)

Column: Bondapak C18 (300
3.9 mm i.d., 10 mm) Mobile phase: phosphate buffer(c) : methanol (70 : 30), flow rate 1 mL/min Detector: UV (l = 215 nm)

HBH

Japp et al. (1988)

Column: ODS (250
5 mm i.d., 5 mm) Mobile phase: methanol : water : phosphate buffer(d) (pH 7.25, 55 : 25 : 20), flow rate 1 mL/min Detector: UV (l = 240 nm)

HBI

Japp et al. (1988)

Column: ODS (250
5 mm i.d., 5 mm) Mobile phase: methanol : water : phosphate buffer(d) (pH 7.67, 70 : 10 : 20), flow rate 1 mL/min Detector: UV (l = 240 nm)

HBJ

CND Analytical (1989)

Column: Bondex C18 (300
3.9 mm) Mobile phase: methanol : water (70 : 30), flow rate 1 mL/min Detector: UV (l = 254 nm)

HL

Baker et al. (1980)

Column: Spherisorb ODS silica (250
4.6 mm i.d., 5 mm) Mobile phase: 0.01 mol/L sulfuric acid : methanol : acetonitrile (7 : 8 : 9) table continued

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Table 11.4 continued Condition code

Source

Details

Thin-layer chromatography TAH

Silica gel plates with hexane : diethyl ether (4 : 1)

TAI

Silica gel plates with acetone

TAJ

Silica gel plates with n-butanol : acetic acid : water (2 : 1 : 1)

(a)

TEAP, triethylammonium phosphate buffer made up by preparing a 1.0 mol/L phosphoric acid (65 mL of 85% phosphoric acid to 1 L of water) titrated to pH 2.5 with triethylamine (approximately 10 mL triethylamine per 100 mL 1.0 mol/L phosphoric acid). A 10 mL quantity of this solution is made up to 1 L of water to give the working solution of 10 mmol TEAP. (b) Phosphate buffer is prepared by adding 10 mL of phosphoric acid to 1 L of water, followed by the addition of sufficient 2 mol/L sodium hydroxide to raise the pH to 6.5. (c) Phosphate buffer is 10 mmol potassium dihydrogen phosphate, adjusted to pH 3 with phosphoric acid. (d) 0.1 mol//L phosphate buffer is prepared by dissolving 14.35 g sodium dihydrogen phosphate dihydrate and 1.14 g disodium hydrogen phosphate in 1 L of water.

Figure 11.2 Gas chromatographic separation.

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Analysis of seized drugs

Figure 11.3 HPLC separation.

197

are suspected of being linked as part of a local distribution chain. This is known as comparison or tactical profiling and may be carried out as a routine requirement in forensic casework. A second stage (intelligence or strategic profiling) exists where answers to wider questions may be sought. These will depend on the drug concerned, but include estimating the number of different profile types in circulation and relating them to the number of active laboratories and how long they have been in operation; determining the extent of importation by comparing the profiles of police and customs seizures; identifying the route of synthesis and types of precursors used; creating large-scale maps of drug distribution and identifying the country or region of origin. Tablet comparisons using general physical features, gross drug content and microscopic examination of defects and punch marks (socalled ballistic analysis) can be of some value, but they suffer from the fact that at any one time a large fraction of illicit tablets in circulation may be almost identical. Such small differences as may exist could simply reflect inherent differences in the punches and dies of a multiple-stage tabletting machine. This can be illustrated by the Mitsubishi logo (Fig. 11.5), which was found on over half of all MDMA tablets seized in Europe in the late 1990s. A similar pattern was also found in the UK for amfetamine in the early 1990s, when nearly half of all samples belonged to one profile type. In these circumstances, any connection between two separate seizures of otherwise identical tablets or powders may be purely fortuitous. This, in turn, raises other problems with profiling. It is necessary to maintain a database of profiles such that the significance of any ‘match’ or ‘non-match’ can be critically assessed. However, in a situation where profiles may change with time, what constitutes a ‘current’ database is not always clear. In the case of determination of country of origin, authentic samples are required in order to provide a statistical ‘training set’, yet such samples may be difficult to obtain and their true provenance uncertain. A general approach to the analysis of unknown substances A general approach to the analysis of unknown substances has been outlined in Fig. 11.1. Different approaches will be required depending on whether the exhibit is a powder, vegetable matter, tablet/capsule or liquid. Powders

When a powder is submitted for analysis, the most likely drugs to be present include heroin, cocaine, amfetamine and metamfetamine. Although others may be present, these are the most common. Initial examination will involve describing/detailing of the packaging material. If there are multiple packs present then a subsample may be removed for analysis. The powder must be weighed before analysis. The powder is then homogenised and an aliquot is taken for analysis. A screen (colour test, TLC, HPLC, GC) will indicate the drug(s) present. Identification of the drug can be achieved using GC-MS or FTIR spectroscopy. GC-MS has an advantage over FTIR spectroscopy because of retention time and a mass-spectral comparison with a known standard.

Figure 11.4 HPLC separation.

cannabis resin). Detection may range from flame-ionisation to isotoperatio mass spectrometry. Non-separation methods, for example using IR, Raman or X-ray diffraction spectroscopy, have only limited scope. Drug profiling may be used for two quite separate purposes. In the first case, it can establish connections between a number of exhibits that

Figure 11.5 Impressions on illicit MDMA tablets.

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Identification of the other components in the powder can be achieved by using a range of analytical techniques including FTIR, X-ray fluorescence (XRF), X-ray diffraction (XRD), NMR spectroscopy and others. The drug content can be quantified by GC or HPLC by preparation of a curve using a range of concentrations of the drug in question. In some circumstances comparison may be required between powders for links in a specific case or for intelligence purposes, and GC and HPLC can be employed to examine some of the minor ingredients of the powder. Vegetable material

This includes cannabis plants, cannabis resin, khat and psilocybe mushrooms. A physical examination would include a description of the material followed by a measurement of the weight of material or the height of the plant. A subsample of the population may be chosen and an aliquot taken for analysis. Homogenisation may be necessary depending on the material. The physical appearance will generally give a very good idea of the drug present, for example cannabis plant or psilocybe mushrooms. We can therefore go directly to a specific test rather than use a screening technique. Identification can be carried out by a combination of microscopic and chemical techniques. The drug content can be quantified by GC or HPLC and comparison/ profiling carried out by chemical and physical comparisons. Tablets and capsules

Tablet or capsules submitted to the laboratory include ecstasy (MDMA, MDEA, etc.), benzodiazepines, steroids, LSD squares and others. An initial examination will include a description of any markings or logos, counting of the items, measurement of the tablet/capsule and possibly a weight. An examination of a tablet/capsule identification database such as TICTAC may give an indication of the drug present (see Chapter 13). Subsampling followed by homogenisation will lead to essentially a powder sample and the procedure for analysis of powder samples may then be followed. For comparison/profiling a physical comparison of the logo or mark may be the most informative piece of information available. Liquids and other forms

These include GHB, steroid oils, cocaine liquor and others. A physical examination may give an indication of the drug likely to be present. The physical measurements to be noted would include the volume, colour, odour and general appearance of the liquid. A subsample may be taken and an aliquot removed for analysis. It may be necessary to (base) extract the drug from the liquid into an organic solvent prior to analysis. The physical examination may allow the analyst to proceed to the identification stage, otherwise a screen may be used to indicate the presence of a certain drug. Identification, quantification and comparison/profiling can be carried out along the lines outlined for powders.

only a small amount (or none) of the final product (i.e. the controlled substance) may be found. In these instances, detailed explanations of the synthetic routes may be required. An explanation of the role of each of the chemicals found at the scene could be required. The scientist must also be aware of alternative explanations for the presence of the chemicals, as this is the likely defence in such cases. In many instances the precursor chemicals themselves are controlled. The forensic investigation of clandestine laboratories has been well documented (Christian 2003) and many of the complications associated with such laboratories have been explored. In the USA, the number of clandestine metamfetamine laboratories has increased enormously in recent times. Illegal seizures increased from 7438 in 1999 to 12 484 in 2005. The hazards and contamination issues associated with such laboratories pose difficulties for those involved in the process and for public health in general (Martyny et al. 2007). Capillary electrophoresis (CE) has been employed to characterise phosphorus species used in the manufacturing process (Knops et al. 2006).

Concealment of drugs The internal concealment of illicit drugs to smuggle quantities across borders has been a phenomenon for decades (Fineschi et al. 2002; Gill, Graham 2002; Takekawa et al. 2007). The ‘body packer’ or ‘mule’ carries quantities of drugs that are generally well wrapped to withstand the biological hazards of internal concealment. Deaths related to such activities are not uncommon. However, it is not known how widespread the practice is as the detection is frequently associated with instances that have adverse medical consequences. Impregnation of items with cocaine is also a novel method of concealment. Cocaine-impregnated silicone in baseball cap parts were detected and chloroform was used to extract the cocaine (Microgram 2003). Items of clothing impregnated with cocaine were extracted with methanol (McDermott, Power 2005) and in this instance ‘moth balls’ containing camphor were added to the suitcase to deter detection by dogs. An unusual way of smuggling cocaine is the use of a cocaine–poly (methylmethacrylate) solid solution (Gostic, Klemenc 2007).

Analysis of the main drugs of abuse In this section, methods are described for the analysis of the main drugs of abuse. In all cases a number of analysis methods are described. The methods chosen will depend on the aim of the analysis, the apparatus/ equipment available, legal aspects and the number of analyses to be performed, and may depend on other details associated with the specific drugs seizure. The analytical data for the various drugs are shown in Tables 11.5 and 11.6.

Clandestine laboratories Because of the increase in abuse of synthetic drugs, clandestine laboratories have become an increased part of forensic investigations. The investigation of such sites is very interesting as they reveal (in situ) the synthetic processes, intermediates, and often notes and chemical equations describing the various reactions used. These laboratories, however, are also very hazardous sites to investigate. The use of the word ‘laboratory’ disguises the more usual scenario of a garage, shed or kitchen. Forensic scientists frequently become involved in an advisory capacity in the initial stages of a clandestine laboratory investigation. Information may come to light about certain chemicals being used at a premises and the scientist is responsible for formulating an opinion as to whether a controlled substance is being produced. The police can then act on the basis of this opinion. Many countries have specially trained police and scientists to deal with the specific problems that clandestine laboratories pose. These problems could be in the form of hazardous chemicals (acids, bases, solvents and reagents) and fire and explosion potentials. Ultimately, if the seizure results in a court case, the testimony in these cases can be technically demanding for the scientist. In many situations,

Amfetamine and metamfetamine Amfetamine (a-methylphenethylamine; 1-phenylpropan-2-amine) and metamfetamine (N-methyl-a-methylphenethylamine; N-methyl1-phenylpropan-2-amine) in free base form are both liquids. Amfetamine is normally produced as amfetamine sulfate or phosphate and is common in Europe. Metamfetamine is normally produced as metamfetamine hydrochloride and is more popular in North America and Japan. Street-level amfetamine and metamfetamine are normally submitted to the laboratory as white/off-white powders with relative low purity (e.g. 5%). Synthesis of amfetamine and metamfetamine

Many methods are available for the illicit synthesis of amfetamine, but the Leuckart reaction has been the most popular. This method is simple and rapid, gives a good yield and does not involve any particularly hazardous chemicals or procedures. It may be considered as a three-step reaction involving the condensation of phenyl-2-propanone (P-2-P) with formamide followed by a hydrolysis of the N-formylamfetamine and finally purification by steam distillation.

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Analysis of the main drugs of abuse

Table 11.5 Analytical data using GC Drug

Retention time (min)

GC system

Amfetamine

4.85

G1

Methamfetamine

5.52

G1

MDMA

8.70

G1

Heroin

14.19

G1

Cocaine

199

Metamfetamine can be made by the Leuckart reaction using methylamine and formic acid or N-methylformamide in the condensation step. Analysis of amfetamine and metamfetamine

12.68

G1

LSD

7.28

G2

LAMPA

8.06

G2

Psilocin

4.88

G3

Because many of the street-level samples submitted to the laboratory are relative low in purity (5%), pre-concentration of samples may be required for the analysis to be successful. This may be achieved by base extracting the amfetamine/metamfetamine into ether and evaporating to dryness in an airflow without heat. A few drops of methanol can then be added and the methanolic solution transferred to a plastic insert prior to analysis. Colour test Marquis test gives an orange colour for both amfetamine and metamfetamine.

Psilocybin

6.90

G3

TLC

Cathine

4.06

G4

Cathinone

3.93

G4

GHB

5.60

G5

n n n

GBL

3.98

G5

Diazepam

13.51 (4.47)

G1 (G6)

Flunitrazepam

14.02 (5.24)

G1 (G6)

Flurazepam

14.59 (6.14)

G1 (G6)

Nitrazepam

14.48 (5.94)

G1 (G6)

Fluoxymesterone

9.04

G7

Nandrolone

7.16

G7

Testosterone

7.98

G7

Methyltestosterone

8.33

G7

Cannabinol

13.86

G1

Tetrahydrocannabinol

13.64

G1

Cannabidiol

13.32

G1

Table 11.6 Analytical data using HPLC Drug

Retention time (min)

HPLC system

Amfetamine

2.06 (3.68)

H1 (H2)

Methamfetamine

2.42 (4.67)

H1 (H2)

MDMA

2.42 (5.31)

H1 (H2)

Heroin

2.88

H1

Cocaine

5.21

H1

LSD

5.34

H3

LAMPA

5.66

H3

Psilocin

6.80(a)

H4

Psilocybin

3.20(a)

H4

GHB

3.5

H5

GBL

4.0

H5

Diazepam

2.29(a) (10.41)(a)

H6 (H7)

Flunitrazepam

0.86(a) (3.34)(a)

H6 (H7)

Flurazepam

3.12(a) (12.98)(a)

H6 (H7)

Nitrazepam

0.87(a) (3.22)(a)

H6 (H7)

Fluoxymesterone

5.5

H8

Nandrolone

6.0

H8

Testosterone

7.5

H8

Methyltestosterone

9.5

H8

Cathine Cathinone

Cannabinol

11.77(a)

HL

Tetrahydrocannabinol

13.35(a)

HL

7.47(a)

HL

Cannabidiol (a)

k values.

TA: amfetamine Rf ¼ 0.43; metamfetamine Rf ¼ 0.31 TB: amfetamine Rf ¼ 0.15; metamfetamine Rf ¼ 0.28 Visualisation is with acidified iodoplatinate solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) In addition, it is common practice with primary amines to prepare derivatives such as N-methylbis(trifluoroacetamide) (MBTFA) or trifluoroacetic anhydride (TFAA) derivatives. It is good practice to analyse both derivatised and underivatised samples since N-hydroxylamines may give the same product as the parent amines. Using a concentrated/base-extracted sample, the molecular ion peaks m/z 134 and m/z 148 for amfetamine and metamfetamine, respectively, can readily be achieved in an underivatised sample. Both amfetamine and metamfetamine have one asymmetrical carbon atom, resulting in a pair of enantiomers in each case. Depending on the synthetic route l-, d- and dl-amfetamine or metamfetamine could be encountered in samples submitted to the laboratory for analysis. These optical isomers differ in their pharmacological activity and are subject to different regulatory measures in certain countries. In those countries where the specific optical isomer needs to be identified, chiral analysis can be undertaken by derivatisation/GC, by the use of chiral columns (GC and HPLC) and by the use of CE (Anastos et al. 2005; Fanali et al. 1998; Lebelle et al. 1995; Sellers et al. 1996). Enantiomeric analysis of metamfetamine samples (Lee et al. 2007) showed that up until 1997 the vast majority of the metamfetamine encountered in the Republic of Korea was the S-(þ)-enantiomer, but from 1997 onwards the R()-enantiomer began to appear and increased continuously until 2005 when 50% of the metamfetamine samples seized contained the R-()-enantiomer. Quantitative analysis and profiling of amfetamine/ metamfetamine

Amfetamine and metamfetamine can be quantified by HPLC or GC. Normally if GC is used the samples are base extracted into an organic solvent and either run directly or derivatised and then run. Using HPLC there is no need to extract and, in many cases, this is the preferred method for quantitative analysis of amfetamine/metamfetamine. Amfetamine produced illicitly often contains impurities that are a result of the manufacturing process. The presence of these impurities can be used to compare and distinguish samples of amfetamine since material used in the same manufacturing batch would almost certainly have the same number and relative amount of identical impurities. Samples from the same illicit laboratory produced at different times may show strong similarities, whereas samples from unrelated laboratories are expected to show major qualitative and quantitative differences. Basic extracts into organic solvents are subjected to GC or GC-MS analysis. Samples are compared by visual inspection of the GC trace and by quantitative comparisons. Metamfetamine impurity profiling is also carried out by GC analysis, with the impurities also giving information on the synthetic route (Seta et al. 1994). A recent study (Lee et al. 2006) of the impurities present as a result of the synthetic process showed that the selected impurity peaks may be utilised as the indicators of synthetic conditions and analysis of their patterns can supply valuable information for understanding the

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conditions of clandestine synthesis of metamfetamine. The use of thermal desorption followed by GC-MS was found to be an efficient method for impurity profiling of metamfetamine (Kuwayama et al. 2007). The purpose of the comparison/profiling is: to identify dealer–user links; to establish possible sources, i.e. the clandestine laboratory; and to build up databases to allow interpretation in comparison casework. This approach has been used in Australia to examine samples over the period 1998–2002 (Qi et al. 2006). A series of studies were undertaken to develop a harmonised method for the profiling of amfetamine synthesised by three different methods. The study covered the sample preparation, extraction procedure, optimisation of GC-MS method, selection of target compounds and numerical comparison of amfetamine impurity profiles (Aalberg et al. 2005a, b; Andersson et al. 2007a,b,c; Lock et al. 2007). This inter-laboratory study found that the variation from laboratory to laboratory was affected by such factors as homogeneity of samples and concentration effects due to dilution. Cannabis, cannabis resin and cannabinoids Herbal cannabis (marijuana) means all parts of the plant Cannabis sativa L., but excludes the seeds and mature/woody stalk material. Cannabis sativa L., which can be grown in all parts of the world, is an annual plant and attains a height of 1–5 m. When it is planted for the production of hemp fibre, the stalks are crowded and without foliage except near the top. The wild growing plant, in contrast, has numerous branches. The resin of the plant occurs mainly in the flowering area, the leaves and the stem, particularly at the top of the plant. The highest amount of resin is found in the flowering part. Up to the time of flowering, the male and female plants produce resin almost equally. After shedding their pollen the male plants soon die. The resin is found in the glandular trichomes. The leaves of Cannabis sativa L., are compound and consist of 5 to 11 separate leaflets. Each leaflet is characteristically hair covered and veined and has serrated edges (Fig. 11.6). Cannabis herbal material may be encountered in blocks of dried flowering tops and dried leaves. Cannabis resin (hashish) is a compressed solid made from the resinous parts of the plant and is usually produced in 250 g blocks. Herbal cannabis imported into Europe may originate from West Africa, the Caribbean or South East Asia, but cannabis resin derives largely from either North Africa or Afghanistan. Cannabis and cannabis resin are normally mixed with tobacco and smoked, but can be ingested. The average ‘reefer’ ‘joint’ cigarette contains around 200 mg of herbal cannabis or cannabis resin. The main psychoactive compound in cannabis and cannabis resin is D9-THC. Cannabinol (CBN) and cannabidiol (CBD) are among the other main components.

Figure 11.7 Microscopic examination of cannabis: (a) cystolith hair; (b) large glandular hair with several cells in the head and stalk; (c) head of one of the large glandular hairs; (d) calcium carbonate.

A sample of the material is extracted with petroleum ether. The petroleum ether is removed and evaporated to dryness. The addition of Duquenois reagent followed by concentrated hydrochloric acid will yield a purple colour after a few minutes. The addition of chloroform should result in a purple colour in the chloroform layer. This result can be taken positive for cannabinoids. TLC n n

TAH: THC Rf ¼ 0.50; CBD Rf ¼ 0.60; CBN Rf ¼ 0.45 Visualisation is by fast blue BB with THC showing a red colour, CBD showing an orange colour and CBN showing a purple colour.

Microscopic examination of cannabis/cannabis resin

The most characteristic botanical feature visible under the microscope is the hairs. There are two types of hair: n n

Cystolithic hairs Glandular hairs.

Colour test The presence of cannabinoids in suspect material can be

The cystolithic hairs contain a deposit of calcium carbonate at their base. These hairs are mostly single celled. The glandular hairs are most important since they contain and secrete the resin. They are short and may be unicellular or multicellular. The bigger glandular hairs have a multicellular stalk with heads containing 8–16 cells (Fig. 11.7). The microscopic test is carried out by putting a small portion of the dry material (cannabis herbal material or cannabis resin) on a microscope slide. A few drops of Duquenois reagent are added followed by a few drops of concentrated hydrochloric acid. The cystolithic hairs contain a deposit of calcium carbonate at their base and a characteristic effervescence can be observed. The heads at the end of the glandular hairs will become a red/purple colour. An alternative method is to add a few drops of chloral hydrate solution to the dry material. This is particularly useful for getting more detailed information on the structure of the plant tissue since it removes coloured materials such as chlorophyll.

indicated by the Duquenois–Levine test.

Quantitative analysis and comparison of cannabis/cannabis resin

Figure 11.6 Cannabis plant.

As already stated, cannabis resin is normally produced in 250 g blocks. Frequently these blocks carry an impression, e.g. a number or a letter or a symbol. Comparison can be made between different blocks on the basis of similar impressions, though unrelated blocks often have the same impression. The street-level deal of cannabis resin is typically a finger-sized portion (normally 1–10 g) possibly wrapped in tinfoil or plastic. It may be possible to link a smaller piece of cannabis resin to its original block by a physical fit between the smaller and larger pieces. GC or HPLC may be used to obtain a chemical profile of the cannabis/cannabis resin (see Tables 11.5 and 11.6). The THC content can be calculated and comparison can be made on that basis. It must be noted that variations can occur in the THC content of a single block of cannabis resin as the THC content decreases with age and storage conditions. The outer material in a block of cannabis resin can differ from that in the centre. The variation of distribution of cannabinoids within blocks of resin was studied and it was noted that composition of the resin changed even within a 12-month period (Lewis et al. 2005). The main chemical constituents of cannabis have been well documented (Elsohly, Slade 2005).

Analysis of cannabis/cannabis resin

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Analysis of the main drugs of abuse The THC contents of the various forms of cannabis can vary enormously. For example, recent seizures of cannabis in the UK had the following THC contents: sensemilla (indoor intensively cultivated herbal cannabis), mean 16.2%, range 4.1–46%; imported herbal material, mean 8.4%, range 0.3–22%; resin (mostly from North Africa), mean 5.9%, range 1.3–28% (Hardwick, King 2008). Short tandem repeat (STR) DNA markers have been used to indicate the likely origin of a cannabis crop and STR markers can permit the identification of hydroponically propagated clonal drug lines, providing evidence to link illegal operators (Gilmore et al. 2007). Cocaine Cocaine is a naturally occurring alkaloid found in certain varieties of plants of the genus Erythroxylum. Coca cultivation is distributed throughout the central and northern Andean Ridge, with approximately 60% in Peru, 30% in Bolivia, and the remainder in Columbia, Ecuador, Venezuela, Brazil, Argentina and Panama. Cocaine production

Production of illicit natural cocaine involves three steps: 1. Extraction of crude coca paste from the coca leaf. 2. Purification of the coca paste to cocaine base. 3. Conversion of cocaine base to cocaine hydrochloride. Cocaine is normally encountered in the laboratory in paper or plastic packs of white powder and can be analysed without extraction. Analysis of cocaine Colour test Cobalt thiocyanate test or modified cobalt thiocyanate test (Scott test): a blue colour indicates the presence of cocaine. Odour test A 5% methanolic solution of sodium hydroxide added to the test sample and warmed gives a characteristic odour in the presence of cocaine. TLC n n

TA: Rf ¼ 0.65; TB: Rf ¼ 0.47 Visualisation is with acidified iodoplatinate solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) In addition to GC and HPLC, IR spectroscopy is routinely used in cocaine cases if a distinction is to be made between cocaine as a salt, e.g. cocaine hydrochloride, and cocaine in base form. The cocaine base is known as crack and, unlike cocaine hydrochloride, can be consumed by smoking. The differences in their IR spectra are shown in Fig. 11.8. The differences in the spectra at 1736 and 1709 cm1 for the base and 1729 and 1711 cm1 for the hydrochloride are explained (Elsherbini 1998) by the effect of the hydrochloride ion on the C¼O stretching bands. A simple laboratory test also exists for the determination of the chemical form of cocaine (Logan et al. 1989). Quantitative analysis and profiling of cocaine samples

Quantitative analysis of cocaine samples may be carried out by GC or HPLC. The general quantitation method previously described may also be used for cocaine. In some jurisdictions, preparations containing less than 0.1% cocaine base are exempt from certain controls and analytical procedures must be designed to accommodate this. Because of the unsophisticated nature of the cocaine manufacturing process, a multitude of trace-level alkaloid impurities are present in illicit cocaine. Many of these impurities are naturally occurring alkaloids that originate from the coca leaf and are carried through the manufacturing process. In addition, cocaine is also contaminated with a variety of manufacturing by-products. The relative amounts of these compounds can be used to compare cocaine samples (Moore, Casale 1998). The alkaloid impurities, which originate in the coca leaf, include cis- and trans-cinnamoylcocaine, tropacocaine, truxillines and hydroxycocaines. Manufacturing by-products found in illicit cocaine include hydrolysis products such as benzoylecgonine, ecgonine methyl ester, ecgonine and benzoic acids.

201

Oxidation by-products also arise and these include N-norcocaine and N-norecgonine methyl ester. New impurities arising from the oxidation of crude cocaine base have been reported recently (Casale et al. 2007). In addition to the above, solvent residues may be detected by NMR or headspace GC. The solvents detected include acetone, methyl ethyl ketone, benzene, toluene and diethyl ether (Cole 1998). A comparison of cocaine samples can be achieved by a combination of qualitative analysis for the presence/absence of certain trace impurities and by quantitative analysis of the cocaine and other ingredients. Isotope ratio analysis has also been used in the profiling of cocaine (Benson et al. 2006). A recent study explored the optimisation and harmonisation of a profiling method for cocaine in two separate laboratories using eight main alkaloids as the comparators (Lociciro et al. 2007). Heroin Street-level heroin (diamorphine, diacetylmorphine) is normally encountered in the laboratory in paper or plastic packs containing typically 100–200 mg of brown (and sometimes white) powder. The street-level purity varies depending on availability and other factors, but values of 40–60% are common. Production of heroin

The raw material for the production of heroin is opium. Opium is a naturally occurring product of the plant Papaver somniferum L. (opium poppy). Opium is purified to form crude morphine. The morphine is acetylated with acetic anhydride to produce diacetylmorphine, the primary constituent of illicit heroin samples. Sometimes known as ‘Chinese heroin’, heroin from south east Asia is a white powder consisting of heroin hydrochloride and minor amounts of other opiate alkaloids, but adulterants are unusual. This material is ideally suited to injection. Heroin from south west Asia is a much cruder product. Typically seen as a brown powder containing heroin base, it has variable amounts of other opiate alkaloids (e.g. monoacetylmorphine, noscapine, papaverine and acetylcodeine) as well as adulterants such as caffeine and paracetamol. It is believed that these cutting agents are added to heroin either at the time of manufacture or during transit. Brown heroin may be ‘smoked’ by heating the solid on a metal foil above a small flame and inhaling the vapour. Those intending to inject brown heroin must first solubilise it with, for example, citric acid or ascorbic acid. Analysis of heroin Colour test Marquis reagent gives a purple/violet colour.

Other opiate alkaloids (morphine, codeine, monoacetylmorphine and acetyl codeine) give the same positive reaction to the Marquis test. TLC n n

TA: Rf ¼ 0.47. TB: Rf ¼ 0.15 Visualisation is with acidified iodoplatinate solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) Quantification and profiling of heroin

Heroin may be quantified by either GC or HPLC. A problem associated with GC analysis is that heroin may hydrolyse to 6-O-monoacetylmorphine and/or transacetylation may occur of the common cutting agent paracetamol by heroin in the injection port of the GC column. The use of fresh samples and of chloroform as solvent can avoid these problems. By examining the amount of heroin, papaverine, noscapine and acetylcodeine in the samples it is possible to discriminate between or show a link between samples (Besacier, Chaudron-Thoxet 1999; Dufey et al. 2007; Seta et al. 1994; Stromberg et al. 2000). This method of comparison was used successfully on 500 heroin samples that were divided into nine groups (Zhang et al. 2004). It may be further possible to examine heroin samples and show potential links between samples by the presence (and amount) of adulterants such as caffeine or by the presence of less common adulterants such as diazepam, phenobarbital

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Figure 11.8 Infrared spectra of (A) cocaine base and (b) cocaine hydrochloride.

or mannitol hexa-acetate (El Haj et al. 2004). In large seizures, differences may be found between various samples originating from the seizure that indicate that it includes more than one batch of heroin. Chemical profiling was used to determine the country of origin of heroin samples in Australia (Collins et al. 2006) and stable isotope analysis was used to complement the chemical profiling on the same

samples (Casale et al. 2006). Isotope ratio analysis has increasingly been used as a comparison tool for heroin (Carter et al. 2005; Zhang et al. 2005). In addition to examining the relative ratios of the main components, it is possible to analyse for solvent residues (Cole 1998; Dams et al. 2001).

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Analysis of the main drugs of abuse LSD LSD is one of the most potent hallucinogenic substances known. Its properties were first discovered in the 1930s and its popularity as a drug of abuse was very high during the 1960s and 1970s when it was associated with the hippy movement. Synthesis of LSD

LSD can be produced by several different methods, the majority of which use lysergic acid as the starting material. Lysergic acid itself is also produced in clandestine laboratories using, most commonly, ergometrine or ergotamine tartrate as the starting material. Ergotamine refluxed with potassium hydroxide and hydrazine in an alcohol–water mixture produces lysergic acid. The methods used for the production of LSD yield a crude product, which is then cleaned up and converted to a more stable form, e.g. the tartrate salt. In the past, LSD was encountered in a variety of substrates including powder in gelatine capsules, gelatine squares, sugar cubes and ‘microdots’. Nowadays LSD is encountered mostly in paper dose form. The paper dosages are produced by soaking pre-printed paper in a solution of LSD. These sheets are then perforated into squares (typically 5
5 mm) with each square (‘tab’) containing approximately 50 mg of LSD. The designs on the paper can vary from one design per square to one large design covering many squares (Fig. 11.9).

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Quantitative analysis and comparison of LSD

HPLC is the method of choice for quantitative analysis of LSD. Using the solvent mixture methanol–water (1 : 1), quantitative extraction of LSD from paper squares is normally achieved after sonication for 20 min (McDonald et al. 1984). In some instances, a comparison is requested between one square of LSD and a large sheet of perforated squares. This can be an easy matter if the design on the large sheet spreads over the whole sheet and the ‘missing’ square fits neatly into the pattern. In other instances, the design may be on each individual square (or there may be no design). In such a case it is necessary to examine the colour/design/dimensions of the squares and the perforation pattern. Chemical comparisons can also be undertaken, but squares from the same larger sheet can vary in the amount of LSD on each square. MDMA

The only analogue of LSD to receive widespread interest is lysergic acid N-(methylpropyl)amide (LAMPA) and any analytical technique should be capable of separating LAMPA from LSD. The presence of LSD may be signalled early by placing the suspect paper under long-wavelength UV light. The presence of LSD is indicated by a blue fluorescence. Colour test Van Urk reagent gives a purple colour.

MDMA is the prototypical member of a large series of phenethylamine designer drugs and has become one of the main drugs of abuse in many countries in northern Europe. Clandestine production is largely centred in Europe. A number of homologous compounds with broadly similar effects, e.g. MDA (methylenedioxyamfetamine), MDEA (methylenedioxyethamfetamine) and MBDB (N-methyl-1-[1,3-benzodioxol-5yl]-2-butanamine) have also appeared, but have proved less popular. These substances are collectively known as the ‘ecstasy’ drugs. MDMA is the most common drug encountered in ‘ecstasy’ tablets. The tablets are typically 10 mm in diameter, either flat or biconvex, and weigh approximately 300 mg. The MDMA content varies but is generally in the range 80–100 mg per tablet. The tablets normally carry a characteristic logo/imprint. These designs are not restricted to MDMA tablets, but may be found on amfetamines and other illicit products. In other words, the logo and other physical characteristics provide no reliable information on the drug content. Many hundreds of different impressions have been found and several examples are shown in Fig. 11.5.

TLC

Synthesis of MDMA

Analysis of LSD

n n n

TAI: LSD Rf ¼ 0.58; LAMPA Rf ¼ 0.49 Visualisation: observe the plate under UV light (254 and 365 nm) Spray with Van Urk reagent and heat to give a blue spot.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) Some difficulty may be encountered in obtaining an unequivocal identification of LSD is because of its low dosage (50 mg or less). However, if the sample is concentrated, a satisfactory analysis can be achieved. Place a suspect LSD square in a glass vial and cover with methanol. After soaking (or sonication) for 10–20 min, the methanol can be transferred to a plastic insert for analysis. Another method is to add concentrated ammonia (2 drops) to the methanol. In addition to chromatographic separation, LSD can be discriminated from other ergot alkaloids by its MS fragmentation pattern; for example, the presence in the LSD spectrum of a m/z 100 fragment nearly as intense as the as the m/z 111 fragment differentiates LSD from other di-substituted amides (Clarke 1989).

Figure 11.9 Examples of LSD paper squares.

Several methods of synthesis can be employed including: 1. Amine displacement method using safrole as the starting material. 2. Via the intermediate 1-(3,4-methylenedioxyphenyl)-2-propanone (MDP2P) with isosafrole or a nitrostyrene as starting material. Analysis of MDMA Colour test Marquis test gives a blue/black colour. TLC n n

TA: Rf ¼ 0.31; TB: Rf ¼ 0.23 Visualisation is with acidified iodoplatinate solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) Base extraction into an organic solvent and/or derivatisation prior to GC-MS analysis is common with MDMA. Quantitative analysis and profiling/comparison

In order to perform a quantitative analysis on ‘ecstasy’ tablets they must first be ground to produce a homogeneous powder and the MDMA content determined by either GC (either directly or base extracted) or HPLC. Chemical profiling of tablets containing MDMA involves the examination/quantification of the drug and main adulterants present such as caffeine, sugars and binding agents. In addition to the main ingredients, many trace-level impurities from the synthetic process can be present and these can be used for comparison (Bohn et al. 1993; Renton et al. 1993). A cross-laboratory study on organic impurity profiling of MDMA tablets based on 46 organic impurities yielded separation between population of samples from the same synthesis batch and samples from different batches (Weyermann et al. 2008). Trace metal analysis has proved to be a useful method for comparing MDMA tablets (Koper et al. 2007; Waddell et al. 2004). The metals arise in the tablets as a result of the catalyst or reducing agent in the synthesis. Isotope ratio analysis has also been used to discriminate between different tablets

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containing MDMA (de Korompay et al. 2008; Palhol et al. 2004). Recent advances in impurity profiling in MDMA tablets have employed a variety of techniques, with multiple techniques being used in many situations (Waddell-Smith 2007). As already mentioned, tablet comparisons can also be made using (so-called) ballistic analysis. In this, general physical features and microscopic examination of defects and punch marks are used for comparison. The difficulty is that, at any one time, a large fraction of illicit tablets in circulation may be almost identical. Anabolic steroids Anabolic steroids may be abused by ‘body builders’ and athletes. In the UK, 48 steroids are listed specifically and generic legislation covers certain derivatives of 17-hydroxyandrostan-3-one or 17-hydroxyestran-3-one. Methandienone, nandrolone, oxymetholone, stanozolol, and testosterone and its esters account for most cases. Further nonsteroidal anabolic compounds are also controlled, i.e. human chorionic gonadotrophin (HCG), clenbuterol, non-human chorionic gonadotrophin, somatotropin, somatrem and somatropin. Certain anabolic steroids are scheduled in the US Controlled Substances Act, but these drugs are not listed in the UN Conventions. A large number of the anabolic steroids encountered in seizures have been found in counterfeited packaging and the drug content may differ qualitatively or quantitatively from what is indicated in the information on the product label. This mislabelling can be particularly frustrating to the forensic chemist trying to identify the particular steroid in the product. Formulations may be either as tablets or as steroid esters dissolved in vegetable oil suitable for injection. The oils may be extracted using a steroid : hexane : methanol ratio of 1 : 2 : 1 (Chiong et al. 1992) with the methanol layer being used for analysis. Because of the large number of steroid products available, analytical information is presented here on only four: fluoxymesterone, nandrolone, testosterone and methyltestosterone. Further information on the analysis of anabolic steroids in urine is to be found in Chapters 6 and 7 on Drugs in sport.

TLC n n n

TA: diazepam Rf ¼ 0.75; flunitrazepam Rf ¼ 0.63; nitrazepam Rf ¼ 0.62; flurazepam Rf ¼ 0.68 TB: diazepam Rf ¼ 0.23; flunitrazepam Rf ¼ 0.10; nitrazepam Rf ¼ 0.30; flurazepam Rf ¼ 0.00 Visualisation is with acidified iodoplatinate solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) GHB and analogues g-Hydroxybutyric acid (GHB) was originally developed as an anaesthetic drug and is still used for that purpose in some countries. It acts as a central nervous system depressant and hypnotic, and is chemically related to the brain neurotransmitter GABA (g-aminoxybutyric acid). Synonyms include sodium oxybate, g-OH, somatomax, ‘GBH’ and ‘liquid ecstasy’. The effects of GHB have been likened to those produced by alcohol and there are claims that it has anabolic properties. GHB is easily manufactured by adding aqueous sodium hydroxide to g-butyrolactone (GBL) to leave a weakly alkaline solution. Not only is the precursor GBL widely used as an industrial solvent, it can also be ingested directly to produce the same effects as GHB. Illicit GHB is normally sold in solution as a clear liquid in 30 mL opaque plastic bottles. The typical dose is around 10 mL equivalent to about 1 g or more of GHB. The sodium and potassium salts of GHB are hygroscopic. This property mean that GHB is almost never found as a powder or in tablets. Analysis of GHB

The legal distinction between GHB and GBL, coupled with the potential for GBL to undergo interconversion with GHB, raises important issues in the analytical approach to GHB analysis. The potential exists for aqueous-based GBL products to undergo conversion to GHB in the time between manufacture and consumption. Some of the factors affecting this interconversion have been explored (Ciolino et al. 2001). Colour test With 1% cobalt nitrate a pink to violet colour is indicative of GHB.

Analysis of steroids

Separation/identification

TLC

(For analytical data see Tables 11.5 and 11.6.) GC analysis of GHB samples will result in conversion of GHB to GBL, thus necessitating the need for derivatisation prior to analysis (Blackledge, Miller 1991). Test samples are taken to dryness under a stream of dry air. Samples are then derivatised with 99 : 1 N,O-bis(tri-methylsilyl)trifluoroacetamide (BSTFA) : trimethylchlorosilane (TMCS) in the presence of pyridine and incubated at 70 C for 30 min. GHB is detected as the di-trimethylsilyl (TMS) derivative and GBL does not form a silyl derivative. HPLC can be used without derivatisation (Mesmer, Satzger 1998). In a study of the reaction of GHB with alcohol it was found that an ester was formed under certain conditions and this can be separated from both GHB and GBL by HPLC and GC (Hennessy et al. 2004). Capillary electrophoresis has been used to separate GHB, GBL and 1,4butanediol (Dahlen, Vriesman 2002). Carbon isotope ratio analysis has been used to examine the discrimination between endogenous and exogenous GHB (Saudan et al. 2007).

n n n

TP: fluoxymesterone Rf ¼ 0.51; nandrolone Rf ¼ 0.87; testosterone Rf ¼ 0.60; methyltestosterone Rf ¼ 0.70 TQ: fluoxymesterone Rf ¼ 0.09; nandrolone Rf ¼ 0.48; testosterone Rf ¼ 0.07; methyltestosterone Rf ¼ 0.16 Visualisation is with ethanol–sulfuric acid or p-toluene sulfonic acid solution.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) Benzodiazepines There are 34 benzodiazepines listed in Schedule 4 of the UN 1971 Convention. Most are now rarely prescribed and abuse is largely restricted to pharmaceutical preparations containing diazepam, flunitrazepam, nitrazepam, flurazepam and temazepam. They may be used in conjunction with opiates (e.g. heroin) or in their own right. A particular problem occurred in Scotland during the mid-1990s when the contents of gel-filled temazepam capsules were injected. Abuse of temazepam declined following the withdrawal of capsules from the market and their replacement with tablets. In other countries, flunitrazepam became the most widely abused benzodiazepine. This drug also gained a certain notoriety for its association with ‘date-rape’. For these reasons, flunitrazepam was moved to Schedule 3 of the UN 1971 Convention and is therefore subject to more stringent controls. Analytical information is presented here only for diazepam, flunitrazepam, nitrazepam and flurazepam. Analysis of benzodiazepines Colour test Zimmerman’s test: reddish-purple or pink indicates the possiblility of some of the benzodiazepines.

Khat Catha edulis is a flowering evergreen shrub cultivated in East Africa and the Arabian Peninsula. The leaves and fresh shoots are commonly known as khat, qat or chat. Khat can be used by chewing the leaves or by brewing as a ‘tea’ and daily consumption can be up to several hundred grams. Khat has stimulant effects similar to those of amfetamine. Alcoholic extracts (tinctures) of khat have been noted especially in ‘herbal high’ sales outlets and at music festivals. The active components of khat, cathinone (()-1-aminopropiophenone) and cathine ((þ)-norpseudoephedrine), are usually present at around 0.3–2.0% (Lee 1995). Both substances are close chemical

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Analysis of the main drugs of abuse relatives of synthetic drugs such as amfetamine and methcathinone. Khat must be used fresh as the more active cathinone begins to deteriorate rapidly after harvesting. Both cathine and cathinone are scheduled under the UN 1971 Convention, but khat itself is only specifically listed in a few jurisdictions. Analysis of khat

Approximately 5–6 g of plant material is cut into small pieces. Methanol (15–20 mL) is added and sonicated for 15 min. The green methanolic solution is filtered/decanted and condensed to near dryness. Approximately 20 mL of 0.2 mol/L sulfuric acid is added and the solution acquires a reddish hue. A chloroform extract will remove the neutral organic compounds. The aqueous layer (red layer) is basified with saturated sodium bicarbonate solution. Methylene chloride (20 mL) is added to extract the cathinone and cathine. A stream of air is used to reduce the volume to approximately 1 mL. Colour test Cathinone gives no reaction with Marquis reagent, but does produce a slow-forming yellow/orange colour with Chen’s reagent. TLC n n

TE: cathinone Rf ¼ 0.46; cathine Rf ¼ 0.25 Visualisation: UV (254 nm) and 0.5% ninhydrin with cathinone showing an orange colour and cathine a purple colour.

Separation/identification

(For analytical data see Tables 11.5 and 11.6.) Psilocybe mushrooms The hallucinogenic substances psilocin and its phosphate ester psilocybin occur in a number of fungi, particularly those of the genus Psilocybe. These are small grey mushrooms which grow wild over large areas. Although such material is not in itself controlled, and neither is its cultivation, it has been held in UK case law that the deliberate drying or processing of these mushrooms constitutes preparation of a controlled drug. Analysis of psilocybe mushrooms

A small quantity (approximately 1 g) of the dried mushrooms is sonicated with methanol (approximately 5 mL) for 10 min. The liquid is removed and reduced in volume at room temperature in an air flow. Psilocybin can be converted to psilocin by heating. This conversion can also occur if the mushrooms are not dried prior to or when they arrive into the laboratory. Colour test Ehrlich reagent: a violet colour is indicative of psilocybin and psilocin TLC n n n

TAN: psilocybin Rf ¼ 0.34; psilocin Rf ¼ 0.59 TA: psilocybin Rf ¼ 0.05 psilocin Rf ¼ 0.39 Visualisation is with Van Urk’s reagent, with both compounds showing a blue/violet colour.

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the 1980s that the phenomenon of so-called designer drugs was fully recognised. Starting in the late 1980s, a large series of designer drugs began to appear, all of which were based on the phenethylamine nucleus. Just as with the production of the major illicit phenethylamines (e.g. MDMA), much of this synthetic activity took place in Europe. Table 11.7 lists a number of designer drugs that have appeared in Europe and the USA since the mid-1990s. This list, which may not Table 11.7 Designer drugs reported in Europe and the USA since the mid-1990s Compound/drug

Acronym

UN/UK

Ring-substituted phenethylamines 3,4-Methylenedioxyamfetamine

MDA

þ/þ

3,4-Methylenedioxymetamfetamine

MDMA

þ/þ

3,4-Methylenedioxyethylamfetamine

MDE(A)

þ/þ

4-Bromo-2,5-dimethoxyamfetamine

DOB (Bromo-STP)

þ/þ

4-Methoxyamfetamine

PMA

þ/þ

N-Hydroxy-MDA

N-OH MDA

þ/þ

3,4-Methylenedioxypropylamfetamine

MDPA

/þ

N-Methyl-1-(1,3-benzodioxol-5-yl)-2butanamine

MBDB

/þ

1-(1,3-Benzodioxol-5-yl)-2-butanamine

BDB

/þ

4-Bromo-2,5-dimethoxyphenethylamine

2C-B

/þ

3,4-Methylenedioxydimetamfetamine

MDDM

/P

2,5-Dimethoxy-4-(n)propylthiophenethylamine

2C-T-7

/þ

4-Allyloxy-3,5-dimethoxyphenethylamine

AL

/þ

3,5-Dimethoxy-4methylallyloxyphenethylamine

MAL

/þ

N-Hydroxy-MDMA

FLEA

/P

2,5-Dimethoxy-4-chloroamfetamine

DOC

/þ

4-Methylthioamfetamine

4-MTA

/P

2,5-Dimethoxy-4-ethylthiophenethylamine

2C-T-2

/þ

4-Methoxy-N-metamfetamine

Me-MA

/þ

6-Chloro-MDMA

—

/þ

N-(4-Ethylthio-2,5-dimethoxyphenethyl)hydroxylamine

HOT-2

/þ

2,5-Dimethoxy-4-iodo-phenethylamine

2C-I

/þ

4-Methoxy-N-ethylamfetamine

—

/þ

N-Substituted amfetamines without ring substitution /P

Separation/identification

N-Hydroxyamfetamine

N-OHA

(For analytical data see Tables 11.5 and 11.6.) Direct injection of psilocybe mushroom extracts onto a GC column will convert psilocybin to psilocin by thermal dephosphorylation and only psilocin will be detected. Thus prior derivatisation is necessary if psilocybin is to be detected. To eliminate sugars that may interfere with derivatisation, 1 mL of acetone is added to the methanolic solution and the mixture allowed to stand for 30 min and then filtered. The solution is taken to dryness in a stream of air. Pyridine (15 mL), TMCS (15 mL) and BSTFA (100 mL) are added and heated at 100 C for 30 min. Psilocin is converted to psilocin di-TMS and psilocybin to psilocybin tri-TMS. LC-MS and LC-MS-MS have also been used to analyse the constituents of magic mushrooms (Kamata et al. 2005).

N,N-Dimetamfetamine

—

/

N-Acetylamfetamine

—

/

Di-(1-phenylisopropyl)amine

DIPA

/

N,N-Dimethyl-5-methoxytryptamine

5-MeO-DMT

/þ

N,N-Di-(n)-propyltryptamine

DPT

/þ

4-Acetoxy-N,N-di-isopropyltryptamine

—

/

a-Methyltryptamine

a-MT

/

1-PEA

/

'Designer Drugs' Although a few ring-substituted phenethylamines (e.g. 2,5-dimethoxy4-bromoamfetamine (DOB), 4-bromo-2,5-dimethoxyamfetamine) had been subject to limited abuse since the 1960s, it was not until

Tryptamines

Other phenylalkylamines and miscellaneous 1-Phenethylamine N-Methyl-1-phenethylamine

N-Me-PEA

/

4-Methyl-1-phenethylamine

4-Me-PEA

/

1-Phenyl-3-butanamine

—

/

N-Benzylpiperazine

BZP

/

Methcathinone

—

þ/þ

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necessarily be complete, shows that the phenethylamines comprise the largest group. Ring-substituted compounds were more common than N-substituted homologues without ring substitution. The substances shown in Table 11.7 have appeared both as powders and as tablets, often manufactured, packaged or marked in such a way that they may appear to the user to be amfetamine or MDMA. Considerable scope exists to develop further series of phenethylamine-related ‘designer drugs’. Thus ring-substituted analogues of cathinone and methcathinone might have MDMA-like activity. As mentioned earlier, the EU, via Europol and EMCDDA, carry out risk assessments on new synthetic drugs. Since 1997 ten risk assessments have been undertaken on the following drugs: MBDB, 4-MTA, GHB, ketamine, p-methoxymethamphetamine (PMMA), 2C-1, 2C-T-2, 2CT-7, TMA-2 and benzylpiperazine (BZP). Various studies have outlined the analytical methods available to separate and identify these and other designer drugs (Blachut et al. 2002; Furnari et al. 1998; Poortman, Lock 1999; Tsai et al. 2006) A detailed chemical identification of DOB has been described using CE, IR spectroscopy and GC-MS (da Costa et al. 2007). Raman spectroscopy has been used as a screen for tablets containing DOB (Bell et al. 2007). Piperazines have increasingly become an alternative to amfetaminederived drugs of abuse (EMCDDA 2005). Among the most prominent of these have been N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP) and 1-(3-chlorophenyl)piperazine (mCPP). The legal status of piperazines is very inconsistent and many European countries are considering control measures, while others already control mCPP (EMCDDA 2005). In the USA, BZP and TFMPP were temporarily placed into Schedule 1 of the Controlled Substances Act in 2002 followed by final placement of BZP into Schedule 1 2004. In 2003 both BZP and TFMPP were controlled in Japan. The analysis of aryl-piperazines has been carried out by HPLC and GC-MS with and without derivatisation (de Boer et al. 2001; Tsutsumi et al. 2005). Analytical profiles covering a range of techniques have been reported for a number of tryptamines (Spratley et al. 2005). The approach to the analysis of these compounds, especially the phenethylamine-related ‘designer drugs’, could be in line with the general procedure outlined for powders and with specific reference to the analytical procedures employed to analyse MDMA or amfetamine. Those substances listed in UN 1971 or which are controlled in UK by the Misuse of Drugs Act 1971 are shown by (þ) in Table 11.7. Substances pending control in the UK are shown by ‘P’. In the USA, unscheduled substances may still be deemed to be controlled by virtue of the Controlled Substances Analogue Enforcement Act 1986.

Conclusion Many other compounds are encountered in the laboratory as ‘drugs of abuse’ such as opium, phencyclidine and analogues, tryptamines, barbiturates, methadone, morphine, dihydrocodeine, ephedrine, ketamine and alkyl nitrites. However, the general approach to the analysis of an unknown substance previously outlined should pose no difficulty to the identification of any of these drugs. Analytical information and background information on many of the drugs can be found in some of the general texts in the area (CND Analytical 1994; Cole, Caddy 1995; Gough 1991; Karch 1996, 1998; Klein et al. 1989; Redda 1989; Shulgin, Shulgin 1991; Smith 2005; UN 1994; Weaver, Yeung 1995).

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McDonald PA et al. (1984). An analytical study of illicit lysergide. J Forensic Sci 29: 120–130. Mesmer MZ, Satzger RD (1998). Determination of gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) by HPLC/UV-VIS spectrophotometry and HPLC/thermospray mass spetrometry. J Forensic Sci 43: 489–492. Microgram (2003) Cocaine impregnated silicone in baseball cap parts in Peru. Microgram Bulletin 36: 271. Moore JM, Casale JF (1994). In-depth chromatographic analyses of illicit cocaine and its precursor, coca leaves. J Chromatogr A 674: 165–205. Moore JM, Casale JF (1998). Cocaine profiling methodology – recent advances. Forensic Sci Rev 10: 13–15. Palhol F et al. (2004). 15N/14N isotope ratio and stratistical analysis: an efficient way of linking seized ecstasy tablets. Anal Chim Acta 510: 1–8. Poortman AJ, Lock E (1999). Analytical profile of 4-methylthioamphetamine (4MTA), a new street drug. Forensic Sci Int 100: 221–233. Qi Y et al. (2006). Australian Federal Police seizures of illicit crystalline methamphetamine (‘ice’) 1998-2002: impurity analysis. Forensic Sci Int 164: 201–210. Redda KK (1989). Cocaine, Marijuana, Designer Drugs, Chemistry, Pharmacology and Behaviour. Boca Raton, FL: CRC Press. Renton RJ et al. (1993). A study of the precursors, intermediates and reaction byproducts in the synthesis of 3,4-methylenedioxymethylamphetamine and its application to forensic drug analysis. Forensic Sci Int 60: 189–202. Saudan C et al. (2007). Carbon isotopic ratio analysis by gas chromatography/ combustion/isotope ratio mass spectrometry for the detection of gammahydroxybutyric acid (GHB) administration to humans. Rapid Commun. Mass Spectrom 21: 3956–3962. Sellers JK et al. (1996). High performance liquid chromatographic analysis of enantiomeric composition of abused drugs. Forensic Sci Int 8: 91–108. Seta S et al. (1994). Impurity profiling analysis of illicit drugs. Forensic Sci Int 69 (special issue): 1–102. Shulgin A, Shulgin A (1991). PiHKAL: A chemical love story. Berkeley, CA: Transform Press. Smith FP (2005). Handbook of Forensic Drug Analysis. New York: Elsevier. Spratley TK et al. (2005). Analytical profiles for five "designer" tryptamines. Microgram 3: 54–68. Stromberg L et al. (2000). Heroin impurity profiling. A harmonization study for retrospective comparisons. Forensic Sci Int 114: 67–88. Takekawa K et al. (2007). Methamphetamine body packer: acute poisoning death due to massive leaking of methamphetamine. J Forensic Sci 52: 1219–1222. Tsai CC et al. (2006). Optimization of the separation and on-line sample concentration of phenethylamine designer drugs with capillary electrophoresis-fluorescence detection. J.Chromatogr A 1101: 319–323. Tsutsumi H et al. (2005). Development of simultaneous gas chromatography–mass spectrometric and liquid chromatography–electrospray ionization mass spectrometric determination method for the new designer drugs, N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl)piperazine (TFMPP) and their main metabolites in urine. J Chromatogr B Analyt Technol Biomed Life Sci 819: 315–322. Tzidony D, Ravreby M (1992). A statistical approach to drug sampling: a case study. J Forensic Sci 371549. UN (1994). Rapid Testing Methods of Drugs of Abuse. New York: United Nations. UN (1998). Recommended Methods for Testing Opium, Morphine and Heroin. New York: United Nations. Waddell RJ et al. (2004). Classification of ecstasy tablets using trace metal analysis with the application of chemometric procedures and artificial neural network algorithms. Analyst 129: 235–240. Waddell-Smith RJ (2007). A review of recent advances in impurity profiling of illicit MDMA samples. J Forensic Sci 52: 1297–1304. Weaver K, Yeung E (1995). An Analyst’s Guide to the Investigation of Clandestine Laboratories. Ottawa, ON: Drug Analysis Service. Weyermann C et al. (2008). Drug intelligence based on MDMA tablets data I. Organic impurities profiling. Forensic Sci Int 177: 11–16. Zhang D et al. (2004). Component analysis of illicit heroin samples with GC/MS and its application in source identification. J Forensic Sci 49: 81–86. Zhang D et al. (2005). Origin differentiation of heroin sample and its acetylating agent with (13)C isotope ratio mass spectrometry. Eur J Mass Spectrom 11: 277–285.

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CHAPTER

12

Medicinal Products AC Moffat and AG Davidson

Introduction Medicinal products should be safe and efficacious. Manufacturers of medicinal products are required by law to possess marketing authorisations from government regulatory agencies in countries in which their products are marketed and to manufacture their products in compliance with current Good (Pharmaceutical) Manufacturing Practice (cGMP) standards. These requirements include conducting appropriate quality control tests to check that the product conforms to a specification that assures its safety and efficacy. Typical specifications include tests to verify the chemical composition and physical properties of the medicine and to ensure that the medicine is not contaminated by microorganisms or other substances. Although authentic medicinal products are subject to quality control testing by the manufacturer, they may have been counterfeited, adulterated or stored poorly, and therefore need to be checked for the following purposes: n n n n n n n

Determine or confirm their composition Assess their suitability for use Investigate defects Identify unknown medicinal products Identify contaminants Determine whether the products have been adulterated Establish whether the products are counterfeit.

Independent quality checks of medicinal products may be carried out by official medicines control laboratories as part of a government surveillance programme, and by hospital quality control laboratories, public analyst laboratories and forensic laboratories. This chapter describes the philosophies, strategies and methodologies for the analysis and testing of medicinal products by laboratories that may not have access to the manufacturers’ research and development data or to the manufacturers’ test methods, specifications and reference materials for the products. It focuses on the information about the medicinal product that can be derived from other sources and on the conclusions that can then be made about the quality of the product.

Submission of samples and choice of tests A wide range of tests that utilise a variety of analytical techniques are available to laboratories. Samples submitted for testing should therefore be accompanied by a clear written request about the nature of the investigation required. The provision of relevant background information about the sample, including the reason for the request, allows the laboratory to choose the most appropriate tests, analytical techniques and, where relevant, acceptance criteria (i.e. criteria that allow an objective assessment to be made about the quality of the product). In most cases that involve checking of the quality of a medicinal product, it is preferable to use the test methods in the product specification defined in the manufacturer’s marketing authorisation for the product. This is because the product specifications, including the test methods and acceptance criteria, have been assessed by the relevant government regulatory agency as being valid and justified. Some manufacturers publish their analytical methods in the scientific literature. Consequently, it may be worthwhile to check the literature or maintain a 208

library of published methods for widely used medicinal products. Alternatively, in certain circumstances, manufacturers may provide the laboratory with the test methods, acceptance criteria and reference materials. If the laboratory does not have access to the authorised finished product specification, including the test methods and acceptance criteria, pharmacopoeial monographs can be used, where applicable (see later). In the absence of a pharmacopoeial monograph, alternative approaches are required to obtain as much information as possible about the quality of the medicinal product. This requires the development of valid test methods to measure relevant quality parameters and the application of generally accepted criteria to assess the quality. However, if the laboratory uses analytical techniques different from those used by the manufacturer (e.g. a high performance liquid chromatographic (HPLC) technique instead of an ultraviolet (UV) spectrophotometric technique), the results may differ significantly from those obtained by the manufacturer, particularly if high levels of impurities are present. This is because analytical techniques differ in their accuracy, precision, selectivity, and/or specificity and sensitivity. It is therefore important to consider the findings in relation to the technique used. This chapter describes the tests that should be carried out to provide information about different aspects of medicinal products. A summary of the principal tests and techniques for checking known products and investigating unknown products is given in Table 12.1.

Counterfeit medicines The World Health Organization (WHO) describes counterfeit medicines as part of the broader phenomenon of substandard pharmaceuticals – medicines manufactured below established standards of quality and therefore dangerous to patients’ health and ineffective for the treatment of diseases. The difference is that counterfeits are deliberately and fraudulently mislabelled with respect to identity or source. Counterfeiting occurs with both branded and generic products. Counterfeit medicines may: n contain no active ingredient n contain the wrong active ingredient (e.g. a cheap antibiotic instead of an expensive antibiotic) n contain an incorrect (usually low) quantity of the active ingredient n be in low-quality packaging n be manufactured using low-quality active ingredient or excipients n be manufactured under poor standards of cGMP compliance. Counterfeit products that contain no active ingredient and those that contain the wrong active ingredient or the correct active ingredient in the wrong amount can be detected by carrying out appropriate identification and quantitative tests (see below). In situations where resources for accurate and precise quantitative testing are limited, for example in developing countries, basic tests, including semi-quantitative tests, may be sufficient to detect these types of counterfeit medicines (see below). It may also be possible to identify products as being counterfeit by their general appearance (colour, markings, etc.), particularly when the appearance differs from a genuine batch of the product. For this reason, laboratories that regularly undertake checks for counterfeit medicines maintain a stock of genuine reference products for comparison. Examination of the labelling should also be carried out to check the

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Direct comparison of products

209

Table 12.1 Summary of tests and techniques Purpose

Test(s)

1. General check of quality

Test to full specification

Analytical technique(s)(a)

Comment Refer to typical monographs for dosage forms in pharmacopoeias

Identity

See 2 below

Assay

See 5 below

Homogeneity

Uniformity of content

Contaminants

HPLC, GC, TLC, CE, etc.

Release of active ingredient

Dissolution test

Microbial quality

TVAC, specific microorganisms, sterility test

2. Confirm identity of product

Instrumental tests for active ingredient (s)

NIR, IR or combination of UV, HPLC, TLC, colour reaction, melting point, etc.

WHO basic tests

Colour reactions, TLC

3. Rapid identification of unknown product

Visual comparison of physical characteristics (size, colour, shape, etc.) against library

TICTAC, Identidex, etc.

See Chapter 13

Instrumental checks provide greater assurance about identity

Simple screening tests

UV, TLC, HPLC, colour tests

See Chapters 32, 39, 41 and 30

4. Unambiguous identification of unknown products

Specific identification tests

IR, NMR, LC-MS, GC-MS, CE, chiral HPLC (for enantiomeric substances), AAS (for inorganic moieties)

See relevant chapters on these techniques

5. Quantification of active ingredient(s)

Assay

HPLC, GC, UV, CE

See relevant chapters on these techniques

6. Homogeneity of active ingredient

Uniformity of content (or mass)

Assay of several (e.g. 10) dosage units or subsamples of the product

Uniformity of content preferred, particularly for unit doses with a content of active substance 3000

37.5

Interpretation

Although there is a complex relationship between bromide ion concentration and signs and symptoms of toxicity, measurement of bromide is helpful in the diagnosis and management of cases of intoxication. Table 17.7 shows reference values of bromide and typical concentrations after therapeutic use and for cases of bromide intoxication (Maes et al. 1985; Vaiseman et al. 1986). Elimination of bromide from the body may be extremely slow in cases of intoxication without the use of active elimination therapy (e.g. diuresis, haemodialysis or haemofiltration). Chlorate Introduction

Sodium chlorate (NaClO3) is an effective, inexpensive, non-selective herbicide. Potassium chlorate is used in the manufacture of matches and some explosives. Both compounds are powerful oxidising agents. Early signs and symptoms of chlorate poisoning include nausea, vomiting and abdominal pain (Steffen and Seitz 1981; Vaiseman et al. 1986). Systemic absorption leads to substantial oxidation of haemoglobin to form methaemoglobin, which may cause cyanosis, dyspnoea, anuria and coma (Ellenhorn 1997). Intravascular haemolysis and severe metabolic acidosis may also occur (Ellenhorn 1997). Ingestion of potassium chlorate may cause hyperkalaemia. Specimen collection

Specimens are collected as follows: n n n n

Blood – 5 mL, lithium–heparin tube Urine – 20 mL, sterile plastic universal container Stomach contents – useful in investigation of fatalities Scene residues – useful for identification purposes.

Cyanide Introduction

Severe or fatal cyanide poisoning is relatively rare and mostly involves suicidal ingestion of cyanide salts. Hydrogen cyanide (HCN; prussic acid) is a highly toxic volatile liquid. Fumes of hydrogen cyanide are given off when cyanide salts are mixed with acids or produced in the stomach following oral ingestion. Although HCN has a characteristic almond-like odour, up to 50% of the population are unable to smell it. Surprisingly, it was not a characteristic feature post mortem of a large series of cyanide suicide deaths, although this could have been related to air-flow ventilation systems in postmortem rooms (Gill et al. 2004). Soluble salts of cyanide include potassium and sodium cyanide, which are used industrially in electroplating and metal processing and as laboratory reagents. Less soluble salts of cyanide include silver and gold cyanide, and mercuric cyanide, which also release HCN on contact with strong acids. As a general rule most cyanide suicide deaths are reported in those individuals, mostly male, who have occupational access to cyanide salts. HCN may also be formed as a combustion product in fires from nitrogen-containing materials such as wool and silk or synthetic polymers such as polyurethanes, polyamides and polyamides (Barillo et al. 1994; Baud et al. 1991; Chaturvedi et al. 2001). Less common sources of cyanide include the accidental or intentional ingestion of cyanogenic plants or their seeds. The signs and symptoms of cyanide toxicity appear rapidly after inhalation of HCN or ingestion of cyanide salts; the estimated fatal doses are approximately 100 mg HCN or 300 mg potassium cyanide (Ellenhorn 1997). Early neurological signs include headache, dizziness, anxiety and confusion. In severe cases there may be a rapid loss of consciousness, respiratory failure and convulsions that lead to cardiorespiratory arrest and death. The mechanism of cyanide toxicity is to block electron transport in the cytochrome a-a3 complex; this leads to a dramatic fall in oxidative metabolism and cellular hypoxia, which most directly affects the brain and heart. Cyanide is metabolised rapidly in the liver by an enzyme (rhodanase) to thiocyanate (SCN), which is largely non-toxic. As a consequence, blood cyanide concentrations decline rapidly after exposure or ingestion, with an estimated elimination half-life of 1–2 h. A number of antidotes are useful in the treatment of cyanide poisoning (e.g. cobalt EDTA and hydroxocobalamin). The measurement of blood cyanide concentrations is rarely useful in a medical emergency when dealing with the acute management of a patient. It can be useful in the retrospective evaluation of the severity of cyanide ingestion and in assessment of the efficacy of different antidotes. Measurement of blood cyanide is most useful in the investigation of suspected cyanide poisoning and in fire-related deaths, including aircraft accidents. Specimen collection

Specimens are collected as follows: n n

Analysis

In clinical cases of chlorate poisoning the diagnosis rests on measurement of methaemoglobin, which is easily carried out as part of a routine biochemical analysis. A method that involves ion chromatography for the direct analysis of chlorate in body fluids and tissues has been described by Eysseric et al. (1999). Ion chromatography can resolve chlorate from closely related anions such as nitrate, bromate and phosphate. Interpretation

Serious and sometimes fatal poisoning can occur after the ingestion of 15 g or more of sodium or potassium chlorate. The case described by Eysseric et al. (1999) involved a 49-year-old man who was admitted to hospital with severe features associated with chlorate poisoning. He had acute haemolysis with a plasma haemoglobin of 3.9 g/L and 30% methaemoglobin formation. High concentrations of chlorate ion were found in the admission plasma (54 mg/L), gastric contents (1300 mg/L) and urine (4300 mg/L). The patient died 12 h after admission.

n

Blood – 10 mL, lithium–heparin or K-EDTA container Stomach contents – useful as part of postmortem examination, but great care is required because of the risk of secondary cyanide exposure Cyanide in blood is stable if kept at 4
C for up to 1 week, but is unstable if frozen or kept at room temperature. However, contradictory findings regarding cyanide stability in whole blood have often been reported (Lindsay et al. 2004).

Analysis

Numerous methods have been described for the analysis of cyanide in biological fluids. These involve colorimetric, fluorimetric or chromatographic techniques (Cruz-Landeira et al. 2000; Dunn and Siek 1990; Felscher and Wulfmeyer 1998; Gambaro et al. 2007; Lindsay et al. 2004; Lundquist et al. 1987; Odoul et al. 1994; Suzuki et al. 1982). Some of these methods can be laborious and are generally unsuited to the emergency diagnosis and management of patients (Lindsay et al. 2004). Many described methods are devoted to the forensic examination of postmortem blood and stomach contents. Some methods are based on the formation of the purple-coloured complex 2-nitrophenylhydroxylamine (Guilbart and Kramer 1966).

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Anions This complex is formed by the reaction of cyanide in alkaline solution with 4-nitrobenzaldehyde and 1,2-dinitrobenzene, and the reaction has been adapted for quantitative Conway diffusion methods (Dunn and Siek 1990; Flanagan et al. 1995). As this method has an unstable endpoint, great care is required in standardising the analytical procedure to ensure accurate results. However, a significant innovation described by Vesey et al. (1999b) has made major improvements. The new procedure is very much faster (12 or 20 kg BW: 20 mg, once daily

20 mg once daily

Phenytoin

18 mg/kg (IV loading), then 2.5–5 mg twice daily (oral)

1.5–2.5 mg/kg twice daily (oral)

1.5–2.5 mg/kg twice daily (oral)

75–150 mg twice daily (oral)

Cyclophosphamide (IV)

500 mg/m2 BSA once a month

500 mg/m2 BSA once a month

500 mg/m2 BSA once a month

Propofol (IV)

2.5–4 mg/kg

2.5–4 mg/kg to 8 years, then 2.5 mg/kg

1.5–2.5 mg/kg

45 mg/kg in 3 divided doses for 24 h then 10 mg/kg daily

Up to 5 years: 35 mg/kg in 3 divided doses for 24 h then 10 mg/kg daily From 5 to 10 years: 25 mg/kg in 3 divided doses for 24 h then 6 mg/kg daily After 10 years: 0.75–1.5 mg in 3 divided doses for 24 h then 62.5–250 mg daily

0.75–1.5 mg in 3 divided doses for 24 h then 62.5– 250 mg daily

Digoxin (oral)

Neonate 2.5 kg: 45 mg/kg in 3 divided doses for 24 h then 10 mg/kg daily

BSA, body surface area; BW, body weight; IV, intravenous. (a) Doses taken from BNF for Children: these are examples of starting doses for common drugs and are not intended as a dosing recommendation; see BNF for Children for complete dosing information.

with generalised symptoms such as lethargy, vomiting, mild pyrexia and neurological signs (e.g. paralysis and fixed, dilated pupils). There may be discoloration of the urine due to myoglobinuria. Children are at greater risk from envenomation because of the relatively greater dose of toxin. Fatality rates for children are higher than for adults following snake bite (McGain et al. 2004). The risk of incurring a snake bite may also be greater in children. Children may develop signs of envenomation sooner than adults and require a higher dose of antivenom.

consistent with poisoning, increases the positive predictive value to 100%, 86% and 92%, respectively (Hwang et al. 2003). Although routine urine drug screens are not useful, urine drug screens may be useful in detecting toxins in symptomatic patients without a history of ingestion or when ingestion is denied (Belson et al. 1999), in particular where drugs of abuse, e.g. cocaine or methadone, may have been ingested by small children. Examination

Clinical aspects Assessment History

A thorough history should be recorded for each episode of poisoning. The history should include time of ingestion, probable dose, the poisons the child was exposed to and the circumstances at the time (including extent of supervision, where the episode happened, who was there, where the poisons were stored and whose medicines were ingested). It is important to establish whether the history is consistent with the patient’s developmental age. A child who is not rolling will not be able to move towards a poison hazard. A child who is not feeding himself or herself will not be able to take a poison independently. Preformatted charts aid in the collection of clinical data, improving both accuracy and completeness of the data (Buckley et al. 1999). An example of a preformatted chart is presented in Figure 27.1. Data can be collected for patient management but also for epidemiology/accident prevention purposes. Electronic databases are useful for recording the data and for performing audits. Patient management software, as is commonly found in emergency departments and hospital in-patient departments, can be modified to improve data capture for toxicology (e.g. disease-specific data entry screens). The clinical history of an episode of poisoning in young children is extremely unreliable (Hwang et al. 2003). Of children presenting with poisoning, around 60% have actually been exposed to the putative poison (Sugarman et al. 1997; Belson et al. 1999; Hwang et al. 2003). A history from an adult or another child is not more reliable than that from the child him-or herself. Physical evidence, such as an abnormal smell of the breath, staining of the clothes and symptoms/clinical signs

The initial step in the physical examination of the poisoned child is the measuring and recording of the vital signs: temperature, pulse rate, respiratory rate, blood pressure and oximetry. Temperature is an important component of the paediatric general examination as it may indicate alternative diagnoses, such as infection. There are some poisonings that affect temperature, such as hypothermia with organophosphate poisoning, and hyperthermia with serotoninergic syndrome or the anticholinergic toxindrome. Pulse rate in children should be interpreted in relation to age-appropriate norms (Wallis et al. 2005). Abnormal pulse (or heart) rate is an important sign in toxicology, indicating cardiotoxicity. Bradycardia and tachycardia are defined at relatively lower pulse rates in children than in adults. Hence, there are different intervention points. Similarly, both diastolic and systolic blood pressure are lower in children than in adults. This results in different values for defining hypotension and hypertension, and the treatment decision points. Respiration rate is also higher in infants and children than in adults (Wallis et al. 2005). Oximetry is useful in assessing respiratory function, particularly when the poison is a sedative or decreases neuromuscular function (e.g. organophosphate poisoning). Arguably, in the context of child poisoning, blood sugar level is also a vital sign. Children are at greater risk of hypoglycaemia than adults (hypoglycaemia may be a differentiating feature in some childhood poisonings compared with adults). Hypoglycaemia, if uncorrected, may lead to tissue/organ injury and long-term morbidity. It is therefore important to identify and correct hypoglycaemia in childhood poisoning. An assessment of conscious state is important in any toxicological examination because many poisons impair consciousness and this may lead to secondary injury due to impairment of respiration and airway. The Glasgow Coma Scale (GCS) is the most widely applied measure of

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Figure 27.1 Preformatted chart for the collection of clinical data in cases of poisoning. (Continued overleaf)

state of consciousness, and can be modified for paediatric use. However, the GCS was developed for the assessment and monitoring of patients with head injury and is less useful in poisoning: A neurological examination focusing upon pupils, nystagmus, muscle tone and reflexes should be performed. Pupil size and response can be important indicators of poisoning: dilated pupils occur particularly in anticholinergic poisoning, while contracted pupils occur in opioid poisoning. Unresponsive pupils can indicate profound coma, such as with barbiturate-induced coma. Horizontal nystagmus is commonly seen in sedative poisoning, and vertical nystagmus may be seen in addition to this with some anticonvulsants, e.g. barbiturates and carbamazepine. An oscillation of horizontal gaze (differentiable from

nystagmus by a lack of directional component) may be seen in serotonin syndrome. Hypotonia can also indicate sedative poisoning or neuromuscular blockade (such as organophosphate poisoning). Deep tendon reflexes may be increased as a result of serotonin syndrome, while they are decreased with sedative poisoning or neuromuscular blockade. Routine examination of the chest should be performed by listening to the sounds of the heart and breathing. Abdominal examination should be performed to determine liver size and condition, and whether there is any abdominal tenderness or rigidity and presence of bowel sounds. Presence of any signs indicating blockage of the intestine or perforation of the viscera would preclude the use of any gastrointestinal decontamination. The skin, scalp and mouth should be examined for any signs of

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Figure 27.1 Continued.

injury. The general state of care of the child should be recorded, e.g. lack of hygiene, general state of hair, nails and clothing. Use of laboratory analyses Considerations for blood sampling

Children have smaller blood volumes than adults and there is greater difficulty in collecting serum, plasma and urine samples. The amount of blood that can be sampled from a child without the need for transfusion depends upon the initial haematocrit (Lister et al. 2008). Recommendations for paediatric clinical trials are for no more

than 3% of blood volume to be sampled on any one study day in a child with a normal red cell mass (haematocrit) (Kauffman 2000). In an infant or toddler this would represent 2.4 mL/kg body weight. As a guide, children can tolerate sampling of 0.25 mL/kg per day without a fall in haematocrit (Lister et al. 2008). In critically ill neonates, it is standard practice to transfuse after 10–15 mL/kg has been sampled. In critically ill patients, there may be other influences decreasing the haematocrit, such as blood loss, haemolysis, and expanded intravascular and/or extracellular volume. The potential clinical effects of the poisoning upon blood volume, haematocrit and oxygen transfer will also need to be considered.

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Consequently, smaller volumes are available for analysis. More sensitive assays are therefore required for the analysis of samples from children. Capillary electrophoresis (CE), and liquid chromatography– mass spectrometry (LC-MS) are newer methods that enable highly sensitive assays to be performed on small sample volumes. Topical anaesthetics (lidocaine, prilocaine, tetracaine or amethocaine) are effective in reducing the pain of venepuncture in children aged from the neonatal period through to adolescence. Distraction through the use of play therapy and/or music therapy is an effective means for reducing the stress of venepuncture (Caprilli et al. 2007). However, it should be considered whether sampling is necessary and sampling should be avoided when the results will not contribute to patient management. Samples obtained by heel-prick sampling yield results comparable with those obtained by venepuncture (Webb et al. 2007). Heel-prick samples are capillary blood as opposed to venous blood. Therapeutic drug monitoring of drugs such as ciclosporin and tacrolimus can be performed using capillary blood specimens because of a high correlation between capillary and venous concentrations. A pitfall in capillary blood sampling arises if the skin is contaminated with the analyte, e.g. if investigating an exposure where the child has had the putative agent on his or her hands and then a finger-prick specimen is obtained. In the majority of cases of paediatric poisons exposure, blood sampling will not be necessary. However, where blood concentrations are decision criteria, e.g. paracetamol poisoning, or where intentional poisoning is suspected, then blood sampling will be necessary. Blood concentrations give information about the magnitude of exposure (i.e. they are quantitative), unlike urine concentrations which give less information about the magnitude of exposure (i.e. they are qualitative). Blood concentrations can be used to determine whether to intervene, (e.g. paracetamol, lead, theophylline) or whether to monitor treatment (e.g. phenobarbital).

plasma concentration of the drug and the characteristics of the saliva (production rate, pH, salivary binding proteins and salivary enzymes) (Aps, Martens 2005). Hence, salivary assays may be more useful in documenting exposure than in determining management criteria or the extent of exposure (see Chapter 18).

Urine drug screens

Treatment of poisoning in children requires dose adjustment for size and development. This is complicated by the lack of paediatric dosing information for many antidotes. Dosing information can be obtained from the BNF for Children, as well as from electronic resources such as Toxbase, TOXINZ and Poisindex. These electronic resources require a subscription in order to obtain access, but the poisons information services will invariably have access. Decontamination, although once standard practice, is increasingly being matched to the risk posed by the exposure. Charcoal is used for carbon-based poisons, but is not effective for metals, strong acids or alkalis. Whole-bowel lavage can be used for slow-release preparations and for metals (e.g. lithium) (Buckley et al. 1995). However, there is a significant risk of aspiration with paediatric decontamination. Gastric lavage can result in aspiration in around 4% of children (Tibballs et al. 1985). Charcoal aspiration, particularly when nasogastric or orogastric tubes are used, is a hazard and can lead to respiratory failure and death (Golej et al. 2001). Whole-bowel lavage, with polyethylene glycol electrolyte lavage solutions, requires large volumes of fluid, beyond the capacity of a child to ingest orally. Hence, nasogastric or orogastric tubes are required. Care is also required for the correct placement of these tubes in order to avoid aspiration. It is accordingly important to consider the risk–benefit ratio of decontamination, and to avoid decontamination when no significant toxicity is predicted. When considering decontamination, the hazard posed by the exposure should be assessed by determining the following:

Urine drug screens may be used to confirm exposure to poisons but are of limited utility in the immediate management of acute poisoning. Urine can be collected by the clean catch method in young children, and by using collection bags in infants. Bacterial contamination of the sample is common, and if a sterile sample is required from an infant then a catheter or suprapubic aspirate sample may be collected. Many of the agents involved in paediatric poisons exposure may not be detectable by routine urine drug screens (Hwang et al. 2003), the results of the test may not be available until after the clinical presentation has resolved, and the vast majority of paediatric poisons exposures do not pose a significant hazard. Hence, performing urine drug screens routinely is not justifiable. However, where there is a diagnostic dilemma, or where deliberate poisoning by a third party is suspected, urine drug screens are warranted. When intentional poisoning is suspected it will be necessary to inform the clinicians responsible for the care of the patient, and the police or the coroner (depending on the circumstances). In most jurisdictions there is a legal requirement to report assaults on children to either the police or child protection authorities. Intentional poisoning would constitute an assault under most legal systems. It is the responsibility of the health professional who suspects that the assault has occurred to report the incident to the police/child protection authorities. Hence, reports should be made directly to the police/child protection authorities rather than to other health professionals. The police will be responsible for documenting the chain of evidence. However, the procedures will vary between legal jurisdictions and it will be necessary for the medical scientist to be familiar with procedures in each state or country in which they work. Saliva

The rate of production of saliva is influenced by hydration status, food, drugs and diurnal variations. In general, drugs are transferred into saliva by passive diffusion and the saliva : plasma ratio of drugs is influenced by the physicochemical properties of the drug (such as pKa, lipid solubility, molecular weight, spatial configuration and charge), the unbound

Hair

Hair analysis can be used to document exposure and give an indication of the timing of exposure in the medium term. When the timing of sampling is late, it may not be possible to detect drugs in blood or urine samples, but it may be possible to detect their presence in hair (Kintz et al. 2007). This approach has been used to detect drugs such as benzodiazepines, zopiclone, barbiturates, methadone, glibenclamide, trimeprazine and diphenhydramine (Kintz et al. 2006, 2007; Villain et al. 2006). More commonly, hair analysis has been used to demonstrate exposure to arsenic or heavy metals (such as mercury, lead, cadmium, chromium, and manganese). Samples of fingernail clippings and teeth (‘milk’ or deciduous teeth are shed naturally in children) can also be analysed to demonstrate heavy-metal exposure (see Chapter 19). Neonatal hair can be analysed to determine intrauterine exposure to ethanol and nicotine as well as illicit drugs such as cocaine, methamfetamine, opioids, cannabinoids and benzodiazepines (Koren et al. 2008). Meconium

In utero exposure to opioids, cocaine, benzodiazepines and ethanol can be determined from the analysis of meconium passed by the newborn (Lopez et al. 2009; Moller et al. 2010; Wang et al. 2010). ELISA and LCMS methods have been reported, as has good correlation between meconium and hair samples. The results have been used by child protection authorities to establish in utero exposure to substance misuse, and also for population studies.

Treatment of poisoning

n n n n

Does the agent have known toxicity? Has the child been exposed to a significant amount of the poison? Are there any confirmatory signs of exposure? Are there alternative safer treatments than decontamination, e.g. antidotes, intravenous fluids?

Some treatment protocols need to be modified for children. The administration of N-acetylcysteine requires relatively high amounts of dextrose solutions which for a child may result in water intoxication. For children, these administration protocols need to be modified to deliver smaller volumes of dextrose.

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Conversely, some antidote doses are based upon the toxin load rather than the size of the patient. For example, with snake or spider bite envenomation, the dose of antivenom is based on the bite/quantity of venom rather than the size/age of the patient. In this case the dose should not be modified for children. However, there should be a lower threshold for treatment in children because the amount of toxin delivered by a snake bite is the same as that for an adult but the dose is proportionately higher because of their smaller body size (White 1995). When a medicine is used in an off-label or unlicensed manner, it is important to make a clinical assessment of its appropriateness. For medicines that are in common use (i.e. when it is the recognised standard of care) or when good-quality evidence is available, the medicine should be prescribed in the normal manner (Gazarian et al. 2006). Where such evidence is lacking, the use of the medicine could be considered experimental or innovative, and measures such as peer-review and informed consent should be conducted prior to use. Response to poisoning (pharmacodynamics) Pharmacodynamic differences between children and adults have been described, e.g. prepubertal children show enhanced response to warfarin compared with pubertal children and adults (Takahashi et al. 2000). The insulin requirements of children with established type 1 diabetes (after the remission phase) are around 0.7–1.0 units/kg per day, increasing at puberty to around 1.4–1.6 units/kg per day in boys and around 1 unit/kg per day in girls, and then decreasing a few years after puberty to adult requirements of around 0.7–0.8 units/kg per day. These insulin dosing requirements are influenced by rates of growth. Selective serotonin reuptake inhibitors (SSRIs) appear to have less efficacy in children and adolescents than in adults, but this may reflect difficulties in diagnosing depression in this age group. Differences in drug response between children and adults may also parallel different susceptibilities to poisoning, e.g. with aspirin causing Reye syndrome and SSRIs causing aggressive behaviour (see below). Aspirin

A 14-year-old boy presented with encephalopathy following the use of aspirin for symptom control during a mild influenza-like illness. Laboratory investigations demonstrated an elevated plasma ammonium concentration (>700 mmol/L), elevated AST (3355 U/L), elevated ALT (2488 U/L), elevated serum lactate (108 mmol/L) and a prolonged prothrombin time (23.6 s). He had a serum salicylate concentration of 232 mg/L. Despite intensive support he died as a consequence of cerebral oedema and tonsillar herniation. Postmortem findings included diffuse microvesicular steatosis and some macrovesicular steatosis (He et al. 2007). The presentation is consistent with a diagnosis of Reye syndrome, which has been linked with aspirin. The mechanism of the interaction between aspirin and a viral illness to produce this condition has been linked with b-oxidation of fatty acids, whereby some individuals are predisposed to aspirin-inhibiting fatty acid metabolism (Deschamps et al. 1991; Glasgow et al. 1999; He et al. 2007). Cytokines can also downregulate some enzymes involved in exogenous and endogenous substrate metabolism. Reye syndrome was described predominantly in children, and this resulted in recommendations to avoid aspirin in children under the age of 12 years. Selective serotonin reuptake Inhibitors

An 11-year-old boy was initially started on atomoxetine 25 mg/day, which was subsequently increased to 60 mg/day after 2 weeks, for the treatment of aggressive behaviour and attention deficit hyperactivity disorder (ADHD). After the increase in dose, he was noted to be increasingly agitated and to have an increase in mood swings. He was reported to have thrown an object at a teacher, and was uncontrollably agitated, crying and threatening to kill himself. These behaviours ceased when his medications were withdrawn (Paxton, Cranswick 2008). When treated with SSRIs, adolescents and young adults are susceptible to treatment-emergent suicidal ideation and suicide-related behaviour (Reith, Edmonds 2007). This risk appears to be age related, with the

vulnerable window being from adolescence through to early adulthood. Although this appears to be a class effect, the risk varies between individual chemical entities. Atomoxetine has its primary mode of action via the inhibition of presynaptic noradrenaline (norepinephrine) reuptake but it also has some effects on serotonin reuptake. Treatment-emergent suicidal ideation and suicide-related behaviour have also been reported with atomoxetine. Other examples of different susceptibilities to poisoning include response to hypnosedatives and ethanol. Children are more susceptible to the respiratory suppressant effects of hypnosedatives. Ethanol intoxication in children has a higher risk of hypoglycaemia than in adults.

Conclusion The approach to poisoning in childhood is determined by the nature and circumstances of the exposure, the stage of development of the child and the legal jurisdiction. Apart from the approach to sample collection, the toxicological analysis is similar to that for adults. For example, in a child who presents with suspected chloroquine ingestion (see the monograph on Chloroquine), a 1 g dose can cause death in a child compared with 3 g in an adult. Exposure to chloroquine could be confirmed by clinical signs (hypotension, QRS prolongation). Analysis of blood concentrations might be used to further confirm exposure, to make an assessment of dose, to predict outcome and to inform management (duration of observation, need for elimination enhancement). In paraquat poisoning the ingested dose might be relatively small in a child because of the unpleasant taste, and larger in an adult because of suicidal intent (see the monograph on Paraquat). Serum paraquat concentrations can be used as a guide to prognosis and as intervention criteria. Hence, the interpretation of the laboratory data can be placed within the paediatric context.

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Reith DM et al. (2003b). Repetition risk for adolescent self-poisoning: a multiple event survival analysis. Aust N Z J Psychiatry 37: 212–218. Reith DM et al. (2003c). Adolescent self-poisoning: a cohort study of subsequent suicide and premature deaths. Crisis 24: 79–84. Rey E et al. (2001). Isoniazid pharmacokinetics in children according to acetylator phenotype. Fundam Clin Pharmacol 15: 355–359. Risk Watch (2003). Let the Clinician Beware! A death caused by a ’safe’ drug. Melbourne, Victoria: The Office of the Chief Clinical Advisor Department of Human Services. www.health.vic.gov.au/clinrisk/downloads/riskwatchedition6.pdf (accessed 28 July 2010). Rivenes SM et al. (1997). Intentional caffeine poisoning in an infant. Pediatrics 99: 736–738. Senst BL et al. (2001). Practical approach to determining costs and frequency of adverse drug events in a health care network. Am J Health Syst Pharm 58: 1126–1132. Shuman RM et al. (1974). Neurotoxicity of hexachlorophene in the human: I. A clinicopathologic study of 248 children. Pediatrics 54: 689–695. Sorbo S et al. (1984). The pharmacokinetics of thiopental in pediatric surgical patients. Anesthesiology 61: 666–670. Southall DP et al. (1997). Covert video recordings of life-threatening child abuse: lessons for child protection. Pediatrics 100: 890–891. Stevens JC et al. (2003). Developmental expression of the major human hepatic CYP3A enzymes. J Pharmacol Exp Ther 307: 573–582. Sugarman JM et al. (1997). Utility of toxicology screening in a pediatric emergency department. Pediatr Emerg Care 13: 194–197. Takahashi H et al. (2000). Developmental changes in pharmacokinetics and pharmacodynamics of warfarin enantiomers in Japanese children. Clin Pharmacol Ther 68: 541–555. Tateishi T et al. (1997). A comparison of hepatic cytochrome P450 protein expression between infancy and postinfancy. Life Sci 61: 2567–2574. Tennefors C, Forsum E (2004). Assessment of body fatness in young children using the skinfold technique and BMI vs body water dilution. Eur J Clin Nutr 58: 541–547. Tibballs J et al. (1985). Drug overdose in children. Aust Paediatr J 21: 7–11.

Tiras S et al. (2006). Nonketotic hyperglycemic coma in toddlers after unintentional methadone ingestion. Ann Emerg Med 48: 448–451. Treluyer JM et al. (1997). Developmental expression of CYP2C and CYP2C-dependent activities in the human liver: in-vivo/in-vitro correlation and inducibility. Pharmacogenetics 7: 441–452. van der Marel CD et al. (2003). Paracetamol and metabolite pharmacokinetics in infants. Eur J Clin Pharmacol 59: 243–251. Vanamo T et al. (2001). Intra-familial child homicide in Finland 1970–1994: incidence, causes of death and demographic characteristics. Forensic Sci Int 117: 199–204. Villain M et al. (2006). Hair to document exposure to glibenclamide. J Chromatogr B Analyt Technol Biomed Life Sci 842: 111–115. Wahl EF et al. (2003). Estimation of glomerular filtration rate and bladder capacity: the effect of maturation, ageing, gender and size. BJU Int 91: 255–262. Wallis LA et al. (2005). Age related reference ranges for respiration rate and heart rate from 4 to 16 years. Arch Dis Child 90: 1117–1121. Wang P et al. (2010). In utero drugs of abuse exposure testing for newborn twins. J Clin Pathol 63: 259–261. Webb NJ et al. (2007). Correlation between finger-prick and venous ciclosporin levels: association with gingival overgrowth and hypertrichosis. Pediatr Nephrol 22: 2111–2118. Wells JC et al. (2005). Prediction of total body water in infants and children. Arch Dis Child 90: 965–971. White J (1995). CSL Antivenom Handbook. Melbourne, Victoria: CSL Limited. Wong IC et al. (2004). Incidence and nature of dosing errors in paediatric medications: a systematic review. Drug Saf 27: 661–670. Woolf AD, Lovejoy FH (1993). Epidemiology of drug overdose in children. Drug Saf 9: 291–308.

Further reading Costello I et al. (2007). Paediatric Drug Handling. London: Pharmaceutical Press. Paediatric Formulary Committee (2009). BNF for Children. London: Pharmaceutical Press.

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CHAPTER

28

Sampling, Storage and Stability S Kerrigan

Specimen selection, sampling, storage and stability Appropriate selection, sampling and proper storage of biological evidence are important, yet sometimes overlooked, steps in forensic toxicology. These factors, in combination with drug stability, can profoundly impact the interpretation of results and the outcome of forensic casework. Criteria surrounding each of these are presented and discussed in the material that follows. Further reference to tissue sampling will also be found in other chapters in this book and will be cross-referenced within the text where appropriate.

Specimen selection Selection of the appropriate specimen is a critical component of any toxicological investigation. Circumstances surrounding the case, the availability of specimens, the nature of the investigation and even legal or statutory issues may dictate which specimens are selected, and for what purpose. Timing is an important factor in specimen collection, particularly in antemortem cases where some drugs have short detection times and therefore limited detection windows; examples include detection of an elevated concentration of D9-tetrahydrocannabinol (THC) in blood from an impaired driver, or of g-hydroxybutyric acid (GHB) following an alleged drug-facilitated sexual assault. Postmortem specimens pose additional challenges owing to autolytic and putrefactive changes. Timing is also important in death investigations because it becomes increasingly difficult to obtain good-quality specimens as the time between death and sampling (postmortem interval) increases. Factors such as embalming of the body, decomposition or burial can further complicate interpretation if tissues have been preserved, if specimens are putrefied, or if exhumation is necessary. In order to be able to select the appropriate specimen(s) the toxicologist should have access to the case history, autopsy records/pathologist’s report and all other relevant documents. Some of the important specimen selection considerations are listed in Table 28.1.

temperatures. In order to minimise sample loss, glass containers are preferred if volatile analytes such as solvents or anaesthetic gases are suspected. Plastic containers are more susceptible to interferences by plasticisers such as phthalates that might interfere with the analysis. The use of an inert plastic such as Nalgene decreases the likelihood of chemical interference, but it is good laboratory practice to evaluate all new specimen containers prior to routine use in the laboratory. If plastic containers are chosen, their integrity at low temperatures should be evaluated. Polystyrene is more susceptible to cracking at frozen temperatures than polypropylene vessels. Rubber septa or liners in screwcap containers should be avoided and replaced with inert liners (e.g. polytetrafluoroethylene (PTFE), or Teflon) to reduce leakage and minimise drug adsorption. Antemortem blood samples are generally collected into evacuated glass tubes such as Vacutainer or Venoject for forensic toxicology purposes. Collection of blood into similar glass collection vessels is also good practice in postmortem blood sampling. These tubes allow the sample to be collected into a vessel that already contains necessary additives to stabilise and preserve the matrix. Proper mixing is necessary when sodium fluoride or other additives are used to ensure that dissolution is complete. Blood Blood is one of the most important specimens of toxicological interest as it provides unique advantages over other matrices in terms of the wide variety of analytical methodologies available, the vast amount of published reference data for both antemortem and postmortem drug concentrations, and the interpretive value of the matrix from a pharmacological standpoint. However, antemortem and postmortem blood samples are notably different, and the site of the postmortem blood draw (central or peripheral) can be of critical importance. Determination of parent drug and metabolite concentrations (and their ratios) may also yield useful information pertaining to acute or chronic use. A summary of the common advantages and disadvantages of various specimens is given in Table 28.3.

Collection and sampling Specimen containers

Antemortem blood

It is important that the specimen container is appropriate for the intended use and does not compromise the analytical findings. Container size should be appropriate for the volume or weight of the specimen so that headspace is minimised. Typical specimen collection quantities are given in Table 28.2. Excessive headspace in the container can increase the chance of oxidative loss, volatilisation of analyte (e.g. ethanol and other low-boiling-point compounds) or salting out, which may occur if preservatives are present. Some analytes have a tendency to adhere to plastic or glass surfaces depending on their physicochemical properties. Silanisation of glassware can reduce adsorptive losses for drugs that are present at trace levels (10 mg/L or less). Although glass containers are preferred by many, disposable plastic containers are used routinely for a wide variety of postmortem tissues and antemortem samples, particularly urine. If glass containers are used, it is important to make use of appropriate racks for storage and transportation. One of the major disadvantages of glass is the possibility of breakage, particularly during storage at low

Antemortem blood is collected by venepuncture, typically from the antecubital region of the arm, using a syringe or evacuated container (e.g. Vacutainer, Venoject). Prior to collection, an antiseptic wipe is often used to clean the collection site. Non-alcohol-containing antiseptic wipes such as Betadine (povidone–iodine) are preferred to avoid any contamination that could interfere with alcohol analysis (see Chapter 4). Although evacuated blood tubes are typically glass, plastic tubes have also been evaluated (Karinen et al. 2010). Postmortem blood Postmortem blood collected at autopsy is quite different from antemortem blood collected by venepuncture from both qualitative and quantitative standpoints. Postmortem blood may be more viscous, may contain numerous small clots or sedimented cells, has a lower pH (as low as 5.5 owing to protein degradation), may contain 60–90% water, and is subject to varying degrees of haemolysis. The site of blood 445

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Table 28.1 Specimen selection considerations n n n n n n n n n n n n n

Ease of use Ease of specimen collection Presence of interferences Matrix effects Parent drug and/or metabolites Detection time Stability of the drug(s) in the specimen Putrefaction Potential for automated analysis Sample volume Indication of short-term or long-term drug use Reference data Interpretive value

collection should be clearly identified on postmortem specimens and blood from different sources should never be combined. Central blood Cardiac blood samples are ideally collected following opening of the pericardial sac, removal of the pericardium, and removal of the blood from the left or right chamber after the heart has been dried. Collection of central blood by insertion of a needle through the chest wall (‘blind stick’) is practised but is discouraged. Although central blood collected in this manner may be identified to the laboratory as ‘heart blood’, it may be contaminated with pericardial fluid, fluid from the pleural cavity, or blood that has drained from the pulmonary vein or artery or the inferior vena cava (Jones 2007). Blood collected in this manner is considered non-homogeneous. Central blood may contain elevated drug concentrations as a result of postmortem redistribution or contamination (diffusion) from other body compartments (Prouty, Anderson 1990; Yarema, Becker 2005), especially following blunt force trauma. Passive drug release from reservoirs such as the gastrointestinal tract, liver, lungs and myocardium may occur immediately after death; later, cell autolysis and the putrefactive process participate in redistribution (Pelissier-Alicot et al. 2003). Drug properties such as volume of distribution, lipophilicity, protein binding and pKa play a role in the

site- and time-dependent mechanisms responsible for postmortem redistribution. Drugs with high volumes of distribution and basic character appear particularly susceptible to postmortem redistribution and their cardiac blood concentrations should be interpreted accordingly. Postmortem redistribution can account for central/peripheral blood drug concentrations that differ by 10-fold or more. Redistribution is time and concentration dependent and is very difficult to predict. Cardiac blood is typically more plentiful than peripheral blood. Although cardiac blood can be a very useful specimen for screening purposes, the relationship between cardiac blood drug concentrations and antemortem blood drug concentrations is complex. Many toxicologists therefore advise against the use of cardiac blood for quantitative and interpretative work. Peripheral blood Femoral blood is the best specimen for use in postmortem testing (Chapter 10) and should be sampled wherever possible. Blood collection from a ligated vein that has been ‘tied off’ is least likely to be contaminated by other sources of blood or a result of release of drug from tissues and organs. Typically, however, a ‘femoral stick‘ involves the collection of femoral blood from an unligated femoral vein in the groin area. Only a small volume of blood should be collected to avoid ‘milking’ the vein and drawing blood from other sources. Typically 10–20 mL of femoral blood can be collected. Over-sampling of blood from the femoral vein will draw blood from the inferior vena cava, and hence the liver, and from the larger iliac vein. Although sampling from a ligated vein is generally preferred, a comparison of drug concentrations in clamped and unclamped femoral vessels showed good correlation for eight drugs including selective serotonin reuptake inhibitors, benzodiazepines, antihistamines and one opioid (Hargrove, McCutcheon 2008). If femoral blood is not available, subclavian or iliac blood may be an alternative. Blood clots Following a fall or blunt trauma to the head, a victim may survive with circulation intact for several hours. Owing to the decreased circulation in the damaged region of the brain, drug or alcohol concentrations in blood clots (e.g. subdural, subarachnoid and/or epidural) may be influenced by incomplete metabolism. It has been suggested that intracranial

Table 28.2 Typical specimen collection quantities Postmortem

Antemortem

Specimen

Quantity

Specimen

Quantity

Blood, heart

25 mL

Blood

10–20 mL

Blood, peripheral

10–20 mL

Urine

25–100 mL

Urine

All

Amniotic fluid

5–30 mL

Bile

All

Breast milk

10–20 mL

Vitreous humour

All

Meconium

All

Cerebrospinal fluid

All

Hair

Pen-size lock

Gastric contents

All

Saliva

1–5 mL

Liver (remote proximity from liver)

All

Sweat

Microlitres (insensible sweat); 1–5 mL (sensible sweat)

Kidney

50 g

Spleen

50 g

Brain

50 g

Lung

50 g

Hair

50 g Pen-size lock (150–200 hairs or 50 mg)

Sources: Dinis-Oliveira et al. (2010); Hepler, Isenschmid (2007); Kerrigan (2002); Kidwell et al. (1998); Skopp (2004); SOFT/AAFS (2006).

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Table 28.3 Advantages and disadvantages of antemortem and postmortem biological specimens Specimen

Advantages

Disadvantages

Amniotic fluid

n n n n

n n n n

Invasive collection Risk of complications Limited reference data Collection by medical personnel

Bile

n Ease of detection of certain drugs (accumulation) n Particularly useful for conjugated drugs

n n n n

Complex matrix Interferences due to bile salts and fats Requires sample preparation/pretreatment Limited reference data

Blood (AM)

n n n n n

Widely accepted matrix Determines recent drug use (hours–days) Related to pharmacological effect Not readily adulterated Extensive reference data

n Invasive collection n Collection by medical personnel n Shorter detection time

Blood (PM)

n n n n

See above (AM) Reference data widely available Central/peripheral blood drug ratios known for some drugs Cardiac blood typically in plentiful supply but requires caution with interpretation

n n n n n

Susceptible to postmortem redistribution (central) Susceptible to postmortem artefacts and interferences Susceptible to contamination (e.g. trauma) Quality of specimen highly dependent on collection protocol Limited volume of peripheral blood

Brain

n Particularly useful for lipophilic drugs, volatiles

n n n n n

Non-homogeneous matrix Drug concentrations vary by region Complex matrix Requires sample preparation/pretreatment Limited reference data

Breast milk

n Determination of neonatal drug exposure n Not readily adulterated n Many drugs present

n n n n n

Privacy, invasive collection Limited reference data Interferences due to high lipid content Drug content varies with milk composition Variable matrix

Cerebrospinal fluid

n Determines recent drug use (hours–days) n Minimal sample preparation n Relatively few interferences

n Invasive collection n Limited reference data

Gastric contents

n Identification of acute ingestion/delayed absorption n Identification of pill fragments possible n Particularly useful for orally administered drugs/poisons

n n n n

Non-homogeneous matrix Complex matrix Requires sample preparation/pretreatment Requires total specimen collection for interpretation

Hair

n n n n

n n n n n

New technology Recent drug use not detected Environmental contamination Potential for ethnic bias Limited reference data

Kidney

n Particularly useful for non-drug analytes, e.g. metals

n Complex matrix n Requires sample preparation/pretreatment

Liver

n Ease of detection of certain drugs (accumulation) n Interpretive value for some drugs n Reference data available

n Complex matrix n Requires sample preparation/pretreatment

Lung

n Particularly important for volatile analyses

n Complex matrix n Requires sample preparation/pretreatment

Determination of prenatal drug exposure Not readily adulterated Minimal sample preparation Relatively few interferences

and centrally acting drugs

History of drug use (months) Readily available, easy collection Low potential for donor manipulation Useful for drug and non-drug analytes, e.g. metals

table continued

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Table 28.3 continued Specimen

Advantages

Disadvantages

Meconium

n Long-term window of drug exposure n Non-invasive sample collection

n n n n n

Non-homogeneous matrix Complex matrix (waxy) Interferences Requires sample preparation/pretreatment Limited reference data

Nails

n Easy collection n History of drug use (months) n Particularly useful for metals

n n n n n

Limited data New technology Not yet widely accepted Recent drug use not detected Environmental contamination

Saliva

n n n n n n

n n n n n

New technology Short drug detection time Small sample volume (1–5 mL) Potential for oral contamination Collection method influences specimen pH and drug content

Spleen

n Particularly useful for certain analytes if no blood

n Complex matrix n Requires sample preparation/pretreatment n Limited data for most analytes

Sweat

n n n n n n

History of drug use (weeks) Cumulative measure of drug use Parent drug present Non-invasive collection Less frequent drug testing required Not readily adulterated

n n n n n n n n

Newer technology Potential for environmental contamination High intersubject variability Requires special collection device Skin irritation and discomfort Small sample volume No pharmacological interpretation possible Non-homogeneous matrix (sweat/sebum)

Urine

n n n n n

Widely accepted matrix Easy collection Plentiful supply Amenable to automated analysis Longer detection window than blood (days–weeks)

n n n n

Potential for donor manipulation Minimal parent drug Not useful for quantitative analysis Not related to impairment or pharmacological effect

Vitreous humour

n n n n n n n

Determines recent drug use (hours–days) Related to pharmacological effect Resistant to putrefaction Interpretive value for ethanol-related investigations Minimal sample preparation Relatively few interferences Useful for postmortem chemistry

n Limited data compared with blood n Small sample volume

Readily available, easy collection Parent drug present Related to free drug concentration in plasma Minimal sample preparation Many drugs determined Indicates recent drug use

is available

blood clots may serve as ‘time capsules’ prior to death, because they may reflect drug concentrations several hours prior to death, when an injury may have taken place. Vitreous humour Direct aspiration of vitreous humour using a hypodermic syringe may yield 2–3 mL of fluid per eye. The needle should be placed in the central globe and aspirated with gentle suction. Preservation with sodium fluoride is generally recommended. The eye is located within the protective environment of the orbit and, being essentially outside the body, is remote from other tissues. Vitreous fluid is therefore a particularly useful specimen owing to its anatomical isolation, affording it notable

resistance in terms of microbial invasion and degradation, as well as being remote from the central organs and subsequently less susceptible to postmortem redistribution phenomena. Vitreous humour is particularly useful for cases involving digoxin or hydrophilic analytes including paracetamol (acetaminophen) and salicylates. The equilibrium that exists between blood and vitreous fluid is slower than with other extracellular fluids, which can result in a slight delay in uptake. Furthermore, only free drugs are able to leave the blood and enter the vitreous humour. Since eye fluid is sterile and less susceptible to microbial contamination and hence postmortem alcohol production, it is routinely used for ethanol determination owing to its interpretive value from the standpoint of postmortem alcohol production and the determination of the pre- or post-absorptive phase of ethanol use

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Collection and sampling (Honey et al. 2005). Vitreous humour is particularly useful for postmortem analysis of glucose, urea nitrogen, uric acid, creatinine, sodium and chloride. These are important analytes for the evaluation of diabetes, degree of hydration, electrolyte imbalance, postmortem interval and the state of renal function prior to death (Coe 1977, 1993). Sodium, calcium and chloride concentrations in vitreous humour during the early postmortem interval can be used to estimate antemortem serum concentrations. It is therefore important that sodium fluoride is not added to specimens requiring vitreous chemistries. For that reason, vitreous humour is frequently collected into two separate containers: one preserved (for drug and alcohol testing) and one unpreserved (for clinical purposes). Cerebrospinal fluid Cerebrospinal fluid (CSF) can be collected either by lumbar puncture at the base of the spine using a hypodermic syringe or by withdrawal of cisternal fluid by puncturing the base of the neck. Although there are limited published reference data for quantitative drug concentrations in CSF, this clear fluid comprising mostly water is amenable to most routine methods of toxicological analysis. CSF may be of particular importance in alcohol-related cases where no vitreous humour is available, particularly if postmortem alcohol production is suspected. Like vitreous humour, CSF is anatomically isolated and less prone to contamination and bacterial invasion. Although it is more plentiful than vitreous humour, the lack of plentiful reference data limits its usefulness. However, CSF may be particularly useful in surgical death investigations. Bile Bile is generally aspirated from the gallbladder using a hypodermic syringe. It may be necessary to tie off the gallbladder prior to collection if contamination appears to be an issue. Bile should be collected prior to the liver specimen to avoid contamination. Many drugs of forensic interest accumulate in the bile, particularly those that are heavily conjugated, such as opiates, benzodiazepines and cannabinoids. Bile may also be used in cases where chronic heavy-metal poisoning is implicated. However, owing to the presence of bile salts and fats, drug extraction from this matrix can be complicated and extensive extraction and cleanup procedures are often required.

449

stomach content collected and retained by medical staff may provide valuable information concerning drugs or poisons consumed. Urine In antemortem settings, a mid-stream urine sample is usually collected into a plastic container containing sodium fluoride as preservative (Chapter 9). In some settings it may be necessary to take precautions against specimen adulteration. In postmortem settings, urine is collected by insertion using a hypodermic syringe directly into the bladder under visualisation. Puncture of the abdominal wall should be avoided to reduce the possibility of contamination. Urine is a valuable specimen for both antemortem and postmortem drug testing because it is a relatively uncomplicated matrix. However, the multiplicity of factors influencing urine drug concentrations (e.g. urine volume, clearance, metabolism, pH and time of last void) generally means that, in isolation, these results have limited quantitative value. Exceptions to this rule may include ethanol determination in a second void. Care must be exercised when considering the interpretation of urine GHB concentrations as GHB is present as an endogenous compound formed as a by-product of metabolism and may also be produced as a postmortem artefact as a consequence of the breakdown of succinic acid semialdehyde. Tissues When tissues are sampled they should be collected quickly and placed immediately into airtight containers. This is particularly important if volatiles or inhalants are suspected. Liver, kidney, brain, lung and spleen are the most frequently collected postmortem tissues. Liver

Liver is a particularly important organ because of the very large number of drugs that undergo hepatic metabolism and the fairly extensive published reference data that exist. To reduce the possibility of drug diffusion from the small bowel, tissue from deep within the right lobe is preferred (Drummer 2004). The concentrations of drugs and metabolites in liver are often elevated, hence this specimen has limited interpretive value. However, liver is particularly useful for highly protein-bound drugs and the comparison of liver/blood drug ratios may allow the differentiation of acute overdose from chronic drug use for some drugs. Kidney

Gastric contents Gastric content is a potentially valuable specimen for analysis in postmortem and clinical cases. Unabsorbed drug or tablet fragments in the gastric contents may provide valuable information concerning ingested compounds and provide an excellent material for preliminary screening (Chapter 9) owing to the potentially large amounts of drug that may be present. The absence of a drug in the gastric contents does not necessarily preclude oral administration. Odours emanating from the gastric content can provide valuable clues about what may have been consumed, e.g. pesticides and cyanides. The entire contents of the stomach should be collected and weighed. Gastric contents are non-homogeneous and should be homogenised prior to sampling. Quantitative drug determinations should be interpreted within the context of the entire contents (total quantity, rather than concentration) and it is important to take into consideration the differing absorption rates of drugs based on their physicochemical properties as well as their formulations and coatings. The presence of a drug in gastric contents, particularly at low concentration, does not necessarily indicate oral administration. Drugs may be absorbed into the stomach via gastric juices that are in equilibrium with blood or as a result of intranasal drug use. Basic drugs are more susceptible to this because they have a tendency to become trapped in the gastric compartment owing to the low pH. If heavy metals are suspected, gastric contents should be collected, together with intestinal contents. In cases of suspected poisoning where the patient may have survived for a few days in hospital prior to death and where drugs may have been metabolised and eliminated from the body prior to death, any

Most drugs pass through the kidney as a result of urinary elimination. Kidney is an important specimen in cases of suspected heavy-metal poisoning owing to accumulation in this tissue. The presence of heavy metals or ethylene glycol during toxicological tests may be accompanied by structural changes to the kidney that can be documented using histological tests. Spleen

Spleen is an important specimen for cyanide or carbon monoxide analyses, particularly in fire-related deaths where blood may be compromised or unavailable. Lung and brain are valuable specimens in cases involving volatiles or inhalants. Brain

Brain tissue is lipid rich and has a tendency to concentrate some drugs, particularly lipophilic analytes, narcotics and halogenated hydrocarbons (Skopp 2004). If quantitative drug brain concentrations are used, it is important to know the location of the specimen because the brain is a non-homogeneous matrix. Drug concentrations within the brain may vary several-fold from one region to another owing to its complex structure and differing composition. Brain is not widely used in routine toxicological analysis. Muscle

Muscle is not routinely encountered, despite the fact that it frequently contains relatively high drug concentrations, particularly for substances with high volumes of distribution. Perfusion rates between sites and drug concentrations are not consistent, and drug concentrations must be interpreted accordingly. Muscle is encountered more frequently for

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ethanol determination in the absence of blood, or during the investigation of a suspected injection site. Hair

Hair has been used in a variety of antemortem toxicology settings to provide a history of drug exposure and has therefore found applications in workplace drug testing, in monitoring of persons on probation or on parole for drug use, in insurance testing to verify the truthfulness of statements made by applicants relating to whether they use drugs or are smokers, in child endangerment, in drug-facilitated sexual assault and in other types of criminal casework (Nakahara 1999; Kintz et al. 2006; Curtis, Greenberg 2008). Further discussion relating to the scenarios in which hair testing is employed is provided in Chapters 8, 19 and 27. One of the major advantages is the long drug detection window compared with many other specimens. Hair may allow drug exposure over several weeks or months to be determined, depending on the length of the hair. Segmenting the hair by length may allow an approximate timeline for exposure to be determined based on head hair growth rates of approximately 1 cm per month (Clauwaert et al. 2000). Hair should be cut as close as possible to the scalp from the posterior vertex region of the head, since this region shows least variation in growth rate. Typically a lock of hair equivalent to the thickness of a pen or pencil is collected. The colour, length, sampling site and any obvious cosmetic treatment of the hair should be recorded. The root (proximal) and tip (distal) sections of the hair should be clearly identified. Although head hair is the preferred specimen, hair from other sites (e.g. pubis, axillae) may be used, but interpretation of analytical findings may be more complex. The lock of hair is typically tied, wrapped in aluminium foil and stored under dry conditions in the dark at room temperature. Hair is also a useful specimen in postmortem investigations where arsenic or heavy metals are suspected. Although postmortem hair analysis is not yet widespread, there is growing interest because it may provide valuable interpretive information pertaining to the chronological sequence of toxin exposure (Cirimele et al. 2002). Hair has also proved to be useful in cases where exhumation is necessary (Tsatsakis et al. 2001). If hair is collected post mortem, it should be sampled at the very beginning of the examination to reduce the risk of contamination. Hair can provide complementary toxicological information. Issues with drug testing in hair include external contamination, ethnicity and pigmentation, chemical treatment and the use of appropriate cut-off concentrations. Contamination of the hair with drugs from other sources (external deposition, environmental contamination, sweat or sebum) is generally minimised by pretreatment of the sample using a variety of aqueous and organic rinses or wash steps prior to analysis. Other keratinised specimens such as nails can also be used to determine long-term exposure to drugs or poisons, in particular heavy metals such as thallium, arsenic or lead. However, drugs are deposited into nails at a much slower rate. External decontamination procedures should be performed prior to analysis (see Chapter 19). Injection sites Excision of skin and tissue (muscle) may be necessary in postmortem investigation of a suspected injection site. Typically a cube of muscle and skin is removed for this purpose. However, it is important to compare the drug concentrations in the suspected injection site with those in a control specimen from the same individual where there is no evidence of injection. Injection sites are not always reliable indicators of drug administration since the presence or absence of drugs in injection site tissue is dependent on the type and depth of the injection. If the injection is made directly into a blood vessel, little drug is likely to remain in the surrounding tissue. Entomological specimens The potential use of insects for detecting drugs and other toxins in decomposing tissues has been demonstrated and reviewed (Introna et al. 2001). If insects or larvae are collected from human remains they should be frozen as soon as possible. Larvae rapidly eliminate drugs when removed from the food source. Drugs, metals and pesticides have been identified in

entomological specimens including larvae and pupae. Following wash steps to remove external contamination, entomological specimens are homogenised and analysed in a manner similar to that for tissues. Saliva Saliva or oral fluid can be collected non-invasively by expectoration, by aspiration, by vacuum or by saturation of an absorbent swab (Kidwell et al. 1998). Detection times are comparable to those in blood. As much as 1.5 L of saliva per day is produced by the submandibular, parotid and sublingual glands inside the mouth. Secretions from a specific gland may be collected using a special device or by cannulation, but this is uncommon. Although specific gland secretions are advantageous from a standpoint of saliva : plasma ratio and reduced oral contamination, mixed saliva is typically collected for routine drug-testing purposes. Oral fluid can be collected non-invasively, conveniently and without invasion of privacy. Chewing an inert substance, such as Teflon tape or a rubber band, may increase salivation for the purpose of specimen collection. It should be verified that no adsorption takes place between the drug and the chewed substance. Acidic sweets or citric acid has also been used to stimulate glandular secretions. Care must be taken that residual food, drink or interfering substances inside the mouth do not interfere with the analysis. This is particularly important for drugs that are ingested orally or smoked. Owing to the ease and non-invasive nature of specimen collection, saliva is of particular interest in workplace drug testing, for insurance testing and, more recently, for roadside impairment testing. Saliva contains serous fluid derived from plasma. This ultrafiltrate of interstitial fluid contains the unbound fraction of drug at concentrations that are typically proportional to those measured in plasma. However, the predictable relationship that theoretically exists between saliva and plasma drug concentrations is influenced by many factors such as saliva flow rate, which can complicate pharmacological interpretation (Crouch 2005). A more detailed discussion on saliva and oral fluid testing is provided in Chapter 18. Sweat Moisture loss via the skin and elimination of insensible (non-visible) sweat take place during normal breathing at a rate of 0.3–0.7 L/day. Sensible sweat refers to perspiration that is actively excreted during stress, exercise or extreme temperature, at rates of 2–4 L/h. About half the total volume of sweat is eliminated from the trunk of the body. The remaining fluid is lost from the legs or upper extremities and head in approximately equal amounts (Kidwell et al. 1998). Sweat is usually collected using an adhesive absorbent patch that is placed on the surface of clean skin or by wiping the skin with a swab or gauze. Careful preparation of the skin is necessary prior to placement of a sweat patch to minimise external drug contamination or bacterial degradation of the drug once it has been retained. Use of a semi-permeable membrane to cover the absorbent pad prevents non-volatile components in the environment from penetrating the pad externally, but allows oxygen, water and carbon dioxide to diffuse through. Salts, solids and drugs that pass through the skin are trapped in the absorbent pad, where they are temporarily stored in situ, until the patch is removed. Owing to the relatively small volume (mL) of insensible sweat secreted from a small absorbent area (typically 3  5 cm), patches are typically worn for several days on the outer portion of the upper arm or back. In practice most skin wipes or sweat patches contain a mixture of sweat and sebum, the oily secretion from the sebaceous glands. As with saliva, increased flow rates can influence the quantity of drug eliminated into sweat. This specimen is particularly useful for compliance testing or monitoring long-term exposure (weeks), which might be desirable in probation or parole settings. Amniotic fluid Amniotic fluid has been used to investigate prenatal drug exposure. Its collection (amniocentesis) typically takes place between weeks 16 and 20

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Sample handling of pregnancy. A needle is inserted through the abdomen into the uterus where there is the least chance of touching the placenta or the foetus. The collection of amniotic fluid is typically performed in conjunction with ultrasound visualisation in order to reduce the risk of damaging the developing foetus. Although complications are rare, miscarriage occurs in a very small percentage of women. Typically 5–30 mL of amniotic fluid is removed during the procedure. Breast milk During pregnancy, oestrogen and progesterone, secreted in the ovary and placenta, cause milk-producing glands in the fatty tissue of the breasts to develop and become active. The pituitary hormone prolactin stimulates the production of fluid (600–1000 mL/day) by the milksecreting cells. Contraction of the myoepithelial cells surrounding the alveoli allows the milk to be expressed. For specimen collection purposes, a breast pump can be used. The matrix is somewhat nonhomogeneous. Colostrum, a creamy white to yellow pre-milk fluid, may be expressed from the nipples during the last trimester of pregnancy and shortly after delivery. Many drugs are excreted into breast milk and the scientific and medical literature contains numerous citations of the presence of drugs in this matrix (Chapter 27). Drugs that are extensively protein bound may not readily pass into the milk, but emulsified fats contained in the milk may concentrate highly lipid-soluble drugs. The high lipid content and natural emulsifying agents present in breast milk mean that some sample pretreatment is often required. Meconium Meconium formation begins between weeks 12 and 16 of gestation. As the first faecal matter passed by the neonate, it is typically collected within 1–5 days of birth. Analysis of drugs in meconium may provide a relatively long-term history of drug exposure during pregnancy, in particular the last 20 weeks of gestation. It provides more complete and long-term information on drug exposure than neonatal urine or cord blood. The specimen is complex and non-homogeneous. All available samples should be collected and homogenised prior to analysis. Meconium and other important matrices involved in maternal–foetal medicine have been reviewed (Gray, Huestis 2007; Lozano et al. 2007).

Sample handling Sample handling is an important consideration during the pre-analytical phase. Unlike a clinical setting, where the time between sample collection and testing is often very short, significant delays are common in a forensic setting. The pre-analytical phase may be considerable, spanning the time of death and/or discovery of a victim, autopsy and collection of specimens, sample storage, transport to the laboratory and subsequent storage prior to analytical testing. In antemortem toxicology settings, the time delay between an alleged offence and specimen collection may be short (e.g. minutes to hours in the case of most impaired driving cases) or long (e.g. hours to days in the case of some sexual assault cases). Following collection, antemortem specimens may be subject to similar delays due to shipping or transport of specimens, requests for testing made by the submitting agency and storage of samples prior to actual testing. Although the toxicologist must consider the time delay between the event (i.e. death, or committing or being the victim of an offence) and collection of a specimen for interpretation purposes, these delays are beyond the control of the laboratory. Measures can be taken, however, to preserve and maintain the integrity of specimens after collection. Sample quality plays an important role in the validity or usefulness of subsequent analytical determinations. Inappropriate sample preservation or storage may have a deleterious effect on qualitative and quantitative determinations. Preservation and storage Specimens should be stored at appropriate temperatures, with adequate preservative and in an environment accessible only to authorised

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personnel to ensure security and integrity. Short-term storage at refrigerated temperature (4 C) is recommended for most samples, or frozen ( 20 C or lower) during long-term storage (more than 2 weeks). Exceptions to this include hair, nails or dried blood swatches on filter paper, which can be stored at ambient temperatures. Whereas clinical specimens are typically unpreserved, the use of a chemical preservative is often warranted in forensic specimens. Preservation of blood samples with sodium fluoride (2% w/v) is routine in most laboratories. Commercial evacuated blood collection tubes (e.g. grey-top tubes) contain sodium fluoride as the preservative and potassium oxalate as the anticoagulant. These are the preferred evacuated blood tubes for antemortem forensic toxicology casework. Inhibition of microorganisms and enzymes with sodium fluoride is important for commonly encountered analytes such as ethanol, cocaine and others. Fluoride acts as an enzyme inhibitor and helps prevent glycolysis. Commercial blood tubes may contain a wide variety of additives (citrate, heparin, EDTA, thrombin, acid citrate dextrose mixtures, clot activator, etc.). Although these tubes are designed for a variety of clinical uses, they are not the preferred specimen containers for drug-testing purposes. Laboratories frequently encounter these blood tubes when they are submitted from a hospital setting and special care must be taken when interpreting their results (LeBeau et al. 2000; Toennes, Kauert 2001). If an anticoagulant is to be used, potassium oxalate is preferred rather than alternatives such as EDTA, heparin or citrate. Antioxidants such as ascorbic acid (0.25% w/v) or sodium metabisulfite (1% w/v) are sometimes used to prevent oxidative losses, but these agents have the potential to act as reducing agents towards some drugs, in particular N-oxide metabolites, which may be transformed into the parent drug. In a similar fashion, adjustment of specimen pH is not generally favoured routinely, because, just as some drugs are alkali labile (e.g. cocaine, 6-acetylmorphine), others are acid labile. Sodium azide (0.1% w/v) is sometimes used as a preservative and antimicrobial agent in urine samples. Sodium azide should not be used if samples are to be analysed by enzyme-linked immunosorbent assay because it can interfere with horseradish peroxidase-mediated colorimetric detection. Although the addition of preservative should be routine for most antemortem and postmortem blood samples, an aliquot of unpreserved postmortem blood is sometimes collected. For example, fluoride preservatives should not be used if organophosphorus chemicals are suspected since this accelerates chemical degradation (Skopp, Potsch 2004). Some drugs are known to be photolabile (e.g. ergot alkaloids such as lysergic acid diethylamide and the phenothiazines). Specimens known to contain photolabile drugs should be stored in amber vials or foilcovered containers, or otherwise protected from direct sources of light. Storage of tightly sealed appropriate containers at low temperature further inhibits sample loss. Short-term storage at refrigerated (4 C) and frozen ( 20 C) temperatures is commonplace in most laboratories and repeated freeze–thaw cycles should be avoided. Labelling and specimen transfer All samples should be properly marked for identification with the case number, donor name, date and time of collection, signature or initials of the collector and specimen description. Tamper-proof containers and/ or tape bearing the collector’s initials and date of collection should be used. Specimens should be forwarded to the laboratory in appropriate leak-proof and tamper-proof packaging/shipping materials with all appropriate documentation (chain-of-custody forms, requisitions for testing, special requests, case information, medications list, police report, donor information/identifier such as date of birth or social security number, agency case number, pathologist/police officer name and contact information). Improperly packaged or identified materials should be returned to the submitting agency. Documentation accompanying the specimen(s) should list all of the specimens that were collected or available for testing. Once received by the laboratory, the specimens should be inspected and appropriately documented in terms of condition and quantity during the accessioning process.

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Sampling, Storage and Stability

Contamination There are a variety of contamination sources for both antemortem and postmortem specimens. In addition to the potential contamination issues that may result from the use of containers and external factors, a number of important exogenous and endogenous sources of contamination should be considered. Exogenous contaminants Specimens collected into plastic containers are sometimes susceptible to phthalate interferences. Numerous plasticiser interferences such as dibutylphthalate may co-extract and interfere with analytical detection by gas-chromatographic or mass-spectrometric techniques, yielding characteristic phthalate ions. All plastic containers should be evaluated prior to widespread implementation. It should be noted that contamination from phthalates may occur during the analytical process through use of disposable pipette tips, solvent containers, solid-phase extraction cartridges, tubing and numerous other sources. However, environmental exposure to these substances from household items, food, beverages and other sources can produce detectable quantities of phthalate esters or their metabolites in biological specimens including blood, serum, urine and breast milk (Silva et al. 2005; H€ ogberg et al. 2008). Embalming fluids, which typically contain a variety of alcohols and aldehydes, are a potential source of contamination in postmortem casework. These fluids not only dilute any remaining fluid in the body, but also alter drug distribution in remaining tissues. Another potential source of contamination comes from reusable syringes and containers for postmortem specimen collection. Some cleaning fluids that are used for syringes may contain alcohols that can compromise the analysis of volatiles. This highlights the importance of analysing specimens from multiple sites and using disposable syringes wherever possible. The principal concern with antemortem contamination arises from the intentional manipulation of the sample to mask the presence of drugs.This typically involves the substitution, dilution or adulteration of the biological specimen with a foreign substance. Donor manipulation occurs most frequently with urine samples in workplace drugtesting situations (see Chapter 3). As a result, specimen validity testing is required in some drug-testing programmes such as for federal employees under US Department of Health and Human Services (DHHS) guidelines. Initially, adulteration of urine for drug-testing purposes involved the use of crude household items such as soap, bleach, vinegar, ammonia or cleaning fluids. Although these substances met with some success, a wide variety of commercial adulteration reagents and kits is now widely available (Dasgupta 2007). A summary of in-vitro adulteration agents is provided in Table 28.4. Some of the most popular commercial products contain glutaraldehyde (fixative), pyridinium chlorochromate (PCC) or chromium(VI)-containing species (oxidant), nitrite (oxidant) or peroxide/peroxidase. In general, in-vitro adulterants can interfere with presumptive immunoassay tests, with the intention of producing false-negative results. However, some agents have the potential to interfere with confirmatory tests such as gas chromatography/ mass spectrometry (GC-MS) as well. Although this is less likely, studies have shown that some reagents may produce lower than expected or negative results for some analytes. Adulteration detection products are available commercially. On-site or dipstick tests are available for nitrite, glutaraldehyde, pH, specific gravity, creatinine, bleach, PCC and oxidants. Specimen dilution or in-vivo adulteration by ingestion of a substance to mask the presence of drugs is also encountered. This is commonly achieved by the ingestion of large quantities of fluid prior to the test or by administration of a diuretic. Examples of in-vivo adulteration agents are given in Table 28.5. Urine specimen substitution or dilution can be detected if specimen validity tests are performed. A specimen may be considered invalid if the pH is between 3 and 4.5 or between 9 and 11. It may be adulterated if the pH is less than 3 or greater than 11. The normal temperature range is 32–38 C. A specimen is considered dilute if the

Table 28.4 In-vitro adulteration agents n n n n n n n n n n n n n n n n n n n n n n n n n n n

Ascorbic acid Alcohols Amber-13 (hydrochloric acid) Ammonia Bleach Clear Choice (glutaraldehyde) Detergent or soap (surfactant) Drano Ethylene glycol Gasoline Glutaraldehyde Hydrogen peroxide Klear (potassium nitrite) Lemon juice Liquid soap Lime-A-Way Mary Jane Super Clean 13 (surfactant) Salt Stealth (peroxide/peroxidase) THC-Free (hydrochloric acid) UrinAid (glutaraldehyde) Urine Luck (chromium VI, oxidant) Vanish Vinegar Visine Water Whizzies (sodium nitrite)

Source: Kerrigan, Goldberger (2005).

Table 28.5 In-vivo adulteration agents Diuretics Prescription n Thiazides and thiazide-like drugs (e.g. hydrochlorothiazide, metolazone) n Carbonic anhydrase inhibitors (e.g. acetazolamide) n Loop diuretics (e.g. bumetanide, furosemide, torsemide) n Osmotic diuretics (e.g. mannitol) Over the counter (OTC) Aqua-Ban Diurex Fem-1 Midol Pamprin Premsyn PMS

n n n n n n

Other n Alcoholic beverages n Xanthines (e.g. caffeine, theophylline, 8-bromotheophylline) n Herbals and aquaretics (e.g. golden seal root, juniper) Source: Kerrigan, Goldberger (2005).

creatinine concentration is less than 200 mg/L and the specific gravity is less than 1.003. Other sources of contaminants or unexpected analytes include pyrolytic breakdown products due to thermal degradation of drugs. These may be present due to pyrolysis during administration of the drug (e.g. anhydroecgonine methyl ester following crack cocaine use) or occasionally in situ during analysis if conditions are not properly controlled or evaluated. Other sources of contamination may arise from pharmaceutical impurities or adulterants and cutting agents that are incorporated into illicit drugs prior to sale.

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Stability Medical artefacts Clinical therapy can sometimes produce medical artefacts that complicate toxicological findings. Medical artefacts are most common in postmortem cases where infusion pumps may continue to run after death, introducing high concentrations of drug in local body compartments. Access to hospital records and case information, and collection of peripheral blood, vitreous fluid and liver are particularly important in these types of cases. Other sources of medical artefacts may include organ harvest drugs such as the calcium-channel blocker verapamil, or papaverine, which is used to inhibit vasoconstriction during transplant surgery. If living patients are administered fluids (e.g. saline) during clinical care, blood is only contaminated (diluted) with the infusion solution if it is collected downstream from the intravenous line. Blood circulation and equilibrium with tissues is rapid, so the administration of fluids does not usually influence drug or alcohol concentrations in blood if normal precautions are taken. If downstream collection is suspected, careful review of the medical records and/or measurement of the haematocrit to determine specimen dilution may be necessary. Endogenous contaminants, artefacts and interferences By their very nature, all biological specimens are subject to endogenous interferences, regardless of whether or not they are derived from living or deceased persons. More complex biological specimens such as blood, tissue or meconium will require more extensive sample preparation to remove these interferences than less complex matrices such as vitreous humour, or cerebrospinal or oral fluid. In general, however, antemortem specimens are somewhat less susceptible to endogenous artefacts or contaminants. Ethanol, GHB, carbon monoxide, cyanide and other short-chain alcohols can be metabolically produced post mortem (Skopp 2004). The formation of toxicologically significant concentrations of cyanide in postmortem tissue (Lokan et al. 1987) has been attributed to the conversion of thiocyanate to cyanide and the breakdown of protein (Curry et al. 1967). Although in some circumstances ethanol can be produced in situ in unpreserved antemortem fluids, the same is true to a far greater extent in postmortem specimens, particularly blood. Likewise, GHB is present in antemortem fluids at very low concentrations in the absence of a serious genetic disorder such as GHBuria (Knerr et al. 2007). Differentiation of exogenous and endogenous GHB is complicated by specimen type, storage conditions, preservative and other factors. Many laboratories use a cut-off concentration to help differentiate the two, for example 10 mg/L in urine (Kerrigan 2002; LeBeau et al. 2007). Concentrations of GHB may increase in urine during storage, upon collection and storage of unpreserved blood, or in citrate-buffered antemortem blood (LeBeau et al. 2000). Although preserved antemortem blood GHB concentrations are typically lower than those in urine, numerous studies have shown forensically significant concentrations of GHB in postmortem blood. Postmortem urine and vitreous fluid appear to be less susceptible to this increase. Major changes that occur after death produce autolytic changes and putrefaction by microorganisms. Invasion of microorganisms, particularly from the gastrointestinal tract into tissues and body fluids, occurs within hours at ambient temperature. Lipids, carbohydrates and proteins are hydrolysed by microbial enzymes, the pH of blood steadily increases, and the putrefactive amines, tyramine, tryptamines, phenethylamines and other endogenous substances are liberated. Trauma is a non-preventable source of contamination in postmortem forensic toxicology. Rupture of organs or compartments within the body can compromise quantitative drug analyses owing to the mixing of fluids (e.g. of gastric contents with blood) or from the microbial action that occurs as a result. Postmortem alcohol production can also result in detectable quantities of ethanol as an artefact. Glycolysis and the presence of yeasts and microorganisms can convert a variety of postmortem substrates to ethanol. Although concentrations are typically low (18) can be extracted as non-ionised molecules at a pH range around their isoelectric zone, where the ionisation of the basic group has ended and the ionisation of the acidic group has not yet begun. In the special case that pKa þ pKb ¼ 18, the isoelectric zone shrinks to a particular point (isoelectric point) at which the molecule is not ionised. Example: n

Sulfacetamide: pKa ¼ 5.4 and pKb ¼ 12.2 (pKa þ pKb ¼ 17.6). To a great extent the molecule is not ionised at pH ¼ 3.6. (This value lies about two pH units above pKa ¼ 1.8, corresponding to pKb ¼ 12.2, and about two pH units below pKa ¼ 5.4.)

H

O

O

N S

O

For acidic substances, the maximum extraction yield is observed two pH units or more below their pKa values. Thus to extract mefenamic acid (pKa 4.2) from an aqueous solution, it should be acidified with sulfuric acid to give a pH value of 13. Ammonia solution will give an aqueous solution pH of only about 10, which is not sufficiently basic for a successful extraction. H

H

N

N

H

Sulfacetamide

Amphoteric substances with one weaker and one stronger acidic and basic function (pKa þ pKb ¼ between 18 and 10) can be extracted as non-ionised molecules at a pH range around their isoelectric point with a lower extraction yield because the molecule is also partly a zwitterion (i.e. the molecule has both its acidic and its basic functions ionised). Example: n

N

Mefenamic acid n

459

Morphine: pKa ¼ 9.9 and pKb ¼ 6.0 (pKa þ pKb ¼ 15.9). The molecule is partly non-ionised at pH ¼ 8.9 (this value lies about one pH unit above pKa ¼ 8.0, corresponding to pKb ¼ 6.0, and about one pH unit below pKa ¼ 9.9); at this pH value there also exist zwitterions that will not be extracted and which therefore reduce the overall extraction yield. Thus morphine is best extracted from aqueous media by using a freshly prepared, saturated sodium hydrogencarbonate solution, or similar compound, to give a pH value between 8 and 9. The high salt concentration of the saturated aqueous solution reduces the drug’s aqueous solubility and therefore improves extraction efficiency. H O

H O H N

H H O

H

H

H Morphine Amfetamine

The distribution behaviour of amphoteric organic electrolytes (e.g. with one acidic and one basic function) also depends on their state of ionisation, but extraction yields are more difficult to predict.

For amphoteric substances with one strong acidic and one strong basic function (pKa þ pKb ¼ 10 or < 10), zwitterions are to be expected, which results in a limited extraction yield.

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Extraction

Ion-pair extraction Strong electrolytes (e.g. compounds with a quaternary ammonium function, such as paraquat) and very hydrophilic compounds (e.g. sulfonamides) tend to stay in the aqueous phase and will not be sufficiently extracted by organic solvents. Liquid–liquid extraction can be accomplished only by ‘ion-pair’ formation with appropriate counterions. These ion pairs are much more soluble in organic solvents than the drugs themselves and so assist an efficient extraction. Anionic dyes such as bromothymol blue, bromocresol green and bromophenol blue are widely used for this purpose. Moreover, solvents such as chloroform and dichloromethane can extract basic substances as hydrohalogenides, perchlorates, nitrates, phosphates, sulfates or thiocyanates. Conversely, the extraction of acidic drugs can be improved by using quaternary ammonium compounds such as cetyltrimethylammonium bromide as ionpairing compounds. Adsorption Extraction of non-volatile drugs and poisons from biological specimens can also be accomplished by adsorption and the fundamental principles discussed above form the basis for adsorption methods as well. When adsorption is used, intermolecular interactions (van der Waal’s forces, aromatic interaction) or electrostatic interactions will enable the isolation of target compounds from the biological matrix. Hydrophobic interactions between neutral or non-polar substances are based on dispersion forces and are short-ranged, weak interactions between molecules or parts of molecules (bond strength 4–20 kJ/mol). They result from random fluctuations in local electron density distribution within molecules. Hydrophilic interactions between polar functional groups or two dipoles are based either on hydrogen bonding (e.g. hydroxyls, carboxylic acids, amines), or on dipole–dipole interactions. Like dispersion forces, they too are short ranged and relatively weak forces (bond strength 10– 40 kJ/mol). Aromatic interactions are caused by attractive forces between diffuse electron clouds in p systems; their bond strength is similar to dispersion forces. Electrostatic interactions between oppositely charged molecules have the highest bonding energies (200–1000 kJ/mol) and ionic bonding can be used for extraction via ion exchange. Extraction efficiency, recovery and internal standards On the basis of the fundamental principles described above, and on the data that can be found in the individual monographs of this book, extraction methods can be developed and optimised with respect to extraction efficiency. With a high extraction efficiency for the target compound, it should then be possible to create a complete analytical procedure with sufficient overall recovery (a complete procedure includes sample pre-treatment, extraction, fractionation, purification, evaporation, chromatographic separation, detection, identification and quantitative determination). The overall recovery can be calculated as the percentage of the analyte response after sample work-up compared with that of a solution containing the analyte at a concentration corresponding to 100% recovery. A low overall recovery can be tolerated only as long as the data for limit of quantification (LOQ) and limit of detection (LOD), precision and bias are acceptable (this is why recovery is not an essential part of method validation). However, it is good practice to determine recovery at high and low concentrations and ensure that it is greater than 50% to confer robustness to the analytical procedure. For the quantitative determination of the analyte, and in order to monitor the whole procedure when biological specimens are extracted, an internal standard has to be added at the earliest possible stage. The internal standard must mimic the physicochemical properties of the analyte as closely as possible and must follow the target compound through the entire process of extraction and subsequent analytical steps in order to compensate for any loss of the target compound.

The selection of the internal standard can be a difficult task and each individual step of the whole analytical procedure should be carefully considered when making this decision. Even minor differences in the physicochemical properties between the target compound and the internal standard can result in errors, because of their different behaviour during extraction, fractionation, purification and concentration towards the applied chromatographic system, the reagents for derivatisation or their response to the detection system. When mass spectrometry is used, stable-isotope-labelled analogues of the target compounds are the best choice as internal standards. For other analytical techniques or, if stable-isotope-labelled analogues are not available, alkyl analogues (because of their similar structure) can be used as an alternative; but it should be kept in mind that some alkyl analogues – such as morphine and codeine (methylmorphine) – can show significant differences in their physicochemical properties (the additional methyl group in codeine inactivates the phenolic function that is active in morphine). If alkyl analogues are also not available, the internal standard should be chosen on the basis of similarity in structure and functional groups (preferably from the same substance class, e.g. benzodiazepines). When glucuronides are analysed, the internal standard should also be a glucuronide. Only if there are no alternatives should active drugs be used as internal standards; in this case the presence of the active drug used as the internal standard would have to be tested for separately in the specimen. The following examples illustrate some specific considerations that should be kept in mind when choosing the internal standard: when GC is used, attention should be paid to volatility, thermal stability and reactivity with the possible derivatisation reagent; for LC, the solubility in the injection solvent (e.g. mobile phase of the LC) and the response to the detection system (e.g. UV spectrum), as well as the applicability of the ionisation technique for LC-MS should be considered. Sometimes simple problems at the beginning of the procedure can cause errors, such as the solvent used to add the internal standard to the sample (e.g. problems because of protein precipitation, or different extraction of the previously dissolved internal standard and the target compound from the sample matrix). Moreover, the added concentration of the internal standard should be similar to the expected concentration of the analyte. In conclusion, stable-isotope-labelled analogues of the target compounds mimic the analyte very closely and therefore they should be used whenever available. But it should be kept in mind that the high separation power of modern chromatographic systems can separate stableisotope-labelled analogues from the targeted analytes (stable-isotopelabelled analogues elute slightly earlier), leading to the possible influence of, for example, ion suppression on only one of the compounds. Moreover, stable-isotope-labelled analogues and targeted analytes can produce identical ion fragments, which can lead to problems during quantification. To produce accurate quantitative results, the analytical procedure should be validated (see Chapter 20). If a certified reference material to validate the analytical procedure is not available (e.g. for postmortem specimens), then the ‘method of standard addition’ can be used, where calibration and quantification are performed directly in the sample matrix, compensating for matrix effects. In this procedure different concentrations of the target compound are spiked to aliquots of the homogeneous sample prior to work-up and the detector responses are plotted as a graph (‘standard addition plot’). The initial concentration of target compound can then be calculated via extrapolation.

Practical aspects of extraction The isolation of the compounds of interest from the biological matrix is essential for their successful detection, identification and quantification. The strategy applied and the effort invested in the development of an extraction procedure depend not only on the physicochemical properties and the expected concentration of the target compounds but also on the nature of the specimen and the available equipment in the analytical laboratory. Sometimes the physicochemical properties of the target compounds allow for their direct detection after digestion of the sample matrix (e.g. metals), or for an easy separation from the less-volatile

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Practical aspects of extraction matrix components (e.g. gases and volatile compounds via headspace analysis). However, there is an important group of less-volatile drugs and poisons that demand more complex extraction procedures (such as liquid–liquid or solid-phase extraction) to ensure their isolation from the biological matrix. A literature review of the Journal of Analytical Toxicology over the past 20 years shows that the number of publications using liquid–liquid extraction has been quite constant and has covered a wide range of analytes, whereas, for solid-phase extraction, the number of publications has continuously increased and, in recent years, the focus has switched from drugs of abuse to applications with a wider range of target analytes. It can be concluded that, although the importance of liquid–liquid extraction in analytical toxicology has already been established, solid-phase extraction is gaining increasing importance in this field. These two important techniques for the extraction of less-volatile drugs and poisons will be discussed in more detail in this chapter. The final decision about which of these two techniques should be chosen for the particular challenge at hand is mainly based on the practical experience of the analyst and a careful consideration of the objective and the intended use of the extraction method, as well as the availability of analytical techniques. Preconditions of extraction Frequent problems in analytical toxicology include the lack of control over the sampling process and, sometimes, a limited availability of specimens. Such problems can result in insufficient or even inadequate samples. This challenge is partially resolved by close cooperation with the person responsible for sampling (e.g. the physician or pathologist) as well as appropriate quality control and educational measures. In most cases, the appropriate preparation of the specimen is a fundamental precondition for successful sample extraction. Protein-free samples (such as urine) or liquid samples with low protein content (such as serum or plasma) that are frequently used in clinical toxicology are comparable to a purely aqueous phase and therefore direct extraction can be tried. If metabolites should also be detected, hydrolytic cleavage of the conjugate bond (deconjugation) with strong acids, bases or enzymes prior to extraction can increase the recovery of these metabolites from biological fluids. This approach is particularly useful for urine and essential for drugs (e.g. laxatives) that are excreted almost exclusively as conjugated metabolites. For the differentiation of the amount of conjugated and unconjugated metabolite present in the sample, either LC-MS is required or two analyses have to be performed. In the first step the unconjugated metabolite is extracted and quantified and then the sample is re-analysed after hydrolysis, resulting in ‘total’ metabolite concentration (conjugated plus unconjugated). To obtain reliable quantitative results, appropriate standards (also conjugated) must be carried throughout the procedure to monitor the efficiency of the hydrolysis step. The use of an enzyme to cleave chemical bonds is the more specific of the different approaches mentioned above. It incurs additional cost and is more time intensive, but cleaner extracts can be achieved, which reduces the ‘down-time’ of analytical instruments. For the different preparations of purified glucuronidase and sulfatase that are available, it is crucial to pay attention to their pH and temperature optima to obtain reproducible results. A typical procedure for the enzymatic hydrolysis of glucuronides is as follows. Mix 1 mL of blood or urine with an internal standard and 1.5 mL of appropriate buffer and then add 100 mL of b-glucuronidase obtained from Helix pomatia. Mix the solution and incubate it at 37 C overnight (approximately 16 h). After incubation, the pH of the solution is adjusted appropriately for solvent or solid-phase extraction of the compounds of interest (see also Chapter 10). Acid or basic hydrolyses (Dubost, Pascal 1955), although faster and less expensive, tend to produce more artefacts due to the vigorous hydrolysis conditions and are therefore more demanding in terms of necessary clean-up procedures. Typically, strong mineral acids or alkalis are used, often with boiling or treatment in a microwave or pressure

461

cooker. To protect the analytical instruments in subsequent analyses, the extracts must be neutralised and organic solvents have to be dried prior to injection, otherwise chromatographic performance deteriorates quickly. Moreover, care should be taken to ensure the stability of the analytes under these harsh conditions of hydrolysis. If several compounds can be hydrolysed to an identical, single compound, the accurate identification of the original substance present can be precluded. For example, both the acid and the enzymatic hydrolysis of benzodiazepines result in the cleavage of conjugates, but acid hydrolysis also converts different drugs to the same benzophenone compound (e.g. diazepam, temazepam, ketazolam, medazepam and camazepam are all converted into 2-methylamino-5-chlorobenzophenone). Although the resulting compound has good chromatographic characteristics, the approach is unsuitable for those applications (such as forensic analysis) that require absolute identification of the drug ingested (see also Chapter 40). Biological materials that are not homogeneous, protein rich or degraded (such as tissue samples and postmortem samples) need homogenisation (e.g. with a blender to disrupt the cellular structure) and sometimes further sample preparation, such as deproteinisation, before extraction from the aqueous phase is possible. Homogenisation can be performed directly in a buffer solution with a physiological pH of 7.4, to avoid protein precipitation. A high dilution ratio of blood (up to 1 : 10) and tissue (up to 1 : 50) results in samples that tend not to clog tightly packed extraction cartridges and are therefore suitable for direct solid-phase extraction. Automation of the extraction process will enable the analyst to handle the large sample volumes and at the same time secures a uniform and efficient extraction by providing a homogeneous flow of the sample through the extraction cartridge. For liquid–liquid extraction of complex sample matrices, protein precipitation is generally needed before extraction. Deproteinisation can be performed with solvents such as ethanol or acetone, or with dimethylformamide, which is particularly well tolerated by most GC stationary phases. Moreover acetonitrile is frequently used for procedures where high performance liquid chromatography (HPLC) systems are applied. It is usual to use two volumes of organic phase to one volume of blood. The following procedure of a combined protein precipitation and subsequent extraction with acetonitrile under alkaline conditions works for target analyses of neutral and basic compounds in specimens of low collagen content (such as blood or brain tissue): to 0.5 mL of blood or 0.5 g of homogeneous brain tissue, add an internal standard and a freshly prepared mixture of 1 mL acetonitrile and 0.1 mL of a saturated aqueous solution of disodium hydrogenorthophosphate. After shaking, centrifugation and evaporation of the supernatant the reconstituted residue can be extracted. For solid-phase extraction, the dilution of the supernatant (to achieve a concentration of acetonitrile below 20%) is in most cases sufficient and the resulting solution can be applied directly to the sorbent. Protein precipitation may also be accomplished with acids (e.g. hydrochloric acid, perchloric acid, trichloroacetic acid and tungstic acid) and salts (e.g. sodium tungstate, ammonium sulfate, cupric sulfate and uranyl nitrate) (Curry 1988). However, all of these procedures are very time-consuming and labour intensive. Protein precipitation also risks loss of the analyte from adsorption and occlusion. When perchloric, trichloroacetic or tungstic acids are applied there is a particularly high chance that the drugs being analysed may also be co-precipitated. For the extraction of trace amounts of drugs and poisons from complex matrices, dilution and homogenisation with water or buffer solutions are therefore preferred and can prevent these problems. To prepare tissue specimens for extraction, enzymatic digestion of these samples is sometimes useful and can be achieved using pepsin, trypsin, enterokinase, lipase and b-glucuronidase. A suitable procedure is as follows: macerate 10 g of liver or other tissue with 40 mL of 1 mol/L tris(hydroxymethyl)methylamine; add 10 mg of subtilisin Carlsberg and incubate in a water bath at 50–60 C for 1 h, with agitation. Filter the digest through a small plug of glass wool to remove undissolved connective tissue. Aliquots of this digest may be substituted for the specified biological fluid in most routine screening procedures

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Extraction

(Osselton 1978, 1979; Osselton et al. 1978). The filtered digest has a pH of 8.0–9.5 (see also Chapter 10). The drawback of this procedure is that the resulting extracts contain undesirable by-products and artefacts, created during the process of digestion, and therefore a screening procedure will become more difficult. The presence of proteases may interfere with antibodies in immunoassay screening procedures. Even after extensive sample preparation, a large number of matrix components are still present in the sample and these will affect the solubility and the sorption of analytes through their association with molecules as well as by their ability to change the ionic strength of a solution. Soluble matrix components are also distributed between heterogeneous phases and can influence the completeness of equilibrium adjustment as well as the overall extraction efficiency. These considerations have to be kept in mind for the subsequent extraction procedure. Liquid–liquid extraction Direct liquid–liquid extraction (LLE) is still predominant in many laboratories when protein-free samples (such as urine) or liquid samples with low protein content (such as serum or plasma) need to be extracted, because this technique is fast, inexpensive and efficient. LLE is based on well-defined thermodynamic relationships and has a wide dynamic range. The extraction yield is strictly determined by: n n n n

The distribution equilibrium (solubility) The equilibrium of electrolytic dissociation (pH dependent) The ratio of phase volumes (organic/aqueous) The number of extraction stages.

In practical applications, the constitution of the sample matrix has an important influence on results and appropriate sample pre-treatment (e.g. protein precipitation) is a fundamental prerequisite for successful LLE. An excess of solvent and/or repetitive extraction will simply increase the amount of co-extracted impurities. Therefore, a phase ratio (organic/aqueous) of 1 to 2 is recommended. For compounds with low extractability, it is better to switch to another solvent instead of increasing the volume of the organic phase and/or to engage in multiple extractions. In order to separate target compounds from interferences as well as from each other, their varied distribution constants and different acidic or basic properties can be used for fractionation or back extraction (see below). Practical experience shows that, in order to achieve sufficient separation, the pKa values of the compounds to be separated must differ by more than four units. Fractionation in more than two or three groups is therefore ineffective, because under such circumstances substances are dragged into several fractions. It should be kept in mind that neutral compounds are to be expected in the first fraction and that co-extraction of endogenous compounds is to be expected in each extract. Therefore, in every extraction scheme, sacrifices must be made when it comes to the question of the cleanliness of extracts. For the adjustment of an appropriate pH in the aqueous solution, acids or bases can be used, but in this case the stability of the analyte and the possibility of protein precipitation should be kept in mind. Thus the use of an appropriate buffer solution for the reliable adjustment of the pH is recommended. The choice of solvent is mainly based on the solubility of the drugs and poisons that have to be extracted and follows the rule of thumb that ‘like dissolves like’. Moreover, the solubility of known interferences from the sample matrix (such as lipids: fatty acids, cholesterol, etc.) that have a negative effect on the identification and/or the quantification of the analyte should also be considered in the choice of the solvent. A major criterion for the solubility of a particular substance is the solvent’s polarity, which results from an unequal sharing of electrons within the molecule and depends on the electronegativity of its atoms and the asymmetry of the molecule. Polarity aside, solvents can also be chosen by their ability or inability to form hydrogen bonds (hydrogen donor or acceptor), their boiling point (solvent removal with the risk of evaporation losses), their pH stability (e.g. ester cleavage), as well as their water miscibility. A low solubility of water in the solvent facilitates

drying of the extract and co-extraction of water-soluble substances (e.g. salts) can be minimised. In the case of larger amounts of water dissolved in the solvent, it is possible to put the extract in a freezer to freeze the water in order to remove it. Small amounts of water can be removed by addition of anhydrous sodium sulfate, but the possible loss of trace amounts of the analyte should be kept in mind. When choosing a solvent, the potential toxicity should also be considered (e.g. chloroform) as well as flammability and explosive potential (e.g. peroxides in diethyl ether). For practical reasons the density (top or bottom layer with water), and the solvent’s UV absorbency (when spectrophotometric detection is used), as well as the grade of purity are important considerations. Before a solvent is used in a new extraction procedure, an aliquot of the solvent should be evaporated and the residue should be analysed to detect any impurities. Moreover, solvents possess different emulsifying potentials (e.g. that of chloroform is greater than that of diethyl ether), and the correct choice of solvent for LLE also includes considerations to avoid emulsion formation. The addition of neutral salts and the use of slow rotation or vortex speeds as well as using larger phase volume ratios can prevent the formation of emulsions. If emulsions are unavoidable, the phases may be separated by centrifugation, by putting the extract in a freezer and by the addition of a small amount of methanol. If emulsions are routinely obtained, the extraction method should be changed. A promising approach to avoiding emulsions, which also works well for solvents with a high density (such as dichloromethane) that are difficult to isolate from the bottom layer with water, is immobilisation of the aqueous phase (e.g. on diatomaceous earth) prior to extraction. This technique of a supported LLE can increase the extraction yield for the analyte. Some solvents can possess potential reactivity with certain analytes (e.g. some pesticides react with ethanol or acetone) and also ion-pair extraction can occur in certain solvents. This can be exploited to extract analytes (e.g. paraquat), but sometimes ion-pair extraction is an unwanted sideeffect (e.g. in the case of extraction of hydrochlorides with chloroform or dichloromethane). Finally, the analytical method applied may also influence the choice of the solvent. For example, in GC, chlorinated solvents would not be chosen if a halogen specific detector were going to be used. However, if the extract is to be evaporated to dryness and then reconstituted in another solvent before being injected into the chromatographic system (e.g. the eluent of an HPLC system), the choice of the original solvent can be wider. The boiling point and expansion volume of the solvent may also be issues (see also Table 40.6 in Chapter 40). Adding a high concentration of neutral salts (e.g. sodium chloride) to the aqueous phase can support the extraction process (the ‘salting-out effect’), depressing the mutual solubility of phases and simultaneously reducing emulsification and foaming. The evaporation of solvents (for enrichment) demands particular care in order to avoid the loss of volatile analytes (e.g. amfetamines: b.p. 200 C). To prevent the loss of volatile analytes, a small volume of acidified methanol can be added to the extract prior to evaporation. The quality and the volume of the glassware should be chosen with care, to avoid loss of the analyte by adsorption and to enable the reconstitution of the extract in a small amount of solvent (to optimise the concentration step) for further analyses. The appropriate solvent should extract as much of the target compound as possible while at the same time co-extracting only a minimum of interferences. To identify which solvents are frequently used in analytical toxicology, a literature review of the Journal of Analytical Toxicology over 5 years (2004 to 2008) was performed (Table 29.1). The most frequently used solvents were ethyl acetate, followed by 1chlorobutane, hexane and dichloromethane. Isopropyl alcohol was mainly used as a modifier. Chloroform and diethyl ether were frequently used in the past as versatile solvents and, although the severe health risks of chloroform and the fire and explosion hazards of diethyl ether are well known today, they are still in use and have not been totally substituted by dichloromethane and methyl t-butyl ether, respectively. Finally, acetonitrile was also used on a regular basis. Other solvents (such as toluene, methanol, pentane, butyl acetate, acetone and ethanol) were used only occasionally.

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Practical aspects of extraction

Table 29.1 Number of times a solvent was used in a paper published in the Journal of Analytical Toxicology between 2004 and 2008 inclusive Solvent

Number of citations

Ethyl acetate

38

1-Chlorobutane

28

Hexane

27

Dichloromethane

25

Isopropyl alcohol

22

Chloroform

18

Diethyl ether

15

Acetonitrile

11

Methyl t-butyl ether

7

Toluene

6

Isoamyl alcohol

6

Methanol

5

Pentane

3

n-Heptane

3

Octane

2

Dichlorothane

2

n-Butyl acetate

2

Diisopropylether

1

Isobutyl alcohol

1

Propyl acetate

1

Acetone

1

Cyclohexane

1

Tricholoroethanol

1

Ethanol

1

TOTAL

227

When a new extraction procedure is developed, and there are no data about an appropriate solvent available, the following solvents should be tested after adjusting the aqueous solution to an appropriate pH value (in order of increasing polarity): hexane, 1-chlorobutane, methyl t-butyl ether, dichloromethane, 1-butanol, ethyl acetate and acetonitrile. The basic properties of these solvents can be found in Table 29.2. If the results are not sufficiently good using a pure solvent, then modifiers can be added, such as in chloroform–2-propanol (9 : 1; e.g. for morphine), or mixtures can be used. Although so-called all-purpose extraction solvents such as ethanol–hexane–acetone (1 : 2 : 2) or dichloromethane–2-propanol–ethyl acetate (1 : 1 : 3) are used, it should be kept in mind that, in general, the more substances with different physicochemical properties are extracted, the larger the amount of interferences from the matrix that will be co-extracted. These mixtures are therefore

primarily used in the screening for a ‘general unknown’. For target analysis, a toxicologist would choose a more refined procedure with a more selective solvent. If the target compound is known, data about appropriate solvent and pH value for extraction can be found in the monographs in this publication or through a literature review. A frequently used solvent that co-extracts a relatively low amount of interferences from biological samples when compared with other solvents is 1-chlorobutane. For this solvent, extraction yields of 331 toxicologically relevant compounds are available from aqueous media at pH 9.0 (Table 29.3 and in the relevant monographs). At this pH, 228 of the 331 compounds were extracted with an extraction yield equal to or higher than 80% (represented in Table 29.3 by a yield in the organic phase with a value equal to or higher than 0.8). For the remaining compounds, sufficient extraction yields could be reached by changing the pH to acidic (e.g. for phenobarbital) and/or through the addition of a modifier (e.g. for morphine). If a target compound has to be extracted, the database gives a quick overview of the extractability of the compound with 1-chlorobutane at a defined pH value. If the extraction yield from aqueous solution is good, it can be expected that the extraction is also possible from serum. Example protocols for the extraction of various analytes can be found in various chapters of this book (e.g. Hospital Toxicology (Chapter 1), Postmortem Toxicology (Chapter 10) and High Performance Liquid Chromatography (Chapter 41)). The extraction procedure will become more complex with an increase in the complexity of the sample matrix because additional steps for purification (back extraction) have to be added. As an example of a complete extraction scheme for the extraction of bases, neutrals and acids, and to illustrate the theoretical background discussed above, the following is reproduced from Chapter 10. The pH of the specimen influences the extent to which acid and basic drugs are extracted. Addition of a weakly basic buffer, such as sodium borate (pH 9), favours the extraction of weakly basic drugs, as well as most neutral substances. Similarly, the addition of an acidic buffer, such as sodium dihydrogenphosphate, favours the extraction of acidic as well as neutral drugs. The majority of drugs of forensic interest are ‘basic’ in character, but are often present at relatively low concentrations in blood. It is, therefore, desirable to have an extraction scheme that incorporates a back-extraction step to eliminate or minimise the extraction of endogenous molecules. An example extraction scheme is shown in Fig. 29.1. The saturated sodium borate solution will force the basic drugs into the lipid-soluble un-ionised form, allowing extraction into the chlorobutane. The chlorobutane is transferred to a fresh tube and the drugs are back extracted into sulfuric acid. Neutral or acidic substances will remain in the upper chlorobutane layer, which may be pipetted or aspirated to waste. The remaining acid layer is then made basic by addition of sodium hydroxide, and the now un-ionised basic drugs are re-extracted with chlorobutane. The upper solvent layer may then be removed and concentrated under nitrogen, prior to analysis by a suitable chromatographic method. This extraction scheme will give extracts that are relatively free of interfering substances. However, it should be noted that morphine and other amphoteric drugs cannot be detected by this method since the phenolic functional group will

Table 29.2 Properties of solvents Solubility (g/100 mL water at 20 C)

Polarity index (water = 10.2)

Hydrogen acceptor

Hydrogen donor

69

0.01

0.1

No

No

78

0.07

1.0

No

No

0.74

55

0.5

2.5

Yes

No

Dichloromethane

1.34

40

2

3.1

No

No

1-Butanol

0.81

118

7.9

3.9

Yes

Yes

Ethyl acetate

0.90

77

8.6

4.4

No

Yes

Acetonitrile

0.78

82

Miscible

5.8

No

Yes

Solvent

Density (g/mL)

Hexane

0.66

1-Chlorobutane

0.89

Methyl t-butyl ether

Boiling point ( C)

463

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Extraction

Table 29.3 Extraction yields of 331 compounds of clinical or forensic interest using 1-chlorobutane from water at pH 9 (multiple observations in different laboratories were averaged)

Table 29.3 continued Compound

Extraction yield

Extraction yield

Amfetamine

0.5

0

Apomorphine

0.8

0.8

Aprindine

0.95

0.7

Articaine

1

0.9

Atenolol

0

0.8

Atropine

0.6

0.5

Azathioprine

0

2-Methylamino-1-(3,4-methylenedioxyphenyl)butane (MBDB) 1

Azinphosethyl

1

2,3-Methylenedioxyamfetamine

0.6

Azinphosmethyl

1

2,3-Methylenedioxymethamfetamine

0.9

Benperidol

1

0.6

Benserazide

0

0.3

Benzatropine

1

0.2

Benzoylecgonine

0

0.5

Betaxolol

1

0.8

Biperiden

1

0.8

Bisacodyl

0.7

2,5-Dimethoxy-4-methyl-phenethylamine (2C-D)

0.5

Bisoprolol

0.9

2,5-Dimethoxy-phenethylamine (2C-A)

0.3

Bromazepam

0.9

3-(2,3-Methylenedioxyphenyl)pentane-2-amine

0.95

Bromocriptine

1

0.6

Bromophosethyl

1

0.7

Bromophosmethyl

1

0.9

Bromperidol

1

0.5

Brotizolam

1

0.3

Budipine

1

1

Bupivacaine

1

4-Methoxyamfetamine (PMA)

0.5

Bupranolol

1

4-Methyl-2-pyrrolidinopropiophenone (MPPP)

1

Buprenorphine

1

4-Methylthioamfetamine (4-MTA)

0.6

Buspirone

0.95

1

Caffeine

0.3

1

Carazolol

0.9

1

Carbamazepine

0.95

1

Carbamazepine epoxide

0.6

0.7

Carbidopa

0

N,N-Diethyltryptamine (DET)

0.95

Carteolol

0.06

Acebutolol

0.05

Carvedilol

1

Acetaminophen (paracetamol)

0

Cathine

0.07

0

Celiprolol

0.10

0.5

Chlordiazepoxide

0.95

1

Chlormezanone

0.9

1

Chloroquine

0.95

0.95

Chlorovinphos

1

1

Chlorpromazine

1

Amantadine

0.5

Chlorprothixene

1

Amfebutamone

1

Citalopram

1

Amfepramone

1

Clobazam

1

1

Clobutinol

1

0.95

Clomethiazole

1

0.6

Clomipramine

1

1

Clonazepam

1

0.10

Clopamide

0.06

1

Clopenthixol

1

Compound 10-Hydroxycarbazepine (metabolite of oxcarbazepine) 2-(2,3-Methylenedioxyphenyl)butane-1-amine 2-(2,3-Methylenedioxyphenyl)propane-1-amine 2-(3,4-Methylenedioxyphenyl)2-methylpropane-1-amine 2-(3,4-Methylenedioxyphenyl)butane-1-amine 2-(3,4-Methylenedioxyphenyl)propane-1-amine

2,3-Methylenedioxo-N-methylphenethylamine 2,3-Methylenedioxo-N-phenethylamine 2,4,5-Trimethoxyamfetamine (TMA-2) 2,4,6-Trimethoxyamfetamine (TMA-6) 2,5-Dimethoxy-4-brom-phenethylamine (2C-B) 2,5-Dimethoxy-4-metamfetamine (DOM)

3,4-Methylenedioxyamfetamine (MDA) 3,4-Methylenedioxymetamfetamine (MDMA) 3,4-Methylenedioxy-N-ethylamfetamine (MDE) 3,4-Methylenedioxy-N-methylphenethylamine 3,4-Methylenedioxy-N-phenethylamine 4-Methoxy-2-pyrrolidinopropiophenone

N-(1-Phenylcyclohexyl)-3-ethoxypropylamine (PCEPA) N-(1-Phenylcyclohexyl)-3-methoxypropylamine (PCMPA) N-(1-Phenylcyclohexyl)-1-propylamine N-(1-Phenylcyclohexyl)-2-methoxyethylamine (PCMEA) N-Methyl-4-methoxyamfetamine (PMMA)

Adenosine Ajmaline Alfentanil Alimemazine Alprazolam Alprenolol

Amfetaminil Amiodarone Amisulpride Amitriptyline Amitriptyline oxide Amlodipine

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465

Table 29.3 continued

Table 29.3 continued Compound

Extraction yield

Compound

Extraction yield

Clotiazepam

1

Gabapentin

0

Clozapine

1

Gallopamil

1

Cocaine

1

Gamma-hydroxybutyric acid

0

Codeine

0.8

Glibenclamide

0.2

Colchicine

0.13

Glutethimide

1

Cotinine

0.10

Haloperidol

1

Cyamemazine

1

Heptenophos

0.8

Deanol

0.4

Hydrochlorothiazide

0

Demelverine

1

Hydromorphone

0.10

Desipramine

1

Hydroxyzine

1

Detajmium

0.9

Ibuprofen

0

Dialifos

1

Imipramine

1

Diazepam

0.95

Indometacin

0

Dibenzepin

1

Ipratropium

0

Dichlorvos

1

Isofenphos

0.95

Diclophenac

0.2

Kavaine

0.7

Dihydrocodeine

0.7

Ketamine

1

Dihydroergocryptine

1

Lamotrigine

0.17

Diltiazem

1

Levetiracetam

0

Dimethoate

0.7

Levodopa

0

Dimetindene

0.98

Levomepromazine

1

Diphenhydramine

1

Lidocaine

1

Disopyramide

0.8

Lisinopril

0

Dosulepin (dothiepin)

1

Lofepramine

0.9

Doxazosin

0.95

Oprazolam

1

Doxepin

1

Lorazepam

0.85

Doxylamine

1

Lormetazepam

1

Droperidol

0.9

Loxapine

1

Enalapril

0

Lysergide (LSD)

0.95

Entacapone

0

Maprotiline

1

Ephedrine

0.2

Medazepam

1

Esmolol

0.8

Mefenorex

0.95

Ethosuximide

0.2

Melperone

1

Etomidate

1

Mepindolol

0.4

Felodipine

0.95

Mepivacaine

1

Fenethylline

0.9

Meprobamate

0.10

Fenofos

1

Meptazinol

0.9

Fentanyl

1

Mesuximide

1

Fenthion

1

Metamizole

0.4

Flecainide

0.95

Methadone

0.95

Fluconazole

0.10

Methamfetamine

0.7

Flumazenil

0.8

Methaqualone

1

Flunitrazepam

1

Methohexital

0.95

0.8

Methylphenidate

0.8

Flupenthixol

1

Metixene

1

Fluphenazine

1

Metoclopramide

0.9

Flupirtine

1

Metoprolol

0.8

Flurazepam

0.95

Mevinphos

0.9

Fluspirilene

0.9

Mexiletine

0.9

Fluvoxamine

0.8

Mianserin

1

Furosemide

0

Midazolam

0.9

Fluoxetine

table continued

table continued

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Extraction

Table 29.3 continued

Table 29.3 continued Compound

Extraction yield

Compound

Extraction yield

Mirtazapine

0.9

Piritramide

0.9

Moclobemide

0.9

Piroxicam

0

Modafinil

0.4

Prajmalium

0.9

Morphine

0

Pramipexole

0

Nadolol

0

Prazepam

1

Nalbuphine

0.8

Prilocaine

0.95

Nalorphine

0.3

Primidone

0

Naloxone

0.9

Procainamide

0.10

Nefazodone

1

Procaine

0.9

Nefopam

0.95

Procyclidine

1

Nicotine

0.9

Promazine

1

Nifedipine

1

Promethazine

1

Nicotinamide

0

Propafenone

0.95

Nimodipine

1

Propofol

0.95

Nisoldipine

1

Propoxyphene

1

Nitrazepam

1

Propranolol

1

Nordiazepam

0.95

Propyphenazone

1

Nortriptyline

1

Prothipendyl

0.95

Noscapine

1

Pseudoephedrine

0.2

Olanzapine

1

Quetiapine

1

Opipramol

1

Quinidine

0.95

Orciprenaline

0

Quinine

1

Oxazepam

0.85

Ranitidine

0

Oxcarbazepine

0.9

Reboxetine

1

Oxitriptan

0

Remifentanil

1

Oxprenolol

0.9

Risperidone

1

Oxycodone

0.95

Ropivacaine

1

Paraoxon

0.9

Salicylate

0

Parathion ethyl

1

Scopolamine (hyoscine)

0.7

Parathion methyl

1

Sertindole

1

Paroxetine

1

Sertraline

1

Pemoline

0

Sildenafil

1

Penbutolol

1

Sotalol

0

Pentazocine

0.8

Strychnine

0.9

Pentobarbital

0.2

Sulfentanil

1

Pentoxyverine

1

Sulfotep

1

Perazine

1

Sulpiride

0

Perphenazine

1

Sultiame

0

Pethidine

1

Talinolol

0.2

Phenazone

0.4

Temazepam

1

Phencyclidine

0.9

Terbufos

1

Phenobarbital

0.10

Tertatolol

1

Phenolphthalein

0.7

Tetrazepam

0.95

Phenprocoumon

0.2

Theobromine

0

Phenytoin

0.5

Theophylline

0

Pholedrine

0

Thiopental

0.9

Phosphamidon

0.8

Thioridazine

1

Phoxime

1

Tiagabine

0.5

Pimozide

1

Tiapride

0.4

Pindolol

0.4

Ticlopidine

1

Pipamperone

1

Tilidine

1

Pirimiphos

1

Timolol

0.6

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Practical aspects of extraction

467

Table 29.3 continued Compound

Extraction yield

Tocainide

0.3

Tolperisone

1

Topiramate

0.2

Tramadol

1

Tranylcypromine

1

Trazodone

1

Triazolam

1

Trichlorophos

1

Trifluperidol

1

Triflupromazine

1

Trihexyphenidyl

1

Trimipramine

1

Tryptophan

0

Valproic acid

0.07

Venlafaxine

0.95

Verapamil

1

Vigabatrin

0

Viloxazine

0.85

Zaleplon

1

Ziprasidone

1

Zolpidem

1

Zopiclone

0.9

Zotepine

0.95

Zuclopenthixol

1

Extraction yield = extraction yield in the organic phase (e.g. 0.3 representing an extraction yield in 1-chlorobutane of 80%). The database was compiled by the Committee on Extraction of the Society of Toxicological and Forensic Chemistry under the supervision of Dr U. Demme, and is reproduced here with the kind permission of the Society of Toxicological and Forensic Chemistry.

ionise at high pH, therefore precluding extraction into the solvent. For amphoteric drugs, the basic phase should be less than pH 9.0, and preferably pH 8.0–8.5. Although a similar extraction scheme to that used for basic drugs (but with the additions of acid and base reversed) could be used for strongly acidic drugs, such a method does not efficiently extract weakly acidic drugs, such as the barbiturates, and neutral drugs, such as meprobamate. Conversely, simple addition of an acidic buffer to whole blood and extraction with a solvent results in the co-extraction of large amounts of endogenous lipid substances. Such extracts may be ‘cleaned up’ by partitioning between immiscible solvents of different polarities, such as acetonitrile and hexane, as shown in Fig. 29.2. The more polar drugs tend to partition into the acetonitrile, whereas the endogenous lipids (fatty acids, sterols) tend to partition into the hexane. Solid-phase extraction Although method development for solid-phase extraction (SPE) is not as straightforward as for LLE, this technique offers appealing advantages. Because of SPE’s high extraction efficiency, even very small sample sizes are sufficient, thereby reducing solvent consumption. Additionally, there is no emulsion formation. Past problems with inconsistent quality of the extraction cartridges have been overcome by manufacturers’ implementation of extensive quality control measures. The increasing interest in SPE by toxicological laboratories lies mainly in its compatibility with automation. The need for automated extraction procedures is directly related to the

Figure 29.1 Extraction scheme for strong bases.

expectation that toxicological laboratories become economically selfsufficient as well as an increased demand for quality assurance and reproducibility and, therefore, comparable results between different laboratories. In SPE the analytes are isolated from the aqueous sample by adsorption onto a solid sorbent, followed by washing and elution steps. In each of these steps a mechanism of total retention or total release of the target compounds is desired and, as with LLE, this technique is based on the fundamental principles of extraction discussed above. Depending upon the choice of sorbent, the extraction of toxicologically relevant compounds is achieved via hydrophobic, hydrophilic, aromatic or electrostatic interactions; often a combination of several mechanisms is involved. In the field of analytical toxicology non-modified silica, surface-modified silica and polymer resins are used, and a more detailed description of these sorbents can be found in Chapter 41 (packing materials). To date, both selective procedures (for target analysis) and non-selective SPE procedures (for the screening for a ‘general unknown’) have been developed. Because a wide variety of different analytes can be extracted using a combination of hydrophobic and electrostatic interactions, these socalled mixed-mode sorbents (or functionalised sorbents, or hybrid extraction sorbents) are widely used in analytical toxicology. A practical

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Dilution (phosphate buffer pH 7.4), homogenisation and centrifugation of the sample

Pre-treatment of the sorbent with ethyl acetate–2-propanol (3 : 1) followed by phosphate buffer pH 7.4

Application of the sample onto the ‘mixed-mode’ sorbent (max. 1 mL/min)

Washing with water and pH change by applying an acidic solution

Elution of acidic and neutral analytes with ethyl acetate–2-propanol (3 : 1) (max. 1 mL/min)

Figure 29.2 Extraction scheme for acids and neutrals.

example of such a procedure for the extraction and fractionation of neutral and basic metabolites of cyclizine in the urine of racehounds can be found in Chapter 7. To illustrate the fundamental principles and possible pitfalls of SPE, the following section includes a step-by-step discussion of the development of a non-selective ‘general unknown’ screening procedure for a wide variety of analytes. An overview of the procedure can be found in Fig. 29.3. The first step is the appropriate pre-treatment of viscous specimens so that the samples do not cause flow problems when passing through the tightly packed cartridges and choosing the sorbent. Tissue homogenates, especially, tend to clog the polyethylene frit (pore diameter 20 mm) holding the sorbent in place. Practical experience has shown that diluting the specimens with ten times the amount of phosphate buffer before homogenisation provides samples that can be extracted with either negative or positive pressure. Some tissues (e.g. putrefied liver) have to be diluted even more (up to 50 times). A physiological pH of 7.4 prevents protein precipitation in biological specimens and therefore avoids an unpredictable loss of analytes. After homogenisation (e.g. with a blender), and before the sample is applied to the extraction cartridge, remaining particles and cellular structures must be removed by centrifugation (cooling can prevent the loss of volatile analytes). For the handling of the sometimes large sample volumes, automation of the whole extraction process is highly recommended. The controlled and therefore homogeneous flow of the sample through the extraction cartridge in such an extraction device secures a uniform and efficient extraction, leading to more reproducible results. For this ‘general unknown’ screening procedure, a mixed-mode sorbent based on a combination of a polar modified polystyrene resin and a cationic exchanger is used. In this way analytes can be extracted by non-selective hydrophobic and aromatic interactions and, in a second step, acidic and neutral compounds can be separated from basic compounds that are retained by electrostatic interactions through their amine functionality. Polystyrene resins do not possess residual silanol groups like silica-based sorbents do (due to varying degree of

Elution of basic analytes with ethyl acetate–2-propanol–triethylamine (75 : 25 : 3) (max. 1 mL/min) Figure 29.3 Overview of a 'mixed-mode' solid-phase extraction procedure.

endcapping) Polystyrene resins have the additional advantage of being stable across the entire pH range, in contrast to the limited stability (pH 2–8) of silica-based sorbents. Moreover, polystyrene resins offer a higher capacity than silica-based sorbents and sufficient retention of a wide variety of different analytes can be achieved at a pH of 7.4. At this pH value an equilibrium of electrolytic dissociation will be reached for acidic and basic analytes in the aqueous phase. By adsorption of the non-ionised analyte to the sorbent, equilibrium must be continuously re-established and a nearly complete extraction will be reached if sufficient contact time with the sorbent is allowed and if the capacity of the sorbent is high enough. Additionally, the porous structure of polystyrene resins can be used to exclude large molecules (e.g. proteins and lipids bound in micelles) from the extraction process. The amount of sorbent that has to be used is determined by the amount of interferences that are expected in the sample matrix analysed. The second step is the pre-treatment of the sorbent. It should be washed with the strongest solvent applied in the procedure and then it must be conditioned – in the procedure described here, with a phosphate buffer of pH 7.4 – in order to make the sorbent compatible with the sample. Silica-based bonded sorbents should not dry out between the applications of different solutions. This is not an issue when working with polar modified polystyrene resins because of their additional hydrophilic properties. The third step is the application of the sample. Competitive influences of matrix components displacing the analytes from the limited surface area of the sorbent have to be considered and ‘overloading’ the extraction cartridge must be avoided. Because the mass transfer of analyte to sorbent is determined by kinetic functions and, because the intermolecular interactions (van der Waals’ forces, aromatic interactions, etc.) are short-ranged and relatively weak forces, sufficient contact

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References

469

Table 29.4 Possible pitfalls in the process of developing a mixed-mode solid-phase extraction procedure Steps of the mixed-mode procedure

Possible pitfalls

Specimen pre-treatment

Protein precipitation (loss of analytes), flow problems

Choice of sorbent

Capacity, undesired secondary interactions, stability

Washing and conditioning sorbent

Improper conditioning (drying of silica-based sorbents, capacity problems)

Sample application

Insufficient contact time, 'break-through', clogging

Washing and changing pH to acidic

Protein precipitation on the sorbent, insufficient acidic capacity

Elution of acidic and neutral compounds

Insufficient contact time, inappropriate solvent (solvent strength, water miscibility)

Elution of basic compounds

Insufficient contact time, inappropriate solvent (solvent strength, water miscibility), insufficient basic capacity, sorbent stability (silica-based sorbents)

time is a key factor for ensuring reproducible results. To avoid ‘breakthrough’ due to ineffective retention of analytes, the adsorption flow rate should be kept below 1 mL/min. In the fourth step, protein and other interferences are washed away with water (to avoid protein precipitation on the sorbent when subsequently applying organic solvents) and the pH is changed by applying an acidic solution so that analytes with amino functions are protonated and establish their electrostatic interactions with the charged sorbent. In the fifth step, because ionic interactions are relatively strong forces, acidic and neutral analytes as well as hydrophobic and polar interferences can be eluted with strong organic solvents (with different polarity and/or solvent strength; the first solvent should be miscible with water to be able to reach the ‘inner surface’ of the sorbent, which is loaded with water comparably to a sponge; flow rates should be kept below 1 mL/min). In the procedure described here, ethyl acetate–2propanol (3 : 1) is used. In the sixth and final step, all bonding mechanisms for the remaining basic analytes (ionic and hydrophobic interactions) have to be disrupted simultaneously and this can be done using an appropriate basic organic solvent (in the procedure described, ethyl acetate–2-propanol–triethylamine (75 : 25 : 3) is used). Elution will be incomplete if the basic solution is not strong enough to completely disrupt the electrostatic interaction. Again, flow rates should be kept below 1 mL/min, because ion-exchange binding kinetics are even slower than with hydrophobic and aromatic interactions. Volatile organic bases (such as triethylamine) have the advantage that they can be removed easily from the extract by evaporation. After evaporation of the eluent from step 5, the extract can then be analysed for neutral and acidic compounds (in the case of a very complex matrix and/or a very low concentration of the analyte, a further back-extraction step might be necessary). Simultaneously, after evaporation, the extract from step 6 can be analysed for basic compounds. The aqueous phase of the sample should not be discarded in this ‘general unknown’ screening procedure, because strong electrolytes (e.g. quaternary ammonium compounds) will not be sufficiently extracted onto the sorbent. Extraction of these compounds can be accomplished by ionpair formation (see Ion-pair extraction above). The possible pitfalls in using a mixed-mode SPE procedure are given in Table 29.4. Microextraction Miniaturisation of the extraction process simplifies the entire analytical procedure by having the extraction carried out in one vessel, eliminating the evaporation step and possible loss of volatile materials, reducing solvent use, and considerably saving in extraction time. It usually involves vigorous mixing of a small volume of solvent with a large volume of biological material (e.g. urine), centrifuging to separate the solvent layer and direct injection into a chromatograph. For example, amfetamines and related compounds may be extracted from 5 mL of urine into 100 mL of chloroform and this is injected into a GC apparatus (Ramsey, Campbell 1971). Some microextraction methods such as hollow fibre-based liquidphase extraction (Esrafili et al. 2007) and fibre-in-tube solid-phase

microextraction (Yazdi et al. 2008) have been successfully used for the analysis of antidepressant drugs in biological fluids. Microextraction by packed sorbent (MEPS) with on-line connection to GC or LC has been applied for the analysis of local anaesthetics in plasma samples (Abdel-Rehim 2004) and the analysis of amfetamines in hair (Miyaguchi et al. 2009).

Conclusions The ongoing development of more powerful and sensitive analytical instruments can only be fully utilised after the successful isolation of the target compounds from the biological specimens. Without sufficient sample extraction, either these sensitive instruments cannot be used at all or the results are unreliable owing to interferences. Although many optimised procedures for the extraction of specific target compounds (in most cases from urine or blood) can be found in the literature, nonselective, reliable and robust procedures for the simultaneous extraction of a wide range of analytes with different physicochemical properties (e.g. for the ‘general unknown’ screening) are rare, especially when more complex matrices have to be extracted. Unfortunately, the greatly desired ‘universal standard extraction procedure’ for all possible constellations of target compounds and specimens does not (yet) exist. The selection or development of a proper extraction procedure and the procedure’s adaptation to specific cases – based on analytical data of targeted compounds, available specimens, advantages and disadvantages of the various extraction techniques, and the time and resources at hand – are key tasks of the analytical toxicologist. Special knowledge and experience are needed to meet these challenges. Analytical results usually have serious medical or legal consequences: toxicologists therefore carry a high level of responsibility. To ensure the integrity of analytical results, stringent quality control measures have been implemented in modern toxicological laboratories. But such measures should not lead to overly rigid structures that limit the toxicologist to a few strictly defined extraction methods and target analytes, as this would narrow a laboratory’s flexibility to an unacceptable level. The analytical toxicologist must stay open to new extraction technologies, improvements in sample preparation and developing trends. Among these improvements is automation, which enables the unattended, reproducible extraction of samples; advances in this application of technology are expected soon.

References Abdel-Rehim M (2004). New trend in sample preparation: on-line microextraction in packed syringe for liquid and gas chromatography applications. I. Determination of local anaesthetics in human plasma samples using gas chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 801: 317–321. Curry AS (1988). Poison Detection in Human Organs, 4th edn. Springfield, IL: Charles Thomas. Dubost P, Pascal S (1955). Determination of chlorpromazine in biological fluids; additional note. Ann Pharm Fr 13: 56–57. Esrafili A et al. (2007). Hollow fiber-based liquid phase microextraction combined with high-performance liquid chromatography for extraction and determination of some antidepressant drugs in biological fluids. Anal Chim Acta 604: 127–133.

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Miyaguchi H et al. (2009). Rapid identification and quantification of methamphetamine and amphetamine in hair by gas chromatography/mass spectrometry coupled with micropulverized extraction, aqueous acetylation and microextraction by packed sorbent. J Chromatogr A 1216: 4063–4070. Osselton MD (1978). The release of basic drugs by the enzymic digestion of tissues in cases of poisoning. J Forensic Sci Soc 17: 189–194. Osselton MD (1979). The use of proteolytic enzymes to release high levels of drugs from biological materials submitted for toxicological analysis. Vet Hum Toxicol 21(Suppl): 177–179. Osselton MD et al. (1978). Enzymic digestion of liver tissue to release barbiturates, salicylic acid and other acidic compounds in cases of human poisoning. Analyst 103: 1160–1164. Ramsey J, Campbell DB (1971). An ultra rapid method for the extraction of drugs from biological fluids. J Chromatogr 63: 303–308. Yazdi AS et al. (2008). Separation and determination of amitriptyline and nortriptyline by dispersive liquid–liquid microextraction combined with gas chromatography flame ionization detection. Talanta 75: 1293–1299.

Further reading Franke JP, De Zeeuw RA (1998). Solid-phase extraction procedures in systematic toxicological analysis. J Chromatogr B Biomed Sci Appl 713: 51–59. Leo A et al. (1971). Partition coefficients and their uses. Chem Rev 71: 525–616. Lide RL (1996). Properties of Organic Solvents. Boca Raton, FL: CRC Press. M€ uller RK (1991). Extraction from aqueous phase. In: M€ uller RK, ed. Toxicological Analysis. Berlin: Verlag Gesundheit, 66–83. Pawliszyn J (2003). Sample preparation: quo vadis? Anal Chem 75: 2543–2558. Siek TJ (1978). Effective use of organic solvents to remove drugs from biologic specimens. Clin Toxicol 13: 205–230. Stimpfl T, Vycudilik W (2004). Automatic screening in postmortem toxicology. Forensic Sci Int 142: 115–125. Telepchak M et al. (2004). Forensic and clinical applications of solid phase extraction. In: Karch SB, ed. Forensic Science and Medicine. Totowa, NJ: Humana Press. Wille SM, Lambert WE (2007). Recent developments in extraction procedures relevant to analytical toxicology. Anal Bioanal Chem 388: 1381–1391.

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CHAPTER

30

Colour Tests B Widdop

Introduction The bulk of the material in this chapter derives from that contributed by the late HM Stevens to the second edition of this publication, which was modified and expanded by Wayne Jeffrey in the third edition. The main addition in this latest version is an appendix dealing with tests for metals and anions that are not covered by the general scheme. Colour tests (sometimes referred to as chemical spot tests) provide toxicologists and drug analysts with one of the first tools for the presumptive identification of drugs and poisons. These colour tests are most usefully applied to pharmaceuticals and scene residues, and to a lesser extent to biological fluids such as stomach contents, urine. They are used to place the unknown into a specific class of compounds or to eliminate categories or classes of compounds. These colour tests remain popular for many reasons. They are simple to perform, use minimal reagents, are inexpensive and give results that can be viewed by the naked eye. They appeal particularly in parts of the developing world where laboratory facilities tend to be very limited. In many instances they can also be used as thin-layer chromatography (TLC) location reagents applied by spraying or dipping (see Chapters 1, 11, and 39). This chapter describes the colour tests cited in the monographs. For some substances, the colour reaction with a particular chemical reagent may be quite specific, but it is much more common for the colour to be produced by a class of compounds. Moreover, compounds that do not fall into the class may also give colours. For some of the tests, the colour reactions can be correlated with certain aspects of the chemical structure of a compound or group of compounds. However, anomalous responses often occur that cannot be explained on that basis. Some of these are noted in the colour tests described below, but it should be borne in mind that many others may be found. It follows that colour tests are only an indication of the presence of a compound or class of compounds and that all tests must be confirmed by more specific methods. This is especially important in forensic cases! The colour tests included here range from those that rely on reactions with certain functional groups (e.g. Folin–Ciocaltaeu for phenols), those that are almost specific for a given group (e.g. FPN reagent for phenothiazines) through to those that give diagnostic colours with a wide range of compounds (e.g. Mandelin’s test and the Marquis test).

Interpretation of colour tests Colour descriptions Colours exhibited by these tests cannot be described with any accuracy. They may vary in intensity or tincture with the concentration of compounds in the test samples and the presence of extraneous material. In addition, their assessment is always a subjective one, even in people with normal colour vision. Some of the complexes formed are unstable such that the colour changes or fades with time. Effects of ionic form Salts may give colours different from those of the corresponding acid or base. In general, free acids or bases that have been isolated from the test material by an extraction process give better colours than their salts. The colour of a salt may be modified by the nature of the other ions present. For example, all hydrochloride salts give a red colour in Mandelin’s test

and a blue colour with Koppanyi–Zwikker reagent (prior to adding pyrrolidine). Basic salts of weak acids may produce different colours because of a change in pH. Where a compound has been extracted from biological material, these factors should not create any difficulty, since it will be present in the form of the base. However, when applying the tests to pharmaceutical preparations, where the compounds are usually present as salts, this can cause problems. To overcome this, the material can be extracted in much the same way as for biological samples to derive the free base. Bromide and iodide salts can be converted into the nitrate before testing, which gives the same colour as the base, by the following method: n

n

To 0.5 mL of a 1% (w/v) solution of the salt in dilute acetic acid, add one drop of an 8% (w/v) solution of silver nitrate followed by one drop of a 2% (w/v) solution of sodium chloride to remove excess silver. Centrifuge to separate the precipitated silver halide and use the supernatant liquid, either as a solution or evaporated to dryness where necessary, for the colour tests.

The colours that are recorded in the tables and monographs are usually those obtained by testing either the free acid or the free base. Use of the colour tests lists The system adopted uses ten basic colours: the spectral colours (red, orange, yellow, green, blue and violet), together with pink, brown, grey and black. Where there is a variation in hue, this is indicated by combining two colours (e.g. red–brown). The second-named colour is considered to be the dominant one and is the main colour used in the lists. For example, red–brown is listed under brown, whereas brown–red is listed under red. When interpreting results, it is often necessary to search the lists given under two main colours (e.g. for red–brown, the lists under both red and brown should be consulted). This takes account of the subjective nature of colour assessment. An arrow between two colours (e.g. red!brown) indicates that the colour changes during the course of the test. In the monographs, the notation brown/red is used where there are two parts to a test that produce two colours. Occasionally, the colour displayed by a test solution in reflected light may be different from that in transmitted light, in which case the solution is described as dichroic. A combined colour may be obtained when more than one drug is present or the drug itself is coloured, which limits the value of the tests for biological samples.

Practical points Performing the colour tests The tests are carried out either in clear glass test-tubes or on white glazed porcelain tiles (spotting tiles), which give a uniform background against which the colours can be assessed. For drugs, the tests are designed to work on about 1 mg, either as the solid form or as a dried extract of this amount (see below), unless stated otherwise. Solutions should be made in water unless otherwise stated. Where an instruction, time, temperature, etc., appears in brackets after the drug name, such as (add water), (15 s) or (slowly at 100
C), this indicates a change in the test procedure for that particular drug. 471

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Colour Tests

The following recommendations are most important: n

n

A sample known not to contain the compound of interest should be tested at the same time as the test sample. This enables a comparison of the colours produced by the sample and by the reagent blank. Ideally, the blank sample should have the same matrix as the test sample (e.g. for urine tests use analyte-free urine), since this takes account of the effects of extraneous materials. Otherwise, water is usually adequate. Before making a final decision on the result of a test, the reaction of the unknown should be compared with that of a reference substance tested under exactly the same conditions.

Table 30.2 is formulated to give a quick lead to those tests that can be applied to detect some of the most important drug groups and other poisons.

Table 30.2 Indication of which tests can be applied to detect some of the most important drug groups and other poisons Substance/functional group

Useful tests

Alcohols

Potassium dichromate

Alkaloids and nitrogenous bases

Dragendorff's reagent

Amides (aliphatic)

Nessler's reagent

It is essential to validate all tests and test reagents for sensitivity and specificity; O’Neal et al. (2000) have outlined a suitable method for a chemical spot test.

Aldehydes (aliphatic)

Schiff's reagent

Amfetamines

See Appendix 30.1

Application of colour tests to sample extracts

Antidepressants

Marquis test

Several solvent extraction schemes have been devised to fractionate compounds on the basis of their acidic, neutral or basic characteristics (see Chapters 1 and 10). The tests listed in Table 30.1 can be applied to the evaporated extracts.

Barbiturates

Dille–Koppanyi reagent

Validation of a colour test

Table 30.1 Tests that can be applied to the evaporated extracts Fraction

Test

Strong acid

Aromaticity Ferric chloride

Sodium nitroprusside–acetone

Koppanyi–Zwikker reagent Mercurous nitrate Vanillin reagent Zwikker reagent Benzodiazepines

Formaldehyde–sulfuric acid

Cannabis

Duquenois reagent

Carbamates (non-aromatic)

Furfuraldehyde

Cocaine

Cobalt thiocyanate

Folin–Ciocaltaeu reagent

p-Dimethylaminobenzaldehyde

Liebermann's reagent

Mandelin's test

Millon's reagent Nessler's reagent Weak acid

Scott's test Chlorinated phenols

Nitric acid (fuming) Nitric–sulfuric acid

Coniferyl alcohol

Chlorinated hydrocarbon insecticides

Diazotisation

Cyanide

Ferrous sulfate (B)

Aromaticity

Ferric chloride

Neutral

Sodium picrate

Folin–Ciocaltaeu reagent

Cyanide groups

Sodium picrate

Koppanyi–Zwikker reagent

Dithiocarbamates

Sodium nitroprusside

Liebermann's reagent

Ergot alkaloids

p-Dimethylaminobenzaldehyde

Mercurous nitrate

Halogenated hydrocarbons

Fujiwara test

Millon's reagent

Imides

Koppanyi–Zwikker test

Nessler's reagent

Ketones

Sodium nitroprusside

Aromaticity

Methadone

Cobalt thiocyanate

Furfuraldehyde

Mandelin's test

Koppanyi–Zwikker reagent

Marquis test

Liebermann's reagent

Tetrabromophenolphthalein ethyl ester

Mercurous nitrate Basic

Nessler's reagent

Mono-substituted pyridine ring

Cyanogen bromide

Amalic acid test

Nitrates and nitrites

Ferrous sulfate

p-Dimethylaminobenzaldehyde

Opiates

See Appendix 30.2

Ferric chloride

Oxidising agents

Diphenylamine

Formaldehyde–sulfuric acid

Paraquat/diquat

Sodium dithionate

Forrest reagent

Phencyclidine

Cobalt thiocyanate

FPN reagent

p-Dimethylaminobenzaldehyde

Liebermann's reagent

Tetrabromophenolphthalein ethyl ester

Mandelin's reagent Marquis reagent Nessler's reagent Sulfuric acid

Phenols

p-Dimethylaminobenzaldehyde Ferric chloride Folin–Ciocaltaeu reagent Millon's reagent

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Colour test methods

Table 30.3 Colours with amalic acid

Table 30.2 continued Substance/functional group

Useful tests

Colour

Compound

Phenothiazines

Ferric chloride

Red (!violet)

Bufylline, caffeine

Formaldehyde–sulfuric acid

Pink (!violet)

Pentoxifylline

Forrest reagent

Orange (!violet)

Acepifylline piperazine, bamifylline, xanthinol nicotinate

FPN reagent Phenylpyrazolines Primary aromatic amines Primary and secondary amines

473

Nitrous acid

Pink–orange (!violet)

Fenetylline, pentifylline

Coniferyl alcohol

Yellow (!pink)

Etamiphylline

Diazotisation

Yellow (!violet)

Diprophylline, proxyphylline, theobromine, theophylline

Dragendorff's reagent Simon's test

Propoxyphene

Cobalt thiocyanate

Indications

Froehde's reagent

A red, pink, orange or yellow residue, which changes to pink, red or violet after the addition of ammonium hydroxide, indicates the presence of a xanthine (Table 30.3).

Liebermann's test Tetrabromophenolphthalein ethyl ester Quaternary ammonium compounds

Tetrabromophenolphthalein ethyl ester

Ammoniacal silver nitrate

Quaternary amines

Dragendorff's reagent

Quinines

Cobalt thiocyanate

To 20 mL of 0.1 mol/L silver nitrate add sufficient strong ammonia solution to dissolve the initial precipitate.

Reagent

Thalleioquin

Method

Quinones

Methanolic potassium hydroxide

Reducing agents

Benedict's reagent Ferric chloride

Dissolve the sample in a minimum amount of water, with the addition of ethanol if necessary, add an equal volume of the reagent and note any colour that develops. Heat the mixture in a water-bath at 100
C for 30 s.

Trinder's reagent

Indications

Salicylates Steroids

Antimony pentachloride Naphthol sulfuric acid Sulfuric acid

Sulfonamides

Copper sulfate Koppanyi–Zwikker reagent Mercurous nitrate Nitrous acid

Sulfur containing

Palladium chloride Sodium nitroprusside

Tertiary amines

Red, yellow, brown or black colours (especially at room temperature) indicate potent reducing power, which occurs when adjacent carbon atoms in a ring each bear a hydroxyl group (Table 30.4). There is no response when the hydroxyl groups are meta to each other, but there is some restoration of reducing power when they are para to each other. Some colour production is also obtained with ethynyl bonds, but not with ethylenic bonds. Ethchlorvynol and ethinylestradiol both give a white precipitate that turns yellow on heating. Carbidopa gives a silver mirror on heating.

Dragendorff's reagent Tetrabromophenolphthalein ethyl ester

Table 30.4 Colours with ammoniacal silver nitrate Colour at room temperature

Compound

Colour at 100
C

Red

Isoetharine

Brown–orange

Hexoprenaline (!brown!black)

Additional information

Isoprenaline (!red–brown)

Brown

Colour reactions given by narcotics and amfetamines with four of the so-called ‘alkaloid colour reagents’ (Marquis, Mecke’s, Froedhe’s and Mandelin’s) are listed in Appendix 30.1 and Appendix 30.2. Three colour tests have been developed for gamma-hydroxybutyric acid (GHB) and its precursor gamma-butyrolactone (GBL; see Appendix 30.3).

Rimiterol

Brown

Yellow Grey–yellow

Ethinamate

Brown

Levodopa (!brown)

Black

Hydroquinone

Brown

Brown Red–brown

Colour test methods Orange–brown

Caution: the following lists of colour tests and drugs tested are not exhaustive; the omission of a compound from a list does not indicate that no response is given, but that it may not have been tested.

Grey

Amalic acid test (test for xanthines)

Black

Red–grey

Adrenaline Methyldopa

Black

Dopamine

Black

Methyldopate

Orange–brown

Protokylol

Brown

Ascorbic acid

Method

Benserazide

–

Add to the sample a few drops of 10 mol/L hydrochloric acid followed by a few crystals of potassium chlorate, and evaporate the mixture to dryness. Observe the colour of the residue then add 2 or 3 drops of 2 mol/L ammonium hydroxide and again observe the colour.

Dobutamine

–

Dodecyl gallate

–

Noradrenaline

–

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Colour Tests

Antimony pentachloride

Table 30.6 Colours with aromaticity test, method 2, obtained on addition of alkali to acid solution

Reagent

Colour of acid solution

Colour after addition of alkali

Compound

Colourless

Red

Clobazam (heat for 3 min with acid)

Orange

Butanilicaine, tolazoline

Method

Yellow

Place a drop of an ethanolic solution of the sample on a filter paper, add a drop of the reagent and dry in a current of warm air. Alternatively, the test may be carried out by adding a drop of the reagent to the sample on a white tile.

Amprolium, atropine methobromide, hyoscine butylbromide, hyoscine methobromide, ketoprofen, pipazethate, tetrahydrozoline, tetramisole, trimetaphan

Violet

Atropine methonitrate (transient), hyoscine methonitrate (transient)

Brown

Isopropamide

Red

Aminacrine, benzonatate, tetracaine (amethocaine), trimethoprim

Dry some antimony trichloride over phosphorus pentoxide, melt the dried material (m.p. 73
C), and pass dry chlorine gas into the melt until a yellow fuming liquid is obtained. Add this liquid to about 10 times its volume of chloroform, filter the solution into a dark glass-stoppered bottle and store in a desiccator.

Indications

Various colours are obtained with the cardiac glycosides, their aglycones and certain oestrogens and corticosteroids (Table 30.5). No colour is obtained with beclometasone, cortisone, fluocinolone, fludroxycortide, prednisolone, prednisone, progesterone, testosterone or triamcinolone.

Yellow

Orange–red

Amicarbalide, carbocromen, dyclonine, glibenclamide, levallorphan, metocurine, padimate, propanidid, quinuronium, salinazid

Brown

Dibromopropamidine, dichlorophen tubocurarine

Brown–violet

Dequalinium

Aromaticity Method 1

Place a portion of the sample in each of two ignition tubes, and to one tube add some solid sodium hydroxide. Heat both tubes carefully, allow the water vapour to escape, insert into the vapours in each tube an open capillary tube that contains Marquis reagent, and observe the colour of the reagent.

Indications

Indications

Red or orange colours indicate that the sample is aromatic in nature. The colours probably result from the liberation of traces of aromatic hydrocarbons, phenols, etc. Colours obtained after heating with sodium hydroxide generally indicate the presence of aromatic acids. Colours obtained after heating without sodium hydroxide generally indicate the presence of phenols, phenolic acids and aldehydes that contain more than one hydroxyl group. A negative result does not necessarily imply that the substance is nonaromatic. Method 2

Add 2 or 3 drops of concentrated nitric acid to the sample, heat in a water-bath at 100
C for 1 min, cool the mixture, dilute 3–4 times with water and make the solution alkaline by the addition of a 40% (w/v) solution of sodium hydroxide.

Table 30.5 Colours with antimony pentachloride Colour

Compound

Red

Dienestrol, diethylstilbestrol

Orange

Cholesterol (!brown), desoxycortone, dydrogesterone, fludrocortisone, hydrocortisone, hydroxyprogesterone, strophanthin-K (!red)

Yellow

Red

Dextromethorphan, haloperidol

Orange

Alfadolone

(!brown)

Androsterone, digitoxigenin, digoxigenin

(!brown!black–violet)

Digitoxin, digoxin, lanatoside C, ouabain (very weak)

Green–yellow

Fluocortolone

Green

Betamethasone (!brown), dexamethasone, mestranol, pancuronium

Brown

Carbenoxolone, dimethisterone, estradiol, estriol, estrone, ethinylestradiol (!black), fluoxymesterone, norethandrolone, norethisterone, oxymetholone, rotenone

Orange–brown

Enoxolone (!violet)

Green–brown

Noretynodrel

A change from colourless or yellow in acid solution to darker colours (e.g. orange or red–orange) after the addition of sodium hydroxide indicates the presence of a benzene ring in the molecule, probably though the production of a nitrophenol or other nitro compound. Certain compounds (e.g. diazepam, methaqualone) give a negative result. Orange colours are given by certain non-aromatic corticosteroids (e.g. cortisone), by substances that contain sulfur and by compounds that already contain an aromatic nitro group (e.g. nifursol). Colour changes are given in Table 30.6. Certain substances give distinct colours with cold nitric acid, but the colours fade on heating; these are listed in Table 30.7. Benedict's reagent Reagent

Dissolve 1.73 g of copper sulfate in 10 mL of water. Dissolve 17.3 g of trisodium citrate and 10 g of anhydrous sodium carbonate in 80 mL of water with the aid of heat; pour this solution into the copper sulfate solution and dilute the mixture to 100 mL. Method

Add 0.5 mL of the reagent to the sample and heat in a water-bath at 100
C for 3 min. Indications

The formation of red cuprous oxide occurs with strong reducing agents, such as ascorbic acid, dithionites, certain phenolic compounds that contain two hydroxyl groups para to each other, and compounds that contain at least four hydroxyl groups on a non-aromatic ring (e.g. glucose, tetracyclines). Table 30.7 Colours with aromaticity test, method 2, obtained on addition of cold nitric acid to sample, which fade on heating Colour

Compound

Red

Aminacrine (15 s), clozapine, dropropizine, medazepam, trimethoprim

Brown

Metocurine

Pink–brown

Diethylthiambutene (changing to green)

Black

Tubocurarine

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:43

Colour test methods A weak response (orange–brown or brown colours) is given by streptomycin, hydroxylamine and substituted hydrazines (e.g. phenelzine). No colour is obtained with beclometasone, cardiac glycosides and estriol (two hydroxyl groups) or clindamycin (three hydroxyl groups). Carbon disulfide Method

Mix the sample with 1 mL of water and 0.1 mL of a 1% (w/v) solution of sodium tetraborate, add 0.2 mL of a 10% v/v solution of carbon disulfide in ethanol and heat in a water-bath at 100
C for 3 min; cool the solution and add 3 drops of 0.1 mol/L silver nitrate. Indications

A brown colour indicates the presence of a dithiocarbamate, which suggests that the original substance was an aliphatic or heterocyclic primary or secondary amine. The original sample should be tested to ensure that it does not give a brown colour with silver nitrate alone. Chromotropic acid Reagent

1. Dissolve 20 mg of chromotropic acid in 10 mL of concentrated sulfuric acid. 2. Dissolve 1 g of sodium nitrite in 10 mL of concentrated sulfuric acid.

Cobalt thiocyanate (see Scott's test) Reagent

1. A 2% (w/v) solution of cobalt thiocyanate in water 2. Phosphoric acid 3. 1 g H2PtCl66H2O in 20 mL of H3PO4. Mix 9 parts of solution 1 and 3 parts of solution 2, add 1 part of solution 3 and mix well. Add 9 parts distilled water and mix. When the solution turns pink it is ready for use. Method

Add a few drops of the reagent to the sample to be tested. Indications

See Table 30.10. Limit of detection (LOD): cocaine-HCl 60 mg, methadone-HCl 15 mg. Table 30.10 Colours with cobalt thiocyanate Colour

Compound

Blue (flaky precipitate)

Cocaine

Brilliant greenish blue

Benzfetamine-HCl, brompheniramine maleate, chlordiazepoxide-HCl, chlorpromazine-HCl, doxepin-HCl, hydrocodone tartrate, methadone-HCl, methylphenidate-HCl

Strong greenish blue

Diacetylmorphine-HCl, ephedrine-HCl, phencyclidine-HCl, procaine-HCl, propoxyphene-HCl, pseudoephedrine-HCl

Strong blue

Quinine-HCl

Method 1

Add a small amount of sample, either solid or in solution, to 1 mL chromotropic acid reagent. Note any colour that may be produced, and then add the solution dropwise to 0.5 mL of water, with cooling. Substances that give a colour with cold sulfuric acid must be excluded. Indications

475

Coniferyl alcohol (primary aromatic amines) Reagent

See Table 30.8.

Warm 0.1 g of coniferyl alcohol until it melts (m.p. 74
C), dissolve in 3 mL of ethanol and dilute to 10 mL with ethanol.

Table 30.8 Colours with chromotropic acid

Method

Colour

Compound

Place 1 drop of a solution of the sample on a filter paper, add 1 drop of the reagent and expose the paper to hydrochloric acid fumes.

Red (before dilution)

Formaldehyde, paraformaldehyde (reacts slowly)

Violet (after dilution)

Hydrochlorothiazide, hydroflumethiazide

Method 2 (for chlorophenoxy herbicides)

Add 1 mL of 1 mol/L hydrochloric acid to 10 mL of sample and extract with 20 mL of toluene for 5 min. Centrifuge for 5 min and remove the toluene layer. Repeat with a further 20 mL of toluene and evaporate the combined extracts to dryness. Dissolve the residue in 0.2 mL of concentrated sulfuric acid and divide between two wells of a spotting tile. Add 0.1 mL of sodium nitrite solution to one well and 0.1 mL of chromotropic acid reagent to the other. Heat the tile at 80
C and observe any colour development.

Indications

An orange colour indicates the presence of an aromatic primary amine in which the amino group is attached directly to a benzene ring. An anomalous reaction is obtained with diphenylamine (bright orange). Copper sulfate Method 1

Dissolve the sample in a minimum volume of 0.1 mol/L sodium hydroxide and add a 1% (w/v) solution of copper sulfate, drop by drop, until the colour change is complete. Indications

Green, blue or brown colours indicate the presence of a sulfonamide (Table 30.11).

Indications

See Table 30.9.

Table 30.11 Colours with copper sulfate, method 1 Colour

Compound

Green

Phthalylsulfathiazole, succinylsulfathiazole (!violet), sulfachlorpyridazine, sulfadimethoxine, sulfadimidine (!brown), sulfadoxine, sulfaethidole, sulfamerazine (!brown), sulfamethizole, sulfamethoxazole, sulfametopyrazine, sulfapyridine (!brown–green), sulfaquinoxaline, sulfasomidine

Blue

Phthalylsulfacetamide, sulfacetamide, sulfaguanidine, sulfanilamide, sulfaphenazole, sulfaurea, sulfinpyrazone, sulthiame

Table 30.9 Colours with sodium nitrite and chromotropic acid Compound

Sodium nitrite

Chromotropic acid

2,4-Dichlorophenoxyacetic acid

Brown

Purple

2,4-Dichlorophenoxypropionic acid

Dark brown

Light purple

4-Chloro-2-methylphenoxyacetic acid

Light brown

Light purple

2-(4-Chloromethylphenoxy)propionic acid

Light brown

Purple

2,4,5-Trichlorophenoxyacetic acid

No reaction

Purple

2-(2,4,5-Trichlorophenoxy)propionic acid

No reaction

Light pink/ purple

Brown Orange–brown

Sulfafurazole Sulfasalazine

Green–brown

Sulfamethoxypyridazine, sulfamoxole

Violet–brown

Sulfadiazine, sulfamethoxydiazine, sulfathiazole

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Colour Tests

Method 2

Method

Add 1 or 2 drops of a 1% (w/v) solution of copper sulfate to the sample on a white tile.

Add 2 drops of solution 1 to the drug, followed by 1 drop of solution 2.

Indications

A light purple (blue–violet) colour indicates the presence of a barbiturate. Other reacting compounds are hydantoins, sulfonamides, pyrimidine, piperidine, methyprylon. The LOD is 25 mg or lower.

A blue colour indicates the presence of an alkali salt of a fatty acid, such as sodium cromoglicate (1–2 min) or valproate. The colours are not produced by a change of pH (some of the alkali salts will change the pH), as negative results are obtained with sodium bicarbonate. Cyanogen bromide

Indications

p-Dimethylaminobenzaldehyde (Wasicky reagent or Van Urk reagent; a general test for ergot alkaloids) Reagent

Reagent

1. Decolorise bromine solution by the addition of solid potassium cyanide and then add more bromine solution until the solution is pale yellow. 2. Prepare a saturated solution of aniline in water. Solutions 1 and 2 are stable for 1 week. Mix equal volumes of the two solutions immediately prior to the test. Method

Add 1 drop of the mixed reagent to the sample on a white tile. Indications

Red, orange or yellow colours indicate the presence of a mono-substituted pyridine ring. Increasing chain length of the substituent group weakens the response; a delayed response is obtained when the pyridine ring is substituted by nitrogen adjacent to the ring nitrogen; a weak response is obtained where there is a C¼O substituent adjacent to the ring nitrogen. There is no response to the test if the pyridine ring is bound to another ring, if it is substituted in more than one position or if the nitrogen in the ring is substituted. Anomalous results are obtained with azatadine (pink), bisacodyl (no response) and tropicamide (violet– pink) (Table 30.12).

Dissolve 2.0 g of p-dimethylaminobenzaldehyde (p-DMAB) in 50 mL of 95% ethanol and 50 mL of concentrated hydrochloric acid. The reagent should be freshly prepared. Method

Add the reagent to the sample in a test-tube, warming if necessary. Observe any colour produced, then carefully dilute with water or spray dried spots on filter paper and heat. Indications

Colours are given by a number of substances, which include ergot alkaloids, dimethyltryptamine, psilocin, psilocybine (gives a violet colour), cannabinols and certain indoles in which the indole ring is not bonded to another conjugated ring (red changing to violet on dilution), and certain phenols and phenolic amines (red or orange, usually changing to violet on dilution). Some other types of compound also respond. See Table 30.13. The LOD for lysergide (LSD) is 6 mg. Diphenylamine test Reagent

Diazotisation

Mix 0.5 g of diphenylamine in 20 mL of water and dilute to 100 mL with concentrated sulfuric acid.

Method

Method

Dissolve the sample in 2 mol/L hydrochloric acid, and to 1 drop on a white tile add 1 drop of a 1% solution of sodium nitrite, and 1 drop of a 4% solution of naphth-2-ol in 2 mol/L sodium hydroxide.

Indications

Indications

A bright red or orange–red colour indicates the presence of a primary aromatic amine. Diphenylamine does not give a reaction; aminonitrothiazole (solid) gives a violet colour. Dille–Koppanyi reagent modified (a general test for barbiturate-like compounds) Reagent

1. Dissolve 0.1 g of cobalt(II) acetate dihydrate in 100 mL of methanol. Add 0.2 mL of glacial acetic acid and mix. 2. Add 5 mL of isopropylamine to 95 mL of methanol.

Apply the reagent to the sample on a white tile or in a test-tube. A blue colour indicates the presence of an oxidising agent such as bromate, chlorate, chromate, dichromate, iodate, lead(IV), manganese (III, IV, VII), nitrate, nitrite, permanganate or vanadate. This test has been modified for use on blood samples to detect ethchlorvynol (Caughlin 1991). Blood (0.5 mL) is mixed with 1.0 mL of acetone and vortex mixed. The sample is centrifuged and 50 mL of the supernatant is added to 50 mL of diphenylamine reagent and 25 mL of chloroform. The mixture is vortex mixed and allowed to stand. A pink colour that develops in the chloroform layer indicates ethchlorvynol. Dragendorff reagent (a general reagent for nitrogenous bases) Reagent

Table 30.12 Colours with cyanogen bromide

(a)

Dissolve 1 g of bismuth subnitrate in 3 mL of 10 mol/L hydrochloric acid with the aid of heat. Dilute to 20 mL with water and dissolve 1 g of

Colour

Compound

Red

Zimeldine (30 s)

Table 30.13 Colours with p-dimethylaminobenzaldehyde

Pink

Azatadine

Colour

Compound

Orange–pink

Carbinoxamine, dimetindene, doxylamine, iproniazid, phenyramidol, triprolidine (1–2 min)

Red (changing to violet on dilution)

Cannabinols, phenazone (100
C, 5 min), pindolol, psilocin, psilocybine, tryptamine

Violet–pink

Tropicamide

Red (no violet on dilution)

Benserazide, cocaine (100
C, 3 min), feprazone, harmine, phencyclidine (100
C, 3 min)

Orange (changing to violet on dilution)

Dobutamine, dopamine, diamorphine, morphine, orciprenaline, phenol, terbutaline, tyramine

Violet

Ergot alkaloids (dihydroergotamine, ergometrine, ergotamine, ergotoxine, lysergide, methysergide), dimethyltryptamine, psilocin, psilocybine

Yellow

Primary aromatic amines, e.g. aminosalicylic acid, anileridine, aniline, procaine, benzocaine

Orange

Red–orange Yellow (a)

Azaperone, brompheniramine, chlorphenamine, isoniazid, metyrapone, nicametate, nicotinamide, nicotine, nicotinic acid, nifenazone, nikethamide, pheniramine, xanthinol nicotinate Benzyl nicotinate Halopyramine, mepyramine, tripelenamine

Anomalous results are obtained with azatadine (pink), bisacodyl (no response) and tropicamide (violet–pink).

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:44

Colour test methods potassium iodide in the mixture. If black bismuth triiodide separates, add 2 mol/L hydrochloric acid and more potassium iodide to dissolve it.

Table 30.15 Colours with ferric chloride Colour

Compound

Method

Red

Acetates, phenazone, propionates

Dissolve the sample in 3 drops of 2 mol/L hydrochloric acid, add 2–3 mL of the reagent and dilute to 10 mL with water.

Brown–red Orange

Indications

An orange, red–orange or brown–orange precipitate suggests the presence of an alkaloidal base (precipitated as the alkaloidal bismuth iodide). Primary, secondary, tertiary and quaternary amines give positive results. This reagent is commonly used as a spray or locating agent to detect alkaloids on TLC plates.

Green–yellow Green

Blue–green

1. Add 2.5 mL of acetaldehyde and 2.0 g of vanillin to 100 mL of 95% ethanol 2. Concentrated hydrochloric acid 3. Chloroform. Method

Place the solid sample, or an evaporated petroleum ether (or other organic solvent) extract of the sample, in a test-tube and add 3 drops of solution 1. Shake for 1 min and add 3 drops of solution 2. Agitate gently and observe the colour produced. Add 9 drops of solution 3, vortex mix gently and note whether the colour is extracted from the mixture. Indications

Ferric chloride (general reagent for phenols, e.g. salicylates) Reagent

Dissolve 5 g of anhydrous ferric chloride, or 8.25 g of ferric chloride hexahydrate, in 100 mL of distilled water.

Chlorpromazine, hexoprenaline, propyphenazone, valproate Paracetamol Adrenaline, betanaphthol, dobutamine, dopamine, etamivan, ethylnoradrenaline, hexylresorcinol, hydroquinone, hydroxyquinoline, isoetarine, isoprenaline, levodopa, methyldopa, methyldopate, noradrenaline, paraphenylenediamine, phenothiazine, protokylol, rimiterol, thioridazine Chlorquinaldol

Blue

Apomorphine, dodecyl gallate, gallic acid, morphine, parachlorophenol, pethidine, phenol, tannic acid

Violet

Aminosalicylic acid, diflunisal, dipyrone, hexachlorophene (transient), labetalol, salicylaldehyde, salicylamide, salicylic acid, salicyluric acid

Blue–violet Brown

Aminophenazone, salicylamide (after hydrolysis), salicylic acid Aloin, carbidopa

Yellow–brown

Salinazid

Green–brown

Benserazide

Black Violet–black

A colour change from grey to green through blue to violet–blue suggests the presence of cannabis, but differentiation from roasted coffee and patchouli oil is required. The colour change is best seen with fresh drug material. The violet colour is extracted into the chloroform layer only when cannabis is present (Table 30.14). The LOD is 350 mg of tetrahydrocannabinol (THC). No colour is obtained with other natural products, such as basil, bay leaf, eucalyptus oil, mace, marjoram, rosemary, sage, thyme or tobacco.

Nifenazone

Yellow

Duquenois reagent, modified Reagent

477

Ethyl gallate (!blue–black)

Ferrous sulfate A (test for nitrates and nitrites) Reagent

To 1 volume of a 10% (w/v) solution of ferrous sulfate (FeSO4,7H2O) add 5 volumes of concentrated sulfuric acid with cooling. Method

Add the sample to 0.5 mL of the reagent. Indications

A red or pink colour is given only by nitrates and nitrites (e.g. glyceryl trinitrate). Ferrous sulfate B (test for cyanide)

Method

Add ferric chloride solution to the sample or an ethanolic solution of the sample. Indications

Red, orange, green, blue, violet or brown colours indicate the presence of a phenolic compound, fatty acid or a phenylpyrazoline. High quantities of phenothiazines can also cause this test to be positive. Salicylates give a violet colour. Many phenols give no colour with ferric chloride when water is used as a solvent, but give positive tests when anhydrous solvents such as chloroform are used. Aspirin (acetylsalicylic acid) does not give a positive result unless first hydrolysed with concentrated sodium hydroxide to give salicylate. Colours are listed in Table 30.15.

Reagent

Dissolve 10 g of ferrous sulfate in 100 mL of freshly boiled and cooled water (prepare fresh). Method

Dilute 1 mL of sample with 2 mL of 10% (w/v) sodium hydroxide solution and add 2 mL of ferrous sulfate solution. Add sufficient 10% (v/v) hydrochloric acid to dissolve the ferrous hydroxide precipitate. Indications

A blue colour is given by cyanide. There are no common sources of interference. Folin–Ciocaltaeu reagent (test for phenolic compounds)

Table 30.14 Colours with modified Duquenois reagent

Reagent

Compound

Initial colour

Colour extracted by chloroform

Cannabis

Violet–blue

Violet

For the stock solution, dissolve 100 g of sodium tungstate and 25 g of sodium molybdate in 800 mL of water in a 1500 mL flask, add 50 mL of phosphoric acid and 100 mL of concentrated hydrochloric acid, and reflux for 10 h. Cool, add 150 g of lithium sulfate, 50 mL of water and 4–6 drops of bromine, and allow to stand for 2 h. Boil for 15 min to remove the excess bromine, cool, filter and dilute to 1000 mL with water. This stock solution should be stored at a temperature not exceeding 4
C and used within 4 months of its preparation; it has a yellow colour and must not be used if any trace of green colour is present.

Coffee (roasted)

Violet–brown

Nil

Nutmeg

Pale reddish purple

Nil

Patchouli oil

Violet

Nil

Tea (leaves)

Green–blue

Nil

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Colour Tests

For use, dilute 1 volume of this stock solution with 2 volumes of water.

Table 30.17 Colours with Forrest reagent Colour

Compound

Method

Red

Acepromazine, carfenazine, chlorpromazine, diethazine, dimetotiazine, mequitazine, mescaline, mesoridazine, piperacetazine, prochlorperazine, promethazine, propiomazine, thiazinamium, thiopropazate, thioproperazine, thioridazine

Add the diluted reagent to the sample and make the mixture alkaline with 2 mol/L sodium hydroxide. Indications

A blue colour indicates the presence of a phenolic compound. The reaction is progressively inhibited with increased halogenation of the phenol nucleus. Formaldehyde–sulfuric acid Reagent

To 4 volumes of concentrated sulfuric acid add 6 volumes of formaldehyde solution (using a pipette with the tip below the surface of the acid) with stirring and adequate cooling. When the reagent is warm it remains clear for about 1 h. If turbidity develops, this may be dispelled by heating in a water-bath at 100
C for about 1 min (note that this reagent is not the same as that used in the Marquis test). Method

Mix the sample with the reagent and heat at 100
C for 1 min. Indications

Benzodiazepines generally give an orange colour with the exception of bromazepam and clozapine (a benzodiazepine-like compound), which both give yellow, and flurazepam (pink). Other indications include phenothiazines, tetracyclines and thioxanthenes. Tryptamine (brown) and zomepirac (red) also react. Those marked with an asterisk in Table 30.16 fluoresce orange under ultraviolet (UV) light (l ¼ 350 nm). No response is obtained with chlordiazepoxide, dimethoxanate or proquamezine. Some of the newer benzodiazepines have not been tested.

Violet–red

Perphenazine

Brown–red

Alimemazine

Pink

Profenamine

Orange

Fluphenazine, phenothiazine, trifluoperazine, triflupromazine

Pink–orange

Acetophenazine

Red–orange

Methdilazine

Brown–orange

Perazine, pericyazine

Green Blue–green

Clomipramine, desipramine, imipramine, ketamine, opipramol, thiethylperazine, thioridazine, trimipramine

Violet

Levomepromazine, proquamezine (!red!orange)

Brown

Metopimazine

Red–brown

Promazine

Forrest reagent Reagent

Mix together equal volumes of a 0.2% (w/v) solution of potassium dichromate, a 30% (v/v) solution of sulfuric acid, a 20% (w/w) solution of perchloric acid and a 50% (v/v) solution of nitric acid. Method

Table 30.16 Colours with formaldehyde–sulfuric acid Colour

Compound(a)

Red

Chlorprothixene*, clopenthixol*, flupentixol*, fluphenazine, metopimazine, pericyazine, promazine, thiothixene, triflupromazine, zomepirac

Brown–red

Lymecycline, oxytetracycline, tolmetin

Pink

Flurazepam, thioproperazine, trifluoperazine

Orange

Clonazepam, clorazepic acid, demeclocycline (!brown–red), demoxepam, diazepam, flunitrazepam, ketazolam, lorazepam, lormetazepam, medazepam (add water), metixene, nitrazepam, nordazepam, oxazepam, prazepam, temazepam, tetrazepam

Red–orange Yellow Green–yellow

Methacycline Bromazepam, clozapine, dimethothiazine, doxycycline Rolitetracycline (!yellow–brown), tetracycline (!yellow–brown)

Green

Thiethylperazine

Blue

Carphenazine, levomepromazine, thioridazine

Violet

Mesoridazine, perphenazine

Red–violet

Indications

Red, pink, orange, blue or violet colours are obtained with phenothiazines. A blue colour is obtained with certain dibenzazepines. The blue colour is inhibited by the presence of phenothiazines, so an excess of reagent must be added to overcome this. Colours are listed in Table 30.17. FPN reagent (general reagent for phenothiazines) Reagent

Mix together 5 mL of 5 % (w/v) ferric chloride solution, 45 mL of a 20% (w/w) solution of perchloric acid and 50 mL of a 50% (v/v) solution of nitric acid. Method

Dissolve the sample in a minimum volume of 2 mol/L hydrochloric acid (or use 1 mL of urine) and add an equal volume of the reagent. Indications

A variety of colours, from pink, red, orange, violet to blue, indicate the presence of phenothiazines (Table 30.18). Froehde reagent Reagent

Blue–violet

Acepromazine, acetophenazine, piperacetazine, promethazine, propiomazine

Brown–violet

Thiazinamium

Dissolve 1.0 g of molybdic acid or sodium molybdate in 100 mL of hot concentrated sulfuric acid.

Tryptamine

Method

Chlortetracycline, clomocycline

Add a drop of the reagent to the sample on a white tile.

Brown Orange–brown (a)

Alimemazine, chlorpromazine, diethazine, mequitazine, methdilazine, perazine, phenothiazine, prochlorperazine, profenamine, thiopropazate

Dissolve the sample in a minimum volume of 2 mol/L hydrochloric acid and add an equal volume of the reagent. To test urine, add 1 mL of reagent to 0.5 mL of urine.

Compounds giving colours that fluoresce under UV light (l = 350 nm) are indicated by an asterisk.

Indications

Colours are listed in Table 30.19.

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:45

Colour test methods Table 30.18 Colours with FPN reagent

Furfuraldehyde (general reagent for carbamates)

Colour

Compound

Reagent

Red

Chlorpromazine, dimetotiazine, mesoridazine, methdilazine, prochlorperazine, thiazinamium, thiopropazate

A 10% (v/v) solution of furfuraldehyde in ethanol.

Orange–red

Mequitazine

Violet–red

Perphenazine, proquamezine (!red!orange)

Brown–red

Alimemazine

Orange

Acetophenazine, diethazine (!yellow), fluphenazine, metopimazine, morphine, pericyazine, phenothiazine, profenamine, promethazine, thioproperazine, trifluoperazine, triflupromazine

479

Method

Dissolve the sample in ethanol, place a drop of the solution on a filter paper, add 1 drop of the reagent and expose the paper to hydrochloric acid fumes for 2–3 min. Indications

A black spot indicates the presence of non-aromatic carbamates. N-Substituted carbamates do not react. The LOD is 1 mg.

Red–orange

Carfenazine

Iodine test

Pink–orange

Propiomazine (!red!fades)

Method

Brown–orange

Acepromazine, perazine, piperacetazine

Blue

Clomipramine, imipramine, thiethylperazine, thioridazine, trimipramine

Mix the sample with an equal volume of manganese dioxide and heat the mixture carefully to dull redness over a small flame. Repeat the test by heating the sample alone.

Violet

Levomepromazine

Indications

Promazine

The appearance of violet vapour indicates the presence of iodine in the molecule. Better results are sometimes obtained when the manganese dioxide is omitted (e.g. with amiodarone).

Brown Red–brown

Table 30.19 Colours with Froehde reagent Colour Yellow Blue–yellow

Compound

Iodoplatinate test (general test for alkaloids and nitrogenous heterocyclic compounds)

Hydrocodone, pethidine

Reagent

Oxycodone-HCl

Add 2 mL of a 5% (w/v) solution of platinic chloride and 5 g of potassium iodide to 98 mL of water and shake until dissolved. This reagent is often used as a locating agent in TLC.

Orange

Diphenhydramine, flurazepam, promazine, trifluoperazine, triflupromazine

Green

Chlorphentermine, codeine, mescaline, oxycodone, phenyltoloxamine

Yellow–green

Lysergide

Method

Dissolve the sample in 2 drops of 2 mol/L hydrochloric acid, add 2–3 mL of the reagent and dilute to 10 mL with water.

Blue

Pentazocine

Indications

Red

Amfetamine, chlorpromazine-HCl

A violet, blue–violet, brown–violet or grey–violet precipitate suggests the presence of an alkaloidal base (precipitated as the alkaloid–iodoplatinate complex). The clearest colours are obtained with tertiary and quaternary amines; primary amines give indistinct colours and amines of small relative molecular mass generally do not react.

Grey–red

Propoxyphene-HCl

Purple–red

Alimemazine, diacetylmorphine, promethazine, propylhexadrine, salicylic acid, tetracycline, thioridazine

Brown Red–brown

Ephedrine, mescaline Doxepin-HCl

Koppanyi–Zwikker test

Black Brown–black

Opium

Reagent

Green–black

Methylenedioxyamfetamine (MDA)-HCl

A 1% (w/v) solution of cobalt nitrate in ethanol. Method

Fujiwara test (general reagent for halogenated hydrocarbons)

Dissolve the sample in 1 mL of ethanol, add 1 drop of the reagent followed by 10 mL of pyrrolidine and agitate the mixture.

Reagent

Indications

Freshly prepared 20% (w/v) sodium hydroxide solution.

A violet colour is given by substances that contain the following structures:

Method

Mix together 2 mL of the reagent and 1 mL of pyridine. Add the sample (1 mL of urine) and heat in a water-bath at 100
C for 2 min with shaking. Indications

A red–pink colour in the pyridine layer indicates the presence of compounds that possess at least two halogen atoms bound to one carbon atom. These include chloramphenicol, chlorbutanol, chloroform, dichlorophenazone, trichloroethane, trichloroethanol, trichloroacetic acid and trichloroethylene. Chloral hydrate and dichlorophenazone do themselves react but are excreted in urine as trichloroacetic acid. No colour is given by dicophane (DDT) or carbon tetrachloride, although massive exposure to the latter solvent may lead to a positive urine test because of the presence of chloroform as a contaminant. 2,2,2-Trichloroethanol gives a yellow colour. The LOD is 1 mg/L.

n n

Imides, in which C¼O and NH are adjacent in a ring (e.g. barbiturates, glutethimide, oxyphenisatine and saccharin). Sulfonamides and other compounds with free –SO2NH2 on a ring (e.g. clopamide, furosemide, sulfanilamide and thiazides), or with –SO2NH2 in a side-chain (e.g. chlorpropamide), or with –SO2NH2 that links a benzene ring with another ring other than a pyrazine, pyridazine, pyridine or pyrimidine ring (e.g. sulfafurazole and sulfamethoxazole). These latter structures give pink or red–violet colours (e.g. sulfadiazine and sulfadimethoxine).

No response is obtained with compounds with other substituents on the nitrogen atom. Anomalous responses are obtained with paramethadione and theophylline (violet), and with cycloserine, idoxuridine, mephenytoin, niridazole and riboflavin (no response). Note that hydrochlorides give a blue colour before the addition of pyrrolidine.

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:45

480

Colour Tests

Liebermann's reagent Reagent

Add 1 g of sodium or potassium nitrite to 10 mL of concentrated sulfuric acid with cooling and swirling to absorb the brown fumes. Method

Add 2 or 3 drops of the reagent to the sample on a white tile. Occasionally it is necessary to carry out the test in a tube and heat in a water-bath at 100
C. Many substances give colours with sulfuric acid alone and the test should be repeated using sulfuric acid instead of the reagent. Indications

the response to the reagent at the violet end of the spectrum are, in decreasing order of efficacy: ring sulfur (with or without aromatic ring); ring oxygen (with aromatic ring); extra-ring oxygen or sulfur (with aromatic ring); aromatic compounds that consist entirely of C, H, N. Thus, there is a tendency for the response to the Marquis reagent to move gradually towards longer wavelength (i.e. through green to orange and red) as the ratio of C, H, N to the other groups in the molecule rises (Table 30.22). The LOD values are: 1 mg for codeine sulfate, mescaline sulfate, methadone-HCl; 5 mg for lysergide tartrate, metamfetamine-HCl and morphine; 10 mg for amphetamine-HCl and diamorphine-HCl.

This test was originally developed to give intense colours with phenols:

McNally's test

n

Reagents

n

n

Orange colours are given by substances that contain a monosubstituted benzene ring not joined to C¼O, N–C(¼O)– or to a ring that contains a C¼N–O– group. Orange or brown colours are given by some substances that contain two monosubstituted benzene rings (or some disubstituted compounds in which fluorine is the second substituent) that are joined either to one carbon atom or to adjacent carbon atoms. A wide range of colours is given by compounds that contain –OH, O–alkyl or –O–CH2O– groups attached to a benzene ring or to a ring in a polycyclic structure that contains a benzene ring. The benzene ring must not bear –NO2, or be halogenated, or contain an –O– substituent ortho to the oxy groups. Compounds that contain ring sulfur give a similar range of colours.

Colours are listed in Table 30.20. Note that a yellow colour is given by a variety of other compounds.

1. A 0.5% solution of copper sulfate in 10% acetic acid. 2. A freshly prepared 2% (w/v) solution of sodium nitrite. Method

Dissolve the sample (1 mg) in a few drops of acetone, and add 1–2 mL of water. Add 3 drops of solution 1 and an equal volume of solution 2. Shake and heat in a water-bath at 100
C for 3 min. Indications

A red colour indicates the presence of free salicylic acid. Aminosalicylic acid gives a brown precipitate, and diflunisal gives a violet colour. Certain acids produced during the putrefaction of tissues also give red colours in this test: p-hydroxyphenylacetic acid, p-hydroxyphenylpropionic acid and p-hydroxyphenyl-lactic acid. Mecke's reagent (useful test for opium alkaloids)

Mandelin's test (useful test for amfetamines and antidepressants)

Reagent

Reagent

Method

Dissolve 1.0 g of ammonium vanadate in 1.5 mL of water and dilute to 100 mL with concentrated sulfuric acid.

Add a drop of the reagent to the sample on a white tile.

Method

An immediate blue or green colour is indicative of opiates (see Table 30.23).

Add a drop of the reagent to the sample on a white tile.

Dissolve 1.0 g of selenious acid in 100 mL of concentrated sulfuric acid.

Indications

Indications

When interpreting the result of this test, account should be taken of the colour given by sulfuric acid and by Liebermann’s test. Hydrochlorides give a red colour with this reagent. When the colours differ from those given with sulfuric acid or Liebermann’s test, this indicates an aromatic ring together with a saturated 5-, 6- or 7-membered ring that contains only one nitrogen atom. The heterocyclic ring must not contain a second nitrogen atom or an oxygen atom. It must not be substituted or bound by –CONH– to the aromatic ring. The aromatic ring must not have –CF3 as a substituent. Colours are also produced if sulfur is in a ring, provided that the ring does not contain more than one nitrogen atom (Table 30.21). LOD values are: codeine sulfate 5.0 mg, amphetamine-HCl 10.0 mg, diamorphine-HCl 20 mg, metamfetamine 150 mg, morphine 5 mg and strychnine 0.05 mg.

Melzer's reagent (general reagent for hallucinogenic mushrooms)

Marquis test

Mercurous nitrate (general reagent for barbiturate-like compounds)

The Marquis test is a useful broad-spectrum test used mostly for opium alkaloids and amfetamines. Reagent

Carefully mix 100 mL of concentrated sulfuric acid with 1 mL of 40% (v/v) formaldehyde solution (stable for several weeks if protected from light). Method

Add a drop of the reagent to the sample on a white tile. Indications

Various colours that represent the whole of the visible spectrum are given by a large number of compounds. Structures that tend to maintain

Reagent

Dissolve 1.5 g of iodine in 100 mL of an aqueous solution that contains 5 g of potassium iodide and 100 g of chloral hydrate. Method

Place a few drops of the reagent on the mushroom spores or mushroom tissue to be tested. Indications

A blue, bluish-grey or black–grey colour indicates amyloid mushrooms. A slight yellow or no change indicates that the mushrooms are nonamyloid. Psilocybes are always non-amyloid.

Reagent

To a saturated solution of mercurous nitrate, add solid sodium bicarbonate until effervescence ceases and the precipitate formed becomes yellow. The precipitate then changes to a biscuit colour. This reagent should be freshly prepared and should be shaken immediately before use, and should not be kept for more than 1 h. Method

Dissolve the sample in the minimum amount of ethanol, add 1 drop of the opaque reagent, shake and examine at intervals during 2 min. A blank solution that contains only ethanol and reagent should be treated similarly at the same time.

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:45

Colour test methods

481

Table 30.20 Colours with Liebermann's reagent Colour

Compound

Red

Acepromazine, ajmaline, alprenolol, aminacrine (100
C), antazoline, brucine, chlorprothixene, clopenthixol, flupentixol, mestranol, oxytetracycline, prajmalium, thiazinamium, tiotixene, tolmetin (100
C), trifluoperazine, xylazine

Violet–red

Indapamide

Brown–red

Methylchlorophenoxyacetic acid

Pink Brown–pink Orange

Trichlorophenoxyacetic acid (!brown) Prazosin (100
C !red–orange) Aletamine, alverine, ampicillin, atropine methobromide, atropine methonitrate, baclofen, benactyzine (!brown), bethanidine (!brown), broxyquinoline, butanilicaine, chloroquine (100
C), clidinium (!brown), cyclandelate, cyclizine, dazomet, decoquinate (slow), diethylthiambutene (100
C), dimefline, diuron, doxapram, dyclonine (100
C), fenclofenac (100
C, !brown), fenitrothion, fenpipramide, glibenclamide (100
C, 15 s), hyoscine butylbromide, hyoscine methonitrate, linuron, loxapine (50–60
C), metindizate (!brown), methylphenidate, metolazone (!green–brown), monolinuron, nomifensine, phenazone (100
C), phenelzine, propham, salinazid, sulfinpyrazone, tolazoline, trimetaphan, tripelennamine (!brown), triprolidine, xipamide, zomepirac (100
C)

Red–orange

Acetanilide, amfetamines, aniline, atropine, bamipine, beclamide, benethamine, caramiphen, carbetapentane, chlorcyclizine, cinchophen, cycrimine, diphenylpyraline, doxylamine, dropropizine, ephedrines, famprofazone, fencamfamin, glutethimide, hyoscine, hyoscyamine, isoaminile, isocarboxazid, levamisole, meclozine, mephentermine, methoin, methyl benzoquate, methylphenobarbital, metixene, metomidate, morazone, nialamide, pentapiperide, pethidine, phenacemide, phenbutrazate, phendimetrazine, phenglutarimide, pheniramine, phenmetrazine, phenobarbital, phensuximide, phenylmethylbarbituric acid, phenytoin, prolintane, tofenacin, tranylcypromine, triamterene, triphenyltetrazolium, warfarin

Brown–orange

Ambutonium, bumetanide, diphenhydramine, fenuron, feprazone (100
C, !brown), ibuprofen, labetalol, mepivacaine, methadone, nefopam (!brown), tetrahydrozoline

Yellow Brown–yellow Green Blue–green

Amicarbalide (100
C), clonidine (100
C, !orange), dequalinium (100
C, !orange), diethylpropion, diloxanide, ethoxzolamide, fenfluramine (100
C), flavoxate, gliclazide, metoclopramide, nifenazone (100
C), piroxicam, propachlor, tropicamide Amiodarone Bialamicol, chlorotrianisene, colchicine, dextromoramide (100
C), diamthazole, hydrastine, mequitazine, naphthols, phenol, phenothiazine, thiocarlide Hydrochlorothiazide, hydroflumethiazide, pindolol

Brown–green

Cyclopenthiazide

Grey–green

Azapropazone

Black–green Blue

Green–blue Violet Red–violet Black–violet Brown

Red–brown

Naproxen Amidopyrine (100
C), bendroflumethiazide, benzonatate (100
C), chromonar (100
C, 3 min), clomipramine, diphenylamine, dipyrone (100
C), imipramine, mefenamic acid, mefruside, oxypertine, padimate (100
C), procarbazine (100
C, 15 s), propyphenazone (100
C; red with water), tetracaine (100
C), yohimbine Amiphenazole (100
C) Methocarbamol, mianserin, paracetamol, penthienate methobromide, phenacetin, propiomazine, resorcinol, timolol (100
C), trazodone (100
C; transient) Chloroxuron Methoxychlor Acepromazine, acetophenazine, adiphenine, azacyclonol, barban, benzilonium, benzyl nicotinate, biperiden, clemastine, clofenotane, clomifene, cyclothiazide, dextropropoxyphene, dichlorprop, diperodon, diphemanil, difenidol, emepronium, etenzamide, fenpiprane, flurbiprofen, haloperidol, mepenzolate, methylpiperidyl benzilate, mexiletine, nadolol, penfluridol, phenaglycodol, phenylbutazone (100
C), phosalone, pimozide, pipazethate (100
C, !red), pipoxolan, pyrrobutamine, rotenone, sotalol (100
C), sulindac, veratrine, zimeldine Benzthiazide, bisacodyl, carfenazine, chlorpromazine, diclofenac, dosulepin, profenamine, etisazole, fenbufen, fenoprofen, methapyrilene, perphenazine, polythiazide

Pink–brown

Metoprolol

Orange–brown

Benazolin, diphenadione, maprotiline, methiocarb, piperidolate

Green–brown

Methdilazine, norbormide, promazine, thiopropazate

Violet–brown

Bamethan, clofibrate, dichlorophen

Black–brown

Mecoprop

Grey

Isopropamide

Black

Acetomenaphthone, aloin, aminophenols, amodiaquine, apomorphine, atenolol, benorilate, benzquinamide, buprenorphine, butorphanol, carbaryl (!green), carbidopa, cepha€ eline, chloroxylenol, chlorphenesin, clomocycline, clorgyline, codeine, cotarnine, cresol, cyclazocine, dextromethorphan, diamorphine, dibromopropamidine, diprenorphine, doxepin, emetine, ethamivan, ethinylestradiol, estradiol, estriol, estrone, etilefrine, furosemide, glycopyrronium, guaiphenesin, hexobendine, hydroxyephedrine, hydroxystilbamidine, ibogaine, indometacin, levallorphan, mebeverine, mescaline, methylchlorophenoxyacetic acid, methylenedioxyamfetamine, morantel, morphine, naloxone, 1-naphthylacetic acid, narceine, nicergoline, normetanephrine, noscapine, noxiptiline, octafonium, oxprenolol, oxyphenisatine, papaverine, pholcodine, pizotifen, practolol, profadol, propanidid, protokylol, pyrantel, rimiterol, ritodrine, rotenone, salbutamol, terbutaline, tetrabenazine, tetracycline, thymol, trimethobenzamide, trimetozine, tubocurarine, verapamil, viloxazine

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:46

482

Colour Tests

Table 30.21 Colours with Mandelin's test Colour

Compound

Red

Ajmaline, amfetamine, azacyclonol, chlorprothixene, diperodon (!green), dofamium (!brown), flupentixol, gelsemine (!green), indapamide, mequitazine, methotrexate, nialamide, pericyazine, prajmalium, prolintane, sodium cromoglicate, tiotixene, xylometazoline

Brown–red Orange

Diacetylmorphine-HCl, doxepin-HCl, nadolol, propoxyphene-HCl Brompheniramine, dropropizine (slow), ethylnoradrenaline, hydrastinine (!green), lachesine (!green), levamisole (!grey–green), methanthelinium, metixene, methyldopa, methyldopate, methylpiperidyl benzilate (!brown!green), noradrenaline, orphenadrine, pipenzolate (!green), poldine metilsulfate (!green!violet), procaine-HCl, propantheline, proquamezine (!violet), solanidine (!violet!blue), solanine (!violet!blue), strychnine (blue!purple!violet!red!red–orange), sulindac, thenalidine (!brown)

Red–orange

Cotarnine (!brown), doxepin

Green–orange

5-Methyltryptamine

Brown–orange Yellow

Mexiletine Azaperone, benzatropine, broxaldine, chelidonine (!green), conessine, deptropine, desipramine (!blue), dihydralazine, diphenhydramine, difenidol, diphenylpyraline, dropropizine (!orange), halquinol, homidium, lidoflazine, methacycline (!orange– violet), paraphenylenediamine, penicillamine, protokylol (!brown), tofenacin, tylosin (!yellow–brown), veratrine (!orange!violet– brown), viprynium

Orange–yellow

Cocaine-HCl, hexoprenaline, methaqualone, methylphenidate-HCl

Green–yellow

Methoxamine, oxycodone-HCl

Yellow–brown

Mescaline-HCl

Green

Acepromazine (!red), adiphenine (!blue), amfetamine, benorilate, bephenium hydroxynaphthoate, bibenzonium, buclosamide (blue rim), bunamidine, chlorpromazine (!violet), clefamide (!brown), codeine (green!blue), colchicine, cyclazocine, cyclomethycaine (!brown), debrisoquine, diaveridine, dibenzepin, diethazine (!violet with excess reagent), diethylthiambutene (!green–blue), dimethindene, dimethoxanate (!brown), dimoxyline, dipipanone (!blue), dosulepin, doxorubicin, doxycycline (!yellow), fenpiprane, guanoxan, harman, hydroxyephedrine, isoxsuprine, metanephrine, methadone (!blue), methdilazine (!violet), methocarbamol, methoxyamfetamine, methylenedioxyamfetamine (!blue), a-methyltryptamine (!orange), metopimazine, monocrotaline, niclosamide, nitroxoline, norharman (!yellow), normetanephrine, obidoxime (!blue), oleandomycin, oxymetazoline, paracetamol, pecazine (!violet), pentazocine, perazine (!violet), phenazone, phenazopyridine, phenformin, phenindamine, phenoxybenzamine (!violet), phenyltoloxamine, pindolol, piperacetazine (!red!violet), pipoxolan (!brown), prenylamine, profenamine (!violet), proflavine, promazine (!violet), promethazine (!violet), propranolol, reserpine, ritodrine, salicylic acid, thenium, thenyldiamine, thiocarlide (!yellow), tranylcypromine (!violet), trifluomeprazine (!red–violet), trihexyphenidyl

Yellow–green

Benzfetamine-HCl, metamfetamine-HCl, normethadone, opipramol

Blue–green

Amfetamine-HCl, benzoctamine, berberine (!brown), edrophonium, hydroxystilbamidine, ketobemidone, methoxyphenamine, phentolamine, profadol (!green), viloxazine

Brown–green

Benzydamine, chlorphenesin

Grey–green

Alverine, azapropazone, diamphenethide, diethyltryptamine (!yellow), dihydrocodeine, guaifenesin, hordenine, levomethadyl acetate, normorphine, oxyphencyclimine, papaverine, terbutaline, trihexyphenidyl

Blue

Green–blue Violet

Bamethan (!green), clomipramine, deserpidine (!green), desferrioxamine (!violet), doxapram, droperidol (!green), harmine (!green), imipramine (add water), maprotiline, mebhydrolin, metaraminol, phenaglycodol, phenyramidol, pyridoxine (!grey–green), salbutamol (blue rim!brown rim), strychnine (blue!purple!magenta!red!red–orange!orange), thioridazine (!violet), trimipramine (add water), triphenyltetrazolium (slow), xipamide, xylazine, yohimbine (!green) Chlophedianol, labetalol Alimemazine, amidefrine, benperidol, bezitramide (!orange), bisacodyl, captodiame, cefaloridine, chloropyrilene (!orange), clomifene (!orange–brown), clomocycline (!brown), denatonium, dipyridamole, guanoclor (!orange!brown–yellow), guanoxan, hexobendine, hydromorphone (!orange), mepacrine (!yellow), mepyramine, metisazone (!yellow), mianserin, morantel, naloxone (!brown), oxyclozanide (!orange), oxyphenisatine, oxytetracycline (!red!orange), penthienate, perphenazine, phenylbutazone, pizotifen (!green), prilocaine, primaquine (!orange), propiomazine, pyrantel, pyrrobutamine, rolitetracycline (!red!orange), tetracycline (!red!orange), thiethylperazine, thiopropazate, triacetyloleandomycin (slow), tridihexethyl, trimetazidine

Red–violet

Antazoline, carfenazine, dimethothiazine, histapyrrodine, thonzylamine

Blue–violet

Alcuronium, hexocyclium, levomepromazine

Brown–violet

Alprenolol, bitoscanate, butaperazine, naphazoline

Grey–violet

Methadone-HCl, methoserpidine, oxprenolol, tricyclamol

Black–violet

Methylenedioxyamfetamine-HCl, methapyrilene

Brown

Amitriptyline (!green), azapetine, bamipine, carbetapentane (slow), clidinium (!green), cyclopentolate, diphemanil, dipyrone, doxepin, embutramide, fluanisone, fluphenazine, isoetarine, isometheptene, isoprenaline, metindizate, methyl benzoquate, methysergide, metoclopramide, norpipanone (!blue), nortriptyline (!green), opium, phenelzine, phenylephrine, pimozide, piperidolate, prochlorperazine (!violet), propoxycaine, rescinnamine, salinazid, stanozolol, tetrabenazine, thioproperazine (!green!violet), tolnaftate, tolpropamine, tramazoline, tubocurarine

Red–brown

Benzthiazide, clioxanide, cycrimine, decoquinate, diclofenac, ethomoxane, hydrastine (!red), trifluoperazine, triflupromazine

Pink–brown

Metoprolol

Orange–brown

Rifampicin, spiramycin, thebaine

Yellow–brown

Clemastine, clofazimine, physostigmine, rifamycin SV, trimethoprim, tripelennamine

Green–brown

Etenzamide, harmaline, lysergic acid, mesoridazine, narceine, pentazocine, phenyltoloxamine, syrosingopine

Violet–brown

Chlortetracycline (!yellow), cyproheptadine, demeclocycline, dihydroergotamine, ergotamine, lymecycline (!yellow), methylergometrine, nicergoline (!brown), octaphonium, oxethazaine, protriptyline, trimethobenzamide

Grey–brown

Dextropropoxyphene, mephenesin carbamate

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:47

Colour test methods

483

Table 30.21 continued Colour

Compound

Grey

Dihydromorphine, diprenorphine, etilefrine (!green!brown), ibogaine (!violet), indometacin, lobeline, lysergide, morphine, oxypertine, propranolol, trazodone (!violet)

Blue–grey Black Grey–black

Alphaprodine, diamorphine, morphine Procyclidine Flurazepam

Table 30.22 Colours with the Marquis test Colour

Compound

Red

Alprenolol, benzylmorphine (!violet), buphenine, dimethothiazine, etenzamide, etilefrine, fenclofenac (slow), fenpiprane, fluphenazine, flurbiprofen, hexoprenaline, labetalol (!brown–red), maprotiline, mephenesin carbamate, mequitazine (slow), mesoridazine (!violet), methoxyphenamine, metopimazine, mexiletine, nadolol, pentazocine (!green), pericyazine, phenazopyridine, phenoperidine, phenylephrine, piperacetazine, prenylamine, thebaine (!orange), thiethylperazine (!green), thioproperazine, tiotixene, tolpropamine, tranylcypromine (!brown), vinblastine

Orange–red

Alverine, amfetamine-HCl, bethanidine, diphemanil, flupentixol, metamfetamine-HCl

Violet–red

Thioridazine (!blue–green)

Black–red

Doxepin-HCl

Brown–red

Alphaprodine, doxepin, trihexyphenidyl

Pink

Alimemazine, fenoprofen, fluopromazine, metoprolol, promazine, promethazine, trifluoperazine

Orange

Adrenaline (!violet), aletamine, amfetamine (!red!brown), anileridine (slow), benactyzine (!green!blue), benzethonium, benzilonium (!green!blue), benzfetamine, bunamidine (!red), carbetapentane (slow), carfenazine (!red–violet), chlorphentermine, clidinium (!blue), cyclandelate (slow), cycrimine (!red), dehydroemetine, dimethyltryptamine, dipyridamole, ethacridine (!red), ethoheptazine, ethylnoradrenaline (!brown), famprofazone, fenbufen (!brown), fencamfamin, fenethylline, fentanyl, harmine, indapamide (!violet), indometacin, isothipendyl, ketobemidone, lachesine (!green!blue), lymecycline, mepenzolate (transient), mephentermine (!brown), mescaline, metamfetamine, metanephrine (!violet–brown), methacycline, methanthelinium, methindizate (!green), methylphenidate, methylpiperidyl benzilate (!green!blue), 5-methyltryptamine (!brown), a-methyltryptamine (!brown), N-methyltryptamine, nefopam (!brown), nomifensine (slow), normetanephrine (!violet–brown), oxeladin, oxytetracycline, pentapiperide, pethidine, phenethylamine, phenformin (!brown), phentermine, piminodine, pipenzolate (!green!blue), piperidolate, pizotifen (!red), poldine methylsulfate (!green!blue), primaquine, profadol (!red–brown), prolintane (!brown), propantheline, prothipendyl, psilocybine, rolitetracycline, spiramycin, tetracycline, trimethoprim, trimethoxyamfetamine, tryptamine, veratrine, xylometazoline

Red–orange

Chlorprothixene

Pink–orange

Diuron

Yellow–orange

Orphenadrine, pipradrol

Brown–orange Yellow

Amitriptyline Acriflavine (!red), amiloride, azacyclonol, benzquinamide, benzatropine, bromazine, broxaldine, broxyquinoline, caramiphen, chlordiazepoxide, chlorphenoxamine (!green), chlortetracycline (!green), chlortalidone, cinchophen, clefamide, clemastine (green rim), colchicine, conessine (!orange), cyclizine, demeclocycline (!green), deptropine, diethyltryptamine (!brown), 2,5-dimethoxy-4methylamfetamine, diphenhydramine, diphenidol, diphenylpyraline, doxycycline, ethoxzolamide, ethylmorphine (!violet!black), furaltadone, halquinol, hydrocodone (!brown!violet), hydromorphone (!red!violet), hydroxyephedrine, isoetarine (!orange), lidoflazine, lorazepam, mepacrine, methyldopa (!violet), methyldopate (!violet), norcodeine (!violet), orciprenaline, oxycodone (!brown!violet), oxyphenbutazone, phanquone, phenbutrazate (slow), phentolamine, phenyramidol, pindolol (!brown), pramoxine (!green), proflavine (!orange), salbutamol, salinazid, sodium cromoglicate, solanine (!violet), terbutaline, tetrabenazine, thebacon (!violet), tofenacin, triamterene, trimetazidine (fades), vancomycin, viprynium embonate, zomepirac (100
C, !orange)

Orange–yellow

Methylphenidate-HCl, stanozolol

Pink–yellow

Methadone-HCl

Green

Berberine, carbaryl, chelidonine, harman, norharman, oleandomycin, propranolol, protriptyline, pseudomorphine, sulindac (slow)

Yellow–green

Acepromazine (!red), verapamil (!grey)

Blue–green

Tolnaftate

Brown–green

Harmaline

Grey–green Blue Grey–blue Violet

Cyproheptadine, deserpidine, naphazoline, oxypertine, phenindamine, protokylol, rescinnamine, reserpine (!brown) Clofibrate, embutramide, nicergoline (!grey) Mebhydrolin, 1-naphthylacetic acid Alimemazine, apomorphine (!black), azatadine, benorilate, bisacodyl, buprenorphine, butriptyline, captodiame, chloropyrilene, chlorpromazine, clofazimine, codeine, diamorphine, diethylthiambutene, dihydrocodeine, dimethindene (!blue), dimethoxanate, doxorubicin, doxylamine, etoxazene, guaifenesin, guanoxan, hexocyclium metilsulfate, mepyramine, 6-monoacetylmorphine, morphine, nalorphine, normorphine, oxprenolol, oxycodone-HCl, oxyphenisatine, pecazine, penthienate, pentazocine, perazine, perphenazine, phenoxybenzamine, phenyltoloxamine, pholcodine, pimozide, pipoxolan (!grey), prochlorperazine, procyclidine, profenamine, promazine, promethazine, proquamezine, solanidine, thenium, thiopropazate, tricyclamol, viloxazine

Red–violet

Acetophenazine, benzoctamine, bephenium hydroxynaphthoate, cefaloridine, chlophedianol (!brown), dihydromorphine, ethomoxane, isoxsuprine, lobeline, methdilazine, propiomazine, tramazoline, trifluomeprazine, trifluoperazine, triflupromazine, trimeperidine

Blue–violet

Methocarbamol, levomepromazine, morantel, neopine, noscapine (fades), pyrantel table continued

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Colour Tests

Table 30.22 continued Colour

Compound

Brown–violet

Butaperazine, dopamine, methylenedioxyamfetamine, tridihexethyl

Grey–violet

Diprenorphine, oxymorphone, pyrrobutamine, thenalidine, trihexyphenidyl

Black–violet

Dextropropoxyphene (!green), methapyrilene, thenyldiamine

Brown

Bibenzonium, carbidopa, cyclazocine (!green), diclofenac (slow), dimoxyline, dosulepin, doxepin, ergometrine, ergotamine, erythromycin, hordenine (!green), ibuprofen (100
C, !orange), isoprenaline (!violet), lysergamide, methadone, naloxone (!violet), naproxen, narceine (!green), noradrenaline, pethidine-HCl, phentermine, phenazocine, rimiterol (!black), serotonin (slow), syrosingopine, tyramine (!green)

Red–brown

Biperiden, debrisoquine, methyl benzoquate, oxetacaine, phenprobamate, trimetozine, tripelennamine

Orange–brown

Benethamine (!brown), clomocycline, nortriptyline

Yellow–brown

Moxisylyte, ritodrine, triacetyloleandomycin, tylosin

Green–brown

Alcuronium, bufotenine, psilocin

Violet–brown

Clomifene, diethazine, levomethadyl acetate (!grey–green), methoxamine (!green)

Grey–brown Grey Blue–grey Black

Dihydroergotamine, methylergometrine, octafonium Butorphanol, diaveridine (!violet–brown), ibogaine (!orange), lysergide, methoserpidine, methysergide, pholedrine (!green) Acetorphine (!yellow–brown), etorphine (!yellow–brown) Methylenedioxyamfetamine

Blue–black

Methylenedioxyamfetamine

Green–black

Lysergide

Table 30.23 Colours with Mecke's reagent Colour

Compound

Green

Diacetylmorphine, mescaline-HCl, morphine, oxycodone-HCl

Blue–green

Codeine, diacetylmorphine-HCl, hydrocodone tartrate, methylenedioxyamfetamine-HCl

Brown–green

Methadone

Orange

Alimemazine, diphenhydramine, fluopromazine, pethidine, phenyltoloxamine, promazine, promethazine, propoxyphene, trifluoperazine, triflupromazine

Indications

A change from colourless or from a pale colour to red, orange, yellow, green or blue is given by quinones, diones that possess an aromatic ring, phenols with adjacent hydroxy groups and compounds that contain nitro groups on a ring (Table 30.24). Many of these compounds are coloured already and give pale or colourless solutions in methanol. Millon's reagent (general reagent for phenols) Reagent

Dissolve 3 mL of mercury in 27 mL of fuming nitric acid and add an equal volume of water with stirring.

Yellow

Amfetamine, procaine

Method

Red

Doxepin-HCl

Add 0.5 mL of reagent to the sample and warm the mixture.

Black–red

Chlorpromazine-HCl

Purple–red

Tetracycline

Violet (dark blue)

Methylenedioxyamfetamine, thioridazine

Brown

Ephedrine

Red–brown

Propoxyphene-HCl

Black Green–black

Table 30.24 Colours with methanolic potassium hydroxide Colour

Compound

Red

Benserazide, isoetarine (!orange!yellow), metronidazole, nitrofurazone, phenindione

Orange–red Lysergide, opium

Indications

A dark grey or black colour indicates a ring imide group or sulfonamides with an additional ring. The speed and intensity of the reaction varies between different compounds. The following ring imides react in decreasing order of intensity: barbiturates, bemegride, phenytoin > benperidol, cycloserine, pimozide > glutethimide, oxyphenisatine > saccharin, sulfinpyrazone. In the case of sulfonamides, succinylsulfathiazole, sulfamoxole, sulfanilamide, sulfasomidine and sulfathiazole react with greater intensity than all others. Chlorpropamide and tolbutamide give a moderate response. If used as a spray, the LOD is 1–5 mg for barbiturates.

Pink

A 20% (w/v) solution of potassium hydroxide in methanol. Method

Add a few drops of the reagent to a solution of the sample in methanol and heat if necessary to boiling point to develop the colour.

Levodopa (!red–brown)

Orange–pink

Rimiterol

Brown–pink

Dobutamine

Orange

Acinitrazole, barban, carbidopa, dinitro-orthocresol (100
C), dinobuton, dinoseb, dodecyl gallate, hexoprenaline (!brown), isoprenaline (!yellow), nifedipine, nifuratel, nifursol, obidoxime, protokylol (!yellow)

Pink–orange

Adrenaline(!brown)

Yellow–orange

Nitrofurantoin

Yellow

Methanolic potassium hydroxide Reagent

Fenitrothion, quintozene, tecnazene, trifluralin

Green–yellow

Acebutolol, diphenadione, methyldopa (!orange), metolazone, niclosamide, niridazole, nitroxoline, nitroxynil, phanquone (!brown–violet), sodium cromoglicate Phytomenadione (!violet!brown)

Green

Apomorphine (!red), dinitolmide

Blue

Dopamine (!orange!brown), methyldopate (!orange), noradrenaline (!orange)

Violet

Dimetridazole (when boiled)

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:48

Colour test methods Indications

A red or orange–red colour indicates the presence of a phenolic substance. Primary aryl amines also react. Some basic compounds that contain a phenolic group do not react to this test; a combination of this test with the Folin–Ciocaltaeu reagent is therefore advised for phenolic compounds. Phenols that contain more than one hydroxyl group do not give the typical red colour. This reagent does not react with phenols substituted with Cl, Br or I. Naphthol–sulfuric acid This test should be carried out in conjunction with the sulfuric acid test. Reagent

Mix 1 g of naphth-2-ol with 40 mL of concentrated sulfuric acid and heat in a water-bath at 100
C, with occasional stirring, until the naphth2-ol is dissolved. Method

Table 30.25 Colours of steroids with naphthol–sulfuric acid Colour with hot reagent

Steroid

Colour after dilution

Red

Mestranol

Red

Orange–red

Desoxycortone

Blue–black

Dydrogesterone

–

Hydroxyprogesterone

Blue, violet (dichroic)

Noretynodrel

Brown–red

Brown–red

Ethinylestradiol

Pink

Orange

Norethisterone

Orange–brown

Orange, green (dichroic)

Norethandrolone

Red–orange

Yellow

Diethylstilbestrol

Orange

Testosterone

Green, brown (dichroic)

Green–yellow

Fluoxymesterone

Yellow

Green

Beclometasone

Brown–yellow

Fluocinolone

Yellow

Mix the sample with 1 mL of the reagent, heat in a water-bath at 100
C for 2 min and note any colour produced. Cool, add 1 mL of water and note the colour again.

Yellow–green

Dexamethasone

Yellow

Indications

Green, yellow (dichroic)

Estriol

Orange

Estrone

Orange

Triamcinolone

Yellow

A range of colours is obtained with steroidal structures (Table 30.25). A positive response to this test combined with a positive response to the sulfuric acid test is indicative of the presence of a steroid. Compounds other than steroids that give colours with this test include chloral hydrate and chloramphenicol (brown–yellow), starch and tartaric acid (green). Nessler's reagent

Green, brown (dichroic)

Fludroxycortide

Yellow

Blue–green, yellow (dichroic)

Estradiol

Orange

Violet

Fludrocortisone

Brown

Brown

Oxymetholone

Pink–orange

Prednisolone

Brown

Prednisone

Orange

Reagent

1. Dissolve 50 g of mercuric chloride and 35 g of potassium iodide in 200 mL of water and cool. 2. Dissolve 50 g of sodium hydroxide in 250 mL of water and cool. Add the cold solution 2 to the cold solution 1 and make up to 500 mL. Allow the mixture to stand and decant the clear supernatant (stable for many months) for use. Store in dark brown bottles away from the light.

Red–brown

Orange–brown

Method

Add the reagent (3 drops) to the sample (3 drops), agitate and heat the mixture to 100
C in a water-bath, examining it every minute for 10 min. A blank solution should be treated similarly at the same time. Indications

A brown–orange colour is produced quickly by aliphatic amides and thioamides. The presence of an aromatic ring slows the reaction. The nearer the amide group is to the ring, the more the reaction is inhibited. Substituents in the ring may cause a weak reaction. An immediate black colour is produced by substances that contain ortho- or para-hydroxy groups and by substances that contain an –NH–NH– or –NH–NH2 group in an aliphatic side-chain. Some compounds must be heated to 100
C to produce blackening. Colours are given in Table 30.26.

485

Yellow–brown

Green–brown

Progesterone

Yellow

Dimethisterone

Brown–green

Enoxolone

Orange

Fluocortolone

Red–brown

Alfadolone

Orange

Androsterone

Orange

Cortisone

Orange

Dienestrol

Yellow

Carbenoxolone

Orange

Cholesterol

Violet

Hydrocortisone

Yellow–brown

Betamethasone

Orange–brown

obtained (amantadine, rimantadine). Gentamicin gives a violet colour after heating for 4 min. Nitric acid, fuming

Ninhydrin Reagent

Dissolve 0.5 g of ninhydrin in 40 mL of acetone. Method

Dissolve the sample in methanol, place a drop of the solution on a filter paper, add 1 drop of the reagent and dry in a current of hot air. Indications

A violet colour that appears rapidly indicates the presence of an aliphatic primary amine or an amino acid group. The presence of an aromatic ring inhibits the response, and the inhibition increases the nearer the amino group is to the ring, as for amfetamine (pink–orange), procainamide and proxymetacaine (both yellow). If the amino group is associated with a saturated ring, a positive but weak pink–violet colour is

Method

Mix the sample with 3 drops of fuming nitric acid, heat at 50
C for 30 s and observe any colour produced. Cool the mixture, add 2 drops of it to 2 mL of concentrated sulfuric acid and observe the colour. To the remainder of the cooled mixture, add 2 mL of water followed by 2 mol/L sodium hydroxide, dropwise, until pH 8 is reached (use an indicator paper). Indications

Chlorinated phenols give a series of colours in the three parts of this test (Table 30.27). Nitric acid–sulfuric acid (Erdmann's reagent) Mix 1 mL of concentrated nitric acid with 30 mL of concentrated sulfuric acid.

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Colour Tests

Table 30.26 Colours with Nessler's reagent

Table 30.28 Colours with nitrous acid

Colour

Compound

Colour

Compound

Orange

Acebutolol (slow), carbidopa (!black), methotrexate

Orange

Sulfafurazole

Acetylcarbromal (slow), bromvaletone, carbromal, chloramphenicol, dinitolmide, etenzamide (weak), ethionamide, fluoroacetamide, nicotinamide, phenacemide (slow), pheneturide (slow), protionamide, pyrazinamide, salicylamide (weak), urea

Yellow

Sulfadoxine, sulfachlorpyridazine, sulfadimidine, sulfaethidole, sulfamethizole, sulfamethoxazole, sulfamethoxydiazine, sulfamethoxypyridazine, sulfametopyrazine, sulfamoxole, sulfaphenazole, sulfapyridine, sulfasomidine, sulfathiazole, sulfinpyrazone

Green

Phenazone

Blue

Dipyrone (transient)

Violet

Amidopyrine (transient)

Brown–orange

Yellow

Dihydrostreptomycin (!brown), penicillamine

Brown

Demeclocycline, mebutamate (slow), nadolol, paracetamol (slow)

Yellow–brown

Atenolol (slow)

Black (immediate)

Adrenaline, apomorphine, ascorbic acid, benserazide, dihydralazine, dobutamine, dodecyl gallate, dopamine, ethylnoradrenaline, hexoprenaline, hydralazine, iproniazid, isocarboxazid, isoetarine, isoniazid, isoprenaline, levodopa, mebanazine, methyldopa, methyldopate, nialamide, noradrenaline, phenelzine, procarbazine, protokylol, rimiterol

Black (at 100
C)

Cimetidine, gentamicin, labetalol, meprobamate (grey–black), methallibure, salinazid, thiacetazone

Table 30.27 Colours with fuming nitric acid Colour Part 1

Part 2

Part 3

Compound

Orange–red

Orange

Orange–brown

Hexachlorophene

Red

Red

Brown–violet

Pentachlorophenol

Method

Mix the sample with 1 mL of the reagent and heat at 100
C in a waterbath for 2 min. A blank solution should be treated similarly at the same time. Indications

Red, orange, yellow, brown or black colours are given by aliphatic compounds that have a sulfur atom in the chain, and by aromatic compounds that have a sulfur atom in the side-chain. However, no colour is given when an S-alkyl chain is present, unless the chain is terminated by an halogenated group. No response is obtained if the sulfur is in a group that links two rings. Reducing agents such as ascorbic acid, chloral hydrate, chloroform and glucose, and compounds that contain a chain with a hydrazine link (–NH–NH–, –NH–NH2), give a translucent dark-grey or black colour, but do not give the gradual yellow to orange to brown colour seen with sulfur-containing compounds. Compounds that contain adjacent hydroxyl groups on an aromatic ring give orange colours that turn brown (Table 30.29).

Method

Dissolve the sample in 1 mL of ethanol, add a pellet of potassium hydroxide and evaporate to dryness (100
C in a water-bath). To the residue add 0.5 mL of water and 1 mL of carbon tetrachloride, shake and allow to separate. Decant the lower carbon tetrachloride layer and shake it with 1 mL of the reagent. Indications

A red colour in the acid layer suggests the presence of clofenotane or its metabolite, dichlorodiphenyldichloroethylene (DDE). The red colour changes to orange and then to green. Weak pink colours are given by aldrin, dieldrin and endrin. A red colour is also given by dichlorodiphenyldichloroethane (DDD, mitotane), but the colour does not change. Note that the substance should be tested to ensure that it does not give a colour with sulfuric acid alone.

Phosphorus test Method

To the sample add 0.5 mL of concentrated nitric acid and 0.2 mL of concentrated sulfuric acid and heat at 100
C in a water-bath for 30 min. Cool, add 1 mL of a 10% (w/v) solution of ammonium molybdate and replace in the water-bath at 100
C for 5 min. A blank solution should be

Table 30.29 Colours with palladium chloride Colour

Compound

Red

Gloxazone

Orange

Adrenaline (!brown), benserazide (!brown), bitoscanate, captopril, carbidopa (!brown), carbimazole, disulfiram, dobutamine (!brown), ecothiopate, isoetarine (!brown), levodopa (!brown), methallibure, thiamazole, polythiazide, rimiterol, thiacetazone, thiopental

Nitrous acid Method

Dissolve the sample in a minimum volume of water, and add an amount of solid sodium nitrite equal in volume to the sample followed by a few drops of 2 mol/L hydrochloric acid.

Brown–orange Yellow

Indications

Orange or yellow colours are given by certain sulfonamides, and green, blue or violet colours by certain phenylpyrazolines (Table 30.28). No response is obtained with succinylsulfathiazole, sulfacetamide, sulfadiazine, sulfadimethoxine, sulfaguanidine, sulfamerazine, sulfaquinoxaline, sulthiame or propyphenazone.

Orange–yellow Brown

Orange–brown

Palladium chloride Reagent

Dissolve, with the aid of heat, 0.1 g of palladium chloride in 5 mL of 2 mol/L hydrochloric acid and dilute the solution to 100 mL with water. Mix together equal volumes of this solution and 2 mol/L sodium hydroxide. The mixed reagent is stable for several weeks.

Black–brown

Demeton-S Clindamycin, dazomet, dimercaprol, dimethoate, methisazone (!orange!brown), penicillamine Thialbarbital Ambazone, azinphos-methyl, dihydrostreptomycin (slow), ethionamide, malathion, noxythiolin, parathion, phosalone, protionamide, spironolactone, thiram Chlorthiamid, diazinon, disulfoton, fenitrothion, formothion, phorate, vamidothion Di-allate, dichlofluanid, tri-allate

Grey

Chlorfenvinphos

Black

Ascorbic acid, captan, chloroform, cloral hydrate, mebanazine, nifuratel, phenelzine, procarbazine, sulfasalazine, sulfaurea, trichlorfon

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Colour test methods Table 30.30 Colours with potassium dichromate

487

once. Add 1 drop of solution 2 and shake (the blue colour disappears and a clear pink solution develops). Add several drops of solution 3.

Colour

Compound

Red

Carbidopa

Yellow (!brown)

Phenol (2 min)

Green (!brown)

Adrenaline, dopamine, hexoprenaline, isoetharine, isoprenaline, levodopa, methyldopa, methyldopate, noradrenaline, rimiterol

Simon's test (modified sodium nitroprusside test)

Aniline (2 min)

Reagent

Benserazide, o-cresol (30 s), m-cresol (2 min), orciprenaline (slow), protokylol (!red–brown on warming), terbutaline (slow)

1. Dissolve 1 g of sodium nitroprusside in 100 mL of water and add 2 mL of acetaldehyde to the solution with thorough mixing. 2. Freshly prepared 2% sodium carbonate in distilled water.

Dobutamine

Method

Blue–green Brown

Green–brown

Indications

The chloroform layer develops an intense blue colour if cocaine is present. Methadone also reacts. The LOD is 60 mg cocaine-HCl and 15 mg methadone-HCl.

Add 1 drop of solution 1 to the sample, followed by 2 drops of solution 2. treated at the same time. For some compounds, the reaction may occur after shorter heating times than those stated above. Indications

A bright yellow solution or precipitate indicates the presence of phosphorus and suggests an organophosphorus pesticide, especially if the sample is a water-immiscible liquid. Cyclophosphamide and triclofos also react. Potassium dichromate

Indications

A dark-blue colour indicates a secondary amine (e.g. metamfetamine, ephedrine, 3,4-methylenedioxymetamfetamine (MDMA)) or an unsubstituted heterocyclic amine as its free base. A deep blue colour indicates the presence of metamfetamine. Primary amines (e.g. amfetamine, methylenedioxyamfetamine (MDA)) yield a slow pink to cherry-red colour. Sodium dithionite Reagent

Method 1

A 5% (w/v) solution of sodium dithionite in a 10% (w/v) solution of sodium hydroxide.

Dissolve the sample by shaking in 0.5 mL of 2 mol/L hydrochloric acid and add a few crystals of potassium dichromate.

Method

Indications

An immediate brown colour, or a green colour that changes to brown, indicates the presence of an aminophenol or of a phenol that has two or more hydroxyl groups in adjacent positions on the ring (Table 30.30). Monophenols, halogenated phenols and phenols with hydroxyl groups meta to each other react more slowly or not at all. Method 2

If the sample is a liquid, add 1–2 drops to 1 mL of water followed by 1 mL of a saturated solution of potassium dichromate in 50% v/v sulfuric acid. Indications

A green colour is given by acetaldehyde, ethanol, methanol, propan-1-ol and propan-2-ol. Schiff's reagent Reagent

Dissolve 0.2 g of basic magenta (fuchsin, CI 42510) in 120 mL of hot water, cool, add 20 mL of a 10% (w/v) solution of sodium hydrogensulfite and 2 mL of concentrated hydrochloric acid, and dilute to 200 mL. Store at 4
C and protect from light.

Apply the reagent to the sample, either on a white tile or as a solution in a test-tube. A blank solution should be treated similarly at the same time. Indications

Colours are produced by bis(pyridyl) compounds (Table 30.31). Dark colours are likely to be given by certain metallic solutions because of reduction. Sodium hypobromite test (for carbamazepine) Reagent

Dissolve 0.5 mL of bromine in 5 mL of a 40% w/v solution of sodium hydroxide with shaking and cooling. (this should be freshly prepared). Method

Add 1 mL of 2 mol/L hydrochloric acid to 5 mL of sample and 5 mL of chloroform. Vortex mix for 1 min and centrifuge for 5 min. Remove the upper layer, add 1 mL of the chloroform extract to 0.2 mL of sodium hypobromite reagent and mix for 30 s. Indications

Carbamazepine forms a blue to violet colour in the chloroform layer. The test has a sensitivity of 250 mg/L.

Method

Add the sample to 1 mL of the reagent.

Sodium nitroprusside

Indications

Reagent

A violet colour indicates the presence of an aliphatic aldehyde. The longer the carbon chain, especially if it is branched, the weaker the response to the test.

A 1% (w/v) solution of sodium nitroprusside.

Scott's test (see also Cobalt thiocyanate) Reagent

1. Cobalt thiocyanate dissolved in water (2% w/v) and then diluted 1 : 1 with glycerine 2. Concentrated hydrochloric acid 3. Chloroform. Method

Add a small amount of the sample to be tested to a test-tube, add 5 drops of solution 1 and shake. If cocaine is present a blue colour develops at

Method 1

Add the sample to 2 mL of the reagent followed by a drop of 2 mol/L sodium hydroxide. Indications

Orange colours are given by ketones and red colours by acetaldehyde. Table 30.31 Colours with sodium dithionate Colour

Compound

Green

Diquat

Blue

Paraquat

Clarke's Analysis of Drugs and Poisons Chapter No. 30 Dated: 10/3/2011 At Time: 13:2:49

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Colour Tests

Method 2

Indications

Mix the sample with a minimum volume of 2 mol/L sodium hydroxide, evaporate to dryness, dissolve the residue in 2 drops of water and add 0.5 mL of the reagent.

A red colour that appears in the lower acid layer indicates the presence of dieldrin (colour develops quickly) or aldrin (colour develops slowly). A pink–orange colour is obtained with endrin.

Indications

A violet colour is given by substances that contain labile sulfur in the molecule and by unsubstituted dithiocarbamates.

Tetrabromophenolphthalein ethyl ester

Method 3

Dissolve 50 mg tetrabromophenolphthalein ethyl ester (TBPE) in 100 mL chloroform, shake the solution for 2 min with 1 mL of 10% (v/v) hydrochloric acid and discard the aqueous phase. Dry the organic layer with anhydrous sodium sulfate. Separate the drying agent by filtration. Store the reagent in an amber bottle at 4
C.

Carry out Method 2 above, but after evaporation to dryness heat the residue until it is yellow or orange in colour before proceeding. Indications

A violet colour is given by certain substances that contain labile sulfur and do not react to method 2 (e.g. clomethiazole, lincomycin and monosulfiram). Sodium nitroprusside–acetone Reagents

1. Dissolve 2 g of sodium nitroprusside in 5 mL of water and add 45 mL of methanol 2. 2% (w/v) sodium carbonate 3. Acetone 4. 10% acetaldehyde. Method 1

Add a drop of solution 1 followed by a drop of solution 2 to 3–4 mg of sample dissolved in solution 3 on a spot plate. A purple colour is indicative of amfetamine. The LOD is 30 mg. Method 2

Reagent

Method

Place 0.5 mL of sample to be tested in a conical test tube, add 100 mL phosphate buffer (10 mmol/L, pH 8.0) and vortex mix. Add 50 mL of the TBPE reagent and vortex mix. After 2–3 min note the colour of the chloroform layer. If the sample to be tested is a solid, dissolve 1–2 mg of the material in 0.5 mL of buffer and proceed. Indications

A deep blue colour indicates quaternary ammonium compounds. An orange, brown, red or purple colour indicates the presence of basic drugs. This test is most sensitive to tertiary amines (e.g. tricyclics, propoxyphene, phenothiazines, diphenhydramine, phencyclidine, methadone, pethidine). Its LOD is 1 mg/L. Thalleioquin test

Add a drop of solution 1 followed by a drop of solution 4 to 1–2 mg of sample dissolved in solution 3. An immediate blue colour is indicative of metamfetamine. The LOD is 5 mg.

Method

Sodium picrate (Steyn test)

Indications

Reagent

A green colour indicates the presence of a quinine-type structure (e.g. hydroquinidine, hydroquinine, quinidine, quinine). Cinchonidine and cinchonine do not respond.

Prepare a solution of 5 g sodium bicarbonate and 0.5 g picric acid in 100 mL of water.

Dissolve the sample in a minimum volume of 2 mol/L hydrochloric acid, add 2 drops of bromine solution, place 1 drop of the mixture on a piece of filter paper and expose the paper to ammonia fumes.

Method

Mix the sample with a few drops of chloroform and concentrated sulfuric acid to hasten the reaction while holding a piece of filter paper, impregnated with the reagent, in the vapours that issue from the tube, and heating the contents to 30
C. Indications

The yellow colour of the filter paper changes from orange to brown– orange and then to orange–red or red in the presence of cyanide. Positive results are given by compounds that contain cyanide groups (e.g. cimetidine, diphenoxylate and isoaminile). Sulfuric acid Method

Apply concentrated sulfuric acid directly to the sample on a white tile or in a test-tube. Indications

A range of colours is obtained with compounds of various types. Steroids give orange or yellow colours, many of which fluoresce under UV light (l ¼ 350 nm) either immediately or after dilution (Table 30.32). Thioxanthenes give red or orange colours that fluoresce under UV light (l ¼ 350 nm) (Table 30.33). Sulfuric acid–fuming sulfuric acid

Trinder's reagent (see Ferric chloride) Reagent

The solution is prepared as follows: 40 g of mercuric chloride and 40 g of ferric nitrate are dissolved in 850 mL of distilled water; 10 mL of concentrated HCl is added and the solution is diluted to 1 L. This solution is stable for 1 year. Method

A few drops of the reagent are added to a few drops of urine. A purple colour indicates the presence of a salicylate. This test was devised for the quantitative assay of salicylates in serum, with the mercuric chloride serving as a protein precipitant. The ferric chloride test has been modified for use on blood samples (Asselin, Caughlin 1990). Blood (0.5 mL) is mixed with 1.0 mL of acetone and vortex mixed. The sample is centrifuged, and 50 mL of the supernatant is added to 50 mL of ferric chloride. A purple colour at the interface indicates salicylates. Vanillin reagent Reagent

Dissolve 1 g of vanillin in 20 mL of concentrated sulfuric acid, warming if necessary. Method

Mix together 7 mL of concentrated sulfuric acid and 3 mL of fuming sulfuric acid.

Add 2 drops of the reagent to the sample, heat in a water-bath at 100
C for 30 s and note any colour that is produced. Dilute the cooled mixture by adding a few drops of water and note any change of colour.

Method

Indications

Dissolve the sample in a minimum volume of toluene and add 1 or 2 drops of the reagent.

Many compounds of different chemical structure react with this reagent. However, for barbiturates, the reaction appears to be a steric

Reagent

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Colour test methods

489

Table 30.32 Reactions of steroids with sulfuric acid Initial colour

Compound

Fluorescence at 350 nm

Fluorescence after dilution

Dienestrol

Nil

Nil

Dimethisterone

Nil

Yellow

Mestranol

Yellow

Orange (pink in daylight)

Dexamethasone

Nil

Nil

Prednisolone

Nil

Green (red in daylight)

Beclometasone (slow)

Nil

Nil

Cholesterol

Nil

White

Dydrogesterone

Green–yellow

Green–yellow

Fludrocortisone

Green

Green (dichroic in daylight)

Norethandrolone

Green–yellow

–

Norethisterone

Orange

Orange (violet in daylight)

Norethynodrel

Orange

Orange

Oxymetholone

Nil

Nil

Spironolactone (!yellow–green)

Yellow–green

Green

Diethylstilbestrol

Nil

Nil

Triamcinolone

Nil

Nil!green (slow)

Red–orange

Ethinylestradiol

Orange

Orange (red in daylight)

Pink–orange

Betamethasone

Nil

Nil

Green, orange (dichroic)

Hydrocortisone

Green

Green

Alfadolone

Nil

Nil

Androsterone

Nil

White

Carbenoxolone

Nil

Yellow

Cortisone

Green

Green

Desoxycortone

Green–yellow

Yellow (violet in daylight)

Red Orange–red

Pink Orange–pink Orange

Yellow

Enoxolone

Nil

Green–yellow

Fluocinolone

Green

Quenched

Fluoxymesterone

Green

Quenched

Fludroxycortide

Green

Quenched

Hydroxyprogesterone

Green

Quenched

Estradiol

Green

Green (orange in daylight)

Prednisone

Green

Green

Progesterone

Green

Quenched

Orange–yellow

Fluocortolone

(Weak)

(Weak)

Green–yellow

Estrone

Green

Green–yellow (orange in daylight)

Estriol

Yellow–green

Quenched (orange in daylight)

Testosterone

Green

Nil

No colour

phenomenon that depends on the structure of the side-chain at the 5-position. Dark colours, which are either dispelled or changed to violet, blue or green by dilution, are produced when either side-chain is greater than two carbon atoms in length or contains a cycloalkene ring. Branching can be proximal to the pyrimidine ring, but not distal. No colour is obtained if both side-chains are less than three carbon atoms in length or if either is branched distally or contains an aryl nucleus. Long, straight, saturated chains also appear to hinder reaction. Hydroxybarbiturates give positive responses (Table 30.34), but bemegride, glutethimide, phenytoin and primidone do not respond. No response is obtained with amobarbital, aprobarbital, barbital, butobarbital, enallylpropymal, hexethal, ibomal, idobutal, metharbital, methylphenobarbital, nealbarbital, phenobarbital or phenylmethylbarbituric acid. With cold reagent, an orange colour is produced by pentobarbital, secobarbital and thiopental, and a brown colour by cyclopentobarbital.

Zwicker reagent (alkaline cobalt test) This is a general test for barbiturate-like compounds. Reagent

1. Dissolve 0.5 g of copper(II) sulfate pentahydrate in 100 mL of distilled water. 2. Add 0.5 mL of pyridine to 95 mL of chloroform. Method

Add a few drops of solution 1 to the sample to be tested, followed by a few drops of solution 2 and then heat. Indications

The presence of a violet–blue colour indicates barbiturates (Table 30.35). The LOD is 1000 mg for phenobarbital.

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Table 30.33 Colours of thioxanthenes with sulfuric acid (a)

Colour

Compound

Purple

Oxytetracycline, tetracycline

Red

Caramiphen (when warmed), dantron, fenitrothion, methenamine (when warmed), mequitazine (slowly at 100
C), methacycline, methylprednisolone (after 1 min), metopimazine, nuarimol, ouabin, phenothiazines, prednisolone (after 1 min), pipoxolan

Table 30.34 Reactions of barbiturates with vanillin reagent Colour after heating

Compound

Colour after dilution

Red

30 -Hydroxybutobarbital

Violet (transient)

Violet–red Brown–red

Heptabarbital

Colourless

30 -Hydroxyamylobarbital

Colourless

Cyclobarbital

Green

Orange–red

Oxprenolol*, quinomethionate

Cyclopentobarbital

Green

Violet–red

Morantel, oxytetracycline

Pentobarbital

Violet

Pink

Doxylamine, indapamide (slow)

Secobarbital

Violet

Orange

Alprenolol, amitriptyline, benactyzine, benzilonium, benzquinamide, benzyl nicotinate, chlorprothixine*, clidinium, clopenthixol*, cyclothiazide (!red–brown), diethylthiambutene, diphemanil, diphenhydramine, diphenidol, doxepin, flupentixol*, indometacin, mazindol, mebanazine, mecoprop, methapyrilene, methindizate, methixene*, methyclothiazide, methylpiperidyl benzilate, naproxen, nefopam, nifedipine, nortriptyline, orphenadrine, penthienate methobromide, polythiazide, pyrantel (!violet), rotenone, tiotixene*, tofenacin

Thiopental

Violet

Butalbital (weak)

Colourless

Yellow

Orange–yellow Green

Acebutolol, amiloride, amiodarone, benzthiazide, broxaldine, broxyquinoline, cinchophen, clefamide, clemastine (green rim), cyclopenthiazide, diphenadione, doxycycline, enoxolone, fenbufen, furosemide, halquinol, hydroquinidine*, hydroquinine*, lorazepam, methyl benzoquate, 5-methyltryptamine, a-methyltryptamine, N-methyltryptamine, metolazone, minocycline, piperacetazine (!red), procyclidine, quinidine*, quinine*, rimiterol, salbutamol, salinazid, sodium cromoglicate, trichlormethiazide, veratrine (!violet), zomepirac

Orange Brown–orange

Brown Violet–brown

Secbutobarbital (weak)

Violet

Allobarbital

Violet (transient)

Brallobarbital

Brown–orange

Talbutal

Violet

Thialbarbital

Violet (transient)

Hexobarbital

Violet

Methohexital

Colourless

Vinbarbital

Colourless

30 -Hydroxypentobarbitone

Colourless

Table 30.35 Colours with Zwicker reagent Colour

Compound

Blue

Diphenylhydantoin

Ethyl biscoumacetate, hexoprenaline, pizotifen (!violet)

Green

Diacetylmorphine, pseudoephedrine

Bromodiphenhydramine, cyclizine, diphenhydramine, diphenylpyraline, phenothiazine, protriptyline

Yellow

Tetracycline

Blue Brown–blue Violet

Red–violet Brown Red–brown

Chlortetracycline, demeclocycline, thioridazine Bendroflumethiazide, chlorotrianisene, chromonar*, clofazimine, cyproheptadine, dosulepin, mesoridazine (!blue), methylenedioxyamfetamine, nicergoline, perazine, phenindione, rolitetracycline, tetracycline Trifluomeprazine Chelidonine, sulindac Lymecycline

Orange–brown

Biperiden, ouabain (slow)

Yellow–brown

Tylosin (slow)

Grey–brown

Octaphonium

Black Blue–black

Clomocycline

(a) Compounds giving colours that fluoresce under UV light (l = 350 nm) are indicated by an asterisk.

Basic tests for drug substances and products The WHO has published texts Basic Tests for Drugs (WHO 1998), which includes pharmaceutical substances, medicinal plant materials and dosage forms, and Basic Tests for Pharmaceutical Substances (WHO 1986). The basic tests described, which are designed to verify the identity of drug substances and medicinal products and to detect gross contamination, use a limited number of readily available reagents and equipment. Overall, the combined texts offer compound-specific tests for approximately 500–600 products that are based on a combination of organoleptic checks and simple physicochemical tests, such as colour reactions and melting-point determinations.

Semi-quantitative TLC methods have been developed as basic tests using a limited number of solvent systems and detection systems. References to these tests are given in (WHO 1998). It should be remembered that basic tests are not, in any circumstances, intended to replace pharmacopoeial requirements, but should be used as a rapid, inexpensive means to verify identity and strength of drugs and medicinal products, and possibly to detect poor-quality counterfeit and other substandard products. In the event that suspect products are detected, these should be tested for compliance against pharmacopoeial requirements. Colour reagents and thin-layer chromatography Many common colour-test reagents are used routinely as spray locating reagents in TLC (e.g. acidified iodoplatinate solution, Dragendorff’s reagent, Marquis reagent, Van Urk’s reagent). It is worth noting that the preparation of the spray equivalent of a colour reagent may differ slightly from that of the colour reagent preparation itself (see Chapters 1 and 11 and Index of Reagents). It is also true that the reaction and resultant colour that marks the presence of a certain substance obtained from spraying a reagent on TLC plates may differ from that obtained from a direct colour test and, in some cases, will not yield any results. This is because of pH effects – that is, whether the TLC plate has been dipped in 0.1 mol/L sodium hydroxide and the substance tested is acidic in character (i.e. the free acid of a salt post extraction). However, this aspect of colour reagents and their use in TLC as sprays can often give clues as to the drug or substance’s chemistry. A list of substances and their colour reactions to various TLC spray reagents is given in the Index of Analytical Data.

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Appendix 30.2. Colour reactions of narcotics

Appendix 30.1. Colour reactions of amfetamine-like compounds Compound

Marquis

Mecke

Froehde

Mandelin O!R!G/Br

D-Amfetamine

R/O!R/Br

Y

Css

Benzfetamine

R!Br

G!Y

Y!G

Diethyltryptamine

Y!Br

B!G/Bk

2,5-Dimethoxyamfetamine

lrY!R/Br

Br/G

ltG

ltG

Dimethoxymethylamfetamine

Y/G

Y!G!dkR/Br

Y/G

Y!G!Br

2,3-Dimethylamfetamine

M R/V

V dkV

V Gy/V

V Gy/G

2,4-Dimethylamfetamine

V O/R

G G/Br

Y Br/Y

Y Br/Y

2,5-Dimethylamfetamine

Y/G

Br/G

Y/G

Y/G

2,6-Dimethylamfetamine

Css

Br/G

Css

Br/Y

3,4-Dimethylamfetamine

Y/Br

G!Br/G

Lt G

NR

3,5-Dimethylamfetamine

Y

O/Br!Br/G

Css

Gy/V

Dimethyltryptamine

Y!G!Br

Y!G/Bk

ltY

G!Br

Css

ltBr

Ephedrine

Css

Css

Mephentermine

O!RBr

G

Mescaline

O/R

O!G/Br

ltY

Y!G!Br

Metamfetamine

R/O!R/Br

NR

NR

G!B/G

3-Methoxy-4,5-methylenedioxyamphetamine

O!M

dkB

B!dkB

O!O/R

3,4-Methylenedioxyamfetamine

B/BK!dkV

G!dkB

G!dkV

R/V!dkV

3,4-Methylenedioxymethylamfetamine

B!V!Bk

G!dkB

Y/G!dkB

B!V!Bk

Paramethoxyamfetamine

Effervescence

ltG

G!R/Br

Phendiamine

V!Bk

ltG

ltV

G

Phendimetrazine

NR

NR

NR

NR

Pheniramine

NR

NR

NR

NR

Phenmetrazine

NR

ltY

NR

NR

Phentermine

ltO

ltO

Br

G

Phenylephrine

O

O/Br

B

G

Phenylpropano lamine

ltY

ltY

Br

NR

Pseudoephedrine

Css

Css

Css

ltBr

3,4,5-Trimethoxyamfetamine

R/O!O

G/Br!Br

Y

NR

B, blue; Bk, Black; Br, brown; Css, colourless; dk, dark; G, green; Gy, grey; lt, light; M, magenta; NR, no reaction; O, orange; R, red; V, violet; Y, yellow.

Appendix 30.2. Colour reactions of narcotics Compound

Marquis

Mecke

Froehde

Mandelin

Anileridine

O

NR

NR

NR

Codeine

V

dkG

ltG

ltG

Dihydromorphinone

V/Br

Br

V

Css

Ethylmorphine

O!V

dkG

Y!ltG!B

G

Fentanyl

O!Br

G

Diamorphine

V

ltG

M

NR

Hydrocodone

V

G!B

ltY

NR

dkB!Gy

Hydromorphine

Y!R!V

Y!O!G

Levorphanol

Gy!Bk

ltGy!Bk

B

G

Meperidine (pethidine)

O(slow)

ltY(slow)

NR

NR

Methadone

ltV

G!Br

B

Morphine

M!V

G!B

V!Gy

ltGy

Nalorphine

O!R!V

ltG!dkG

M

G

Oxycodone

O/Y!ltV

Y!dkG

Y!YBr

Y!G

Oxymorphone

M!V

Y!Br

B!V

Bk

Papaverine

M

dkG

G

G/Br

Pentazocine

M

ltGy!V

dkB

G!Br

Thebaine

V/Br

Br

Br

V/Br

B, blue; Bk, black; Br, brown; Css, colourless; dk, dark; G, green; Gy, grey; lt, light; M, magenta; NR, no reaction; O, orange; R, red; V, violet; Y, yellow.

491

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Appendix 30.3. Chemical test for gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL)

Appendix 30.4. Tests for metals and anions not covered by the general scheme

Reagents

Metals

Chlorophenol red

Reinsch test (for antimony, arsenic, bismuth and mercury)

0.04 g chlorophenol red in 100 mL water, adjust to pH 7 with 0.01 mol/L sodium hydroxide.

Method

Modified Schweppes

1. 2 g of dextrose in 20 mL of water 2. 2.4 g of aniline hydrochloride in 20 mL of ethanol. Mix solutions 1 and 2 together and dilute to 80 mL with methanol. Bromocresol purple

0.04 g bromocresol purple in 100 mL of water. Adjust to pH 7 with 0.01 mol/L sodium hydroxide. Bromothymol blue

Use a 5  5 mm piece of copper foil or mesh. Clean the copper in 50% (v/v) nitric acid until it develops a shiny surface, rinse with water and proceed as follows: 1. Place 20 mL of sample and 10 mL of concentrated hydrochloric acid in a 100 mL conical flask and add the copper foil or mesh. 2. Heat on a boiling water-bath for 1 h and add as necessary dilute hydrochloric acid to maintain the volume of the solution. 3. Cool, remove the copper, wash gently with water and examine the surface. Indications

Bromocresol green

n n n n

0.03 g bromocresol green in 100 mL of methanol–water 4 : 1. Adjust to pH 7 with sodium hydroxide 0.01 mol/L.

Note that other elements, (e.g. selenium and tellurium) also give black deposits and sulfur compounds may give a speckled discoloration.

Methyl orange

Confirmatory test

0.04 g bromothymol blue in 100 mL of water. Adjust to pH 7 with 0.01 mol/L sodium hydroxide.

0.01 g methyl orange in 100 mL of methanol. Adjust to pH 7 with sodium hydroxide 0.01 mol/L.

A purple–black stain indicates antimony. A dull black stain indicates arsenic. A shiny black stain indicates bismuth. A silvery deposit suggests mercury.

This is applied to the stained copper foil or mesh derived as described above. Reagents

Colour test 1 Reagent

Mix chlorophenol red and modified Schweppes reagent in a 3 : 1 ratio. Method

Place 0.5 mL of a liquid sample in a test tube. Adjust to pH 5–8 with 0.01 mol/L sodium hydroxide. Add 2 drops of the test reagent and swirl. An immediate colour change (orange–red to dark red) indicates GHB. GBL gives a yellow colour. Colour test 2 Reagent

Mix bromocresol purple and bromothymol blue in a 1 : 1 ratio and mix the combined reagent with modified Schweppes reagent in a 7 : 1 ratio. Method

Same procedure as for colour test 1. GHB gives a purple colour and GBL gives a yellow colour. Colour test 3 Reagent

Mix bromocresol green and methyl orange in a 1 : 1 ratio and mix the combined reagent with modified Schweppes reagent in a 3 : 1 ratio. Method

Adjust the pH of the test solution to neutral if necessary and add 2 drops of test reagent. A dark-green colour indicates GHB; GBL gives a yellow– orange colour. Colour test 4 Reagent

1% cobalt nitrate solution. Method

Place 0.5 mL of a liquid sample in a test tube. Add a few drops of the test reagent. A pink-to-violet colour indicates GHB.

1. 2. 3. 4.

Aqueous potassium cyanide solution (100 g/L) Freshly prepared aqueous sodium sulfite solution (50 g/L) 3 mol/L nitric acid Quinine/potassium iodide reagent. Dissolve 1 g of quinine in 100 mL of water containing 0.5 mL of concentrated nitric acid. When the quinine has dissolved add 2 g of potassium iodide.

Method

1. Leave the copper in potassium cyanide solution for 10 min. 2. Wash any undissolved stain with water and add 1 mL of sodium sulfite solution followed by 1 mL of 3 mol/L nitric acid. 3. Shake the mixture frequently for 5 min and add 1 mL of water and 1 mL of potassium iodide reagent. Indications Stains caused by the presence of arsenic dissolve in potas-

sium cyanide solution whereas stains due to antimony and bismuth remain. The slow formation of an orange–brown suspension is seen with the quinine/potassium iodide reagent if a stain due to bismuth is present. The LOD for arsenic is about 5 mg/L and for antimony and bismuth is 2 mg/L. A more specific test for arsenic, the ‘Gutzeit test’, involves the conversion of arsenic to arsine and subsequent reaction of the gas with reagents such as silver diethyldithiocarbamate to give a coloured product. A modified version of the Gutzeit apparatus is available from Fischer Scientific which allows quantitative measurement of arsenic in stomach contents, food, water and other materials. Confirmatory test for mercury

This is applied to the silver stained foil or mesh from the Reinsch test. Reagent Cuprous iodide suspension. Dissolve 5 g of copper sulfate and 3 g of ferrous sulfate in 10 mL of water with continuous stirring and add 7 g of potassium iodide dissolved in 50 mL of water. Allow the cuprous iodide precipitate to form, filter, and wash with water. Transfer the precipitate as a suspension in water to a brown glass bottle. Method Add 0.1 mL of copper(I) iodide suspension to a filter paper, place the foil on the suspension, cover and leave for 1–12 h. Indications A salmon-pink colour due to the formation of cuprous mercuric iodide suggests the presence of mercury and positive results may appear within 1 h, but with low concentrations colour development may take up to 12 h. The LOD for mercury is about 5 mg/L.

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Appendix 30.4 Barium

Thallium

Reagents

Test 1

1. Concentrated hydrochloric acid 2. Platinum wire.

Reagents

Method

1. Dip the end of the platinum wire into the hydrochloric acid and then into the test material. 2. Insert the wire into the hot area of a micro-burner flame and observe any changes in the flame colours. Indications

Barium salts impart a green flame; copper and thallium salts also give a green flame in this test.

1. Cyanide reagent: dissolve 1.6 g of sodium hydroxide, 1.2 g of potassium sodium tartrate and 1.36 g of potassium cyanide in 10 mL of water. 2. Prepare a fresh solution of dithizone (250 mg/L) in chloroform. Method Add 1 mL of cyanide reagent to 5 mL of urine in a stoppered glass test-tube and vortex mix for 20 s. Then add 2 mL of dithizone solution, vortex mix for 1 min and centrifuge (5 min). Indications The presence of thallium is indicated by a pink–red colour in the chloroform layer. The test will detect thallium at 0.1 mg/L. A number of other metal ions give colours with this test.

Confirmatory test

Test 2

Reagents

Reagents

1. 2. 3. 4.

1 mol/L sulfuric acid 100 g/L aqueous lead acetate solution 50 mL/L aqueous acetic acid Solid ammonium acetate.

Method

1. To a mixture of 2 mL of lead acetate solution and 2 mL of dilute sulfuric acid add enough ammonium acetate to dissolve the lead acetate precipitate. 2. Add 0.1 mL of dilute acetic acid to 1 mL of sample followed by 1 mL of the lead sulfo-acetate solution (from step 1) and vortex mix for 5 s. 3. Centrifuge for 2 min and observe the tube against a dark background. Indications Barium salts yield either a white turbidity or white precip-

itate. Calcium and strontium salts interfere. The LOD for barium is approximately 100 mg/L. Copper Reagents

1. 10 g/L solution of dithiooxamide in methanol 2. Concentrated ammonium hydroxide. Method

Place 0.1 mL of sample onto a filter paper to give a spot of around 1 cm in diameter. Expose the paper to ammonia fumes and add 0.1 mL of the dithiooxamide reagent to the spot. Indications

An olive green stain is seen with copper salts. A green stain due to chromium is usually visible before addition of the dithiooximide reagent. Yellow–brown or yellow–red colours are given by several other metals. The LOD for copper is approximately 1 mg/L.

493

1. 2. 3. 4. 5.

Bromine water (saturated) 6 mol/L hydrochloric acid 20% (w/v) aqueous sulfosalicylic acid solution 0.1% (w/v) aqueous methyl violet solution Toluene.

Method To 1 mL of urine carefully add 2 drops of hydrochloric acid and 5 drops of bromine water. Leave to stand for 5 min, add 5 drops of sulfosalicylic acid solution and 0.5 mL of toluene, and shake gently. Indications A transient blue–green colour in the toluene layer suggests the presence of thallium. The LOD for thallium is 1 mg/L.

Anions Borate Reagents

1. 10 g/L solution of turmeric in methanol 2. 1 mol/L hydrochloric acid 3. 4 mol/L ammonium hydroxide. Method

1. Prepare turmeric test papers by soaking strips of filter paper (1  5 cm) in the turmeric solution and drying at room temperature. 2. Acidify a portion of the sample with the dilute hydrochloric acid and apply to a strip of turmeric paper. 3. When the paper is dry moisten it with dilute ammonium hydroxide. Indications

A positive sample will impart a red–brown colour to the turmeric paper which strengthens as the paper dries. When the paper is moistened with dilute ammonium hydroxide a green–black colour is produced. Oxidising agents that bleach turmeric (e.g. bromates, chlorates, iodates and nitrites) interfere with the test. The LOD for borate is 50 mg/L. Confirmatory test

Iron

Reagent 0.5 g/L solution of carminic acid in concentrated sulfuric acid.

Reagents

Method Add 0.5 mL of filtered stomach contents or scene residue to a

1. 2 mol/L hydrochloric acid 2. 10 g/L aqueous potassium ferricyanide solution 3. 10 g/L aqueous potassium ferrocyanide solution. Method

1. Add 0.1 mL of 2 mol/L hydrochloric acid and 0.05 mL of potassium ferricyanide solution to 0.1 mL of sample. 2. Add 0.1 mL of 2 mol/L hydrochloric acid and 0.05 mL of potassium ferrocyanide solution to a further 0.1 mL of sample. 3. Agitate both mixtures for 5 s, leave for 5 min at room temperature and centrifuge for 5 min. Indications

Ferrous salts give a deep blue precipitate with potassium ferricyanide and ferric salts give a deep blue precipitate with potassium ferrocyanide. The LOD for both ferrous and ferric salts is about 10 mg/L.

10 mL glass tube and slowly pour 0.5 mL of carminic acid solution down the inside of the tube so that a layer is formed underneath the sample. Indications Borate is indicated by the formation of a blue–violet ring at the junction of the two layers. Note that strong oxidising agents (e.g. bromates, chlorates, iodates and nitrites) also give positive results. Bromides Reagents

1. 2 mol/L nitric acid 2. 10 g/L solution of silver nitrate 3. Concentrated ammonium hydroxide. Method

1. To 1 mL of clear tests sample add 0.5 mL of 2 mol/L nitric acid and mix for 5 s.

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2. Add 0.1 mL of silver nitrate solution. 3. Centrifuge any precipitate and decant off the supernatant. 4. Add 0.1 mL of concentrated ammonium hydroxide.

3. Calcium chloride (solid) 4. Powdered silica. Method

1. Saturated solution of fluorescein in aqueous acetic acid (600 mL/L) 2. Concentrated sulfuric acid 3. Solid potassium permanganate.

1. To 5 mL of sample in a porcelain crucible add 100 mg of calcium chloride and gently evaporate to dryness over a burner. 2. Destroy the organic material by strong heating to leave a white ash. 3. Mix the residue with 200 mg of powdered silica. 4. Apply a drop of sodium chloride solution to a microscope slide, add 1 mL of concentrated sulfuric acid to the contents of the crucible and immediately position the slide such that the sodium chloride drop is suspended over the crucible. 5. Rest a small beaker of ice on the slide and heat the crucible gently for 5 min over a burner. 6. Remove the slide after 5 min and examine the sodium chloride solution under a microscope.

Method

Indications In the presence of fluorine-containing compounds, silicon

Indications

Bromide gives an off-white precipitate that is slightly soluble in ammonium hydroxide. A white precipitate that dissolves in ammonium hydroxide indicates chloride and a yellowish precipitate insoluble in ammonium hydroxide suggests iodide. The LOD for bromide is 50 mg/L. Confirmatory test Reagents

1. Soak a strip of filter paper in fluorescein solution. 2. Transfer 2 mL of test solution to a 10 mL glass tube and add about 2 mg of potassium permanganate. 3. Add 0.2 mL of concentrated sulfuric acid and hold the fluorescein paper at the mouth of the tube. Indications Any bromide is oxidised to free bromine, which then reacts

with the fluorescein dye (yellow) to give tetrabromofluorescein (eosin) which has a pink–red colour. The LOD for bromide is 50 mg/L. Chlorate Chlorates and other oxidising agents can be detected by the diphenylamine test described previously. The following tests can also be applied. Test 1 Reagents

1. 6 mol/L sulfuric acid 2. 1% w/v aqueous solution of indigo carmine 3. Solid sodium sulfite. Method To 1 mL of sample add 4 mL of 6 mol/L sulfuric acid followed by 1 mL of indigo carmine reagent. Indications A deep blue colour indicates the presence of chlorates. The colour fades after adding several crystals of sodium sulfite. The same reactions are given by bromates and hypochlorites.

tetrachloride is produced, which dissolves in the sodium chloride solution to form sodium silicon tetrafluoride. This forms small hexagonal crystals as the water evaporates from the slide, which sometimes have a pink hue. The crystals are seen at the edge of the drop and appear before the larger cubic crystals of sodium chloride. Test 2 Reagents

1. Concentrated sulfuric acid 2. Calcium hydroxide (solid) 3. Paraffin wax. Method

1. Repeat steps (1) and (2) described for test 1. 2. Smear a film of paraffin wax on one side of a glass microscope slide and expose part of the surface by making an identifiable sign on the paraffin film. 3. Add 5 mL of concentrated sulfuric acid to the crucible and cover it with the slide, with the paraffin film on the inside. 4. Heat the crucible gently for 20 min and then remove the slide. 5. Remove the paraffin film with toluene and examine the slide. Indications Hydrogen fluoride is generated from fluorine-containing compounds and etches the glass to give a mark corresponding to that made in the paraffin film. Both tests have a LOD of approximately 100 mg/L of fluoride.

Test 2 Reagents

1. Manganous sulfate reagent: a 1 : 1 mixture of saturated aqueous manganous sulfate and orthophosphoric acid 2. A 10 g/L solution of diphenylcarbazide in methanol. Method

1. Add 0.2 mL of manganous sulfate reagent to 0.1 mL of test sample and warm the mixture gently over a burner. 2. Cool and add 0.1 mL of diphenylcarbazide solution. Indications Chlorate yields a purple colour that intensifies after cooling

and adding diphenylcarbazide. A similar reaction is given by persulfates and periodates. Note: after ingestion of chlorate the blood turns brown owing to the formation of methaemoglobin by oxidation of the ferrous ion of haemoglobin. Add 0.2 mL of a 10% (w/v) of aqueous potassium cyanide solution to 1 mL of blood and an immediate red colour confirms the presence of methaemoglobin. Exposure to a wide range of other substances (e.g. aniline, nitrites, nitrates, aniline, dapsone, benzocaine, urea herbicides) causes methaemoglobinaemia.

Hypochlorite Test 1 Reagents

1. Glacial acetic acid 2. 50 g/L aqueous lead acetate solution. Method

1. To 1 mL of test solution add acetic acid dropwise to reach a pH of approximately 6 (test with universal indicator paper). 2. Add 0.5 mL of lead acetate solution and boil for 3 min. Indications Hypochlorite forms a brown precipitate. An immediate

brown to black precipitate is given by sulfides with lead acetate. Test 2 Reagents

1. 100 g/L aqueous potassium iodide solution 2. Glacial acetic acid 3. Solid starch. Method

Test 1

1. To 0.1 mL of test solution add 0.1 mL of acetic acid followed by 0.1 mL of potassium iodide solution. 2. Mix and add about 20 mg of starch.

Reagents

Indications Hypochlorite gives a blue colour. (Note: hypochlorite also

1. A 50 g/L aqueous solution of sodium chloride 2. Concentrated sulfuric acid

gives a positive reaction in the diphenylamine test for oxidising agents described above.)

Fluoride

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Appendix 30.4 Iodides Reagents

1. 10 g/L aqueous silver nitrate solution 2. 2 mol/L nitric acid 3. Concentrated ammonium hydroxide. Method

1. To 1 mL of clear test solution add 0.1 mL of nitric acid and 0.1 mL of silver nitrate solution. 2. Centrifuge down any precipitate, remove the supernatant and add to it 0.1 mL of ammonium hydroxide. Indications

A curdy yellow precipitate that is insoluble in ammonium hydroxide is given by iodides. Chlorides give a white precipitate that dissolves in ammonium hydroxide, and an off-white precipitate that is sparingly soluble in ammonium hydroxide suggests bromides. Confirmatory test

495

2. To 1 mL of test sample in a test-tube add 3 mL of sulfuric acid and insert the lead acetate paper into the neck of the tube. 3. Heat the tube in a boiling water-bath for 5–10 min. Indications

Hydrogen sulfide fumes that turn lead acetate paper black are produced by sulfides. The LOD for sulfide is 50 mg/L. Thiocyanates Reagent

A 50 g/L aqueous solution of ferric chloride. Method

Mix 0.5 mL of ferric chloride solution with 0.5 mL of sample. Indications

A deep red colour is given by thiocyanates. The LOD for thiocyanate is 50 mg/L.

Reagents

1. 2 mol/L hydrochloric acid 2. 100 g/L freshly prepared sodium nitrite solution 3. Solid starch. Method Mix thoroughly about 20 mg of starch with 0.1 mL of test solution, 0.1 mL of hydrochloric acid and 0.1 mL of sodium nitrite solution in a test-tube. Indications A blue colour confirms the presence of iodide.

Oxalates

References Asselin WMA, Caughlin JD (1990). A rapid and simple colour test for detection of salicylate in whole hemolyzed blood. J Anal Toxicol 14: 254–255. Caughlin JD (1991). A rapid colour test for detection of ethchlorvynol in whole hemolyzed blood. Can Soc Forensic Sci J 24: 111–114. O’Neal CL et al. (2000). Validation of twelve chemical spot tests for the detection of drugs of abuse. Forensic Sci Int 109: 189–201. WHO (1986). Basic Tests for Pharmaceutical Substances. Geneva: World Health Organization. WHO (1998). Basic Tests for Drugs: Pharmaceutical substances, medicinal plant materials and dosage forms. Geneva: World Health Organization.

Reagents

1. 100 g/L aqueous calcium chloride solution 2. 30% (v/v) solution of acetic acid 3. 2 mol/L hydrochloric acid. Method

1. Add 1 mL of calcium chloride solution to 2 mL of test solution and mix. 2. If a precipitate forms, add 1 mL of acetic acid. 3. If the precipitate does not dissolve, separate it by centrifugation and add 1 mL of dilute hydrochloric acid. Indications

A white precipitate that is insoluble in acetic acid but dissolves in hydrochloric acid indicates the presence of oxalates. Confirmatory test Reagents

1. Thiobarbituric acid 2. Concentrated ammonium hydroxide. Method

1. Add 50 mL of test solution to 100 mL of ammonium hydroxide in a micro test-tube and mix thoroughly. 2. Gently evaporate the mixture to dryness over a micro-burner. 3. Add about 200 mg of thiobarbituric acid and reheat gently to about 150
C. Indications Oxalates give a bright red product that is soluble in methanol. The LOD for oxalate is 250 mg/L. Sulfides Reagents

1. 10% (v/v) aqueous solution of sulfuric acid 2. 10% (w/v) solution of lead acetate in boiled and purified water 3. 2 mol/L acetic acid. Method

1. Immerse a strip of white filter paper in a mixture of 10 volumes of lead acetate solution and 1 volume of acetic acid and allow to dry.

Further reading Bamford F (1951). Poisons, Their Isolation and Identification, 3rd edn. London: Churchill. Bentley KW (1954). The Chemistry of Morphine Alkaloids. Oxford: Clarendon Press. Clarke EGC (1962). The isolation and identification of alkaloids. In: Lundquist F, eds. Methods of Forensic Science, Vol. 1. London: Wiley, 1–241. Enders PW (1985). A simple color test on quaternary ammonium compounds. In: Brandenberger H, Brandenberger R, eds. Reports on Forensic Toxicology. Mannedorf: Branson Research, 195–198. Fiegl F (1966). Spot Tests in Organic Analysis, 7th edn. Amsterdam: Elsevier. Fiegl F, Anger V (1972). Spot Tests in Inorganic Analysis, 6th edn. New York: Elsevier. Flanagan RF et al. (1995). Basic Analytical Toxicology. Geneva: World Health Organization. Gonzales TA et al. (1954). Colour reactions for the identification of non-volatile organic poisons. In: Legal Medicine, Pathology and Toxicology, 2nd edn. New York: Appleton–Century–Crofts, 1191–1255. Johns SH et al. (1979). Spot tests: a colour chart reference for forensic chemists. J Forensic Sci 24: 631–649. Johnson CA, Thornton-Jones AD, eds. (1966). Drug Identification. London: Pharmaceutical Press. Kaye S (1980). Handbook of Emergency Toxicology, 4th edn. Springfield, IL: Charles C Thomas. Musshoff F et al. (2000). Hallucinogenic mushrooms on the German market – simple instructions for examination and identification. Forensic Sci Int 113: 389–395. Saker EG, Solomons ET (1979). A rapid inexpensive presumptive test for phencyclidine and certain other cross-reacting substances. J Anal Toxicol 3: 220–221. Sangalli BC (1989). A new look at qualitative toxicology. Spot tests in the emergency department. Vet Hum Toxicol 31: 445–448. Stair E, Whaley M (1990). Rapid screening and spot tests for the presence of common poisons. Vet Hum Toxicol 32: 564–566. Sunshine I, ed. (1975). Methodology for Analytical Toxicology. Cleveland: CRC Press. US Department of Justice (1978). NILECJ Standard for Chemical Spot Tests for Preliminary Identification of Drugs of Abuse. Washington DC: US Department of Justice. US Department of Justice (1981). NILECJ Standard for Chemical Spot Tests for Preliminary Identification of Drugs of Abuse. Washington DC: US Department of Justice.

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CHAPTER

31

Immunoassays RS Niedbala and JM Gonzalez

Introduction The first immunoassay principles were published decades ago (Yalow, Berson 1959). Since that time immunoassays have become a routine part of day-to-day life for scientists in various disciplines as well as for general consumers. For example, every over-the-counter pregnancy and fertility test is based upon immunoassay results. The impact has been even greater in broader fields where analysts use immunoassays for the routine screening of samples for a host of target analytes. Fields such as veterinary medicine, environmental testing, anti-terrorism and human clinical diagnostics all use various types of immunoassays (Huckle, Wild 2005). For instance, all the applications that exist, immunoassays have limitations that are generally created by the antibodies used. Antibodies may have poor specificity that results in cross-reactivity with structurally similar compounds, creating false results. Antibodies may also be affected by the matrix used. For example, hair testing for drugs of abuse is challenged by the treatments that consumers use on their hair and scalp. It is also difficult to generate antibodies reliably against target compounds. It requires a ‘make it and test it’ scenario that can take years. Nevertheless, immunoassays have become an integral part of routine clinical testing. When developed carefully, immunoassays can detect minute amounts of target analyte in complex samples. They can be robust and capable of working in harsh conditions along the roadside or in remote areas. Immunoassays will remain an important tool for anyone who needs to detect analytes reliably in a cost-effective and rapid manner. The focus of this chapter will be to explain the principles of immunoassays and practical considerations in applying them in the field of toxicology. Immunoassays can generally be constructed for target molecules with molecular weights as low as 250, as well as for large, complex proteins and cellular components. Each immunoassay is formatted to the usage situation and its analytical requirements. Some are designed to be highly sensitive in detecting and quantifying extremely small amounts of target analyte. Others are designed only for qualitative analysis to detect the presence or absence of analytes. Thus, researchers and developers have refined immunoassays to become reliable, robust, accurate tests that can be manufactured on a large scale. Parallel to its development, and in response to it, societies have embraced the use of immunoassay technologies. For example, many countries have enacted legislation defining cut-offs as well as specific targets for immunoassays aimed at the detection of drugs of abuse in various situations where individuals or the general public may be at risk (US Department of Health and Human Services 2006; US Department of Transportation 2000). These legislated assay parameters regulate the implementation of millions of tests that use urine, saliva, hair or blood as matrices. The results from an immunoassay test followed by confirmation using alternative technologies can be used as forensic evidence in a court of law. In several countries individuals are tested for drug use prior to starting a new job, for suspicion of use during employment (Chapter 3) or following treatment or incarceration. Other countries are seeking to allow police to perform roadside testing of suspected drivers who may be under the influence of drugs of abuse (Parliament of Victoria, Australia 2003) (Chapter 5). Efforts are under way to develop and validate robust on-site immunoassays that can meet specific requirements of law and technical performance. Although they are not 496

yet available for all desired target drugs, much progress has been made (Maes et al. 1999; Moeller et al. 1999; Samyn et al. 1999b; Verstraete, Puddu 2000; Walsh et al. 2004). Immunoassays may take a variety of formats, although they all contain common elements regardless of design (Table 31.1) The first element is an antibody that has been targeted against a specific analyte. A target analyte can be a large molecule, an infectious agent or a small molecule that can elicit an immune response. The quality of the antibody will ultimately determine the potential performance of any immunoassay. The antibody is relied upon to be specific for the desired target analyte, insensitive to the test matrix being used, and stable over long periods of storage when packaged into an immunoassay test kit. The second element in an immunoassay is the reporter that will generate a signal that can be used to determine the immunoassay results. A variety of systems based on radioactive, enzymatic and chemiluminescent labels as well as colloidal particles are available for this task (Table 31.2). Each has advantages and disadvantages in immunoassay design and use. These strengths and weaknesses will be discussed later in this chapter. Once a quality antibody and a reporter have been identified, the platform for performing the immunoassay may be selected. There are many commercial platforms that are proprietary to corporations. However, basic researchers can utilise microtitre plates or rapid test formats such as lateral flow (Peace 2000; Niedbala et al. 2000; Perrigo, Joynt 1995; The Walsh Group 2002; Verheijen 2002; Volkov et al. 2009). Finally, the buffers and packaging for any immunoassay must meet the requirements for stability and storage of the test. Buffers also serve another key purpose, which is to prepare the sample matrix to be tested for compatibility with the antibody and test format being used. Various issues confront an immunoassay test developer when trying to use samples as diverse as urine or saliva. Each matrix has unique characteristics that can interfere with an immunoassay causing falsenegative or false-positive results. Immunoassay developers have the challenge first of developing and then of thoroughly validating any test before it is used by collaborative researchers or the general public. When specifically evaluating the performance of a toxicology assay it is worth examining what makes a good immunoassay test. First of all the purpose of the test should be considered. For instance, is the test to be used for qualitative screening or for monitoring concentrations of a particular drug in biological fluids? The difference in analytical requirements between, for example, a test for therapeutic drug monitoring (TDM) purposes and a qualitative screening test can be substantial. A TDM test is performed to optimise the level of drug therapy and therefore the immunoassay test result must be very accurate. In some cases, changing the dose of the drug incorrectly can result in serious or fatal outcomes (Baselt 2005; Hardman et al. 2001; Porter 2006; Uhlenhuth et al. 1990) (Chapter 2) Conversely, a screening test must simply detect the presence or absence of a target compound or a class of compounds reliably above or below a designated cut-off concentration. In such tests the cut-off concentration is specified; however, the actual performance of the test will have some variation around the cut-off. It is worth discussing two aspects of immunoassay design and performance. The terms sensitivity and specificity are often used and require some explanation. These terms relate to analytical sensitivity/specificity or clinical sensitivity/specificity. The term ‘analytical sensitivity’ is used to describe the assay’s ability

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Table 31.1 Elements of an immunoassay Element

Purpose

Representation

Antibody

Targets analyte

497

principles described below should allow researchers to achieve optimal performance for any immunoassay they may develop or evaluate.

Basic principles and issues of immunoassays Antibody development and production

Reporter

Amplifies result

Hapten derivative

Drug derivative to link to reporter

Analyte

Target for assay

Buffers

Conditions, pH Sample

Solid phase

Location to immobilise antibody

Matrix

Describes type of sample being analysed

to detect the lowest level of target drug while ‘analytical specificity’ describes how precise it is at targeting a compound. ‘Clinical sensitivity’ describes how reliably a test identifies positives among a tested population, while ‘clinical specificity’ is a measure of whether or not identified positives are truly positive. For example, in the case of a screening test for drugs of abuse, a test that was clinically 100% sensitive and specific would have correctly identified all presumptive positives and all of them would have been confirmed positive for the target drug by an alternative and completely specific technique. The reality is that no screening immunoassay test is 100% accurate in identifying positives or negatives. In the case of drugs of abuse assays, positives are confirmed by other techniques such as gas chromatography–mass spectrometry (GC-MS). This is often expensive, but necessary, since antibody-based immunoassays are often subject to interferences or lack target analyte specificity. A well-designed screening immunoassay will therefore have a high degree of clinical specificity, meaning that positive immunoassay results are likely to be confirmed quickly and cheaply by GC-MS, thereby minimising costs by identifying samples that do not contain the analyte of interest. A final consideration before exploring the specifics of immunoassay is the metabolism and disposition of any target analyte drug and the matrix in which it is to be detected or quantified. Toxicological assays are increasingly performed on a variety of matrices including hair, saliva, blood or urine. The reason for choice of matrix may be ease of collection (saliva), a long window of detection (hair), compliance with regulations (urine) or forensics (stool or blood). Prior to developing an assay, it is critical to know which metabolites are the most prevalent and reliable as target analytes in a given type of sample. References are available to help any immunoassay developer outline their strategy for development (Baselt 2005). As immunoassay use has expanded, so have the tools and the understanding of the principles for developing them. Application of the Table 31.2 Various reporters used in immunoassays Reporter

Sensitivity achieved in immunoassay (molecules detected)

Enzymes

107

Fluorescence

106

Chemiluminescence

105

Radioimmunoassay

105

The development of an immunoassay for opiates (diamorphine, morphine, etc.) serves as a good example with which to describe in detail the principles and issues of immunoassay development. Diamorphine, morphine and related opiates are used throughout the world and their benefits and deleterious effects on health are well known (Baselt 2005; Hermes 1993). However, developing an immunoassay to identify specific opiates can be tedious and requires an appropriate strategy. The first step when developing such an assay is to consider the target molecule and the human matrix that will be tested. Diamorphine and morphine have been thoroughly studied and their pharmacokinetics are well understood (Moore et al. 1984). Figure 31.1 shows the metabolism of diamorphine and the major metabolites formed. It can be seen that morphine is a primary metabolite in urine, saliva and blood. It also has a relatively long half-life and is known to be stable in biological matrices. Therefore, morphine becomes the target analyte against which antibodies will be needed. Antibodies are proteins in mammals with a primary purpose to fight infection. They are generated by beta-lymphocytes following exposure to an immunogen. Immunogens are materials such as foreign proteins or cells that trigger the immune response. There are several antibody subtypes including IgG, IgA, IgM, IgE and IgD. Each functions within various compartments of the human body and may act to attack new infections or as sentinels if an infection reappears. For our purpose, antibodies are deliberately generated by introducing a designed immunogen (or antigen) into a host cell or animal. Beta cells within the host respond to the antigen by producing antibodies with affinity for the target antigen. Antibodies may be generated using a number of methods. The two major techniques produce monoclonal or polyclonal antibodies (Howard, Kaser 2007; Levine 2003). Polyclonal antibodies are often produced in rabbits, sheep, chickens and even llamas (Frenken et al. 2000). Each species has particular characteristics. Sheep, for example, may be bled to yield substantial quantities of blood for large-scale needs. Chickens are easy to use since antibodies may be isolated from eggs. No matter what the choice of species, the disadvantage of polyclonal antibodies is that there may be a mixture of antibody subtypes isolated with varying performance. In contrast to polyclonal antibodies, monoclonals are produced following fusion of a polyclonal cell with bacterial cell lines (Kohler, Milstein 1975). The fused line will produce a highly specific antibody that is immortalised in the cell line. Thus, supply is never an issue as long as the cell line is carefully maintained. Newer methods that are alternatives to traditional polyclonal and monoclonal antibody production include phage display as well as targeted engineering of protein domains with antibody-like attributes (Binz et al. 2005; Chiswell, McCafferty 1992; Ryan 2003). These newer techniques have primarily been used against large proteins and have not yet been developed sufficiently for use against small drug molecules. Following the example of morphine, an immunogen must first be developed that will be used to trigger production of antibodies. Morphine is a small molecule and, by itself, is not immunogenic. Usually a compound must have a molecular weight greater than 2000 before it will trigger an immune response. To solve the problem of immunogenicity, a morphine derivative is first linked to a carrier protein by a process known as haptenisation. The aim of this haptenisation process is to conjugate multiple, chemically modified, or derivatised, drug molecules (the hapten) to the carrier protein. Figure 31.2 shows the structure of morphine. Examining the chemical structure, it can be seen that morphine has amino or hydroxyl groups that can be used as sites for conjugation. The specificity of the antibodies generated can be determined by the position on the molecule used for conjugation. The site of conjugation is usually hidden from the immune system, so that changes made to the molecule at this position will have no effect on antibody binding. In the case of morphine, if an

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Immunoassays O CH3C N

HO

CH3

N

O

CH3

O

CH3C

CH3C

O

O

heroin

6-acetyl morphine Glucuronide

HO

HO NH

O

HO normorphine

N

CH3

O

HO O morphine

Figure 31.1 Structure of diamorphine (heroin) and its major metabolites.

immunogen is produced via conjugation with the hydroxyl group at position 3, this ceases to be a determinant against which antibodies are generated. As a result, the antibodies are likely to have cross-reactivity towards the major urinary metabolite of both diamorphine and morphine, morphine-3-glucuronide, and they will also recognise codeine (3-O-methylmorphine). Urine screening immunoassays to detect abuse of diamorphine or morphine usually employ antibodies raised by this means. Conversely, conjugation via the hydroxyl group at position 6 will yield antibodies that have a greater specificity towards morphine relative to morphine-3-glucuronide and that also display good cross-reactivity towards 6-monoacetylmorphine and morphine-6-glucuronide. These antibodies are well suited to formulating saliva assays for detecting diamorphine abuse, since this sample contains large amounts of 6monoacetylmorphine as well as of morphine itself. Various methods are available to covalently couple through these groups or to add an extension linker prior to linking it to a carrier protein (Van Regenmortel et al. 1988). Carriers include large proteins such as bovine serum albumin (BSA), bovine gamma globulin or keyhole lymphocyte cyanin. A plethora of other possible carrier proteins exists, but in general the selected carrier proteins are dissimilar in structure to the reporter to be used in the assay and large enough to trigger the immune response. Once antibodies are generated they are usually purified using simple precipitation techniques or by isolation using protein-A. Protein-A is a lectin that specifically binds to antibodies and allows rapid isolation of the purified proteins (Hober et al. 2007). Immunoassay design The antibody is a key element in the construction of any immunoassay and requires detailed consideration. Once an antibody is available it must be evaluated in the format in which it will be used. Assay formats are generally divided into two categories. The first is called a heterogeneous immunoassay, in which the differentiating characteristic is the requirement of a step

to separate bound from free material. The alternative is called a homogeneous immunoassay and is distinguished from heterogeneous assays by the fact that it does not require a separation step. Figure 31.3 shows the basic elements of a heterogeneous immunoassay. Heterogeneous immunoassay/examples The simplest form of heterogeneous immunoassay involves a competitive assay using a solid phase. Most commonly, a microtitre plate coated with immobilised antibodies against the target drug can be used. In this assay format, sample, buffer and a hapten–drug–reporter conjugate are added to a microtitre well. The mixture is incubated for some time during which the conjugate and free drug compete to bind to the solid phase. The solid phase is then washed to remove unbound material. Substrate is added, after which the signal is measured in each reaction well. The signal generated is inversely proportional to the concentration of free drug in the sample. Enzyme-linked immunosorbent assay (ELISA) is the most common format used for the detection of large molecules or proteins, but it has also been adapted to detect small-molecule drugs. These types of heterogeneous assay are sometimes called ‘sandwich’ assays because the target analyte is captured between two antibodies. Figure 31.4 shows a diagram of a typical heterogeneous sandwich assay. In the first step, an aliquot of sample or calibrator is mixed with buffer and incubated with the capture antibody that has been conjugated or adsorbed onto a solid surface such as a microtitre plate. After Analyte

Reporter–hapten conjugate Antibody

Solid phase Figure 31.2 Structure of morphine.

Figure 31.3 Basics of an immunoassay.

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Step 1

Large molecular weight analyte

Step 1

Capture antibody on solid phase

Antibody

Analyte

Hapten on solid phase

Step 2

Incubate + Wash Step 2

499

Reporter Anti-antibody

Incubate + Wash

Enzyme Secondary antibody

Incubate + Wash

Step 3 Add substrate

Colour is produced

Step 3

Incubate + Wash

Add substrate

Color is produced Step 4

Measure signal generated

Figure 31.4 The basic scheme for an ELISA assay. Note the wash between steps. This is the distinctive feature of a heterogeneous assay.

incubation, the surface is washed to remove whatever material did not specifically bind to the antibodies on the solid surface. In the second step, another solution is added that contains a secondary antibody, often labelled with a reporter. Following this incubation, the surface is again washed to remove unbound material. Next, a substrate solution or developer solution is added to generate a signal. Once sufficient signal has been generated, the reaction is stopped and the signal is measured. The amount of signal generated is proportional to the amount of target material that was captured on the surface. Heterogeneous assays are often extremely sensitive and may be titred by adjusting the concentration of reagents and sample. The disadvantages of heterogeneous assays include the long incubation periods and multiple wash steps. In the field of toxicology a number of heterogeneous assay techniques have become available and are in routine use. These techniques include ELISA, radioimmunoassay (RIA), chemiluminescent immunoassay (CIA), fluorescent immunoassay (FIA) and finally lateral flow assay (LFA). Figure 31.5 shows a scheme for a typical heterogeneous assay targeting a small-molecule drug. In the first step, sample and buffer are mixed with anti-drug antibodies. If target drug is absent, the antibody will bind to the drug linked to the solid phase. The solid phase can be a microtitre plate, bead or membrane made of nitrocellulose, polymers or glass. Once this initial incubation is complete, the surface is washed and a

Step 4 Measure signal generated Figure 31.5 The scheme shows a typical competitive solid-phase immunoassay commonly used to detect drugs of abuse. It is a heterogeneous format using washing steps to remove materials that did not bind immunologically. Signal is inversely proportional to the concentration of free drug in the sample.

second reagent is added containing reporter-labelled antibodies against the first antibody targeting the drug of interest. A sandwich is formed between the drug conjugate on the solid phase and the secondary antibody–reporter conjugate. After incubation, unbound secondary antibody is washed away and substrate or signal is measured in the reaction mix. The signal detected is inversely proportional to the concentration of drug in the sample. There are a number of variations on the two assay schemes described above. In most cases the differences are in either the reporter used or the solid surface (see Table 31.2). Various reporters used include radioactive (RIA), fluorescent (FIA) and chemiluminescent (CIA) labels. The key to each heterogeneous assay is the wash step that removes excess reagent and lowers the background signal from the sample or other interferents. In this way heterogeneous assays achieve maximum analytical sensitivity. Lateral flow is a type of heterogeneous assay that has become prevalent in emergency rooms and laboratories and in law enforcement to test on-site for drugs of abuse (Ulti-Med 2002). These tests are capable of multiplexed detection of a panel of drugs of abuse from a single aliquot of urine, blood or saliva (Inoue, Seta 1992; The Walsh Group 2002). The basic scheme for a multiplexed lateral flow assay is shown in Fig. 31.6. A lateral flow assay is constructed from various materials that are assembled to form a test strip. A sample pad is the first component and is where a small aliquot, usually 50–100 mL, of sample is placed onto the strip. The sample pad often contains buffer salts to condition the sample to the correct pH and sometimes to remove cells or debris. These pads are often glass fibre or cellulose (Verheijen 2002; Volkov et al. 2009). The next material beyond the sample pad is the conjugate pad. It will usually hold the reporter–antibody conjugate targeting each drug of interest. The reporter most commonly used is colloidal gold. Gold particles are small, usually 10–100 nm in size, but are easily seen with the naked eye as they accumulate in a detection zone on a strip. Purified antibodies can easily be adsorbed onto the surface (Verheijen 2002; Volkov et al. 2009). Once a sample is added to a test strip it flows through the sample pad, the conjugate pad and then onto a nitrocellulose strip. Nitrocellulose as a raw material acts to bind capture

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Immunoassays Sample applied to absorbent pad Assay reagents

Sample wicked up into absorbent pad

Lateral flow membrane Control lines Test lines (target capture) Figure 31.6 The figure shows the basic design of a lateral flow assay. It contains a series of material in linear order designed to contain reagents that flow along with the sample past capture zones. The immunological result is read in each capture zone usually by visual interpretation.

reagents and to allow liquids to flow by capillary action along a test strip. The nitrocellulose strip can vary in length according to the desired goals of the test. For example, a longer strip will allow multiple analyte test lines (see Fig. 31.6). However, it will take longer for fluids to move along the strip, thus increasing the time of the assay. The nitrocellulose is also where the immunological reaction takes place. Capture zones at the control line and test lines are created by spraying a solution of the capture protein derivative onto the nitrocellulose. The proteins then adsorb onto the nitrocellulose and become immobilised (see Fig. 31.6). The usual capture zone material is target hapten derivatised and conjugated to a carrier protein such as BSA. As the buffered sample and reporter antibody reach the reaction zone there is competition between drug in the sample and hapten conjugate on the strip to bind the reporter antibody. If there is a large concentration of drug, there will be little reporter conjugate available to bind to the reaction zone and the line will have no colour. If the sample is negative for drug, the line at the capture zone will be intense and easily seen with the naked eye. Use of lateral flow assays has grown exponentially in the last decade. Once the basic chemistry has been developed, the system must be packaged into a housing (Fig. 31.7). The housing may be integrated into a urine cup, dip stick or a saliva collector (Draeger Safety UK Ltd 2008; Orasure Technologies 2008). The challenge for lateral flow assays used in toxicology is that they must reliably detect a panel of drug targets using dynamic flow, and binding of reagents all moving through a series of striped materials is governed solely by capillary forces. From a purely analytical perspective, lateral flow assays are often imprecise and insensitive. It is not uncommon for lateral flow assays to have relative standard deviations (RSDs) of 15–30% when testing replicates of sample or calibrator. Also, numerous field evaluations have highlighted performance shortcomings (Peace 2000; The Walsh Group 2002). Even so, lateral flow is an accepted method used by those performing on-site testing of drugs of abuse. It is expected that, as commercial use continues to grow, new instrumentinterpreted methods will improve the performance of lateral flow assays (Alverix Inc. 2010; Faulstich et al. 2007). Visual QC window (control lines)

Sample port

Test window (test lines) Side view Figure 31.7 Lateral flow assay in a housing.

Homogeneous immunoassay/examples Homogeneous immunoassays, by definition, do not require a step to separate bound from free target analytes and reagents. In most cases, homogeneous assays are used on automated platforms that require little operator interaction. This is something of a disadvantage since such equipment is not easily portable. Thus, homogeneous assays are used routinely on large laboratory-based instrument platforms capable of analysing hundreds or thousands of samples per day. The availability of these assays at a very low cost has revolutionised immunoassay-based drugs of abuse testing. Homogeneous drug assays are often competitive assays where free drug in a calibrator or sample competes with a reporter conjugate to bind to a specific antibody on the target. After a brief incubation, the change in signal is measured spectrophotometrically, fluorescently or through chemiluminescence. The following are descriptions for some homogeneous assay methods available for drugs of abuse testing. The EMIT (enzyme-multiplied immunoassay technique) has been a staple in drug testing since the 1970s when the method was first developed (Kabakoff, Greenwood 1981; Rubenstein et al. 1972; Ullman 1994; Ullman, Maggio 1980). An EMIT assay is a competitive format wherein hapten-labelled glucose-6-phosphate dehydrogenase (G6PDH) competes with free drug in a sample or calibrator to bind to a specific antibody raised against the hapten target. If the hapten-G6PDH binds to the antibody, there is a conformational change in the enzyme that leads to a decrease in activity (Fig. 31.8). This change in activity is proportional to the concentration of free drug present in the sample. EMIT assays have been developed for a large number of drugs of abuse and TDM analytes. Over time a wide variety of instrumentation has also been made available to laboratory workers, allowing EMIT to quickly become a method of choice. However, it is worth noting that, as with all immunoassays, EMIT assay performance is dependent upon the antibody used. In addition, EMIT assays are subject to interference from sample adulteration and non-specific cross-reactivity (Colbert 1994; Rollins et al. 1990; Rossi et al. 2006). Somewhat comparable in operating principles, CEDIA (cloned enzyme donor immunoassay) relies on the modulated activity of mutant beta-galactosidase. (Henderson et al. 1986) developed a genetically engineered form of beta-galactosidase which exists as two components. One fragment is called the enzyme acceptor (EA) and the other is called the enzyme donor (ED). The mutant enzyme becomes enzymatically active when both the ED and the EA are present. A homogeneous assay can be developed by conjugating a hapten to the ED. The hapten-ED will compete with free drug in the presence of antibody against the hapten (Fig. 31.9). When high concentrations of target hapten-drug are present, the hapten-ED is available to bind to the EA and an increase in betagalactosidase will be detected. Thus, like EMIT, the amount of enzymatic activity is proportional to the concentration of free drug in the sample. Additionally, CEDIA assays can be run on the same automated spectrophotometric equipment that also runs EMIT assays. Other types of homogeneous assays exist and are based upon reporters that are non-colorimetric. In this category, fluorescence

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In the presence of free drug Substrate

Antibody Product

Hapten

Free drug

G6DPH

In the absence of free drug Antibody Substrate Hapten

G6DPH Figure 31.8 EMIT assay scheme; signal is directly proportional to the concentration of analyte.

polarisation, microparticle agglutination and new techniques such as LOCI (luminescent oxygen channelling immunoassays) (Ullman 2005) have all been demonstrated and used routinely in commercial laboratories. Fluorescence polarisation is a relatively simple technique that utilises the competitive binding to antibodies between a hapten–fluoroscein In the presence of free drug

Hapten ED

conjugate and free drug (hapten; Fig. 31.10) (Abbott Laboratories 2005; Colbert et al. 1985; Dandliker et al. 1973). The hapten–fluorescein conjugate rotates rapidly when not bound to an antibody. When it is bound to the antibody, the rotation is slowed dramatically compared with the unbound molecule. To generate the assay signal, a fluorimeter shines light at the excitation wavelength for fluorescein through a vertical polarising filter. Rapidly rotating unbound hapten–fluorescein molecules emit light in a different plane to the incident light. The

In the presence of free drug

Product

Antibody

High rotation/low polarisation Antibody

EA

F Hapten–fluorescein Free drug Substrate Free drug In the absence of free drug In the absence of free drug

Low rotation/high polarisation

Antibody

F Substrate

Hapten ED

Hapten–fluorescein

EA

Antibody Figure 31.9 CEDIA assay scheme; signal is directly proportional to the concentration of analyte.

Figure 31.10 Fluorescence polarisation immunoassay assay scheme; signal is inversely proportional to the concentration of analyte.

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Immunoassays In the presence of free drug Low agglutination/low light scattering Antibody

Micro particle

Hapten

Free drug

In the absence of free drug High agglutination/high light scattering

Micro particle

Hapten

Figure 31.11 KIMS assay scheme signal is inversely proportional to the concentration of analyte.

relatively stationary antibody-bound hapten–fluorescein molecules, however, return light in a similar plane. This is detected via the polarising filter. Drug added via the sample competes for binding to the antibody with the fluorescein-labelled hapten, thereby reducing the amount of fluorescein bound to the antibody, resulting in less emitted fluorescence being detected via the polarised filter. Fluorescence polarisation assays require specialised equipment and are the basis of the Abbott ADx system. The background fluorescence found with many biological samples means that it is necessary to take a blank reading of the sample and reagents before the addition of the fluorescent tracer to the mixture. Another popular homogeneous technique used for drugs of abuse assays is based upon microparticle agglutination. The KIMS assay (kinetic interaction of microparticles in solution) relies on the old but reliable principles of latex particle agglutination (Fig. 31.11) (De Giovanni, Fucci 2006; Feldman et al. 2004). In the KIMS technology, polystyrene latex microparticles have been coated with a drug-conjugate. These particles are dispersed in solution, after which sample and antibodies to the drug (hapten)-conjugate are added. In the presence of high drug concentrations, the antibody-binding sites are occupied by free drug and a low amount of agglutination takes place. In the presence of low target drug concentrations, the antibodies bridge between the particles and begin to agglutinate particles into large clusters. These clusters scatter light. Therefore, the change in signal is inversely proportional to the concentration of free target drug in the sample. KIMS is the basis of the Roche Abuscreen Online system, which involves monitoring the rate of agglutination by spectrophotometric means. The challenge to any homogeneous assay technique is sensitivity. These assays usually take seconds to minutes to perform and are often limited by kinetics or antibody quality. LOCI is a chemiluminescent system that overcomes many limitations of other assay reporters (Ullman 2005). The reagents used to produce a signal in the assay are encapsulated in latex spheres which prevent interference from sample matrices. One population of latex spheres contains a chemiluminescer and the second a photosensitiser capable of exciting oxygen when exposed to 680 nm light. When the two beads are in close proximity, excited oxygen diffuses out of the bead and into the chemiluminescer bead, producing detectable photons. The LOCI assay has been tested with a variety of analytes and each one has shown superior sensitivity to other competing technologies (Ullman 2005).

Immunoassay optimisation Earlier in this chapter the methods used to develop an antibody against a chosen hapten drug target were reviewed. Assuming that a viable candidate antibody has been generated and an assay format chosen, the next step is to begin developing and optimising the immunoassay. The goal of this section is to outline some of the key experiments followed by an explanation of important assay parameters that should be considered. Again the target analyte to be used as an example in this section will be morphine. The structure for morphine is shown in Fig. 31.1. Morphine is one of a large class of opiate compounds that are often abused. Therefore, the goal of this hypothetical assay is to detect a broad class of compounds in a single sample. The format for the assay to be developed is lateral flow and the sample matrix will be saliva. Studies of the metabolism of opiates have shown that both the parent drug and its major conjugated metabolites appear in oral fluids and these have previously been shown to be detectable using a laboratory-based immunoassay (Niedbala, Kardos 2005). This suggests that a lateral flow assay is plausible. To start developing the lateral flow assay, morphine is derivatised through the hydroxyl group before being conjugated to BSA (Verheijen 2002; Volkov et al. 2009). Using a Kinematic Linomat striper, the morphine–BSA conjugate can be adsorbed onto the surface of a 10 mm pore size nitrocellulose sheet by sequentially dispensing small amounts of conjugate to form a capture zone (Verheijen 2002; Volkov et al. 2009). Additionally, a reporter such as colloidal gold, fluorescent latex particles or upconverting phosphors can be labelled with purified anti-morphine as described elsewhere (Niedbala et al. 2000, 2001). Finally, phosphatebuffered saline, N-(2-hydroxyethyl)piperazine-N 0 -2-ethanesulfonic acid (Hepes), or an alternative buffer may be chosen as long as it adequately controls the pH of the final assay mixture without negatively affecting the assay. Once all the reagents are prepared and a standard curve showing a response over various concentrations of morphine has been run, experiments can be conducted to optimise the concentration of each assay component. Usually the components that are varied are the loading of reagents onto the capture zone, the level of reporter conjugate to control detectable signal, and finally the sample volume to obtain the best precision and optimal dynamic range.

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503

80,000 Average peak area

70,000 60,000 50,000

0 ng/mL

40,000

10 ng/mL

30,000

20 ng/mL

20,000

40 ng/mL

10,000 0 A

B

C

Coupling protocol ID Figure 31.12 This graph shows the results from three experiments loading various amounts of morphine-BSA onto a lateral flow strip. The optimum level was reached when the signal at zero concentration of morphine was highest.

For example, Fig. 31.12 shows the effect of three different line striping protocols for the capture zone on the nitrocellulose strip. As the loading of morphine-BSA increased there was a maximum amount of signal that could be generated. Note, however, that the overall displacement between zero and the various concentration of free drug remained about the same. Next, the concentration of reporter conjugate may be optimised. Figure 31.13 shows an example of a lateral flow assay utilising up-converting phosphor conjugates as the reporter. Note that, as the level of reporter increased, so did the signal at 0 ng/mL free morphine. This large separation helps to ensure discrimination between negative and positive samples. Finally, the assay sample size was adjusted and the assay was tested for precision by running replicates at a number of morphine levels. As shown in Fig. 31.14, the assay developed was capable of easily discriminating between 0 and 40 ng/mL morphine, the target concentrations appropriate for an oral fluid-based test. Immunoassay performance parameters

immunoassays for toxicological investigations (Green, Isenchmid 1995; Kwong et al. 1988; Linnet, Brandt 1986). Parameter 1: Precision

Precision is a measure of the variation that occurs either when replicate samples are tested in a single run (intra-assay precision) or when replicate samples are tested and compared between runs (inter-assay precision). Whether intra- or inter-assay precision, the value is always reported as the RSD. It is important to plot the RSDs of any test and look for the deviation around each point tested. In the author’s laboratory it is usual to plot the mean plus 2 standard deviations above and below each assay point on the curve. If it overlaps the standard deviation from the calibrator above, optimisation of the assay is continued to improve performance. It is important that precision data should be examined as either the precision of the signal generated in an assay or transformed numbers on a standard curve. Most toxicology assays are qualitative and therefore use signal only. However, transformed numbers are more stringent and are an excellent way to show robust assay performance. Parameter 2: Limit of detection

Having discussed the major aspects of test reagents and development, it is appropriate now to turn to parameters used to evaluate immunoassay performance. It is important to note that there are a number of ways to determine each of the parameters considered. Those discussed here are relevant to scientists developing or evaluating commercial

Many toxicology screening assays require cut-off levels that challenge the development of any immunoassay. Analytes such as LSD, buprenorphine or tetrahydrocannabinol (THC) must be detected at extremely low concentrations in oral fluids. The assay parameter that indicates the lowest level detectable is the limit of detection (LOD). The LOD for an

Integrated peak area response

80,000 70,000 60,000 50,000 0 ng/mL 40,000

10 ng/mL

30,000

20 ng/mL

20,000

40 ng/mL

10,000 0 4 µg

8 µg

12 µg

16 µg

Amount of phosphor per test Figure 31.13 This graph shows results of varying the conjugate reporter in the lateral flow assay being designed to detect morphine. Signal increased as more conjugate was added. This produced a steeper curve and improved the performance of the assay.

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Immunoassays

Average peak area (n=3)

18,000 16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0 0

10

20

30

40

50

60

70

80

90

Drug concentration [ng/mL] Figure 31.14 Once optimised, the morphine lateral flow assay was tested for precision. This graph shows that the optimised assay had little variation around each calibrator concentration. There was also a large separation in signal between calibrators.

immunoassay is often determined in one of two ways. The first approach is to run replicates of the zero calibrator in an assay and then take the mean plus 2 or 3 standard deviations and plot it onto the assay standard curve. This concentration is then assigned as the LOD. The second method, which is more robust, is to run replicates of a variety of concentrations along the assay curve and, similarly to determining precision, plot 2 standard deviations above and below each point. The lowest concentration that can separate 2 standard deviations above zero and 2 standard deviations below the replicate concentration is then denoted the LOD. Parameter 3: Specificity/cross-reactivity

This parameter is extremely important for toxicology assays. As stated earlier, some assays are targeted for certain compounds, such as THC, while others will ideally detect a broad class of compounds, such as benzodiazepines. In addition, assays should also be free from interference from more common over-the-counter medications. Using the example of a morphine assay, Table 31.3 lists a number of opiates of interest along with their cross-reactivities in an immunoassay. In addition to cross-reactivity, the table also shows calculations for morphine equivalents and the concentration that may produce a positive result. Each of these values for cross-reactivity, equivalents and concentration that may produce a positive result is derived by running various concentrations of potential cross-reactants in the assay of interest and comparing the results against the cut-off calibrator, which, in this case, is morphine. Each of the parameters can be determined by comparing concentrations against the cut-off.

Parameter 4: Interferences/adulteration

Even if an immunoassay is performed correctly, it can provide an incorrect result if the sample was somehow adulterated or contained an interfering substance. The literature or product package inserts for assays that use blood or urine will frequently warn that haemolysis, lipaemia or high levels of protein in a sample can interfere with homogeneous immunoassays. More recently, orally based diagnostic tests have looked at the food or drinks that can interfere with assays, while hair testing has considered the effects of various treatments, soaps or hair colorants. In all of these cases the assay designers must be prepared to realistically evaluate such interferences. At a minimum, immunoassay developers should document and inform users of their assays about potential interferents. Adulterants are related to, but different from, interfering compounds. Interferents are defined as normal day-to-day materials used by individuals that can cause aberrant results. Adulterants are materials wilfully added to a sample in order to disrupt a toxicology immunoassay. Such materials have been used in recent years to target many of the homogeneous assays used to initially screen urine samples for drugs of abuse (Wu et al. 1999). Materials such as bleach (sodium hypochlorite), salts or detergents have been reported to be very effective and their potential for disruption should be evaluated in any newly developed assay. Parameter 5: Stability

Any immunoassay is expected to perform over a relatively long period. Commercial immunoassay kits are usually stable from 6 months to

Table 31.3 Morphine immunoassay cross-reactants Compound

Concentration (ng/mL)

Morphine equivalents (ng/mL)

Percentage cross-reactivity (%CV)

Concentration that may produce a positive result (ng/mL)

6-Acetyl morphine

40

34.8

87.0

46

Codeine

40

24.1

60.3

66

Diacetylmorphine

100

>73.7

>73.7

54

Hydrocodone

100

60.5

60.5

66

Hydromorphone

100

>55.9

55.9

72

10 000

68.8

0.7

5,714

100

80.0

80.0

50

Meperidine b–Morphine-3-glucuronide

1

36.8

>100

1

Normorphine

100

34.3

34.3

117

Oxycodone

100

43.1

43.1

93

Oxymorphone

100

36.3

36.3

110

Nalorphine

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References several years. Laboratory-based tests are somewhat easier to maintain and control, since cold-room storage is not a problem. Point-of-care tests that are used in remote locations without room temperature control or perhaps no facilities at all face some of the greatest challenges to stability. In these cases the stability of a test must always be related to the functional temperature range that can be tolerated. Any immunoassay developer should field test their immunoassay under various conditions of humidity, temperature and even altitude. Limitations can then be reported to potential users of the test. Such studies are difficult and often highlight the limitations of an immunoassay. However, they are essential so that the incidence of false results is minimised in the field or in the laboratory. Parameter 6: Quality control

There are various ways to approach quality control for any immunoassay. Some laboratory methods look at the precision of controls from one run to another. Other immunoassays use the standard curve generated to see whether the spread between calibration points is appropriate. These internal assay controls are all good measures that ensure that the final result is correct. Additional quality control steps should include external controls. Some vendors sell urine, blood or saliva samples that have been spiked at various concentrations. Proficiency testing programmes are a very useful means by which an individual laboratory can ensure that its assays are performing correctly and for detecting any problems with a particular method. These schemes regularly send samples to a large number of participant laboratories to be tested on a blind basis. A number of organisations exist that can supply urine, saliva, hair or sweat proficiency samples. Examples include RTI International, the College of American Pathologists or Cardiff Bioanalytical Services Ltd. Once tested, the results for each proficiency sample are returned to the co-coordinators of the scheme and each participant laboratory receives a report showing how its findings have compared with those of the others (Cone 1992). Immunoassay automation Great strides have been made over the last few decades in the automation of immunoassays. The use of homogeneous assays such as EMIT, CEDIA or KIMS for the rapid analysis of large numbers of samples for drugs of abuse has been described above. In addition, specialised instrumentation has been developed by commercial companies to utilise reporters such as fluorescence, radiolabels and chemiluminesence. In every case these immunoassay platform instruments allow for precise pipetting and incubation of samples. This degree of control is essential in assuring consistent and reliable results. It is interesting to see the overall sensitivity of each system (see Table 31.2). Future systems will be required not only to perform rapid analyses on large numbers of specimens but also to test different specimen types. This is an enormous challenge for immunoassay design and matching instrumentation. Future systems may be as much as 1000 times more sensitive than current techniques. Alternative fluids for toxicology immunoassays The latest area of drug immunoassay innovation has been concerned primarily with applications to alternative matrices. Traditionally, toxicological assays for drugs of abuse have been developed for use with urine, whereas TDM assays almost always utilise blood, serum or plasma. Urine testing remains the dominant matrix for drugs-of-abuse screening, although it is now recognised that this sample can be easily adulterated, requires private collection facilities, and is affected by overhydration (Cone et al. 2003). In recent years, more assays are being performed using fluids or matrices such as hair, sweat and oral fluid. Although considered several years ago, it is only recently that interest in using alternative fluids for routine screening of drugs of abuse has arisen (Cone 1992; Malamud, Niedbala 2007; Samyn et al. 1999a; Schramm et al. 1992; Wong 2008) There are several reasons behind this trend and they vary for different countries. For some there is a desire to allow law enforcement to screen for drugs of abuse at the roadside (Parliament of

505

Victoria, Australia 2003). This is not practical with any specimen except an oral sample (Verstraete et al. 1999c). In other situations, the drug concentration in the sample collected must have some correlation with that in blood so that some idea of impairment can be demonstrated. Again, this has led to the increased use of saliva testing (Cone et al. 2002; Thompson et al. 1987; Toennes et al. 2005) (Chapter 18). Where the goal is to have the longest window of detection to identify drug abuse, hair is the specimen of choice (Chapter 19).

Conclusions Immunoassays for the detection or measurement of drugs and their metabolites are routinely used in commercial laboratories worldwide. Automated immunoassay instrumentation capable of analysing thousands of samples per hour has now become routine. Although there are ways in which immunoassays can be flawed, the great majority perform reliably when used correctly. Future directions in immunoassay development are expected to improve upon methods to develop antibodies and format assays. However, these improvements will still utilise the basic principles outlined in this chapter.

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Kohler G, Milstein C (1975). Continuous culture of fused cells secreting antibody of predefined specificity. Nature 256: 495–497. Kwong TC et al. (1988). Critical issues in urinalysis of abused substances: Report of the substance-abuse testing committee. Clin Chem 34: 605–632. Levine B (2003). Principles of Forensic Toxicology, 2nd edn. Washington, DC: AACC Press. Linnet K, Brandt E (1986). Assessing diagnostic tests once an optimal cutoff point has been selected. Clin Chem 32: 1341–1346. Maes V et al. (1999). Drugs and medicines that are suspected to have a detrimental impact on road user performance. Roadside Testing Assessment (ROSITA) D1 DG VII PL98-3032. www.rosita.org. Malamud D, Niedbala RS (2007). Oral-based Diagnostics. New York: New York Academy of Sciences. Moeller M et al. (1999). Operational user and legal requirements across EU member states for roadside drug testing equipment. Roadside Testing Assessment (ROSITA) D3, DG VII 98-SC.3032. www.rosita.org. Moore RA et al. (1984). Sensitive and specific morphine radioimmunoassay with iodine label: pharmacokinetics of morphine in man after intravenous administration. Ann Clin Biochem 21(Pt4): 318–325. Niedbala RS et al. (2001). Detection of analytes by immunoassay using up-converting phosphor technology. Anal Biochem 293: 22–30. Niedbala RS, Kardos K (2005). Oral fluid drug testing using the Intercept device. In: Wong R, Tse H, eds. Drugs of Abuse: Body Fluid Testing. Totowa, NJ: Humana Press. Niedbala RS et al. (2000). Multiphoton Up-Converting Phosphors for Use in Rapid Immunoassays, 7th edn. Proceedings of SPIE, Vol. 3913. Bellingham, WA: SPIE. Orasure Technologies (2008). OraQuick Advance. Bethlehem, PA: Orasure Technologies. Parliament of Victoria, Australia (2003). Road Safety (Drug Driving) Act. 31-102003. Peace MR et al. (2000). Performance evaluation of four on-site drug-testing devices for detection of drugs of abuse in urine. J Anal Toxicol 24: 589–594. Perrigo BJ, Joynt BP (1995). Use of ELISA for the detection of common drugs of abuse in forensic whole blood samples. Can Soc Forensic Sci J 28: 261– 269. Porter WH (2006). Clinical toxicology. In: Burtis CA et al., eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, 4th edn. St Louis, MO: Elsevier Saunders, 1287–1369. Rollins DE et al. (1990). Investigation of interference by nonsteroidal antiinflammatory drugs in urine tests for abused drugs. Clin Chem 36: 602–606. Rossi S et al. (2006). Characterization of interference with 6 commercial delta9tetrahydrocannabinol immunoassays by efavirenz (glucuronide) in urine. Clin Chem 52: 896–897. Rubenstein KE et al. (1972). “Homogeneous” enzyme immunoassay. A new immunochemical technique. Biochem Biophys Res Commun 47: 846–851. Ryan TA (2003). Fluorescent proteins with ties that bind. Nature Biotechnol 21: 1447–1479. Samyn N et al. (1999a). Analysis of drugs of abuse in saliva. Forensic Sci Rev 11: 1–19.

Samyn N et al. (1999b). Inventory of state-of-the-art road side drug testing equipment. Roadside Testing Assessment (ROSITA) D2, DG VII PL98-3032. www. rosita.org. Schramm W et al. (1992). Drugs of abuse in saliva: a review. J Anal Toxicol 16: 1–9. Thompson LK et al. (1987). Confirmation of cocaine in human saliva after intravenous use. J Anal Toxicol 11: 36–38. Toennes SW et al. (2005). Screening for drugs of abuse in oral fluid – correlation of analysis results with serum in forensic cases. J Anal Toxicol 29: 22–27. Uhlenhuth EH et al. (1990). International study of expert judgement on therapeutic use of benzodiazepines and other psychotherapeutic medications IV: Therapeutic dose dependence and abuse liability of benzodiazepines in the long-term treatment of anxiety disorders. J Clin Psychopharmacol 19: 23S–29S. Ullman EF (1994). Homogenous immunoassays. In: Wild D, ed. The Immunoassay Handbook. New York: Stockton Press, 212-230. Ullman EF (2005). Homogenous immunoassays. In: Wild D, ed. The Immunoassay Handbook, 3rd edn. New York: Elsevier. Ullman EF, Maggio ET (1980). Principles of homogenous enzyme-immunoassay. In: Maggio ET, ed. Enzyme Immunoassay. Boca Raton, FL: CRC Press, 105–134. Ulti-Med (2002). Assay for the qualitative detection of drug of abuse in saliva. St Paul, MN: Ulti-Med. US Department of Health and Human Services (2006). National Survey on Drug Use and Health: National findings. Office of Applied Studies NSDUH Series H-32. DHHS publication No. SMA 07-4293. US Department of Health and Human Services, Substance Abuse and Mental Health Service Administation (SAMHSA) (2008). Mandatory guidelines for fedral workplace drug testing programs. Fedral Registry 73: 71858–71907. US Department of Transportation Procedures for transportation workplace drug and alcohol programs: Final rule 49 CFR Part 40. Federal Register 65: 79462–75579. Van Regenmortel MHV et al. (1988). Laboratory Techniques in Biochemistry and Molecular Biology, Synthetic Polypeptides as Antigens. Amsterdam: Elsevier. Verheijen R (2002). Immunological strip tests. Methods and tools in biosciences and medicine. Anal Biochem 4: 134–166. Verstraete A, Puddu M (2000). General conclusions and recommendations. Roadside Testing Assessment (ROSITA) D5, DG VII 98-SC.3032. www.rosita.org. Volkov A et al. (2009). Rapid prototyping of lateral flow assays. In: Rasooly A, Herold K, eds. Methods in Molecular Biology: Biosensors and Biodection, vol 504. New York: Humana Press, 217–235. Walsh JM et al. (2004). Developing global strategies for identifying, prosecuting and treating drug-impaired drivers. Bethesda, MD: The Walsh Group, sponsored by the US Office of National Drug Control Policy, June 2004. The Walsh Group (2002). An Evaluation of Oral Fluid Point of Collection Testing Devices. Bethesda MD: The Walsh Group. Wong DT (2008). Salivary diagnostics. Am Sci 96: 37–43. Wu AH et al. (1999). Adulteration of urine by “Urine Luck”. Clin Chem 45: 1051–1057. Yalow RS, Berson SA (1959). Assay of plasma insulin in human subjects by immunological methods. Nature 184(Suppl 21): 1648–1649.

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CHAPTER

32

Ultraviolet, Visible and Fluorescence Spectrophotometry J Cordonnier and J Schaep

Introduction and theoretical background General introduction Analytical absorption spectroscopy in the ultraviolet (UV) and visible regions of the electromagnetic spectrum has been widely used in pharmaceutical and biomedical analysis for quantitative purposes and, with certain limitations, for the characterisation of drugs, impurities, metabolites and related substances. By contrast, luminescence methods, and fluorescence spectroscopy in particular, have been less widely exploited, despite the undoubted advantages of greater specificity and sensitivity commonly observed for fluorescent species. However, the wider availability of spectrofluorimeters able to present corrected excitation and emission spectra, coupled with the fact that reliable fluorogenic reactions permit non-fluorescent species to be examined fluorimetrically, has led to a renaissance of interest in fluorimetric methods in biomedical analysis. UV and visible spectrophotometry: theoretical background General considerations

Molecular absorption in the UV and visible regions arises from energy transitions that involve the outer orbital or valence electrons. Spectra in liquid media are usually broad, relatively featureless bands, a result of the large number of closely spaced vibrational and rotational transitions. The fundamental band shape approximates Gaussian or lognormal Gaussian curves. Given the broad, overlapping profiles commonly encountered, the shape and precise location of individual bands are of limited usefulness in qualitative analysis. However, any fine structure detected in the spectra, coupled with solvent and pH effects, can be of diagnostic value. More informative spectra can be obtained for some volatile molecules of toxicological interest, such as benzene and polynuclear aromatic hydrocarbons; when examined in the vapour phase, vibrational and rotational fine structure can readily be seen superimposed on the broad spectral profiles. This is illustrated in Fig. 32.1 for 1,2,4,5-tetrazine. However, most drugs, metabolites and related compounds are relatively non-volatile; their spectra are observed necessarily in solution, or possibly in the solid phase by reflectance, or by compression to form a KBr disc, as used in infrared spectrophotometry. UV and visible spectrophotometry find their primary application in quantitative analysis. The scope of absorption spectroscopy can be extended significantly by the use of colour reactions, often with a concomitant increase in sensitivity and/or selectivity. Such reactions are used to modify the spectrum of an absorbing molecule so that it can be detected in the visible region, well separated from other interfering components in the UV spectrum. Moreover, chemical modification can be used to transform an otherwise non-absorbing molecule into a stable derivative that possesses significant absorption. Spectral selectivity can be enhanced further by a number of chemical or instrumental techniques, which include difference, higher-derivative and dual-wavelength spectrophotometry. Such methods, and certain graphic techniques such as the Morton–Stubbs method, can contribute in different ways to reducing the general problem of spectral interference in quantitative spectroscopy. Spectral interference can arise from socalled ‘irrelevant’ non-specific absorption, and also from absorption by other materials and impurities that may be present. When interference

arises specifically from the spectral overlap of two or more well-defined components, a number of methods can be applied to measure the individual concentrations. These methods include the Vierordt multiwavelength technique, least-squares deconvolution and second- or higher-derivative spectrophotometry. Spectral selectivity, and in some cases detection sensitivity, can be enhanced significantly by the various chemical and instrumental techniques outlined above. Such methods should, of course, be validated by applying the conventional analytical criteria of accuracy (against a reference method), linearity, precision and independence from interfering substances. The scope of UV and visible spectrophotometry can be further extended when combined with a chromatographic separation step, such as high performance liquid chromatography (HPLC). The development of rapid-scanning detectors based on the linear photodiode array permits spectra to be acquired during the elution of peaks. Computer-aided manipulation of these spectra has led to new strategies for the examination of chromatographic peak homogeneity, based on classic techniques in spectroscopy. The use of microcomputers enables the development of archive-retrieval methods for spectral characterisation (Fell et al. 1984). Nomenclature

In the UV and visible spectrum, the energy of photons associated with electronic transitions lies in the range 147–630 kJ/mol. This energy (DE) can be expressed in terms of the principal parameters that define electromagnetic radiation, namely frequency m (Hz), wavelength l (nm) and wavenumber n (cm
1): DE ¼ hm ¼

hc ¼ hcn l

ð32:1Þ

where h is Planck’s constant and c is the velocity of radiation in vacuo. The positions of peaks are sometimes described in terms of wavenumber, which has the advantage of being a linear function of energy, but this term is much more frequently used in infrared spectrophotometry. The practical unit most often used in UV and visible spectrophotometry is wavelength, usually expressed in nanometres (nm). The units that have previously been used for wavelength – millimicron (mm)   and angstr€ om (A ) – are not recommended terms. The position of maximum absorbance of a peak is designated lmax. The wavelength span is conventionally divided into two ranges: the UV extends from 200 nm to about 400 nm; the visible range extends from about 400 nm to 800 nm. Outside these limits, the ‘far UV’ or ‘vacuum UV’ extends from 100 nm to 200 nm, and the ‘near infrared’ from 1 mm to about 3 mm. A molecular grouping specifically responsible for absorption is described as a chromophore, and is usually a conjugated system with extensive delocalisation of electron density. Any saturated group with little or no intrinsic absorption of its own, but that modifies the absorption spectrum when attached directly to a chromophore, is described as an auxochrome, examples being –OR, –NR2, –SR. Auxochromes are considered to exert their effect through partial conjugation of their polarisable lone-pair electrons with those of the adjacent chromophore. If, however, the lone pair of electrons is involved in bonding as, for example, in the case of a protonated quaternary ammonium group, the 507

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508

Ultraviolet, Visible and Fluorescence Spectrophotometry The logarithmic term is linearly related to concentration and path length, and is referred to as absorbance (A). The older terms extinction (E) and optical density (OD) are not recommended, although they are often found in the literature. Transmittance (T ¼ I/I0) and percentage transmittance (%T ¼ 100(I/)0)) are not linear functions of concentration and path length, and can be related readily to absorbance: A ¼ log

Io 1 ¼ log ¼ 2
logð%TÞ T I

ð32:3Þ

The molar absorptivity, e, is a fundamental property of a molecule that tells how much light is absorbed at a particular wavelength. It has two connotations in European usage, and a third according to American convention. If the concentration is expressed in g/L, e is described as the absorptivity (k, L/g per cm). When concentration is expressed in g/100 mL, k is described as the specific absorbance and given the symbol A1% 1 cm or A(1%, 1 cm), defined as ‘the absorbance of a 1% w/v solution in a cell of 1 cm path length’. It is usually written in the shortened form A11 and is widely used in analytical chemistry. It was formerly known as the ‘specific extinction coefficient’, symbol E1% 1 cm or E(1%, 1 cm). American convention recognises the constant k as ‘absorptivity’ (a, L/g/cm) defined as ‘the absorbance of a 1 g/L solution in a cell of 1 cm path length’. These terms for absorptivity can readily be interconverted: a¼ Figure 32.1 UV absorption spectra of 1,2,4,5-tetrazine.

auxochromic effect vanishes. This property can be used for molecular characterisation, as discussed below. Laws of absorption spectrophotometry

The extent of absorption of radiation by an absorbing system at a given monochromatic wavelength is described by the two classic laws of absorptiometry, which relate the intensity of radiation incident on the absorbing system (I0) to the transmitted intensity (I) (Fig. 32.2). Lambert’s (or Bouguer’s) law concerns instrumental factors, and states that, at a given concentration (c) of a homogeneous absorbing system, the transmitted intensity (I) decreases exponentially with increase in path length (b). The complementary Beer’s law deals with concentration and states that, for a layer of defined path length (b), the transmitted intensity (I) decreases exponentially with the increase in concentration (c) of a homogeneous absorbing system. Combination of these observations gives the familiar Beer–Lambert law: log

I0 ¼ cb I

ð32:2Þ

where e is the molar absorptivity or molar extinction coefficient of the system, defined as ‘the absorbance of a one molar solution in a cell of 1 cm path length’. The concentration c is here expressed in mol/L.

A11 ¼ 10 Mr

ð32:4Þ

where Mr is the relative molecular mass. Thus, a compound with an Mr of 100 and absorptivity a of 20 at wavelength l in a particular solvent at a defined pH (if aqueous) and at a specified temperature, has a corresponding specific absorbance A11 of 200 and a molar absorptivity e of 2000. Absorbance and absorptivity are often expressed in logarithmic form in cases where spectra are to be compared. The logarithmic form of the Beer–Lambert law expresses the effects of the molar extinction coefficient (e), concentration (c) and path length (b) as additive terms logA ¼ logþlogcþlogb

ð32:5Þ

The value for log e is typically in the range 1–5. Since only the molar extinction coefficient (e) is a function of l, the shape of a logarithmic absorption curve is independent of concentration and path length. Their only effect is to shift the log A spectrum along the log A axis. A disadvantage of the log A plot is that fine structure near the top of the peak is compressed. Validity of the Beer–Lambert law

The validity of the Beer–Lambert law is affected by a number of factors. If the radiation is non-monochromatic, i.e. if its spectral bandwidth is greater than about 10% of the drug absorption bandwidth at halfheight, the observed absorbance will be lower than the ‘true’ limiting value for monochromatic radiation. Thus, sharp bands are more susceptible than broad bands to absorbance error on this account. Moreover, if the absorbing species is non-homogeneous, or if it undergoes association, dissociation, photodegradation, solvation, complexation or adsorption, or if it emits fluorescence, then positive or negative deviations from the Beer–Lambert law may be observed. Stray-light effects and the type of solvent used may also lead to non-compliance with the Beer–Lambert law. Stray-light effects

Figure 32.2 Attenuation of a beam of radiation by an absorbing solution.

Stray light is radiation at wavelengths different from those desired. It may arise from light scattering or other defects within the instrument, or it may be caused by external radiation. If the stray light is not absorbed, the observed absorbance tends to a constant value as the concentration of drug is increased, yielding a negative deviation from the Beer– Lambert law.

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Introduction and theoretical background

509

Stray-light errors are more likely to be observed near the wavelength limits of an instrument, at which the radiation intensity of the source and the efficiency of the optical system are reduced, especially below 220 nm and at the crossover point between the UVand the visible lamps (about 320–400 nm). Errors may become serious if the solvent absorbs strongly or if a strongly absorbing sample is measured by difference spectrophotometry. Solvent effects

The solvent often exerts a profound influence on the quality and shape of the spectrum. For example, many aromatic chromophores display vibrational fine structure in non-polar solvents, whereas in more polar solvents this fine structure is absent because of solute–solvent interaction effects (see also Fig. 32.1). A classic case is phenol and related compounds, which have different spectra in cyclohexane and in neutral aqueous solution. In aqueous solutions, the pH exerts a profound effect on ionisable chromophores because of the differing extent of conjugation in the ionised and the non-ionised chromophore. The quality of spectral measurement is affected directly by the type and purity of the solvent used. Each solvent has a cut-off wavelength (which corresponds to about 10% transmittance) and this varies with solvent purity (Table 32.1). A solvent should not be used below its cutoff wavelength, even though reference-cell compensation is employed, because of the greater risk of stray-light effects. The UV spectra of some solvents are illustrated in Fig. 32.3. Some cautionary comments may be appropriate at this point. It is better to use single- or double-distilled water, and to avoid deionised water, which can be contaminated with absorptive fragments of ionexchange resin or contain bacterial metabolites; these can contribute significantly to non-specific absorption at low wavelengths. Ethanol is normally used as the 96% v/v strength, since dehydrated alcohol is usually contaminated with traces of benzene added to form the azeotropic mixture for distillation. Acetonitrile can vary noticeably in quality, depending on the supplier; the grade supplied for use in HPLC is usually to be recommended. Acetone, sometimes used to clean cells, is highly absorptive and not always easily removed, despite its volatility and aqueous solubility. Chloroform and carbon tetrachloride absorb strongly at about 250 nm and should therefore be used only for

Table 32.1 Cut-off points equivalent to 10% transmittance for spectroscopic solvents

Figure 32.3 UV absorption of solvents (HPLC grade unless otherwise stated): A, acetonitrile (far-UV grade); B, methyl t-butyl ether; C, acetonitrile; D, 1chlorobutane; E, methylene chloride; F, acetic acid (AR grade); G, ethyl acetate; H, acetone; I, hexane; J, iso-octane; K, methanol; L, tetrahydrofuran; M, chloroform; N, diethylamine (AR grade).

measurements at wavelengths above about 280 nm. Given the safety considerations of chlorinated solvents, use of these is best avoided if possible. Ether, although transparent down to 220 nm, presents particular problems because of its volatility (unstable standard solutions) and inflammability. Although absorptivity is considered to be relatively insensitive to temperature changes, organic solvents in general suffer from high temperature coefficients of expansion, so that for ultimate precision a cell provided with a thermostat may be required.

Solvent

Wavelength (nm)

Water (distilled) or dilute inorganic acid

190

Acetonitrile (HPLC, far-UV grade)

200

Acetonitrile

210

Butyl alcohol

210

Cyclohexane

210

Fluorescence spectrophotometry: theoretical background

Ethanol (96% v/v)

210

General considerations

Heptane

210

Hexane

210

Isopropyl alcohol

210

Methanol

210

Ether

220

Sodium hydroxide (0.2 mol/L)

225

Ethylene dichloride

230

Methylene chloride

235

Chloroform (stabilised with ethanol)

245

Carbon tetrachloride

265

N,N-Dimethylformamide

270

Benzene

280

Pyridine

305

Acetone

330

Molecular fluorescence is an emission process in which molecules are excited by the absorption of electromagnetic radiation. The excited species then relax to the ground state, giving up their excess energy as photons. There are several ways in which an excited molecule can give up its excess energy and relax to its ground state. Two of the most important of these mechanisms are non-radiative relaxation and fluorescent relaxation. Non-radiative relaxation can occur through collisions between excited molecules and molecules of the solvent, giving excess energy to solvent molecules. When relaxation takes place by fluorescence, bands of radiation are produced as the excited molecules relax to several energy states, which are very close in energy level and thus in wavelength (Fig. 32.4). Fluorescence occurs only from the lowest vibrational level of an excited electronic state. Note that molecular fluorescence bands are made up largely of lines that are longer in wavelength (lower in energy) than the band of absorbed radiation responsible for their excitation. This shift to

Clarke's Analysis of Drugs and Poisons Chapter No. 32 Dated: 11/3/2011 At Time: 8:39:18

510

Ultraviolet, Visible and Fluorescence Spectrophotometry

Figure 32.4 Energy-level diagram showing some of the energy changes that occur during (A) absorption, (B) non-radiative relaxation and (C) fluorescence by a molecular species.

longer wavelength is sometimes called the Stokes shift. For that reason, the absorption or excitation spectrum and the fluorescence spectrum for a compound often appear as approximate mirror images of each other. The most useful region for the fluorescence technique is 200– 800 nm. Fluorescence spectrophotometry is usually the method of choice for quantitative analytical purposes if applicable. It has assumed a major role in analysis, particularly the determination of trace contaminants in our environment, industries and bodies, because for applicable compounds fluorescence spectrometry gives high sensitivity and high specificity. The selectivity of fluorescence methods is greater than that of absorption methods, as fewer substances fluoresce than absorb radiation in the UV or visible region. Furthermore, fluorescence is more selective because both the emission and the absorption spectra can be obtained. Fluorescence is usually also more sensitive than absorption methods, as it is always easier to measure a small signal against a very small zero background than to measure a small difference between large signals. However, the phenomenon of fluorescence itself is subject to more rigorous constraints on molecular structure than is absorption.

fluoresces whereas nitrobenzene does not). The molecular grouping responsible for fluorescence is sometimes described as a fluorophore. Fluorescence is particularly favoured in rigid molecules, as molecular rigidity reduces deactivation by non-radiative processes (there being fewer internal vibrations). This is also why certain organic chelating agents are more fluorescent when complexed with a metal ion. Laws of fluorescence spectrophotometry

The power of fluorescent radiation If is proportional to the radiant power of the excitation beam absorbed by the system: 0

I f ¼ K ðI 0
IÞ

ð32:6Þ

The constant K0 depends upon the quantum efficiency of the fluorescence. Beer’s law can be used to relate If to the molar concentration c of the fluorescing molecule: I ¼ 10
bc I0

ð32:7Þ

Nomenclature

The term quantum efficiency used in fluorescence is quantified by the quantum yield (i.e. the ratio of the number of molecules that fluoresce to the total number of excited molecules). Highly fluorescent molecules can have quantum efficiencies that approach unity. Many drugs possess rather high quantum efficiencies for fluorescence, such as quinine and lysergic acid diethylamide (LSD). All absorbing molecules have the potential to fluoresce. They do so if fluorescent emission occurs at a greater rate than relaxation by nonradiative pathways. The kind of relaxation process is highly dependent on the molecular structure. Compounds that contain aromatic rings give the most intense and most useful fluorescence emission. Substitution on an aromatic ring causes shifts in the excitation wavelength spectrum and in fluorescence efficiency. Substituents such as –NH2, –OH, –OCH3 and –NHCH3 groups often enhance fluorescence, while –Cl, –Br, –I, –NO2 or –COOH are electron-withdrawing groups that can lead to reduction or absence of fluorescence (e.g. aniline

Substituting equation (32.7) into (32.6) we obtain:
 0 I f ¼ K I 0 1
10
bc

ð32:8Þ

After expansion of the exponential term, and provided that ebc 10 mm can be achieved. Because of the nature of NIR diffuse reflection, it is most probably not meaningful to work at spatial

Clarke's Analysis of Drugs and Poisons Chapter No. 34 Dated: 10/3/2011 At Time: 14:9:54

Resources

Figure 34.19 NIR image of a cross-section of a ranitidine tablet. Each pixel is 30  30 mm2. The image is 60  60 pixels (i.e. 1.8  1.8 mm2). The map shows the value (coded in different shades of grey) of the correlation coefficient of the NIR spectrum at each pixel when compared with that of a pure sample of
ska.) ranitidine. (Reproduced with permission of RA Watt and A Duszyn

resolutions of 99

—

Relative sensitivity — —

1

1/2

99.98

600.00

1.00

3

1/2

0.0

639.98

1.21

C

1/2

1.11

150.86

15

N

1/2

0.37

60.80

19

F

1/2

H H

13

29 31 2

Si P

H

14

1/2 1/2 1

N

1

23

Na

3/2

35

Cl

3/2

O

5/2

17

1.59
102 1.04
103

100.0

564.46

8.30
101

4.7

119.19

7.84
10

100.0

242.88

6.63
10

0.015 99.63 100.0 75.53 0.037

92.10 43.34 158.71 58.79 81.34

565

Fragment

d(1H)

Cyclopropyl CH2

–0.2–0.8

CCH2C

0.4–2.4

CH3C

0.5–2.0

CNH

0.5–3.0

COH

0.5–5.0

CH3C=C

1.5–2.3

CCH2C=C

1.6–2.0

CH3S; CH3CO

1.7–2.7

3

CH3Ph

20–30

2

CH3N

22–31

CCH2S

23–30

9.65
104 1.01
101 9.25
102 4.70
103 2.91
102

important in biology, including many studies in vivo. Many other spin-1/2 nuclei, such as 29Si, 119Sn, 129Xe, 195Pt and 199Hg, have found much use in specialist applications. Nuclei with I > 1/2 are quadrupolar and, in general, give broad NMR lines, but in some cases useful information can be gleaned. Examples include 2H NMR in liquid crystals, 14 N NMR in heterocyclic chemistry, and 23Na NMR studies of intraand extracellular sodium ions.

C=CCH2C=C

23–30

CCH2CO

23–35

CCH(Ph)C

25–30

CCH2Ph

25–32

CCH2N

25–33

C=CCH2CO

28–37

C=CCH2Ph

30–38

CH3O

32–41

NCH2CO

32–45

CCH2Cl; CCH2O

34–43

CH3OCO

35–38

COCH2Ph

35–42

CHNCO

35–43

Parameters from an NMR spectrum

CCH2OCO

35–45

Chemical shifts

NCH2N

35–51

As mentioned earlier, not all nuclei of a given isotope resonate at exactly the same frequency. This is because, in a molecule, a given atomic nucleus is surrounded by electrons which also possess a magnetic moment, and these provide a fluctuating magnetic field that opposes the main field of the NMR magnet. As a consequence, the nuclei are shielded from the main magnetic field and require a higher field to bring them to resonance and thus they can be considered to have higher Larmor frequencies. The degree of shielding depends on the electron distribution around the nucleus and hence on the chemical environment. The different degrees of shielding are known as chemical shifts. Thus, interpretation of chemical shift values allows identification of molecular structural fragments. Chemical shifts are measured relative to that of a reference substance usually placed into the sample. For 1H and 13C shifts in organic solvents, this is tetramethylsilane (TMS). The chemical shift is then defined as d(H) ¼ (difference in the resonance frequency in hertz between the analyte and TMS)
106/(operating frequency of the spectrometer). Chemical shifts are thus quoted in parts per million (ppm) and are independent of the operating frequency of the spectrometer, which allows comparisons irrespective of magnetic field strength. For aqueous samples, an alternative reference compound is used, of which trimethylsilyl [2,2,3,3-2H4]propionic acid sodium salt (TSP) is the most common example. The chemical shifts for TMS and TSP are set arbitrarily to zero. Typical 1H and 13C NMR chemical shifts of a variety of important molecular fragments are shown in Tables 36.2 and 36.3, respectively.

NCH2Ph

36–46

C=CH

36–85

C=CCH2O

40–52

Indirect (J) spin–spin coupling

The resonance lines of individual nuclei can show further splitting because of indirect spin–spin coupling. Given the symbol J, this is measured in hertz and is independent of the observation frequency. Such spin coupling arises from a magnetic interaction between NMRactive nuclei and is transmitted via the intervening electrons, hence the term ‘indirect’. Coupling is observed only within a molecule. Thus for

COCH2O

40–55

OCH2O

45–63

OCH2Ph

47–56

NCH2O

48–59

NHCO

50–90

Aromatic H

60–90

OCHO

80–83

C=NOH

80–110

CHO

95–100

COOH

95–130

two spin-1/2 nuclei, such as protons, the resonance line for each proton is split into a doublet, the two lines corresponding to the two possible orientations of the adjacent proton relative to the magnetic field. For extended coupling chains, each component of a doublet can be split further into doublets of doublets and so on. If a given proton is adjacent to two equivalent other protons (as in a CH2 group) then, of the four possible spin orientations of the two protons, two of them are identical and a 1 : 2:1 triplet results. For such ‘first-order’ systems, the multiplicity can be deduced on the basis of Pascal’s triangle according to the number of equivalent coupled nuclei. In situations where the chemical shift difference between the protons is large compared with the J-coupling, this simple rule applies. For situations where the chemical shift in hertz between coupled partners is not large compared with the magnitude of the coupling constant (d/J 1.0 to be able to quantify either peak reliably in the presence of the other. Sometimes the parameter percentage resolution is used, as this concept is easier to visualise (calculated by dividing the height of the valley between the peaks by the total peak height). equations (40.5a) and (40.5b) allow the operator to calculate resolution directly from the chromatogram, but they give little indication of the factors that contribute to it, or the parameters that can be modified by the analyst. A preferred relation is equation (40.7c), which shows that resolution is a product of three parameters, selectivity, capacity and the square root of the efficiency.
 pffiffiffiffi a1 k N R¼ ð40:5cÞ a kþ1 4

b¼

643

ð40:6bÞ

where k ¼ retention factor. Thus, to increase solute retention the phase ratio must be decreased, which can be brought about either by decreasing column diameter or by increasing film thickness. Sometimes it will be necessary to alter either the column diameter (e.g. to reduce flow though a detector) or to alter film thickness (e.g. to increase efficiency), but as long as the phase ratio remains constant then these changes can be accomplished without compromising separation. Table 40.8 shows calculated phase ratios for the most common sizes of capillary columns and loadings. Peak shape or asymmetry

A well-designed GC system should give symmetrical peaks, as tailing or fronting adversely affects resolution. Tailing may result from non-swept

volume in the system or from component–stationary phase or component–support interactions. Tailing of polar compounds can often be remedied by the use of a more polar stationary phase. Fronting (shark’s fin peaks) is usually caused by overloading, particularly with capillary columns, and can be resolved either by making a smaller injection or by using a column with a higher stationary phase ratio. Column capacity is the maximum amount of a solute that can be chromatographed successfully without loss of peak shape. Table 40.4 shows the relationship between column capacity, film thickness and column diameter. Peak fronting caused by thermal decomposition can be reduced by either lowering the injection temperature or using a cold on-column injector system. Peak shape is usually expressed by the peak asymmetry (As). In Fig. 40.3, the peak asymmetry factor for substance B is given by equation (40.7): As ¼

YZ XY

ð40:7Þ

where a vertical line is drawn through the peak maximum and XYZ is drawn at 10% of the peak height. A symmetrical peak has As ¼ 1. Use of retention indices If gas chromatographic retention data are to be exchanged between laboratories, they must be independent of the instrument used. The concept of retention index (RI) has been shown to be more reliable than that of relative retention time (i.e. the retention time of the solute relative to that of a reference compound). The RI system uses a homologous series of compounds (i.e. a series of compounds that increase in size by an additional methylene unit) to provide the reference points on the scale. The most commonly used is the system described by Kovats (1961) using straight-chain saturated hydrocarbons (n-paraffins or nalkanes). For any column temperature and stationary phase, the elution times of members of a series of n-alkane homologues are assumed to increase by an index of 100 for each additional methylene unit. On this scale, H2 has an index of zero, methane has an index of 100, ethane 200, and so on up the scale of alkanes. The RIs of unknown substances are measured against this scale, obviating the need to correct data between laboratories because of variations in retention time. The method is illustrated in Fig. 40.4, in which phenobarbital has a retention time of 4.5 min and an RI of 1957. RI ¼ 100ðPzþn  Pz Þ 

log t R ðxÞ  log t R ðPz Þ log t R ðPzþn Þ  log t R ðPz Þ

ð40:9aÞ

where tR is the retention time, Pz is the carbon number of the smaller nalkane, Pzþn is the carbon number of the larger n-alkane and x is the unknown solute.

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Figure 40.4 Calculation of the RI of phenobarbital from a plot of a series of homologous n-alkanes. (A) Packed column, isothermal conditions, retention time for phenobarbital is 4.5 min, RI = 1953; (B) capillary column, temperature programmed to run at 10
/min, retention time for phenobarbital is 7.01 min, RI = 1960.

Retention indices collected from many sources show remarkable agreement, even when measurements were made on different (though equivalent) phases and at different temperatures (Ardrey, Moffat 1981). Most capillary GC is performed in temperature-programmed mode and the relationship between the retention time and chain length is almost linear, provided that the ramp rate is constant throughout the run and n >7. The simplified equation (40.8b) is used to calculate RI (see Fig. 40.4 for an illustration). RI ¼ 100ðPzþn  Pz Þþ

t R ðxÞ  t R ðPz Þ t R ðPzþn Þ  t R ðPz Þ

ð40:8bÞ

Other homologous series have been proposed and good results were found with the alkan-2-ones and n-aldehydes. Use of other series (nalkyl esters, n-alkylbenzenes, n-alkyl iodides) has been less successful. When using a specific detector to identify compounds, the accuracy is increased by splitting a small percentage of the column effluent to a flame ionisation detector to give a ‘real-time’ calculation of RI, rather than relying on either historical retention times for n-alkanes or using an alternative homologous series. Alternatively, Franke et al. (1993) have proposed the use of an RI reference mix (a selected group of compounds that structurally resemble those under investigation) rather than a homologous series. Retention indices for unknown compounds can then be normalised relative to the known RI values of the reference compounds. The main advantage of this approach is that both temperature-dependent and column-ageing effects on RI, which arise from polarity mismatch between the homologous series and the investigated solutes, are somewhat reduced and the data are therefore more reproducible over time. As would be expected, there is better agreement for non-polar (most hydrocarbon-like) compounds than for polar compounds, and also for non-polar versus polar stationary phases where there is less difference in retention time between the n-alkane index markers. Using capillary columns, reproducibility can be as good as to within one RI unit for a non-polar phase, and to within a few units for a more polar one. Good temperature, pressure and flow control, and precise measurement of the injection time and peak elution, are essential for accurate measurement of RIs. The carrier gas flow rate and the polarity of the stationary phase are temperature dependent. Thus, the partitioning of polar compounds into the stationary phase is affected by temperature to a greater extent than that for the n-alkanes. Differences between constant-flow and constant-pressure modes of operation are exaggerated when the column is ramped in several stages. It is common practice to have an initial fast rate of increase in temperature followed by one or more slower ramp rates, since the number of low-boiling compounds of

interest is usually lower than the number of higher boiling compounds. Inaccuracies in RI calculation can also arise with high solute concentrations because of problems identifying the crest of the peak. This can also result in a trace component that elutes on the back of the concentrated one, as the major component begins to take part in the separation process by acting as a ‘dynamic stationary phase’. This delays the elution of the trace component. Column deterioration with use can lead to a preferential destruction or loss of the more labile component of a mixed phase. For example, in columns that contain mixed cyanopropyl- and methyl-PSX phases, the cyanopropyl group is preferentially lost, so that the column polarity is reduced. The elution of the index markers (n-alkanes) remains unchanged, but the progressive loss of cyanopropyl substituents results in a poorer interaction with polar compounds and an apparent decrease in their RI. With single-component stationary phases, the effect is still present, though less noticeable, as there is loss of retention of both RI markers and polar compounds.

Inlet systems The inlet system provides the means of introducing the specimen into the GC. Obtaining a narrow sample band at the start of the chromatographic process is critical to achieve good resolution, since broad sample bands usually produce broad peaks, especially for analytes that elute early. The choice of injector depends on the characteristics of the specimen or residue, the quantity and characteristics of the analytes to be separated, and the temperature and nature of the stationary phase and the column. Solids may be dissolved in a suitable solvent and injected with a micro-syringe. It is best to keep the solution as concentrated as possible to reduce the size of the solvent peak. Liquids can be injected using a micro-syringe, but with sensitive detection systems the sample should be dissolved in a suitable solvent to reduce the sample size and avoid overloading the detector. Gases and vapours may be introduced by injection through the inlet port septum using a gas-tight syringe. The three common types of GC injectors are split, splitless and cold on column. In reality, splitless injection is an extreme example of split injection and both are carried out using the same hardware. Conventional glass syringes of 1–10 mL volume with stainless-steel needles can be used on all but cold-on-column injectors, and the injection is made by piercing a silicone rubber septum. Care must be taken to select septa that have low bleed characteristics at the operating temperature, and those with Teflon backs are most reliable in this respect. Unstable materials can be decomposed by the high temperature of the injection system, particularly if the system is constructed of metal. For labile substances, cold-on-column injection is preferred, but clean extracts must be used to minimise column contamination.

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Inlet systems Split and splitless injectors Split injectors are used for more concentrated samples, since only a fraction of the sample actually enters the column. An inlet splitter allows a high flow of carrier gas through the injector while maintaining a low flow (1–4 mL/min) through the column: the excess gas and associated sample components are vented to the atmosphere through the split line. The ratio of these two flows (the split ratio) controls the proportion of the injected sample that reaches the column. The total flow through the injector may be from 10 mL/min to 100 mL/min, which gives split ratios of 10 : 1 to 100 : 1. A good splitter should be linear, i.e. it should split high- and low-boiling point compounds equally. The function of the splitter is not primarily to reduce sample volume, but rather to ensure that the sample enters the column as a compact plug. Split injections, therefore, produce some of the most efficient chromatographic separations, and allow the use of very narrow capillary columns. A lower split ratio channels a larger fraction of the injected sample down the column and may result in column overload. High split ratios waste large amounts of carrier gas and insufficient analyte may reach the column. In splitless injection, all the carrier gas passes to the column. This is useful for very volatile compounds, for low sample concentrations or for trace analysis. The flow rate in the injector is the same as that in the column (1–4 mL/min), and the only path for the injection to take is into the column, since the split vent is closed. At a fixed time after injection (usually 15–60 s), the injector is purged by opening the split vent to introduce a much larger flow of carrier gas through the injector (typically 20–60 mL/min) and any remaining sample in the injector is discarded through the split vent. Since the rate of sample transfer onto the column is so slow (because of the low gas flow), peaks are usually somewhat broader than for split injections. Care should also be taken to ensure that the volume of the injector liner is not exceeded by the expanding solvent injected (Table 40.5), otherwise splitting of early peaks will be observed. Temperature conditions can be adjusted to narrow or focus the sample band at the top of the column. Splitless injections should therefore be made with the initial column temperature at least 10
C below the boiling point of the solvent (Table 40.5), and the initial temperature should be held at least until after the purge activation time. Solvent condenses on the front of the column and traps the solute molecules, which focuses the sample into a narrow band (known as the solvent effect). Individual solutes with a boiling point 150
C above the initial column temperature condense and focus at the top of the column in a process known as cold trapping. Either the solvent effect or cold trapping must occur before efficient chromatography can be obtained. Some newer chromatographs have the option of a pulsed splitless injection. In this mode, the column head pressure is increased immediately upon injection (typically to 174 kPa) and held there

Table 40.5 Boiling points and expansion volumes for commonly used injection solvents Solvent

Boiling point (
C)

Expansion volume (mL) per L of solvent(a)

Suggested GC oven starting temperature (
C)

Methylene chloride

40

330

15–30

Carbon disulfide

46

355

15–30

Acetone

56

290

30–45

Methanol

65

525

40–55

n-Hexane

69

165

40–60 45–65

Ethyl acetate

77

215

Acetonitrile

85

405

55–75

iso-Octane

99

130

70–90

Water

100

1180

70–90

Toluene

111

200

80–100

Values are given at 250
C and 105 kPa head pressure.

(a)

645

for 30–60 s, before returning to the normal operating pressure. This facilitates band sharpening and, while the process is not guaranteed to increase the fraction of the injection delivered onto the column, sensitivity is often improved because of improved chromatography. Glass liners for split and splitless injectors come in a variety of shapes and volumes and it is prudent to start with a straight liner and to investigate some of those that cause turbulence (e.g. the inverted cup style) later if this is unsatisfactory. A plug of deactivated glass wool in the liner helps prevent the deposition of non-volatile or particulate material on the column, but may cause some peak deterioration, and for the best results needs to be placed at a consistent position in the liner. Packing of splitless injection liners with deactivated glass wool may decrease the chromatographic performance, but this must be weighed against the potential for damage to the stationary phase from the repeated injection of non-volatile or particulate material. Large-volume injectors The analysis of trace amounts of components or contaminants in complex matrices such as foods, beverages and environmental samples is difficult. Adequate sensitivity to detect trace components is provided by specific detectors such as the NPD or ECD, but regulatory standards require positive identification of these compounds by mass spectroscopy (MS). To overcome the inferior sensitivity of MS, large-volume injectors have been developed. Examples include the Apex pre-column separating inlet (PSI), the temperature-programmed sample inlet (PTV) from Gerstel and time-coupled time-resolved chromatography (TCRC). The inlet typically consists of a length (10–50 cm) of standard (2 mm i.d.) glass chromatography column that can be deactivated or packed with traditional materials. The first two injectors are mounted directly in the GC injector port; the latter is a free-standing column coupled by a four-way valve into the GC inlet. Injection volumes range from 125 mL for the PSI, 1 mL for the PTV and up to 20 mL for the TCRC. Injection of larger volumes (up to 60 mL) is possible for some applications, but result in discrimination in favour of high-boiling components and loss of volatiles. Large-volume injectors remove the solvent from the sample prior to its introduction onto the capillary column, typically by low-temperature evaporation through the split vent. As the sample is concentrated towards the bottom of the injector, the injector is heated, the split vent is closed and the analytes are introduced onto the GC column in splitless mode. Those injectors that can be heated selectively and cooled allow the precise introduction of selected components only from the sample, and thus reduce the quantity of non-volatile components (e.g. sugars) that might overload or destroy the analytical column. The TCRC has a small mobile oven (2–8 mm width) that can be scanned along the length of the column to produce band compression. Prior to the next injection, the injector columns are usually baked to vent high-boiling compounds to waste. Sensitivity can often be improved 50- to 100-fold and time is saved in sample preparation, since extensive clean-up or extraction procedures are no longer required. Cold-on-column injection Cold-on-column injection is most suited to compounds that are thermally labile. The injection needle must be fine enough to enter the column bore, usually fused-silica or stainless steel with a fusedsilica insert. The top of the column is held at a temperature low enough for the solvent that contains the sample to condense, usually by an air- or carrier gas-cooled sleeve. The solvent temporarily swamps the stationary phase and ensures that the sample components concentrate in a narrow band. Any solvent or sample that remains in the injector is backflushed with carrier gas, often by automatic valves. The proximal end of the column is then brought rapidly to the operating temperature, when the solvent vaporises and chromatography begins. The potential for rapid column contamination or deterioration means that cold-on-column injection is usually restricted to those applications where its use is essential.

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Volatiles interface The volatiles interface allows automated analysis of gaseous samples. The interface is a low-volume highly inert switching block, and is ideally suited to trace-level detection. A portion of the carrier gas supply is diverted through the specimen sampler and released under controlled conditions onto the column. The remainder of the carrier gas goes to a flow sensor, which prevents fluctuations in column gas flow that would otherwise occur when the switching valves are opened and closed. The interface can be run in split, splitless or cold-on-column modes as described in the sections above. Samples may be introduced from external devices, such as air samplers or purge-and-trap devices (see section below), or from headspace analysis, which permits analysis of volatile substances in a liquid sample while minimising contamination of the column. This technique is used in the assay of ethanol and other solvents in blood and for complex household preparations, such as polishes, which contain volatile substances (see Chapter 14).

pressure. If the volume of the liner is smaller than the expanded solvent volume (see Table 40.5), some of the sample is propelled out of the injector in a process known as backflush. This can appear as a broad tailing solvent front, since it now takes longer to flush the expanded solvent out of the injector and carrier gas line. Backflush can also cause injector contamination, since the analytes condense in the cooler carrier gas line, from where they may bleed continuously into the injector and cause high background or spurious peaks. Carryover or peak ghosting can occur when the next injection backflushes and carries previously condensed compounds back into the vapour phase and onto the column. Backflush can usually be solved by using a smaller injection volume, a less expansive solvent, a lower injector temperature, a liner with an upper restrictor, or a faster carrier gas flow. The use of an adjustable septum purge gas (usually 0.5–1 mL/min) also decreases the potential for backflush, as components that would normally condense on the cooler septum and travel into the carrier gas lines are swept away by the septum purge. Too high a purge flow results in loss of highly volatile components.

Thermal desorption and purge-and-trap injection The analysis of samples that have been pre-concentrated onto solid adsorbents is common in the fields of industrial air monitoring, analysis of residues in food, soil and water, petrochemical analysis and environmental monitoring. The methods of preparing samples for analysis are described in the section on specimen preparation. These samples require special interfaces with GCs to ensure good chromatography. In some instances the sample preparation device and injector are manufactured as stand-alone pieces of equipment that require very little modification of conventional injectors, while others must be dedicated pieces of equipment. Once collected, the concentrated sample must be desorbed into the chromatograph using the heated injector port. The major problem here is the possible introduction of water into the chromatograph from moisture adsorbed during collection from high-humidity samples. Release of solutes from the adsorbent should be as rapid and complete as possible to allow for rapid and sensitive analysis and for a narrow sample band to be introduced into the chromatograph. This is achieved either by cooling the column oven cryogenically to refocus the sample in the injector prior to injection or by using a dry purge system coupled to the gas chromatograph via a volatile interface (see above) designed to operate above ambient temperature. Here, the specimen is thermally desorbed from the collection tube onto a narrower (1 mm i.d.) tube of the same adsorbent material. The concentrated solute is then released into the chromatograph, ensuring rapid and complete sample introduction. Adsorbents must be thermally stable to reduce interference from background contaminants. With solid phase microextraction (SPME) the adsorbed sample is introduced into the heated injector port via a special sleeved needle (see under Specimen preparation). This technique requires the injector liner to be narrow (usually 0.75 mm as opposed to 2 or 4 mm) to increase the linear velocity of carrier gas through the liner and ensure that a narrow band of sample is introduced onto the column. Solid injection When solvent interference is serious the sample may be injected as a solid. The ‘moving needle’ injector has found application in steroid analysis and for the determination of anticonvulsant drugs. A solution of the material to be injected is placed on the tip of the glass needle with a syringe. A small flow of carrier gas sweeps the solvent out of the top of the device to waste. The dry residue is then introduced by moving the needle into the heated injection zone of the chromatograph with a magnet. This form of injection can be used only with compounds that do not volatilise with the solvent. Backflush Upon vaporisation, the injected sample undergoes considerable expansion, sometimes up to 100 to 1000 times its original volume, which creates a pulse of pressure that often exceeds the column carrier gas

Injector discrimination Injector discrimination occurs because not all the compounds in the sample vaporise at the same rate. Since the sample remains in the liner for a limited time, this usually results in some loss of higher-boiling solutes. This can be alleviated by increasing the residence time of the sample within the injector, or by using a higher injector temperature or smaller injection volume. However, there is usually a compensatory loss in lower-boiling compounds. Discriminating behaviour can usually be managed by making reproducible injections. Gas pressure and flow control For accurate and reproducible GC, either a constant carrier gas flow or a constant carrier gas pressure must be maintained. Under isothermal conditions, simple pressure control is adequate for packed or capillary columns and back pressure can be monitored by a pressure gauge between the flow controller and the injector. A decrease indicates a leaking septum and an increase suggests contamination of the injector liner or the top of the column. This also ensures that the flow controller is performing correctly. Since the back pressure rises to equal the supply pressure, flow becomes pressure controlled. Flow control is highly desirable, if not essential, during temperature programming with packed columns and can be used to advantage with capillary columns. The added convenience of a digital (electronic) flow controller may be worthwhile. Since the carrier gas becomes less viscous as the column oven temperature rises, the gas pressure must be increased as the run progresses to maintain constant velocity (or constant flow) throughout the analysis. Fig. 40.5 shows the effects of increasing the column temperature on the carrier gas flow and velocity if the head pressure is held constant during the run. As flow and velocity do not respond identically to increasing temperature (see Fig. 40.5D), late-eluting analytes are recovered more quickly using constant flow than under constant-pressure conditions. Furthermore, since column efficiency is a function of the carrier gas velocity (Fig. 40.6), resolution at the end of the chromatogram is improved under constant flow conditions. Switching between conditions of either constant flow or constant pressure can sometimes resolve otherwise co-eluting compounds. Table 40.6 shows the relationship between flow and pressure for various lengths and diameters of capillary columns. It shows the calculated head pressure (kPa) required to achieve the stated gas velocity or flow through a 25 m column operating at 150
C. Note that head pressure values above 280 kPa are not usually practicable using standard pressure regulators. Increasing the column length has a direct and proportional increase on head pressure for both velocity and flow calculations. The way in which carrier gas velocity affects column efficiency is best demonstrated by reference to the van Deemter curves in Fig. 40.6. These demonstrate that the optimum column efficiency (minimum height equivalent of a theoretical plate, HETP) occurs at intermediate

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Detector systems

647

Figure 40.5 Effect of temperature on carrier gas flow and velocity. (A) and (B) are under conditions of constant carrier gas head pressure (140 kPa). (A) shows the change in column flow (mL/min) with change in temperature from 50
C to 300
C. (B) shows the change in velocity (cm/s) with change in temperature from 50
C to 300
C. (C) and (D) are under conditions of constant carrier gas flow (1 mL/min). (C) shows the change in carrier gas velocity (cm/s) with change in temperature from 50
C to 300
C. (D) shows the change in column head pressure (kPa) with change in temperature from 50
C to 300
C. All calculations are for a 25 m column of 0.25 mm i.d. operating at atmospheric pressure and 150
C.

velocity, and that column efficiency is compromised at both low and very high velocities. A small loss in efficiency for a shorter analysis time is usually tolerated. Curves are shown for the three most common carrier gases (helium, nitrogen and hydrogen), and it can be seen that the chromatography is much less tolerant to changes in nitrogen

velocity than to helium. Helium is favoured by most users, as analysis times are half that with nitrogen, with only a slight loss in efficiency. While hydrogen gives the best dynamic range and shortest analysis times, there are safety issues relating to its use. While the gas used for the carrier gas should always be of the highest purity available, a lower-quality gas can sometimes be used for the makeup or detector, since these do not contribute to column deterioration by oxidation. Regardless of quality, it is advisable always to use a scrubber (to remove oxygen and hydrocarbons) followed by a dryer (to remove water vapour) between the supply and the instrument. Metal trap bodies are recommended, as plastics are permeable to impurities in laboratory air, especially when large amounts of organic solvents are used. Most traps have an indicator to show when they are saturated, and they can be changed without interruption to the gas flow. Stainless steel or copper tubing is recommended for plumbing of all gases, as plastics are permeable to moisture and oxygen, and Teflon, nylon, polyethylene, polypropylene and PVC contain contaminants that degrade gas purity.

Detector systems

Figure 40.6 Van Deemter plots for a 25 m  0.25 mm i.d. WCOT OV-101 column. HETP = height equivalent of a theoretical plate.

The choice of chromatography detector for an application depends on factors such as cost, ease of operation, consumables supply, sensitivity, selectivity and the linear working range. Some detectors respond to almost all solutes, while others (selective detectors) respond only to solutes with specific functional groups,

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Table 40.6 Relationship of gas chromatography column diameter to column flow, velocity and head pressure (kPa) Column internal diameter (mm) 0.20

0.25

0.32

0.53

93.2

10.8

74.5

28.7

10.4

654.1 181.5

144.9

90.4

54.2

19.5

724.5 201.5

160.0

100.1

60.0

215.3

Hydrogen

675.5 157.3

115.9

58.2

24.8

3.66

Nitrogen

959.1 240.8

181.5

95.9

43.2

6.76

193.2

102.8

46.9

7.38

0.10

0.18

Hydrogen

329.1

Nitrogen Helium

Velocity 30 cm/s

Flow 1 mL/min

Helium

1007.4 254.6


Data for a 25 m column operating at 150 C.

atoms or structural configurations. Additional functional groups can often be added to solutes, generally after extraction (see below under Derivatisation), to achieve a response from a selective detector and gain additional sensitivity and selectivity. The use of detectors such as the ECD to identify amenable compounds, and the NPD to detect compounds that contain phosphorus and nitrogen, removes many of the extraneous peaks frequently observed when using non-selective detectors, such as the FID. However, these selective detectors have also led to the detection of substances such as plasticisers from blood-collection tubes or transfusion lines, which interfere in many toxicological analyses. Detectors that detect the presence of a solute and also give information about its structure are increasingly popular and MS, Fourier transform infrared spectroscopy and atomic emission spectrometry have been invoked to achieve this goal. Detector sensitivity is measured as signal-to-noise ratio, in which the signal corresponds to the height of the peak, and the noise to the height of the baseline variability. A signalto-noise ratio of 8 to 10 is considered sufficient to confirm the presence of a peak. Each type of detector has a linear operating range in which the response obtained is directly proportional to the amount of solute that passes through, although this can be modified slightly by the nature of the solute and the chromatographic conditions (mobile phase type and flow, detector temperature). The linear operating range is considered to be exceeded when the incremental response obtained from the detector varies by more than 5% from that expected. Most detectors (except MS) rely on gas other than the mobile phase (combustion, reagent or purge gas) for their operation. Usually, a total flow of at least 30 mL/min is necessary to sweep the solute molecules physically through the body of the detector at sufficient speed to prevent refluxing and produce narrow peaks. Thus, the addition of a ‘makeup’ gas is invariably required with capillary columns. Recommended gases and their flows for each detector are included in the manufacturer’s instruction manuals, and it is important to follow these guidelines (and those on maintenance) to achieve the stated performance. Here, only the most widely used detectors are considered in detail. Several other types of detectors are available; for a more detailed discussion, the reader is referred to the text by Scott (1996). Flame ionisation detector This is the most widely used of all detectors, since it responds to nearly all classes of compounds. The effluent from the column is mixed with hydrogen and the mixture is burnt at a small jet in a flow of air. A polarising current is applied between the jet and an electrode situated above it. When a component elutes from the column it burns in the flame to create ions that carry a current between the electrodes and provide the signal. The background current and noise are both low. Any of the usual carrier gases can be used and minor changes in gas flow are without effect. Sensitivity is moderate (0.1–10 ng), with linearity extending sometimes as high as six orders of magnitude. The response of the FID is dependent on the number of carbon atoms in the molecule, but the response is lowered if oxygen or nitrogen is also present in the

molecule. It responds to all organic compounds that contain carbon– hydrogen bonds with the exception of formic acid. Both the sensor design and electronics are simple, and manufacturing cost is therefore low. The FID is easy to clean, and when operating with capillary columns it is virtually maintenance free. With packed columns, however, there is a tendency for a build-up of stationary phase bleeding from the column, which must be removed periodically. The insensitivity of the detector to water is a useful feature that allows aqueous solutions to be used. Nitrogen–phosphorus detector or alkali flame ionisation detector The introduction of alkali metal vapours (usually supplied by an electrically heated bead of rubidium chloride or caesium chloride) into the flame or ‘plasma’ of an FID confers an enhanced response to compounds containing phosphorus and nitrogen. By adjustment of the plasma gases the detector can be made virtually specific for phosphorus compounds (e.g. a phosphorus : carbon response ratio of 50 000 : 1 and a phosphorus : nitrogen response ratio of 100 : 1). Even when optimised for nitrogen compounds, it retains its response to phosphorus (e.g. a nitrogen : carbon response ratio of 5000 : 1 and a nitrogen : phosphorus response ratio of 10 : 1). This detector is particularly useful for drug analysis, since most drugs contain nitrogen, while the solvent and the bulk of the co-extracted material from a biological sample do not. The NPD is ideal for detecting pesticides that contain phosphorus, and therefore has wide application in environmental and regulatory analysis (air, soil, water and residues in food). The extreme sensitivity to compounds that contain phosphorus can be further exploited by the preparation of derivatives that contain this element. Sensitivity is excellent (1–10 pg), with a good linear range of up to four or six orders of magnitude. A disadvantage is the need for the supply of three gases and, unlike with the FID, their control is absolutely critical to selectivity. The detecting element (bead) lasts between 1 and 3 months depending on usage. Stationary-phase bleeding from packed columns coats the bead and collector assembly but can be rinsed off using methanol or dilute (0.1 mol/L) sulfuric acid. Most of the early problems that arose from poor reproducibility in bead coating have now been resolved, and the most stable detectors nowadays have a geometry that enables the bead to be located and fixed in its optimal position with relative ease. Electron-capture detector The early form of this detector consists of a small chamber with a pair of electrodes and a radioactive source, usually 63Ni, placed close to the cathode to ionise the carrier gas. Potential applied to the electrodes produces a steady background current. Electron-capturing solutes arriving in the chamber remove some of the electrons and reduce the detector current. The response of the detector is therefore a loss of signal rather than an increase, as is given by most other detectors. Although the ECD can be polarised from a suitable low-voltage direct-current supply, it is more sensitive when a pulsed power supply is used, and in modern detectors the polarising pulses are modulated to maintain a constant current. A voltage that depends on the modulation frequency is generated as the output signal. Additional carrier gas is necessary, even with packed columns, to obtain a flow of at least 60 mL/min to purge the detector adequately and avoid peak broadening and distortion. Sensitivity can also be improved dramatically by raising the operating temperature of the detector, and decreasing the makeup gas flow. The ECD is a selective detector with a very high sensitivity to compounds that have a high affinity for electrons; for many compounds, the sensitivity of the ECD often exceeds that of MS, and sometimes even that of the NPD. Compounds that contain a halogen, nitro group or carbonyl group are detected at 0.1–10 pg, 1–100 pg and 0.1–1 ng, respectively. This makes it very useful for compounds such as the benzodiazepines or halogenated pesticides and herbicides. Alternatively, the great sensitivity of the detector may be utilised by preparing derivatives with halogenated reagents, such as trifluoroacetic, heptafluorobutyric or pentafluoropropionic (PFP) anhydrides. Linearity (at best only two or three orders of magnitude) is a limiting factor for quantitative analysis.

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Detector systems In older models, the addition of a small amount of quench gas, such as methane, improves stability and linearity, and is essential if argon or helium carrier gas is used. Newer models can be operated successfully with helium as both carrier and detector gas. The ECD, because of its high sensitivity, can be contaminated easily: an impure cylinder of gas can damage a detector beyond repair in a matter of only a few hours. Cleaning is difficult, although some material can be removed by heating the detector to its maximum operating temperature overnight, and the injection of water in 100 mL aliquots through an empty glass column can also help. However, if contamination is avoided, it is virtually maintenance free. The radioactive source requires special handling procedures that may be subject to federal legislative regulations. More recently, it has been shown that this detector can work with greater sensitivity and operate over an increased linear range using a helium plasma in place of the radioactive source.

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700 amu) several times per second. The abundance of each mass at a given scan time produces the mass spectrum, which can be summed and plotted versus time to obtain a total ion chromatogram. The MS detector can be operated either in full scan mode (collecting all the ions within a given mass range) or selected-ion monitoring (SIM) mode, which collects only pre-selected masses characteristic for the compound under study. Sensitivities for the two modes of operation are quite different: 1–10 ng for full scan, increasing to 1–10 pg in SIM because of the dramatic decrease in background noise. The linear range is excellent and often spans five or six orders of magnitude. Recent advances in computer technology, coupled with improved detector design, have revolutionised the use of the MS detector from a research tool to one of routine application. This technique is described in more detail in Chapter 37. Ion-trap mass spectrometer

Fourier-transform infrared detector In the Fourier-transform infrared detector (FTIRD), the column effluent is conducted through a light pipe and swept by a scavenging gas into the path of an infrared light beam that has been processed by an interferometer. The interferometer directs the entire source light to a beam splitter, which sends the light in two directions at right angles. One beam takes a fixed path length to a stationary mirror, while the other takes a variable path length to a computerised moving mirror. The two beams are recombined, and the difference in path lengths creates constructive and deconstructive interference, or an interferogram. The recombined beam is then passed through the sample. Analyte molecules absorb light energy of specific wavelengths from the interferogram, and the sensor reports variation in energy versus time for all wavelengths simultaneously. For molecules to be infrared active they must be able to undergo a change in dipole moment with the transition to their excited state. As a result, many compounds that are symmetrical do not respond. Fourier transformation refers to the mathematical computation that converts the data from an intensity versus time plot into an intensity (percentage transmission) versus frequency spectrum. Each dip in the spectrum corresponds to light absorbed, and can be interpreted as characteristic of specific functional groups in the molecule. Computer libraries allow for easy and rigorous comparison of spectra. FTIR can be fully quantitative, but it is relatively insensitive (10 ng range). Its advantages are that it is non-destructive, and it can distinguish between isomers (MS cannot). Because of the logistical difficulties of combining FTIR with GC, this combination of techniques has started to emerge only recently. Atomic emission detector With the atomic emission detector (AED), carrier gas that elutes from the column delivers solutes into a high-temperature helium plasma, where heat energy is absorbed by the constituent elements. In returning to their ground state, they emit energy as light, the wavelength of which is characteristic for each element. Emitted light is focused by a quartz lens and spherical mirror onto a diffraction grating, and the dispersed light is focused onto a diode array that is continuously scanned (wavelength usually 170–800 nm). Typically, some 15 elements can be monitored simultaneously, and each is plotted against time. The composite chromatogram allows the percentage elemental composition of each peak to be determined. Sensitivity is very good, but the detector is complex and expensive to operate and is not widely used. Mass spectrometer A gas chromatograph is an almost ideal inlet device for quadruple MS. The detector is maintained under vacuum, and in the most common technique of electron impact (EI) the column effluent is bombarded with electrons. Compounds absorb energy, which causes them to ionise and fragment in a characteristic and reproducible fashion. The resultant ions are focused and accelerated into a mass filter that allows fragments of sequentially increasing mass to enter the detector stepwise. The mass filter scans through the designated range of masses (usually up to about

As with other forms of mass spectrometers, EI or chemical ionisation (CI) is used to produce an ion source, but this is focused into the iontrap mass spectrometer in pulses rather than continuously. The fundamental difference is that all the solute ions generated over the entire pulse period are trapped in the detector and are then sequentially ejected in increasing mass number from the trap into the electron multiplier. The addition of helium into the trap (133 mPa) contracts the ion trajectory to the centre of the trap, where it is further focused by the ring electrode, to form dense ion packets that are expelled more efficiently than diffuse clouds, and thus greatly improve resolution. The spectral patterns can be quite different from those produced by mass filter spectrometers, and are often characteristic of the conditions under which the instrument is run, which makes comparison difficult between instruments. However, because the ion collection period is longer, the sensitivity of the ion trap in full scan mode is similar to that obtained in SIM on the average MS. Furthermore, an improved mass range (sometimes up to several thousand atomic mass units) gives this type of detector many applications, particularly for quantitative trace analysis, and for higher mass components. This technique is described in more detail in Chapter 37. Dual detector systems The simultaneous use of a combination of a universal detector (FID) with a specific detector to monitor the effluent of a column can provide useful information about the properties of functional groups and substituents in a molecule. The FID response is roughly dependent on the number of carbon atoms in a molecule and is quite predictable. However, the ECD response varies widely for different compounds is dependent on the electron-deficient part of the compound and is difficult to predict. The NPD response of a compound depends to some extent on the number of phosphorus or nitrogen atoms in a molecule, but it also depends on their environment. Thus, by using the FID as a reference, and measuring the ECD or NPD response relative to it, another characteristic for identification is obtained in addition to retention behaviour. Dual detector systems can be used in several ways. The column can be split at the detector end and the effluent passed into two different detectors that operate in parallel. This approach allows the most flexibility, since the choice of detectors is wide, and the effluent can be split in proportion to the sensitivity required from each detector. For capillary columns this is accomplished easily with zero-dead-volume press-fit tee connectors, but it is a more complicated operation for packed columns. Additional makeup gas may be required to ensure a good flow through the detectors, and care should be taken to use tubing of a total area smaller than or equal to the analytical column to avoid loss of peak shape through refluxing at the detector. Alternatively, the GC oven houses two completely separate but identically matched columns, each connected to a single detector. This is not an ideal approach, as matching columns is difficult and has to be checked at frequent intervals. Another approach is to stack the detectors in series, and some manufacturers deliberately provide detectors in identical modules for this purpose. There are limitations to the choice of possible detector combinations, as the first

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detector must always be a non-destructive detector, such as the ECD, AED or FTIRD.

Specimen preparation Prior to chromatography, it is usually necessary to isolate the compound(s) of interest from either a biological matrix (plasma, urine, stomach contents, hair or tissue) or some other matrix, such as soil, air or water. Removal of extraneous material and concentration of the compounds of interest usually take place simultaneously. The high water solubility of some drug metabolites (e.g. glucuronide conjugates) requires chemical conversion to a less polar entity to permit isolation from waterbased samples, and a hydrolysis procedure is often used for this purpose. Isolation and concentration Protein precipitation

If the analyte is present in blood in high concentration, a simple protein precipitation step often provides a suitable extract, although the possibility of losing significant amounts of analyte with the precipitate must be considered. Mixing with a solution of mercuric chloride or barium sulfate readily precipitates plasma proteins, and centrifugation provides a supernatant for direct injection onto the chromatography column. Use of perchloric or trichloroacetic acids (10%) is not advised, unless the resultant solution is neutralised prior to injection. Dimethylformamide is a good organic precipitation reagent that is well tolerated by most GC stationary phases. Other organic precipitating agents are methanol, acetone and acetonitrile, all of which should be added in the proportion of two volumes to each volume of blood. While the extract is still water based, most columns with a high stationary-phase loading (5 mm film thickness) can tolerate the injection of 1 mL of water. If the column is not water tolerant, it is possible to evaporate small volumes of the supernatant to dryness for reconstitution in a more suitable solvent. Liquid–liquid extraction

Liquid–liquid extraction is the most frequently used method to isolate and concentrate solutes for GC. The pH of the specimen is adjusted to ensure that the compounds to be extracted are not ionised (basic for bases, acid for acidic compounds). Bearing in mind that some portion of the aqueous acid or base will dissolve in the solvent, the use of strong mineral acids or alkalis is not advised as this adversely affects column performance. Best results are obtained with acidic buffers (phosphate or acetate) and with ammonium hydroxide or basic buffers (borate), using a 5 : 1 ratio of solvent to specimen. The solvent chosen should be sufficiently polar to partition the compound of interest without co-extracting excessive amounts of polar contaminants. For more water-soluble drugs, such as beta-blockers, the addition of 2–10% of a polar solvent (e.g. isopropanol or butanol) is helpful, or solid sodium chloride can be added to ‘salt out’ the analyte. If a derivatisation step is to be carried out subsequently, the use of a solvent compatible with the derivatisation eliminates the need for an evaporation step. Use of solvents with a higher density than the sample (e.g. dichloromethane) can lead to difficulty in isolation of the organic phase. Purification of extracts by back extraction (re-extraction of the analytes from the organic solvent at the opposite pH followed by re-extraction into solvent at the original pH) may be helpful for trace analysis. The use of a small volume of solvent for the final extraction serves as a concentration step without the need for separation and evaporation of the organic phase. Solid–liquid or solid-phase extraction

Solid–liquid extraction uses a polypropylene cartridge with a small amount (200 mg to 3 g) of high-capacity (1–20 mL) silica-based packing at the base of the reservoir. On introduction of the sample matrix, the compounds of interest are withheld by the packing. Impurities are then rinsed selectively from the column, and the final elution releases the compound of interest. Evaporation followed by reconstitution in a suitable solvent provides a clean, concentrated sample ready for analysis by GC. Bonded-phase packings that have been modified by the addition of various functional groups are available. The mechanisms of interaction for the matrix, analytes and packings are similar to those in LC

(see Chapter 38). Polar stationary phases retain polar analytes (normal phase) and are eluted with organic solvents, while non-polar stationary phases retain non-polar analytes (reversed-phase) and are eluted with aqueous solvents. Ion-pair extraction uses a non-polar stationary phase and polar analyte, with a counter-ion added to the sample solution, and allows retention of the (now neutral) analyte by a reversed-phase mechanism. In ion-exchange extraction, the adsorbent surface is modified with ionisable functionalities. Analytes with ionic charges opposite to those on the packing are retained. Solvents that contain counter-ions of greater strength are used to elute the analytes of interest from the tube. Solid-phase microextraction

Solid-phase microextraction (SPME) requires no solvents or complicated apparatus and can concentrate volatile and non-volatile compounds in both liquid and gas samples. The unit consists of a fusedsilica fibre attached to a stainless-steel plunger coated with a stationary phase (mixed with solid adsorbents as required). The plunger is inserted through a septum into a vial that contains the sample, and the fibre is exposed by depressing the plunger either into the liquid or into the headspace for 20–30 min. The retracted fibre is inserted into the injection port of the GC, and is desorbed when the plunger is depressed. The unit may be reconditioned and used 50 to 100 times. For field analysis, adsorbed samples can be stored and transported in the needle sealed in a special container for subsequent analysis by GC (or LC). Pesticides recovered from water samples have been shown to be more stable when stored in this way than in water. The special small-volume injection liner fits any model of chromatograph, and produces sharper peaks because of the higher linear gas velocity, with little or no backflush. Suitable stationary phases are: n

n n n n

100 mm dimethyl-PSX film for low-molecular-weight compounds or volatiles, or a thinner film (7 mm) for higher-molecular-weight semivolatile compounds 85 mm polyacrylate film for polar compounds 65 mm film of dimethyl-PSX-divinyl benzene for volatile alcohols and amines For surfactants, 50 mm Carbowax-templated resin For trace-level volatiles, a 75 mm Carbowax-carboxen phase is suitable.

An alternative approach uses a small magnetic stir bar encapsulated in glass and coated with a layer of dimethyl-PSX. The bar is left to stir in the sample for 30–120 minutes and then removed and placed in a thermal desorption tube. From there, it is introduced onto the GC as described in the section Thermal desorption. Both approaches give similar performance for higher-boiling compounds (>350
C), but SPME is inferior for lower-boiling compounds such as naphthalene and fluorene (b.p. 218
C and 298
C, respectively). Supercritical fluid extraction

A supercritical fluid (SCF) is a substance that is maintained above its critical temperature and pressure, where it exhibits physicochemical properties intermediate between those of a liquid and those of a gas. Properties of gas-like diffusivity, gas-like viscosity and liquid-like density combined with a pressure-dependent solvating power provided the impetus to apply SCFs to analytical separation. The initial applications most often involved isolation of flavours and contaminant residues from food and soil. These have now been extended to the isolation of drugs from blood and other aqueous-based media by using adsorbents added in-line (such as molecular sieves, diatomaceous earth, silica gel, etc.) to filter proteinaceous material and adsorb water. It is possible, by adding small volumes of co-solvent to the SCF, to extract highly polar solutes with excellent efficiency. In contrast to the conventional extracting solvents, the fluid most often used in supercritical fluid extraction (SFE), supercritical CO2, is non-polluting, non-toxic and relatively inexpensive. Additionally, extractions are carried out quickly at temperatures that avoid degradation of temperature-sensitive analytes and provide clean extracts with extremely high efficiency. Several dedicated SFE analysers are available; each consists of a gas supply, pump and controller used to pressurise the gas, temperature-controlled oven, extraction vessel, internal diameter regulator and collection device.

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The CO2 supply is compressed to a selected pressure (e.g. 28 000 kPa) and its is temperature adjusted (e.g. 50
C). As the supercritical CO2 passes through the sample material, the solutes are extracted to an equilibrium solubility level, typically about 10% (w/w). The gaseous solution that leaves the extractor is passed through the pressure-reduction valve, where the pressure (and thus the dissolving power) of the CO2 is reduced. The solutes precipitate in the separator, and the CO2 is recycled through the system several times until the extraction is completed, when it is vented to waste.

pyrolysis unit has been devised that sits in the GC oven (Gorecki, Poerschmann 2001). This is a silicosteel metal capillary (0.53 mm diameter) connected through butt connectors to a fused-silica restrictor inserted in the injection port which prevents backflush, and to the analytical column. The unit can be heated up to 750
C in 13 ms, and can be cooled again to ambient temperature in 4 s. The much reduced discrimination traditionally related to transfer of higher-boiling fractions is thus overcome, and this arrangement greatly extends the application of pyrolysis as a means of sample introduction into GC.

Headspace analysis

Tissues and hair

This method of isolation is used for analytes with volatility higher than that of the common extraction solvents. A detailed description of the technique is given in Chapter 14.

Tissues and hair require treatment prior to drug extraction to break down the biological matrix and enable a good recovery of the drug. For solid tissues, good results are obtained by incubation of a portion of the tissue with a mixture of a collagenase, a protease and a lipase in a buffer of suitable pH. For small amounts of tissues (100 mg), overnight treatment at room temperature suffices, although gentle agitation or occasional mixing speeds up the process. Larger amounts of tissue benefit from mechanical homogenisation prior to incubation. For the analysis of hair, an initial washing to remove residues from cosmetic products or environmental contaminants is recommended, followed by incubation with either caustic alkali (for basic drugs) or mineral acid (for acidic drugs). After adjustment of the pH, drug recovery can proceed by the usual procedures established for the specific compounds under investigation. For additional information see Chapter 10 and Chapter 19.

Purge and trap

Purge and trap is a powerful procedure for extracting and concentrating volatile organic compounds from soil, sediment, water, food, beverages, etc. It is especially useful for poorly water-soluble compounds and those with boiling points above 200
C. The procedure involves bubbling an inert gas (nitrogen or helium) through an aqueous sample or suspension at ambient temperature, which causes volatile organic compounds to be transferred into the vapour phase. During the purge step, purge gas sweeps the vapour through a trap containing adsorbent materials that retain the volatilised compounds. Water vapour may be removed by dry purging. The trap is rapidly heated to 5–10
C below the desorption temperature. The valve is then switched to join the trap flow to the carrier gas flow, and the trap heated to its desorption temperature for a fixed time. Adsorbent tubes are usually packed with multiple beds of sorbent materials, each one more active than the preceding one, which allows compounds with a wide range of boiling points and polarities to be analysed simultaneously. During the purge, the smaller and more non-polar solutes are readily carried down the beds and, since the carrier gas passes in the opposite direction during the desorption phase, the larger and more polar compounds do not come into contact with the innermost active beds, from which their release may be difficult to effect. Thermal desorption

This technique is used extensively for air monitoring in industrial hygiene, environmental air, indoor air or source-emission monitoring. The device may be portable or fixed and of varying size. Air is pumped continuously through the device at a fixed rate, during which time components are extracted gradually and concentrated onto the adsorbent beds; the arrangement of the beds is the same as described above for the purge and trap, and prevents potentially irreversible binding of large molecules. The direction of the flow is simply reversed during desorption. Analysis requires a special interface to the GC, which is described above in the section Thermal desorption and purge-and-trap injection. The adsorbents must have high capacity to remain active during the entire sampling period, and show an acceptable pressure drop during sampling. Ideally, a minimal amount of unwanted analytes should be absorbed, as these will contribute to the background noise. Pyrolysis

Analytical pyrolysis can be a very useful tool for characterization of complex materials, including synthetic polymers (e.g. plastics) and natural organic polymers such as humic organic matter (HOM). Conventional pyrolysis with a unit connected to the exterior of the GC injection port is of only limited use for HOM, first because of the formation of much carbonaceous residue of virtually zero diagnostic value, and second because of loss of high-boiling pyrolysis products with great diagnostic value during the sample transfer from the pyrolysis unit to the GC column. Such products are long-chain alkanes, alkylbenzenes, fatty acids and dicarboxylic acids, as well as steranes and hopanes. These large compounds originate in the HOM as they are not formed during pyrolysis, and are diagnostically distinct from smaller products such as phenols that might originate either from the HOM itself or equally from the breakdown of lignin, carbohydrates and proteins, which are the starting material for HOM. To circumvent this deficiency, an in-column

Hydrolysis Recovery of conjugated drug metabolites from biological fluids can be increased by hydrolytic cleavage of the conjugate bond prior to extraction. This offers a vast improvement in sensitivity for qualitative analysis, particularly from urine, and is essential to identify drugs (e.g. laxatives) that are excreted almost exclusively as conjugated metabolites. However, reliable quantitative analysis of conjugated metabolites requires that the unconjugated metabolite must first be removed or quantified, and then the total (conjugated plus unconjugated) metabolite be measured after hydrolysis in a subsequent separate procedure. For quantitative work, appropriate standards that contain conjugated metabolites must be carried through the procedure to monitor the efficiency of the hydrolysis step. Enzymatic hydrolysis

The use of a specific enzyme to cleave chemical bonds is the more specific of the two approaches but it incurs additional cost and time. It also provides cleaner extracts, and therefore prolongs the life of the chromatography column. There are a number of commercial preparations of purified glucurase and sulfatase harvested from different species. It is important to pay attention to the pH and temperature optima of the specific enzyme preparation. Temperature-tolerant preparations allow heating up to 60
C, which permits relatively short incubation times (2 h). Chemical hydrolysis

This quicker and less expensive approach can provide suitable extracts for chromatography for some analytes, although they are generally more demanding in terms of clean-up procedures. Typically, strong mineral acids or alkalis are used, often with boiling or treatment in a microwave or pressure cooker. Extracts must be neutralised, otherwise the chromatography column deteriorates quickly. Care should be taken to ensure the stability of the analytes to the hydrolysis conditions. Vigorous hydrolysis conditions often yield undesirable by-products or, if several compounds can be hydrolysed to a single entity, preclude accurate identification of the original compound present. For example, both the acid and the enzymatic hydrolysis of benzodiazepines remove glucuronide conjugates, but acid hydrolysis also converts two or three drugs to the same benzophenone compound (diazepam, temazepam and ketazolam are all converted into 2-methylamino-5-chlorobenzophenone). While this compound has good chromatography characteristics, the approach is unsuitable for those applications (such as forensic analysis) that require absolute identification of the drug ingested.

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Derivative formation Derivatisation enables the analysis of compounds that otherwise could not be monitored readily by GC. To some extent the availability of stable polar stationary phases in capillary columns and the use of temperature programming has negated the requirement for derivatisation, although it is still widely used. Choice of reagent is based on the functional group that requires derivatisation, the presence of other functional groups in the molecule and the reason for performing the reaction. Although the retention characteristics are changed, the order of elution of a series of derivatives will be the same as that for the parent compounds. The preparation of derivatives modifies the functionality of the solute molecule to increase (or sometimes decrease) volatility, and thereby shortens or lengthens the retention time of a substance, or to speed up the analysis. Another common reason for derivatisation is to improve resolution and reduce tailing of polar compounds (hydroxyl, carboxylic acids, hydrazines, primary amines and sulfhydryl groups). For instance, hydroxylated compounds often have long retention times and column adsorption causes tailing, which results in low sensitivity. However, they readily form silyl ethers and these derivatives show excellent chromatography, and sensitivity can often be improved by a factor of 10 or more. Derivatisation can also help to remove the substance peak away from interfering material. For example, the reaction of amfetamine with acetone enables successful differentiation from methyl ethyl ketone on most stationary phases. Derivatives may also be used to make the molecule amenable to detection by selective detectors, or can be used to improve the fragmentation pattern of the compound in the mass spectrometer. The reaction may be carried out during extraction (e.g. extractive alkylation), on the dry residue after solvent extraction (e.g. silylation) or during injection (e.g. methylation). In choosing a suitable reagent, certain criteria must be used. A good reagent produces stable derivatives without harmful by-products that interact with the analytical column, in a reaction that is almost 100% complete. Poor reagents cause rearrangements or structural alterations during formation, and contribute to loss of sample during reaction. Most manufacturers of derivatising reagents provide information on the potential uses of each product, along with standard operating instructions. Entire texts, such as that by Blau and Halket (1993), are devoted to this topic. Chiral separations

Chiral compounds can be derivatised to improve their chromatographic characteristics, and the enantiomers separated on a chiral stationary phase. Both enantiomers behave similarly, provided that steric hindrance does not preclude a reaction with one enantiomer. An alternative approach is to use a chiral derivatising reagent which, when reacted with enantiomers, produces diastereoisomers that can then be separated on a conventional stationary phase. As with enantiomers, diastereoisomers still produce similar mass spectra, but are resolved in time by the chromatography column. This approach is less expensive and also less restrictive, since a dedicated column is not required. Care should be taken to ensure the enantiomeric purity of the derivatising reagent, and to guard against racemisation during the reaction. n-Trifluoroacetyl-Lpropyl chloride (TPC) in triethylamine and chloroform (or ethyl acetate) is a commonly used chiral reagent that couples with enantiomeric amines. Excess reagent is washed off with 6 mol/L HCl and the organic phase is dried over magnesium sulfate. For chiral alcohols, (1R,2S,5R)(–)-menthylchloroformate (MCF) reacts well if pyridine is used as a catalyst.

Quantitative determinations Quantitative work usually requires some form of sample preparation to isolate the drug from the bulk of the sample and some degree of concentration or, more rarely, dilution. These processes inevitably introduce a degree of analytical error. A further difficulty is caused by the non-reproducibility of injected volumes. To compensate for these errors, it is usual to compare the response of the unknown with the response of an added internal standard. The internal standard

should be added as early as possible in the assay process and should have chromatographic properties matching the drug’s as closely as possible, preferably with a longer retention time. It is often possible to obtain unmarketed analogues of drugs, or compounds specially synthesised for use as internal standards (e.g. a methyl addition or a halogen substitution). However, the internal standard usually does not behave exactly as the drug and careful control of variables, such as pH, is necessary. If a derivative is to be prepared, the internal standard should also be amenable to derivatisation. Use of an inappropriate internal standard can seriously affect precision (Dudley 1980). If a mass spectrometer is being used as the detector, then the ideal internal standard is a 3H- or 13C-substituted analogue of the drug, a number of which are readily available at reasonable cost. Calibration should include points of higher and lower concentrations than the sample, and quality assurance samples should be included at appropriate concentrations in frequently run assays. Peak measurement may be by peak height or by the peak area obtained by integration. If the peaks show even a modest degree of tailing, use of peak area usually provides a more accurate quantitative result. A plot of the ratio of peak height (or area) of the drug to internal standard versus concentration is a straight line with most detectors. Care should be taken in the preparation of standards to match the matrix to that of the specimens, and to allow for any associated salt or water of crystallisation in the calculation of the concentration. The best results are obtained when the amount of internal standard used produces a peak response ratio of 1 at the mid-point of the calibration range.

Optimising operation conditions to customise applications Additional sensitivity can be achieved by increasing sample size, using a concentration step, derivatisation, injecting a larger sample volume, selecting a different stationary phase or using the detector at a higher sensitivity level. When attempting a new analysis, it is advisable first to review published literature for a method that can be copied or for a method that involves a similar type of compound and can be adapted. Column manufacturers’ catalogues are a useful source of information and invariably show examples of separations performed with their columns. Data on boiling points and RI (see monographs in Volume 2) are also useful indicators. If the review is not helpful, a start can be made with a standard column, such as a 100% methyl-PSX capillary column (25 m with a 0.5 mm film) and using standard flow conditions (1–2 mL/min helium). The oven temperature should be taken from 80
C to 300
C at 10
/min (or started at 200
C or 250
C if only an isothermal oven is available). A solution of the compounds of interest in ethanol or methanol should be injected with the injector temperature set at 250
C. If a peak tails, derivatisation or use of a more polar stationary phase should be considered. Fine-tuning is carried out once some peaks have been obtained. Having established the chromatography, the extraction and concentration steps can be determined. Manufacturers’ catalogues are again a useful source for both derivatisation and solid-phase extraction procedures. Good preventive maintenance is essential. The injector (or liner) should be cleaned periodically, and any glass wool changed regularly (approximately every 100 to 1000 injections, depending on the quality of the extracts). For capillary columns, the performance is improved by periodically removing the first 5–10 cm of capillary tubing, or a retention gap could be considered for dirty samples. It is advisable to monitor performance by selecting certain performance criteria (e.g. a certain response size or amount of acceptable separation between two closely eluting components) to indicate when maintenance is required. The manufacturers’ instructions for cleaning detectors should be followed. The presence of traces of contaminants in the carrier gas supply shortens the column life drastically, and also causes detector deterioration. In-line filters (to remove oxygen, hydrocarbons, etc.) and molecular sieves (to remove water vapour) are strongly recommended, and the use of stainless-steel gas tubing minimises further contamination.

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Optimising operation conditions to customise applications Carrier gas flow should be optimised for a particular column and a particular carrier gas. This is most important for capillary columns. Fig. 40.6 shows the relationship between efficiency expressed as the HETP versus carrier gas velocity (van Deemter plot) for a 28 m  0.25 mm i.d. WCOT OV-101 column. Modifying the mobile phase in GC has very little effect compared with that observed with HPLC or thin-layer chromatography (TLC) and, in general, affects efficiency rather than selectivity. Nitrogen gives higher efficiency but at the expense of longer analysis time, while the less dense, but more hazardous, hydrogen gives lower efficiency but faster analysis. In practice, nitrogen is usually used for packed columns and helium for capillary columns. Certain detectors impose restrictions on the choice of carrier gas, but an additional supply of gas can be added to the column effluent to purge the detector. Experimenting with higher flow and a lower operating temperature (or vice versa) can give rewarding results for the separation of compounds that elute closely. This effect is particularly noticeable for two compounds that have different polarities, as the retention of the more polar compound is influenced to a greater extent the longer it resides in the column (non-polar compounds elute in boiling point sequence). Conditions of constant flow improve the efficiency of late-eluting peaks and produce faster chromatography than do constant pressure conditions. For a particular separation, the lowest temperature compatible with a reasonable analysis time should be used. In general, retention times double with each 20
C decrease in temperature. If the time is excessive, it is generally better to reduce the stationary phase loading or use a shorter column than to increase the column operating temperature. There is a maximum temperature at which a column can be operated and there is also a minimum temperature below which efficiency drops sharply. Manufacturers give the temperature operating ranges for each of their stationary phases (see Table 40.3). The stationary phase must be a liquid at the temperature of operation, and if a column is run at too low a temperature to obtain longer retention times the stationary phase may still be in the solid or semi-solid form. When using temperature programming, experimentation with a faster initial ramp followed by a slower subsequent ramp or an isothermal period can help resolve problematic separations. Efficiency can also be improved by decreasing the column diameter or increasing the column length. The resultant increase in analysis time (particularly if the flow must be reduced to accommodate the increased pressure demand imposed by a narrower column) can usually be offset by using a slightly higher operating temperature (temperature increases

affect retention time much more than do increases in gas flow). As shown in Table 40.7, reducing the diameter of a capillary column markedly increases efficiency, but the retention time remains constant only as long as the same phase ratio is maintained. Therefore, unless there is a simultaneous reduction in film thickness, retention increases in direct proportion to the phase ratio. The solvent used for the sample can sometimes produce unexpected derivatives that give different retention times (traces of acetic anhydride that remain in butyl acetate avidly derivatise primary amines at room temperature). An inert non-polar solvent should be used if possible to minimise the co-extraction of unwanted contaminants. Acetone, other ketones, ethyl acetate and carbon disulfide readily form derivatives with primary amines and should be avoided. The choice of injector type and injection solvent also play an important part in the chromatography. A solvent volume should be chosen that does not expand to exceed the capacity of the injector (see Table 40.5), otherwise backflush and irreproducible results are obtained. Split injection significantly reduces the amount of solvent and associated contaminants that enter the column and, although the analyte response is reduced, the improvement in the signal-to-noise ratio often results in enhanced sensitivity. The use of a selective detector, such as an ECD (with the preparation of a strongly responsive derivative if appropriate), can improve sensitivity typically up to 100-fold. Similarly, switching from full scan to SIM in MS improves the sensitivity, usually by a factor of 10. However, selective detectors should not be used as a substitute for cleaning up of sample extracts, as loading contaminants onto the column affects the chromatography adversely, even if the selective detector does not respond to the compounds. Increasing the detector temperature may also improve sensitivity. Fronting or splitting of peaks indicates column overload. If the detector sensitivity permits, the best option here is to inject a smaller sample volume (or a more dilute sample), rather than to increase the column loading or diameter, otherwise efficiency is also affected. If trace impurities are sought in the presence of a preponderant component, a number of stationary phases of differing polarities should be tried. Trace impurities are seen easily if they emerge before the main component of a mixture, while they may be lost completely in the tail if they elute just after the large peak. Early peaks are also sharper and thus, for the same peak area, higher – an effect that can contribute enormously to the successful detection of trace substances.

Table 40.7 Relationship of film thickness, phase ratio (b)(a), efficiency (N)(b) and column diameter Film thickness d (mm)

Column internal diameter (mm) 0.10

0.10

0.18

250(a)

450

0.20 500

0.25 625

0.32 800

0.45

0.53

1125

1325

0.18

139

250

278

347

444

625

736

0.25

100

180

200

313

400

450

663

0.40

63

113

125

156

200

282

331

0.42

—

107

119

149

190

265

315

0.50

—

90

100

125

160

225

265

0.83

—

—

60

75

96

136

160

0.85

—

—

59

74

94

133

156

1.00

—

—

50

63

80

113

133

1.27

—

—

—

49

63

88

104

1.50

—

—

—

42

53

75

88

2.55

—

—

—

25

31

44

52

3.00

—

—

—

21

27

38

44

5.00

—

—

—

13

16

23

27

12 500

6600

5940

4750

3710

2640

2240

Efficiency N (b)

Phase ratio b = r/2d, where r = column radius (mm), d = film thickness (mm). N, theoretical plates per metre; maximum efficiency calculated for a solute with k = 5.

(a)

(b)

653

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:23:8

654

Gas Chromatography

Two-dimensional GC

For most quantitative applications in drug analysis the chromatogram contains only two or three compounds, while some qualitative applications may contain 20 or more peaks of interest. Using an efficient column maximises the probability that a peak in a given time window consists of only one compound and that it is indeed the compound of interest. Other separations are far more complex, and the compound of interest may be present at minute concentrations relative to the background (e.g. flavours in foods or trace residue analysis in foods or groundwater). While these analyses can be fine-tuned to a limited extent by the use of element-specific detectors, the problem of obtaining a clean peak for positive identification and quantitation often remains. Consideration of equation (40.5c) shows column resolution to be related to two terms that can be varied by the analyst. The first of these is N, the number of theoretical plates, which in a typical capillary column is a few more than 100 000 plates. However, since R increases with the square root of N, a substantial increase in resolution can be obtained only by a very large increase in column length, and with a correspondingly large increase in analysis time. Some 500 million theoretical plates would be needed to separate 99 compounds out of a 100 component mixture. The other term is a, which describes the selectivity of a stationary phase for a particular pair of analytes. A modest increase in a can have a significant impact on resolution. This same philosophy was applied by analysts in the 1970s and 1980s who used two or more complementary stationary phases in parallel housed in different packed columns to make positive peak identifications (Moffat et al. 1974b). These columns were selected to have chemical properties as different as possible, but were limited by temperature compatibility since they were often placed in the same GC oven, and sometimes were split off from the same injector to allow reproducible temperature programming. However, recent advances in electronic pressure control and electronic proportional back-pressure regulators with pressure sensing, the manufacture of inert connector fittings and improvements in cryogenic focusing devices are enabling analysts to contemplate using two different analytical columns in series to achieve a satisfactory result. Two main multidimensional approaches are receiving attention for routine use: two-dimensional gas chromatography (GC  GC) and 2DGC with heartcutting. Instruments are now commercially available with two independently operated and controlled column ovens in a variety of injector and detector configurations. 2D-GC techniques began with heartcutting, in which only a timed portion of the chromatographed effluent from the first column was diverted from the detector or waste line into a second column of different polarity. A connector (or modulator) was employed to trap and focus the first eluent into narrow bands and transfer it to the second column at a rate that preserved the separation already achieved. In GC  GC the entire chromatography effluent from the first column is introduced to the second one. Difficulties arise when the retention of a solute on the second column exceeds the modulation cycle and ‘wraparound peaks’ appear with the solutes in subsequent cycles. A more recent modification, aimed at preventing this phenomenon, is called stopped-flow GC  GC. Here, the flow through the first column is stopped for a brief period, typically a few seconds, during each modulation cycle. This allows not only for better preservation of the separation of the first column, but also for longer separation times on the second column because subsequent bands are held up. This renders the secondary separation time independent of the modulation cycle, and increases the options for varying the chromatography conditions. A disadvantage is the longer run time and associated larger data file. Time-of-flight (TOF)-MS is almost mandatory to de-convolute the rapid analysis in the second column. These techniques have been demonstrated across many areas of industry, for example for identification of flavours in liquors, pesticide residues in foods and essential oils, and oxygenates in gasoline. The topic was recently reviewed by Pierce et al. (2008), and detailed examples showing hardware configurations can be found at www.chem.agilent.com/cag/prod/GC/Simplified_2DGC.

Specific applications The systems given below are applicable to the routine screening, separation and identification of groups of drugs and chemicals. They are not

exhaustive lists and references to specific systems for individual drugs and chemicals are given in the relevant monographs. Some of these systems use columns that are identical or very similar in terms of discriminating power (see Table 40.3), but are operated with different temperature programmes for specific groups of compounds. Moreover, some groups of substances are chromatographed as derivatives rather than as the parent compounds. The most commonly used general screening system is a 100% dimethyl-PSX (methyl-PSX or X-1) capillary column (for packed columns, SE-30, OV-1 or OV-101 is equivalent). This should always be used for screening purposes, since it has the best chance of eluting any compound of interest. Analysts have collaborated to compile comprehensive lists of retention indices using this system (De Zeeuw 2002), some of which are included in the Index of Gas Chromatographic Data. Most of the data are for the drugs themselves, but thermal decomposition may occur and the peak observed may be for the decomposition product (referred to as ‘artefact’) rather than the original drug. Where the drug is known to chromatograph badly, or to decompose, data are given for suitable derivatives (e.g. methyl or ethyl esters for the sulfonamides, and TMS derivatives for hydroxides). Wherever possible, the RI of the drug is given, since this is a more reproducible parameter than retention time or relative retention (see discussion above). However, if a laboratory prefers routinely to use the latter parameters, the RI data can be converted easily after chromatography of a few representative drugs and using a regression analysis of RI against either retention time or relative retention. RIs for some additional non-drug substances that might interfere with toxicological analyses, but are not included in the monographs. A nitrogen–phosphorus (alkali flame ionisation) detector is the best detector for nitrogenous drugs and phosphorus-containing pesticides, but an FID should also be used, since some drugs do not contain nitrogen (e.g. some anti-inflammatory agents). ECDs are excellent for benzodiazepines and halogen-containing compounds, such as some phenothiazines and herbicides. Extra selectivity can always be obtained by using element-specific detectors (e.g. those for phosphorus and sulfur for compounds that contain these elements). Additional specificity or confirmation of identity can be obtained by using a mass-selective detector, such as MS or an ion-trap detector. Where improved fragmentation can result from the use of derivatisation, data for suitable derivatives have been included. As mass spectrometry has matured as a technique, significant improvements in detection of higher-mass fragments have enabled the use of larger derivatising reagents such as heptafluorobutyrate (HFB). In any analysis for an unknown compound, the data obtained from complementary techniques, such as TLC, HPLC or colour tests, should always be assessed for compatibility with the GC result. (In the tables of retention indices given here, a dash indicates that no value is available for the compound, not that it does not elute.) General screen, systems GA and GB Both systems use standard columns that are able to chromatograph a wide variety of drugs and chemicals. System GB uses a slightly more polar column, which gives better peak shapes for hydroxylated compounds (many drug metabolites are hydroxylated), better resolution between structural isomers and improved peak shape for primary amines over the less polar GA. However, the retention indices are very similar for GA and GB, and can be interconverted using the equations: GB RI ¼ 1:079  ðGA RIÞ  66 or GA RI ¼ ðGB RIþ66Þ=1:079 As the stated values for drugs are retention indices, the operating conditions for the columns may be varied to suit particular laboratory situations.

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:24:1

Specific applications n

System GA

Details are taken from the TIAFT book (De Zeeuw 2002) and the PMW Spectral Library (Pfleger et al. 2004). Chromatography details are given for both systems below. n

n

n n n n n n n n

Column: 3% SE-30 or OV-1 on 80 to 100 mesh Chromosorb G HP (acid washed and dimethyldichlorosilane treated) glass (2 m  2 mm i.d.); it is essential that the support be fully deactivated. Temperature: Normally between 100
C and 300
C; for isothermal conditions, an approximate guide to temperature is to use the RI divided by 10. Carrier gas: N2, 45 mL/min. Capillary column: 100%-dimethyl-PSX (X-1) (10–15 m  0.32 or 0.53 mm i.d., 1.5–3 mm). Carrier gas: He. Temperature programme: 135
C for 4 min to 200
C at 13
/min to 312
at 6
/min for 6 min. Column: HP1 (100%-dimethyl-PSX) fused-silica capillary (12 m  0.2 mm i.d., 0.33 mm). Injector: 280
C splitless mode. Temperature programme: 100
C for 2 min to 310
C at 30
/min for 8 min. Carrier gas: He, 1 mL/min.

System GB

Data generated by the author. n n n n

Column: 5% phenyl–95% dimethyl-PSX (X-5) capillary (20–30 m  0.2 or 0.25 mm i.d., 0.5–1 mm). Carrier gas: He, 1 mL/min. Temperature programme: 90
C for 0.7 min to 240
C at 35
/min to 290
C at 8
/min to 325
C at 25
/min for 6 min. Reference compounds: n-Alkanes with an even number of carbon atoms, or a reference drug mix that contains amfetamine (1125), ephedrine (1365), benzocaine (1545), methylphenidate (1725), diphenhydramine (1870), tripelennamine (1976), methaqualone (2135), trimipramine (2215), codeine (2375), nordazepam (2490), prazepam (2648), papaverine (2825), haloperidol (2930) and strychnine (3116). (RI values for system GA are given in parentheses for the drug mix.)

655

Retention indices: Values for drugs in these systems are found in drug monographs and in the Indexes of Analytical Data; they are also included in the systems for specific groups of drugs that follow. The search window should be 50 RI units if hydrocarbons are used to calculate RI, or 30 RI units if a reference drug mixture is used for the RI calculation.

Amfetamines and other stimulants Amfetamines are basic drugs that require strongly alkaline conditions to be extracted from aqueous solution. The conditions are too basic to extract the phenolic metabolites, but these can be recovered at pH 8 or 9 and the extracts combined prior to chromatography. For high sensitivity, back extraction into dilute sulfuric acid (0.05 mol/L) is a useful clean-up procedure. When using packed columns, derivatives are almost always required for the primary and secondary amines, since the peaks tail badly. Suitable derivatives are acetyl, trifluoroacetyl, pentafluoropropionate or TMS (see Derivative formation). With capillary columns, derivatives are used most often to improve mass spectral patterns or to modify the separation of compounds that elute closely. For hydroxylated metabolites, derivatisation is invariably required to achieve acceptable chromatography. Data for the most commonly used derivatives are given in Table 40.8. Care must be taken to avoid drug loss during solvent evaporation, which can be obviated by adding a small amount of concentrated aqueous acid (20 mL 6 mol/L HCl) to the organic solvent. Unless otherwise stated, GC retention data and mass spectral data are identical for both D- and L- (þ and –) enantiomers. To differentiate enantiomers (such as D- and L-metamfetamine or amfetamine), a chiral column or chiral derivatising reagent is required (Cody, Schwarzhoff 1993). At present, all amfetamine- or metamfetamine-producing drugs (aminorex, amfetaminil, clobenorex, ethylamfetamine, fencamine, fenethylline, fenproporex, mefenorex, prenylamine, benzfetamine, dimethylamfetamine, famprofazone, furfenorex) are racemates (with the exception of L-selegiline, L-metamfetamine and dexamfetamine). Stereo-inversion does not occur in humans (Nagai, Kamiyama 1991). Drugs that are metabolised to amfetamines, but are not themselves classified as such, are also listed. System GA or GB, previously described, may be used as well as system GC.

Table 40.8 GC retention data and mass spectral data for the amfetamines and derivatives (reference compounds are n-alkanes with an even number of carbon atoms; AC, acetyl; ET, ethyl; HFB, heptafluorobutyrate; PFP, pentafluoropropionate; TFA, trifluoroacetyl; TMS, trimethylsilyl) Compound

System

Principal ions (m/z)

GA

GB

GC

Amfetaminil (metabolised to amfetamine, see below)

1755

—

—

132

105

133

89

77

65

Amfetamine (D or L)

1125

1150

—

91

65

51

63

89

120

Amfetamine-TFA

1095

—

1536

140

118

91

69

65

117

Amfetamine-PFP

1330

—

—

118

190

91

119

65

117

Amfetamine-TMS

1190

—

—

116

73

100

91

117

192

Amfetamine-AC

1501

—

—

44

86

118

91

117

65

Art (formyl)

1100

1142

—

56

91

125

146

147

132

M (3OH-)-PFP2

1520

—

—

190

280

119

253

69

M (3OH-)-TMS2

1850

—

—

116

73

100

280

117

— 179

M (3OH-)-AC2

1930

—

—

86

134

176

107

77

235

M (4OH-)

1480

—

—

56

107

77

108

91

151

M (4OH-)-AC

1890

—

—

134

107

86

77

133

193

M (4OH-)-AC2

1900

—

—

134

86

176

107

77

133

M (3,4-di-OH-)-AC3

2150

—

—

86

150

234

192

137

123

M (OH-methoxy-)

1465

—

—

138

137

122

123

94

181

M (OH-methoxy-)-AC2

2065

—

—

164

86

206

137

165

265

M (desamino-oxo-OH-)-AC

1520

—

—

107

149

150

176

192

—

M (desamino-oxo-OH-methoxy-)

1510

—

—

137

180

94

122

138

107

M (desamino-oxo-OH-methoxy-)-AC

1600

—

—

137

180

138

109

122

222 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:24:22

656

Gas Chromatography

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

1735

—

—

123

166

208

150

124

250

Aminorex (metabolised to amfetamine)

2065

—

—

56

118

162

91

119

145

Amiphenazole-AC2

2575

—

—

191

233

121

275

149

257

3,4-Benzodioxazol butanamine (BDB)

1570

1622

—

58

136

77

135

164

193

BDB-TFA

1705

—

—

135

176

154

77

161

289

BDB-PFP

1700

—

—

135

176

119

204

126

339

BDB-AC

1950

—

—

58

176

162

100

135

235

M (desamino-oxo-di-OH-)-AC2

Art (formyl)

1585

—

—

70

135

77

205

176

92

M (desmethylenyl-methyl-)-AC2

2140

—

—

58

178

220

100

137

279

2235

—

—

58

100

164

248

123

307

2,3-Benzodioxazol butanamine (2,3-BDB)

1550

1602

—

58

77

135

83

164

193

2,3-BDB-TFA

1705

—

—

176

154

135

77

136

289

2,3-BDB-PFP

1615

—

—

135

176

119

204

136

339

2,3-BDB-TMS

1670

—

—

130

73

135

236

250

77

M (desmethylenyl-)-AC3

1895

—

—

58

176

100

135

235

131

1575

—

—

70

77

135

205

176

105

Bemegride

—

—

1253

—

—

—

Benzfetamine (metabolised to metamfetamine and amfetamine)

1855

1899

2172

2,3-BDB-AC Art (formyl)

— 91

—

—

148

65

149

92

56

N-Benzylpiperazine (BZP)

—

1530

—

91

134

176

56

120

146

BZP-AC

1920

—

—

91

146

85

134

132

218

BZP-HFB

1730

—

—

91

281

372

175

146

295

BZP-TFA

1665

—

—

91

181

272

195

146

132

BZP-TMS

1860

—

—

102

248

157

86

116

233

M (4-OH-) isomer 1-AC2

2275

—

—

107

85

149

192

204

276

M (4-OH-) isomer 2-AC2

2245

—

—

107

149

204

85

190

276

M (OH-methoxy-)-AC2

306

2380

—

—

137

85

127

179

234

4-Bromo-2,5-dimethoxyamfetamine (DOB)

1804

1875

—

44

77

230

232

105

91

DOB-TFA

1935

—

—

229

231

256

258

69

369

DOB-PFP

1905

—

—

229

231

119

256

258

419

DOB-TMS

1920

—

—

116

73

117

229

272

201

2150

—

—

86

256

258

162

315

317

1790

—

—

56

254

256

285

229

199

4-Bromo-2,5-dimethoxyphenethylamine (2C-B, BDMPEA)

1785

1867

—

230

232

215

217

259

261

2C-B-AC

303

DOB-AC Art (formyl)

2180

—

—

242

244

229

148

301

Art (formyl)

1840

1860

—

242

240

229

231

271

273

M (O-desmethyl-) isomer 1-AC2

2410

—

—

228

230

287

289

215

329

M (O-desmethyl-) isomer 2-AC2

2440

—

—

228

230

287

289

215

329

M (O-desmethyl deamino-OH-)-AC2

2160

—

—

228

230

288

290

213

329

Cathinone

—

—

—

—

—

—

—

—

—

Cathinone-TFA

1350

—

—

105

77

69

106

140

78

Cathinone-PFP

1335

—

—

190

119

105

280

253

225

Cathinone-TMS

1590

—

—

116

73

77

117

191

206

Cathinone-AC

1610

—

—

86

77

105

191

134

132

1-(3-Chlorophenyl)-piperazine (mCPP)

—

1806

—

154

196

138

111

156

75

mCPP-AC

2265

—

—

166

238

138

154

168

195

M (pOH-) isomer 1-AC2

2515

—

—

182

254

169

184

211

296

M (pOH-) isomer 2-AC2

2525

—

—

182

254

169

184

296

211

M (desethylene-)-AC2

2080

—

—

140

195

153

142

111

169

M (chloroaniline)-AC

1580

—

—

127

129

169

171

99

100

Cathine (see [D+]-norpseudoephedrine)

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:24:41

Specific applications

657

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

M (OH-chloroaniline) isomer 1-AC2

1980

—

—

143

145

167

185

227

169

M (OH-chloroaniline) isomer 2-AC2

2020

—

—

143

185

145

187

78

114

Chlorphentermine

1355

1393

1725

58

107

108

125

168

89

Chlorphentermine-AC

1730

—

—

58

100

86

166

167

125

Chlorphentermine-HFB

1560

—

—

254

125

166

214

169

255

Chlorphentermine-PFP

1515

—

—

204

166

164

154

125

119

Chlorphentermine-TFA

1520

—

—

154

125

114

166

89

69

Chlorphentermine-TMS

1520

—

—

130

73

114

125

89

240

Clobenzorex (metabolised to amfetamine and norephedrine)

1940

—

—

168

125

170

127

89

244

Clobenzorex-AC

2290

—

—

168

125

210

170

91

266

Clobenzorex-PFP

2040

—

—

125

127

118

91

314

316

Clobenzorex-TFA

2075

—

—

125

127

91

118

264

266

M (chlorobenzyl-OH-)-AC2

2565

—

—

226

141

183

200

268

324

M (OH-) isomer 1-AC2

2585

—

—

168

125

210

107

272

364

M (OH-) isomer 2-AC2

2630

—

—

168

125

210

170

134

176

M (OH-methoxy-)-AC2

2690

—

—

168

125

164

137

206

210

M (OH-chlorobenzyl-OH-) isomer 1-AC3

2705

—

—

226

141

183

161

215

268

M (OH-chlorobenzyl-OH-) isomer 2-AC3

2725

—

—

141

226

268

183

125

150

M (OH-chlorobenzyl-OH-) isomer 3-AC3

2775

—

—

226

141

183

161

215

268

M (OH-chlorobenzyl-OH-) isomer 4-AC3

2795

—

—

141

226

183

134

107

268

M (OH-alkyl-OH-)-AC3

2725

—

—

168

125

210

192

150

220

M (di-OH-)-AC3

2765

—

—

168

125

210

192

150

234

Diethylpropion (amfepramone)

1486

1532

1715

100

44

72

101

77

56

M (phenylpropanolamine)

1360

1352

—

44

77

79

51

45

M (diethylnorephedrine)

—

1599

—

Dexamfetamine (see Amfetamine)

—

—

—

—

—

42 —

M (ethylnorephedrine)

—

1457

—

—

—

—

—

—

—

M (N-desethyl-)

—

1423

—

—

—

—

—

—

—

M (N-didesethyl-)

—

1338

—

—

—

—

—

—

—

M (norephedrine)

—

—

1383

—

—

—

—

—

—

2,5-Dimethoxyamfetamine (DMA)

1546

1601

—

44

152

137

121

195

91

DMA-AC

1870

—

—

44

178

86

121

237

91

1550

—

—

56

176

151

121

207

91

2C-E-AC

2000

—

—

192

177

149

179

91

251

2C-E-TFA

1770

—

—

179

192

305

177

149

193

M (O-desmethyl-) isomer 1-AC2

2210

—

—

178

237

165

163

179

279

M (O-desmethyl-) isomer 2-AC2

2240

—

—

178

237

165

163

179

279

M (OH-) isomer 1-AC2

2340

—

—

250

191

207

309

175

237

M (OH-) isomer 2-AC2

2420

—

—

190

191

250

309

164

295

M (OH-) isomer 3-AC2

2500

—

—

196

195

250

208

309

212

M (desamino-OH-)-AC

1850

—

—

192

177

149

91

252

179

M (O-desmethyl-desamino-OH-) isomer 1-AC2

1990

—

—

178

163

145

165

238

280

M (O-desmethyl-desamino-OH-) isomer 2-AC2

2000

—

—

178

163

220

238

154

280

M (O-desmethyl-OH-) isomer 1-AC3

2430

—

—

176

235

177

277

309

337

M (O-desmethyl-OH-) isomer 2-AC3

2460

—

—

176

177

235

277

161

337

2,5-Dimethoxy-4-ethylthio-b-phenethylamine (2C-T-2)

1980

—

—

212

211

183

241

153

197

2C-T-2-AC

2310

—

—

224

211

283

209

153

181

Art (formyl) 2,5-Dimethoxy-4-ethyl-b-phenethylamine (2C-E)

2C-T-2-TFA M (N-acetyl-)

2210

—

—

211

337

224

181

151

222

2310

—

—

224

211

283

209

153

181 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:24:49

658

Gas Chromatography

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

M (N-acetyl-)-AC

2400

—

—

224

211

209

181

153

M (desamino-OH-)-AC

2050

—

—

224

284

209

167

225

100

M (O-desmethyl-)-AC2 or (O-desmethyl-N-acetyl-)-AC 2120

—

—

269

210

197

252

311

297

M (OH-N-acetyl-)-TFA

2270

—

—

259

427

260

428

367

M (O-desmethyl-)-TFA2

1980

—

—

306

293

419

209

294

325

307

2290

—

—

306

323

293

307

355

197

2,5-Dimethoxy-4-iodo-b-phenethylamine (2C-I)

2330

—

—

278

263

307

247

279

232

2C-I-AC

2260

—

—

290

349

275

277

148

247

2C-I-TFA

2100

—

—

290

277

247

275

231

403

M (O-desmethyl-) isomer 1-AC2

2480

—

—

276

335

233

263

259

377

M (O-desmethyl-) isomer 2-AC2

2500

—

—

276

335

263

261

377

358

M (desamino-OH-)-AC

2150

—

—

290

275

148

350

247

277

M (O-desmethyl-desamino-OH-) isomer 1-AC2

2240

—

—

276

336

134

261

191

378

M (O-desmethyl-desamino-OH-) isomer 2-AC2

2275

—

—

276

261

336

263

150

378

—

—

M (O-desmethyl-N-acetyl-)-TFA

2,5-Dimethoxy-4-methyl-b-phenethylamine (2C-D) 1940

—

—

178

135

163

165

179

237

M (O-desmethyl-) isomer 1-AC2

2130

—

—

164

223

151

149

265

165

M (O-desmethyl-) isomer 2-AC2

2200

—

—

164

223

151

149

265

206

M (OH-)-AC2

2390

—

—

236

295

193

177

235

223

M (desamino-OH-)-AC

1740

—

—

178

163

135

238

79

104

M (O-desmethyl-desamino-OH-) isomer 1-AC2

1880

—

—

164

149

72

224

182

266

M (O-desmethyl-desamino-OH-) isomer 2-AC2

2C-D-AC

1890

—

—

164

149

121

224

266

206

2,5-Dimethoxy-4-propylthio b-phenethylamine (2C-T-7) 2470

—

—

226

183

225

153

169

255

2C-T-7-AC

2410

—

—

238

255

181

297

153

183

2C-T-7-TFA

2170

—

—

225

351

181

153

238

183

M (OH-N-acetyl-)-AC and (OH-)-AC2

2590

—

—

296

236

101

355

356

283

M (desamino-OH-)-AC

2080

—

—

238

298

181

239

255

299

M (OH-)-TFA2

2105

—

—

337

463

350

181

2350

—

—

350

409

351

337

181

2,5-Dimethoxy-4-methylamfetamine (STP or DOM)

1612

1652

—

44

166

151

135

91

209

DOM-PFP

1730

—

—

165

192

135

119

91

355

DOM-AC

2020

—

—

44

192

86

165

166

251

DOM-AC2

2090

—

—

192

165

86

135

177

293

Art (formyl)

1565

—

—

56

190

165

135

221

91

M (O-desmethyl-)-PFP2

1780

—

—

324

297

190

119

325

487

M (OH-)-PFP2

1830

—

—

354

327

190

119

355

517

M (OH-)-AC2

2260

—

—

250

309

86

164

191

91

M (desamino-oxo-OH-)-PFP2

2045

—

—

353

326

516

233

206

396

M (desamino-oxo-OH-)-AC2

2560

—

—

164

249

206

233

308

91

1235

—

1429

72

91

73

44

42

56

M (OH-N-acetyl-)-TFA

Dimethylamfetamine (trimethylbenzeneethenanamine; metabolised to metamfetamine) Ephedrine (D or L)

1365

1410

—

58

77

105

146

131

—

Ephedrine-TFA2

1345

—

—

154

110

69

115

244

338

Ephedrine-PFP2

1370

—

—

204

119

95

160

294

338

Ephedrine-TMS2

1620

—

—

130

73

147

149

163

294

Ephedrine-AC2

1795

—

—

58

100

148

117

249

122

M (nor-)

1360

1356

—

77

79

91

107

118

132

M (nor-)-TFA2

1355

—

—

140

69

230

203

105

175

M (nor-)-PFP2

1380

—

—

190

119

105

117

280

253

M (nor-)-TMS2

1555

—

—

116

73

117

147

280

163

M (nor-)-AC2

1805

—

—

86

87

107

134

176

235

M (OH-)

1875

—

—

58

71

77

95

148

107

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:9

Specific applications

659

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

2145

—

—

58

100

205

247

123

Etafedrine

1519

1510

1737

86

58

87

42

56

Ethylamfetamine (etilamfetamine; also metabolised to amfetamine)

1230

—

—

72

91

117

148

162

—

Ethylamfetamine-AC

1675

—

—

72

114

91

205

148

119

Ethylamfetamine-HFB

1485

—

—

268

240

91

118

269

169

Ethylamfetamine-PFP

1450

—

—

218

190

118

91

119

117

Ethylamfetamine-TFA

1450

—

—

168

69

140

118

91

83

M (OH-methoxy-)

1640

—

—

72

94

137

122

77

209 263

M (OH-)-AC3

307 77

M (4OH)-AC2/(PHEA)-AC2

1995

—

—

72

114

134

176

107

M (OH-methoxy-)-AC

2000

—

—

72

164

114

137

251

—

M (OH-methoxy-)-AC2

2080

—

—

72

114

164

206

137

293

2200

—

—

72

114

150

234

192

321

2965

—

—

286

229

91

135

377

—

M (3-OH-methylpropylphenazone)

2410

—

—

231

246

232

215

77

M (3-OH-methylpropylphenazone)-AC

2240

—

—

245

232

273

288

190

274

Fencamfamin (also metabolized to amfetamine)

1675

1723

—

98

58

84

215

71

186

Fencamfamin-AC

2085

—

—

170

142

58

97

91

84

Fencamfamin-HFB

1795

—

—

170

142

67

117

129

280

Fencamfamin-TFA

1970

—

2180

142

170

91

180

115

242

Fencamfamin-TMS

1780

—

—

170

258

287

272

259

130

Fencamfamin-PFP

1755

—

—

170

142

91

230

105

292

M (di-OH-)-AC3 Famprofazone (also metabolised to metamfetamine)

154

M (desethyl-)-AC

2005

—

—

170

142

91

171

115

229

M (desethyl-OH-)-AC2

2305

—

—

142

168

228

91

119

287 148

Fenethylline (fenetylline; also metabolised to amfetamine) 2830

2900

—

250

70

207

91

119

Fenethylline-AC

3110

—

—

250

207

91

292

180

383

Fenethylline-HFB

2815

—

—

91

446

419

266

180

118

Fenethylline-PFP

2790

—

—

91

396

369

207

339

217

Fenethylline-TFA

2840

—

—

91

166

346

319

207

170

M (N-desalkyl)-AC

2480

—

—

206

180

193

265

122

86

M (etophylline)

2125

—

—

180

95

224

109

122

194

M (etophylline)-TMS

2160

—

—

180

73

281

296

252

123

M (etophylline)-AC

2200

—

—

87

266

206

180

122

223

Fenfluramine

1230

1252

—

72

44

159

73

58

42

Fenfluramine-TFA

1455

—

1621

168

140

159

169

186

308

Fenfluramine-PFP

1455

—

—

218

190

159

119

168

358

Fenfluramine-AC

1580

—

—

72

114

159

216

58

254

M (desethyl-, norfenfluramine)

1133

1157

—

44

42

159

43

45

184

M (desethyl-)-AC

1510

—

—

86

159

186

109

226

245

Fenproporex (metabolised to amfetamine)

1585

1648

—

97

56

91

68

132

173

Fenproporex-TFA

1705

—

—

193

118

140

91

56

152

Fenproporex-PFP

1685

—

—

243

118

190

56

91

202

Fenproporex-AC

1915

—

—

97

56

139

91

118

65

4-Hydroxyamfetamine (PHA)

1480

—

—

56

107

77

108

91

151

4-Hydroxyamfetamine-AC

1890

—

—

134

107

86

77

133

193

4-Hydroxyamfetamine-AC2

1900

—

—

134

86

176

107

77

133

M (methoxy-)

1465

—

—

138

137

122

123

94

181

M (methoxy-)-AC2

2065

—

—

164

86

206

137

165

265

M (1-OH-)/(4-OH-norephedrine)-AC3

2150

—

—

86

150

234

192

137

123 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:14

660

Gas Chromatography

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

Mebeverine (metabolised to PMEA)

3045

—

—

Meclofenoxate

1770

1804

2200

Mefenorex (also metabolised to Amfetamine)

1719

1602

Mefenorex-TFA

1715

Mefenorex-PFP Mefenorex-AC

Levamfetamine (see Amfetamine) 308

165

309

121

55

58

111

71

42

75

98 59

—

120

122

91

56

65

121

—

—

216

218

140

118

91

154

1710

—

—

266

190

91

118

268

119

1935

—

—

120

122

162

164

91

147

M (OH-dechloro-)-AC2

2060

—

—

144

186

101

91

84

118

M (OH-) isomer 1-AC2

2300

—

—

120

121

58

162

107

77

M (OH-) isomer 2-AC2

2230

—

—

120

121

58

162

107

77

M (OH-methoxy-)

2145

—

—

120

84

122

137

107

256

M (OH-methoxy-)-AC

2360

—

—

120

122

162

164

137

257

M (OH-methoxy-)-AC2

2410

—

—

120

164

162

206

137

341

M (dechloro-di-OH-)-AC3

2400

—

—

144

84

101

107

134

186

M (dechloro-tri-OH-)-AC4

2630

—

—

144

150

186

192

234

84

M (di-OH-)-AC3

2510

—

—

120

122

162

150

192

234

M (dechloro-di-OH-methoxy-)-AC3

2520

—

—

144

164

186

206

101

137

Mephentermine (metabolised to phentermine)

1240

1250

—

72

91

56

65

115

148

Mephentermine-TFA

1335

—

—

168

110

91

56

117

122

Mephentermine-AC

1501

—

—

72

114

91

132

148

117

Mescaline

1680

1737

—

182

167

211

151

148

—

Mescaline-TFA

1830

—

—

181

194

179

307

148

151

Mescaline-PFP

1835

—

—

181

194

357

179

119

151

Mescaline-TMS

1745

—

—

102

73

181

182

268

283

Mescaline-TMS2

1990

—

—

174

73

175

86

340

100

Mescaline-AC

2160

—

—

194

179

181

253

151

148

Art (formyl)

1700

—

—

181

223

182

148

167

77

4-Methoxyamfetamine (PMA)

1412

1410

—

122

121

77

78

91

107

PMA-AC

1720

—

—

44

148

121

86

77

207

4-Methoxyethylamfetamine (PMEA; metabolised to PMA 1660 and 4OH-ethylamfetamine)

1512

—

72

121

91

149

77

192

PMEA-TFA

1775

—

—

168

148

121

140

149

289

PMEA-PFP

1765

—

—

218

148

121

190

149

339

PMEA-TMS

2065

—

—

144

73

145

250

121

264

PMEA-AC

1855

—

—

72

148

141

121

77

235

Methoxyphenamine

1361

1416

—

58

91

59

56

42

121 134

Methylamfetamine (see Metamfetamine) Metamfetamine (also metabolised to amfetamine)

1175

1200

—

58

91

65

56

77

Metamfetamine-AC

1575

—

—

58

100

91

117

191

Metamfetamine-HFB

1460

—

—

254

210

118

91

169

Metamfetamine-PFP

1415

—

—

204

160

118

119

91

69

Metamfetamine-TFA

1300

—

1722

154

110

118

91

69

245

Metamfetamine-TMS

206

— 69

1325

—

—

130

73

59

91

131

M (4-OH-, pholedrine)

1885

—

—

58

77

107

135

150

—

M (4-OH-, pholedrine)-TFA2

1585

—

—

154

110

69

230

155

357

M (4-OH-, pholedrine)-PFP2

1605

—

—

204

160

119

280

154

253

M (4-OH-, pholedrine)-TMS2

1620

—

—

179

206

73

154

110

309

M (4-OH-, pholedrine)-AC2

1995

—

—

58

100

134

176

107

—

M (OH-methoxy-)

1810

—

—

58

137

94

122

65

195

M (OH-methoxy-)-AC2

2115

—

—

58

100

164

206

136

279

M (di-OH-)-AC3

2190

—

—

58

100

150

123

234

307

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:23

Specific applications

661

Table 40.8 continued Compound

System GA

Principal ions (m/z) GB

GC

4-Methoxy-metamfetamine (PMMA) (also metabolized to PMA, MDMA and MDA) 1820

—

—

58

100

148

121

77

221

M (O-desmethyl-, pholedrine)-AC2

1995

—

—

58

100

134

176

249

107

M (1-OH-pholedrine)-AC2

2095

—

—

58

100

164

206

137

279

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)

1965

—

—

114

72

160

174

144

274

Methyl-3,4-benzodioxazol butanamine (MBDB; also metabolised to BDB, see above)

1630

1690

—

72

57

135

77

178

207

PMMA-AC

MBDB-AC

1995

—

—

72

176

114

135

77

249

MBDB-PFP

1785

—

—

218

176

135

160

119

353

MBDB-TFA

1800

—

—

168

176

135

110

303

140

2170

—

—

72

114

178

220

137

293

M (desmethylenylmethyl-)-AC2

2295

—

—

72

114

164

248

123

321

Methyl-2,3-benzodioxazol butanamine (2,3-MBDB; metabolised to 2,3-BDB)

1610

1660

—

72

57

89

135

178

120

2,3-MBDB-PFP

1710

—

—

218

176

160

135

129

353

2,3-MBDB-TFA

1725

—

—

168

176

110

135

303

140

2,3-MBDB-AC

1965

—

—

72

114

176

135

249

77

2,3-MBDB-TMS

1730

—

—

144

73

135

145

250

264 161

M (desmethylenyl-)-AC3

2,3-Methylenedioxyamfetamine (2,3-MDA)

1470

—

—

44

77

51

135

179

2,3-MDA-AC

1770

—

—

162

77

135

105

86

51

2,3-MDA-HFB

1595

—

—

162

135

240

77

163

375

2,3-MDA-PFP

1545

—

—

162

135

119

190

77

325

2,3-MDA-TFA

1585

—

—

162

135

140

275

77

136

2,3-MDA-TMS

1655

—

—

116

73

77

135

236

251

1490

—

—

56

135

77

191

105

176

1480

1512

—

44

136

135

51

77

179

Art (formyl) 3,4-Methylenedioxyamfetamine (MDA; metabolised to amfetamine metabolites) MDA-AC

1860

—

—

44

162

135

77

86

221

MDA-HFB

1650

—

—

135

162

169

77

240

375

MDA-PFP

1605

—

—

135

162

119

190

136

325

MDA-TFA

1615

—

—

135

162

77

105

136

275

1520

1689

—

56

77

135

191

136

105

3,4-Methylenedioxyethylamfetamine (MDEA; metabolised 1560 to MDA and ethylamfetamine metabolites)

1630

—

72

77

135

105

163

207

MDEA-TFA

1770

—

—

168

162

140

135

125

303

MDEA-PFP

1755

—

—

218

190

162

135

119

353

MDEA-TMS

1825

—

—

144

73

135

264

100

77

MDEA-AC

1985

—

—

72

162

114

135

77

249

3,4-Methylenedioxymetamfetamine (MDMA; metabolised 1585 to MDA, and amfetamine metabolites)

1572

—

58

135

77

177

105

193

MDMA-HFB

1770

—

—

254

162

135

210

77

389

MDMA-PFP

1830

—

—

204

162

160

135

119

339

MDMA-TFA

1720

—

—

154

162

135

110

77

289

MDMA-TMS

1710

—

—

58

100

162

77

135

235

—

—

Art (formyl)

—

1735

—

—

—

—

Methylephedrine (metabolised to ephedrine)

1405

1451

—

72

77

105

115

161

—

Methylephedrine-AC

1495

—

—

72

77

91

105

117

162

Methylephedrine-TFA

1185

—

—

72

134

91

162

115

117

Art (formyl)

—

Methylephedrine-TMS

1485

—

—

72

149

163

236

117

251

2,3-Dimethylbenzodioxazolbutanamine (MMBDB; also metabolised to MBDB)

1660

1700

—

86

71

96

135

192

105

1890

—

—

86

87

123

180

222

264

M (desmethylenyl-methyl-)-AC

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:26

662

Gas Chromatography

Table 40.8 continued Compound

System GA

GB

GC

3,4-Methylenedioxy-5-methoxyamfetamine (MMDA)

1690

1743

—

44

166

165

77

65

MMDA-AC

2050

—

—

44

192

165

86

166

77

1685

—

—

56

165

221

120

77

166

4-Methylthioamfetamine (MTA)

1300

1610

—

44

138

137

122

91

78

MTA-TFA

1750

—

—

137

164

122

69

277

140

MTA-PFP

1760

—

—

137

164

122

190

327

91

MTA-TMS

1750

—

—

116

73

117

100

137

238

MTA-AC

Art (formyl)

Principal ions (m/z)

209

1760

—

—

164

86

137

122

117

265

Art (formyl)

1560

—

—

56

137

193

122

78

91

Methylphenidate

1725

1793

2200

84

56

91

115

77

Methylphenidate-AC

2085

—

—

84

126

112

56

275

—

Methylphenidate-TFA

1730

—

—

180

67

150

91

181

126

—

—

—

84

91

56

55

136

175

2328

—

—

—

—

M (ritalinic acid) Methylpiperidyl benzilate 4-Methoxy-a-pyrrolidinopropiophenone (MOPPP) M (desmethyl-)-ET

—

—

—

—

85

1705

—

—

98

77

92

135

233

107

1955

—

—

98

99

121

69

149

247

M (desmethyl-)-TMS

2005

—

—

98

73

56

135

193

276

M (desmethyl-3-OH-)-ET2

2165

—

—

98

99

56

137

165

193

M (desmethyl-3-methoxy-)-ET

2135

—

—

98

99

56

151

123

179

M (desamino-oxo-)

1440

—

—

135

77

92

107

136

178

M (desmethyl-desamino-oxo-)-ET

1530

—

—

149

121

93

65

150

192

M (desmethyl-3-methyoxy-desamino-oxo-)-ET

1680

—

—

179

151

123

108

73

222

M (oxo-)

2120

—

—

112

121

135

164

150

246

M (dihydro-)-TMS

1880

—

—

98

121

135

209

218

292

4-Methyl-a-pyrrolidinopropiophenone (MPPP) M (carboxy-)-ET

1725

—

—

98

56

65

91

119

216

2320

—

—

98

177

149

230

104

275

M (carboxy-)-TMS

2195

—

—

98

290

135

99

—

—

M (oxo-)

1920

—

—

112

69

119

84

113

231

M (OH-)-TMS

2095

—

—

98

290

135

90

99

M (dihydro-)-TMS

1730

—

—

98

73

163

193

276

202

M (desmethyl-3-methoxy-desamino-oxo)-ET

1680

—

—

179

151

123

108

73

222

105

202

—

1960

—

—

98

223

306

321

1595

—

—

98

56

77

69

M (oxo-)

1820

—

—

112

69

77

84

105

217

M (4-OH-)-ET

1955

—

—

98

99

121

69

149

247

M (desmethyl-3-methoxy-)-TMS a-Pyrrolidinopropiophenone (PPP) (also metabolized to cathinone and norephedrine)

M (4-OH-)-TMS

2005

—

—

98

73

56

135

193

276

M (dihydro-)-TMS

1665

—

—

98

73

105

56

188

262

1995

—

—

98

56

99

121

149

178

3,4-Methylenedioxy-a-pyrrolidinopropiophenone (MDPPP) M (desmethylene-)-ET2

2165

—

—

98

99

56

137

165

193

M (desmethylene-3-methyl-)-ET

2135

—

—

98

99

56

151

123

179

321

M (desmethylene-3-methyl-)-TMS

1960

—

—

98

223

306

M (desamino-oxo-)

1525

—

—

149

121

192

65

91

150

M (desmethylene-desamino-oxo-)-ET2

1720

—

—

193

165

137

109

194

136

M (desmethylene-3-methyl-desamino-oxo-)-ET

1680

—

—

179

151

123

108

73

222

M (desmethylene-3-methyl-oxo-)-ET

2290

—

—

112

179

151

208

123

290

M (oxo-)

2290

—

—

112

149

178

121

175

261

M (desmethylene-oxo-)-ET2

2325

—

—

112

151

69

193

222

305

1965

—

—

98

121

149

232

306

223

4 0 -Methyl-a-pyrrolidinohexanophenone (MPHP)

1965

—

—

140

141

91

119

84

202

a-Methyltryptamine (AMT)

1740

—

—

44

131

77

103

174

M (dihydro-)-TMS

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:30

Specific applications

663

Table 40.8 continued Compound

System

Principal ions (m/z)

GA

GB

GC

Phendimetrazine (metabolised to phenmetrazine)

1334

1504

1735

57

85

191

77

91

Phenmetrazine

1432

1483

—

71

56

177

77

91

105

Phenmetrazine-TFA

1530

—

1873

70

167

98

105

134

273

Phenmetrazine-TMS

1620

—

—

100

73

114

115

143

249

Phenmetrazine-AC

1810

—

—

71

113

86

85

176

219

105

M (OH-) isomer 1

1830

—

—

71

56

193

121

107

105

M (OH-) isomer 2

1865

—

—

71

56

193

106

163

121

M (OH-methoxy-)

1900

—

—

71

56

223

151

107

137

M (OH-) isomer 1-AC2

2150

—

—

71

70

113

85

234

277

M (OH-) isomer 2-AC2

2200

—

—

71

70

113

85

234

277

M (OH-methoxy)-AC2

2320

—

—

71

70

113

86

265

307

Phentermine

1155

1191

—

—

—

—

—

Phentermine-TFA

1100

—

1450

154

59

91

132

114

230

Phentermine-PFP

1305

—

—

204

91

132

164

129

280

Phentermine-TMS

1195

—

—

130

73

91

114

206

221

Phentermine-AC

1510

—

—

58

100

91

117

134

191

N-(1-Phenylcyclohexyl)-2-ethoxyethenamine (PCEEA)

1825

—

—

159

91

204

188

247

218

N-(1-Phenylcyclohexyl)-2-methoxyethenamine (PCMEA) 1770

—

—

91

190

159

283

188

218

—

—

N-(1-Phenylcyclohexyl)-3-ethoxypropanamine (PCEPA)

1915

—

—

218

91

117

261

232

174

N-(1-Phenylcyclohexyl)-propanamine (PCPR)

1630

—

—

174

91

58

217

159

188

Phenylephrine

1606

0000

—

—

—

—

—

—

—

Phenylephrine-TFA

1755

—

—

95

141

123

77

140

136 359

Phenylephrine-TFA2

1755

—

—

140

69

232

121

219

Phenylephrine-TMS3

2110

—

—

116

73

368

146

267

383

Phenylephrine-AC3

2110

—

—

86

87

115

129

165

220

Phenyl-1-ethylamine

—

1078

—

—

—

—

—

—

—

Phenyl-2-ethylamine

1111

1122

—

—

—

—

—

—

— 167

Phenylpropanolamine (see Norephedrine) Prenylamine (also metabolised to amphetamine)

2555

—

—

58

238

91

45

239

Prenylamine-AC

2925

—

—

58

91

100

280

238

164

Prolintane

1634

1660

1849

—

—

—

—

—

M (oxo-)

1895

—

—

140

98

91

86

188

231

M (OH-phenyl-)

2135

—

—

126

127

96

107

190

232

M (OH-phenyl-)-AC

2110

—

—

126

127

107

190

232

274

M (OH-methoxy-phenyl-)-AC

2115

—

—

126

127

137

55

262

304

M (oxo-OH-alkyl-)

2200

—

—

86

71

156

91

188

—

M (oxo-OH-alkyl-)-AC

2255

—

—

138

86

198

156

91

71

M (oxo-OH-methoxy-phenyl-)

2240

—

—

140

98

192

86

163

277

—

M (oxo-OH-methoxy-phenyl-)-AC

2360

—

—

140

192

98

77

234

319

M (oxo-di-OH-)-AC2

2485

—

—

198

156

128

162

279

107

M (oxo-di-OH-phenyl-)

2475

—

—

140

98

86

178

123

263

M (oxo-di-OH-phenyl)-AC2

2450

—

—

140

98

77

141

178

220

M (oxo-OH-phenyl)-AC

2275

—

—

140

98

86

162

204

289

M (oxo-di-OH-methoxy-)AC2

2560

—

—

198

192

156

234

128

377

M (di-OH-phenyl-)-AC2

2295

—

—

126

123

248

150

290

232

M (tri-OH-)-AC3

2630

—

—

198

156

128

178

151

123

Propylhexedrine

1175

1192

—

—

—

—

—

—

—

Propylhexedrine-TFA

1385

—

—

154

182

69

110

155

251

Propylhexedrine-PFP

1385

—

—

204

182

160

119

124

205

M (OH-)

1475

—

—

58

156

81

171

138

108

M (OH-)-AC2

1915

—

—

58

100

74

240

195

255 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:25:31

664

Gas Chromatography

Table 40.8 continued Compound

Propylhexedrine-AC

System

Principal ions (m/z)

GA

GB

GC

1570

—

—

100

1450

1453

—

—

58

182

140

101

114

—

—

—

—

Pseudoephedrine (see Ephedrine) Selegiline (also metabolised to L-metamfetamine and L-amfetamine)

—

M (OH-)

1580

—

—

96

56

97

107

76

171

M (OH-)-AC

1860

—

—

96

56

97

107

77

—

M (nor-)

1350

—

—

82

91

65

67

115

128

M (nor-)-AC

1735

—

—

82

124

91

65

118

214

M (nor-OH-)

1550

—

—

82

107

77

67

135

—

M (nor-OH-)-AC2

2030

—

—

44

182

167

151

107

225

1890

—

—

200

56

174

188

172

272

M (OH-)-AC2

2275

—

—

216

288

56

203

188

330

M (desethylene-)-AC2

1865

—

—

174

187

229

73

175

145

M (OH-desethylene-)-AC3

2275

—

—

190

203

245

287

191

232

M (trifluoroaniline)-AC

1400

—

—

161

203

184

142

114

111

M (OH-trifluoromethylaniline)-AC2

1840

—

—

157

177

219

117

129

261

1,(3-Trifluoromethylphenyl)piperazine (TFMPP) TFMPP-AC

3,4,5-Trimethoxyamfetamine (3,4,5-TMA) 3,4,5-TMA-AC Art (formyl) 2,3,5-Trimethoxyamfetamine (2,3,5-TMA)

1740 2020

—

—

44

208

193

86

181

267

1680

1745

—

56

181

237

148

77

222

2040

—

—

182

167

181

151

107

225

2,3,5-TMA-AC

2285

—

—

208

181

193

86

267

167

2,3,5-Trimethoxymethamfetamine (TMMA)-AC

2310

—

—

58

208

100

224

281

177

1-(1,3-Benzodioxol-5-yl)-butan-2-one

1525

—

—

135

57

77

192

136

105

1-(1,3-Benzodioxol-5-yl)-butan-1-ol

1560

—

—

151

93

65

123

194

Benzylmethylketone (BMK)

1110

1153

—

—

—

—

—

2,5-Dimethoxybenzaldehyde

1345

1381

—

166

63

95

120

151

123

2,5-Dimethoxyphenyl-2-nitroethene

1900

—

—

209

77

133

147

148

162

2,5-Dimethoxyphenylethylamine

1630

1689

—

152

44

137

121

162

181

2,5-Dimethoxytoluene

1020

—

—

137

152

77

109

65

Dimethylphenylethylamine

—

1954

—

—

—

—

—

Isosafrole

1215

—

—

162

104

103

131

3,4-Methylene dioxymethylbenzylamine

—

1423

—

—

—

—

—

—

—

Methylene dioxyphenylacetone

—

1530

—

—

—

—

—

—

—

Methylphenylethylamine acetate

—

1593

—

—

—

—

—

—

—

2-Methylhydroquinone

1210

—

—

124

123

67

95

77

107

Piperonal

1302

1373

—

149

150

63

121

91

—

Piperonylacetone

1315

1357

—

135

77

178

79

105

3,4,5-Trimethoxybenzaldehyde

1550

1630

—

196

181

125

118

93

95

3,4,5-Trimethoxyphenylacetonitrile

1610

—

—

192

207

164

78

149

124

Precursors and intermediates of synthesis of illicit amfetamines

System GC

DA Cowan, personal communication (2003). In this system, the drugs are chromatographed as tertiary bases or trifluoroacetyl derivatives. n n n n

Column: 3% OV-17 on 80–100 mesh Chromosorb W HP glass (2 m  3 mm i.d). Temperature programme: 170
C for 2 min to 270
C at 16
/min for 8 min. Carrier gas: N2, 30 mL/min. Reference compounds: n-Alkanes with an even number of carbon atoms.

n

—

—

— 77

77

94 — 65

136

Retention indices: The retention indices given for system GC are those of the tertiary bases or of trifluoroacetyl derivatives.

Analgesics (non-narcotic) and non-steroidal antiinflammatory drugs Analgesics (non-narcotic) and non-steroidal anti-inflammatory drugs (NSAIDs) are acidic and/or neutral drugs and, although water soluble, are readily extracted at pH 5 (sodium acetate or phosphate buffer) into polar solvents, such as ethyl acetate or diethyl ether. Recovery can be improved by ‘salting out’ using excess solid sodium chloride. Many are arylacetic (indometacin), arylpropionic (ibuprofen), salicylic

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:3

Specific applications (diflunisal) or fenamic (mefenamic acid) acid derivatives, and thus require the formation of suitable derivatives prior to GC (data for methyl derivatives are given in Table 40.9; the values given for system GD are retention times of methyl derivatives relative to n-C16H34). This is most pertinent at low concentrations or with packed columns, while at higher concentrations peaks may tail, and there is thus a tendency for retention time to increase with concentration. Capillary columns, especially those with higher phase ratios, give better peaks for underivatised phenols. The arylpropionic acid derivatives are chiral, with the nonsteroidal anti-inflammatory (NSAI) activity usually residing in the S-enantiomer, but these are usually marketed as racemates and undergo enantiomeric inversion in vivo. Separation of NSAI enantiomers has been reviewed (Davies 1997). Systems GA or GB, described above, may be used, or systems GD and GL. System GD

In this system, the substances are chromatographed as their methyl derivatives. n n n n

Column: 3% SE-30 on 80–100 mesh Chromosorb G (acid-washed and dimethyldichlorosilane-treated) glass (2 m  3 mm i.d). Temperature programme: 120
C for 2 min to 260
C at 10
/min for 5 min. Carrier gas: N2, 40 mL/min. Reference compound: Hexadecane (n-C16H34). Note: Free carboxylic acids and phenols will not generally give peaks, although large quantities may give tailing peaks.

n

665

Retention indices and relative retention times: The values given for system GD are retention times of methyl derivatives relative to nC16H34.

System GL

In this system (Maurer et al. 2001), the substances are chromatographed as their methyl derivatives after extractive methylation. Equal volumes of urine and phase-transfer reagent (0.02 mol/L tetrahexylammonium hydrogensulfate in 1 mol/L phosphate buffer, pH 12) are incubated with three volumes of 1 mol/L methyl iodide in toluene on a shaker at 50
C for 30 min. The organic phase is eluted on a diol solid-phase cartridge (conditioned with methanol, then toluene) using diethyl ether–ethyl acetate (95 : 5 v/v). After evaporation to dryness at 60
C, the residue is reconstituted in ethyl acetate for injection. n n n n n

Column: HP1 (methyl-PSX) fused-silica capillary (12 m  0.2 mm i.d. 0.33 mm). Injector: 280
C splitless mode. Temperature programme: 100
C for 2 min to 310
C at 30
/min for 8 min. Carrier gas: He, 1 mL/min. Retention indices are given in Table 40.9.

Anticholinergics The anticholinergics comprise a chemically diverse group of drugs, although most can be extracted successfully from biological specimens under mildly alkaline conditions. Some (atropine, hyoscine) are

Table 40.9 GC retention data and mass spectral data for analgesics (non-narcotic) and NSAIDs (ET, ethyl; Me, methyl) Compound

System GA

Principal ions (m/z) GB

GD

GL

Acemetacin (metabolised to indometacin)

Not eluted

—

—

—

Acemetacin-Me

3150

—

—

—

139

429

141

431

312

430

Acemetacin-ET

3220

—

—

—

139

443

141

312

445

442 131

Art-Me2

2390

Acetanilide (also metabolised to paracetamol) 1368 Alclofenac

—

—

—

—

174

233

291

175

159

1400

—

—

93

135

43

66

65

39

—

—

—

41

226

77

143

181

141

Aletamine

1293

—

—

—

70

120

43

91

39

103

Amidopyrine/aminophenazoine

1895

1992

—

—

56

231

97

111

112

42

M (nor-)

1980

—

—

—

—

—

—

—

—

—

M (bis-nor-)

1955

—

—

—

—

—

—

—

—

—

—

—

—

—

—

M (desamino OH-) Aspirin (metabolised to salicylic acid and salicylamide)

1855

—

—

—

—

1545

—

—

—

120

43

138

92

121

64

Aspirin-Me

1394

1430

—

—

135

194

179

136

91

76

Azapropazone (metabolised to paracetamol and aspirin)

2461

—

—

—

160

300

189

145

188

301

1804

1779

—

—

—

—

—

—

—

—

1840

—

—

—

121

163

151

109

43

122 65

Art Benorilate (metabolised to paracetamol and aspirin) Benoxaprofen

2550

Not eluted —

—

256

301

91

258

119

Benoxaprofen-Me

2485

—

1.98

—

256

315

91

119

258

65

Benzydamine

2380

—

—

—

85

58

86

91

84

70

Bufexamac

—

—

—

—

—

—

—

—

—

Celecoxib

—

—

—

—

381

300

382

301

281

140

Clonixin-Me

—

—

1.61

—

—

—

—

—

—

—

Dexketoprofen (see Ketoprofen)

—

—

—

—

—

—

—

—

—

—

Diclofenac

2271

2231

—

—

214

216

242

295

215

297

Diclofenac-Me

2195

—

1.42

2200

214

242

309

216

311

179

Diclofenac-Me2

2220

—

—

—

228

323

229

325

214

264

2322

2418

—

—

320

355

357

322

228

292

Art

—

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:7

666

Gas Chromatography

Table 40.9 continued Compound

System

Principal ions (m/z)

GA

GB

GD

GL

Art (-H2O)

2135

2231

—

—

214

277

242

279

179

Art (-H2O)-Me

2300

2436

—

—

228

230

200

263

291

164

M (OH-)-Me2

2460

—

—

2460

244

339

272

341

201

246

M

—

2592

—

—

214

216

242

277

179

294

M (OH-) isomer 1

—

2600

—

—

230

293

232

295

258

195

M (OH-) isomer 2

—

2941

—

—

230

293

232

271

158

310

Diflunisal

2095

—

—

—

232

250

175

204

176

233

Diflunisal-Me

2050

—

1.20

—

—

—

—

—

—

—

278

247

245

175

188

204

56

42

83

57

77

Diflunisal-Me2

1990

—

—

1990

Dipyrone

—

2069

—

—

M (bisdesalkyl-)

1955

—

—

—

M (desalkyl-)-AC

2395

Etenzamide (also metabolised to salicylamide) — M (desethyl-)-AC

—

—

—

—

—

216

51 —

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

—

120

92

105

148

150

121

—

228

258

287

198

57

229

1660

—

Etodolac

2333

—

—

Etodolac-Me

—

—

—

2225

228

272

301

198

229

115

Famprofazone (also metabolised to metamfetamine)

2965

—

—

—

286

229

91

81

287

77 229

2410

2850

—

—

244

273

44

302

214

Fenbufen

3078

—

—

—

181

152

153

254

182

151

Fenbufen-Me

2315

—

1.79

1975

181

152

153

182

268

237

2190

M (OH-)

—

—

—

197

256

154

128

152

198

Fenclofenac-Me

—

—

1.55 and — 1.26

—

—

—

—

—

—

Fenoprofen

2016

2040

—

—

197

241

198

77

242

104

Fenoprofen-Me

1906

—

1.31

1970

197

256

198

257

91

103

—

—

—

2130

286

227

287

123

91

152

Feprazone

2380

—

—

—

183

77

252

320

184

41

Feprazone-Me

—

—

1.81

Floctafenine

3132

—

—

—

—

—

—

—

—

M (acetic acid OH-)-Me2

M (OH)-Me2

—

—

Floctafenine-Me

2433

—

—

—

—

—

—

—

—

Flufenamic acid

1950

—

1.26

—

263

281

166

92

145

167

Flufenamic acid-Me

—

—

—

1875

263

295

235

166

264

92

—

—

—

2115

325

293

278

250

223

202 —

M (OH-)-Me2 Flunixin-Me

—

—

1.39

—

—

—

—

—

—

Flupirtene

2603

—

—

—

109

304

231

124

258

110

Flurbiprofen

1900

—

—

—

199

244

200

178

179

184

Flurbiprofen-Me

1885

—

1.3

1880

199

178

183

258

170

200

M (OH)-Me2

2180

—

—

2180

229

288

230

289

214

183

2310

—

—

—

318

259

319

260

215

303

2770

—

—

—

—

—

—

—

—

—

Ibuprofen

1615

1637

—

—

163

161

119

91

206

117

Ibuprofen-ME

1510

—

0.89

1505

161

177

220

119

91

117

—

2096

—

—

177

117

119

91

118

221

M (OH-methoxy-)-Me2 Glafenine-ME

M (2OH-) M (2OH-)-Me

1750

—

—

—

177

117

159

145

131

236

M (3OH-)-Me

1630

—

—

1680

119

118

91

59

178

162

1765

—

—

—

205

145

177

117

121

264

Indometacin

2550

—

—

—

139

141

357

111

359

140

Indometacin-Me

2770

—

1.55 and 2770 0.49

139

141

111

371

312

114

M (HOOC-)-Me2

—

—

—

139

141

140

401

111

262

Indoprofen-Me

2708

—

2.27 and — 2.07

236

295

237

296

218

206

Isopropylaminophenazone

2033

—

—

—

—

—

—

—

—

M (OH)-Me2

2880

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:12

Specific applications

667

Table 40.9 continued Compound

System

Principal ions (m/z)

GA

GB

GD

GL

—

—

—

2510

77

336

105

183

266

117

—

—

—

2690

77

107

366

367

213

296

Ketoprofen

2245

—

—

—

105

177

77

209

254

210

Ketoprofen-Me

2090

—

1.45

2090

209

105

77

268

191

210

—

—

—

2250

239

298

135

191

107

103

—

2686

—

—

105

210

77

44

132

254 328

Kebuzone-Me M (OH)-Me2

M (OH)-Me2 Ketorolac

—

—

—

2685

267

326

77

232

269

—

—

—

2875

356

297

262

358

299

247

Meclofenamic acid

2420

—

—

—

—

—

—

—

—

—

Meclofenamic acid-Me

—

—

1.62

2240

242

244

309

311

277

214

Mefenamic acid

2201

2370

—

—

223

241

208

222

194

180

Mefenamic acid-Me

2069

—

1.45

2110

223

255

208

180

194

222

—

—

—

2400

209

271

221

224

180

194

Methyl salicylate (metabolised to salicylic acid 1195 and salicylamide)

1228

—

1210

92

120

152

65

121

93

Methylsalicylate-Me

1200

—

—

—

135

133

77

92

166

104

Mofebutazone-Me2

—

—

—

1955

121

77

204

260

83

105

—

—

—

2075

77

276

121

71

128

220

Morazone (metabolised to phenazone and phenmetrazine)

3130

—

—

—

201

56

176

202

70

258

Nabumetone

—

2084

—

—

171

228

172

185

128

115

M (6-methoxy-2-naphthyl acetic acid, 6-MNA)

—

2322

—

—

171

215

128

172

44

102

M (O-desmethyl-)

Lonazolac-Me M (OH)-Me2

M (OH)-Me

M (OH)-Me2

—

2385

—

—

157

201

128

158

127

44

Naproxen

2045

2337

—

—

185

230

141

186

184

115

Naproxen-Me

1980

—

1.37 and 2120 1.18

185

244

170

141

115

186

M (ET)

1830

2115

—

—

185

258

186

170

153

141

M (O-desmethyl-)

—

2396

—

—

171

215

115

141

153

130

M (O-desmethyl-)-Me2

1980

—

—

2120

185

244

170

141

115

186

M (OH)-Me2 Nifenazone

1800

—

—

1800

274

215

259

171

184

275

3080

—

—

—

—

—

—

—

—

—

1955

—

—

—

—

—

—

—

—

—

Niflumic acid

2085

—

1.35

—

282

236

237

281

263

145

Niflumic acid-Me

1955

—

—

1960

236

295

296

263

237

145

—

—

—

2140

326

325

251

293

294

266

Not eluted

Not eluted —

—

199

324

93

77

65

55

Art (phenyldiazophenol)

2070

—

—

—

93

77

65

198

121

51

Art (phenyldiazophenol)-Me

2020

—

—

—

77

107

212

135

64

51

Oxyphenbutazone-Me2 (isomer 1)

2545

—

2.11

—

352

213

77

107

118

135

Oxyphenbutazone-Me2 (isomer 2)

2720

—

—

—

352

77

160

190

309

278

Paracetamol (acetaminophen)

1665

1722

—

—

109

51

43

80

108

81

1253

1280

—

—

109

52

53

80

81

108 122

M (desacyl-)

M (OH-) Me2 Oxyphenbutazone

Art (p-aminophenol) Paracetamol-Me Art (p-aminophenol)-Me2

1512

—

—

1630

108

123

165

80

95

1220

—

—

—

136

137

121

120

94

65

1730

—

—

108

109

179

137

43

81

Phenacetin (also metabolised to paracetamol) 1675 Phenacetin-Me M (hydroquinone)

—

—

—

—

122

193

151

56

123

94

1240

—

—

—

110

81

55

53

82

39

M (p-phenetidine)

1275

—

—

—

108

137

109

80

53

65

Phenazone/antipyrine

1835

1951

—

—

188

96

77

56

105

189

M (4-OH) Phenazopyridine (also metabolised to paracetamol) M (aniline)

1855

—

—

—

2245

2370

—

—

1158

—

—

—

85 — 93

56 — 66

84 — 65

204 — 92

77 — 46

120 — 41

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:13

668

Gas Chromatography

Table 40.9 continued Compound

Phenylbutazone (also metabolised to oxyphenbutazone) Art Phenylbutazone-Me

System

Principal ions (m/z)

GA

GB

GD

2367

2472

2.05 and — 1.81

GL —

2435

2550

—

—

183

2290

—

—

2290

183

—

—

—

—

—

77

184

324

325

119

77

322

266

118

323

266

184

—

—

—

—

—

2500

183

338

162

Phenyramidol

1960

—

—

—

—

—

—

Piroxicam

1413

—

—

—

173

117

145

78

104

94

Propyphenazone

1920

2030

—

—

215

230

56

77

216

96

M (OH-alkyl)-Me

77 —

M (nor-)

1772

—

—

—

174

216

77

173

129

145

M (OH-methyl-)

2410

—

—

—

231

246

232

77

215

154

M (OH-phenyl-)

2300

—

—

—

231

56

246

96

122

217

M (OH-propyl-)

2210

—

—

—

215

56

246

124

77

231

M (isopropanolyl-)

2020

—

—

—

231

246

213

56

232

61

M (isopropenyl-)

1970

—

—

—

136

228

95

77

108

106

M (nor-OH-)

1780

—

—

—

93

232

77

190

120

174 161

M (nor-OH-phenyl-)

2080

—

—

—

190

232

93

121

65

M (nor-di-OH-)

2090

—

—

—

248

109

136

206

121

232

M (nor)-Me

1735

—

—

—

215

77

230

51

200

185

M (OH-phenyl)-Me

2310

—

—

—

56

246

260

122

96

77

M (COOH)-Me

2160

—

—

—

215

274

56

77

105

165

M (nor-OH-phenyl isomer 1)-Me2

2030

—

—

—

245

230

260

215

77

92

M (nor-OH-phenyl isomer 2)-Me2

2060

—

—

—

245

230

260

77

92

215

M (nor-di-OH)-Me3

2240

—

—

—

275

290

260

252

236

276

—

3119

—

—

257

314

178

131

176

165

Rofecoxib Salicylamide

1414

1489

—

—

120

92

137

65

121

64

Salicylic acid

1307

1340

—

—

120

92

138

64

63

121

120

43

138

92

121

64

92

120

152

65

121

93

Salicylic acid-AC

1545

—

—

—

Salicylic acid-Me

1195

1228

—

1210

Salicylic acid-Me2

1200

—

—

—

135

133

77

92

166

104

—

—

—

1530

196

165

163

181

107

151 149

M (5-OH)-Me3 M (glycine conj)

1825

—

—

—

120

121

92

65

195

M (glycine conj)-Me

1810

—

—

—

121

120

209

119

65

92

M (glycine conj)-Me2

1845

—

—

—

135

90

77

105

223

121

Salsalate (metabolised to salicylic acid)

—

Not eluted —

—

121

120

92

65

138

258

Sulindac

2890

—

0.49

—

341

233

356

246

247

281

Sulindac-Me

3220

—

—

—

233

354

355

370

248

247

M (sulfide-)

2896

2959

—

—

328

233

234

313

159

247

M (sulfone)

—

3029

—

—

328

233

234

329

220

246

—

2715

—

—

58

143

142

115

156

295

Tenoxicam-Me2

2690

—

—

—

—

—

—

—

—

—

Tiaprofenic acid

1976

—

—

—

216

139

201

77

173

105

Tiaprofenic acid-Me2

2180

—

—

2175

229

288

230

77

105

201

Tolfenamic acid

—

—

—

—

—

—

—

—

—

—

Tolfenamic acid-Me

—

—

—

2255

208

243

275

180

89

Tolmetin

1890

—

—

—

212

213

122

198

44

91

Tolmetin-Me

2247

—

1.77 and 2235 1.36

212

271

256

119

270

91

M (COOH)

—

2615

—

—

212

91

256

119

44

65

Sumatriptan

245

—

—

—

2600

256

315

242

197

135

314

Zomepirac-Me

2343

—

—

—

246

305

248

304

139

111

M (-CO2)

2025

—

—

—

246

247

248

211

230

136

M (COOH)-Me2

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:16

Specific applications relatively unstable and produce several artefacts, either from hydrolysis during extraction or from thermal degradation in the GC. The quaternary ammonium compounds (e.g. emepromium bromide) are not amenable to gas chromatography. Systems GA or GB, previously described, may be used. Retention indices and relative retention times are given in Table 40.10.

Anticonvulsants and barbiturates Most barbiturates and anticonvulsants are acidic or mildly basic drugs and are extracted readily from aqueous medium into organic solvents. Although phenobarbital requires pH 7 or less for good recovery in liquid–liquid extractions, the other barbiturates and anticonvulsants (such as carbamazepine and phenytoin) may be extracted from aqueous

Table 40.10 GC retention data and mass spectral data for anticholinergics (AC, acetyl) System GA

Principal ions (m/z) GB

Adiphenine

2200

—

86

167

99

87

58

Atropine

2190

2293

124

82

94

83

42

96

Art (-CH2O)

1980

2051

124

259

140

94

221

178

Art (-H2O)

165

2085

2250

124

271

96

82

140

94

Benzatropine (benztropine)

2302

2423

83

140

82

124

96

42

Biperiden

2276

—

98

218

99

55

41

77

2645

—

98

218

114

327

284

85

Caramiphen

—

—

86

99

91

144

58

56

Chlorphenoxamine

2080

2190

58

59

179

42

178

72

2470

—

58

152

165

181

195

231

M (OH-)

M (OH-) M (nor-)

2094

2205

—

—

—

—

—

—

M (OH-methoxy-carbinol)-H2O

2220

—

260

262

210

245

227

181

Cyclopentolate

2022

2092

58

71

72

207

42

91

Cyclopentolate-H2O

2000

1551

58

71

91

115

129

273

Cycrimine

2114

—

98

41

42

55

99

77

Dicycloverine (dicyclomine)

2111

2175

86

71

99

58

55

56

Diethazine

2377

—

86

298

87

58

299

212

Eucatropine

2026

—

124

276

58

140

56

72

Homatropine

2072

2165

124

107

82

83

42

77

Hyoscine (scopolamine)

2300

2427

94

138

42

108

136

41

Art (H2O)

2230

2255

94

103

138

154

108

285

M (desacyl-)

1210

—

96

94

155

126

110

70

Metixine (methixine)

2480

2596

99

197

44

58

112

309

Orphenadrine

Hyoscyamine (see Atropine) 1935

2014

58

73

44

45

165

181

M (nor-, tofenacin)

1900

2007

180

179

86

255

165

240

M (methylbenzophenone)

1700

1827

195

196

77

105

119

165

M

1560

1630

167

182

107

108

165

119

1661 and 2250

—

105

129

112

77

42

313

111

96

167

112

165

71

84

204

205

85

42

—

—

Oxyphencyclimine Piperidolate

2318

—

Procyclidine

2156

2261

—

2487

M (OH-isomer 1)

—

—

—

55 —

M (OH-Isomer 2)

—

2517

—

—

—

—

—

—

M

—

2548

—

—

—

—

—

—

M

—

2603

—

—

—

—

—

—

M (oxo-) art-H 2O

2490

2669

200

115

86

98

198

283

Tigloidine

1687

—

124

82

83

94

55

42

Trihexyphenidyl (benzhexol)

2245

2354

98

105

55

99

77

218

2500

2618

Scopolamine (see Hyoscine)

M (OH-) Tropicamide Art (-CH2O)

—

669

—

—

—

—

—

2335

2442

92

91

65

103

93

163

2230

—

92

91

163

65

254

107

Art (-H2O)

2250

—

103

266

92

251

77

265

Tropicamide-AC

2410

—

92

104

266

65

163

326

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:26:53

670

Gas Chromatography

solutions at pH values as high as 11. There is little to be gained in terms of sensitivity by using NPD over FID, especially for barbiturates, but the former is more specific and excludes fatty acids, which can be problematic in decomposing samples. For GC, some investigators prefer to methylate, either during the extraction (iodomethane–tetramethylammonium hydroxide in dimethyl sulfoxide) (Liu et al. 1994), or by flash methylation in the injection port (trimethylphenylammonium hydroxide in ethyl acetate) (Brugmann 1981). This method is sometimes considered unreliable for quantitative analysis, and is arguably only necessary when using packed chromatography columns. Data for the methyl derivatives are therefore given where they are available. When using packed columns for GC, the medium- and higher-polarity phases, such as *-1310, *-2100, DEGS and waxes, are useful, although methylation allows the use of some more non-polar phases such as *-1 (Stern, Caron 1977). Systems GA and GB, previously described, may be used, or systems GE, GF and GAJ. System GE

System GF

(Flanagan, Berry 1977). n n n n

System GAJ

Data generated by the author. n n n n n n

Quantitative analysis of underivatised antiepileptic drugs (Supelco 1979). n n n n n

Column: 2% SP-2110 and 1% SP-2510-DA on 100–120 mesh Supelcoport glass (1 m  2 mm i.d.). Temperature programme: 120
C to 250
C at 16
/min. Carrier gas: N2, 50 mL/min. Reference compound: Phenytoin. Note: This system separates cholesterol from all drugs in the group. Retention indices and relative retention times: The values given for system GE are retention times relative to phenytoin.

Column: 3% Poly A103 on 80–100 mesh Chromosorb W HP glass (1 m  4 mm i.d.). Temperature: 200
C. Carrier gas: N2, 60 mL/min. Reference compounds: n-Alkanes with an even number of carbon atoms.

Column: DB1301 capillary (25 m  0.32 mm i.d., 0.25 mm). Temperature programme: 100
C to 235
C at 35
/min for 3.6 min to 290
C at 8
/min for 3.5 min. Carrier gas: He, 1.5 mL/min. Reference compound: Methylphenobarbital. Detection by FID and NPD split. Retention indices and relative retention times are given in Table 40.11. The values given for system GAJ are retention times relative to methylphenobarbital.

Antidepressants Antidepressants (tricyclics, selective serotonin reuptake inhibitors (SSRIs), monoamine oxidase inhibitors (MAOIs)) can be extracted readily under mildly basic conditions (pH 10) into many solvents, such as ethyl acetate, hexane, diethyl ether. Less polar solvents, such as hexane, limit the extraction of hydroxylated metabolites. An acidified (0.05 mol/L H2SO4) back extraction is a useful clean-up procedure

Table 40.11 GC retention data and mass spectral data for anticonvulsants and barbiturates (Me, methyl; ET, ethyl) Compound

Allobarbital

System

Principal ions (m/z)

GA

GB

GE

GF

GAJ

1600

1636

—

2340

—

41

167

124

80

53

68 236

Me2

1505

—

—

—

—

195

138

194

110

221

M

1785

—

—

—

—

—

—

—

—

—

1710

1742

—

2430

0.794

156

141

157

41

55

142 239

Amobarbital (amylobarbital)

—

Aobarbital-Me2

1593

—

—

—

—

184

169

170

185

226

M (3OH-)

1915

2015

—

—

1.138

156

157

141

227

214

195

M (3OH)-Me2

1750

—

—

—

—

137

184

169

185

255

270

M (COOH-)

1960

—

—

—

0.775

156

141

157

183

212

155

M (COOH)-Me3

1850

—

—

—

—

169

184

137

185

240

211

Aprobarbital

1618

—

—

—

—

167

41

124

168

97

39

M (OH-)

1815

—

—

—

—

183

154

184

122

165

226

Aprobarbital-Me2

1540

—

—

—

—

195

196

138

181

111

220

Barbital

1489

—

—

2230

0.612

156

141

55

155

98

82

Barbital-Me2

1420

—

—

—

—

184

169

126

112

183

83

Barbituric acid

0000

—

—

—

—

42

128

85

44

70

69

Barbituric acid-Me3

1645

—

—

—

—

170

55

82

98

113

155

Beclamide

1720

1778

—

—

—

91

106

197

162

107

148

1680

1539

—

—

—

55

161

106

116

79

77

1853

—

—

2765

1.000

207

41

39

124

91

165

Art Brallobarbital Brallobarbital-Me2 M (2OH-) M (desbromo-OH-) Butalbital Butalbital-Me2 M (OHButobarbital (butobarbitone)

1725

—

—

—

—

235

193

136

194

236

121

2040

—

—

—

—

223

165

180

136

152

122

167

124

141

98

181

224

41

167

168

124

97

141

1795

—

—

—

—

1665

1698

—

2395

0.778

1655

—

—

—

—

196

195

138

209

169

237

1940

2016

—

—

—

168

167

153

141

222

240

1660

—

—

2390

0.732

141

156

41

55

98

142

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:27:6

Specific applications

671

Table 40.11 continued Compound

Butobarbital-Me2 M (3 0 -OH-)

System

Principal ions (m/z)

GA

GB

GE

GF

1565

—

—

—

GAJ —

169

184

112

170

183

211

1920

—

—

—

1.053

156

141

157

199

181

213

M (3 0 -oxo-)

1880

—

—

—

—

156

141

198

157

199

211

Carbamazepine

2285

2435

0.83

—

1.716

193

192

236

191

194

165

—

—

1905

—

—

—

—

—

—

—

—

M (10,11-epoxide)

2220

—

—

—

1.188

180

179

178

152

44

181

M (iminostilbene)

1998

2064

—

—

0.732

193

192

194

165

179

191

Carbamazepine-Me

M (acridine)

1800

1880

—

—

0.591

179

178

151

152

180

177

M (formylacridine)

2025

2158

—

—

0.813

179

207

178

180

151

152

M (methylacridine)

—

2054

—

—

0.699

193

192

165

191

194

167

M

—

2316

—

—

—

180

209

181

210

152

190

M

—

2332

—

—

—

283

180

208

284

192

266

M

—

2387

—

—

—

209

180

208

210

152

167

M

—

2402

—

—

—

180

210

227

209

181

208

M (10,11-di-OH-)

—

2738

—

—

—

180

208

196

270

253

167

Cyclobarbital

1955

—

—

2825

1.142

207

141

81

79

67

80

Cyclobarbital-Me2

1845

—

—

—

—

235

169

236

178

121

264

M (oxo-)

2190

—

—

—

—

221

193

260

179

222

178

M (oxo)-Me2

2050

—

—

—

—

249

221

250

164

192

278

Cyclopentobarbital

1865

—

—

—

—

67

193

66

41

169

39

Cyclopentobarbital-Me2

1775

—

—

—

—

221

196

164

181

111

107

Dimethadione

1060

—

—

—

—

43

59

42

41

58

129

Enallylpropymal-Me2

1520

—

—

—

—

181

41

182

124

53

138

Clobazam (see Benzodiazepine section) Clonazepam (see Benzodiazepine section)

Ethosuximide

1205

1258

0.18

—

0.453

113

70

55

42

41

39

Ethosuximide-Me

1130

—

—

—

—

55

127

70

112

140

155

1370

1436

—

—

—

113

85

98

69

71

142 157

M (OH-ET-) M (3OH-)

1322

1395

—

—

—

71

86

129

139

142

M (oxo-)

1270

—

—

—

—

70

155

55

113

69

98

1800

1751

0.57

—

0.940

104

105

204

77

78

133

Ethotoin

1450

1475

—

—

—

104

103

91

77

121

134

Art (-C2H3NO2)

1890

1854

—

—

—

104

103

91

77

121

134

Art (-CH3NO2)

2210

2212

—

—

—

134

104

103

91

77

177

1750

1633

—

—

—

81

153

152

87

110

96

1560

—

—

—

—

81

67

167

166

124

110

Heptabarb

2055

2110

—

2940

1.282

221

43

78

93

80

141

Heptabarb-Me2

1915

—

—

—

—

249

169

250

133

183

192

2275

—

—

—

—

219

93

141

115

237

157

—

—

—

—

Felbamate(H2O)

Fosphenytoin (see Phenytoin) Gabapentin Art (-H2O) Art (-H2O)-Me

M (OH-) M (3 0 -oxo-)

2320

—

—

—

—

—

—

Hexethal

1850

—

—

—

—

156

141

55

41

157

98

Hexethal-Me2

1745

—

—

—

—

169

184

112

185

170

209

Hexobarbital

1855

—

—

2380

0.940

221

81

157

80

79

155

Hexobarbital-Me

1800

—

—

—

—

235

81

169

171

170

236

M (3 0 -oxo-)

2055

—

—

—

—

95

235

250

156

193

123

M (nor-)

1980

—

—

—

—

81

143

207

123

139

222

M (oxo)-ME

2020

—

—

—

—

249

264

95

221

207

170

M (3 0 -OH-)

1970

—

—

—

—

156

79

219

234

233

191 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:27:12

672

Gas Chromatography

Table 40.11 continued Compound

Ibomal (propallylonal; also metabolised to aprobarbital)

System

Principal ions (m/z)

GA

GB

GE

GF

GAJ

1880

—

—

—

—

167

209

43

124

39

41

1745

—

—

—

—

237

195

138

110

238

196

M (desbromo-OH-)

1770

—

—

—

—

169

141

142

98

184

226

M (desbromo-oxo)-Me2

1720

—

—

—

—

169

197

112

212

140

170

M (desbromo-OH)-Me2

1730

—

—

—

—

169

183

170

112

198

214

1700

—

—

—

—

167

41

168

124

97

141 223

Ibomal-Me2

Idobutal Isobutal-Me2

1610

—

—

—

—

195

196

138

181

169

Lamotrigine

2635

2562

—

—

1.941

185

187

255

257

123

124

Levetiracetam

—

1629

—

—

—

126

41

69

98

44

127

Metharbital (metabolised to barbital)

1470

—

—

—

—

155

170

112

169

55

82

Methohexital

1770

1827

—

—

0.798

41

81

53

221

79

39

Methohexital-Me

1735

1797

—

—

—

235

178

195

247

261

275

M (4 0 -OH-)

1880

—

—

—

—

219

124

181

245

261

278

1785

—

0.55

—

0.918

189

104

190

77

44

105

Methoin (mephenytoin) M (p-OH-)

2400

—

—

—

—

205

120

109

152

176

234

M (OH-methoxy-)

2380

—

—

—

—

235

150

135

137

247

264

M (nor-)

1950

—

—

—

—

104

175

77

132

163

204

1705

—

0.35

—

0.689

181

117

203

103

77

78

1750

—

—

—

0.779

118

117

103

77

189

155

Mesuximide (methsuximide) M (nor-) M (OH-)

2220

—

—

—

—

134

219

119

107

91

204

M (nor-OH-)

2300

—

—

—

—

134

205

119

133

103

165

Methylphenobarbital

1890

2222

—

—

1.000

218

117

118

146

103

77

Methylphenobarbital-Me

1855

—

—

—

—

232

118

117

146

175

260

M (OH-, MHD)

2370

—

—

—

—

134

233

234

262

162

133

M (OH-methoxy-)

2310

—

—

—

—

231

292

263

164

188

174

Nealbarbital

1720

—

—

2460

0.789

57

41

141

167

39

83

Nealbarbital-Me2

1620

—

—

—

—

169

195

209

112

138

250

Oxcarbazepine

—

2266

—

—

—

209

180

208

153

181

210

M (formylacridine)

2025

2158

—

—

—

179

207

178

180

151

152

M (methylacridine)

—

2054

—

—

—

193

192

165

191

194

167

M

—

2204

—

—

—

211

180

194

182

167

152

M

—

2296

—

—

—

193

192

180

165

191

237

M (10-OH-, MHD)

—

2580

—

—

—

193

180

194

210

254

167

M (carbamazepine)

2285

2435

—

—

1.716

193

192

236

191

194

165

Paramethadione

1115

—

0.06

—

—

43

129

57

56

41

72

Pentobarbital

1735

1776

—

2465

0.803

141

156

43

41

157

55

Pentobarbital-Me2

1630

—

—

—

—

184

169

112

225

185

126

M (3 -OH-)

1955

2039

—

—

—

156

141

157

197

195

227

M (3 0 -OH)-Me2

1820

—

—

—

—

169

184

185

223

225

241

Phenacemide

1473

—

—

—

—

91

92

118

44

43

135

Pheneturide

1465

—

—

—

—

91

146

44

119

206

41

Phenobarbital (phenobarbitone)

1953

2031

0.74

2960

1.150

204

117

146

161

77

103

0

Phenobarbital-Me2 M (4-OH-)

1855

—

—

—

—

232

118

117

146

175

260

2295

2378

—

—

—

219

248

148

220

176

204

2200

—

—

—

—

290

261

148

233

262

176

Phensuximide

1634

—

0.39

—

—

104

189

103

78

51

77

Phenylmethylbarbituric acid (heptobarbital)

1880

—

—

—

1.087

104

132

218

51

103

77

M (4-OH)-Me3

Phenylmethylbarbituric acid-Me2

1790

—

—

—

—

132

104

246

103

79

189

Phenytoin

2320

2435

1.00

—

1.773

180

104

223

77

209

252

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:27:15

Specific applications

673

Table 40.11 continued Compound

System

Principal ions (m/z)

GA

GB

GE

GF

GAJ

2795

2910

—

—

—

268

239

196

225

120

180

2245

—

—

—

—

180

266

237

209

189

165

M (p-OH)-Me2

2720

—

—

—

—

296

267

219

210

180

134

M (p-OH-methoxy-)

2770

—

—

—

—

298

269

226

196

211

254

M (OH-) Phenytoin-Me

2740

—

—

—

—

326

397

249

282

210

196

Primidone (also metabolised to phenobarbital)

2250

2384

0.89

—

1.674

146

190

117

118

161

189

Primidone-Me2

246

M (p-OH-methoxy)-Me2

—

2161

—

—

—

146

218

117

118

217

M (phenylethylmalondiamide)

1884

1996

—

—

1.074

148

163

103

120

91

117

M (diamide)

1935

—

—

—

—

163

148

103

118

120

133

M (AC) Secbutabarbital

2115

2189

—

—

—

146

232

117

118

189

218

1655

—

—

—

—

141

156

41

57

39

98

1565

—

—

—

—

—

—

—

—

—

—

1926

—

—

—

—

—

—

—

—

—

—

Secobarbital (quinalbarbitone)

1786

1827

—

—

0.865

167

168

41

43

97

Secobarbital-Me2

1690

—

—

—

—

196

195

138

181

224

237

M (3 0 -OH-)

1865

2029

—

—

1.206

168

167

169

153

209

195

M (3 0 -keto)

—

—

—

—

—

43

168

69

85

167

124

M (desallyl-)

1665

—

—

—

—

129

128

85

86

154

169

M (2,3 0 -diOH-)

—

—

—

—

—

171

143

128

159

198

241

—

—

—

—

—

28

99

56

27

26

55

Secbutabarbital-Me2 M (2 0 -OH-)

Succinimide

124

Sulthiame-Me

2880

—

—

—

—

304

274

226

198

210

211

Sulthiame-Me2

2815

—

—

—

—

318

274

226

210

211

104 168

Sultiame

3000

—

—

—

—

290

184

185

104

77

Talbutal

1703

—

—

—

—

167

168

41

97

124

39

Talbutal-Me2

1600

—

—

—

—

195

196

138

181

111

211

Thialbarbital

2116

—

—

—

—

81

223

79

80

157

185

Thiamylal

1899

—

—

—

—

43

41

184

168

167

97

Thiopental (thiopentone; also metabolised to pentobarbital)

1857

1923

—

2600

0.948

172

157

173

43

41

55

Thiopental-Me2 M (OH-)

1825

—

—

—

—

200

185

201

127

157

167

—

2134

—

—

—

172

173

157

97

258

229

156

157

113

111

358

96

—

2253

—

—

—

324

43

80

110

189

206

Taigabine-Me Topiramate Art (fructopyranose) Troxidone (trimethadione; metabolised to dimethadione) Valproate (valproic acid) M

—

1621

—

—

—

43

245

69

59

127

85

1090

—

0.04

—

—

43

58

143

42

41

128

1064

1098

0.09

—

0.350

73

102

41

57

43

55

—

1195

—

—

—

100

55

41

69

127

113 113

M

1200

—

—

—

—

100

55

41

69

127

M

—

1267

—

—

—

72

101

114

100

55

44

M

—

1312

—

—

—

100

55

41

113

99

69

Vigabatrin

—

Not eluted—

—

Not eluted 56

84

111

69

82

54

Vinbarbital

1753

—

2495

—

41

141

69

152

135

—

195

1670

—

—

—

—

223

224

166

169

138

135

2070

—

—

—

—

167

169

85

211

193

155

Vinylbital

1729

—

—

—

0.798

154

83

71

55

155

67

Vinylbital-Me2

1655

—

—

—

—

182

181

183

97

125

154

83

Vinabarbital-Me2 M (OH-)

M (3 0 -OH-)

1995

—

—

—

—

154

155

M (desvinyl-)

1665

—

—

—

—

—

—

—

2042

—

—

—

132

Zonisamide

77

112

139

195

—

—

—

—

133

104

51

64

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:27:37

674

Gas Chromatography

where sensitivity is important. Chromatography of primary and secondary amines is poor on packed columns, but is adequate on wellmaintained capillary columns, particularly those of low–medium polarity such as PSX-5 (see Table 40.3). Some authors prefer to chromatograph the secondary amines and hydroxylated metabolites as acetylated derivatives, prepared by heating the dried residue with acetic anhydride and pyridine (3 : 2, v/v) (Maurer, Bickeboeller-Friedrich 2000). Others employ an enzymatic hydrolysis procedure to improve recovery of both parent drug and metabolites, although the additional sensitivity gained is often negated by the increased analytical time in the emergency setting. Acid hydrolysis is quicker, but some relevant compounds are destroyed under these conditions. System GA, GF or GB, described above, may be used, or system GM. System GM

System GM (Dawling et al. 1990) is ideal for plasma samples, since the isothermal conditions allow high throughput and the limited resolution of hydroxylated metabolites is not important as these do not constitute a significant fraction of the extract from plasma. Conditions are given for both packed and capillary column systems.

n n n n n n n n n

Column: 3% SP2250 on Supelcoport 80–100 mesh glass (2.1 m  2 mm i.d.). Temperature: 265
C. Carrier gas: He, 25 mL/min. Column: HP-50 þ fused silica capillary (25 m  0.53 mm i.d., 1 mm). Temperature: 250
C. Carrier gas: He, 7 mL/min. Reference compound: Iprindole. Quantification: NPD. Retention indices, relative retention to iprindole, are given in Table 40.12. Only the metabolites known to occur in urine and/ or plasma specimens are included in this list; the list in De Zeeuw (2002) is more extensive.

Antihistamines Antihistamines are a diverse group of drugs that includes the ethanolamines (diphenhydramine), ethylenediamines (pyrilamine), alkylamines (hydroxyzine), phenothiazines (promethazine) and piperidines (chlorphenamine). Many share common metabolites with other members of

Table 40.12 GC retention data and mass spectral data for antidepressants (AC, acetyl) Compound

System

Principal ions (m/z)

GA

GB

GF

GM

Amitriptyline (also metabolised to nortriptyline)

2194

2284

2510

0.723

M (cis-10-OH-)

2348

2454

—

1.149

M (cis-10-OH-N-oxide)

—

2215

—

—

M (trans-10-OH-)

2348

2466

—

1.168

M (trans-10-OH-N-oxide)

—

2239

—

—

M (cyclobenzaprine)

2235

2330

—

0.850

58

59

202

42

203

214

58

202

215

178

189

165

215

229

230

207

248

178

58

202

215

178

189

165

215

229

230

207

248

178

58

215

202

189

176

163

Amitriptyline N-oxide

1975

2051

—

—

232

217

215

202

117

189

Amoxapine

2638

2746

—

2.831

245

257

247

193

56

246

M (7-OH-)

2951

3525

—

—

261

209

273

263

244

329

M (8-OH-)

2959

3546

—

—

261

209

273

263

244

329

Atomoxetine Bupropion M

—

1645

—

—

44

100

111

139

224

57

—

1746

—

—

44

100

77

57

208

113

M

—

1764

—

—

44

100

77

57

208

113

M (OH-)

—

1898

—

—

44

100

116

139

224

110

M

—

1916

—

—

44

100

224

157

57

M

—

2107

—

—

44

100

57

84

260

2181

2288

2465

0.683

58

293

—

2330

—

0.761

Butriptyline M (nor-)

—

—

45 —

59 —

65 —

193

100

—

—

Citalopram

2525

2499

—

1.121

58

238

208

42

324

190

M (nor-)

2500

2526

—

1.232

44

238

208

138

310

190

M

—

2846

—

—

238

81

136

192

265

221

M

—

2987

—

—

238

207

163

254

265

282

Clomipramine

2415

2511

2795

1.172

58

85

269

268

270

271

M (nor-)

2432

2540

—

1.374

268

269

229

227

242

300

M (N-oxide)

2146

2246

—

—

228

193

192

269

230

165

M (ring)

2230

2335

—

—

229

194

193

214

228

231

M (8-OH-)

2727

2843

—

—

58

285

243

209

284

330

M (8-OH-nor-)

2762

2880

—

—

M (2OH-)

2569

2735

—

—

M (10OH-)

2574

2698

—

—

44 — 58

245

243

284

258

316

—

—

—

—

—

268

329

313

85

86

Clorgiline

1883

—

—

—

—

—

—

Desipramine

2235

2338

—

0.896

235

195

208

— 44

—

—

234

193

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:27:52

Specific applications

675

Table 40.12 continued Compound

System

Principal ions (m/z)

GA

GB

GF

M (2-OH-)

2553

2669

—

GM —

44

211

224

250

180

282

M (10-OH-)

—

2521

—

—

44

180

194

206

251

282

M (ring)

1930

2014

—

—

195

194

180

167

97

89

M (ring-OH-)

2240

2335

—

—

211

210

196

180

167

212

M (ring di-OH-)

2600

—

—

—

227

226

157

196

228

183

M (di-OH-)

—

2995

—

—

44

266

240

227

298

225 222

M (AC-)

2670

2811

—

—

208

114

308

193

194

M (OH-methoxy-)

—

2749

—

—

227

241

254

280

312

44

M (ring OH-methoxy-)

2390

—

—

—

241

240

226

180

210

198

Dibenzepin

2450

2566

2885

1.735

M (nor-)

2449

—

—

—

58 —

324

209

—

—

71 —

225 —

72 —

M (di-nor-)

2406

—

—

—

235

234

207

206

179

192

M (ter-nor-)

2680

—

—

—

235

234

206

207

179

192

M (N5-desmethyl-)

2455

—

—

—

Dosulepin (dothiepin)

2380

2486

2770

1.259

58 —

210

211

167

195

223

—

—

—

—

—

M (nor-)

2421

2507

—

1.450

204

281

221

263

238

165

M (N-oxide)

2100

—

—

—

217

235

250

202

221

240

M (OH-N-oxide)

2130

—

—

—

266

165

251

233

237

215

M (sulfoxide)

2392

2533

—

—

—

—

—

—

—

—

—

—

—

—

—

—

220

219

191

189

M (norsulfoxide)

2421

2839

—

—

Doxepin cis-isomer

2220

2301

2570(a)

0.788

Doxepin trans-isomer

58

59

2220

2321

—

0.823

58

220

219

59

191

189

M (cis-N-oxide)

1970

2077

—

—

234

219

165

178

202

189

M (trans-N-oxide)

—

2081

—

—

234

219

165

178

202

189

M (cis-nor-)

2245

2333

—

0.830

44

165

178

189

202

219

M (trans-nor-)

2245

2339

—

0.933

44

165

178

189

202

219

M (cis-OH-)

2535

2528

—

—

58

165

295

152

178

220

M (trans-OH-)

2560

2544

—

—

58

165

295

152

178

220

M (cis-nor-OH-)

2540

2644

—

—

44

220

238

165

152

281

M (trans-nor-OH-)

—

2671

—

—

44

220

238

165

152

281

—

2750

—

—

44

297

265

240

181

115

Duloxetine Escitalopram (see Citalopram) Fluoxetine

1859

1903

—

0.304

44

309

183

104

251

91

M (nor-)

1851

1888

—

0.284

104

134

103

77

162

191 115

M (AC-)

2250

2319

—

—

44

86

190

117

104

M (nor-AC-)

2190

2278

—

—

117

176

72

104

115

91

1885

1911

—

0.295

187

71

45

276

172

145

Fluvoxamine Art (ketone)

1525

—

—

—

173

228

145

159

188

241

Art

1560

1602

—

—

187

172

200

228

244

259

M (AC-)

2240

2284

—

—

86

102

187

258

341

360

Art

1895

1921

—

—

258

71

226

242

311

329

M

—

2200

—

—

71

145

172

198

226

258

M

—

1791

—

—

241

172

212

145

144

198

M

-

Imipramine (also metabolised to desipramine) 2230

1687

—

—

86

257

198

145

281

341

2314

2540

0.784

58

235

85

234

236

195 224

M (2-OH-)

2565

2636

—

—

58

250

251

211

296

M (10-OH-)

—

2494

—

—

58

193

180

232

251

296

M (di-OH-)

—

2962

—

—

58

266

267

227

312

252

—

2715

—

—

58

280

241

326

254

266

Iprindole

2335

2437

—

1.000

58

170

284

213

145

212

Iproniazid

1593

1609

—

—

123

58

106

79

43

78

M (OH-methoxy-)

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:28:1

676

Gas Chromatography

Table 40.12 continued Compound

System

Principal ions (m/z)

GA

GB

GF

GM

Isocarboxazid

1949

0000

—

—

Lofepramine (metabolised to desipramine)

Not elutedNot eluted—

Maprotiline

2390

2440

—

1.086

44

70

59

277

71

191

M (nor-)

2293

2404

—

1.107

56

202

203

178

263

189

M (desamino-di-OH-)

2570

2620

—

—

252

207

280

253

219

195

44

70

203

189

187

293

—

—

—

—

M (OH-)

—

—

—

—

—

Not eluted —

—

—

—

—

2622 1240

—

Mianserin

2210

2302

2595

0.879

193

264

43

72

71

M (nor-)

2235

2348

—

1.105

193

208

250

165

178

220

M (8-OH-)

2495

2628

—

—

280

209

72

236

265

180

M (OH-methoxy-) M (CM30488) Mirtazapine M (nor-)

—

—

—

—

Mebanazine

Minaprine

—

—

220

2530

—

—

—

310

239

266

295

224

72

2855

3023

—

—

100

113

186

56

198

77

3040

3222

—

—

—

—

—

—

—

—

2250

2361

—

—

195

194

208

180

167

265

—

2414

—

—

195

194

209

180

167

251 208

M (oxo-)

—

2665

—

—

195

250

279

180

194

Moclobemide

—

2333

—

0.967

100

56

113

139

111

—

2578

—

2.191

—

—

—

—

—

— 317

M Nefazodone

42

4510

Not eluted—

Not eluted 303

274

260

304

454

M (m-chlorophenylpiperazine, mCPP)

—

1806

—

—

154

196

138

111

156

75

M (mCPP)-AC

2265

—

—

—

166

238

138

154

168

195

M (N-desalkyl-OH-) isomer 2-AC2

2525

—

—

—

182

254

169

184

296

211

M (desamino-OH-)-

2340

120

198

291

127

171

140

M (desamino-OH-)-AC

2500

—

120

240

91

333

77

126 238

—

—

2650

—

—

—

120

298

391

91

101

Nialamide

1500

—

—

—

91

177

44

106

45

78

Nomifensine

2130

2239

2670

0.850

194

195

238

193

72

178

M (OH-ethyl-desamino-OH)-AC2

M (4-OH-)

2450

—

—

—

86

210

211

194

254

228

M (OH,MeO-) isomer 1

2505

—

—

—

284

86

241

210

209

224

M (OH,MeO-) isomer 2

2590

—

—

—

284

86

241

210

209

224 215

2215

2304

—

0.816

44

202

45

220

218

M (cis-10-OH-)

2375

2480

—

1.261

44

218

203

202

178

165

M (trans-10-OH-)

2375

2494

—

1.323

44

218

203

202

178

165

Nortriptyline

M (norcyclobenzaprine)

—

2343

—

0.880

44

215

218

202

189

163

M (AC-)

2660

2774

—

—

44

232

202

217

86

203 178

—

2949

—

—

44

230

215

202

86

2270

—

—

—

58

71

208

72

59

42

M (dibenzocycloheptanone)

1850

—

—

—

208

180

179

178

165

152

M (OH-dibenzocycloheptanone)-H 2O

2200

—

—

—

178

206

176

152

76

89

M (norcyclobenzaprine-AC) Noxiptyline

Opipramol

3050

3219

—

—

363

206

143

M (ring)

1985

—

—

—

—

—

—

42 —

70

193

—

— 109

—

2691

—

2.047

44

329

192

70

138

M (desmethylenyl-3-methyl-)

—

2734

—

—

44

192

140

331

177

70

M

—

2687

—

—

105

210

77

254

132

44

Paroxetine

Phenelzine

1335

1278

—

—

31

45

46

29

59

74

Protriptyline

2253

2329

2590

0.878

70

44

191

192

188

59

M (nor-)

—

2343

—

—

M (10-OH-)

—

2406

—

—

2472

—

—

—

2481

—

1.166

M (10,11-di-OH-) Sertraline

— 70

— 44

—

—

—

—

207

178

279

249

44

70

179

178

207

280

274

276

159

262

239

304

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:28:4

Specific applications

677

Table 40.12 continued Compound

System

Principal ions (m/z)

GA

GB

GF

M (nor-)

—

2468

—

GM 1.218

119

145

274

246

130

290

M (ketone)

—

2496

—

—

227

290

292

199

163

248

M

—

2279

—

—

274

276

128

202

239

259

M

—

2333

—

—

202

272

274

200

236

100

M

—

2619

—

—

131

290

292

189

220

254

M

—

2786

—

—

287

289

217

251

189

108

250

252

305

307

263

214

44

59

165

166

181

179

—

2802

—

—

Tofenacin

1920

2013

—

0.420

Tranylcypromine

1220

1252

1455

—

133

132

56

115

30

117

Trazodone

3330

3564

—

Not eluted 205

70

231

78

135

166

M (mCPP)

—

1806

—

—

154

196

138

111

156

75

M (mCPP)-AC

2265

—

—

—

166

238

138

154

168

195

M (OH-AC-)

3380

3640

—

—

205

336

414

429

231

176

M (desalkyl AC-)

2265

2261

—

—

166

238

56

195

140

153

M (N-desalkyl-OH-) isomer 2-AC2

2525

—

—

—

182

254

169

184

296

211

2215

2302

2505

0.734

58

249

208

99

193

234

M

Trimipramine M (nor-)

—

2335

—

0.858

208

193

44

249

234

280

M (OH-)

2575

2631

—

—

58

265

224

250

209

310

M (nor-OH-)

—

2662

—

—

224

44

209

265

250

296

M (OH-methoxy-)

2590

2715

—

—

58

295

280

254

340

241

M (nor ring) Venlafaxine

1930

2107

—

—

194

249

208

193

167

179

—

2163

—

0.544

58

134

179

119

91

277

M (N-desmethyl-)

—

2196

—

0.570

44

202

134

121

91

263

M (O-desmethyl-)

—

2230

—

0.625

58

120

165

107

91

263

M (N,O-didesmethyl-)

—

2264

—

0.687

44

188

120

107

145

249

M (nor-OH-)

—

2450

—

—

44

134

218

200

121

185

M (O-desmethyl-OH-) isomer 1

—

2373

—

—

58

134

91

179

121

77

M (O-desmethyl-OH-) isomer 2

—

2408

—

—

58

134

91

179

121

77

1855

1923

—

—

56

100

138

110

57

237

2325

—

—

—

56

100

110

138

265

128

Viloxazine M (di-oxo-) Zimeldine

2270

—

—

0.820

58

70

318

316

317

193

M (nor-)

2223

—

—

0.941

302

304

224

193

260

272

(a)

Racemate.

their class, which may compromise the identification of the parent drug ingested. Some authors advocate the preparation of acetylated derivatives, particularly to analyse the hydroxylated metabolites, but to identify them in biological fluids this is an unnecessary additional step. Systems GA, GB, GC or GF, described above, may be used. The retention indices and principal ions are given in Table 40.13. Benzodiazepines The analysis of benzodiazepines in biological specimens is hampered by their high potency and resultant low plasma concentrations, and by their inter-connected metabolic pathways. Several benzodiazepines appear in urine almost exclusively as glucuronide-conjugated metabolites, and these can be hydrolysed with glucuronidase (1000 U glucurase/mL of urine at 60
C for 1–2 h), although some can degrade with prolonged heating. Extraction can be performed at any pH between 3 and 12, but basic extracts (pH 9–11) give cleaner chromatograms. The extraction solvent should be moderately polar (ethyl acetate is appropriate), and TMS derivatives form easily in 20–30 min at 60
C using 50% bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) in acetonitrile. These derivatives markedly improve peak shape

and sensitivity. All compounds except 7-aminonitrazepam show electron-capture responses with high sensitivity. However, quantitation by ECD is problematic as it has a narrow linear range, and a multiple point calibration is essential. Alternatively, for most compounds a nitrogen detector (NPD) gives adequate sensitivity with a much improved linear range, although it is not advisable to make TMS derivatives if using this detector. MS detection is required to confirm the identity. System GA or GB, described above, may be used or system GG. The retention indices and principal ions are given in Table 40.14. System GG

M M€ oller, personal communication n n n

Column: 2.5% OV-17 on 80–100 mesh Chromosorb G, treatment and dimensions as for system GA. Column temperature, carrier gas, reference compounds: As for system GA. Retention indices: The retention indices of benzodiazepines have been shown to be dependent on column temperature (Schuetz, Westenberger 1978, 1979). The values given by these authors are about 50 RI units above those generally quoted. The values given below should therefore be checked before use by chromatographing a few sample compounds.

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:28:29

678

Gas Chromatography

Table 40.13 GC retention data and mass spectral data for antihistamines (BP, benzophenone; CBPH, chlorobenzophenone) Compound

Alimemazine (trimeprazine) M (OH-)

System

Principal ions (m/z)

GA

GB

GC

GF

2315

2402

2646

2715

58

198

298

100

180

84

2650

—

—

—

58

314

100

255

196

281

M (OH-)-AC

2600

—

—

—

58

100

356

269

214

196

M (bis-nor-)-AC

2765

—

—

—

212

312

114

198

199

180 252

M (nor-)

2335

2432

—

—

199

284

212

198

180

M (nor-OH-)-AC 2

2930

—

—

—

128

384

228

214

270

196

M (phenothiazine)

2120

2130

—

—

199

167

198

166

154

139

M (sulfoxide)

2665

2805

—

—

M (norsulfoxide)

—

2829

—

—

M (nor-OH-) Antazoline Astemizole M (N-desalkyl)

58 —

212

199

180

298

299

—

—

—

—

—

—

2845

—

—

—

—

—

—

—

—

2318

2421

2749

—

—

—

—

—

—

—

3900

Not eluted

—

—

96

337

109

338

294

458

2470

—

—

—

109

241

132

83

242

111

2377

2499

—

—

246

280

232

247

291

217

M (OH-alkyl-)-H 2O

2410

—

—

—

244

255

288

230

216

229

M (nor-)-AC

2710

—

—

—

128

326

212

198

180

86 182

Azatadine

2250

—

—

—

97

91

96

70

77

M (OH-)

2580

—

—

—

97

91

296

198

98

96

M (OH-)-AC

2620

97

70

98

96

338

240 183

Bamipine

M (nor-)-AC

2675

91

182

77

217

308

M (nor-OH-)-AC 2

3020

91

366

240

56

199

275

Benzophenone (BPH)

1610

105

77

51

182

106

183

1673

—

—

M (OH-BPH) isomer 1

2065

—

—

—

198

121

77

199

192

151

M (OH-BPH) isomer 2

2080

—

—

—

121

198

77

105

199

122

M (OH-methoxy-BPH) isomer 1

2050

—

—

—

105

151

198

228

77

121

M (OH-methoxy-BPH) isomer 2

2070

—

—

—

105

151

198

228

77

121

M (carbinol)

1670

1722

—

—

79

219

77

218

78

108

Bromodiphenhydramine (Bromazine)

2155

2231

—

2480

58

73

45

165

59

166

Brompheniramine

2092

2184

2457

2470

247

249

58

72

167

168

M (nor-)

—

2219

—

—

247

249

167

44

246

168

M (bis-nor-)

—

2203

—

—

247

249

167

106

260

180

3360

3461

—

—

231

147

285

201

132

165

2520

2355

—

—

85

165

201

241

230

286

Buclizine (also metabolised to chlorobenzophenone) M (desalkyl-) (norchlorcyclizine) Carbinoxamine M (chlorobenzoylpyridine) M (nor-) Chlorcyclizine (also metabolised to chlorobenzophenone) M (nor-) Chlorobenzophenone (CBPH) M (4OH-CBPH)

2080

2147

2430

—

58

71

54

167

72

202

1645

—

—

—

139

189

111

216

217

191

203

167

202

220

205

204

99

56

72

165

300

228

2150

—

—

—

2220

2316

—

2560

2520

2355

—

—

85

165

201

241

230

286

1850

—

—

—

105

139

77

216

218

141

2300

—

—

—

121

232

111

139

234

197

M (4-chloromethylbiphenyl)

—

1688

—

—

167

165

202

152

82

166

M (4-chlorobiphenylmethanone)

—

1862

—

—

105

139

216

111

141

181

M (carbinol)

1750

—

—

—

105

77

139

218

165

111

Chloropyriline

2133

—

—

—

58

131

72

71

79

42 204

Chlorphenamine (see Chlorpheniramine) Chlorpheniramine

1996

2079

2586

2355

203

58

44

205

54

M (nor-)

2014

2115

—

—

203

167

44

205

202

204

M (bis-nor-)

—

2065

—

—

203

167

205

202

204

216

M (nor-AC-)

2530

2563

—

—

203

216

167

205

202

302

M (OH-)-AC

2405

—

—

—

—

—

—

—

—

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:28:42

Specific applications

679

Table 40.13 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

M (bis-nor-)-AC

2535

—

—

—

—

—

—

—

—

—

M (desamino-OH-)-AC

2530

—

—

—

—

—

—

—

—

—

3233

—

—

201

117

167

251

165

202

2120

2128

—

—

167

165

207

85

152

252

2425

2521

—

2710

84

128

179

85

214

2220

—

—

—

—

—

—

Cinnarizine (also metabolised to benzophenone) 3050 M (desalkyl-, norcyclizine) Clemastine M (OH-methoxy-carbinol)-H 2O

—

98

—

— 152

2440

—

—

—

246

248

288

247

330

2620

—

—

—

131

256

125

42

255

89

2965

—

—

—

255

256

214

339

131

186

2025

2104

2348

2320

99

56

167

207

194

266

2120

2128

—

—

167

165

207

85

152

252

2355

2460

2307

2710

287

96

286

215

70

58

M (OH-)

3060

—

—

—

303

203

202

217

304

205

M (nor-)

2400

—

—

—

273

215

216

231

229

272

M (oxo-)

2960

—

—

—

229

215

202

242

301

258

M (nor-OH)-H2O

2450

2608

—

—

271

272

270

165

193

241

2615

—

—

—

83

140

82

124

96

193

M (di-OH-)-H2O Clemizole M (oxo-) Cyclizine (also metabolised to benzophenone) M (nor-) Cyproheptadine

Deptropine Dimenhydrinate (see Diphenhydramine) Dimetindene

2275

2376

2669

—

58

59

72

45

292

218

Dimetotiazine

3060

3096

—

—

72

73

230

56

210

198

M (nor-)

3150

—

—

—

72

320

306

58

198

210

M (OH-)-AC

3200

—

—

—

72

245

59

198

398

263

M (bis-nor-)-AC

3380

—

—

—

319

405

346

211

210

320

M (nor-)-AC Diphenhydramine (also metabolised to benzophenone)

3360

—

—

—

58

114

319

346

72

419

1873

1928

2387

2105

58

73

167

165

166

152

M (nor-)

1520

1922

—

—

44

165

59

167

152

166

M (nor-acetyl-)

2265

2360

—

—

44

101

167

86

165

152

M (di-nor-acetyl-)

2240

2318

—

—

167

165

87

183

72

152

M (desamino-)

—

1883

—

—

214

181

183

153

152

167

M (methoxy-)

2010

2239

—

—

58

73

165

181

152

153

M

—

2190

—

—

183

165

167

105

152

166 115

M

—

2631

—

—

167

165

152

168

253

M (diphenylmethane)

—

1465

—

—

167

168

165

152

153

91

M (diphenylmethanol)

1645

1644

—

—

167

165

152

162

168

115

—

1780

—

—

184

183

165

107

152

115

Diphenylpyraline (metabolised to benzophenone)

2100

2128

2447

2405

99

114

98

167

70

165

Doxylamine

1910

1970

—

2170

58

71

167

182

180

72

M (4-phenylmethylphenol)

M (nor-)

—

1974

—

—

182

183

167

200

149

44

M

1520

1623

—

—

182

173

167

168

94

106

M (carbinol)-H 2O

1560

1670

—

—

180

181

152

77

90

102

M (OH-)-AC

2300

—

—

—

58

71

183

198

72

182

M (OH-carbinol-)-AC

2980

—

—

—

137

257

78

79

106

200

M (OH-methoxy-)-AC

2320

—

—

—

58

71

72

183

198

196

M (bis-nor-)-AC

2280

—

—

—

182

86

167

183

181

198

M (desamino-OH-)-AC

1960

—

—

—

198

87

182

183

167

180

M (nor-)-AC

2340

—

—

—

182

183

167

100

181

58

Histapyrrodine

2240

—

—

—

84

91

196

280

197

65

M (OH-)

1650

—

—

—

84

91

212

296

213

297

M (oxo-)

2570

—

—

—

91

196

209

197

275

294

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:28:49

680

Gas Chromatography

Table 40.13 continued Compound

System

Principal ions (m/z)

GA

GB

GC

M (N-desbenzyl-)

1800

—

—

—

84

190

106

111

122

77

M (N-desbenzyl-oxo-)

2120

—

—

—

106

119

118

98

77

204

M (N-desphenyl-oxo)-H2O Hydroxyzine (also metabolised to chlorobenzophenone)

GF

2100

—

—

—

91

159

216

160

215

84

2880

3000

—

—

201

203

165

299

166

202 286

2520

2355

—

—

85

165

201

241

230

Isothipendyl

2225

—

—

—

72

214

200

285

86

56

M (nor-)

2220

—

—

—

58

214

213

181

199

271

M (desalkyl-) (norchlorcyclizine)

M (OH-)

2450

—

—

—

72

301

218

197

178

228

M (bis-nor-)

2230

—

—

—

214

257

213

58

181

215

382

265

245

280

292

294

91

233

232

276

275

65

189

105

201

285

165

190

—

—

Loratadine

—

3236

—

—

Mebhydrolin

2450

2575

2739

2920

Meclozine (also metabolised to chlorcyclizine)

3035

3193

—

—

2520

—

—

—

—

2220

2328

—

2560

121

M (N-desalkyl-) Mepyramine

58

72

—

—

—

214

122

215

M (N-desalkyl-)

2120

—

—

—

121

214

78

165

136

154

M (N-desalkyl-)-AC

2150

—

—

—

107

214

78

256

163

197

M (N-desmethoxybenzyl-) Mequitazine

1580

—

—

—

58

78

107

60

95

119

2765

2939

—

—

124

322

198

125

212

180

M (phenothiazine)

2120

2130

—

—

199

167

198

166

154

139

M (sulfoxide)

3120

—

—

—

124

198

338

321

180

—

M (sulfone)

3250

—

—

—

124

125

354

180

152

—

Methapyrilene

1981

—

—

2305

58

97

72

71

191

261

Methdilazine

2462

—

—

2920

97

98

296

199

55

212

Phenindamine

2165

2245

2926

2515

260

261

42

57

184

215

M (OH-)

2300

—

—

—

276

277

275

233

234

200

M (N-oxide)

2230

—

—

—

260

277

259

276

215

202

M (nor-)

2210

—

—

—

246

247

202

217

168

215

M (nor-OH-)

2590

—

—

—

262

263

261

184

233

228

1805

1874

—

2100

169

58

168

170

72

167 226

Pheniramine M (nor-) Phenyltoloxamine M (N-oxide)-(CH3) 2NOH

2080

1890

—

—

169

168

167

182

184

1940

2030

—

—

58

255

42

71

59

44

1500

1580

—

—

165

210

181

167

195

152

M (nor-)

2140

2002

—

—

44

58

210

165

241

181

M (O-desalkyl-)

1680

1724

—

—

184

165

78

77

106

183

M (O-desalkyl-OH-)

2220

—

—

—

200

107

94

122

152

181

M (OH-) isomer 1

2280

—

—

—

58

72

271

152

226

197

M (OH-) isomer 2

2300

—

—

—

58

72

271

152

226

197

M (nor-OH-) isomer 1

2320

2398

—

—

58

257

226

107

152

197

M (nor-OH-) isomer 2

2340

2402

—

—

58

257

226

152

197

91

M (OH-methoxy-)

2320

—

—

—

58

72

301

271

152

107

228

183

165

184

106

181

72

284

198

213

199

180

M (desamino-OH-) Promethazine

1830

1928

—

—

2339

2383

2546

2675

M (nor-)

2250

2333

—

—

58

213

180

198

152

270

M (phenothiazine)

2120

2130

—

—

199

167

198

166

154

139

M (sulfoxide)

2710

2797

—

—

72

198

180

152

213

229

M (norsulfoxide)

—

2732

—

—

58

212

180

198

229

152

M (nor-OH-)

2580

2717

—

—

212

58

180

229

198

286

M (OH-)

2590

—

—

—

72

196

300

229

214

288

Propiomazine

2738

—

—

3225

72

73

340

269

197

56

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:29:19

Specific applications

681

Table 40.13 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

2419

—

—

2815

205

240

91

84

125

242

2920

—

—

—

205

115

98

240

200

123

Thenalidine

2318

—

—

—

97

70

99

43

188

44

Thenyldiamine

1999

—

2300

2340

58

97

72

71

203

191

Pyrrobutamine M (oxo-)

Thiazinamium methysulfate Art (promethazine) 2339

2383

2546

2675

72

284

198

213

199

180

Thonzylamine

2203

—

2576

—

58

121

72

71

216

215

Tolpropamine

1900

—

—

—

58

253

165

193

178

117

M (OH-)

2150

—

—

—

58

269

165

115

178

193

M (nor-)

2100

—

—

—

193

239

165

178

208

117

M (nor-OH-)

2200

—

—

—

255

193

167

165

178

115

M (N-oxide)

1750

—

—

—

115

193

208

178

165

116

Trimethobenzamide

3281

—

—

—

58

195

59

72

388

89

Tripelennamine

1976

—

—

—

58

91

72

71

197

185

2420

—

—

—

91

129

197

58

147

241

Trimeprazine (see Alimemazine)

M (nor-) M (OH-)

2400

—

—

—

58

91

72

213

200

271

M (benzylpyridylamine)

1650

—

—

—

184

106

79

183

78

107

2253

2340

2954

2600

208

209

278

207

193

200

Triprolidine

Table 40.14 GC retention data and mass spectral data for benzodiazepines (ET, ethyl; TMS, trimethylsilyl) Compound

System

Principal ions (m/z)

GA

GB

GG

2955

—

—

308

307

309

310

58

280

Alprazolam(a)

3100

3108

—

308

279

204

273

77

307

M (aOH-)

3245

0000

—

287

322

321

323

288

324

M (aOH)-TMS

—

3183

—

381

396

382

383

398

397

M (4OH-)

3045

—

—

—

—

—

—

—

Bromazepam

2665

2760

3280

236

317

318

288

316

286

Bromazepam-TMS

—

2702

—

388

386

387

389

372

374

M (3OH-)

2470

—

—

304

314

331

305

303

302

M (3OH)-TMS2

—

2650

—

388

386

477

475

179

360

M (aminohydroxybromazepam)-TMS

—

2590

—

249

247

366

364

338

336

3070

—

—

394

245

316

210

291

176

Adinazolam

(a)

Brotizolam(a)

3050

—

—

380

378

379

299

301

245

Camazepam(a) (metabolised to oxazepam and temazepam)

2945

3162

—

58

72

43

78

271

44

Chlordiazepoxide (metabolised to nordazepam and oxazepam)

2795

2981 thermally unstable

3065

282

299

284

283

241

253

M (nor-)

2452

2679

—

268

269

270

233

271

205

M (demoxepam)

2529

2806

—

120

285

286

269

241

287

Clobazam(a)

2558

2683

3174

300

258

77

259

283

231

M (nor-)

2747

2759

—

286

218

215

217

216

244

M (OH-)

3000

—

—

316

318

274

271

299

247

M (OH-MeO-)

3255

—

—

346

316

301

348

274

271

Clonazepam

2823

3000

3600

280

314

315

285

234

288

Clonazepam-TMS

M (OH-) Art (-CH2O)

—

2781

—

387

352

306

372

386

388

M (7-amino-)

2890

2996

—

285

256

257

287

250

111

M (7-amino)-TMS2

—

2742

—

429

394

414

430

431

314

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:29:23

682

Gas Chromatography

Table 40.14 continued Compound

System

Principal ions (m/z)

GA

GB

2935

—

—

283

220

225

285

254

284

Clorazepic acid (hydrolysed in vivo and absorbed as diazepam)

2457

2618

3125

242

43

270

269

241

103

Clotiazepam(a)

2532

—

—

289

318

291

320

275

290

M (OH-)

2705

—

—

287

316

318

289

288

317

M (OH-)-AC

2870

—

—

271

316

256

300

273

241

M (di-OH-)-AC2

2995

—

—

332

374

319

291

303

434

M (amino-OH-)

GG

Demoxepam

2529

2806

3043

285

286

269

287

241

242

Diazepam(a) (metabolised to nordazepam, oxazepam and temazepam)

2428

2556

2940

256

283

284

287

257

255

Estazolam(a)

3070

3050

—

259

294

293

205

239

101

Etizolam(a)

2980

—

—

342

266

313

224

239

45

Flunitrazepam(a)

2600

2744

3190

285

312

313

286

266

238

M (nor-)

2720

2816

—

224

299

298

372

271

252

M (nor-)-TMS

—

2622

—

371

370

352

356

324

372

M (7-amino-)

2723

2804

—

283

255

254

282

264

240

M (7-amino-)-TMS

—

2836

—

355

327

326

354

356

312

M (nor-amino-)

2825

—

—

269

240

241

268

270

213

2780

2896

3220

86

87

99

58

84

387

M (desalkyl-)

2470

2559

—

288

260

259

287

261

289

M (desalkyl-)-TMS

—

2350

—

359

360

341

361

345

362

M (2-OH-ET-)

2675

2805

—

288

273

287

332

331

304

M (2-OH-ET-)-TMS

—

2778

—

288

287

273

389

273

360

M (bis-desethyl-)

2694

2739

—

313

315

314

312

250

259

M (desalkyl-OH-)

2255

2373

—

223

286

258

75

257

251

Flutazolam

2460

—

—

289

245

246

210

259

211

Halazepam(a) (metabolised to nordazepam, oxazepam)

2285

—

—

324

352

323

325

351

353

Flurazepam(a)

Ketazolam (hydrolyses to diazepam) Loprazolam(a)

Not eluted Not eluted —

Lorazepam

2410

2528

2910

70

464

42

43

465

394

291

239

274

293

75

302

Lorazepam-TMS2

—

2566

—

429

431

430

347

449

432

Lormetazepam

2660

2770

—

305

307

306

309

308

334

Lormetazepam-TMS (metabolised to lorazepam)

—

2799

—

377

379

391

291

317

406

Medazepam(a) (metabolised to diazepam, nordazepam, oxazepam and temazepam)

2235

2340

2620

242

207

244

270

243

271

2280

—

—

228

193

256

257

165

110

2640

—

—

349

347

321

351

350

394

M (O-desmethyl-)

2730

—

—

321

319

380

378

349

347

M (nor-)

2690

—

—

335

333

349

347

378

380

(a)

Mexazolam

2600

—

—

251

70

253

41

42

139

Midazolam(a)

2575

2722

—

310

312

311

163

325

75

M (a-OH-)

2830

2901

—

310

311

312

341

283

313

M (a-OH-)-TMS

—

2866

—

310

398

413

312

400

415

M (4-OH-)-TMS

—

2775

—

268

269

297

412

298

397

2740

2915

3450

280

253

281

206

234

252

M (nor-) Metaclazepam(a)

Nitrazepam

—

2642

—

352

353

306

338

354

307

M (7-amino-)(b)

2785

2878

—

251

223

222

250

252

235

M (7-amino-)-TMS3

—

2634

—

394

395

396

380

280

322

43

222

Nitrazepam-TMS

—

Not eluted —

293

265

264

292

Nordazepam

2490

2625

3041

—

—

—

—

—

—

Nordazepam-TMS

—

2367

—

341

342

343

327

344

329

Oxazepam

2803

2325

2438

257

77

268

239

205

267

M (7-acetamido-)

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:29:34

Specific applications

683

Table 40.14 continued Compound

System GA

GB

GG

Oxazepam-TMS2

—

2468

—

429

430

431

313

415

401

Oxazolam(a) (metabolised to oxazepam)

2540

—

—

251

70

253

241

105

252

Prazepam(a) (metabolised to nordazepam and oxazepam)

2648

2783

3145

91

269

324

55

296

295

2860

—

—

257

55

311

259

313

340

2440

2576

—

386

359

323

388

245

303

2255

—

—

342

341

343

370

369

259

Temazepam (metabolised to oxazepam)

2595

2727

3125

271

273

300

272

256

77

Temazepam-TMS

—

2713

—

343

344

257

283

357

372

Tetrazepam(a)

2430

—

—

253

288

287

289

225

259

M (nor-)

2530

—

—

239

274

273

275

245

240

M (OH-) isomer 1

2570

—

—

235

375

304

237

261

247

M (OH-) isomer 2

2580

—

—

235

375

304

237

261

247

M (oxo-)

2430

—

—

285

287

267

245

302

247

M (3-OH-) Quazepam(a) (also metabolised to desalkylflurazepam) M (2-oxo-)

Principal ions (m/z)

3080

3219

—

313

238

342

315

75

344

M (a-OH-)

3000

0000

—

328

330

293

265

239

357

M (a-OH)-TMS

—

3308

—

415

417

430

432

416

380

—

2426

—

257

285

267

286

258

145

(a)

Triazolam

Zolazepam(a) (a)

Does not form a TMS derivative. No electron-capture response.

(b)

Hydrolysis of benzodiazepines (preparation of benzophenones)

Boil an aqueous solution (or urine) with concentrated hydrochloric acid (1 part to 10 parts urine or solution) for 30–60 min. Cool, and neutralise with solid KHCO3 or adjust the pH to 8–9 with 10 mol/L KOH. Mix with an equal volume of petroleum ether for 10 min. Centrifuge and evaporate the upper organic phase to dryness at 60
C. The reconstituted extract can be used for GC or other analytical procedures such as TLC (Chapter 39). Data are also presented here for some acetylated hydrolysis products (treatment of the dried residue with acetic anhydride and pyridine (3 : 2) for 30 min at 60
C (Maurer, Pfleger 1987). Not all benzodiazepines make benzophenones when hydrolysed by acid, and a number of other degradation products are furnished. The a-OH-metabolites of alprazolam, brotizolam and triazolam are partly altered by the elimination of formaldehyde. Hydrolysis products of bis-desethylflurazepam and di-OH-tetrazepam are dehydrated; OH-bromazepam, lorazepam and oxazepam form artefacts by rearrangement; the nor-metabolites of

clobazam are cleaved and rearranged to benzimidazole derivatives; tetrazepam, and its two hydroxylated metabolites, are transformed into a pair of cis- and trans-isomeric hexahydroacridone derivatives. Since the metabolism of benzodiazepines is complex, assays that convert drugs and metabolites into hydrolysis products are not ideal, because they do not permit unequivocal identification of the parent compound. After acid hydrolysis, care must be taken to ensure that the acid is neutralised prior to extraction or before injecting the solvent onto the chromatograph, otherwise the column deteriorates rapidly. The retention indices and principal ions are given in Table 40.15. Cardioactive drugs Cardioactive drugs (beta-blockers, calcium channel antagonists, angiotensin-converting enzyme (ACE) inhibitors, etc.) are a diverse group of

Table 40.15 GC retention data and mass spectral data for benzophenones, hydrolysis products (HY) and some acetylated derivatives (AC) of benzodiazepines and their metabolites Abbreviation

Structure

ABP

2-(2-Amino-5-bromobenzoyl)pyridine 2245

ABP-AC

ACB

System GA

Parent compound (in-vivo by metabolism)

Principal ions (m/z)

3-OH-Bromazepam, bromazepam

247

249

276

278

246

248

121

247

249

318

320

289

2490 3OH-bromazepam HY Art 1

2255

(Bromazepam)

285

287

206

286

284

179

3OH-bromazepam HY Art 2

2265

(Bromazepam)

299

301

220

300

298

179

2-Amino-5-chlorobenzophenone

2039

Nordazepam, oxazepam (camazepam, chlordiazepoxide, clorazepic acid, diazepam, halazepam, ketazolam, medazepam, oxazolam, prazepam)

230

77

231

232

233

195

ACB-AC

2245

230

231

232

273

77

105

ACB Art 1

2060

239

205

240

241

163

177

ACB Art 2

2070

253

219

254

255

110

238

ACDP

2-Amino5-chlorodiphenylamine

2210

Norclobazam (clobazam)

242

241

77

166

206

243

Nor-OH-MeO-clobazam HY

2405

(Clobazam)

288

290

289

272

245

281

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:29:36

684

Gas Chromatography

Table 40.15 continued Abbreviation

ACFB

Structure

System GA

Nor-OH-MeO-clobazam HY-AC

2615

Nor-OH-clobazam HY

2650

Nor-OH-clobazam HY-AC

3000

OH-MeO-clobazam HY

2905

2-Amino-5-chloro-2 0 fluorobenzophenone

2030

ACFB-AC

Parent compound (in-vivo by metabolism)

Principal ions (m/z) 288

290

330

332

290

235

258

257

259

260

224

246

258

300

260

259

257

302

(Clobazam)

320

322

206

240

321

207

Desalkylflurazepam, N-desalkylflutoprazepam, oxo-quazepam (flurazepam, flutoprazepam, quazepam)

249

248

250

251

123

124

(Clobazam)

2195

249

248

291

123

95

250

Didesethylflurazepam Art (-H2O)-AC

2460

Didesethylflurazepam (flurazepam) 246

316

245

211

273

275

ACNB

2-Amino-2 0 -chloro5-nitrobenzophenone

2470

Clonazepam, loprazolam

241

276

139

111

195

165

ADCB

2-Amino-5,2 0 -dichlorobenzophenone 2120

Lorazepam, mexazolam (lormetazepam)

230

265

267

232

154

195

ADCB-AC AFMAB

239

265

307

287

232

309

Lorazepam HY Art

2300 2170

Lorazepam (lormetazepam)

239

241

274

273

275

276

5-Amino-2 0 -fluoro-2methylaminobenzophenone

2753

7-Acetamidoflunitrazepam, 7-aminoflunitrazepam (flunitrazepam)

244

227

243

245

211

123

205

286

328

244

243

269

AFNB

2-Amino-2 0 -fluoro-5nitrobenzophenone

2870 2330

Desmethylflunitrazepam (flunitrazepam)

260

259

123

165

213

241

ANB

2-Amino-5-nitrobenzophenone

2388

Nitrazepam

242

241

77

105

195

165

CPMACB

2-Cyclopropylmethylamino-5chlorobenzophenone

2385

3-Hydroxyprazepam, prazepam

56

77

105

285

270

165

AFMAB-AC2

CPMACB-AC

257

256

259

241

283

343

CTFEAB

5-Chloro-2-(2,2,2-trifluoro)ethylaminobenzophenone

2595 2380

Halazepam

313

312

314

315

244

296

DAB

2,5-Diaminobenzophenone

2175

7-Acetamidonitrazepam, 7-aminonitrazepam (nitrazepam)

211

212

77

107

195

183

296

212

211

254

253

297

246

211

245

248

107

247

330

288

246

211

139

332

230

229

211

210

107

231

230

314

272

229

123

201

244

279

229

281

111

75

86

87

57

348

350

109

262

109

166

264

293

275

262

109

166

275

335

264

77

245

244

228

105

246

DAB-AC2 DACB

2985 2,5-Diamino2 0 -chlorobenzophenone

7-Acetamidoclonazepam, 7-aminoclonazepam (clonazepam)

2845

DACB-AC2 DAFB

2305

2,5-Diamino2 0 -fluorobenzophenone

DAFB-AC2

2175

7-Aminodesmethyl flunitrazepam (flunitrazepam)

2715

DCMAB

2 0 ,5-Dichloro-2-(methylamino) benzophenone

2220

Lormetazepam

DEACFB

2-Diethylaminoethylamino5-chloro-2 0 fluorobenzophenone

2505

Flurazepam

HEACFB

2-Hydroxyethylamino5-chloro2 0 -fluorobenzophenone

2400

Hydroxyethylflurazepam, flutazolam (flurazepam, quazepam)

HEACFB-AC

2470

MACB

2-Methylamino-5chlorobenzophenone

2105

Diazepam, temazepam (camazepam, chlordiazepoxide, clorazepate, ketazolam, tetrazepam)

MACDP

2-Methylamino-5chlorodiphenylamine

2220

Clobazam

257

259

77

274

215

231

MANFB

2-Methylamino-5-nitro2 0 -fluorobenzophenone

2385

Flunitrazepam

274

273

257

211

123

275

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:30:23

Specific applications

685

Table 40.15 continued Abbreviation

Structure

System GA

Parent compound (in-vivo by metabolism)

Principal ions (m/z)

a-OH-alprazolam HY Art (-CH2O)

3070

(Alprazolam)

259

294

293

205

239

265

a-OH-alprazolam-AC

3180

(Alprazolam)

323

324

325

366

271

295

OH-brotizolam HY Art (-CH2O-)

3050

(Brotizolam)

380

378

379

299

301

245

OH-brotizolam-AC

3140

(Brotizolam)

409

407

410

450

452

408

a-OH-midazolam-AC

2820

(Midazolam)

310

340

383

312

342

385

a,4-Di-OH-midazolam-AC2

3020

(Midazolam)

310

326

399

340

383

441

Normedazepam-AC

2470

(Medazepam)

228

297

193

256

298

255

Tetrazepam HY (isomer 1)

2220

Tetrazepam

207

249

220

206

209

234

Tetrazepam HY (isomer 2)

2260

Tetrazepam

207

249

220

206

209

234

Tetrazepam M (OH-) HY-AC (isomer 1) 2380

(Tetrazepam)

307

248

234

264

309

220

Tetrazepam M (OH-) HY-AC (isomer 2) 2470

(Tetrazepam)

307

248

234

264

309

220

Tetrazepam M (OH-) HY-AC (isomer 3) 2535

(Tetrazepam)

307

206

309

248

218

220

Tetrazepam M (OH-) HY-AC (isomer 4) 2560

(Tetrazepam)

307

218

220

206

248

264

Tetrazepam M (nor-OH-) HY-AC2

2500

(Tetrazepam)

233

232

196

154

275

335

a-OH-triazolam HY Art (-CH2O)

3000

(Triazolam)

328

293

330

265

239

329

a-OH-triazolam-AC

3200

(Triazolam)

357

359

358

400

402

329

chemicals that require different analytical strategies. The calcium channel antagonists of the phenylalkylamine class (e.g. verapamil) and the benzothiazines (e.g. diltiazem) chromatograph well on standard GC phases (X-1 or X-5) after solvent extraction under mildly basic conditions (pH 10–12). As a rule, the beta-blockers (those with names that end in ‘-olol’) are more water soluble and often require ‘salting out’ of aqueous solution at mildly basic conditions (pH 10–12) with an excess of solid sodium chloride. When subjected to GC they have a tendency to produce artefacts by loss of water and/or their amino-alkyl side-chain. Although they chromatograph reasonably well on capillary columns such as X-1 or X-5, they often give tailing peaks on packed columns, and various derivatisation strategies have been employed to overcome this. Simultaneous preparation of N-TFA and O-TMS derivatives have been described (Leloux et al. 1989; Lho et al. 1990), and cyclic boronates may be formed from phenyl or n-butylboronic acids using either triethylamine or pyridine catalysts (Lee et al. 1998). Acetylation of acid-hydrolysed sample extracts is described using acetic anhydride with a pyridine catalyst; although this process results in the complete destruction of some of the parent compounds, data for many metabolites are given (Maurer, Pfleger 1986). The ACE inhibitors (those with names that end in ‘-pril’) have a free carboxylic acid group, and acquire a second such group by enzymatic hydrolysis of their ethyl ester link (‘prilates’). Neither of these groups of compounds, nor the dihydropyridine calcium channel antagonists (e.g. nifedipine), chromatographs in their native

state, but GC has been applied successfully to their analysis following extractive methylation. Here, equal volumes of urine and phase-transfer reagent (0.02 mol/L tetrahexylammonium hydrogensulfate in 1 mol/L phosphate buffer pH 12) are incubated with three volumes of 1 mol/L methyliodide in toluene on a shaker at 50
C for 30 min. The organic phase is eluted on a diol solid-phase cartridge (conditioned with methanol, then toluene) using diethyl ether–ethyl acetate (92.5 : 7.5, v/v). After evaporation to dryness at 60
C, the residue is reconstituted in ethyl acetate for injection. Systems GA and GB, described above, can be used, and system GP chromatographs many of the drug metabolites as their methyl derivatives (Maurer et al. 1998; Maurer, Arlt 1999). System GP n n n n n

Column: HP1 (methyl-PSX) fused silica capillary (12 m  0.2 mm i.d., 0.33 mm) Injector: 280
splitless mode. Temperature programme: 100
C for 3 min to 310
C at 30
/min for 8 min. Carrier gas: He, 1 mL/min. Retention indices and principal ions are given in Table 40.16.

Coumarins and other anticoagulants Coumarins are extracted fairly readily from acidic solution, especially when ‘salted out’ with solid sodium chloride; the coumarin structure is

Table 40.16 GC retention data and mass spectral data for cardioactive drugs (Me, methyl) Compound

Acebutolol Art

System

Principal ions (m/z)

GA

GB

GP

2811

2926

—

72

43

56

151

221

98

2910

3014

—

151

221

333

98

86

348

Art

—

2761

—

72

193

43

151

136

122

Art (-H2O)

2850

2569

—

303

98

140

318

82

233

M (phenol-)

2450

2056

—

151

221

136

108

43

132

Art (desacetyl-)

—

2089

—

194

209

264

279

166

234 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:30:34

686

Gas Chromatography

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

GP

Acecainide (NAPA)

2550

2724

—

Acetyldigitoxin

Not eluted Not eluted —

162

120

205

—

86

—

99

—

58

—

—

—

Ajmaline

2880

—

—

144

326

173

198

297

237

Alprenolol

1820

—

—

72

56

73

249

98

234

Amiodarone

3335

Not eluted —

2800

—

—

Amlodipine

—

2982

—

297

208

44

254

298

347

Amlodipine-Me

2820

—

—

311

254

88

312

208

238

Art (O-desalkyl)

86

87

84

58

56

44

142

121

294

265

251

237

2815

—

—

72

58

325

165

208

347

M (dehydro-2-HOOC-)-Me

—

—

2430

356

296

224

268

357

391

M (dehydro-desamino-HOOC-)-Me

—

—

2635

260

347

318

316

349

400

2462

—

—

113

84

116

98

117

115

M (N-desalkyl-)

1920

—

—

104

209

77

116

115

94

M (p-aminophenol)

1253

1280

—

109

52

53

80

81

108

Amlodipine-Me2

Amyl nitrite (see Chapter 12) Aprindine

2385

2469

—

72

56

98

43

107

41

Art (-H2O)

2150

2090

—

98

56

107

248

72

190

Art

2400

2648

—

46

86

107

127

72

263

Art (HOOC)-Me

2140

—

—

72

107

116

237

56

266

2175

—

—

278

127

112

56

293

292

—

Not eluted —

—

—

—

—

—

Atenolol

Art (HOOC)-Me Benazepril

—

—

—

3030

365

204

91

366

392

347

—

—

2985

379

204

380

91

144

438

Benziodarone

—

Not eluted —

518

173

264

519

373

376

Betaxolol

2370

2420

—

72

253

292

307

55

107

Art

2410

2508

—

304

319

318

290

127

55

Art (-H2O)

2400

2519

—

72

98

53

56

107

158

Benazepril-Me M (benazeprilate)-Me3

Bethanidine

1925

—

—

71

91

106

177

57

72

Bisoprolol

2378

2427

—

72

116

107

100

281

73

Art

2595

2680

—

127

112

86

322

337

224

Art (-H2O)

2400

2480

—

98

56

307

107

204

220

M (phenol)

1690

—

—

107

123

77

167

103

210 221

Bunazosin

3330

—

—

247

260

233

373

234

Bunitrolol

1960

—

—

86

233

70

71

58

204

1980

—

—

245

70

56

119

158

260

Art Captopril

Not eluted Not eluted —

70

41

69

75

114

217

Captopril-Me

1730

—

—

70

128

172

199

231

198

Captopril-Me2

—

—

—

70

128

89

245

203

130

Carazolol

2810

—

—

183

72

298

154

184

116

2830

—

—

183

127

112

310

222

295

Carazolol-Me

2815

—

—

86

183

154

312

298

268

Carteolol

2588

—

—

86

57

277

70

292

87

Art

2690

—

—

289

141

126

202

304

290

Carvedilol Art 1

—

2056

—

222

223

11

151

98

77

Carvedilol Art 2

—

2224

—

183

154

155

127

77

184

Art

Celiprolol

2610

—

—

86

58

250

44

291

307

Art 1

2350

—

—

333

86

56

96

112

216

Art 2

2650

—

—

86

151

291

277

114

265

2740

—

—

86

209

323

294

114

56

Cilazapril

—

Not eluted —

211

143

91

283

197

344

Cilazapril-Me

—

—

157

225

297

344

91

358

Art 3

3010

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:30:55

Specific applications

687

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

GP

—

—

2960

157

225

297

245

361

2100

—

—

86

57

127

190

243

90

Art (-H2O)

1895

—

—

57

174

175

176

202

258

Art

2160

—

—

99

243

245

187

174

176

M (cilazaprilate)-Me3 Clenbuterol

372

2090

2165

—

229

231

172

194

174

200

M

—

2046

—

192

157

227

194

229

193

M

—

2264

—

243

188

245

186

194

236

Art (dichlorophenylisocyanate)

1350

—

—

187

189

124

159

126

161

Art (dichlorophenylmethylcarbamate)

1500

—

—

184

186

174

133

160

219

Art 5

2110

—

—

248

194

250

229

243

283

44

117

Clonidine

0000

0000

—

132

104

175

103

Debrisoquine-acetylhydrazone deriv.

—

2100

—

—

—

—

—

—

—

M (4OH)-acetylhydrazone deriv.

—

2450

—

—

—

—

—

—

—

—

—

578

195

577

367

351

579

125

189

230

127

191

63

Debrisoquine

Deserpidine

—

Deslanoside (metabolised to digoxin)

Not eluted Not eluted —

Diazoxide

Not eluted Not eluted —

Digitoxin

Not eluted Not eluted —

Digoxin

Not eluted Not eluted —

Diltiazem

2949

3076

—

58

71

72

121

150

136

M (desacetyl-)

2990

3092

—

58

71

121

136

150

109

M (O-desmethyl-)

3050

3147

—

58

71

159

283

207

253

M (N-desmethyl-)

—

3114

—

44

150

283

161

121

136

72

194

2505

2608

—

195

212

114

167

M (N-desalkyl-)

—

2286

—

—

—

—

—

—

—

M

—

2264

—

194

196

167

180

280

252

—

Disopyramide

Doxazosin

—

3054

—

—

—

—

Enalapril-H2O

2770

2864

—

208

254

117

70

—

—

169

358 317

2650

—

2675

234

91

70

130

160

M (enalaprilate)-Me3

—

—

2680

234

130

174

235

331

91

M (enalaprilate-H2O)-Me

2730

—

2735

208

240

91

70

117

344

Enalapril-Me

Encainide

3016

—

—

98

135

70

99

77

352

Enoximone

—

Not eluted —

248

247

151

201

249

200

Esmolol

—

2311

—

72

56

107

116

73

91

Art

—

2395

—

292

306

307

293

278

234

Felodipine

2670

2793

—

238

210

239

354

383

338

Felodipine-Me

2725

—

—

252

224

324

326

164

338

M (dehydro-desethyl)-Me

—

—

2235

322

324

323

300

258

173

M (dehydro-)

—

—

2280

346

318

348

320

286

173

2250

2351

—

84

56

97

301

219

209

Flecainide

2500

2240

—

301

125

42

97

218

343

Heptaminol

1120

—

—

59

56

69

113

95

127

Hexobendine

Not eluted —

—

296

195

58

253

297

Hydralazine

1528

—

—

—

—

—

—

Art (formyl)

1914

Hydroquinidine

2810

—

—

—

Imidapril-Me

2700

—

—

234

M (desethyl-)-Me2

2695

—

—

M (desethyl-)-Me3

2710

—

—

2177

—

—

Indoramin

—

—

—

Isradipine

2680

—

—

Imolamine

—

84 —

—

—

—

—

91

346

159

160

117

220

56

117

70

159

346

234

360

130

235

56

117

—

—

—

—

217

174

105

218

143

130

210

252

77

150

178

284

—

—

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:5

688

Gas Chromatography

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

GP

2670

—

—

298

224

268

56

385

M (dehydro-desiospropyl)-Me

2270

—

2270

309

264

341

310

294

279

M (dehydro-)

2360

—

—

295

327

265

251

369

310

1320

1270

—

44

91

132

117

65

78

Isradipine-Me

Labetalol Art

284

Lacidipine

2955

—

—

252

196

326

169

119

382

Lanatoside c (hydrolyses to digoxin)

—

Not eluted —

41

43

55

57

73

81

Lidocaine

1870

1947

—

86

87

58

44

72

42

M (MEGX)

1800

1870

—

58

121

120

163

91

77

M (GX)

—

1776

—

121

178

120

106

148

77

M (2,6-xylidine)

1180

1195

—

106

121

120

91

77

65

M (3OH-)

2350

—

—

86

58

250

194

110

120

3870

Not eluted —

343

70

344

109

42

113

2230

—

—

—

—

—

—

—

—

2125

—

—

—

—

—

Lidoflazine M (desaminocarboxy-)

—

—

—

Lisinopril

Not eluted Not eluted —

70

91

84

113

245

224

Lorcainide

2810

2923

—

82

56

110

355

124

251

M (N-desmethyl-)

2660

2789

—

—

—

—

—

—

M (desacyl-)

2100

—

—

110

56

125

252

180

254 435

M (desaminocarboxy)-Me

—

Losartan-Me2

—

—

3555

192

201

249

165

450

Mecamylamine

—

—

—

98

84

71

56

99

124

Methoserpidine

—

—

—

608

195

607

397

609

395

Methyldopa

—

Not eluted —

88

42

123

124

89

77

Metipranolol

2220

2320

—

72

152

56

116

102

194

Art

2240

—

—

127

114

112

86

152

306

Art

2190

—

—

72

152

116

56

137

223

2035

2090

—

72

107

56

73

223

100

Art

2120

2176

—

56

127

112

114

264

279

M

2200

2284

—

72

107

116

251

280

145

M (OH-) Art

2240

2355

—

128

127

250

280

295

294

1400

1431

—

58

44

83

77

69

85

M (desamino-oxo-)

1350

1395

—

105

178

135

134

133

121

M

—

1745

44

58

91

77

135

178

—

—

84

209

67

43

110

192

Metoprolol

Mexiletine

Minoxidil

—

—

—

3575

234

305

190

250

91

439

M (moexiprilate)-Me3

—

—

3580

234

305

190

91

220

453

M (moxeprilate-H2O)-Me3

Moexipril-Me

—

—

3775

190

466

449

91

164

290

Moracizine

—

—

—

100

286

142

56

239

70

Nadolol

2540

2658

—

86

57

294

71

310

70

Art

2560

2670

—

306

70

86

141

201

307

3900

Not eluted —

91

134

147

146

148

165

M (dehydro-desbenzylMeNH2)-Me

—

—

2300

327

297

313

312

344

252

M (dehydro-desamino-HOOC)-Me

—

—

2645

312

313

281

371

285

139

M (dehydro-desamino-OH-)

—

—

2665

312

313

299

281

252

374

M

2250

—

—

299

269

316

285

300

241 285

Nicardipine

2545

2708

—

239

284

224

268

330

M (dehydro-)

2250

2370

2255

298

299

252

267

313

344

M (dehydo-HOOC-)

2290

—

—

283

252

224

126

282

298

M (dehydro-2-HOOC)-Me

—

—

2695

342

343

139

195

357

388

Nifedipine

—

Not eluted —

—

—

—

—

—

—

M (dehydro-desisopropyl)-Me

—

—

2565

340

324

355

308

164

341

M (dehydro-)

—

—

2565

324

341

310

383

294

164

Nilvaldipine

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:25

Specific applications

689

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

2929

3096

—

—

—

—

—

—

—

M (dehydro-desisopropyldesmethoxyethyl)-Me2

—

—

2300

327

297

313

312

344

252

M (dehydro-desmethoxyethyl)-Me

—

—

2390

313

298

252

330

283

372

M (dehydro-desisopropyl-O-desmethyl- — HOOC)-Me

—

2645

312

313

281

371

285

139

Nimodipine

GP

—

—

2655

298

299

340

341

342

357

M (dehydro-desisopropyl-O-desmethyl)-— Me

—

2665

312

313

299

281

252

374

—

—

2740

298

340

281

299

341

371

2730

—

—

371

210

270

266

284

254

M (dehydro-desisobutyl)-Me

—

—

2255

298

299

252

267

313

344

M (dehydro-)

—

—

2450

284

340

57

285

236

303

M (dehydro-OH-)

—

—

2615

284

356

59

253

313

267

M (dehydro-desisobutyl-2-HOOC)-Me2 —

—

2695

342

343

139

195

357

388

M (dehydro-)

M (dehydro-O-desmethyl-HOOC)-Me Nisoldipine

2635

—

—

238

210

239

287

360

150

M (dehydro-desethyl-)-Me

—

—

2300

327

297

313

312

344

252

M (dehydro-)

—

—

2370

341

313

358

312

281

252

M (dehydro-desethyl-)-CO2

2275

—

—

269

329

255

286

139

180

Nitrendipine

M (dehydro-desmethyl-)-CO2

2330

—

—

251

139

253

255

283

300

M (dehydro-desethyl-OH-)-H2O

2650

—

—

311

281

328

297

312

250 326

M (dehydro-desethyl-OH-)-H2O Oxprenolol Art

2690

—

—

325

297

342

266

250

1870

1972

—

72

56

221

41

73

57

1985

2062

—

56

262

248

148

277

235

—

—

—

—

—

1700

—

—

Pargyline

1214

1257

—

82

68

91

159

42

Pempidine

—

—

—

140

84

51

41

72

69

Penbutolol

2139

2221

—

86

70

56

276

133

161

M (desamino-OH-desalkyl-)

—

158

M (OH-)

2425

—

—

86

304

56

178

319

292

Art (formyl)

2150

—

—

288

289

303

141

91

159

Perhexiline

2245

—

—

84

194

55

85

56

99

M (OH-)

2485

—

—

84

56

210

192

97

110

M (di-OH-)

2660

—

—

84

56

98

210

70

249

M (di-OH-)-H2O

2510

—

—

84

56

192

208

210

291

Perindopril

—

Not eluted —

—

—

—

—

—

Perindopril-Me

—

—

—

2450

172

98

309

173

124

382

M (perindoprilate)-Me3

—

—

2470

172

112

86

323

173

382

M (perindoprilate-H2O)-Me3

—

—

2560

222

249

277

336

133

294

Phenoxybenzamine

2235

2332

—

91

196

198

92

197

65

Phentolamine

Not eluted Not eluted —

281

120

91

122

280

160

Pindolol

2245

2335

—

72

133

116

248

134

56

Practolol

0000

2440

—

72

151

43

109

56

57

Prajamlium M (OH-) Art

2925

—

—

—

—

—

—

—

—

3130

—

—

224

126

196

384

313

356

M (methoxy-) Art

2895

—

—

254

370

398

255

126

297

M (OH-methoxy-) Art

3200

—

—

224

196

206

414

343

399

Prazosin

Not eluted Not eluted —

233

383

259

245

95

56

Prenylamine (also metabolised to amphetamine)

2555

—

—

58

238

91

45

239

167

Prenylamine-AC

2925

—

—

58

91

100

280

238

164

2320

—

—

73

165

167

193

253

152

M (N-desalkyl-)-AC

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:31

690

Gas Chromatography

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

GP

M (N-desalkyl-OH-)-AC2

2635

—

—

73

183

311

220

239

269

M (N-desalkyl-OH-methoxy-)-AC2

2700

—

—

73

213

299

152

240

341

M (OH-methoxy-)-AC2

3310

—

—

58

326

368

270

459

240

M (OH-)-AC2

3200

—

—

58

296

338

297

100

429

—

—

—

—

—

1940

—

—

2255

2332

—

86

99

120

92

87

58

M (N-acetyl-)

2550

2724

—

86

99

58

162

120

205

M

—

2245

—

120

58

71

92

137

149

M

—

2292

—

86

146

99

117

120

189

M

—

2642

—

58

71

120

162

92

191

M (desamino-OH-)-H2O Procainamide

—

—

2783

—

86

99

120

58

176

219

Propafenone

2730

—

—

72

91

98

297

131

312

Art (-H2O)

2300

—

—

91

98

105

294

323

230

72

M

M (OH-)-H2O

2720

—

—

230

137

339

310

M (O-desalkyl-)

1830

—

—

—

—

—

—

—

—

M (O-desalkyl-OH-) isomer 1

2345

—

—

—

—

—

—

—

—

M (O-desalkyl-OH-) isomer 2

2355

—

—

—

—

—

—

—

—

M (O-desalkyl-OH-methoxy-)

2400

—

—

—

—

—

—

—

—

2147

2234

—

72

56

98

115

144

116

M (4OH-)

—

2546

—

72

116

160

131

275

199

M (1-naphthol)

1505

1534

—

144

115

116

72

89

63

M (desamino-OH-)

2065

—

—

144

115

218

131

116

101

—

Propranolol

98

Protoveratrine

2465

—

—

—

—

Quinapril

—

—

—

316

270

3380

3467

—

91

316

270

130

117

104

—

—

3110

234

91

130

190

160

379

M (quinaprilate)-Me3

—

—

3080

234

91

130

148

174

235

M (quinaprilate-H2O)-Me3

—

—

3310

91

130

270

302

103

132

Art-H2O Quinapril-Me

91

—

—

—

130

117

104

2790

2979

—

136

81

322

188

55

172

M (N-oxide)

2950

3086

—

152

136

189

340

173

324

M

2940

3125

—

152

124

138

338

323

158

—

Not eluted —

294

248

91

110

117

209

2980

—

248

294

117

110

209

297

Quinidine

Ramipril M (-H2O)

—

—

—

2880

234

91

160

235

357

220

M (ramiprilate)-Me3

—

—

2865

234

91

235

130

371

148

M (ramiprilate-H2O)-Me3

Ramipril-ME

—

—

2925

280

248

91

110

193

284

Rescinnamine (Reserpinine)

2180

—

—

221

109

200

186

395

251

Reserpine

Not eluted Not eluted —

608

606

195

609

395

397

M (trimethoxybenzoic acid)

1780

—

—

212

197

141

154

169

111

M (trimethoxyhippuric acid)

2085

—

—

195

251

223

152

122

167

M (trimethoxybenzoic acid)-Me

1740

—

—

226

211

195

155

183

168

M (trimethyoxyhippuric acid)-Me

2350

—

—

283

195

284

152

268

252

Sotalol

2413

2520

—

72

43

122

73

106

121

Syrosingopine

—

Not eluted —

181

395

198

251

397

396

2310

—

—

86

166

251

280

57

151

2400

—

—

292

293

307

141

166

151

Terazosin

—

Not eluted —

233

71

316

245

43

387

Timolol

2266

2373

—

—

—

—

Art

Tertatolol Art

—

—

—

2275

2380

—

86

96

72

142

154

313

Tocainide

1714

1769

—

44

121

77

120

42

106

Trandolapril-H2O

3090

—

—

262

308

91

124

117

223

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:40

Specific applications

691

Table 40.16 continued Compound

System

Principal ions (m/z)

GA

GB

GP

Trandolapril-Me

—

—

2970

Trandolapril-Me2

2995

234

91

235

160

371

130

248

249

91

174

144

385

M (perindoprilate)-Me3

—

—

3005

234

235

91

385

130

174

M (perindoprilate-H2O)-Me3

—

—

3070

294

262

91

398

117

223

Trimetazidine

—

—

—

181

85

56

166

266

182

Valsartan-Me2

—

—

3420

264

378

192

320

249

164

Verapamil

3150

3305

—

303

58

43

304

151

44

M (nor-)

3180

3371

—

289

151

290

152

165

260

M (N-desalkyl-)

2100

2193

—

44

164

203

247

290

57

M (O-desmethyl-didesalkyl-)

—

2169

—

216

233

164

185

276

203

M (O-desmethyl-desalkyl-)

—

2246

—

44

257

212

171

247

290

M (didesalkyl-)

—

2300

—

247

248

275

233

216

290

M

—

2409

—

202

189

230

247

290

203

M (N-desalkyl-acetyl-)

2460

2546

—

247

289

332

216

248

290

M (N-didesalkyl-acetyl-)

2545

2579

—

275

318

233

276

234

170

not amenable to GC without prior derivatisation. Maurer and Arlt (1998) describe an extractive methylation procedure from aqueous alkali (5 mL) using iodomethane in toluene (5 mL of 0.5 mol/L) and 150 mL of a phase-transfer reagent (tetrahexylammonium hydrogensulfate (THA), 4.5 g in 50 mL of 0.5 mol/L NaOH). Removal of excess THA was achieved by passing the toluene layer through a solid-phase extraction (SPE) cartridge and eluting with diethyl ether–ethyl acetate (92.5 : 7.5, v/v), which enables GC with MS. System GA, described

above, is used, and the retention indices and principal ions are given in Table 40.17. Anticoagulants of the heparin family (e.g. enoxaparin) are not included as these peptides cannot be chromatographed. Diuretics As a group, the diuretics chromatograph poorly on packed columns, and only marginally better on capillaries because of the presence of one or

Table 40.17 GC retention data and mass spectral data for coumarins and other anticoagulants (Me, methyl) Compound

System GA

Principal ions (m/z)

Acenocoumarol-Me

3035

324

325

367

189

121

278

M (amino)-Me3

2985

308

365

292

309

293

249

M (acetamido)-Me2

3265

350

351

393

278

56

394

M (OH-) isomer 1-Me2

3350

354

355

397

151

308

219 219

M (OH-) isomer 2-Me2

3500

354

355

397

151

308

Anisindione

2273

252

237

253

181

238

77

Coumachlor-Me

2770

313

315

356

128

189

201

M (OH-) isomer 1-Me2

2990

343

345

386

125

151

231

M (OH-) isomer 2-Me2

3035

343

345

386

125

151

231

M (OH-dihydro)-Me2

3095

388

343

329

245

125

151

M (OH-methoxy)-Me2

3195

373

375

416

372

359

125

M (di-OH)-Me3

3195

373

375

416

372

359

125

2635

292

188

130

128

293

187 189

Coumatetralyl Coumatetralyl isomer 1-Me

2655

306

175

291

115

121

Coumatetralyl isomer 2-Me

2690

306

291

175

115

202

91

M (OH-) isomer 1-Me

2910

203

303

304

187

121

322

M (OH-) isomer 2-Me2

2925

336

205

217

232

302

321

M (OH-) isomer 3-Me2

2935

321

320

336

175

319

305

M (OH-) isomer 4-Me2

2990

336

205

232

217

321

337

3235

—

—

—

—

—

—

Dicoumarol-Me Diphenadione

2934

173

167

340

165

89

Phenindione

2055

222

165

223

194

76

152 90

Phenprocoumon isomer 1-Me

2375

203

279

265

294

249

121

Phenprocoumon isomer 2-Me

2395

91

265

294

203

279

221 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:51

692

Gas Chromatography

Table 40.17 continued Compound

System GA

Principal ions (m/z)

M (OH-) isomer 1-Me2

2655

295

324

233

91

309

251

M (OH-) isomer 2-Me2

2675

295

296

324

121

279

201

M (OH-) isomer 3-Me2

2705

295

324

296

91

233

151

M (OH-methoxy)-Me2

2770

325

354

326

151

279

201 201

2770

325

354

326

151

279

Pyranocoumarin/cyclocumarol

2670

322

72

265

249

275

148

M (O-desmethyl-) Art-Me (warfarin-Me)

2580

279

280

322

91

121

189

M (O-desmethyl-OH-) isomer 1 Art-Me2

2810

309

310

352

91

277

151

M (O-desmethyl-OH-) isomer 2 Art-Me2

2830

309

121

201

295

352

310

M (O-desmethyl-OH-) isomer 3 Art-Me2

2870

309

310

352

91

295

206

M (di-OH)-Me3

Warfarin-ME

2580

279

280

322

91

121

189

M (OH-) isomer 1-Me2

2810

309

310

352

91

277

151

M (OH-) isomer 2-Me2

2830

309

121

201

295

352

310

M (OH-) isomer 3-Me2

2870

309

310

352

91

295

206

more sulfonamide (SO2–NH2) or carboxylic acid groups. However, they can be methylated easily, and then systems GA or GB, described above, may be used effectively. The diuretics can be extractively alkylated from aqueous alkali (5 mL) using iodomethane in toluene (5 mL of 0.5 mol/L) and 150 mL of a phase-transfer reagent. Tetrahexylammonium hydrogensulfate (4.5 g in 50 mL 0.5 mol/L NaOH) is far superior to tetrabutylammonium hydroxide (TBAH) and tetrapentylammonium hydroxide (TPAH) (Carreras et al. 1994). Removal of excess THA was achieved by passing the toluene layer through an SPE cartridge and eluting with diethyl ether–ethyl acetate (92.5 : 7.5, v/v). Alternatively, diuretics can be extracted from aqueous acidic solution into ethyl acetate, and the evaporated residue heated with 10% methyl iodide in acetone and 100 mg solid K2CO3 for 6 h at 60
C, and the resultant extract applied directly to the chromatograph. The former method tends to produce more completely substituted derivatives than the latter, which gives a more varied pattern of substituted derivatives. Although good sensitivity for plasma samples can be obtained with an NPD, the use of a mass spectrometer is required to confirm the identity (Lisi et al. 1991; Yoon et al. 1990).

System GA or GB, described above, can be used, as can systems GX and GY. System GX

The details are taken from Carreras et al. (1994). n n n n

Column: 5%-phenyl-PSX (X-5) (25 m  0.2 mm i.d., 0.33 mm). Temperature programme: 230
C to 320
C at 35
/min (drugs elute isothermally). Carrier gas: He, 1 mL/min. Retention indices and times (min) are given in Table 40.18.

System GY

The details are taken from Lisi et al. (1991). n n n n n

Column: methyl-PSX (X-1) (25 m  0.22 mm i.d., 0.1 mm). Temperature programme: 130
C to 320
C at 40
/min for 3 min. Injection: Split 10 : 1. Carrier gas: H2, 1 mL/min. Retention indices and times (min) are given in Table 40.18.

Table 40.18 GC retention data and mass spectral data for diuretics (Me, methyl) Compound

System GA

Principal ions (m/z) GX

GY

Acetazolamide-N-Me3

1827

3.62

2.90

249

83

108

43

264

265

Acetazolamide-O-Me

1930

—

2.69

70

44

129

236

237

—

—

13.20

—

352

354

244

42

145

—

Art-Me

1840

—

—

202

144

171

116

204

101

Art-Me2

1860

—

—

187

170

189

142

116

101

—

10.70

5.62

386

278

91

42

387

145

Althiazide-Me4

Bendrofluazide-Me4

Bendroflumethiazide-Me3 3344

12.00

—

386

278

42

387

388

—

Benzbromarone

—

—

264

173

424

115

279

423

2760

Benzbromarone-Me

—

2730

—

278

438

173

440

439

—

Benzthiazide

2680

—

—

309

91

311

123

176

121

Bumetanide-Me3

2970

7.71

4.90

254

318

363

406

77

196

Chlorothiazide

1720

—

—

295

268

297

270

64

124

Chlorothiazide-Me3

—

6.55

4.34

337

245

42

339

230

293

Chlortalidone

2145

—

—

148

130

76

321

299

300

Chlortalidone-Me4

2630

7.67

4.81

176

287

363

365

364

289

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:54

Specific applications

Table 40.18 continued Compound

System

Principal ions (m/z)

GA

GX

Clopamide-Me2

2805

6.97

GY —

111

112

127

55

139

Clopamide-Me3

2600

—

—

372

374

387

373

264

245

Art(-SO2NH)

2195

—

—

111

127

139

83

96

251

Cyclopenthiazide-Me4

—

—

6.33

352

354

233

42

145

435

358

Dichlorphenamide-Me4

—

5.15

3.94

44

108

253

255

144

360

Ethacrynic acid-Me

2195

4.02

3.33

261

263

243

245

281

316

Furosemide (frusemide)

Not eluted

—

—

81

300

53

96

82

332

Furosemide-Me

2890

—

—

81

344

346

96

329

311

Furosemide-Me2

2850

—

—

81

358

360

96

325

343

Furosemide-Me3

2800

6.95

4.65

81

372

358

374

339

312

Art (-SO2NH)

2040

—

—

81

53

251

96

253

233

Art (-SO2NH)-Me

2020

—

—

81

265

53

96

232

250

Art (-SO2NH)-Me2

2050

—

—

81

232

279

250

234

204

M (N-desalkyl-)-Me

2750

—

—

264

232

266

234

248

200

M (N-desalkyl-)-Me2

2450

—

—

278

200

280

248

185

169

Not eluted

—

—

269

297

271

221

268

188

9.01

4.99

310

353

218

288

355

202

2170

—

—

139

232

127

63

167

189

Not eluted

—

—

303

239

331

255

266

158

Hydroflumethiazide-Me4 2653

6.30

4.38

387

236

215

344

252

322

Indapamide-Me3

3035

9.01

—

161

132

131

407

409

130

Mefruside-Me2

2860

7.43

4.74

85

43

86

110

325

367

—

9.80

5.14

99

325

327

218

282

326

Methazolamide

2187

—

—

221

43

83

236

223

221

Methazolamide-Me2

—

—

2.90

249

264

43

108

83

265

Hydrochlorothiazide

Hydrochlorothiazide-Me4 2966 Art (-SO2NH)-Me Hydroflumethiazide

M (5-oxo-)-Me2

Methyclothiazide

Not eluted

—

—

310

312

42

311

230

359

Methyclothiazide-Me3

—

9.90

—

352

354

244

246

—

—

Metolazone-Me3

3910

—

6.23

392

394

393

284

118

407

Piretanide-Me3

2965

8.40

—

295

296

404

266

297

—

Polythiazide

2380

—

—

310

312

42

129

311

230 —

Polythiazide-Me3

2985

11.01

—

352

354

244

42

246

Probenecid

2336

—

—

256

185

121

224

257

65

Probenecid-Me

2205

3.90

3.23

270

135

199

271

228

299

Quinethazone

Not eluted

—

—

260

262

180

289

261

145

Quinethazone-Me3

—

—

5.05

316

208

318

42

173

317

Spironolactone

3280

—

—

341

340

374

342

267

359

M (canrenone)

3250

—

5.57

340

267

107

91

341

325

M (canrenoic acid)

3100

—

—

358

84

85

329

359

274

M (canrenoic acid)-Me 3130

—

—

354

355

339

340

356

173

Triamterene

2010

—

—

253

252

43

104

254

235

Triamterene-Me6

2875

9.15

—

336

338

322

308

293

309

Trichlormethiazide-Me4

2810

10.72

—

352

354

244

Trometamol

1645

—

—

—

—

—

42 —

—

—

—

—

Xipamide-Me2

3350

—

—

262

264

382

263

168

223

Xipamide-Me3 isomer 1

2800

8.72

—

396

276

365

395

397

398

Xipamide-Me3 isomer 2

3320

—

—

276

396

278

277

168

233

Xipamide-Me4

2780

—

—

410

290

379

409

411

412

Art (-SO2NH)

2385

—

—

121

155

275

157

106

99

Art (-SO2NH)-Me

2480

—

—

169

170

289

126

290

291

Art (-SO2NH)-Me2

2115

—

—

183

303

272

257

302

304

M (OH-)-Me4

3000

—

—

426

428

395

396

275

262

693

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:31:59

694

Gas Chromatography

Essential oils, flavours and fragrances

System GN

Essential oils, flavours and fragrances are complex mixtures of many components, so Table 40.19 is representative of the most common ones only. For a more extensive list, the reader is referred to specialist texts (Adams 1995). In addition, many small esters and ketones are contributory and can be detected via the system described in the section below on volatile substances. Essential oils, once thought innocuous, are now recognised as a potential cause of serious poisonings, especially in children, and are encountered in highly concentrated forms with increasing frequency in everyday use as vehicles for medicines, aromatherapy and handicraft supplies. Since most natural flavours occur in predominantly one enantiomeric form, and the majority of synthetic flavours are racemates, chiral analysis of enantiomeric proportions is an effective way to determine the authenticity of flavours. The catalogues of chromatography column suppliers show many examples of chiral separations; some examples are found on-line at www.restek.com. Systems GA and GB, described above, can be used, as can systems GN and GO (Supelco 2000).

n n n n

Column: Supelcowax 10 (Wax10) (10 m  0.25 mm i.d., 0.25 mm). Temperature programme: 50
for 2 min, to 280
at 2
/min Carrier gas: He, 25 cm/s. Retention times (min) are given in Table 40.19.

System GO n n n n

Column: SPB-5 (5%-phenyl-PSX, X-5) (30 m  0.25 mm i.d. 0.25 mm). Temperature programme: 75
for 8 min to 200
at 4
/min. Carrier gas: He, 25 cm/s. Retention times (min) are given in Table 40.19.

Narcotic analgesics, opiates and opioids Many laboratories perform specific assays for opiates for federal or legal purposes; these are generally limited to codeine, morphine and more recently 6-monoacetyl morphine (MAM) (Paul et al. 1999).

Table 40.19 GC retention data for oils, flavours and fragrances Compound

System GA

GB

GN (min)

GO (min)

Anethole

1284

1316

—

—

Camphene

—

—

Camphor

1143

—

Carene

—

—

Carvone

—

1275

—

18.8

Cedrol

—

—

64.3

—

Cineole

—

1063

—

11.2

Citronellal

1265

—

—

14.4

Citronellol

—

—

45.8

—

Eugenol

1368

1380

—

—

Geranial

—

—

—

19.4

Geraniol

1192

—

51.7

18.8

Ionine

—

—

50.4

—

Jasmone

—

—

56

—

Lavandulol

—

—

40.5

—

Limonene

1053

1063

11.0

8.9

Linalool

1100

—

32.4

12.0

Menthol

1206

1194

—

—

Menthone

—

—

26.1

—

Methyl salicylate

1195

1228

—

—

Myrcene

—

—

Neral

—

—

Nerol

—

—

48.0

17.6

Nerolidol

—

—

59, 61

—

Patchouli

—

1691

—

—

a-Pinene

—

—

4.9

5.4

b-Pinene

—

—

7.1

6.7

Piperonal

—

1373

—

Sabinine

—

—

—

6.5

a-Terpinene

—

—

—

8.4

g-Terpinene

—

—

18.4

10.2

Terpinen-4-ol

—

—

—

15.5

a-Terpineol

1126, 1176

—

41.5

16.1

Thymol

—

1316

—

—

Vanillin

1630

1632

86.5

—

5.9 — 8.6

9.6 —

5.9 — 8.3

7.1 18.1

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:34:6

Specific applications However, for clinical purposes a wider range of analytes is desirable and can include codeine, dihydrocodeine, hydrocodone, hydromorphone, oxycodone and oxymorphone. All assays involve a hydrolysis step (acidic or enzymatic – see earlier discussion for an evaluation of these) to cleave the glucuronide conjugates, followed by a basic extraction (often using solid-phase or acidic back extraction for cleanliness). Derivatisation is possible with a number of reagents

695

(PFP, TMS, TFA or AC derivatives are the most common) (Chen et al. 1990; Grinstead 1991; Maurer, Pfleger 1984), and retention data for some of these are included in Table 40.20. The derivatising reagent is selected on the basis of personal preference for a desired separation or the formation of unique ions on MS fragmentation. Analysis of hydromorphone, oxycodone and oxymorphone is complicated by the possibility that several structurally different

Table 40.20 GC retention data and mass spectral data for narcotic analgesics, opiates and opiods (AC, acetyl; HFB, heptafluorobutyrate; PFP, pentafluoropropionate; TFA, trifluoroacetyl; TMS, trimethylsilyl) Compound

System GA

Principal ions (m/z) GB

GC

GF

GM

Acetorphine (hydrolyses to etorphine) Acetylcodeine

2503

2645

—

—

1.449

341

282

342

229

204

240

Alfentanil

2970

3108

—

—

Not eluted

289

268

290

140

222

170

—

—

—

Alphameprodine

1850

1927

—

—

—

—

—

Alphaprodine

1792

1862

—

—

—

172

187

84

57

42

— 188

Anileridine

2850

—

3469

—

—

246

247

218

120

277

106

Apomorphine

2715

0000

—

—

Not eluted

266

267

224

220

152

248

Apomorphine-AC 2

2830

—

—

—

—

351

350

266

308

309

292

Apomorphine-TMS2

2715

—

—

—

—

410

411

322

73

368

412

Benzylmorphine

3015

—

—

—

—

284

91

81

375

285

175

Buprenorphine

3360

3610

—

—

Not eluted

378

410

379

435

434

449

Buprenorphine-HFB

2960

—

—

—

—

574

606

575

562

548

607

Buprenorphine-HFB2

2820

—

—

—

—

55

562

83

630

646

604

Buprenorphine-PFP

3040

—

—

—

—

524

556

525

512

498

580

Buprenorphine-PFP2

2775

—

—

—

—

55

512

580

554

513

595

Buprenorphine-TFA

2920

—

—

—

—

55

474

506

475

448

507

Buprenorphine-TFA2

2800

—

—

—

—

55

462

530

463

504

546

Buprenorphine-TMS

3890

—

—

—

—

450

451

482

506

493

424

Art (-H2O)

3240

—

—

—

—

449

434

408

419

435

450

Art (-H2O)-AC

3320

—

—

—

—

491

476

450

434

477

492

Buprenorphine-AC

3410

—

—

—

—

420

452

421

408

394

509

Butorphanol

2761

2902

—

—

—

272

273

411

254

157

327

Butorphanol-TMS

—

2832

—

—

—

344

345

271

326

399

384

Butorphanol-TMS 2

—

2851

—

—

—

416

417

326

270

456

471

Cetobemidone

2045

—

—

—

—

70

71

190

247

119

57

Cetobemidone-AC

2095

—

—

—

—

70

71

232

289

190

247

Cetobemidone-HFB

1915

—

—

—

—

70

71

69

128

96

115

Cetobemidone-PFP

1865

—

—

—

—

70

57

128

336

393

129

Cetobemidone-TFA

1925

—

—

—

—

70

71

69

286

128

129

Cetobemidone-TMS

2070

—

—

—

—

70

71

262

319

191

304

M (nor-)-AC 2

2545

—

—

—

—

261

58

70

218

160

219

M (methoxy-)-AC

2265

—

—

—

—

70

71

188

319

220

262

2375

2511

2681

2860

1.519

299

162

229

300

124

59

Codeine (also metabolised to morphine, O-desmethylcodeine) Codeine-AC

2503

2645

—

—

1.449

341

282

229

342

204

298

Codeine-HFB

2320

—

—

—

—

282

283

169

225

495

266

Codeine-PFP

2430

—

—

—

—

282

445

446

388

266

283

Codeine-TFA

2280

—

—

—

—

282

395

283

225

266

396

Codeine-TMS

2520

2592

—

—

—

371

178

196

234

343

229

M (nor-)

2388

2535

—

—

—

285

215

148

164

200

242 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:34:22

696

Gas Chromatography

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

GM

M (nor)-AC

2945

—

—

—

—

87

223

224

369

209

195

M (nor)-PFP2

2440

—

—

—

—

563

355

209

400

327

387

M (nor)-TMS2

—

2631

—

—

—

429

254

250

292

284

414

2025

2104

—

—

0.514

194

98

165

167

207

208

M (nor-)

2120

2128

—

—

0.610

167

165

207

85

152

252

M (benzophenone) (BP)

1610

1673

—

—

—

105

77

51

182

106

183

M (carbinol)

1750

—

—

—

—

105

77

139

141

165

218

M (OH-BP) isomer 1

2065

—

—

—

—

198

121

77

199

192

151

M (OH-BP) isomer 2

2080

—

—

—

—

121

198

77

105

199

122

M (OH-methoxy-BP) isomer 1

2050

—

—

—

—

105

151

198

228

77

121

M (OH-methoxy-BP) isomer 2

2070

—

—

—

—

105

151

198

228

77

121

2940

3094

3625

—

—

100

128

265

56

165

266

M (OH)

3095

3310

—

—

—

100

128

281

165

194

322

M (OH)-AC

3210

—

—

—

—

100

128

194

323

325

365

M (methoxy-)

3269

—

—

—

—

100

128

194

323

422

423

2188

2268

2173

2370

1.220

58

91

105

115

59

208

M (nor-)

2214

2487

—

—

1.248

44

220

100

205

129

307

M (nor-amide)

2526

2673

—

—

1.969

234

100

105

220

129

94

M (nor-N-proponyl-)

2400

2514

—

—

1.300

44

220

100

205

129

91

M

—

2520

—

—

1.250

205

220

91

126

115

160

M

—

2526

—

—

1.255

205

220

91

126

115

160

M

—

2624

—

—

—

220

91

147

105

135

115

M (nor-)-AC

2365

—

—

—

—

220

205

86

293

129

191

Art

1621

1659

—

—

—

115

208

91

193

130

117

Art

—

1756

—

—

—

115

91

208

193

117

Art

1890

1957

—

—

—

58

91

191

178

130 ̀ 128

Art

—

1987

—

—

—

44

91

178

191

115

129

Art

—

2021

—

—

—

44

178

91

115

129

191

2230

2323

2230

—

—

257

59

150

256

200

157

Cyclizine

Dextromethorphan (see Methorphan) Dextromoramide

Dextropropoxyphene

Dextrorphan

115

Dextrorphan-AC

2280

—

—

—

—

59

150

299

198

231

256

Dextrorphan-PFP

2060

—

—

—

—

150

403

335

402

119

346

Dextrorphan-TFA

2015

—

—

—

—

150

285

353

352

128

296

Dextrorphan-TMS

2230

—

—

—

—

59

150

329

272

328

314

M (nor-)

2241

2328

—

—

—

243

157

136

198

200

242

M (nor)-AC2

2710

—

—

—

—

87

72

198

211

327

285

M (OH)-AC2

2555

—

—

—

—

357

231

356

355

298

315

2615

2769

—

—

—

327

369

310

268

204

215

M (6-MAM)

2525

2646

—

—

—

327

268

215

328

285

310

M (3-MAM)

2495

2625

—

—

—

327

285

162

215

268

310

M (6-MAM)-PFP

2650

—

—

—

—

414

473

361

204

430

454

M (3-MAM)-PFP

2490

—

—

—

—

268

310

431

473

267

211

M (6-MAM)-HFB

2425

—

—

—

—

464

465

480

677

407

411

M (6-MAM)-TMS

2590

2688

—

—

—

399

340

287

204

282

266

M (3-MAM)-TMS

2570

2668

—

—

—

399

357

234

196

164

329

Diethylthiambutene

2008

—

—

—

—

276

111

219

42

277

97

Dihydrocodeine (metabolised to dihydromorphine, hydrocodone and hydromorphone)

2390

2511

2702

2840

1.493

301

164

59

300

301

115

Diamorphine (heroin; metabolised to MAM, morphine and codeine)

Dihydrocodeine-AC

2445

—

—

—

—

343

300

284

344

226

328

Dihydrocodeine-HFB

2315

—

—

—

—

497

284

498

300

185

169

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:34:48

Specific applications

697

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

Dihydrocodeine-PFP

2360

—

—

—

GM —

447

448

284

300

392

432

Dihydrocodeine-TFA

2265

—

—

—

—

397

284

185

300

340

382

Dihydrocodeine-TMS

2480

2496

—

—

—

373

236

282

315

146

178

M (nor-)

—

2599

—

—

—

287

150

242

213

176

132

M (nor)-AC2

2750

—

—

—

—

243

371

225

224

285

285

M (nor)-TMS2

—

2559

—

—

—

431

316

226

294

416

340

Dihydromorphine

2400

2527

2504

—

—

287

70

164

286

230

288

Dihydromorphine-AC2

2545

—

—

—

—

329

371

286

270

212

310

Dihydromorphine-PFP2

2330

—

—

—

—

119

579

416

432

359

560

Dihydromorphine-TMS2

2520

2518

—

—

—

431

236

146

416

373

326

M (nor)-AC3

2790

—

—

—

—

357

399

229

211

272

315

Diphenoxylate

3514

3670

—

—

Not eluted

246

377

193

165

452

184

Dipipanone

2474

2586

2894

2710

1.309

112

113

91

165

334

223

Diprenorphine

—

3385

—

—

Not eluted

—

—

—

—

—

—

Ethoheptazine

1857

1923

1630

2110

—

—

—

—

—

—

—

Ethylmorphine (metabolised to morphine, see below)

2411

2530

—

—

—

—

—

—

—

—

—

Ethylmorphine-AC

2530

—

—

—

—

355

296

327

234

268

204

Ethylmorphine-PFP

2430

—

—

—

—

296

459

280

266

402

430

Ethylmorphine-TFA

2320

—

—

—

—

296

409

380

280

352

266

Ethylmorphine-TMS

2540

—

—

—

—

385

192

146

196

234

357

2930

—

—

—

—

87

209

237

383

341

181

3033

3211

—

—

Not eluted

44

215

411

324

164

216

—

—

—

—

272

396

250

162

354

483

2720

2833

—

—

—

146

245

189

105

202

158

M (nor-)

—

—

—

—

—

—

—

—

—

—

—

M (despropionyl)

—

—

—

—

—

146

189

44

118

132

280

Hydrocodone (also metabolised to hydromorphone, dihydromorphine and dihydrocodeine)

2440

2580

3028

2930

—

299

242

243

96

185

214

Hydrocodone-TMS

387

M (nor)-AC 2 Etorphine Etorphine-TMS Fentanyl

—

2674

—

—

—

297

386

371

329

298

M (nor)

—

2599

—

—

—

285

242

115

214

128

185

M (nor)-AC

2760

—

—

—

—

87

241

327

212

285

228

Hydromorphone (metabolised to dihydromorphine)

2445

2598

—

—

—

285

96

228

229

286

128

Hydromorphone-AC

2595

—

—

—

—

285

327

228

229

214

242

Hydromorphone-enol-AC2

2625

—

—

—

—

327

284

162

228

369

270

Hydromorphone-HFB

2385

—

—

—

—

481

425

424

410

482

452

Hydromorphone-HFB2

2325

—

—

—

—

481

425

424

410

482

452

Hydromorphone-PFP

2250

—

—

—

—

431

375

374

360

346

402

Hydromorphone-enol-PFP2

2320

—

—

—

—

430

308

414

577

372

520

Hydromorphone-enol-TFA2

2230

—

—

—

—

477

473

380

364

258

458

Hydromorphone-TMS

—

2621

—

—

—

357

300

243

342

314

286

Hydromorphone-enol-TMS2

—

2595

—

—

—

429

414

234

184

357

324

Hydromorphone oxime TMS2

—

2678

—

—

—

355

444

429

356

339

372

Ketamine

1840

1939

—

—

0.427

180

182

209

152

138

102

M (nor-)

1810

1907

—

—

0.423

166

168

195

131

138

223

M (nor-OH)-H2O

1960

2058

—

—

—

166

221

168

193

131

138

M (nor-OH)-NH3

1740

1840

—

—

—

187

222

117

159

131

224 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:34:59

698

Gas Chromatography

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

M (nor-di-OH)-2H2O

1920

2009

—

—

GM —

190

219

192

221

156

184

M (nor)-H2O

—

1931

—

—

—

153

138

221

118

155

192

Ketobemidone

2040

—

—

—

—

70

71

42

44

57

190

Levallorphan (metabolised to nordextrorphan)

2355

2460

—

—

—

283

157

282

176

256

84

Levallorphan-AC

2390

—

—

—

—

85

325

298

176

157

257

Levomethadyl acetate (LAAM)

—

2267

—

—

—

72

43

73

91

255

165

M (nor-)

—

2262

—

—

—

58

101

100

208

281

165

M (di-nor-)

—

2255

—

—

—

44

120

193

165

178

208

1910

—

—

—

—

243

67

95

223

245

97

Levopropoxyphene (see Dextropropoxyphene) Levorphanol (see Dextrorphan) Lofexidine Lofexidine-AC

2200

—

—

—

—

86

139

257

265

223

243

Meptazinol

1920

1980

—

—

0.429

58

84

98

71

85

233

M (nor-)

1995

2069

—

—

0.428

70

84

219

107

159

91

M (oxo-)

2410

2600

—

—

—

148

147

247

204

176

133

Methadone

2145

2228

2470

2370

0.606

72

73

294

57

223

91

Methadone-TMS

2260

—

—

—

—

72

73

296

85

165

178

2040

2120

—

—

0.520

277

276

262

220

165

115

M (dinor-;2-ethylidene-1-methyl-3,3- 2021 diphenylpyrrolidine, EMDP)

2069

—

—

—

262

263

248

221

186

165

M (nor-)

2095

—

—

—

—

58

72

224

165

115

178

M (methadol)

2185

—

—

—

—

72

91

165

105

253

193

M (normethadol)

—

—

—

—

—

58

91

115

165

178

193

2138

2237

—

—

—

59

150

271

270

214

171

2193

2244

—

—

—

257

212

171

136

213

214

M (2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidine, EDDP)

Methorphan (dextromethorphan/ racemethorphan/levomethorphan; also metabolised to dextrorphan see above) M (nor-methorphan/norracemethorphan) M (OH-)

—

2420

—

—

—

287

59

230

150

187

228

M (OH)-AC2

2555

—

—

—

—

357

231

356

355

298

315

2775

—

—

—

—

259

260

203

146

91

110

—

—

—

—

—

58

203

146

110

91

118

2445

2564

2542

—

Not eluted

285

162

215

284

124

268

Methylfentanyl (3- or a-) (china white) M (despropionyl-) Morphine Morphine-AC2 (diamorphine)

2615

2769

—

—

—

327

369

268

310

195

162

Morphine-PFP2

2360

—

—

—

—

414

577

415

578

558

430

Morphine-TMS2

2560

2602

—

—

—

429

236

357

414

401

196

Morphine-TFA2

2250

—

—

—

—

364

477

478

365

380

458

M (nor-)

2459

—

—

—

—

271

150

201

148

162

81

M (nor)-AC3

2955

—

—

—

—

87

209

210

355

397

181

M (nor)-PFP2

2440

—

—

—

—

563

355

387

373

400

544

M (nor)-PFP3

2405

—

—

—

—

355

709

367

382

533

546

M (nor)-TMS3

2605

—

—

—

—

222

416

487

472

192

355

Nalbuphine

2960

Not eluted

—

—

—

302

303

357

284

272

254

Nalbuphene-AC2

3110

—

—

—

—

386

387

441

344

296

326

Nalbuphene-AC3

3080

—

—

—

—

428

429

368

483

326

440

Nalbuphene-AC

3030

—

—

—

—

344

345

399

326

302

—

—

3093

—

—

—

573

518

428

468

574

410

M (N-desalkyl-)

2930

—

—

—

—

289

272

271

202

115

242

M (N-desalkyl)-AC2

2970

—

—

—

—

87

331

227

373

228

313

M (N-desalkyl)-AC3

3020

—

—

—

—

87

373

227

228

296

415

Nalbuphene-TMS3

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:16

Specific applications

699

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

Nalmefene

—

—

—

—

GM —

55

339

110

149

82

298

Nalorphine

2620

Not eluted

—

—

—

311

312

310

188

241

294

Nalorphine-AC2

2820

—

—

—

—

353

395

226

294

354

396

Nalorphine-AC

2800

—

—

—

—

353

294

354

241

310

230

Nalorphine-TFA

2403

—

—

—

—

—

—

—

—

—

—

Nalorphine-TMS2

—

2738

—

—

—

455

414

324

260

438

350

Naloxone

2715

Not eluted

—

—

—

327

328

242

96

286

229

Naloxone-AC

2840

—

—

—

—

327

369

328

286

244

310

Naloxone-AC 2

2750

—

—

—

—

369

411

285

310

326

352

Naloxone-enol-AC2

2810

—

—

—

—

411

369

330

270

228

244

Naloxone-enol-AC3

2770

—

—

—

—

327

369

328

411

286

453

Naloxone-enol-TMS3

2645

2787

—

—

—

438

528

543

355

371

461

Naloxone-enol-TMS2

2700

2843

—

—

—

471

456

366

390

229

398

Naloxone-TMS2

2680

2881

—

—

—

399

471

456

314

358

384

Naloxone-oxime-TMS3

—

2892

—

—

—

558

379

313

453

543

469

Naloxone-PFP

2530

—

—

—

—

473

388

432

375

348

446

Naloxone-enol-PFP2

2360

—

—

—

—

619

472

456

428

620

592

Naloxone-enol-PFP3

2270

—

—

—

—

765

602

618

738

519

454

Naloxone-PFP2

2470

—

—

—

—

82

119

619

472

592

456

M (dihydro)-AC2

2820

—

—

—

—

82

83

413

172

214

371

M (dihydro)-AC3

2855

—

—

—

—

82

413

455

327

254

372

Naltrexol

—

3033

—

—

—

343

55

302

110

98

288

Naltrexone

2880

Not eluted

—

—

—

341

55

300

342

110

243

Naltrexone-AC

2980

—

—

—

—

341

383

342

243

300

286

Naltrexone-AC 2

2870

—

—

—

—

383

425

341

324

340

366

Naltrexone-enol-AC2

3060

—

—

—

—

425

383

384

342

286

382

Naltrexone-enol-AC3

2960

—

—

—

—

425

467

408

382

366

324

Naltrexone-TMS2

—

3051

—

—

—

485

486

470

388

412

444

Naltrexone-TMS3

—

2975

—

—

—

540

555

500

288

272

450

Naltrexone-enol-TMS3

—

2945

—

—

—

557

542

355

242

484

452

Naltrexone-oxime-TMS3

—

3071

—

—

—

572

573

475

327

557

499

M (methoxy-)

2920

—

—

—

—

371

286

274

330

356

316

M (methoxy)-AC

3150

—

—

—

—

413

274

372

371

328

358

M (methoxy)-AC2

3130

—

—

—

—

455

412

396

456

413

273

M (methoxy)-enol-AC2

3300

—

—

—

—

455

414

456

384

400

440

M (methoxy)-enol-AC3

3180

—

—

—

—

497

454

498

396

440

412

413

427

469

384

370

426

58

179

180

225

178

165

—

2990

—

—

—

—

2035

2106

—

—

0.586

M (nor-)

—

2116

—

—

—

—

—

—

—

—

M (nor)-AC

2080

—

—

—

—

208

87

194

179

165

281

M (p-OH-)

—

2266

—

—

—

58

195

165

178

210

241

M (OH)-AC isomer 1

2250

—

—

—

—

195

194

238

165

224

311

M (OH)-AC isomer 2

2285

—

—

—

—

178

195

208

179

268

311

M (nor di-OH-)

—

2649

—

—

—

—

—

—

—

—

—

M (nor di-OH)-AC2 isomer 1

2610

—

—

—

—

87

266

337

295

224

252 178

M (dihydro)-AC3 Nefopam

2640

—

—

—

—

87

337

195

295

209

Neopine

2395

2532

—

—

—

299

162

229

123

59

42

Normethadone

2095

—

—

—

—

58

72

59

224

178

165

M (nor di-OH)-AC2 isomer 2

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:22

700

Gas Chromatography

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

GM

Norpipanone

2488

—

—

—

—

98

111

99

112

165

Noscapine

3145

3358

—

—

—

220

221

205

147

42

118

Oxycodone (metabolised to oxymorphone)

2524

2671

—

—

—

315

230

258

70

201

316 212

178

Oxycodone-AC

2555

—

—

—

—

357

314

298

240

230

Oxycodone-enol-AC2

2560

—

—

—

—

399

357

240

314

296

298

Oxycodone-TFA

2290

—

—

—

—

411

314

240

298

396

254

Oxycodone-TMS

—

2703

—

—

—

387

229

230

388

372

214

Oxycodone-enol-TMS2

—

2602

—

—

—

459

460

444

312

297

242

Oxycodone-oxime-TMS2

—

2740

—

—

—

474

229

214

459

295

385

M (nor-)

—

2703

—

—

—

313

187

314

115

214

229

M (nor)-enol-AC3

2680

—

—

—

—

385

427

343

281

326

368

M (nor)-TMS2

—

2763

—

—

—

373

445

288

258

226

240

M (nor)-enol-TMS2

—

2621

—

—

—

445

446

312

430

354

288

M (nor)-enol-TMS3

—

2746

—

—

—

517

518

502

312

342

428

M (dihydro-)

—

2666

—

—

—

317

230

115

242

260

216

M (dihydro)-AC2

2570

—

—

—

—

359

401

242

230

224

282

M (nor-dihydro)-AC3

2935

—

—

—

—

242

387

224

343

284

429

M (nor-dihydro)-AC2

2900

—

—

—

—

343

258

201

239

242

387

Oxymorphone

2538

2723

—

—

—

301

216

44

42

70

302

Oxymorphone-TMS

—

2715

—

—

—

373

288

259

374

316

358

Oxymorphone-TMS2

—

2728

—

—

—

445

446

430

287

331

372

Oxymorphone-TMS3

—

2641

—

—

—

517

502

355

412

518

503

Oxymorphone-oxime-TMS3

—

2748

—

—

—

532

533

517

287

443

459

M (nor-)

—

Not eluted

—

—

—

—

—

—

—

—

—

M (nor)-enol-TMS2

—

2788

—

—

—

431

259

316

346

432

416

M (nor)-enol-TMS3

—

2662

—

—

—

503

488

355

398

504

308

M (nor)-enol-TMS4

—

2773

—

—

—

575

503

355

560

486

242

M (dihydro-)

—

2690

—

—

—

303

286

115

216

315

256

2825

2973

—

—

—

338

324

339

340

308

325

2805

—

—

—

—

324

310

325

294

266

309

Pentazocine

2280

2356

2225

3030

0.870

70

217

110

69

285

202

Pentazocine-AC

2330

—

—

—

—

259

110

327

312

244

217

Pentazocine-PFP

2120

—

—

—

—

363

348

110

416

431

430

Papaverine M (O-desmethyl-)

Pentazocine-TFA

2075

—

—

—

—

69

313

110

298

366

381

Pentazocine-TMS

2320

—

—

—

—

289

244

245

274

342

357

M (desalkyl-)

—

2019

—

—

—

—

—

—

—

—

—

M (desalkyl)-AC 2

2380

—

—

—

—

87

88

301

172

217

218

202

268

301

110

71

70

172

247

246

218

72

73

2545

2649

—

—

—

1754

2025

1995

1809

0.319

M (nor-)

1885

1842

—

—

0.357

57

233

158

103

131

117

M (nor)-AC

2240

2256

—

—

—

187

57

275

158

232

202

M (OH-)

2045

2145

—

—

—

71

140

263

262

189

234

M (OH)-AC

2205

—

—

—

—

71

305

188

230

261

276

M (nor-OH)-AC2

2600

—

—

—

—

203

56

245

333

218

290

M (OH-) Pethidine (meperidine)

Phenazocine

2686

2833

—

—

—

230

231

58

105

158

173

Phenoperidine

2872

2983

—

—

—

246

247

367

91

158

172

Pholcodine

3070

3348

—

—

—

114

100

42

56

398

115

Pholcodine-AC

3260

—

—

—

—

114

100

56

70

115

440

Pholcodine-PFP

2980

—

—

—

—

114

100

277

354

380

544

Pholcodine-TFA

2800

—

—

—

—

114

100

277

354

380

494

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:25

Specific applications

701

Table 40.20 continued Compound

System

Principal ions (m/z)

GA

GB

GC

GF

GM

Pholcodine-TMS

3140

3410

—

—

—

114

100

115

470

196

356

M (nor)-PFP2

3010

—

—

—

—

100

114

380

70

513

676

M (nor)-TMS2

3260

—

—

—

—

114

100

73

468

528

456

M (nor)-PFP

3270

—

—

—

—

100

114

56

530

502

— 181

M (nor)-AC

3620

—

—

—

—

100

114

70

426

340

M (nor)-AC2

3650

—

—

—

—

100

114

56

468

382

70

Piminodine

2884

—

—

—

—

246

366

106

234

247

260

Pipazethate (pipazetate)

200

2037

—

—

—

—

98

111

99

199

288

M (alcohol)

1830

—

—

—

—

98

112

99

156

103

84

M (ring sulfone)

2720

—

—

—

—

232

200

168

184

156

140

—

—

—

2800

—

—

—

—

—

—

—

Piritramide

3560

—

—

—

—

386

138

387

Profadol

1748

—

—

—

—

—

—

—

—

—

—

2533

2559

—

—

—

341

298

299

242

284

162

M (OH-ring)

84

110

42

Propoxyphene (see Dextropropoxyphene) Racemorphan (see Methorphan) Thebacon (metabolised to dihydrocodeine) Thebacon-TMS

2475

—

—

—

—

371

234

356

184

370

313

Thebaine

2517

2672

—

—

—

311

296

312

297

242

139

Tilidine (tilidate)

1838

—

—

—

—

97

82

103

77

132

176

M (nor-)

1827

—

—

—

—

83

68

259

184

157

214

M (nor)-AC

2165

—

—

—

—

125

83

111

155

170

258

M (bis-nor-)

1830 — —

—

—

—

69

83

119

135

170

245

M (bis-nor)-AC

2100

—

—

—

—

69

111

155

170

244

287

1943

2021

—

—

—

58

263

135

77

264

92

M (nor-)

—

2049

—

—

—

44

188

249

135

150

159

M (nor)-AC

2295

—

—

—

—

86

200

58

273

172

184

M (nor-) carbamate Art

—

2065

—

—

—

189

121

135

202

261

188

M (O-desmethyl-)

1995

2093

—

—

—

58

249

121

93

107

131

M (O-desmethyl)-AC

1998

—

—

—

—

58

121

248

163

291

128

M (N,O-didesmethyl-)

—

2122

—

—

—

44

174

235

121

145

159

M (N,O-didesmethyl)-AC2

2464

—

—

—

—

86

186

301

228

107

113

Tramadol

M (didesmethyl-) carbamate Art

2148

—

—

—

—

73

173

174

188

145

247

M (OH-)

2200

2252

—

—

—

58

279

135

77

234

261

Trimeperidine

1808

1895

—

—

—

186

201

187

70

105

91

derivatives will form in non-reproducible proportions from the automerisation of the enol and keto forms. However, these compounds can be stabilised in their keto forms by incubating with hydroxylamine or methoxyamine–pyridine, and then yield only a single derivatised oxime product (Broussard et al. 1997; Meatherall 1999). Systems GA, GB, GC or GF, described previously may be used. For plasma, system GM, described previously, is a rapid isothermal packedcolumn method with good sensitivity on NPD. Table 40.20 gives retention indices, or relative retention times to iprindole for GF. Non-amfetamine stimulants and hallucinogens Non-amfetamine stimulants and hallucinogens have a variety of clinical and toxic actions. Extraction of cocaine is straightforward under basic conditions, and most metabolites, except benzoylecgonine, can be detected in the clinical setting without derivatisation. For

regulated testing, quantification of benzoylecgonine is required, and most laboratories use TMS as the derivatising reagent. As a result of the recent interest in other metabolites that may have clinical importance, data for these and their TMS derivatives are also included in Table 40.21. For analysis of cannabis metabolites, hydrolysis of conjugates with 10 mol/L potassium hydroxide is usually performed on urine prior to weakly acidic extraction (pH 6.5); TMS is the derivative of choice. Phencylidine (PCP) analysis is complicated by the low concentration present, although extraction is straightforward and derivatisation is required only for metabolite measurement (Nakahara et al. 1997). Chromatographic confirmation of lysergide (LSD) is hampered by the low concentrations and acidic nature of the metabolites, which necessitates both derivatisation (TMS) and tandem MS (Nelson, Foltz 1992). Systems GA or GB may be used. Table 40.21 gives the retention indices (reference compounds are the alkanes with an even number of carbon atoms).

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:40

702

Gas Chromatography

Table 40.21 GC retention data and mass spectral data for non-amfetamine stimulants and hallucinogens (AC, acetyl; PFP, pentafluoropropionate; TFA, trifluoroacetyl; TMS, trimethylsilyl) Compound

System GA

Principal ions (m/z) GB

Amiphenazole

2170

—

191

121

77

104

122

43

Bemegride

1367

—

55

83

82

113

70

69

Bufotenine

2057

—

58

204

146

59

160

42

Caffeine

1800

1904

94

109

55

67

82

195

M (1-nor-, theobromine)

1807

1920

180

55

67

109

82

137

M (7-nor-, theophylline)

1925

1990

180

95

68

53

181

96

Cannabidiol

2390

2480

231

232

245

174

314

187

Cannabidiol-TMS2

2330

2510

390

337

301

351

319

324

Cannabigerol

2500

—

193

123

231

316

247

136

Cannabigerol-TMS2

2440

2520

—

—

—

—

—

—

Cannabinol

2535

2644

295

296

310

238

251

223

Cannabinol-TMS

2485

2600

367

368

382

310

295

238

D9-Tetrahydrocannabinol (D9-THC)

2473

2578

299

314

231

271

243

258

D9-THC-TMS

2405

2499

386

371

315

303

343

330

2710

—

—

—

—

—

—

—

M (8a,11-di-OH-D9-THC)-TMS 9

M (8a,OH-D -THC)

2775

2975

271

295

297

311

312

214

M (8a,OH-D9-THC)-TMS

2580

—

—

—

—

—

—

—

M (11-OH-D9-THC)

2775

2975

299

300

330

217

231

193

M (11-OH-D9-THC)-TMS2

2630

2762

371

372

373

474

459

403

M (11-nor-D9-THC-9-carboxylic acid)

—

—

325

268

326

340

281

253

M (11-nor-D9-THC-9-carboxylic acid)-TMS2

2660

2820

371

473

488

372

398

417

Cinnamoylcocaine isomer 1

2345

2489

82

182

96

131

238

329

Cinnamoylcocaine isomer 2

2450

2625

82

182

96

131

238

329

Cocaine

2187

2289

82

182

94

77

83

303 185

M (ecgonine)

—

0000

82

96

83

97

124

M (ecgonine)-TMS2

1680

—

82

83

96

97

314

329

M (anhydroecgonine methyl ester)methylecgonidine

1280

1430

152

181

82

122

166

138

M (anhydroecgonine methyl ester)-TMS

1345

1472

210

239

224

211

122

183

M (norecgonine)

—

1472

82

156

126

96

171

116

M (methylecgonine)

1472

1530

82

96

83

199

168

182

M (methylecgonine)-TMS

1580

1585

82

96

212

271

182

240

M (ethylecgonine)

—

1602

82

96

83

97

168

213

M (ethylecgonine)-TMS

1485

1651

82

96

83

240

285

196

M (nor-)

2162

2259

168

136

105

77

68

289

M (nor-)-TMS

—

2378

105

240

140

152

179

346

M (benzoylecgonine)

2570

2663

124

168

82

105

94

289

M (benzoylecgonine)-TMS

2285

2365

82

240

105

122

256

361

M (benzoylnorecgonine)-TMS2

—

2400

404

140

298

—

—

—

M (m-OH-benzoylecgonine)-TMS2

2505

2600

82

240

193

210

256

449

M (p-OH-benzoylecgonine)-TMS2

—

2650

82

240

193

210

256

449

M (m-OH-)

2460

2608

82

182

94

121

198

319

M (m-OH-)-TMS

—

2550

182

82

94

193

391

198

M (p-OH-)

—

2650

82

182

94

121

198

319

M (p-OH-)-TMS

—

2610

182

82

94

193

391

198

M (OH-methoxy-)

2670

2729

82

182

151

349

18

168 223

M (OH-methoxy-)-TMS

2850

—

82

182

83

421

198

M (OH-dimethoxy-)-TMS

2970

—

82

182

83

94

451

240

M (cocaethylene)

2250

2345

196

82

317

272

94

105

M (norcocaethylene)

2115

2317

182

68

136

105

108

303

M (norcocaethylene)-TMS

—

2385

254

140

360

—

—

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:46

Specific applications

703

Table 40.21 continued Compound

System

Principal ions (m/z)

GA

GB

M (OH-cocaethylene)

—

2709

82

196

94

121

333

M (OH-methoxy-cocaethylene)

—

2779

82

196

151

212

318

363

M (methoxy-cocaethylene)

—

2663

82

196

121

94

267

333

1230

—

58

126

59

69

56

44

Cyclopentamine

288

Cyclopentamine-AC

1680

—

58

100

168

126

104

183

Ethamivan

1900

—

151

72

223

123

222

152

Ethamivan-AC

1970

—

151

222

223

194

195

265

Harmaline (makes harmine on heating)

2430

—

213

214

170

198

169

115

Harmaline-TFA

2525

—

241

310

121

169

184

198

Harmaline-PFP

2540

—

241

360

242

121

198

184 115

Harmaline-AC

2670

—

213

256

214

170

186

Harmaline-AC2

2800

—

255

298

256

241

212

141

Harmine

2291

2322

212

169

197

213

106

211

Harmine-AC

2545

—

212

254

197

169

213

140

2550

—

198

197

170

99

75

199

M (O-desmethyl-)

2600

—

198

240

169

197

199

115

Isometheptene

1052

—

58

55

128

44

59

56

Isoprenaline

1730

—

72

44

124

123

193

70

Isoprenaline-AC4

2460

—

84

193

235

277

319

365

Lobeline

1820

—

96

105

77

97

216

218

Lysergamide

—

—

267

221

207

180

223

154

Lysergic acid

—

—

268

224

154

180

207

223

Lysergide (lysergic acid diethylamide, LSD)

3445

3332

323

221

207

181

196

280 337

M (O-desmethyl)-AC

3595

—

395

253

293

268

279

M (nor-)

—

—

207

309

182

280

128

100

M (nor-)-TMS1

3705

—

381

279

254

100

265

205

LSD-TMS

M (nor-)-TMS2

3515

—

453

253

351

279

326

337

M (2-oxo-)

—

—

—

—

—

—

—

—

3430

—

309

499

235

325

397

409

Iso-LSD-TMS

3515

—

395

293

279

253

268

337

Mazindol

2325

2504

266

268

267

255

231

102

Mazindol-AC

2705

—

256

255

254

326

220

284

Meclofenoxate

1770

—

58

111

71

75

141

113

M (chlorophenoxyacetic acid)

1770

—

186

141

111

128

113

99

M (N-acetyl)

2160

—

194

179

181

253

151

148

Methoxamine

1726

1596

168

137

44

139

152

124

Myristic acid

1755

Not eluted

73

60

57

129

185

228

Myristic acid-Me

1715

—

74

87

143

129

199

242

Myristic acid-TMS

2280

—

73

117

285

149

101

300

M (2-oxo-3-OH-)-TMS2

Myristicin

1400

—

192

91

119

165

161

147

Naphazoline

2100

—

209

210

141

115

153

208

Nicotine

1350

1380

84

133

42

162

161

105

1715

1645

98

176

118

119

58

147

M (cotinine) Nikethamide

1525

1569

106

78

177

51

178

107

M (N-ethylnicotinamide)

1605

—

106

150

78

149

51

135

M (nicotinamide)

1341

1418

122

78

106

51

50

52

Oxymetazoline

2170

2254

245

260

44

217

218

246

Oxymetazoline-AC2

2760

—

302

344

287

320

203

245

Pemoline

1969

2081

107

176

90

77

70

105

Pemoline-Me2

1590

—

118

204

90

105

77

70 table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:56

704

Gas Chromatography

Table 40.21 continued Compound

System

Principal ions (m/z)

GA

GB

M (mandelic acid)

107

79

77

51

152

90

177

105

77

106

51

107

176

90

77

70

105

69

56

71

91

261

84

M (5-phenyloxazolidine-2,4-dione) Pentetrazole

1550

1579

Phenbutrazate (fenbutrazate)

2675

—

Phencyclidine (PCP)

125

1900

1981

200

242

243

91

84

186

M (4-phenyl-4 piperidinocyclohexanol, PPC)

—

—

216

258

259

91

202

182

M (PPC)-TMS

—

—

200

254

331

—

—

—

M (1(1-phenylcyclohexyl)-4-OH-piperidine)TMS

—

—

172

288

331

—

—

—

M (PCA) 5[N-(1 0 -phenylcyclohexyl)-amino]pentanoic acid

—

—

—

—

—

—

—

—

Pipradrol

2145

2242

Pipradrol-AC

2478

—

Psilocin

1985

Psilocin-AC

2270

Psilocin-AC2 Psilocybin

84

56

85

77

105

55

249

248

165

229

291

206

2080

58

204

150

146

205

155

—

58

246

146

130

202

117

2340

—

58

288

80

202

122

246

2046

—

—

—

—

—

—

—

Theobromine (see Caffeine) Theophylline (see Caffeine)

Oral hypoglycaemics

metabolites are not amenable to GC because of their acidity and polarity. Extractive methylation has been employed, but the use of standard conditions leads to the formation of unstable compounds, and in addition there may be thermal decomposition during chromatography. Thus, the formation of common fragments (superscripts a to f in Table 40.22; HH Maurer, personal communication, 2003) complicates

Oral hypoglycaemics constitute three chemical classes: biguanides (metformin), sulfonylureas (chlorpropamide) and thiazolidenediones (rosiglitazone). The latter two classes contain sulfur, and the sulfonylureas also share structural similarities with the thiazide diuretics (see above) and the sulfonamide antibiotics (see later). Sulfonylureas and their

Table 40.22 GC retention data and mass spectral data for oral hypoglycaemics (Me, methyl) Compound

System

Principal ions (m/z)

GA

GB

Acetohexamide

1859

Not eluted

210

56

43

184

211

Acetohexamide-Me

2250

—

183

198

98

119

115

Buformin-nitrobenzoyltriazine

—

3200

—

—

—

—

—

75 91 —

Carbutamide-Me

2300

—

109

156

92

285

—

—

Chlorpropamide

1791

1887

111

175

75

85

276

127

Chlorpropamide-Me

2165

—

58

115

111

175

290

127

Chlorpropamide-Me2

2250

—

109

156

92

304

—

—

Art 1-Me

1825

—

111

175

205

75

113

141

Art 1-Me2

1690

—

111

219

75

175

113

221

Art (chlorosulfonamide)-Me

1740

—

111

75

141

175

205

—

Art (chlorosulfonamide)-Me2

1655

—

111

75

219

175

155

—

Art (chloroamide)-Me

2135

—

111

75

125

248

175

—

Art (chloroamide)-Me2

2150

—

87

111

125

175

262

—

Glibenclamide-Me

3800

—

169

82

97

171

198

381

Glibenclamide-Me2

3840

—

169

289

171

291

353

126

Art 3-Me

3445

—

169

198

126

287

382

—

Art 3-Me2

3355

—

169

289

198

353

396

—

1730

1660

91

65

171

155

107

—

Art(e) (methylsulfonamide)-Me

1740

—

91

65

185

155

121

108

Art(f) (amide)

1620

1695

91

155

197

65

106

—

Glibornuride Art(d) (methylsulfonamide)

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:35:59

Specific applications

705

Table 40.22 continued Compound

System

Principal ions (m/z)

GA

GB

Art 5

1845

1995

95

109

134

164

195

—

Art 5-Me

1715

—

95

209

109

139

150

—

M (OH)-Art

2305

—

95

109

125

181

211

—

M (OH)-Art (sulfonamide)-Me(d)

2265

—

107

89

201

172

141

—

M (OH)-Art (sulfonamide)-Me2(e)

2030

—

107

89

215

171

151

—

M (COOH)-Art(sulfonamide)-Me3(f)

1955

—

135

243

103

199

212

—

1545

—

110

181

125

184

151

—

1620

—

91

155

197

65

106

— —

Gliclazide Art 1-Me Art(c) (amide) Art 3

1670

—

81

110

125

67

169

Art(a) (methylsulfonamide)

1730

1660

91

65

171

155

107

—

Art(b) (methylsulfonamide)-Me

1740

—

91

65

185

155

121

—

M (OH)-Art (sulfonamide)-Me(d)

2265

—

107

89

201

172

141

—

M (OH)-Art (sulfonamide)-Me2(e)

2030

—

107

89

215

171

151

—

1955

—

135

243

103

199

212

—

Glipizide-Me

3420

—

150

111

93

459

98

—

Glipizide-Me2

3455

—

150

121

93

334

392

Art 2-Me

3020

—

150

121

93

197

334

—

Art 2-Me2

3005

—

150

241

121

93

348

—

M (COOH)-Art (sulfonamide)-Me3

(f)

197

Gliquidone

2024

Not eluted

—

—

—

—

—

—

Gliquidone-Me

3850

—

323

220

204

175

176

430

Art 4-Me

3460

—

204

219

176

321

416

—

Art 4-Me2

3415

—

204

219

176

321

416

—

Glymidine

1632

2750

244

59

77

43

245

168

Glymidine-Me

—

—

—

—

—

—

—

—

Metformin-nitrobenzoyltriazine

—

3050

—

—

—

—

—

—

Tolazamide

1651

1720

91

155

114

65

197

Tolazamide-Me

2630

—

113

155

170

241

325

42 —

2540

—

91

155

339

184

229

114

Art 1-Me

1315

—

98

113

59

68

85

172

Art (methylsulfonamide)

1730

1660

91

65

171

155

107

Tolazamide-Me2

—

Art (methylsulfonamide)-Me

1740

—

91

65

185

155

121

—

M (OH)-Art (sulfonamide)-Me(d)

2265

—

107

89

201

172

141

—

M (OH)-Art (sulfonamide)-Me2(e)

2030

—

107

89

215

171

151

—

M (COOH)-Art(sulfonamide)-Me3(f)

1955

—

135

243

103

199

212

—

1683

—

91

30

155

108

65

Art(c) (amide)

1620

—

91

155

197

65

106

Tolbutamide-Me

2320

—

91

129

155

284

269

87

Tolbutamide-Me2

2170

—

91

155

113

121

220

184

Art (methylsulfonamide)

1730

1660

91

65

171

155

107

—

Art (methylsulfonamide)-Me

1740

—

91

65

185

155

121

—

M (OH)-Me

2645

—

129

171

300

285

200

—

M (OH)-Me2

2740

—

134

215

107

197

314

—

M (COOH)-Me3

2590

—

129

135

199

297

328

—

M (OH)-Art (sulfonamide)-Me(b)

2265

—

107

89

201

172

141

—

M (OH)-Art (sulfonamide)-Me2(a)

2030

—

107

89

215

171

151

—

M (COOH)-Art(sulfonamide)-Me3(c)

1955

—

135

243

103

199

212

—

Tolbutamide

(a)–(f)

Hydrolysis artefacts common to several sulfonylureas.

197 —

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:37:0

706

Gas Chromatography

identification of the parent compound and limits the application as a screening tool. For this purpose, HPLC may be more applicable (Maurer et al. 2002). However, stable N-methyl (sulfonamide nitrogen) derivatives can be formed by maintaining the extraction pH below 7, and by using TBAH as the ion-pairing reagent. With the alkylation pH above 10, and using TPAH as the counter-ion, there is almost complete hydrolysis to sulfonamide and amide artefacts (Hartvig et al. 1980). Thermal stability can also be achieved by the judicious choice of pairs of derivatising reagents: methyl iodide plus trifluoroacetic anhydride (TFAA) for parent compounds, and methyliodide plus heptafluorobutyric anhydride (HFBA) for the hydroxy and carboxy metabolites (Braselton et al. 1977). This strategy offers the additional advantage of an improved ECD response. The biguanides also present analytical difficulties, although successful chromatography is achieved by forming a triazine derivative by them reacting with p-nitrobenzoyl chloride, for which the retention data are given in Table 40.22 (Brohon, N€ oel 1978; Paroni et al. 2000). Screening methods are not yet developed for the newer classes of drugs such as the alpha-glucosidase inhibitors, glinides, glitazones and gliptins, although LC-MS methods have been published for some of the individual compounds. Systems GA and GB may be used, and the reference compounds are n-alkanes with an even number of carbon atoms. Pesticides A comprehensive method for screening pesticides using systems GA and GK can be found in Chapter 16, Table 16.1. Systems GKA, GKB, GKC and GKD cater for a smaller set of compounds and these can be found in the Indexes of Analytical Data. System GKA

(Osselton, Snelling 1986). n n n n

Column: Chromosorb W HP 3% SE-30 on 80–100 mesh silanised glass (2 m  4 mm i.d.). Carrier gas: O2-free N2, 50 mL/min. Detector: Flame ionisation and nitrogen–phosphorus. Reference compound: Straight-chain hydrocarbons

System GKB

(Osselton, Snelling 1986). n n n

Column: Chromosorb W HP 3% OV-7 on 80–100 mesh silanised glass (2 m  4 mm i.d.). Carrier gas: O2-free N2, 50 mL/min. Detector: Flame ionisation and nitrogen–phosphorus.

n

Reference compound: Straight-chain hydrocarbons.

System GKC

(Osselton, Snelling 1986). n n n n

Column: Chromosorb W HP 3% OV-17 on 80–100 mesh silanised glass (2 m  4 mm i.d.). Carrier gas: O2-free N2, 50 mL/min. Detector: Flame ionisation and nitrogen–phosphorus. Reference compound: Straight-chain hydrocarbons.

System GKD

(Junting, Chuichang 1991). n n n n

Column: Fused silica HP-1, methyl silica gum (5 m  0.53 mm i.d., 2.65 mm). Temperature programme: 190
C to 235
C at 10
/min. Carrier gas: N2, 20 mL/min flow rate. Detector: Flame ionisation.

Phenothiazines and other tranquillisers Phenothiazines and other tranquillisers can be extracted readily under mildly basic conditions (pH 10) into solvents such as ethyl acetate, hexane, butyl chloride and diethyl ether. An acidified (0.05 mol/L H2SO4) back extraction is a useful clean-up procedure where sensitivity is important. Chromatography of the primary and secondary amines is poor on packed columns, but is adequate on well-maintained capillary columns, particularly those of low-to-medium polarity, such as PSX-5 (see Table 40.3). Meprobamate is unstable in basic solution, and benefits from the use of mildly acidic (pH 5) extraction conditions. Some authors prefer to chromatograph the secondary amines and hydroxylated metabolites as acetylated derivatives, prepared by heating the dried residue with acetic anhydride and pyridine (3 : 2, v/v) (Maurer, Bickeboeller-Friedrich 2000). Others employ an enzymatic hydrolysis procedure to improve the recovery of both parent drug and metabolites, although the additional sensitivity gained is often negated by the increased analytical time in the emergency setting. Acid hydrolysis is quicker, but some relevant compounds are destroyed under these conditions. System GA or GB may be used (Table 40.23), and the reference compounds are n-alkanes with an even number of carbon atoms. Laxatives Table 40.24 lists the stimulant laxatives. Other types of laxatives, such as bulkers (bran), osmotic (PEG and lactulose), stool softeners and saline

Table 40.23 GC retention data and mass spectral data for phenothiazines and other tranquillisers Compound

System

Principal ions (m/z)

GA

GB

Acepromazine

2735

2844

100

72

240

340

44

197

M (dihydro)-H2O

2720

2824

58

86

310

225

224

251

M (7-OH-)

—

3160

58

86

342

326

296

257

2650

—

72

73

255

326

56

240

2920

—

72

56

84

238

270

340

Acetophenazine

—

Not eluted

254

143

42

70

411

113

Alimemazine (trimeprazine)

Aceprometazine M (methoxy-dihydro)-H2O

2305

2402

58

298

212

198

100

299

M (nor-)

2335

2432

199

284

212

198

180

252

M (sulfoxide)

2665

2805

M (norsulfoxide)

—

2817

58 —

199

180

298

297

—

—

—

—

M (OH-)

2650

2829

314

100

255

196

281

M (nor-OH-)

—

2845

—

—

—

—

—

—

—

—

—

—

—

Alpidem

—

3313

Amisulpride

3260

—

M (O-desmethyl-) Apronal

58

212 —

98

99

44

242

—

70

111

2960

—

98

135

182

228

99

107

1331

—

55

44

142

141

61

81

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:37:21

Specific applications

707

Table 40.23 continued Compound

System GA

Principal ions (m/z) GB

Aripiprazole M

—

2108

174

176

218

220

75

44

M

—

2258

213

215

242

244

98

172

Azacyclonol

2243

2361

Azaperone

2705

—

Benactyzine

2255

—

Benperidol

3433

3667

M

1490

M (N-desalkyl-)

2415

Benzoctamine Bromisoval

85

84

183

105

56

107

165

123

95

121

77 —

86

105

77

87

182

99

230

109

82

187

243

363

—

56

125

123

180

136

95

—

134

79

51

106

217

161

2078

2172

218

44

191

221

219

178

1540

—

55

70

163

165

83

222

M (Br-isovaleric acid)

1190

—

136

140

101

59

120

122

M (OH-isovaleric acid)

1140

—

76

73

55

58

57

74

M (iso-valeric acid carbide)

1850

—

102

59

85

57

70

61

Bromperidol

3037

—

42

268

270

281

283

123

Art (-H2O)

3020

—

236

238

252

250

253

265

M

1890

—

233

235

56

94

127

154

M (N-desalkyl-oxo)-2H2O Buspirone

1850

—

233

235

127

154

63

101

3300

3468

177

277

265

122

148

108

—

1558

122

108

96

80

164

134

Butaperazine

3190

—

113

70

409

141

283

127

Captodiame

2774

—

58

165

255

359

166

73

M (1-pyrimidinyl piperazine)

Carbromal M (OH-carbromide)

1513

—

44

69

208

210

55

71

1340

—

150

152

165

167

183

194

M (carbromide)

1215

—

69

165

167

114

71

150

M (desbromo-)

1380

—

87

113

71

130

86

115

Art

1450

—

69

58

70

105

179

97

Art

1470

—

69

70

140

151

193

191

Carphenazine

3590

—

268

143

245

70

269

394

Chlormezanone

2199

2346

98

152

154

42

69

174

Art

1235

1245

152

153

154

89

111

59

M (4-chlorobenzoic acid)

1400

—

139

156

111

75

141

113

M (N-methyl-4-chlorobenzamide)

1555

1596

139

111

75

169

141

168

2495

2618

58

86

318

85

320

272

M (nor-)

2480

2656

44

232

233

196

214

304

M (didesmethyl-)

2480

2646

232

290

233

246

272

214

M (sulfoxide)

2809

3003

58

246

214

232

272

318

M (norsulfoxide)

2900

3046

44

246

232

302

214

196

M (7-OH-)

—

2939

58

86

334

248

288

262

M (N-oxide)

2100

2355

233

198

201

154

166

171

Chlorprothixene

2492

2608

58

59

221

42

222

255

2750

—

58

333

335

247

334

215

Chlorpromazine

M (OH-dihydro-) isomer 1 M (OH-dihydro-) isomer 2

2790

—

58

333

247

335

249

334

M (OH-methoxy-dihydro-)

2810

—

58

363

277

173

262

249

M (N-oxide)-(CH 3)2NOH

2410

—

234

235

270

269

202

255

M (N-oxide sulfoxide)- (CH3)2NOH

2560

—

203

234

202

251

286

269

M (sulfoxide)

2720

—

58

221

189

255

176

331

Art (dihydro-)

2490

—

58

317

231

73

152

195

Art (Cl-thioxanthenone)

2260

—

246

218

248

139

220

183

—

1269

112

161

85

45

163

113

Clomethiazole (chlormethiazole) Clopenthixol (see Zuclopenthixol)

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:37:37

708

Gas Chromatography

Table 40.23 continued Compound

System

Principal ions (m/z)

GA

GB

2712

2833

—

2882

—

Cloral betaine (see Chapter 14) Cloral hydrate (see Chapter 14) Clothiapine (clotiapine) M (nor-)

83

70 —

273

244

209

—

—

—

71 —

3030

—

357

209

244

285

273

291

2895

3024

—

—

—

—

—

—

M (nor-)

3105

3092

192

243

256

56

227

312

M (nor-acetyl-)

3490

3609

396

310

298

192

227

256

M

—

2833

58

300

256

243

299

160

M (oxo-) Clozapine

M

—

2972

243

286

44

256

244

270

M

—

3150

225

238

294

209

250

264

M

—

3264

255

268

192

239

338

280

M

—

3320

255

192

268

324

239

280 256

—

3527

340

192

339

228

243

Dichloralphenazone

1855

—

188

47

82

96

77

84

M (phenazone)

1835

1951

188

96

77

56

105

189

M (4-OH-phenazone; metabolised to chloral hydrate)

1855

—

85

56

84

204

77

120

Diethazine

2377

—

86

298

87

58

299

212

Dimetotiazine

3060

3096

72

73

320

56

210

198

M (nor-)

3150

—

72

58

320

306

198

210

M

Dixyrazine M (phenothiazine) Droperidol

3220

—

212

42

187

70

180

56

2120

2130

199

167

198

166

154

139 247

3430

Not eluted

246

165

42

123

199

1950

—

134

79

106

121

105

67

Emylcamate

1105

—

73

43

84

55

69

44

Ethchlorvynol

1015

1060

115

117

89

53

109

51

Ethinamate

1365

—

91

81

106

78

95

68

M (benzimidazolone)

Ethomoxane

1975

—

86

44

265

180

87

Fluanisone

2785

—

205

218

123

356

219

162

2715

—

194

165

123

342

338

134

M (O-desmethyl-)

—

Fluopromazine (see Triflupromazine) Flupentixol (flupenthixol) cis isomer

3058

3199

143

70

100

144

98

58

Flupentixol trans isomer

—

3217

143

70

100

144

98

58

M (ring)

2190

—

267

235

247

222

198

216

M (N-oxide)

2120

—

304

303

234

235

289

283

M (desalkyl-) cis

—

2832

—

—

—

—

—

—

M (desalkyl-) trans

—

2855

—

—

—

—

—

—

3050

3194

280

143

437

406

113

Fluphenazine

70

M (ring)

2190

—

280

143

113

248

M (7-OH-)

—

3572

—

—

—

—

—

—

—

—

—

M (sulfoxide) Fluspirilene M (N-desalkyl-oxo-)

70

56

—

3752

—

—

—

1017

—

244

42

72

475

109

245

2405

—

57

56

245

68

228

206

M (desamino-OH-)

2120

—

203

201

262

183

216

244

M (desamino-carboxy-)

2230

—

203

201

183

216

276

167

Glutethimide

1830

1910

189

132

117

160

91

115

M (OH-ethyl-)

1865

1958

146

104

233

103

133

117

M (OH-phenyl-)

1875

2040

133

204

233

176

77

205

M (2-phenylglutarimide)

2235

—

104

189

103

117

78

M (desethylphenylgutarimide)

2370

—

—

—

—

—

—

91 —

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:37:57

Specific applications

709

Table 40.23 continued Compound

System

Principal ions (m/z)

GA

GB

2930

3094

224

42

237

226

123

M (reduced)

3152

3152

224

206

226

193

377

139

M (N-desalkyl-oxo)-2H2O

1650

1707

189

154

127

191

126

190

M

1750

1872

56

139

84

223

206

111

Haloperidol

206

M (N-desalkyl)

1800

—

56

84

139

111

133

211

Hydroxyphenamate

1724

—

135

57

91

77

119

105

Hydroxyzine

166

2849

3000

201

203

165

45

299

M (norchlorcyclizine)

—

2355

165

201

166

85

241

230

M (4-chlorobenzophenone)

1850

1862

105

139

216

77

111

218

M (4-chloromethylbiphenyl)

1600

1688

167

165

202

152

82

204

M (OH-chlorobenzophenone)

2300

2230

121

139

95

234

111

152

M

—

2704

201

165

166

228

242

299

M

—

2847

85

165

166

201

242

256

Levomepromazine (methotrimeprazine) M (norsulfoxide)

2514

2641

328

100

228

185

329

—

3088

—

58

—

—

—

—

—

—

—

—

—

—

3114

—

—

2555

2717

70

83

257

193

56

M (8-OH-)

2931

3077

70

83

273

209

260

343

M (7-OH-)

—

3068

70

273

260

209

244

343

M (amoxapine)

2638

2746

245

193

257

247

228

164

M (7-OH-amoxapine)

2951

3525

261

209

273

263

244

329

M (8-OH-amoxapine)

2959

3546

261

209

273

263

244

329

M (sulfoxide) Loxapine

228

Mebutamate

1889

—

97

55

69

72

71

98

Mecloqualone

2255

—

235

111

75

76

236

50

1854

83

84

55

56

43

71

Meprobamate (also carisoprodol metabolite) 1785 Art

1535

1487

84

55

56

44

83

75

M

1720

1763

104

43

45

62

148

86

M

—

1932

104

43

45

71

119

204

M

—

2079

43

111

104

132

172

62

Mesoridazine

3380

3629

98

70

99

386

126

55

Methaqualone

2135

2256

235

250

91

233

236

65

M (2-formyl-)

2240

2370

235

132

264

206

248

192

M (2-OH-methyl-)

2360

2437

235

266

251

175

132

160

M (2-carboxy-)

2400

—

235

146

77

221

252

280

M (2 0 -OH-methyl-)

2410

2500

160

266

235

251

77

247

M (3 0 -OH-)

2490

—

251

266

249

77

148

252

M (4 0 -OH-)

2510

—

251

266

249

77

143

235

M (6-OH-)

2525

—

251

266

249

132

65

92

M (OH-methoxy-)

2560

2698

296

281

143

249

279

266

Methdilazine

2462

—

97

98

296

199

212

198

Methyprylon

1527

1581

155

140

83

98

55

41

M (OH)-H2O

1540

1601

83

55

153

166

84

98

M (oxo-)

1870

1834

83

98

55

168

151

182

Molindone

2465

—

100

56

176

98

120

70

Olanzapine

—

2861

242

229

231

198

312

169

—

2911

229

213

242

298

198

254

Oxypertine

2355

—

175

70

176

132

379

204

Pecazine

2540

2669

310

58

199

112

111

212

Penfluridol

3360

—

42

292

56

294

109

203

M (nor-)

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:2

710

Gas Chromatography

Table 40.23 continued Compound

System

Principal ions (m/z)

GA

GB

M (N-desalkyl-oxo)-2H2O

1920

—

257

259

258

222

167

M (N-desalkyl-)

2210

—

56

261

279

179

260

114

M (desamino-OH-)

2120

—

—

—

—

—

—

—

M (desamino-carboxy-)

2230

—

—

—

—

—

—

—

202

2798

—

113

339

141

340

M (OH-)

3175

—

—

—

—

—

—

—

M (phenothiazine)

Perazine

44

70

2120

2130

199

167

198

166

154

139

Pericyazine

3260

3486

114

44

142

365

223

115

M (ring)

2555

—

224

192

223

120

112

179

Perphenazine

3380

3594

246

143

403

70

404

248

2100

—

—

—

—

—

—

—

Phenothiazine

2120

2130

199

167

198

166

154

139

Phenprobamate

1520

—

118

117

91

92

119

65

Pimozide

42

217

83

461

M (ring)

3870

Not eluted

230

187

M (N-desalkyl-)

2415

—

—

—

—

—

—

—

M (benzimidazolone)

1950

—

—

—

—

—

—

—

M (desamino-OH-)

2120

—

—

—

—

—

—

—

M (desamino-carboxy-)

2130

—

—

—

—

—

—

—

Pipamperone

3040

—

165

138

331

123

110

194

M (OH-)

3250

—

165

154

347

123

292

194

Piperacetazine

0000

—

142

170

44

410

143

42

Pipotiazine (pipothiazine)

2932

Not eluted

142

44

140

198

170

96

Prochlorperazine

2954

3129

113

70

373

141

43

72

M (N-oxide)

2100

2356

233

198

235

218

272

201

M (norsulfoxide)

—

3571

70

113

373

246

319

232

M (sulfoxide)

—

3758

70

113

246

319

373

232

Promazine

2315

2425

58

284

86

238

198

199

M (nor-)

2405

2452

199

270

198

213

238

212

M (sulfoxide)

2705

2840

212

199

300

284

180

M (norsulfoxide)

—

2875

—

—

—

—

—

—

M (phenothiazine)

2120

2130

199

167

198

166

154

139

M (OH-)

2685

2781

58

86

300

215

254

228

M (nor-OH-)

—

2797

—

—

—

—

—

Promethazine

2339

2383

72

284

198

213

199

180

M (nor-)

2250

2333

58

213

180

198

152

270

M (phenothiazine)

2120

2130

199

167

198

166

154

139

M (sulfoxide)

2710

2797

72

198

180

152

213

229

M (norsulfoxide)

—

2732

58

212

180

198

229

152

M (nor-OH-)

2580

2717

212

58

180

229

198

286 288

58

—

M (OH-)

2590

—

72

196

300

229

214

Propiomazine

2738

—

72

340

269

197

73

71

Prothipendyl

2345

—

58

285

214

200

86

227

M (OH-)

2720

—

58

301

86

216

230

243

M (OH-ring)

2800

—

216

187

168

188

200

161

M (ring)

2045

—

200

168

199

156

201

155

M (sulfoxide)

2750

—

58

86

216

179

155

200

Quetiapine M M Remoxipride M

—

3400

210

239

144

251

321

226

—

2745

227

210

239

251

265

295 233

—

2709

195

207

178

151

219

2520

2588

98

99

70

228

230

243

—

2981

—

—

—

—

—

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:4

Specific applications

711

Table 40.23 continued Compound

System GA

Principal ions (m/z) GB

M

—

3022

—

—

—

—

—

—

M

—

3313

—

—

—

—

—

—

Risperidone Art Sulforidazine (also thioridazine and mesoridazine metabolite) M (ring) Sulpiride Art (-SO2NH) Tetrabenazine M (O-desmethyl-OH-)

—

1877

220

191

204

178

192

221

2063

—

—

—

—

—

—

—

3415

3690

197

198

290

98

402

70

3180

—

277

198

154

127

278

263

3102

Not eluted

—

—

—

—

—

—

2295

—

98

70

77

135

99

111

2490

2579

191

261

260

274

316

176

2500

2638

205

191

274

318

319

232

Thalidomide

2440

Not eluted

173

104

76

111

148

170

Thiethylperazine

3226

—

70

113

141

399

72

259

M (ring)

2750

—

259

230

198

186

260

167

M (sulfone)

3400

—

70

113

127

305

431

212

Thiopropazate (metabolised to perphenazine) 3467

—

246

70

185

98

87

213

Thioproperazine

—

70

113

127

212

320

141 154

3552

M (ring)

3200

—

306

198

199

197

277

Thioridazine

3115

3292

98

370

126

99

371

250

—

3275

84

56

112

356

245

185

M (nor-) M (sulfoxide) mesoridazine

3380

3629

98

70

99

386

126

55

M (ring)

2570

2639

245

198

186

230

154

166

384

385

244

112

258

245

98

370

402

244

258

290

M (oxo-)

3500

—

M (ring sulfone)

3420

3626

M (side chain sulfone)

3800

Not eluted

416

112

290

277

276

417

3060

—

—

—

—

—

—

—

Thiothixene Trichloroethanol (see Chapter 14) Triclofos (metabolised to trichloroethanol)

—

1952

31

49

77

113

51

Trifluomeprazine

2250

—

58

366

100

266

248

84

Trifluoperazine

2683

2798

113

70

407

43

141

127

M (phenothiazine)

2120

2130

199

167

198

166

154

139

M (sulfoxide)

2990

3145

113

141

248

266

306

280

M (norsulfoxide)

—

3191

—

—

—

—

—

—

115

2675

—

42

271

258

123

83

240

M (N-desalkyl-oxo-)-2H 2O

1570

—

223

224

154

170

183

204

M (N-desalkyl-)

1970

—

56

227

226

245

223

198

2230

2318

58

352

86

353

306

266

M (phenothiazine)

2120

2130

199

167

198

166

154

139

M (OH-)

2700

—

58

368

86

322

282

323 265

Trifluperidol

Triflupromazine

2730

—

58

398

86

312

313

Trimetozine

2253

—

195

281

196

152

280

81

Tybamate

1725

—

55

72

97

158

118

56

M (OH-methoxy-)

Zolpidem

2715

2941

235

236

307

219

92

65

Zopiclone

2950

3263

143

245

112

99

139

217

1200

1261

128

101

130

73

98

93

Zotepine

Art (amino-chloropyridine)

2660

—

208

199

221

163

231

147

Zuclopenthixol (clopenthixol) cis isomer

3360

3557

143

70

144

100

42

56

Zuclopenthixol trans isomer

3400

3680

143

70

144

100

42

56

M (N-oxide)-C6H14N2O2

2410

—

—

—

—

—

—

—

Art (Cl-thioxanthenone)

2260

—

—

—

—

—

—

—

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:12

712

Gas Chromatography

Table 40.24 GC retention data and mass spectral data for laxatives (AC, acetyl; Me, methyl; TMS, trimethylsilyl) Compound

System GA

Principal ions (m/z) GB

Aloe-emodin

2660

—

—

—

—

—

—

—

Aloe-emodin-AC

2735

—

270

241

312

271

242

225

Aloe-emodin-AC2

3000

—

270

354

312

241

271

224

Aloe-emodin-Me

2900

—

284

266

238

225

209

237

Aloe-emodin-Me2

2705

—

298

267

239

299

240

291

Aloe-emodin-TMS

2685

—

311

312

342

225

296

268

Aloe-emodin-TMS2

2785

—

399

400

184

310

383

325

Aloe-emodin-TMS3

2900

—

471

472

472

399

367

281

Aloin

0000

0000

—

—

—

—

—

—

Arecoline

1195

—

155

96

140

43

81

94

Bisacodyl

2818

2956

361

277

319

276

199

318

M (bismethoxybisdesacetyl)

2820

—

337

322

336

338

259

307

M (bismethoxydesacetyl-)

2890

—

379

322

364

336

378

380

M (desacetyl-)

2750

2876

319

276

277

199

318

246

M (bisdesacetyl-)

2655

2793

277

276

199

183

278

246

M (methoxybisdesacetyl-)

2680

—

307

306

229

292

275

198

M (methoxydesacetyl-)

2810

—

349

306

307

229

292

348

M (bismethoxybisdesacetyl)-AC2

2950

—

379

421

364

322

336

378

M (methoxybisdesacetyl)-AC2

2870

—

349

391

307

306

229

348

M (trimethoxybisdesacetyl)-AC2

3060

—

409

367

451

329

352

203

M (desacetyl)-TMS

—

2830

391

348

349

271

390

392

M (bisdesacetyl)-TMS2

—

2728

421

343

420

422

256

240

Dantron

2330

2450

240

212

241

184

138

92

Dantron-Me

2435

—

254

208

236

225

139

168

Dantron-Me2

2475

—

253

268

139

152

209

180

Dantron-AC

2460

—

240

282

241

212

184

155

Dantron-AC2

2595

—

240

282

241

212

184

155

Dantron-TMS

2465

2574

297

298

253

240

210

312

Dantron-TMS2

2530

2611

369

370

297

371

268

210

Emetine

2505

Not eluted

192

206

272

480

288

246

Emetine-Me

4010

—

206

207

190

272

288

494

Frangula-emodin

2620

—

—

—

—

—

—

—

Frangula-emodin-AC

2740

—

270

312

271

242

213

241

Frangula-emodin-Me2

2775

—

298

252

280

269

237

281

Frangula-emodin-Me3

2845

—

297

312

295

283

266

251

Phenolphthalein

—

3292

274

225

318

273

275

226

Phenolphthalein-AC2

3375

3351

360

318

274

225

257

402

Phenolphthalein-Me2

3060

—

271

302

346

301

239

287

Phenolphthalein-TMS2

—

3205

418

417

419

297

253

329

3395

—

390

273

272

348

304

391

Physcion

2660

2732

284

285

255

128

283

241

Physcion-AC2

2920

—

284

326

285

255

227

184

M (methoxy)-AC2

Physcion-Me2

2845

—

—

—

—

—

—

—

Physcion-Me

2775

—

—

—

—

—

—

—

Physcion-TMS

2150

2247

341

117

129

132

145

356

Picosulfate (hydrolysed to bisacodyl metabolites, see above) Rhein

2675

—

284

285

255

128

241

139

Rhein-Me

2660

—

298

267

239

155

126

284

Rhein-Me2

2740

—

312

266

294

251

235

126

Rhein-Me3

2855

—

311

326

312

309

235

295

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:18

Specific applications

713

Table 40.25 GC retention data for steroids (AC, acetyl; TMS, trimethylsilyl) GA

GAG

GAI

GAR

—

—

—

—

dihydrotestosterone

2510

—

—

—

Androstanolone dihydrotestosterone-AC

2630

—

—

—

dihydrotestosterone-TMS

2485

—

—

—

dihydrotestosterone enol-TMS2

2450

—

—

—

5a-dihydrotestosterone

—

—

0.95

—

2475

—

—

11.9

Androsterone -AC

2580

—

—

—

enol-TMS2

2500

—

—

— 12.8

—

1.05

0.961

undecylenate

—

2.62

—

22.4

5b-androst-1-en-17b-ol-3-one

—

—

0.96

—

acetate

—

—

—

13.6

benzoate

—

—

—

18.7

undecylenate

—

—

—

22.4

2530

—

—

11.8

DHEA-H2O

2595

—

—

—

DHEA enol-TMS2

2580

—

—

—

2985

—

0.974

—

drostanolone

2555

—

—

—

drostanolone-AC

2700

—

—

—

drostanolone-TMS

2575

—

—

—

drostanolone-enol-TMS2

2625

—

—

—

drostanolone propionate

2985

—

0.974

—

2835

1.5

1.155

14.6

2672

1.2

—

13.2

—

—

0.925

—

Boldenone

DHEA

Drostanolone propionate

Fluoxymesterone Methandienone 17a-methyl-5b 17a-androstan-3a,17b-diol

—

—

0.925

—

17a-methyl-5b-androst-1en-3a,17b-diol

—

—

0.921

—

17a-methyl-1,4-androstadien-6b,17b-diol-3-one

—

—

1.117

—

—

0.89

—

12.3

dipropionate

—

1.70

—

—

17a-methyl-5b-androstan-3a,17b-diol

—

—

0.925

—

Methandriol

2645

1.05

—

13.1

-AC

2770

—

—

—

-TMS

2590

—

—

—

enol-TMS2

2665

—

—

—

17a-methyl-5b

—

—

0.925

—

2395

0.91

—

12.5

2760

—

—

—

—

1.17

1.111

13.7

Methyltestosterone

Nandrolone -TMS Oxandrolone Oxymetholone

3005

1.28

—

13.7

enol-TMS3

2870

—

—

—

17a-methyl-5a-androstan-3a,17b-diol

—

—

0.925

—

2-hydroxymethyl-17a-methyl5-androstan-3,17-diol

—

—

1.106

—

2-hydroxymethyl-17a-methyl5-androstan-3,6,17-triol

—

—

1.180

—

Stanozolol

3085

—

1.31

15.4

-TMS2

3025

—

—

—

-AC

2120

—

—

—

3 0 -hydroxystanozolol

—

—

1.38

—

4b-hydroxystanozolol

—

—

1.393

— table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:18

714

Gas Chromatography

Table 40.25 continued GA

GAG

GAI

GAR

2620

—

0.97

12.9

-AC

2750

—

—

—

enol-TMS2

2690

—

—

—

propionate

2815

1.43

—

14.2

dipropionate

3350

—

—

—

methyltestosterone

—

1.05

—

13.1

acetate

—

1.21

—

13.5

isobutyrate

—

1.54

—

—

cipionate

—

2.19

—

18.7

enantate

—

1.92

—

16.7

undecylate

—

2.56

—

—

isocaproate

—

1.77

—

15.9

decanoate

—

2.36

—

19.8

benzoate

—

—

—

18.0

phenylpropionate

—

—

—

20.2

Testosterone

(magnesium and other salts), are detected by other means (Duncan 2000) and are not discussed here. Stools may be analysed after homogenisation, or alternatively purgatives may be detected in urine, both after enzymatic hydrolysis to release conjugated metabolites. Extraction of hydrolysis products with chloroform–isopropanol (9 : 1) or other moderately polar solvents at the hydrolysis pH yields good recovery, and derivatisation (e.g. silylation, methylation or acetylation) improves chromatography, particularly of the more polar compounds such as rhein. Rhein is a product of many vegetable glycoside laxatives, including sennosides, aloes and cascara (except frangula, which is metabolised to emodin), which are hydrolysed by colonic bacteria to active aglycones prior to absorption (this is important to remember when analysing pharmaceutical products). System GA or GB may be used, and the reference compounds are n-alkanes with an even number of carbon atoms.

n n

Column: Fused silica capillary with methylsilicone (12 m  0.25 mm i.d., 0.25 mm). Temperature programme: 70
C to 150
C at 15
/min to 250
C at 25
/ min.

System GAG

(Lurie et al. 1994). n n n n

Column: Bonded DB-1 fused silica capillary, cross-linked (30 m  0.25 mm i.d., 0.25 mm). Split ratio of 30 : 1. Temperature programme: 180
C to 230
C at 10
/min to 245
C at 1
/ min to 295
C at 30
/min for 15 min. Carrier gas: H2. Detector: Flame ionisation.

System GAI

Solvents and other volatile compounds Methods for the analysis and analytical data for solvents and other volatile substances are described in detail in Chapter 16. An ECD run in parallel with an FID produces the optimal detection rates, since many of the compounds of interest are halogenated. The low boiling point of these compounds requires their careful isolation from biological samples by headspace analysis, and the GC may benefit from cryogenic cooling (Flanagan et al. 1997; Sharp 2001). Standard capillary columns can be used (e.g. dimethyl-PSX), but chromatography and durability benefit from the use of a column with a high-phase ratio (3–5 mm film thickness). Other columns, such as an X-wax phase (a 0.25 mm film thickness is adequate here), or those specifically designed for volatiles, such as X-624 (3 mm film thickness), are used commonly (see Table 40.3 for details of these stationary phases). System GA or GI may be used and a more comprehensive method for screening volatiles can be found in Chapter 16, Table 16.3. System GA, previously described, may be used, or system GI, below. System GI

(Ramsey, Flanagan 1982). n n n

Column: 0.3% Carbowax 20M on 80-100 mesh Carbopak C glass, (2 m  2 mm i.d.). Temperature programme: 35
C for 2 min to 175
C at 5
/min for at least 8 min. Carrier gas: N2, 30 mL/min.

Steroids (Table 40.25) System GAR

(CND Analytical, 1989).

(Ayotte et al. 1996). n n n n n

Column: HP-5 5% phenyl polymethyl siloxane capillary (25 m  0.25 mm i.d., 0.33 mm). Temperature programme: 100
for 1 min to 220
at 16
/min to 301
at 20
/min for 5.5 min. Carrier gas: He. Detection: Mass-selective detector. Retention time: Relative to 17a-methyl-5a-androstan-3b,17b-diol.

Sulfonamides Gas chromatography of sulfonamides is only possible after extractive N-methylation of the secondary amino group (Gyllenhaal et al. 1978) and some authors additionally prepare HFB or PFP derivatives of any primary amino groups (Tarbin et al. 1999). These latter derivatives may require the use of positive chemical ionisation MS (Reeves 1999). As with the oral hypoglycaemics, differences in methylation conditions may lead to hydrolysis, and a number of sulfonamides may yield products or metabolites that correspond to sulfanilamide derivatives. The main metabolites are the N4-acetylacted derivatives, which are hydrolysed relatively easily back to the parent compound under acidic conditions. System GA or GJ (Gyllenhaal et al. 1978) may be used. System GJ n n n n

Column: 5% OV-17 on 80 to 100 mesh Gas-Chrom Q glass (1.5 m  2 mm i.d.). Temperature: 250
. Carrier gas: N2, 30 mL/min. Relative retention times are given in Table 40.26 (retention times of methyl derivatives are relative to griseofulvin).

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:24

Specific applications

715

Table 40.26 GC retention data and mass spectral data for the sulfonamides (AC, acetyl; Me, methyl) Compound

System

Principal ions (m/z)

GA

GJ

Mafenide

2340

—

106

77

185

105

104

89

Mafenide-Me2

1920

—

58

214

213

74

89

133

Mafenide-Me3

1900

—

58

89

228

227

133

214

Mafenide-Me4

1870

—

58

242

89

107

134

117

M (AC-)

2425

—

105

106

147

228

185

160

M (AC)-Me

2300

—

119

161

185

242

89

199

Sulfabenzamide-Me2

2770

0.09

118

105

77

304

170

240

Sulfabenzamide-Me (metabolised to sulfanilamide)

2700

—

118

105

77

92

226

290

Sulfacetamide

2132

—

92

109

156

180

65

214

—

—

Phenylbenzenesulfonamide (see Sulfabenzamide) Phthalylsulfacetamide (metabolised to sulfacetamide) Phthalylsulfathiazole (metabolised to sulfathiazole) Succinylsulfathiazole (metabolised to sulfathiazole)

Sulfacetamide-Me

—

0.16

—

—

Sulfadiazine

2502

—

185

186

—

—

92

65

108

170

Sulfadiazine-Me

2625

0.66

199

M (AC)-Me

3710

1.69

241

200

92

108

156

184

242

199

108

92

Sulfadimidine

2613

—

266

—

—

—

—

—

Sulfadimidine-Me

—

0.71

—

—

—

—

—

—

—

Sulfaethidole (also metabolised to sulfanilamide)

2620

—

108

156

220

Sulfadoxine

—

—

—

—

—

—

—

—

Sulfaethidole (also metabolised to sulfanilamide)

2620

—

—

—

—

—

—

—

Sulfaethidole-Me

3060

—

298

92

83

190

234

156

Sulfaethidole-Me 2

2840

—

106

92

65

161

156

234

2490

—

213

108

80

326

136

283

M (AC-) M (AC)-Me2 Sulfafurazole (sulfisoxazole)

92

284

65

3410

—

148

106

203

276

302

354

1212

—

156

92

108

140

65

267

Sulfafurazole-Me

—

0.42

Sulfaguanidine

0000

—

108

214

92

65

148

156

Sulfaguanole-Me (metabolised to sulfanilamide)

2905

—

203

57

323

322

249

204

Sulfamerazine

2566

—

199

200

92

65

108

100

Sulfamerazine-Me

2625

0.69

199

200

65

92

108

140

Sulfamethizole-Me (metabolised to sulfanilamide)

2660

0.98

92

284

108

156

176

220

Sulfamethoxazole-Me (also metabolised to sulfanilamide)

2500

—

92

108

119

162

156

203

Sulfamethoxazole-Me2

2460

—

92

108

119

62

156

188

Sulfamethoxazole-Me

2500

0.40

92

108

119

162

156

188

3255

0.91

161

134

230

245

205

199

Sulfamethoxydiazine-Me3 (also metabolised to sulfanilamide) 2925

1.38

229

230

92

108

138

156

3620

—

271

272

229

65

92

—

0.93

—

—

—

M (AC)-Me M (AC)-Me Sulfamethoxypyridazine-Me

—

—

139 —

Sulfametopyrazine/sulfalene-Me

—

0.69

—

—

—

—

—

—

Sulfamoxole-Me

—

0.40

—

—

—

—

—

—

Sulfanilamide

2185

—

65

92

156

172

108

80

Sulfanilamide-Me

2135

—

92

186

56

65

108

122

Sulfanilamide-Me3

—

—

122

214

170

106

77

79

Sulfanilamide-Me4

2095

—

136

120

228

184

77

105

M (AC-)

2690

—

172

156

92

108

125

214

M (AC)-Me

2600

—

186

156

228

92

108

65

1795

—

213

214

65

92

198

306

3420

—

255

256

65

93

122

213

—

1.71

—

—

—

—

Sulfaperin-Me3 (also metabolised to sulfanilamide) M (AC)-Me2 Sulfaphenazole-Me

—

—

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:24

716

Gas Chromatography

Table 40.26 continued Compound

System GA

Principal ions (m/z) GJ

Sulfapyridine

2600

—

184

185

65

92

108

66

Sulfapyridine-Me

—

0.47

198

199

92

65

78

108

M (AC)-Me

—

1.16

—

—

0.50

—

—

—

—

Sulfasalazine (metabolised to sulfapyridine) Sulfasomidine/sulfaisomidine-Me

—

—

—

—

—

—

—

Sulfathiazole

Not eluted —

—

—

—

—

—

—

Sulfathiazole-Me

—

—

—

—

—

—

—

0.49

Sulfaurea (metabolised to sulfanilamide)

References Adams RP (1995). Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry. Carol Stream, IL: Allured Publications. Ardrey RE, Moffat AC (1981). Gas–liquid chromatographic retention indices of 1318 substances of toxicological interest on SE-30 or OV-1 stationary phase. J Chromatogr 220: 195–252. Ayotte C et al. (1996). Testing for natural and synthetic anabolic agents in human urine. J Chromatogr B Biomed Appl 687: 3–25. Blau K, Halket J (1993). Handbook of Derivatives for Chromatography, 2 edn. New York: Wiley. Braselton WE Jr et al. (1977). Measurement of antidiabetic sulfonylureas in serum by gas chromatography with electron-capture detection. Diabetes 26: 50–57. Brohon J, N€ oel M (1978). Determination of metformin in plasma therapeutic levels by gas–liquid chromatography using a nitrogen detector. J Chromatogr 146: 148–151. Broussard LA et al. (1997). Simultaneous identification and quantitation of codeine, morphine, hydrocodone, and hydromorphone in urine as trimethylsilyl and oxime derivatives by gas chromatography–mass spectrometry. Clin Chem 43: 1029–1032. Brugmann G (1981). Gas chromatographic determination of phenytoin, phenobarbital and primidone: flash-methylation after direct addition of trimethyl-phenyl-ammonium hydroxide to the ethyl acetate extract (author’s transl.). J Clin Chem Clin Biochem 19: 305–306. Carreras D et al. (1994). Comparison of derivatization procedures for the determination of diuretics in urine by gas chromatography–mass spectrometry. J Chromatogr A 683: 195–202. Chen BH et al. (1990). Comparison of derivatives for determination of codeine and morphine by gas chromatography/mass spectrometry. J Anal Toxicol 14: 12–17. CND Analytical (1989). Analytical Profile of the Anabolic Steroids. Auburn, AL: CND Analytical Inc. Cody JT, Schwarzhoff R (1993). Interpretation of methamphetamine and amphetamine enantiomer data. J Anal Toxicol 17: 321–326. Davies NM (1997). Methods of analysis of chiral non-steroidal anti-inflammatory drugs. J Chromatogr B Biomed Sci Appl 691: 229–261. Dawling S et al. (1990). Rapid measurement of basic drugs in blood applied to clinical and forensic toxicology. Ann Clin Biochem 27(Pt5): 473–477. De Zeeuw RA (2002). Gas Chromatographic Retention Indices of Toxicologically Relevant Substances on Packed or Capillary Columns with Dimethylsilicone Stationary Phases, 3rd edn. New York: Wiley. Dudley KH (1980). Trace organic sample handling. In: Reid E, ed. Methodological Surveys Sub-series (A). Chichester: Ellis Horwood, 336. Duncan A (2000). Screening for surreptitious laxative abuse. Ann Clin Biochem 37 (Pt1): 1–8. Flanagan RJ, Berry DJ (1977). Routine analysis of barbiturates and some other hypnotic drugs in the blood plasma as an aid to the diagnosis of acute poisoning. J Chromatogr 131: 131–146. Flanagan RJ et al. (1997). Volatile Substance Abuse. United Nations International Drug Control Programme Technical Series Number 5. Vienna: UNIDCP. Franke JP et al. (1993). An overview on the standardization of chromatographic methods for screening analysis in toxicology by means of retention indices and secondary standards. Fresenius J Anal Chem 347: 67–72. Gorecki T, Poerschmann J (2001). In-column pyrolysis: a new approach to an old problem. Anal Chem 73: 2012–2017. Grinstead GF (1991). A closer look at acetyl and pentafluoropropionyl derivatives for quantitative analysis of morphine and codeine by gas chromatography/mass spectrometry. J Anal Toxicol 15: 293–298. Gyllenhaal O et al. (1978). Electron-capture gas chromatography of sulphonylureas after extractive alkylation. J Chromatogr 156: 275–283. Hartvig P et al. (1980). Electron-capture gas chromatography of plasma sulphonylureas after extractive methylation. J Chromatogr 181: 17–24.

Junting L, Chuichang F (1991). Solid phase extraction method for rapid isolation and clean-up of some synthetic pyrethroid insecticides from human urine and plasma. Forensic Sci Int 51: 89–93. Kovats E (1961). Zusammenh€ange zwischen strucktur und gaschromatographischen daten organischer verbindungen. Fresenius Z Anal Chem 181: 351–366. Lee J et al. (1998). The effect of organic solvents on the determination of cyclic boronates of some beta-blockers by gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom 12: 1150–1160. Leloux MS et al. (1989). Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography-mass spectrometry. J Chromatogr 488: 357–367. Lho DS et al. (1990). Determination of phenolalkylamines, narcotic analgesics, and beta-blockers by gas chromatography/mass spectrometry. J Anal Toxicol 14: 77–83. Lisi AM et al. (1991). Screening for diuretics in human urine by gas chromatography-mass spectrometry with derivatisation by direct extractive alkylation. J Chromatogr 563: 257–270. Liu RH et al. (1994). Improved gas chromatography/mass spectrometry analysis of barbiturates in urine using centrifuge-based solid-phase extraction, methylation, with d5-pentobarbital as internal standard. J Forensic Sci 39: 1504–1514. Lurie IS et al. (1994). The determination of anabolic steroids by MECC, gradient HPLC, and capillary GC. J Forensic Sci 39: 74–85. Maurer HH, Arlt JW (1998). Detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography–mass spectrometry after extractive methylation. J Chromatogr B Biomed Sci Appl 714: 181–195. Maurer HH, Arlt JW (1999). Screening procedure for detection of dihydropyridine calcium channel blocker metabolites in urine as part of a systematic toxicological analysis procedure for acidic compounds by gas chromatography–mass spectrometry after extractive methylation. J Anal Toxicol 23: 73–80. Maurer HH, Bickeboeller-Friedrich J (2000). Screening procedure for detection of antidepressants of the selective serotonin reuptake inhibitor type and their metabolites in urine as part of a modified systematic toxicological analysis procedure using gas chromatography–mass spectrometry. J Anal Toxicol 24: 340–347. Maurer HH, Pfleger K (1984). Screening procedure for the detection of opioids, other potent analgesics and their metabolites in urine using a computerized gas chromatographic–mass spectrometric technique. Fresenius Z Anal Chem 317: 42–52. Maurer HH, Pfleger K (1986). Identification and differentiation of beta-blockers and their metabolites in urine by computerized gas chromatography–mass spectrometry. J Chromatogr 382: 147–165. Maurer HH, Pfleger K (1987). Identification and differentiation of benzodiazepines and their metabolites in urine by computerized gas chromatography–mass spectrometry. J Chromatogr 422: 85–101. Maurer HH et al. (1998). Screening for the detection of angiotensin-converting enzyme inhibitors, their metabolites, and AT II receptor antagonists. Ther Drug Monit 20: 706–713. Maurer HH et al. (2001). Screening procedure for detection of non-steroidal anti-inflammatory drugs and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons by gas chromatography-mass spectrometry after extractive methylation. J Anal Toxicol 25: 237–244. Maurer HH et al. (2002). Screening, library-assisted identification and validated quantification of oral antidiabetics of the sulfonylurea-type in plasma by atmospheric pressure chemical ionization liquid chromatography–mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 773: 63–73. McReynolds WO (1970). Characterization of some liquid phases. J Chromatogr Sci 8: 685–691.

Clarke's Analysis of Drugs and Poisons Chapter No. 40 Dated: 10/3/2011 At Time: 15:38:25

Further reading Meatherall R (1999). GC-MS confirmation of codeine, morphine, 6-acetylmorphine, hydrocodone, hydromorphone, oxycodone, and oxymorphone in urine. J Anal Toxicol 23: 177–186. Moffat AC et al. (1974). Otimum use of paper, thin-layer and gas-liquid chromatography for the identification of basic drugs. I. Determination of effectiveness for a series of chromatographic systems. J Chromatogr 90: 1–7. Moffat AC et al. (1974). Optimum use of paper, thin-layer and gas–liquid chromatography for the identification of basic drugs. III. Gas–liquid chromatography. J Chromatogr 90: 19–33. Nagai T, Kamiyama S (1991). Simultaneous HPLC analysis of optical isomers of methamphetamine and its metabolites, and stereoselective metabolism of racemic methamphetamine in rat urine. J Anal Toxicol 15: 299–304. Nakahara Y et al. (1997). Hair analysis for drugs of abuse. XVII. Simultaneous detection of PCP, PCHP, and PCPdiol in human hair for confirmation of PCP use. J Anal Toxicol 21: 356–362. Nelson CC, Foltz RL (1992). Determination of lysergic acid diethylamide (LSD), iso-LSD, and N-demethyl-LSD in body fluids by gas chromatography/tandem mass spectrometry. Anal Chem 64: 1578–1585. Osselton MD, Snelling RD (1986). Chromatographic identification of pesticides. J Chromatogr 368: 265–271. Paroni R et al. (2000). Comparison of capillary electrophoresis with HPLC for diagnosis of factitious hypoglycemia. Clin Chem 46: 1773–1780. Paul BD et al. (1999). A practical approach to determine cutoff concentrations for opiate testing with simultaneous detection of codeine, morphine, and 6-acetylmorphine in urine. Clin Chem 45: 510–519. Pfleger K et al. (2004). Mass Spectral and GC Data of Drugs, Poisons, Pesticides, Pollutants and their Metabolites, Part 5, 2 edn. Weinheim: Wiley-VCH. Pierce KM et al. (2008). Recent advancements in comprehensive two-dimensional separations with chemometrics. J Chromatogr A 1184: 341–352. Ramsey JD, Flanagan RJ (1982). Detection and identification of volatile organic compounds in blood by headspace gas chromatography as an aid to the diagnosis of solvent abuse. J Chromatogr 240: 423–444. Reeves VB (1999). Confirmation of multiple sulfonamide residues in bovine milk by gas chromatography-positive chemical ionization mass spectrometry. J Chromatogr B Biomed Sci Appl 723: 127–137. Rorschneider L (1966). Eine methode zur charakterisierung von gaschromatographischen trennflussigkeiten. J Chromatogr 22: 6–22. Schuetz H, Westenberger V (1978). GLC-data of 19 hydrolysis-derivatives rised from 12 important benzodiazepines and 17 main-metabolites (author’s transl.). Z Rechtsmed 82: 43–53.

717

Schuetz H, Westenberger V (1979). Gas chromatographic data of 31 benzodiazepines and metabolites. J Chromatogr 169: 409–411. Scott RPW (1996). Chromatographic Detectors: Design, function, and operation. Chromatographic Science Series, 73, Cazes J, ed. New York: Marcel Dekker. Sharp ME (2001). A comprehensive screen for volatile organic compounds in biological fluids. J Anal Toxicol 25: 631–636. Stern EL, Caron GP (1977). Measuring barbiturates, sedatives, and anticonvulsants in serum by gas–liquid chromatography. Am J Med Technol 43: 834–842. Supelco (1979). Supelco Bulletin. Bellefonte, PA: Supelco Inc, p. 779. Supelco (2000). Supelco Catalogue test chromatograms. Supelco Catalogue 2000, pp. 506-508. Tarbin JA et al. (1999). Screening of sulphonamides in egg using gas chromatography–mass-selective detection and liquid chromatography–mass spectrometry. J Chromatogr B Biomed Sci Appl 729: 127–138. Yoon CN et al. (1990). Mass spectrometry of methyl and methyl-d3 derivatives of diuretic agents. J Anal Toxicol 14: 96–101.

Further reading Blau K, Halket J (1993). Handbook of Derivatives for Chromatography, 2nd edn. New York: Wiley. Grob K (1993a). On-Column Injection in Capillary Gas Chromatography. Heidelberg: H€ uthig. Grob K (1993b). Split and Splitless Injection in Capillary Gas Chromatography. Heidelberg: H€ uthig. Hill HH, McMinn DG (1992). Detectors for Capillary Chromatography. New York: Wiley. Jennings W et al. (1997) Analytical Gas Chromatography, 2nd edn. London: Academic Press. Jinno K (1997). Chromatographic Separations Based on Molecular Recognition. New York: Wiley. Rood D (1995). A Practical Guide to the Care, Maintenance, and Troubleshooting of Capillary Gas Chromatographic Systems, 2nd edn. Heidelberg: H€ uthig. Scott RPW (1996). Chromatographic Detectors: Design, Function, and Operation. Chromatographic Science Series, 73, Cazes J, ed. New York: Marcel Dekker. Stevenson D, Wilson ID (1994) Sample Preparation for Biomedical and Environmental Analysis. New York: Plenum Press.

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:1

CHAPTER

41

High Performance Liquid Chromatography T Kupiec and P Kemp

Introduction The ability to separate and analyse complex samples, both small and large molecules, is important to the biological and medical sciences. Classic column chromatography has evolved over the years, with chromatographic innovations introduced at roughly decade intervals. These techniques offered major improvements in speed, resolving power, detection, quantification, convenience and applicability to new sample types. The most notable of these modifications was high performance liquid chromatography (HPLC). Modern HPLC techniques became available in 1969; however, they were not widely accepted in the pharmaceutical industry until several years later. Once HPLC systems capable of quantitative analysis became commercially available, their usefulness in pharmaceutical analysis was fully appreciated. By the 1990s, HPLC had begun an explosive growth that made it a popular analytical method judged by sales of instruments and also scientific importance. During the last decade developments in chromatographic supports and instrumentation for liquid chromatography have continued to evolve. The use of silica-based monolithic supports, elevated mobile phase temperatures and columns packed with particles 50 C) are used for variation and single-nucleotide polymorphism (SNP) discovery and detection are based on DNA fragment sequence as described above. Fully denaturing temperatures (>70 C) can be used for the separation and analysis of oligonucleotides and nucleic acids as illustrated in Figure 41.9. On-line sample preparation The preparation of samples typically demands a large amount of time, work and cost in an analytical laboratory. The innovation of on-line sample preparation makes the process more efficient and reduces the cost. On-line sample preparation techniques usually involve direct elution of the extract from a solid-phase extraction cartridge into the system by the mobile phase. The on-line method gives superior analytical results and can be automated fully. Another benefit is that the sample preparation is reliable, reproducible and robust. This sample preparation method is also discussed in the column-switching section of this chapter. Rapid screening The need for high throughput in a laboratory environment is constantly increasing. The use of short, highly efficient, analytical columns, rapid gradients and column-switching apparatus in HPLC systems is helping to facilitate this. Sample turnaround time can often be reduced to a few minutes or less in highly automated and optimised systems. Other information on this topic is given earlier in this chapter in the sections on gradients and column switching.

Systems for drug analysis Eluent systems A large number of eluent and/or packing material combinations have been used for drug analysis. However, currently most are performed on silica or one of the hydrocarbon-bonded silicas (usually ODS). Other types of packing are employed when these conventional materials fail. The majority of drug analyses can be carried out with the four types of system described next. Silica with non-polar eluents

With silica normal-phase systems the principal mechanism is adsorption chromatography. Separation is controlled by the competition between solute molecules and molecules of the mobile phase for the adsorption sites on the silica surface. Polar groups are attracted most strongly to these sites and hence polar compounds are retained more strongly than non-polar ones. Retention can be decreased by increasing the polarity of the eluent. Adsorption energies of numerous solvents on alumina (e values are given in Table 41.1) have been measured and this scale can be used as a good guide to the elution strengths of eluents on silica as well as alumina (Snyder 1968). Mixtures of solvents can be employed to give elution strengths between those of the pure solvents. Furthermore, different solvent mixtures that have the same e value often give different separations of a group of compounds. Water is strongly bound to silica and thus the water content of the eluent must be controlled strictly to maintain constant activity of the silica surface and hence reproducible retention times. This is most critical when the eluent is of very low polarity. However, because anhydrous systems are difficult to maintain, a low concentration of water can be used in the eluent, sufficient to deactivate the most active sites without deactivating the whole surface. Typical water concentrations range from 0.01% to 0.2% (v/v). The most satisfactory method used to prepare a solvent of known water content is to mix anhydrous and water-saturated solvents in known proportions. Anhydrous hydrocarbon or halo hydrocarbon solvents can be prepared by passing them through a bed of activated silica or alumina (200 mm) in a glass column. The problems associated with the control of water concentration mean

Table 41.1 e values for numerous solvents on alumina (Snyder 1968)

70-80°C Heat

(a) EP

+C

(b)

+T +G

+A

Time

Figure 41.9 Principle of completely denaturing HPLC. (a) At column temperatures >70 C, double-stranded DNA fragments will denature completely. (b) The single-stranded components can then be resolved from each other even if they differ only in sequence and not in size. The chromatogram depicts the separation of an extension primer (EP) and the four possible isomeric products generated by single nucleotide extension sequencing. Resolution for any given pair of alleles can be optimised by varying column temperature. The order of elution of the alleles depends on the stationary phase used. On poly(styrene-divinylbenzene) monoliths, as depicted, extension products elute in the order C < G < T < A. On micropellicular alkylated poly(styrene-divinylbenzene) particles, in contrast, the elution order is G < C < A < T.

731

Solvent

e

Pentane

0.00

Hexane

0.01

Isooctane

0.01

Cyclohexane

0.04

Toluene

0.29

1-Chlorobutane

0.30

Ether

0.38

Chloroform

0.40

Methylene chloride

0.42

Tetrahydrofuran

0.45

Acetone

0.56

Ethyl acetate

0.58

Diethylamine

0.63

Acetonitrile

0.65

Isopropyl alcohol

0.82

Ethanol

0.88

Methanol

0.95

Acetic acid

Large

Water

Large

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:6

732

High Performance Liquid Chromatography

that commonly alcohols, such as methanol (0.01–0.5% v/v), are employed to moderate the silica surface (Engelhard 1977). Silica with polar eluents

Several systems have been described that involve the use of silica with eluents of moderate-to-high polarity containing alcohols and/or water as major components. With such eluents, adsorption chromatography is most probably not the principal mechanism. The mechanisms are poorly understood, which makes the prediction of retention behaviour difficult; nevertheless, many of these systems are very useful for drug analysis. An eluent that consists of methanol–ammonium nitrate buffer (90 : 10) is suitable for a wide range of basic drugs (e.g. amfetamines and opiates). Retention can be controlled by changes to the pH, ionic strength or methanol : water ratio, or by the addition of other organic solvents such as methylene chloride. With these alkaline eluents the silica surface must bear a negative charge and the principal mechanism is probably cation exchange. Benzodiazepines can be chromatographed with methanol eluents that contain perchloric acid (typically 0.001 mol/L). Retention can be modified by the addition of other organic solvents (e.g. ether) or by changes to the acid concentration. Both acidic and basic drugs can be chromatographed on silica using aqueous methanolic eluents that contain cetyltrimethylammonium bromide (Hansen 1981). Hydrophobic quaternary ammonium ions are strongly adsorbed on silica to give a dynamically coated stationary phase. Retention may be controlled by varying the concentration or nature of the quaternary ammonium ion, changing the ionic strength or pH of the buffer, or changing the concentration or nature of the organic component. ODS with polar eluents

Eluents for RPC on ODS are usually mixtures of methanol or acetonitrile with an aqueous buffer solution. Retention is controlled mainly by the hydrophobic interactions between the drugs and the alkyl chains on the packing material. Retention increases as the analytes decrease in polarity (i.e. polar species are eluted first). Hence, the elution time is increased by increasing the polarity of the eluent (i.e. increasing the water content). The pH of the eluent and the pKa of the drug are also important, since non-ionised species show greater retention. Thus, acids show an increase in retention as the pH is reduced, while bases show a decrease. It is important to use a buffer of sufficient capacity to cope with any injected sample size, otherwise tailing peaks can arise from changes in ionic form during chromatography. Phosphate buffers (0.05–0.2 mol/L) are widely used as they have a good pH range and low UV absorbance. Drugs that contain basic nitrogen atoms sometimes show poor efficiencies and give tailing peaks caused by interactions with residual silanol groups on the packing material. This can often be improved by the addition of an amine or quaternary ammonium compound to the eluent, which competes with the analytes for adsorption sites on the silica. Amines of small molecular weight (e.g. diethylamine) can be used as part of the buffer system. Alternatively, low concentrations (0.001 mol/L) of long-chain hydrophobic modifiers (e.g. N,N-dimethyloctylamine) can be added to eluents together with conventional buffers. Other hydrocarbon-bonded packing materials can be used in RPC. A decrease in retention is associated with a decrease in the alkyl chain length. ODS with polar eluents that contain hydrophobic cations or anions

Drugs that bear positive or negative charges are retained poorly in reversed-phase systems. If the pH of the eluent cannot be changed to convert the drug into its non-ionised form, a hydrophobic ion of opposite charge can be added to form a neutral ion-pair and increase retention. Hence, for a basic drug an acidic eluent is chosen and a hydrophobic anion is added. This technique is referred to as reversedphase ion-pair chromatography. The sodium salts of alkylsulfonic acids (RSO3
Naþ, where R ¼ pentyl, hexyl, heptyl or octyl) are used widely as ion-pair reagents for basic drugs, while quaternary ammonium compounds (e.g. tetrabutylammonium salts) are used for acidic drugs. Ion-pair reagents are generally added to eluents in the concentration range 0.001–0.005 mol/ L, and within this range an increase in concentration leads to an increase

in retention. When detergents such as sodium lauryl sulfate or cetyltrimethylammonium bromide are used as the ion-pair reagents, the method is sometimes referred to as ‘soap chromatography’. With these salts, ions build up on the surface of the packing material and produce a stationary phase, which behaves like an ion exchanger. This type of mechanism has been described as ‘dynamic ion exchange’ and probably also occurs with less hydrophobic ion-pair reagents. It is virtually impossible to remove an ion-pair reagent completely from a hydrocarbon-bonded phase, and such columns should therefore not be re-used with other reversed-phase eluents. Selection of chromatographic systems Many different combinations of packing material and eluent may be suitable for the analysis of a particular compound or group of compounds and the final choice can be influenced by many factors. The time required to develop a new system can be shortened if it is possible to predict the way in which changes in eluent composition influence chromatographic retention. Systems that use hydrocarbon-bonded phases are particularly attractive from this viewpoint as a large range of parameters can be adjusted (pH, organic solvent, ionic strength, ion-pair reagents) with largely foreseeable consequences. Predictions for silica are generally less reliable. Silica is good for separating drugs that belong to different chemical classes, while hydrocarbon-bonded silicas are preferred for separations of drugs with closely related structures (e.g. barbiturates). Most of the endogenous materials in biological extracts that can interfere with the analysis of a drug are fairly polar. In reversed-phase systems this material generally elutes before the drug and can obscure the drug peak. In these circumstances, reversed-phase ion-pair chromatography can be valuable to increase selectively the retention of the drug relative to the interfering peaks. Normal-phase systems that use silica do not generally suffer from this problem, as most of the endogenous material usually elutes after the drug. However, these slow-eluting compounds can lead to a noisy baseline or may remain adsorbed to the packing material and thus eventually lead to a loss in column performance. The vast majority of compounds are separated using a silica-based column with C18, and fine-tuning of the separation can be done by selecting a column with a shorter bonded phase, such as C8 (see later). Specially end-capped columns are available that are designed to minimise the tailing that is common with nitrogen-containing weak bases. These are often marketed as a ‘basic’ column (e.g. Metachem’s MetaSil Basic). There are also specially end-capped columns designed to withstand extremely high concentrations of aqueous mobile phase (95–100%). These columns are end-capped with a hydrophilic moiety that ensures proper ‘wetting’ of the silica to prevent bonded-phase collapse. The columns are typically marketed as ‘AQ’ for aqueous (e.g. YMC’s ODS-AQ). Analysis of drugs in pharmaceutical preparations HPLC has found widespread use for the quantitative analysis of drugs in preparations of pharmaceutical and illicit manufacture. Drug concentrations are generally high enough to allow dissolution of the sample (tablet, powder, ointment, etc.) in a suitable solvent followed by injection. UV, visible, FL, RI or mass spectrometric detection methods are used often. These techniques are well suited to provide specific data regarding the chemical composition of the sample in question (e.g. a UV spectrum, mass spectrum). Within the pharmaceutical industry, HPLC is used at various stages of drug development, such as the optimisation of synthetic reactions and stability testing. Furthermore, it is used extensively for quality control during production to monitor the purity of drugs and excipients. HPLC systems can be automated easily (including injection and data handling), which allows large numbers of samples to be analysed rapidly and economically. HPLC is particularly valuable for the analysis of drugs that are polar (e.g. aspirin), thermally unstable (e.g. benzodiazepines) or present in oil-based formulations for which analysis by GC can be very difficult. Similarly, HPLC can be used for the forensic analysis of illicit preparations to aid the identification of an unknown drug by the measurement of retention times and UV spectra and

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Systems for drug analysis comparison to spectral libraries. Furthermore, as the technique can be non-destructive, depending on the detection system used, the eluted compounds can be collected for further analysis. Example of a drug analysis system

Opiates have been separated by many methods in the past, and the system described here was developed for this purpose. The three opiates separated were morphine sulfate, hydrocodone bitartrate and oxycodone hydrochloride. The column used was a Phenomenex Luna C18 (2), 150  4.60 mm  5 mm. The mobile phase was 39 mmol/L dipotassium hydrogenphosphate (K2HPO4) and methanol in a 40 : 60 ratio. The final pH was 10 and the mobile phase flow rate was 1.0 mL/min. The retention times obtained (Figure 41.10) for morphine sulfate, hydrocodone bitartrate and oxycodone hydrochloride were 2.799, 4.696 and 6.143 min, respectively. Analysis of drugs in biological fluids and tissues Several factors determine the ability of HPLC to detect a drug among the endogenous compounds present in biological material. Clearly, selective detection of the drug relative to the endogenous material is advantageous. In addition, the stationary phase and/or mobile phase can be altered to separate the drug peak from interfering peaks (e.g. using ionpair reagents). Finally, the sample may be extracted before HPLC to concentrate the drug relative to the endogenous material. The chromatographic system and detector should always be chosen to minimise the time needed for sample preparation. The complexity of the sample preparation procedure is controlled by several factors, which include the nature of the sample (urine, blood, liver, etc.), the condition of the sample and the concentration of the drug. Interference from endogenous compounds is most acute when drug concentrations are low (e.g. in therapeutic drug monitoring), so more extensive sample preparation and more sensitive and specific detectors are often required. Such assays can be very susceptible to changes in the condition of the sample (e.g. a method developed for fresh blood may not be satisfactory for urine or hair samples), which can present severe difficulties in forensic toxicology. Thus, methods should be tested and validated with the most difficult samples that may be encountered. In contrast, the analysis by HPLC of biological samples that contain high drug concentrations (e.g. in fatal drug overdose) may require much less sample preparation and is less susceptible to changes in sample condition. Sample preparation for HPLC is essentially the same as for other methods of drug analysis. A drug that is physically trapped within solid tissue (e.g. liver), or chemically bound to the surface of proteins, must be

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released; then the protein is precipitated to leave the drug in aqueous solution. The protein may be degraded by strong acids or enzymes, precipitated by various chemicals (e.g. tungstic acid, ammonium sulfate) or removed by ultrafiltration. Some drugs are destroyed by protein degradation methods, while ultrafiltration and precipitation can lead to drug losses through protein binding. No single procedure works well for all drugs and the method should be selected to give the maximum recovery of the drug being analysed. When drug concentrations are high (typically mg/mL) and systems with polar mobile phases are used, the direct injection of deproteinised solutions may be acceptable. Proteins must be removed to protect the column from irreversible contamination. A rapid procedure is to: mix the biological fluid with at least two volumes of methanol or acetonitrile; centrifuge to remove the precipitated protein; evaporate the organic supernatant; and reconstitute the sample in a volume of mobile phase. Urine can be treated similarly to guard against the precipitation of salts on the column. Great care and consideration should be afforded when injecting minimally prepared biological samples on to a HPLC system. Particulates are more likely to become trapped in the system plumbing and a more rapid degradation of column performance may be observed from build-up of contaminants on the head of the column. To help maximise column performance and lifetime, it is good policy to use a guard column between the injector and analytical column. This is packed with the same material as the analytical column and replaced at frequent intervals. The configurations of guard columns range from easily replaceable and relatively inexpensive frit-like filters and/or cartridges to shorter versions of the analytical column itself. All are designed to protect the analytical column by acting as a trap for components that would otherwise irreversibly bind to the analytical column and thus decrease the usable life of the column. Extraction of drugs and other analytes away from endogenous materials prior to analysis is a common procedure for all types of biological samples. This may also entail a concentration step, which increases the sensitivity of the method. Solvent extraction remains the most popular approach, as many factors can be modified to optimise the extraction. These modifications include changing the polarity of the organic solvent, the pH and ionic strength of the aqueous phase, and the use of ion-pairing agents. It is generally recommended that the collected organic phase be evaporated to dryness and the residue dissolved in a suitable solvent, typically something greater than or equal to the polarity and composition of the initial mobile phase before injection. Care must be taken that volatile drugs are not lost by evaporation and that lipid material in the residue does not prevent the drug from dissolving in the new solvent.

Figure 41.10 Separation of opiates by HPLC. Conditions for separation are described in the text.

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Example protocol for the extraction of a wide variety of weak bases n n n n n

n

To 1 mL of plasma, urine or other homogenised matrix add 100 mL concentrated ammonium hydroxide. Extract the sample with 4 mL of a mixture of n-butyl chloride– acetonitrile (4 : 1) for 20 min. Centrifuge at high speed for 20–30 min to partition the phases. Carefully collect the organic phase into a clean tube. Evaporate the organic phase under a stream of air or nitrogen at 25– 40 C, depending on the volatility of the analytes (a small volume of acidified methanol can be added to prevent the loss of amfetaminetype analytes). Reconstitute the residue in an HPLC mobile phase that is more polar than the LC mobile phase to be used for analysis (e.g. if the HPLC elution ratio is 60% aqueous, reconstitute the sample in a phase that is >60% aqueous). This ensures that, when injected, the sample is focused on the front end of the column and minimises band (peak) broadening.

An example of a chromatogram that utilises this extraction technique is shown in Figure 41.11. The urine was fortified with analytes and deuterated internal standards for amfetamine and methamfetamine (dashed chromatograms) and extracted as described above. The sample was eluted using a MetaSil Basic 3  100 mm  3 mm column. The mobile phase was 85% (0.1% formic acid in water), 15% methanol, pumped isocratically at 0.2 mL/min. The instrument used was an Agilent 1100 LC/MSD with ESI. SPE columns are also widely used to extract drugs from biological samples. The column is washed with suitable solvents to remove endogenous material before the drug is removed by passing through a solvent of higher elution strength. Such columns are usually attached to extraction manifolds utilising either positive or negative pressure to draw the liquids through the sorbent beds. Extraction selectivity can be controlled by adjustments to the biological fluid before extraction (e.g. pH, ionic strength) and the choice of washing solvents. Most, if not all, manufacturers of SPE columns offer methods and columns optimised for a particular drug class and/or matrix. As less traditional biological matrices are used for drug analysis (e.g. sweat, hair, oral fluids), some modifications of the sample preparation scheme are needed. Hair requires solubilisation prior to extraction; oral fluids and sweat may need to be isolated from their respective collection devices. Consideration of the pH and solubility may be needed prior to sample preparation, but in general the principles in

place for the extraction of blood, urine, etc. apply to these alternative matrices. Some important issues unique to these matrices are: n n

Sample volume is typically much less than blood or urine. The amount of drug extracted from a particular matrix may be much less than from traditional matrices, so that much more sensitive detectors (e.g. MS or MS–MS) are required.

Identification of drugs by HPLC with photodiode array detection and UV spectra library search HPLC with DAD in combination with a UV spectra library has proved to be a very successful ‘systematic toxicological analysis’ (STA) technique for use in clinical and forensic toxicology (see Chapter 1). Any drugs or other poisons in the sample are identified by coincidence of the UV spectrum and of the retention time or another chromatographic retention parameter with the library data; one system and its use are described below (F Pragst and M Herzler, personal communication). Chromatographic conditions

Since the method is used in combination with a database of UV spectra and retention parameters, the chromatographic conditions must be reproducible and the same as used to generate the database. The mobile phase must be suitable for the separation of a large variety of organic substances and must be transparent in the wavelength range used. These prerequisites are best met by reversed-phase columns (RP8 or RP18) and acidic acetonitrile–buffer mixtures as mobile phases. Systems described in the literature generally use either a gradient elution or two isocratic runs with different buffer : acetonitrile ratios. Gradient elution has the advantages that strongly polar and non-polar substances can be analysed in one run, that peaks are not broadened with increasing retention time, and that the retention times of the toxicologically relevant compounds are distributed more evenly over the run time, but it has some disadvantages (see above). A system of HPLC retention indices was introduced by Bogusz et al. (1993) analogous to the Kovats indices used in GC and based on the retention times of the nitroalkanes. Isocratic HPLC has the advantage of higher reproducibility of the retention times, greater ruggedness and a more economic use of the mobile phase by recycling. Disadvantages are an unfavourable distribution of the retention times of toxicologically relevant compounds with an increased number at the beginning of the chromatogram, and the need for a second mobile phase for non-polar compounds. Nevertheless, isocratic HPLC–DAD procedures are used successfully in many toxicological laboratories for screening purposes. Suitable experimental

Figure 41.11 Separation of amfetamines by HPLC–MS. Conditions for separation are described in the text.

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Systems for drug analysis conditions, also used in the recording of an extensive UV spectra library, were as follows (Pragst et al. 2001): n n n n

HPLC column: RP8, end-capped, 5 mm, 250  4.0 mm Mobile phase A: 0.1 mol/L phosphate buffer pH 2.3–acetonitrile (67 : 33 v/v) Mobile phase B: 0.1 mol/L phosphate buffer pH 2.3–acetonitrile (33 : 67 v/v) Flow rate: 1 mL/min.

Standard compounds are histamine hydrochloride to measure the time of an unretained peak t0 (dead time), 5-(4-methylphenyl)-5-phenylhydantoin (MPPH) to calculate relative retention times (RRTs) in mobile phase A, and 4-phenylbenzophenone to calculate RRTs in mobile phase B. The UV spectra of a large number of compounds listed in this book were measured under these conditions. An overview of HPLC–DAD conditions used for STA is given in Pragst et al. (2001). Retention parameters

Absolute retention times are not suitable for peak identification purposes, since they depend strongly on the configuration and experimental conditions of the HPLC device. Moreover, the capacity ratio kA (see above) is sensitive to small fluctuations of the experimental conditions and is not suitable for an identification system used in different laboratories. Therefore, for gradient elution, retention indices are preferred (Bogusz et al. 1993). Under isocratic conditions RRTs related to a standard compound are more reproducible (Equation 41.6). RRTx ¼

tx
t0 ts
t0

ð41:6Þ

where RRTx is the RRT of compound x, tx is the absolute retention time of compound x, t0 is the retention time of an unretained peak, and ts is the retention time of the standard compound. The relatively small peak resolution of HPLC and the differences between charges of the reversed-phase material mean that the value of retention indices or of RRTs in the identification of a compound from a large number of candidates is rather limited. However, it is very useful for distinguishing between compounds with very similar UV spectra. In this way an RRT window can be chosen as a pre-selection parameter for the spectra library search. UV spectral library search and specificity of UV spectra

Before peak identification a ‘peak purity check’ should be carried out. A pure peak means that it originates only from one compound and that the UV spectrum does not change over the whole peak width. A UV spectral library search is based on the comparison of the spectrum of the unknown peak with all spectra of the library. This comparison is not confined to UV maxima and minima, but can comprise all absorbance–wavelength pairs measured by DAD. Mathematical models to assess spectral similarity use the description of the spectrum as a vector in n-dimensional space, where n is the number of absorbance–wavelength pairs measured. For the complete identity of two spectra, both vectors point in exactly the same direction, that is the angle between them is u ¼ 0 . Different concentrations have an effect on vector length, but not on its direction in space. The similarity index (SI) is defined as cos u and is calculated with Equation (29.7): SIx ¼ cosus1  s2 ¼

s1  s2 js1 j  js2 j

ð41:7Þ

where Si is the vectorised spectrum of compound i. UV spectra can be measured with extremely high reproducibility. Therefore, small differences between spectra measured under identical conditions indicate that they originate from different compounds. SI is 1.000 for completely identical spectra. However, in practice two spectra with SI >0.9990 can be regarded as identical. At small concentrations, and in the case of partly overlapping peaks, SI >0.990 may be a sufficient criterion for identity.

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It was shown in a systematic study on the selectivity of an HPLC– DAD method (Herzler et al. 2003) that, from 2888 toxicologically relevant compounds, 2682 (93%) exhibited UV absorption above 195 nm. Out of these, 1619 (60.4%) had a unique UV spectrum and could be identified unambiguously. By inclusion of the retention time this proportion was increased to 84.2%. Large UV spectra libraries can be divided into sub-libraries, according to the retention parameter or the effect or use of the substance, to facilitate a faster and more specific library search. The result can also be supported by the presence of metabolites, while in doubtful cases complementary methods may be used for confirmation (e.g. MS). As an example, in Figure 41.12 the results of the library search for a peak with RRT ¼ 0.0811 in an intoxication case are shown. In this case a sub-library of all compounds with RRT ¼ 0.601–0.900 was used. Hit 1 was promethazine with SI ¼ 0.9992; hit 2 (promazine, SI ¼ 0.9964) and hit 3 (dixyrazine, SI ¼ 0.9961) also originated from compounds of the phenothiazine type. The small difference between the spectra of hits 1 and 2 may arise because in these two compounds the amino group of the side chain is separated from the phenothiazine ring by two and three saturated carbon atoms, respectively. Dixyrazine could clearly be excluded by the much smaller retention time. However, promethazine and promazine could not be distinguished by the RRT values stored in the database. Therefore, to confirm the library search result, promazine and promethazine standards were measured immediately after the sample, which resulted in an exact agreement with promethazine. As a prerequisite for the optimal use of a commercially available UV spectral library, the same mobile phase must be used and the technical parameters of the DAD (wavelength accuracy and resolution) need to be (and stay) sufficient. This can be controlled by daily measurement of a compound with a vibration fine structure of the UV spectrum, such as benzene. UV spectra and retention times of metabolites

The use of HPLC–DAD has the advantage that in many cases, metabolites can be attributed easily to the parent drug by the UV spectrum. Depending on the site of metabolism, the UV spectrum may be altered significantly (change of the UV-absorbing unsaturated part of the molecule, the chromophore) or it may be the same as (or very similar to) that of the parent drug (reaction at the aliphatic part of the molecule). As an example, in Figure 41.13 the spectrum of flunitrazepam is compared with that of its metabolites, 7-aminoflunitrazepam (strong change of the chromophore by transformation of the aromatically bound nitro group into the amino group) and 3-hydroxyflunitrazepam (no essential change of the chromophore by hydroxylation at the aliphatic carbon atom 3). The retention times of drugs on reversed-phase columns are shifted in a typical way by metabolism. Metabolism to more hydrophilic products (e.g. hydroxylation, reduction of the nitro to an amino group; Figure 41.13) leads to a decrease in retention time, whereas deamination strongly increases retention time, particularly in an acidic mobile phase (removal of the strongly hydrophilic, protonated amino group). For many drugs, the chromatograms obtained from blood or urine extracts have a typical metabolite pattern that supports identification in the context of STA. Sample pretreatment

Tablets, powders or residues in syringes can simply be dissolved in the mobile phase and analysed by HPLC-DAD without further treatment. The investigation of biological samples, such as whole blood (serum, plasma), stomach contents, urine or tissue samples, is more complicated. In these cases the drug must be separated from the biological matrix. Although SPE has been much improved in the past decade, liquid– liquid extraction (LLE) is still preferred if HPLC-DAD is used for toxicological screening, since it is less susceptible to interferences, more reproducible and easier to handle for single samples. An important advantage of UV detection is that cholesterol and fatty acids, coextracted to a high extent from human samples by lipophilic solvents, show no UV absorption and therefore, in contrast to GC-MS, do not interfere with the analysis. Moreover, derivatisation is not necessary. A sample pretreatment method by extraction with n-butyl chloride–acetonitrile (4 : 1), which can be used for a wide variety of basic compounds,

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High Performance Liquid Chromatography

Figure 41.12 Results of the HPLC–DAD library search for a peak in the chromatogram of an alkaline extract of a lethal trimipramine–promethazine intoxication. Hit 1 (promethazine) was confirmed by exact agreement of the retention time with the reference compound measured immediately after the sample. sa, sample; li, library.

Figure 41.13 Change of the UV spectrum and the relative retention time (RRT) of flunitrazepam by metabolism.

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Recommended HPLC systems is given above. For systematic toxicological screening of blood (serum, plasma) samples by HPLC-DAD, the measurement of two extracts obtained at pH 2 and pH 9 with dichloromethane and of the supernatant of a protein precipitation by acetonitrile has proved to be very useful (Pragst et al. 2002). Preparation of a basic and an acidic methylene chloride extract

1. Dispense 500 mL of whole blood, serum or plasma into two 1.5-mL vials. 2. To vial 1 add 100 mL of a 0.2 mol/L solution of tri(hydroxymethyl) amine (basic extract). 3. To vial 2 add 100 mL of 0.1 mol/L hydrochloric acid (acidic extract). 4. To both vials add 400 mL of dichloromethane. 5. Vortex mix the vials for 1 min and centrifuge. 6. Withdraw 200 mL of the dichloromethane extract and evaporate the solvent at room temperature under a stream of nitrogen. 7. Dissolve the residue in 100 mL of mobile phase. 8. Analyse 50 mL of each extract (basic extract in mobile phase A and acidic extract in mobile phase B).

General screens System HA

Jane I et al. (1985). J Chromatogr 323: 191–225. n n

n

Protein precipitation is particularly useful for hydrophilic drugs, which are extracted poorly by the procedure mentioned above. These include paracetamol, salicylic acid and lamotrigine. The limits of detection are between 0.01 and 0.1 mg/mL for dichloromethane extraction (depending on the extinction coefficient and on the extraction yield) and between 0.1 and 1 mg/mL for protein precipitation. Application example

In STA, the library search must be applied to all peaks of the HPLC-DAD chromatogram. As an example, the chromatogram at 225 nm of the basic extract from the blood sample of a lethal drug poisoning case and the UV spectra of the highest peaks are shown in Figure 41.14. To determine RRT, the standard compound (MPPH, peak no. 10, RRT ¼ 1.000) was added. From the remaining 11 peaks of the chromatogram, 7 could be identified by both UV spectrum and RRT. As the result, a high overdose of trimipramine and promethazine was found to be the cause of death. The extensive metabolism indicated that there had been a long survival time after drug ingestion. The similarities between the UV spectra of the parent drugs (peaks 9 and 12) and some of their metabolites (peak 8, and peaks 6 and 11, respectively) are also demonstrated in this case. On the other hand, the sulfoxides of promethazine (peak 3) and desmethylpromethazine (peak 2) show completely changed spectra because of the transformation that takes place directly at the UV-absorbing phenothiazine ring. Caffeine (peak 1) is found in almost all samples. The poor separation of peaks 4, 5 and 7 meant that the UV spectra were not suitable for a library search.

Recommended HPLC systems There are general screening methods based on gradient elution and retention indices that have proven value by many laboratories, and data from these are listed below (systems HA, HX, HZ, HY and HAA). Another (system HBK) is based on a combination of isocratic systems. The tabulated data are derived from systems in which groups of compounds have been chromatographed either as part of a general screening procedure or from systems that have been used specifically for that group of compounds. Other systems for the chromatography of individual compounds, especially those used for quantification, are given in the monographs. Chromatographic retention data are presented as k values as well as retention times (RTs), retention indices (RIs) and relative retention times (RRTs). Note In the tables, a dash indicates that no value is available for the compound, not that it does not elute.

Column: Silica Spherisorb S5W (125  4.9 mm i.d., 5 mm). Mobile phase: Solution containing 1.175 g (0.01 mol/L) ammonium perchlorate in 1 L methanol; adjust to pH 6.7 by the addition of 1 mL 0.1 mol/L sodium hydroxide in methanol. k values: Values for drugs in this system will be found in drug monographs and in the Indexes to Analytical Data; they are also included in the systems for specific groups of drugs that follow.

System HX

J Hartstra, JP Franke, RA de Zeeuw, personal communication. n n n

Protein precipitation by acetonitrile

1. To 500 mL of whole blood, serum or plasma add 500 mL of acetonitrile. 2. Vortex the mixture for 2 min and centrifuge. 3. Separate off the supernatant. 4. Analyse 50 mL in mobile phase A.

737

n n n n

Column: Lichrospher 60 RP-Select B (125  4.0 mm i.d., 5 mm) with precolumn Lichrospher 60 RP-Select B (4  4.0 mm i.d., 5 mm). Mobile phase: (A:B) triethylammonium phosphate buffer (25 mmol/ L, pH 3.0)–acetonitrile. Elution programme: (A:B) (100 : 0) to (30 : 70) in 30 min, hold 10 min, back to initial conditions in 3 min with equilibration for 10 min before next injection. Flow rate: 1 mL/min. Detection: DAD. Standards: Nitro-n-alkanes (C1 to C11) 10 mL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and in the Indexes to Analytical Data; they are also included in the systems for specific groups of drugs that follow.

System HY

RK Watt, RA Waters, AC Moffat, unpublished information. n n n n

n n n

Column: C18 symmetry (250  4.6 mm i.d., 5 mm). Column temperature: 40 C. Mobile phase: (A:B) sulfuric acid (0.5 mL 2.5 mol/L) in water (500 mL)–sulfuric acid (0.5 mL 2.5 mol/L) in acetonitrile (500 mL). Elution programme: (98 : 2) for 3 min to (2 : 98) over 23 min, hold for 10 min, back to initial conditions over 2 min with equilibration of 8 min before next injection. Detection: DAD. Standards: Nitro-n-alkanes (C1 to C16) 10 mL in 10 mL acetonitrile. RI values: Values for drugs in this system will be found in the monographs and in the Indexes to Analytical Data; they are also included in the systems for specific groups of drugs that follow.

System HZ

Conemans JMH et al. http://www.zanob.nl/pages/LSShowElements Page_v2.asp?ListID¼1650&elemid¼29275&articleid¼133751&token¼ (accessed 14 December 2010). n n

n n n

Column: C18 end-capped LiChrospher 100 RP-18e (125  4.0 mm i.d., 5 mm), with precolumn LiChrocart 124-4. Mobile phase: Add 146 mL triethylamine and about 750 mL phosphoric acid to 530 mL water. Adjust pH to 3.3 using a 10% potassium hydroxide solution and finally add 470 mL acetonitrile. Flow rate: 0.6 mL/min. Detection: DAD. Retention times: Values for drugs in this system will be found in the monographs and in the Indexes to Analytical Data; they are also included in the systems for specific groups of drugs that follow.

System HAA

Gaillard Y, Pepin G. (1997). J Chromatogr A 763: 149–163. n n n n

Column: C8 Symmetry (250  4.6 mm i.d., 5 mm) with Symmetry C18 precolumn (20 mm). Column temperature: 30 C. Mobile phase: (A:B) phosphate buffer (pH 3.8)–acetonitrile. Elution programme: (85 : 15) for 6.5 min to (65 : 35) until 25 min to (20 : 80) for 3 min, and back to initial conditions for equilibration for 7 min.

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Figure 41.14 HPLC–DAD investigation of a combined trimipramine–promethazine poisoning. Chromatogram of a basic extract of a venous blood sample, UV spectra of the highest peaks, results of the library search and semiquantitatively determined concentrations.

n

n

Flow rate: 1 mL/min for 6.5 min, then linear increase to 1.5 mL/min for 6.5–25 min and hold for 3 min (re-equilibration is made at 1.5 mL/min). Detection: DAD.

n

Retention times: Values for drugs in this system will be found in the monographs and in the Indexes to Analytical Data; they are also included in the systems for specific groups of drugs that follow.

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:13

Recommended HPLC systems Amfetamines, other stimulants and anorectics

System HBK

Pragst F et al. (2001) UV Spectra of Toxic Compounds. Heppenheim: Verlag Dr Dieter Helm. n n

n n n

739

Column: Lichrospher RP-8ec (250  4.0 mm i.d., 5 mm). Mobile phase: Three different composition are used: A: acetonitrile–phosphate buffer pH 2.3 (33 : 67). Internal standard: 5-(4methylphenyl)-5-phenylhydantoin (for compounds eluting within 30 min). B: acetonitrile–phosphate buffer pH 2.3 (67 : 33). Internal standard: 4-phenylbenzophenone (for compounds eluting after 30 min). C: acetonitrile–phosphate buffer pH 2.3 (20 : 80). Internal standard: salicylamide (for compounds with RRTs below 0.2). Flow rate: 1 mL/min. Detection: DAD. Note: The phosphate buffer is prepared by dissolving 4.8 g phosphoric acid (85%) and 6.66 g potassium dihydrogenphosphate in 1 L water, adjust pH to 2.3. Values for drugs in this system will only be found in the Indexes to Analytical Data.

Systems HA, HX or HY previously described may be used, or Systems HB or HC below. System HB

Gill R et al. (1981). J Chromatogr 218: 639–646. n n

Column: ODS Hypersil (250  5 mm i.d., 5 mm). Mobile phase: Solution containing 19.60 g (0.2 mol/L) phosphoric acid and 7.314 g (0.1 mol/L) diethylamine in 1 L of a 10% v/v solution of methanol; adjust the pH to 3.15 by the addition of sodium hydroxide solution.

System HC

Law B et al. (1984). J Chromatogr 301: 165–172. n n

Column: Silica Spherisorb (250  5 mm i.d., 5 mm). Mobile phase: Methanol–ammonium nitrate buffer solution (90 : 10). To prepare the buffer solution add 94 mL strong ammonia solution and 21.5 mL nitric acid to 884 mL water and adjust to pH 10 by the addition of strong ammonia solution.

Amfetamines, other stimulants and anorectics

Adrenaline

HA

HB

HC

HX

HY

k

k

k

RI

RI

–

–

0.63

–

–

0.98

244

–

Amfetamine

0.9

8.48

1.2

–

0.15

–

–

11.1

–

–

312

267

Caffeine

0.2

–

Cathine

1

Benzfetamine Brucine

4.39

0.26

–

–

0.83

–

–

Chlorphentermine

0.9

–

0.82

–

–

Diethylpropion

1.7

–

0.16

–

230

Dimethylamfetamine

–

11.08

1.89

–

–

DOM

–

–

1.13

–

–

1.79

–

–

–

0.72

354

309

–

0.27

–

–

1.3

–

0.88

371

315

1

–

–

–

–

–

–

–

226 –

Ephedrine

1.0

Fencamfamin

1.3

Fenethylline

–

Fenfluramine Norfenfluramine Fenproporex

–

5.68

Hordenine

–

2.00

–

–

Hydroxyamfetamine

–

2.24

1.11

–

–

Hydroxyephedrine

–

0.73

–

–

–

Mazindol

1.8

–

0.2

357

286

Mephentermine

1.5

–

2.48

–

–

Mescaline

1.3

16.82

2.17

–

–

2

10.52

2.07

262

216

14.95

–

–

–

32.17

–

–

– –

Metamfetamine Methoxyamfetamine

–

Methoxyphenamine

1.7

Methylamfetamine Methylenedioxymethamfetamine

10.52

2.07

–

–

–

278

252

2.3

–

1.83

–

–

1.7

–

2.0 –

Methylephedrine Methylphenidate Noradrenaline

–

Normetanephrine

–

Oxedrine

–

0.10 – 0.27 0.2

–

Phendimetrazine

0.9

–

Phenelzine

1.0

Pemoline

5.91

0.36

–

277

–

–

–

1.08

–

–

–

–

–

0.1

307

271

0.3

263

218

0.37

–

– table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:16

740

High Performance Liquid Chromatography

Amfetamines, other stimulants and anorectics, continued HA

HB

HC

k

k

k

RI

RI

1.31

–

–

3.64

HX

HY

Phenethylamine

1.2

Phenmetrazine

1.7

–

–

258

241

Phentermine

0.6

19.46

0.86

–

245

Phenylephrine

1.3

–

1.64

–

–

Phenylpropanolamine

0.9

0.70

–

–

Pipradrol

1.2

–

0.69

355

–

Prolintane

2

–

1.3

370

–

Pseudoephedrine

1.2

1.77

–

–

3.87

5.90

Tranylcypromine

1.0

–

0.26

–

–

Trimethoxyamfetamine

–

–

1.48

–

–

Tyramine

1.2

1.47

–

–

0.81

Analgesics, non-steroidal anti-inflammatory drugs

Antifungals

System HD

The general screening systems, previously described, may be used.

HM Stevens, R Gill, unpublished data. n n

Column: ODS Hypersil (160  5 mm i.d., 5 mm). Mobile phase: Isopropyl alcohol–formic acid–0.1 mol/L potassium dihydrogenphosphate (13.61 g/L; 540 : 1 : 1000).

Antifungals HX

HY

HZ

HAA

RI

RI

RT

RT

Econazole

526

385

–

20.1

Fluconazole

340

289

–

11.4

Flucytosine

72

–

1.5

3.1

System HW

Griseofulvin

–

488

–

18.4

HM Stevens, R Gill, unpublished data.

Ketoconazole

439

464

5.2

15.7

System HV n n n

n n

Column: ODS Spherisorb (200  4.6 mm i.d., 5 mm). Mobile phase: Acetronitrile–acetic acid (45 : 55) for 2 min, to (75 : 25) at 3%/min, for 6 min. Flow rate: 1.7 mL/min.

Column: As for System HD, above. Mobile phase: Isopropyl alcohol–formic acid–0.1 mol/L potassium dihydrogenphosphate (13.61 g/L; 176 : 1 : 1000).

Analgesics, NSAIDs HD

HV

HW

HX

HY

HZ

HAA

k

RRT

k

RI

RI

RT

RT

0.5

–

2.3

–

281

–

–

0.1

–

0.32

–

–

–

–

Alclofenac

2.6

0.61

–

–

–

Aminophenazone

0.2

–

0.32

262

204

Aspirin

0.5

–

2.7

350

318

0.7

–

4.6

–

–

–

–

0.7

–

22.4

–

–

–

–

Aspirin

0.5

–

2.7

–

–

–

–

Paracetamol

0.1

–

0.32

–

–

–

– –

Acetanilide Paracetamol

Salicylic acid Benorilate

–

– 2.1

–

2.7

–

Benoxaprofen

11.3

0.98

–

–

–

–

Clonixin

–

0.87

–

–

345

–

–

Diclofenac

11.5

0.85

–

616

592

14.8

22.1

Diflunisal

4.1

0.77

–

508

583

5.4

–

Dipyrone

0.1

–

0.45

316

194

1.4

–

Etenzamide

0.55

–

4.6

–

303

–

–

Fenbufen

4

0.81

–

520

461

–

19.3

Fenoprofen

7.9

–

–

574

524

10.9

21.2

Floctafenine

–

–

–

–

–

Flufenamic acid

19.7

1

–

671

667

–

–

Flunixin

–

0.99

–

–

414

–

–

4.4

17.2

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:18

Recommended HPLC systems

Analgesics, NSAIDs, continued HD

Flurbiprofen

HV

HW

HX

HY

HZ

HAA

k

RRT

k

RI

RI

RT

RT

–

0.89

–

585

–

11.8

21.3

Glafenine

–

–

–

372

276

2.3

Ibuprofen

15.1

–

–

616

598

16.5

23.8

–

Indometacin

6.95

0.87

–

607

590

14.4

21.7

Indoprofen

1.2

0.52

–

–

406

–

–

0.66

–

495

–

6.4

Ketorolac

–

–

–

–

–

4.1

Meclofenamic acid

–

–

–

653

690

–

–

Ketoprofen

2.4

19.6 –

Mefenamic acid

21.1

0.95

–

661

686

–

–

Methyl salicylate

3.9

–

–

480

449

–

–

Salicylic acid

0.7

–

–

–

–

–

–

Morazone

0.4

–

–

294

–

Naproxen

3.3

–

–

501

468

–

–

–

313

–

12.7 –

Nefopam

–

Nifenazone Niflumic acid

2.05

–

0.1 –

– –

6.8

310

–

–

0.93

–

595

530

–

–

501

459

6.7

–

0.45

22

Oxyphenbutazone

1.95

0.69

Paracetamol

0.1

–

0.32

264

241

1.9

5.6

Phenacetin

0.6

–

4.4

377

335

3.0

–

Paracetamol

0.1

–

0.3

264

241

1.9

–

Phenazone

0.1

–

0.95

333

299

2.1

–

Phenylbutazone

6.5

0.95

–

672

643

19.5

1.95

0.7

–

501

459

6.7

Oxyphenbutazone Piroxicam M (5-hydroxy)

–

0.6 –

7.7

–

– 11

431

382

–

446

441

370

327

289

– –

4.9

1.3

–

0.4

–

Salsalate

3.6

0.69

–

–

–

Sulindac

1.25

0.78

–

488

462

3.9

–

–

–

–

–

7.2

–

–

–

366

–

Tenoxicam Tiaprofenic acid

–

–

–

484

452

Tolfenamic acid

–

–

–

690

–

Tolmetin

2.05

0.60 and 0.99

–

470

434

Zomepirac

3.7

–

–

–

495

16.6 –

Salicylamide

Sulindac sulfoxide

–

–

Propyphenazone

2.5

24.1

–

4.7

– – 16.6 –

–

12.7 5.8

17.6 –

37.9

–

5.4 –

–

Antibacterials The general screening systems, previously described, may be used.

Antibacterials, continued

HY

HAA

RI

RI

RT

Amoxicillin

–

226

3.1

Ampicillin

–

250

3.8

Azithromycin

–

–

Ceftriaxone

239

–

Chloramphenicol

390

336

14.1

Ciprofloxacin

318

260

9.1

Clarithromycin

–

–

–

Clindamycin

354

291

12

Furazolidone

336

–

12.2

– 5.3

table continued

HY

HAA

RI

RI

RT

Isoniazid

–

246

–

Metronidazole

257

226

6.8

Minocycline

–

240

22.6

Nalidixic acid

–

380

16

Nitrofurantoin

319

288

–

Ofloxacin

314

260

Oxytetracycline dehydrate

299

260

–

Rifampicin

–

417

16.2

Roxithromycin

–

–

15.8

Tetracycline

314

265

9.9

Trimethoprim

299

254

8.3

Antibacterials HX

HX

8.6

741

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:20

742

High Performance Liquid Chromatography

Anticholinergics

Anticonvulsants and barbiturates

The general screening systems, previously described, may be used or systems HAX and HAY below.

System HE

System HAX

n n

Koves EM (1995). J Chromatogr A 692: 103–119. n n n n n n

Column: Supelcosil LC-DP (250  4.6 mm i.d., 5 mm). Eluent: (A : B : C) Acetonitrile–phosphoric acid (0.025% v/v)– triethylamine buffer. Isocratic elution: (25 : 10 : 5). Flow rate: 0.6 mL/min. Detection: DAD (l  229 nm). Note: The triethylamine (TEA) buffer is prepared by adding 9 mL concentrated phosphoric acid and 10 mLTEA to 900 mLwater, adjusted to pH 3.4 with diluted phosphoric acid and made up to 1 L with water.

System HAY

Column: Alkyl-silica SAS-Hypersil (125  4.5 mm i.d., 5 mm). Mobile phase: Acetonitrile–tetrabutylammonium phosphate, 0.005 mol/L, pH 7.5 (20 : 80).

System HG

Gill R et al. (1981). J Chromatogr 204: 275–284. n n

Column: ODS Hypersil (150  4.6 mm i.d., 5 mm). Mobile phase: Methanol–0.1 mol/L sodium dihydrogenphosphate (11.998 g/L) (40 : 60); adjust to pH 3.5 by the addition of phosphoric acid.

System HH

Koves EM (1995). J Chromatogr A 692: 103–119. n n n n n

Christofides JA, Fry DE (1980). Clin Chem 26: 499–501.

Gill R et al. (1981). J Chromatogr 226; Biomed Appl 15: 117–123.

Column: LiChrospher 100 RP-8 (250  4.0 mm i.d., 5 mm). Eluent: (A : B : C) as for System HAX. Isocratic elution: (60 : 25 : 15). Flow rate: 0.6 mL/min. Detection: DAD (l  229 nm).

n n

Column: As for System HG, above. Mobile phase: As for System HG except that the mixture is adjusted to pH 8.5 by the addition of sodium hydroxide solution.

Anticholinergics HA

HX

HY

HZ

HAA

HAX

HAY

k

RI

RI

RT

RT

RT

RT

Adiphenine

1.8

422

–

–

–

–

Atropine

3.9

306

251

2.2

10.4

– 7

3.8

Biperiden

–

–

–

6.4

14.8

–

–

Chlorphenoxamine

2.9

–

346

–

–

–

–

Clidinium

–

379

–

–

–

–

–

Clidinium bromide

–

–

–

–

13.3

–

–

Cyclopentolate

1.6

353

287

3.2

–

–

–

Dicycloverine

1.1

–

575

–

–

–

–

Diethazine

3.4

–

–

–

–

15.1

7.4

Emepronium bromide

5.2

420

–

–

–

–

–

–

Homatropine

4.2

272

223

–

Hyoscine

1.1

270

253

–

Hyoscyamine

3.7

–

–

–

Isopropamide Iodide

2.4

379

–

–

7.4 9.7 –

6.8

3.6

7

3.7

–

–

–

–

Metixene

3.6

451

–

–

–

–

–

Orphenadrine

3

418

323

6

–

–

–

1.7

–

–

–

–

–

–

N-Monodesmethylorphenadrine N-Oxide Oxyphencyclimine

1.1

–

–

–

–

–

–

2.8

424

–

–

–

–

–

Oxyphenonium bromide

2.6

424

–

–

–

–

–

Piperidolate

1.7

429

–

–

–

–

–

Procyclidine

2

406

–

6.2

–

>20

4.7

Profenamine

2.4

444

338

–

–

16.6

8.3

Propantheline bromide

4.4

454

–

–

–

–

–

–

499

–

–

–

–

–

1.8

429

381

7.6

15.3

–

–

Xanthanoic acid Trihexyphenidyl

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:22

Recommended HPLC systems

743

Anticonvulsants, barbiturates and antiepileptics HG

HH

k

k

Allobarbital

2.46

1.33

HX

HY

HZ

RI

RI

RT

346

–

2.7

Amobarbital

10.91

7.05

424

374

4

Aprobarbital

3.42

2.22

357

319

2.8

Barbital Benactyzine

1.11 –

0.63 –

308

258

2.2

382

–

–

Brallobarbital

3.09

1.72

371

336

3

Butalbital

6.17

3.48

394

342

3.4

390

–

–

3.42

384

355

3.2

Butetamate

–

Butobarbital

– 5.43

Carbamazepine

–

–

418

368

–

Clonazepam

–

–

465

403

4.6

Cyclobarbital

5.25

2.61

384

352

3.2

Cyclopentobarbital

6

3.84

391

352

–

Enallylpropymal

8.65

Ethosuximide

–

Flavoxate

–

Heptabarb Hexethal

6.96 – –

9.9 34.28

4.93 20.39

–

394

–

301

276

2.3

–

–

–

416

377

3.9

–

451

–

Hexobarbital

7.37

5.67

419

242

4.3

Ibomal

4.01

2.58

379

352

–

Idobutal Mebeverine

8.12 –

4.77 –

–

357

–

448

–

7.1

Mephenytoin

–

–

–

366

3.7

Mesuximide

–

–

–

387

4.8

Metharbital

2.69

1.99

435

324

–

Barbital

1.11

0.63

–

–

–

7.27

3.84

435

395

4.6

Nealbarbital

Methylphenobarbital

10.22

6.19

417

382

–

Papaverine

–

363

295

–

–

Pentobarbital

10.96

Phenacemide

–

Phenobarbital

8.07 –

3.09

Phenytoin

–

Primidone

–

Secbutabarbital

1.23 – –

4.9

3.3

424

383

4.1

339

266

–

379

335

3

431

381

3.7

322

288

2.1

377

331

–

Secobarbital

16.28

11.47

437

407

4.7

Sultiame

–

–

344

275

–

403

370

–

Talbutal

7.2

4.7

Thiamylal

–

–

516

476

–

Thiopental

–

–

485

433

6.9

Vinbarbital Vinylbital

4.83 –

2.32 –

Antidepressants The general screening systems, previously described, may be used or systems HF and HAZ below. System HF

R Gill, unpublished data, after Kabra PM et al. (1981). Clin Chim Acta 111: 123–132.

379

363

–

424

–

4.1

n n

Column: ODS Hypersil (160  5 mm i.d., 5 mm). Mobile phase: Acetonitrile–phosphate buffer (pH 3.0; 30 : 70). To prepare the phosphate buffer, add 0.6 mL nonylamine to 1 L 0.01 mol/L sodium dihydrogenphosphate (1.1998 g/L) and adjust the pH to 3.0 by the addition of phosphoric acid.

System HAZ

Chiba K et al. (1995). J Chromatogr B 668: 77–84.

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:26

744 n n

High Performance Liquid Chromatography

Column: C18 (250  4.0 mm i.d., 5 mm). Mobile phase: (A : B : C) Water–methanol–triethylamine adjusted to pH 5.5 with phosphoric acid.

n n n

Isocratic elution: (70 : 30 : 0.1). Flow rate: 0.7 mL/min. Detection: UV (l ¼ 240 nm).

Antidepressants and antipsychotics HA

HF

HX

HY

HZ

HAA

HAX

HAZ

k

k

RI

RI

RT

RT

RT

k

440

375

15.9

15.8

1.76

10-Hydroxyamitriptyline

2.9

–

–

–

–

–

–

–

10-Hydroxynortriptyline

1.8

–

–

–

–

–

–

–

Nortriptyline

2

–

–

–

–

–

1.71

398

–

–

14.2

–

–

Amitriptyline

3.3

5.42

4.58

–

–

Benperidol

1.1

–

Butriptyline

2.7

Amoxapine

Norbutriptyline Citalopram Desmethylcitalopram Clomipramine Monodesmethylclomipramine Desipramine

7.33

7.5

393

324

–

–

–

–

369

–

–

–

–

–

–

–

– –

3.6

1.7

–

–

–

–

–

403

–

4.5

–

–

–

–

–

–

3.7

–

–

–

9.92

462

405

10.2

16.4

–

–

–

–

–

3.6

424

361

3.4 2

–

2.1

5.9

–

–

–

14.9

13

1.52

Didesmethylimipramine

1.3

–

–

–

–

–

–

–

2-Hydroxydesipramine

1.2

–

–

–

–

–

–

–

M (2-OH-)

–

–

–

–

–

–

–

0.39

0.5

361

300

–

–

–

–

3.6

Dibenzepin

2.8

Dosulepin

3.2

428

367

–

–

–

M (sulfoxide)

4.6

–

–

–

–

–

–

–

M (nor-)

2.2

–

–

–

–

–

–

–

404

316

14.1

12.9

–

Doxepin M (nor-) Fluoxetine

3.7

2.27

5.7

5

2.2

–

–

–

4.6

–

–

–

–

–

–

400

7.6

16.2

12.2

–

–

–

–

–

6.7

–

–

–

Fluvoxamine

–

–

430

363

5.6

15.3

10

–

Imipramine

6.7

Desmethylfluoxetine

4.2

4.17

437

335

15.1

14.7

1.62

Desipramine

2.1

3.6

–

–

–

–

–

–

2-Hydroxydesipramine

1.2

–

–

–

–

–

–

–

2-Hydroxyimipramine

3.1

–

–

–

–

–

–

–

M (10-OH-)

–

–

–

–

–

–

–

0.39

M (2-OH-)

–

–

–

–

–

–

–

0.39

M (N-oxide)

–

–

–

–

–

–

–

1.85

Iprindole

4.1

10.83

–

–

–

–

–

–

Isocarboxazid

–

–

392

353

–

–

–

–

Maprotiline

2.2

4.92

15.5

–

1.44

–

–

–

13.8

–

1.18

–

–

–

–

–

–

0.88

–

–

–

–

0.53

–

–

–

438

389

1.1

–

–

–

Mianserin

1.8

–

391

342

M(nor-)

2.4

–

–

–

–

M (nor-)

–

–

–

–

M (N-oxide)

–

–

–

M (8-OH-)

Desmethylmaprotiline

6.6 – 4.6

–

–

–

–

Moclobemide

–

–

295

–

Nialamide

1.2

–

334

–

– –

2.4

10.2 –

0.19 6.9

–

– –

–

–

–

15.6

13.7

1.71

Nomifensine

0.9

0.42

349

296

Nortriptyline

2

4.58

–

338

–

–

–

–

–

–

1.63

–

330

–

–

–

–

377

340

3.9

14.2

–

–

10-Hydroxynortriptyline

1.8

Noxiptiline

–

Opipramol

2.2

– 1.63

6.6

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:30

Recommended HPLC systems

745

Antidepressants and antipsychotics, continued HA

Paroxetine

HF

HX

HY

HZ

HAA

HAX

HAZ

k

k

RI

RI

RT

RT

RT

k

–

–

426

337

5.6

15.3

11.1

–

Phenelzine

1

–

184

–

–

–

–

–

Protriptyline

2.1

3.6

418

362

–

–

–

–

Remoxipride

–

–

334

–

3

–

M (FLA-838)

–

–

316

–

–

–

–

8.8 –

–

M (NCM-001)

–

–

364

–

–

–

–

–

M (NCM-009)

–

–

341

–

–

–

–

–

Sertraline

–

–

460

–

8.2

–

14.5

–

–

–

–

–

7.0

–

–

–

1.7

–

–

–

5.3

–

–

–

(desmethylsertraline) Tofenacin Trazodone

0.6

–

378

305

3.3

12.7

–

–

Trimipramine

2.7

6.17

454

345

8.3

15.9

15.5

–

1.8

–

–

–

–

–

–

–

Viloxazine

–

2.7

325

273

–

11

–

–

Zimeldine

3.2

0.67

–

270

–

–

–

–

M (nor-)

2.9

–

–

–

–

–

–

–

M (nor-)

Antihistamines

Antimalarials

The general screening systems, previously described, may be used.

The general screening systems, previously described, may be used.

Antihistamines HA

HX

HY

HZ

HAA

HAX

HAY RT

k

RI

RI

RT

RT

RT

Alimemazine

3.1

420

–

–

–

14.9

Antazoline

1.8

383

294

–

–

–

–

7.1

–

–

286

13.2

–

–

(astemizole)

–

383

–

–

–

–

–

(M-nor)

Astemizole

3.9

–

361

–

–

–

–

–

Bromazine

2.7

444

–

–

–

–

–

Brompheniramine

4.1

–

267

–

13.9

–

–

Buclizine

0.7

–

454

–

–

–

–

Carbinoxamine

4.7

359

–

–

12.8

–

Cetirizine

–

–

–

Chlorcyclizine

2.3

–

340

Chlorphenamine

3.9

356

264

Cinnarizine

0.8

560

–

Clemastine

3.7

501

–

Clemizole

4.8

420

–

Cyclizine

2.9

405

–

Norcyclizine

2.2

–

–

Cyproheptadine

3.2

–

354

Deptropine

5

471

–

10.3

Dimetindene

5.1

338

288

Diphenhydramine

3.3

393

Diphenylpyraline

3.3

Doxylamine

4.4

Hydroxyzine

3.6 –

15.7

– 8.89

5.29

–

–

12.9

10.8

22

19.3

–

–

14

–

–

–

–

–

–

–

–

12.4

–

–

–

15

–

–

–

–

–

–

–

–

–

336

–

–

12.2

401

–

–

–

–

–

–

259

–

11.1

–

–

1.4

437

326

15.3

11.4

Isothipendyl

3.8

390

–

–

13.5

–

–

Loratadine

–

523

362

14.6

22.9

10.9

13.3

3.5

4.8 – 6.5

5.7

– 5.3

5.8

6

6.3

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:32

746

High Performance Liquid Chromatography

Antihistamines, continued HA

HX

HY

HZ

HAA

HAX

HAY

k

RI

RI

RT

RT

RT

RT

Mebhydrolin

3

411

–

5.3

–

–

–

Meclozine

0.7

587

398

–

20

–

–

Mepyramine

3.9

448

257

–

–

–

–

Methapyrilene

4.1

342

197

–

–

–

–

Methdilazine

6

–

–

–

–

15.2

6.7

Phenindamine

2.5

397

–

–

–

–

–

Pheniramine

4.1

283

206

–

–

Phenyltoloxamine

3.1

415

–

–

–

–

–

Pizotifen

3.4

435

–

6.6

15.2

–

–

Promethazine

5

409

324

5.7

14.5

13.2

6.4

Propiomazine

2.1

440

359

–

–

14.1

7.1

Pyrrobutamine

2.8

477

–

–

–

–

–

Thenyldiamine

4

317

–

–

–

–

–

Thiazinamium metilsulfate

–

–

–

6.4

–

–

–

Trimethobenzamide

4.7

347

–

–

–

–

–

Tripelennamine

3.6

336

265

–

–

–

–

Triprolidine

3.2

388

270

–

13.1

–

–

9.5

4.5

Antimalarials HA

HX

HY

HZ

HAA

HAX

HAY

RT

RT

RT

k

RI

RI

RT

Chloroquine

15.2

282

246

2.1

Cinchonidine

3.1

306

214

–

5.4

12.7

3.6

–

–

– –

Cinchonine

–

304

209

–

10.2

–

Halofantrine

–

800

–

–

23

–

Hydroxychloroquine

–

280

–

1.9

–

1.4

–

276

–

–

379

–

3.8

13.6

–

–

Pyrimethamine

1

–

289

–

12.5

–

–

Quinine

2.4

327

246

2.6

11.3

Primaquine Proguanil

–

– 9.6

3.2

–

–

8.3

4.5

Antitussives

Antineoplastics

HA

HX

HY

HAA

The general screening systems, previously described, may be used.

k

RI

RI

RI

Antineoplastics

Bromhexine

0.4

417

334

–

Dextromethorphan

5.6

377

298

13.3

HX

HAA

4.7

–

–

–

RI

RT

Dextrorphan

–

325

–

–

Dextrorphan

Diethylstilbestrol

592

20.9

Dropropizine

–

240

–

Doxorubicin

370

12.1

Guaifenesin

–

328

262

11.4

3.4

Noscapine

0.3

368

289

12.8

Pholcodine

6

65

92

2.7

Pipazetate

5.4

385

–

Fluorouracil

70

Methotrexate

292

Vinblastine

–

– 8.4

7.2

–

Antivirals Antitussives The general screening systems, previously described, may be used.

The general screening systems, previously described, may be used or systems HAB and HAC below.

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:37

Recommended HPLC systems System HAB

Sparidans RW et al. (2000). J Chromatogr B Biomed Sci Appl 742: 185–192. n n n n n

Column: C18 Symmetry (100  4.6 mm i.d., 3.5 mm) with Symmetry C18 precolumn (20  3.8 mm, 5 mm). Mobile phase: Acetonitrile–sodium phosphate buffer (25 mmol/L, pH 6.8) (40 : 60). Flow rate: 1.5 mL/min. Detection: Fluorescence (lex ¼ 270 nm, lem ¼ 340 nm). Note: 8 min after each injection, flush column for 5 min at 1.5 mL/ min with acetonitrile–water (30 : 70). Equilibrate for about 8 min with the original eluent before injecting the next sample.

System HAC

Aymard G et al. (2000). J Chromatogr B Biomed Sci Appl 744: 227–240. n n n n n n

Column: C18 Symmetry (250  4.6 mm i.d., 5 mm) with C18 precolumn (Guard-Pak, mBondapak). Column temperature: 37 C. Mobile phase: (A : B) Disodium hydrogenphosphate (0.04 mol/L) with 4% (v/v) octane sulfonic acid (0.25 mol/L)–acetonitrile. Isocratic elution: (50 : 50). Flow rate: 1.3 mL/min. Detection: DAD (l ¼ 261 nm between time 0 and 9 min; l ¼ 241 nm between time 9 and 20 min; l ¼ 254 nm between time 20 and end of the run (32 min).

Cannabinoids System HL

Baker PB et al. (1980). J Anal Toxicol 4: 145–152. n n

Column: ODS Spherisorb (250  4.6 mm i.d., 5 mm). Mobile phase: 0.01 mol/L sulfuric acid–methanol–acetonitrile (7 : 8 : 9).

Cannabinoids System HL k Cannabichromene

19.09

Cannabicyclol

14.78

Cannabidiol

7.47

Cannabidiolic acid

8.76

Cannabigerol

8.18

Cannabinol

11.77

Cannabivarin

7.47

D8-Tetrahydrocannabinol

14.07

D9-Tetrahydrocannabinol

13.35

Tetrahydrocannabinolic acid

25.83

Tetrahydrocannabivaric acid

14.64

Tetrahydrocannabivarin Antivirals HAB

HAC

RT

k

Abacavir

1

–

Amprenavir

4

2.5

Efavirenz

–

8.5

Indinavir

4.2

2

747

8.18

Cardiac glycosides System HM

Cobb PH (1976). Analyst (Lond) 101: 768–776. n n

Column: Silica LiChrosorb SI60 (250  4 mm i.d., 10 mm). Mobile phase: Cyclohexane–ethanol–acetic acid (60 : 9 : 1).

Cardiac glycosides System HM k

Benzodiazepines System HI

Digitoxigenin

2.0

R Gill, unpublished data.

Digitoxigenin bisdigitoxoside

3.9

Digitoxigenin monodigitoxoside

2.8

Digitoxin

5.4

Digoxigenin

4.5

Digoxigenin bisdigitoxoside

8.2

Digoxigenin monodigitoxoside

5.5

n n

Column: ODS Hypersil (200  5 mm i.d., 5 mm). Mobile phase: Methanol–water–phosphate buffer (55 : 25 : 20). To prepare the phosphate buffer, dissolve 11.038 g (0.092 mol/L) sodium dihydrogenphosphate and 1.136 g (0.008 mol/L) disodium hydrogenphosphate in sufficient water to produce 1 L.

System HJ

R Gill, unpublished data.

Digoxin

11.3

n n

Gitaloxin

6.8

Gitoxigenin

3.7

Gitoxigenin bisdigitoxoside

6.5

System HK

Gitoxigenin monodigitoxoside

4.5

R Gill, unpublished data, after RJ Flanagan et al. (1980). J Chromatogr 187: 391–398.

Gitoxin

n n

Column: As for System HI, above. Mobile phase: Methanol–water–phosphate buffer (as in System HI) (70 : 10 : 20).

Column: Silica Spherisorb (250  5 mm i.d., 5 mm). Mobile phase: Methanol to which has been added 100 mL perchloric acid per litre.

8.6

Lanatoside A

17.9

Lanatoside B

31.8

Lanatoside C

39.5

Benzodiazepines HI

HJ

HK

HX

HY

HZ

HAA

HAX

HAY

k

k

k

RI

RI

RT

RT

RT

RT

Acecarbromal

–

–

–

429

374

–

–

–

–

Alprazolam

–

–

2.79

–

–

–

–

–

–

Bromazepam

–

–

2.99

–

–

–

–

–

– table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:42

748

High Performance Liquid Chromatography

Benzodiazepines, continued HI

HJ

HK

HX

HY

HZ

HAA

HAX

HAY

RT

k

k

k

RI

RI

RT

RT

RT

Bromisoval

–

–

–

365

307

2.9

–

–

–

Brotizolam

–

–

–

484

–

4.6

–

Carbromal

–

–

–

410

377

3.9

–

–

–

Chlordiazepoxide

–

–

2.87

–

–

–

–

–

–

Clobazam

–

–

0.03

–

–

–

–

–

–

7.4

7.9

Clomethiazole

–

–

–

395

292

–

16

–

–

Clonazepam

–

–

0.35

–

–

–

–

–

–

Clorazepic acid

–

–

2.00

–

–

–

–

–

–

Demoxepam

–

–

0.03

–

–

–

–

–

–

Diazepam

–

–

2.49

–

–

–

–

–

–

Flumazenil

–

–

–

387

327

2.6

–

–

–

Flunitrazepam

3.15

–

0.47

483

305

5.6

18.6

–

–

Flurazepam

–

3.19

6.5

397

305

4.2

–

10.5

5.5

Glutethimide

–

–

–

436

401

4.8

–

6.6

6.2

Ketazolam

–

–

0.04

–

–

–

–

–

–

Loprazolam

–

–

–

388

–

–

13.4

–

–

Lorazepam

–

–

0.14

–

–

–

–

–

–

Lormetazepam

6.32

–

0.08

487

463

–

–

–

Medazepam

–

–

4.44

–

–

–

–

Methaqualone

–

–

–

459

400

Methyprylon

–

–

–

347

302

Midazolam

9.75

2.1

5.9

399

306

Nitrazepam

2.96

–

1.49

448

370

6.2 – 5.4 –

–

– 6.8

–

–

4.2

14.9

10.2

4.2

16.9

6.3

7.4 – 6.3 6

Nordazepam

–

–

1.99

–

–

–

–

–

–

Oxazepam

4.62

–

0.73

–

–

–

–

–

–

Prazepam

–

–

2.19

–

–

–

–

–

–

Quazepam

–

–

–

–

766

37.5

–

11.9

Temazepam

5.68

–

0.6

472

438

5.5

–

–

0.73

–

–

Oxazepam

4.38

–

1.83

476

390

–

–

–

–

–

Zolpidem

–

–

–

–

291

Zopiclone

–

–

–

331

269

Triazolam Not detected

–

18.6 –

4.2 –

17.7

8.9

6.7

–

17.4

– 6.4

6.7

–

–

–

3.2

11.9

–

–

2.3

–

7.5

3.8

Cardioactive drugs The general screening systems, previously described, may be used.

Cardioactive drugs, continued

Cardioactive drugs

HA HA

HX

HY

HZ

HAA

k

RI

RI

RT

RT

Ajmaline

2.8

–

277

Alfuzosin

–

–

–

Amiodarone

2.4

683

476

90.4

–

1.8

–

–

–

–

–

433

–

–

17

0.9

250

–

–

415

–

Monodesethylamiodarone Aprindine Bamethan Benzthiazide

–

–

– 2.4

Betahistine

3.1

–

–

–

Bretylium tosilate

4.3

–

275

–

Buphenine

HX

HY

HZ

k

RI

RI

RT

0.9

370

–

–

HAA RT –

–

Captopril

–

316

283

2.1

9.7

10.4

Cilazapril

–

420

–

4.5

14.4 –

–

–

1.7

1.2

258

194

2.5

Clopamide

–

377

310

–

–

Debrisoquine

1.2

–

245

–

–

Diltiazem

–

–

361

4.5

14

Deacetyldiltiazem

–

–

–

–

–

Desmethyldiltiazem

–

–

–

–

–

Cilazaprilate

5.9 – 3.2 –

–

Clonidine

6.1

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:46

Recommended HPLC systems

Cardioactive drugs, continued

Desacetyldiltiazem Disopyramide N-Monodesisopropyldisopyramide

HA

HX

HY

HZ

HAA

k

RI

RI

RT

RT

–

–

–

–

–

2.4

345

281

1.8

–

–

n n

Column: ODS Hypersil (160  5 mm i.d., 5 mm). Mobile phase: Acetonitrile–water containing 10 mL/L acetic acid (30 : 70).

Diuretics HN

3 –

11.4

–

201

–

Encainide

–

363

–

– 25.8

HX

HY

HAA RT

k

RI

RI

Acetazolamide

–

268

226

–

Enalapril

749

6.9

Amiloride

–

257

190

–

Bendroflumethiazide

15.35

508

–

18.6

1.5

3.4

3.6

Felodipine

–

690

–

24.4

Benzthiazide

9.32

–

415

–

Flecainide

–

419

355

5.2

–

Chlorothiazide

0.54

–

239

–

Hydralazine

–

193

132

1.9

–

Chlortalidone

1.28

367

308

–

Isoxsuprine

0.8

353

301

–

Clopamide

4.01

377

310

–

Labetalol

1.7

365

290

–

Clorexolone

7.26

–

391

–

Lidoflazine

0.6

530

–

–

Cyclopenthiazide

16.45

–

453

–

Lisinopril

–

271

250

1.5

–

Cyclothiazide

–

433

–

–

10.78, 11.91, and 12.81

Etacrynic acid

–

521

497

–

Furosemide

–

435

380

15.2

– 3 –

Lorcainide

1.8

425

–

6.6

Methyldopa

–

69

–

1.4

Mexiletine

1.2

329

278

–

3 11.5

Hydrochlorothiazide

0.7

294

255

–

Mefruside

8.67

–

417

–

Methyclothiazide

3.82

–

364

15.4

19.5

Metolazone

4.89

–

371

–

Minoxidil

–

297

–

Naftidrofuryl oxalate

–

–

409

Nifedipine

0.2

527

464

Pargyline

0.2

–

203

–

–

Spironolactone

–

592

539

20.7

Pentaerithrityl tetranitrate

–

663

–

–

–

Triamterene

–

298

263

8.7

–

–

–

–

–

341

14.9

23.1

355

274

–

18.8

Pentoxifylline

–

488

Perindopril

–

–

–

13.7

Drugs of abuse

–

314

–

–

–

0.1

396

–

–

–

A comprehensive HPLC method for the screening of common drugs of abuse is described in Chapter 1, Table 1.24. Furthermore, an additional eight systems (HBC, HBD, HBE, HBF, HBG, HBI and HBJ) are provided in Chapter 11, Table 11.4.

(pentaerithrityl)

(perindoprilat) Phenoxybenzamine

2.4 –

9.8 15.8

7.2

Trichlormethiazide 2.1 1.6

3.1 –

Xipamide

11.5

Phentolamine

1.7

368

–

Prajmalium bitartrate

2.2

–

340

Prazosin

0.8

352

–

2.5

10.6

Procainamide

1.3

208

160

1.9

–

HA

HC

HX

HY

HZ

HAA

3

–

–

1.8

–

k

k

RI

RI

RI

k

Quinapril

–

–

–

5.4

16.8

–

–

–

–

–

–

Quinidine

2.1

322

245

2.6

11

Amfetamine

0.9

0.98

244

–

–

–

–

–

Ramipril

4.2

15.7

Benzfetamine

1.2

0.15

–

0.6

496

407

–

–

Rescinnamine

–

–

Benzoylecgonine

0.9

1.7

467

351

–

–

236

–

–

Reserpine

16.4

Bufotenine

3.1

–

181

–

–

Sotalol

1.2

226

–

–

2

3.8

Cannabidiol

–

–

990

902

–

–

Tocainide

1.2

247

208

2.1

Tolazoline

2.1

225

179

Trandolapril

–

–

–

–

–

–

Trimetazidine

3

–

–

Verapamil

2.6

447

386

7

15.4

M (nor-)

1.7

–

–

6.6

–

N-Acetylprocainamide

3 –

– –

Drugs of abuse

5-Methyltryptamine

3.7 – 9.7

–

Cannabinol

–

–

1080

1028

–

–

–

Cocaine

2.8

–

348

289

3.3

11.9

6.1

17 –

D9-THC

–

–

–

–

–

–

2.1

Diamorphine

3

0.66

340

282

–

–

Diethyltryptamine

–

–

–

–

–

–

Dimethyltryptamine

–

–

–

228

–

–

Diuretics

DOM

–

1.13

340

–

–

–

System HN

Ketamine

–

–

311

262

2.4

R Gill et al., unpublished data, after Tisdall PA et al. (1980). Clin Chem 26: 702–706.

Lysergic acid

0.8

–

–

236

–

–

Lysergide

0.7

–

362

–

–

12

Trandolaprilat

–

–

6.1

9.6

table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:48

750

High Performance Liquid Chromatography Local anaesthetics

Drugs of abuse, continued HA

HC

HX

HY

HZ

HAA

k

k

RI

RI

RI

k

The general screening systems, previously described may be used, as well as system HQ or HR, below.

Mescaline

1.3

2.17

272

243

–

–

System HQ

Metamfetamine

2

2.07

262

216

2.4

8.4

Methadone

2.2

1.03

440

343

8.5

15.8

Methylenedioxyamfetamine

–

0.98

266

248

2.1

8.1

Methylenedioxymethamfetamine

–

–

278

252

2.2

9.1

Monoacetylmorphine

3.6

0.8

–

–

–

7.3

Morphine

3.8

1.3

200

182

1.8

N-Methyltryptamine

–

–

–

–

–

–

p-Methoxyamfetamine

–

–

–

–

–

–

Psilocin

3.1

–

240

226

–

–

Psilocybine

–

–

–

185

–

–

System HA, previously described, may be used or System HP, below. System HP

R Gill et al., unpublished data, after Twitchett PJ et al. (1978). J Chromatogr 150: 73–84. Column: ODS Hypersil (100  5 mm i.d., 5 mm). Mobile phase: Methanol–phosphate buffer (60 : 40). To prepare the phosphate buffer, dissolve 3.43 g (0.022 mol/L) sodium dihydrogenphosphate and 10.03 g (0.028 mol/L) disodium hydrogenphosphate in sufficient water to produce 1 L.

Ergot alkaloids HA

HP

k

k

Bromocriptine

–

44.3

Dihydroergocristine

–

18.3

Dihydroergocryptine

–

15.9

Dihydroergotamine

0.6

11.4

Ergocornine

0.4

10.2

Ergocristine

0.3

17.3

Ergocryptine

0.4

15.2

Ergometrine

0.4

Ergosine

0.3

Ergosinine

0.3

Ergotamine

0.4

n n

0.50 7.08 17.7 9.58

Column: ODS Hypersil (160  5 mm i.d., 5 mm). Mobile phase: Methanol–water–1% v/v solution of phosphoric acid– hexylamine (30 : 70 : 100 : 1.4).

System HR

Gill R et al. (1984). J Chromatogr 301: 155–163. n n

3.3

Ergot alkaloids

n n

Gill R et al. (1984). J Chromatogr 301: 155–163.

Column: As for System HQ above. Mobile phase: Methanol–1% v/v solution of phosphoric acid–hexylamine (100 : 100 : 1.4).

Local anaesthetics HA

HQ

HR

HX

HY

HZ

k

k

k

RI

RI

RT

Benzocaine

0.1

20.06

1.61

404

358

4.3

Bupivacaine

0.9

7.19

0.86

366

310

4.1

Butacaine

1.2

8.97

–

392

331

–

Butanilicaine

–

4.42

–

–

280

–

Chloroprocaine

–

0.24

–

–

250

–

Cinchocaine

1.9

–

371

–

Cocaine

2.8

2.68

–

348

289

3.3

Benzoylecgonine

0.9

5.68

–

–

–

–

Ecgonine

1.1

–

–

–

–

–

Cyclomethycaine

–

–

10.31

–

413

–

Dyclonine

–

–

2.78

–

347

–

Etomidate

–

–

475

417

–

Ketamine

–

–

–

311

262

Lidocaine

0.6

–

288

258

2.6

–

–

–

–

–

296

260

2.6

–

503

484

–

405

–

–

357

312

–

415

–

6.5

–

–

–

2.7 –

M (monoethylglycinexylidide)

1.2

–

5.51

0.79 – 1.09

2.4

Mepivacaine

0.9

Methohexital

–

–

Oxybuprocaine

–

16.25

Piperocaine

–

4.59

Pramocaine

0.6

Prilocaine

1

Procaine

1.9

–

–

264

225

Propofol

–

–

–

–

–

35

Proxymetacaine

2.1

–

–

269

–

Quinisocaine

2.2

–

–

–

–

Tetracaine

2

16.25

389

321

4.4

0.86 –

–

2.48 1.38

1.38

11.24 1.33

Iso-lysergic acid

–

0.83

Iso-lysergide

2.6

0.0

Lysergamide

0.5

0.33

Narcotic analgesics

Lysergic acid

0.8

0.0

Lysergic acid methylpropylamide

–

1.98

Systems HA or HC, previously described, may be used or System HS, below.

Lysergide

0.7

1.83

System HS

Lysergol

1.1

0.83

Baker PB, Gough TA (1981). J Chromatogr Sci 19: 483–489. n

Methylergometrine

0.4

0.83

Methysergide

0.4

2.33

2-Oxylysergide

–

0.92

n

Column: Aminopropyl-bonded silica Spherisorb S5NH2 (250  4 mm i.d., 5 mm). Mobile phase: Acetonitrile–tetrabutylammonium phosphate, 0.005 mol/L, pH 7.5 (85 : 15).

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:52

Recommended HPLC systems

Narcotic analgesics and narcotic antagonists HA

HC

HS

HX

HY

HZ

HAA

HAX

HAY

k

k

k

RI

RI

RT

RT

RT

RT

Alphaprodine

2.8

–

–

363

317

–

–

–

–

Bezitramide

0.2

–

–

564

–

22.5

–

–

–

Buprenorphine

0.4

0.05

–

397

339

5

14

–

Codeine

4.8

1.21

1.9

266

237

1.9

Morphine

3.8

1.3

5.16

–

–

–

–

–

–

M (nor-)

3.1

3.51

–

–

–

–

–

–

–

Cyclazocine

2.1

–

–

–

289

–

–

–

–

Dextromoramide

0.7

0.09

–

440

390

–

15.8

–

–

Dextropropoxyphene

1.9

0.19

–

–

374

15.8

–

–

Norpropoxyphene

1.3

–

–

–

–

–

–

–

7.6 –

5

– 6.1

3.4

3

0.66

0.35

340

282

–

–

6-Monoacetylmorphine

3.6

0.8

1

–

–

–

–

–

–

Morphine

3.8

1.3

5.16

–

–

–

–

–

–

Dihydrocodeine

7.2

2.5

–

261

208

–

–

Dihydromorphine

5.7

2.75

–

237

156

–

–

–

–

Dipipanone

2.2

1.61

–

500

363

–

–

–

–

Ethoheptazine

3.3

1.55

–

359

–

–

–

–

Ethylmorphine

3.7

1.06

1.45

291

244

–

–

Fentanyl

0.8

1.11

–

373

299

–

14.2

Hydromorphone

7.9

–

–

240

187

–

–

Ketobemidone

2.8

–

–

294

245

–

–

–

–

Levallorphan

1.9

1.46

–

356

291

–

–

–

–

Levorphanol

4.4

3.2

–

–

265

–

–

–

–

Meptazinol

3.1

–

–

–

269

–

–

–

–

Methadone

2.2

1.03

–

440

343

15.8

16.5

8.4

M (EDDP)

2.8

–

–

–

–

–

–

–

–

M (EMDP)

0.2

–

–

–

–

–

–

–

3.8

1.30

5.16

200

182

Morphine3-glucuronide

–

1.56

–

–

–

–

–

N-oxide

–

Diamorphine

Morphine

2

8.5

7.9

4.7

1.8

– 6.7

11.4 5.8

3.3

4.1

3.6 6 3.4

– 5.6

3.2

–

–

–

–

–

–

14

–

–

3.2

–

–

–

–

–

Nalorphine

1

0.29

–

260

237

–

Naloxone

1.4

0.17

–

–

238

Norcodeine

3.1

3.51

–

–

235

–

–

–

–

Normethadone

–

0.53

–

–

366

–

–

–

–

Normorphine

2.9

3.92

–

–

133

–

–

–

–

Norpipanone

–

0.35

–

466

–

–

–

–

Oxycodone

6.9

0.85

–

277

246

–

–

6.7

–

–

–

–

–

–

–

Oxymorphone

6.7

–

–

217

184

–

–

–

Pentazocine

1.8

0.67

–

372

288

3.8

12.5

9.9

5.5

Pethidine

2.8

0.55

–

345

281

3.2

11.8

9.2

4.8

M (nor-)

1.7

2.04

–

–

–

–

–

–

–

Pethidinic acid

2.8

–

–

–

–

–

–

–

–

Phenazocine

1.3

0.3

–

409

299

–

–

–

–

Phenoperidine

Oxymorphone

4.8 2

– 6.5

5.8 – –

0.8

0.1

–

434

–

–

–

–

–

Norpethidine

1.7

2.04

–

–

–

–

–

–

–

Pethidine

2.8

0.55

–

–

–

–

–

–

–

Piritramide

0.6

0.1

–

377

343

–

–

–

–

Thebacon

3.7

0.85

–

333

–

–

–

–

–

Tramadol

–

–

–

328

267

–

–

–

2.9

751

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:10:55

752

High Performance Liquid Chromatography

Oral hypoglyacemics and antidiabetics

Pesticides

The general screening systems, previously described, may be used.

System HAO

Osselton MD, Snelling RD (1986). J Chromatogr 368: 265–271.

HX

HY

HZ

HAA

Column: ODS Hypersil (160  5 mm i.d., 5 mm), stainless steel. Mobile phase: Acetonitrile–water (60 : 40). Flow rate: 2 mL/min. Detection: DAD (200–450 nm).

n n n n

Oral hypoglyacemics and antidiabetics

RI

RI

RT

RT

Carbutamide

–

321

–

14.5

Osselton MD, Snelling RD (1986). J Chromatogr 368: 265–271.

Chlorpropamide

450

411 and 413

17.7

Glibenclamide

637

571

14.4

Gliclazide

536

483

8.8

20.5

n n n n

Glipizide

478

423

4.5

17.6

Metformin

60

–

1.7

2.8

Tolazamide

452

445

6.8

–

Tolbutamide

477

424

5.9

–

5

System HAP

22

Column: Silica Spherisorb S5W (250  5 mm i.d.). Mobile phase: Dichloromethane–isoctane (60 : 40). Flow rate: 2 mL/min. Detection: DAD (200–450 nm).

For more information on screening pesticides, see Chapter 16, Table 16.1. Phenothiazines and other tranquillisers The general screening systems, previously described, may be used.

Phenothiazines and other tranquillisers HA

Acepromazine

HX

HY

HZ

HAA

HAX

HAY

HAZ

k

RI

RI

RT

RT

RT

RT

k

4.1

–

350

–

10.8

–

–

–

Azacyclonol

1.2

–

–

–

–

4.5

–

Benzoctamine

1.7

380

322

–

–

–

–

–

8.7

Butaperazine

3.4

464

406

–

–

–

–

–

Captodiame

–

561

–

–

20.2

–

–

–

Chlordiazepoxide

–

363

285

Chlormezanone

–

–

334

Chlorpromazine

3.2 –

6.9

5.3

1.68

6

5.3

–

4.1

456

350

16

17

BASE

2.64

M (nor-)

2.2

–

–

–

–

–

–

–

M (sulfoxide)

–

–

–

–

–

8.4

4.3

0.62

3

459

353

10.1

–

17.6

8.3

–

–

Chlorprothixene Clopenthixol

–

448

411

Clorazepic acid

–

475

388

9.1

15.2 15.5

5.6

–

–

–

–

–

–

–

–

–

–

–

–

18.4

–

–

–

–

423

349

–

–

–

–

–

Flupentixol

1.2

475

435

10.7

17.4

13.7

7.5

–

Sulfoxide

1.3

–

–

–

–

–

–

–

Fluphenazine

1.2

462

471

10.1

17.4

13.6

7.2

–

–

Clorazepate Fluanisone

Fluspirilene

–

538

–

Haloperidol

1.2

421

316

Levomepromazine

3.2

435

381

Loxapine

1.1

407

336

5.8 7.5 –

–

18.3

9.8

–

14.4

11.1

6.2

0.72

–

15.2

7.2

1.82

14.6

–

–

–

Mesoridazine

5

–

337

Oxypertine

0.7

402

–

Pecazine

3.9

443

382

–

–

15.3

7

–

Penfluridol

–

659

656

43.4

20.2

–

–

–

3.4 –

–

10.1

5

–

–

–

–

–

Perazine

–

403

371

6.3

–

–

–

–

Pericyazine

1.3

410

356

4.4

–

10.2

5.1

–

Perphenazine

1.9

428

395

3.28

Pimozide

0.7

504

–

Pipamperone

–

299

241

16

13.1

6.3

11.9

7.2

17.2

–

–

–

2.7

10.9

–

–

–

Pipotiazine

–

431

–

–

14.7

–

–

–

Prochlorperazine

3.9

450

323

10.4

–

–

–

–

Promazine

5.9

407

326

5.9

–

–

–

–

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:11:0

Recommended HPLC systems

753

Phenothiazines and other tranquillisers, continued HA

Prothipendyl

HX

HY

HZ

HAA

HAX

HAY

HAZ

k

RI

RI

RT

RT

RT

RT

k

4.4

388

–

–

–

–

–

–

Sulforidazine

–

421

–

4.8

Sulpiride

–

259

235

2

–

–

–

–

3.9

–

–

0.02

Thiopropazate

1

483

–

–

–

–

–

–

Thioproperazine

4.1

427

305

15.4

15.2

–

–

– 3.88

5.2

490

427

13.5

17.2

–

9.8

5

–

–

–

–

–

–

–

Tiotixene

3.8

442

374

6.8

–

–

–

–

Triflupromazine

2.7

484

454

12.3

–

17.3

8.9

–

Thioridazine Mesoridazine

n n

Steroids System HATa

System HAR

Walters MJ et al. (1990). J Assoc Off Anal Chem 73: 904–926. n n n n n

Column: ODS Zorbax (250  4.6 mm i.d., 5 mm), stainless steel. Eluent: (A) Methanol. Isocratic elution: (100). Flow rate: 1.5 mL/min. Detection: UV (l ¼ 240, 210 and 280 nm).

System HATb

Walters MJ et al. (1990). J Assoc Off Anal Chem 73: 904–926. n n n

Flow rate: 1.5 mL/min. Detection: UV (l ¼ 240, 210 and 280 nm).

Lurie I et al. (1994). J Forensic Sci 39: 74–85. n n n n n

Column: ODS Zorbax (250  4.6 mm i.d., 5 mm). Mobile phase: (A : B) Water–methanol. Gradient elution: (30 : 70) to (0 : 100) over 15 min with 15 min hold. Flow rate: 1.0 mL/min. Detection: DAD.

System HT

Column: ODS Zorbax (250  4.6 mm i.d., 5 mm), stainless steel. Eluent: (A : B) Methanol–water. Isocratic elution: (75 : 25).

Rose JQ, Jusko WJ (1979). J Chromatogr Biomed Appl 162: 273–280. n n

Column: Silica Zorbax SIL (250  4.6 mm i.d., 5 mm). Mobile phase: Methylene chloride–methanol (97 : 3).

Steroids

Beclometasone Dipropionate Betamethasone Betamethasone valerate Boldenone Undecylenate Cortisone

HT

HX

HY

HZ

HAA

HAR

HATa

HATb

k

RI

RI

RT

RT

RRT

RRT

RRT

4.2

444

–

–

–

–

–

–

–

–

711

–

–

–

–

– –

–

–

–

14.2

13.3

–

–

–

–

584

–

–

–

–

–

–

–

–

–

–

0.74

–

0.76

–

–

–

–

–

–

1.94

–

2.4

–

372

–

–

–

–

–

Dexamethasone

4.8

–

381

Fluoxymesterone

–

–

427

3.4 –

13.1

–

–

–

–

0.78

–

0.7

Hydrocortisone

5.8

403

349

–

17.7

–

–

–

Hydroxyprogesterone

–

1054

–

–

–

–

–

–

Metenolone

–

–

–

–

–

–

–

–

Acetate

–

–

–

–

–

–

1.26

3.54

Enantate

–

–

–

–

–

–

1.87

–

Methandienone

–

–

–

–

–

0.86

–

0.87

Methandriol

–

–

–

–

–

1.25

–

1.29

–

–

–

–

–

–

–

2.75

7.5

426

390

–

18.9

–

–

–

Dipropionate Methylprednisolone Methyltestosterone

–

–

587

–

–

1.17

–

1.27

Nandrolone

–

–

–

–

–

0.84

–

0.92

–

Norethisterone

–

536

676

Prednisolone

8.4

401

361

Prednisone

3.4

250

340

Progesterone

–

672

698

2.5 2.6 –

24

–

–

–

14.1

–

–

–

14.2

–

–

–

23.8

–

–

– table continued

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:11:2

754

High Performance Liquid Chromatography

Steroids, continued HT

HX

HY

HZ

HAA

HAR

HATa

HATb

k

RI

RI

RT

RT

RRT

RRT

RRT

Testosterone

–

534

508

–

–

–

–

–

Acetate

–

–

894

–

–

1.76

–

2.59

Propionate

–

–

1003

–

–

2.01

1.31

4.06

Methyltestosterone

–

–

–

–

–

1.17

–

1.27

Isobutyrate

–

–

–

–

–

2.17

–

–

Cipionate

–

–

–

–

–

2.63

–

–

Enantate

–

–

–

–

–

2.6

1.8

–

Undecanoate

–

–

–

–

–

3.18

–

–

Phenylpropionate

–

–

–

–

–

–

1.48

–

Isocaproate

–

–

–

–

–

–

1.62

–

Cipionate

–

–

–

–

–

–

2.05

–

Undecenoate

–

–

–

–

–

–

2.53

–

Decanoate

–

–

–

–

–

–

2.78

–

Undecylate

–

–

–

–

–

–

3.27

– –

–

438

312

–

–

–

–

2.5

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

Hexahydrobenzylcarbonate

–

–

–

–

–

–

1.65

–

Acetate

–

–

–

–

–

–

–

1.71

Triamcinolone Acetonide Trenbelone

Sulfonamides

Xanthine stimulants

System HU

Cobb PH, Hill GT (1976). J Chromatogr 123: 444–447. n n

Column: Silica Spherisorb (250  4 mm i.d., 5 mm). Mobile phase: Cyclohexane–ethanol–acetic acid (85.7 : 11.4 : 2.9).

Sulfonamides HU k

HA

HX

HY

HZ

HAA

k

RI

RI

RT

RT

Caffeine

0.2

305

259

1.9

6.7

Diprophylline

–

275

227

–

3.6

Fenetylline

–

336

277

–

–

Proxyphylline

0.1

293

–

–

–

Theobromine

0.1

262

201

1.6

3.8

Theophylline

0.1

276

249

1.7

4.9

Phthalylsulfathiazole

14.0

Additional systems

Succinylsulfathiazole

16.8

System HAD

Sulfadoxine

4.4

Aymard et al. (2000). J Chromatogr Biomed Sci Appl 744: 227–240.

Sulfamerazine

8.1

n

Sulfaquinoxaline

4.8

Sulfacetamide

7.7

Sulfachlorpyridazine

3.3

Sulfadiazine

8.7

Sulfadimidine

7.1

Sulfafurazole

6.0

Sulfamethoxazole

4.8

Sulfamethoxydiazine

8.2

Sulfamethoxypyridazine

7.5

Sulfamoxole

12.6

Sulfanilamide

8.9

Sulfapyridine

3.8

Sulfathiazole

13.4

Xanthine stimulants The general screening systems, previously described, may be used.

Column: C18 Symmetry Shield (250  4.6 mm i.d., 5 mm) protected by 2 mm Upchurch filter. Column temperature: 30 C. Mobile phase: (A : B) M/15 potassium dihydrogenphosphate with 1% (v/v) octane sulfonic acid : acetonitrile. Mobile phase (MP) 1: (95 : 5) at flow rate 1 mL/min; MP 2: (80 : 20) at flow rate 1 mL/min; MP 3: (30 : 70) at flow rate 1.2 mL/min. Eluent switching programme: At injection, MP1 to the column. From time 12 min to 30 min, MP2 to the column. From time 30 min, MP3 to the column to rinse it. From time 35 min to 40 min, equilibration with MP1. Detection: DAD (l ¼ 260 nm).

n n

n

n

k

Compound

2.7

Lamivudine

3.2

Didanosine

3.8

Stavudine

6.6

Zidovudine

8.1

Abacavir

11.1

Nevirapine

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:11:3

Recommended HPLC systems

755

System HAF

System HBB

Tanaka E et al. (1996). J Chromatogr B Biomed Sci Appl 682: 173–178.

Taninaka C et al. (2000). J Chromatogr B Biomed Sci Appl 738: 405–411.

n n n n n

Column: ODS TSK-gel Super (100  4.6 mm i.d., 2 mm). Mobile phase: (A : B) Acetonitrile–5 mmol/L sodium dihydrogenphosphate (pH 6). Isocratic elution: (45 : 55). Flow rate: 0.65 mL/min. Detection: UV (l ¼ 254 nm).

Retention time (min)

Compound

n n n n n

Retention time (min)

Clonazepam

6.8

Clarithromycin

6.6

Bromazepam

6.8

Erythromycin

9.1

Nitrazepam

9.6

Azithromycin

16.3

Roxithromycin

Triazolam

15.0

Lorazepam

18.4

Etizolam

21.0

Chlordiazepoxide

29.8

Diazepam

32.2

Flutazolam

System HAE

Proust V et al. (2000). J Chromatogr B Biomed Sci Appl 742: 453–458. n n

System HAV

Rutledge DR et al. (1994). J Pharm Biomed Anal 12: 135–140.

n n n n

Column: RP-short alkyl chain, silanol-deactivated (SCD 100; 250  4.6 mm i.d.), stainless steel. Mobile phase: (A : B) Methanol–0.04 mol/L dibasic potassium phosphate (pH 5.5). Isocratic elution: (50 : 50). Flow rate: 1 mL/min. Detection: UV (l ¼ 237 nm).

k

n n n

Celiprolol

2.3

Propranolol

3.6

Diltiazem deacetyldiltiazem

5.1

Diltiazem desmethyldiltiazem

6.1

Diltiazem

6.4

Imipramine

8.2

Verapamil

System HBA

n n n n

Column: C18 base-deactivated silica (125  4.6 mm i.d., 5 mm) with base-deactivated C18 precolumn (20  4.6 mm i.d., 5 mm). Eluent: (A : B) Acetonitrile–50 mmol/L potassium dihydrogenphosphate (pH 7.5, containing 500 mL triethylamine). Isocratic elution: (60 : 40). Flow rate: 2 mL/min. Detection: Fluorescence (lex ¼ 255 nm, lem ¼ 315 nm).

Retention time (min) 8.8

Compound

Compound

6.3

Delavirdine

7.0

Saquinavir

8.0

Nelfinavir

9.4

Amprenavir

22.2

Ritonavir

28.6

Efavirenz

System HAK

Le Guellec C et al. (1988). J Chromatogr Sci Appl 719: 227–233. n n

Sastre-Toran˜o J, Guchelaar H-J (1998). J Chromatogr B Biomed Sci Appl 720: 89–97.

Column: C18 (Lichrospher, 100 RP-18, 5 mm) with C18 precolumn (Lichrospher RP-18, 5 mm). Mobile phase: (A : B) Acetonitrile–25 mmol/L sodium phosphate modified with diethylamine (0.9%) and tetrahydrofuran (1%), pH 3.0. Isocratic elution: (44.8 : 55.2). Flow rate: 0.5 mL/min. Detection: UV (l ¼ 260 nm).

Retention time (min)

Compound

2.2

n

Compound

5.3

13.7

n

Column: C18 (250  6.0 mm i.d., 5 mm). Eluent: (A : B) Acetonitrile–50 mmol/L phosphate buffer (pH 7.2). Isocratic elution: (43 : 57). Flow rate: 1.7 mL/min. Detection: Electrochemical (working electrode: glassy carbon; reference electrode: Ag/AgCl).

n n n

Column: C18 Symmetry (250  4.6 mm i.d., 5 mm) with C18 precolumn Symmetry sentry. Mobile phase: (A : B) Acetonitrile–20 mmol/L potassium dihydrogenphosphate. Elution programme: (50 : 50) to (70 : 30) in 15 min. Flow rate: 1 mL/min. Detection: UV (l ¼ 313 nm).

Retention time (min)

Compound

4.7

Carbamazepine

6.2

Clonazepam

7.6

Nordazepam

9.3

Clobazamm

Not detected

Phenobarbital

Not detected

Phenytoin

Erythromycin

15.7

Clarithromycin

17.1

Roxithromycin

20.7

Azithromycin

System HAL

Boukhabza A et al. (1990). J Chromatogr 529: 210–216. n

Column: C18 Novapak (150  4.6 mm i.d., 5 mm).

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:11:4

756 n n n n n

High Performance Liquid Chromatography

Mobile phase: (A : B : C) Acetonitrile–methanol–6 mmol/L phosphate buffer (pH 5.7). Isocratic elution: (30 : 10 : 60). Flow rate: 1.3 mL/min. Detection: DAD (l ¼ 242 nm). Note: The phosphate buffer stock solution is prepared using 94 mL 0.2 mol/L sodium dihydrogenphosphate added to 6 mL 0.2 mol/L disodium phosphate heptahydrate.

9.1

Compound Desipramine Diazepam

Not detected

Alprazolam

Not detected

Bromazepam

Not detected

Clobazam

Not detected

Codeine

Compound

Not detected

Ephedrine

1.4

Barbital

Not detected

Levomepromazine

1.45

Clonazepam 7acetamidoclonazepam

Not detected

Lidocaine

Not detected

Medazepam

1.55

Clonazepam 7-aminoclonazepam

Not detected

Nortriptyline

2.0

Aprobarbital

Not detected

Propranolol

2.4

Hexobarbital

Not detected

Thioridazine

3.7

Flunitrazepam M (nor)

Not detected

Triazolam

4.4

Nordazepam oxazepam

4.4

Oxazepam

4.6

Nitrazepam

4.33

Clonazepam

5.1

Lorazepam

6.2

Flunitrazepam

6.3

Alprazolam

6.6

Triazolam

7.7

Chlordiazepoxide

7.8

Clobazam

7.9

Nordazepam

8.1

Bromazepam

8.2

Medazepam

13.2

Diazepam

System HAM

de Carvalho D, Lanchote VL (1991). Ther Drug Monit 13: 55–63.

n n n n

Retention time (min) 10.3

Retention time (min)

n

, continued

Column: C18 (150  4.0 mm i.d., 3 mm) with C18 precolumn (40  4.0 mm i.d., 3 mm). Mobile phase: (A : B ) Water–acetonitrile. Isocratic elution: (50 : 50). Flow rate: 0.7 mL/min. Detection: UV (l ¼ 313 nm).

Retention time (min)

Compound

1.8

Theophylline

1.98

Caffeine

2.0

Paracetamol

2.2

Primidone

2.7

Sulfamethoxazole

2.8

Phenobarbital

3.1

Chlordiazepoxide

3.4

Diazepam

3.4, 4.4

Oxazepam

3.5

Phenytoin

4.2

Lorazepam

4.3

Clonazepam

4.5

Nitrazepam

9.0

Imipramine

References Aquilante CL et al. (2006). Common laboratory methods in pharmacogenomic studies. Am J Health Syst Pharm 63: 2101–2110. Atanassova A et al. (2004). A high-performance liquid chromatography method for determining transition metal content in proteins. Anal Biochem 335: 103–111. Badoud F et al. (2010). Fast analysis of doping agents in urine by ultra-high pressure liquid chromatography quadrupole time of flight mass spectrometry II: Confirmatory analysis. J Chromatogr A 1217: 4109–4119. Bijlsma L et al. (2009). Simultaneous ultra-high pressure liquid chromatographytandem mass spectrometry determination of amphetamine and amphetaminelike stimulants, cocaine and its metabolites, and a cannabis metabolite in surface water and urban wastewater. J Chromatogr A 1216: 3078–3089. Bogusz M et al. (1993). An overview on the standardisation of chromatographic methods for screening analysis in toxicology by means of retention indices and secondary standards. Part II. High performance liquid chromatography. Fresenius Z Anal Chem 347: 73–81. Budowle B, van Daal A (2009). Extracting evidence from forensic DNA analyses: future molecular biology directions. Biotechniques 46: 339–340,342–350. Cheng X, Kaplan LA (2003). Simultaneous analysis of neutral carbohydrates and amino sugars in freshwaters with HPLC-PAD. J Chromatgr Sci 41: 434–438. Costabile M (2006). Molecular approaches in the diagnosis of primary immunodeficiency diseases. Hum Mutat 27: 1163–1173. Danielson PB (2005). Separating human DNA mixtures using denaturing highperformance liquid chromatography. Expert Rev Mol Diagn 5: 53–63. Engelhardt H (1977). The role of moderators in liquid-solid chromatography. J Chromatogr Sci 15: 380–384. Ettre LS (1980). Relative retention expressions in chromatography. J Chromatogr 198: 229–234. Hansen SH (1981). Column liquid chromatography on dynamically modified silica. J Chromatogr 209: 203–210. Halasz I et al. (1975). Ultimate limits in high-pressure liquid chromatography. J Chromatogr A 112: 37–60. Herzler M et al. (2003). Selectivity of substance identification by HPLC–DAD in toxicological analysis using a UV spectra library of 2682 compounds. J Anal Toxicol 27: 233–242. Huber S, George A, eds (1993). Applications of diode-array detection in HPLC. In: Chromatographic Science Series 62. New York: Marcel Dekker. Ibanez M et al. (2009). Screening of antibiotics in surface and wastewater samples by ultra-high pressure liquid chromatography coupled to hybrid quadrupole timeof-flight mass spectrometry. J Chromatogr A 1216: 2529–2539. Kosaki K et al. (2005). DHPLC in clinical molecular diagnostic services. Mol Genet Metab 86: 117–123. Lin SY et al. (2008). Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM. BMC Med Genet 20(9): 53. Liu W et al. (1998). Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Acids Res 26: 1396–1400. Luquin N et al. (2010). DHPLC can be used to detect low level mutations in amyotrophic lateral sclerosis. Amyotroph Lateral Scler 11(12): 76–82. Macnair JE et al. (1997). Ultra high-pressure reversed-phase liquid chromatography in packed capillary columns. Anal Chem 69: 983–989.

Clarke's Analysis of Drugs and Poisons Chapter No. 41 Dated: 10/3/2011 At Time: 16:11:4

Further reading Miura Y, Hamada H (1999). Ion chromatography of nitrite at the ppb level with photon measurement of iodine formed by post-column reaction of nitrite with iodide. J Chromatogr A 850: 153–160. Pragst F et al. (2001). UV Spectra of Toxic Compounds. Data Base of Photodiode Array UV Spectra of Illegal and Therapeutic Drugs, Pesticides, Ecotoxic Substances and Other Poisons. Heppenheim: Verlag Dieter Helm. Pragst F et al. (2002). Suchverfahren (General unknown). In: K€ ulpmann WR, ed. Klinisch-Toxikologische Analyse. Weinheim: Wiley-VCH Verlag GmbH, 49–124. Pragst F et al. (2004). Systematic toxicological analysis by high-performance liquid chromatography with diode array detection (HPLC-DAD). Clin Chem Lab Med 42: 1325–1340. Martin AJP, Synge RLM (1941). A new form of chromatogram employing two liquid phases. Biochem J 35: 1358–1368. Rozing G et al. (2001). A system and columns for capillary HPLC. Am Lab 33: 26–38. Small H et al. (1975). Novel ion exchange chromatographic method using conductrimetric analysis. Anal Chem 47: 1801–1809. Snyder LR (1968). Principles of Adsorption Chromatography. New York: Marcel Dekker, 194–195. Snyder LR et al. (1997). Practical HPLC Method Development. New York: Wiley. van Deemter JJ et al. (1956). Longtitudinal diffusion and resistance to mass transfer as cuases of non ideality in chromatography. Chem Eng Sci 5: 271–289. Ventura R et al. (2008). High throughput and sensitive screening by ultra-performance liquid chromatography tandem mass spectrometry if diuretics and other doping agents. Eur J Mass Spectrom (Chichester) 14: 191–200. Wells MJM, Clark CR (1981). Liquid chromatographic elution characteristics of some solutes used to measure column void volume on C18 bonded phases. Anal Chem 53: 1341–1345.

Further reading Aldridge AA et al. (2009). Ultra high performance liquid chromatography in the contract manufacturing environment. http://pharmtech.findpharma.com/ pharmtech/AnalyticsþArticle/Ultra-High-Performance-LiquidChromatography-in-th/ArticleStandard/Article/detail/584973 (accessed 14 December 2010). Aquilante CL et al. (2006). Common laboratory methods in pharmacogenomic studies. Am J Health Syst Pharm 63: 2101–2110. Armstrong D, Zhang B (2001). Chiral stationary phases for high performance liquid chromatography. Anal Chem 73: 557A–561A. Ayrton J et al. (1998). Use of generic fast gradient liquid chromatography– tandem mass spectroscopy in quantitative bioanalysis. J Chromatogr B 709: 243–254. Bobzin SC et al. (2000). LC–NMR: a new tool to expedite the dereplication and identification of natural products. J Ind Microbiol Biotechnol 25: 342–345. Budowle B, van Daal A (2009). Extracting evidence from forensic DNA analyses: future molecular biology directions. Biotechniques 46: 339–340, 342–350. Dai J (2009). Fast liquid chromatography for method development. Pharm Rev 12: 12–17. Fornstedt T, Guiochon G (2001). Nonlinear effects in LC and chiral LC. Anal Chem 73: 609A–617A. Fritz JS (2004). Early milestones in the development of ion-exchange chromatography: a personal account. J Chromatogr A 1039: 3–12. Gao VCX et al. (1998). Column switching in high performance liquid chromatography with tandem mass spectrometric detection for high-throughput preclinical pharmacokinetic studies. J Chromatogr A 828: 141–148. Guillarme D et al. (2010). New trends in fast and high-resolution liquid chromatography: a critical comparison of existing approaches. Anal Bioanal Chem 397: 1069–1082.

757

Haddad PR et al. (2008). Recent developments and emerging directions in ion chromatography. J Chromatogr A 1184: 456–473. Hamilton RJ, Sewell P (1977). Introduction to High Performance Liquid Chromatography, 2nd edn. London: Chapman & Hall. Heinig K, Bucheli F (2002). Application of column-switching liquid chromatography–tandem mass spectrometry for the determination of pharmaceutical compounds in tissue samples. J Chromatogr B 769: 9–26. Henion J et al. (1998). Sample preparation for LC–MS–MS: analyzing biological and environmental samples. Anal Chem 70: 650A–656A. Hicks RP (2001). Recent advances in NMR: expanding its role in rational drug design. Curr Med Chem 8: 627–650. Jerkovich AD et al. (2003). The use of micrometer-sized particles in ultrahigh pressure liquid chromatography. LC/GC North Am 21: 60–61. Johns D (1987). Resolving isomers on HPLC columns with chiral stationary phases. Am Lab Jan.: 72–76. Karnes HT, Sarkar MA (1987). Enantiomeric resolution of drug compounds by liquid chromatography. Pharm Res 4: 285–292. Lo´pez-Ruiz B (2000). Advances in the determination of inorganic anions by ion chromatography. J Chromatogr A 881: 607–627. Lunn G, Schmitt NR (1997, 2000). HPLC Methods for Pharmaceutical Analysis. Vols 1, 2–4. New York: Wiley. Majors RE (1997). New chromatography columns and accessories at the 1997 Pittsburgh Conference Part 1. LC–GC 15: 220–237. Majors RE (1998). New chromatography columns and accessories at the 1998 Pittsburgh Conference Part 1. LC–GC 16: 228–244. Majors RE (1999). New chromatography columns and accessories at the 1999 Pittsburgh Conference Part 1. LC–GC 17: 212–220. Majors RE (2000). New chromatography columns and accessories at the 2000 Pittsburgh Conference Part 1. LC–GC 18: 262–285. Meyer VR (1979). Practical High Performance Liquid Chromatography, 2nd edn. New York: Wiley. Peng SX et al. (1999). Direct determination of stability of protease inhibitors in plasma by HPLC with automated column-switching. J Pharm Biomed Anal 25: 343–349. Plumb RS et al. (1999). The application of fast gradient capillary liquid chromatography–mass spectrometry to the analysis of pharmaceuticals in biofluids. Rapid Commun Mass Spectrom 13: 865–872. Sch€ ufer C et al. (2001). HPLC columns: the next great leap forward – Part 1. Am Lab Feb.: 40–41. Sch€ ufer C et al. (2001). HPLC columns: the next great leap forward – Part 2. Am Lab Apr.: 25–26. Simpson CF (1976). Practical High Performance Liquid Chromatography. London: Heyden. Snyder LR (2000). HPLC past and present. Anal Chem 72: 412A–420A. Tanaka N et al. (2001). Monolithic LC columns. Anal Chem 72: 420A–429A. The MHE Research Foundation (2008). DHPLC Genetic Testing. Available at: www. mheresearchfoundation.org/DHPLC_Genetic_Testing.html (accessed 30 October 2008). Transgenomic Inc. (2008). Wave
Systems for mutation detection. Available at: www.transgenomic.com/lib/br/602077.pdf (accessed 30 October 2008). Wehr T (2000). Configuring HPLC systems for LC–MS. LC–GC 18: 406–416. Weiss J (2005). Handbook of Ion Chromatography, 3rd revised updated edn. Weinheim: Wiley-VCH. Wilson I et al. (2000). Analytical chemistry: advancing hyphenated chromatographic systems. Anal Chem 71: 534A–542A. Wolfender JL et al. (2001). The potential of LC–NMR in phytochemical analysis. Phytochem Anal 12: 2–22. Yang LY et al. (2001). Applications of new liquid chromatography–tandem mass spectrometry technologies for drug development support. J Chromatogr A 926: 43–55.

Clarke's Analysis of Drugs and Poisons Chapter No. 42 Dated: 10/3/2011 At Time: 16:24:6

CHAPTER

42

Capillary Electrophoresis F Tagliaro, A Fanigliulo, J Pascali and F Bortolotti

Introduction

Theoretical aspects

Since its introduction in the early 1980s, and particularly in the first decade of this century, capillary electrophoresis (CE) has established a prominent role in many areas of applied analytical chemistry. This is demonstrated by an increasing number of applications and scientific papers in biomedical sciences, toxicology, biopharmaceutics, biotechnology, and environmental and forensic science. The spread of this application relies on its peculiar features:

Capillary electrophoresis can be defined as high-voltage electrophoresis (10–30 kV) carried out in a capillary-shaped separation compartment (typical dimensions: internal diameter 20–100 mm, length 20–100 cm). The geometry of this set-up, with respect to traditional slab gel electrophoresis, offers the neat advantages of minimal band diffusion and improved joule heating control, thus permitting the application of high voltages. The reduced zone broadening results in excellent separation efficiency, reaching up to 106 theoretical plates. The CE separation mechanism, as in any form of electrophoresis, is based on the principle that charged species subjected to an electric field tend to migrate, driven by electrostatic force, towards the electrode with the opposite charge. Their velocity depends on their electrophoretic mobility (m), which is specific for each individual ionic species on the basis of the mass-to-charge ratio, as described by Equation (42.1):

n n n n

n

Wide analytical applications (from inorganic ions to large DNA fragments and even viruses and cells) Variety of separation modes (electrophoretic, electrokinetic, chromatographic and more) Variety of detection systems (from ultraviolet (UV) spectroscopy to mass spectrometry (MS)) High separation efficiency (up to millions of theoretical plates) and mass sensitivity (from femtomoles (10
15 moles) down to yoctomoles (10
21 moles)); minimal consumption of samples (in the order of nanolitres) and solvents (a few millilitres per day) Simple and inexpensive operation coupled with instrumental ruggedness.

Moreover, being based on specific separation mechanisms, CE has established itself as an independent analytical technique complementary to chromatography. Capillary electrophoresis originated from the optimisation of basic electrophoretic principles traditionally applied using slab gels. The range of applications of CE soon extended to include hybrid separation mechanisms, partially borrowed from chromatography, and it has become a highly versatile and flexible tool in the hands of separation scientists. CE has been recognised as potentially admissible in the US courts as a form of evidence in accordance with the Daubert Standard (Kuffner et al. 1996). Although CE has received major attention in molecular biology, clinical chemistry, analytical toxicology and other areas of forensic analysis, it is still ‘in its infancy’. Forensic science laboratories have to deal with a range of diverse analytical problems involving, for example, gunshot residues, explosives, inks, dusts, soils, illicit drugs, poisons, DNA fragments and proteins and others, and CE has particular characteristics that make it applicable to all these areas. Forensic samples are often limited in quantity and heavily contaminated and must be conserved as far as possible in order to allow for further investigations. CE has great potential as a practical and productive investigation tool for analytical toxicologists and other forensic scientists dealing with such samples. In addition, scientists working in the fields of clinical and biochemical analysis, where analytical versatility, minimum use of sample volume and low operative costs are extremely important, derive considerable benefits from the technique. This chapter builds on that contributed to the third edition of this publication by Professor David Perrett in that it aims to illustrate the basic principles of CE by giving a description of its instrumentation and of the different modes of separation and detection. A major addition is the presentation of recent review papers that cover important topics of CE applications in analytical toxicology and to which the readers are referred for more detailed information. Further, a selected number of applications of particular interest are discussed.

758

mi ¼

qi 6phri

ð42:1Þ

where mi ¼ ion mobility, qi ¼ ion charge, h ¼ electrolyte solution viscosity, and ri ¼ ion effective radius. When an electric field of strength E is applied, the ion migration velocity (vi) will equal the product mi  E, causing the physical separation of the components of a mixture of molecules with different mi (Equation 42.2): vi ¼ mi  E

ð42:2Þ

where the electric field strength E is given by Equation (42.3): Electric field ðEÞ ¼

Applied voltage V ¼ Distance between electrodes d

ð42:3Þ

An additional phenomenon known as electroosmosis takes place inside the separation capillary and results from the double electrical layer that builds up at the solid–liquid interface whenever a solid surface is in contact with a solution of ions. This ionic double layer is described by the so-called zeta potential z. The inner wall of capillaries, silica being the most common material of which the CE capillaries are made, exhibits an excess of surface charge, since it contains a great number of silanol groups (SiOH), which at pH values higher than 2 are ionised as SiO
. The resulting negative charge of the wall surface tends to be compensated for by cations attracted from the solution, thus building up a double electric layer. When an electric potential difference is established between the ends of the capillary (i.e. when electrophoresis starts), all the cations in the solution migrate towards the cathode (the negative electrode) and anions move in the opposite direction. The migration towards the cathode of excess cations close to the capillary wall (being not compensated by migration of the corresponding anions stationary in the wall) drags water in the same direction, thus producing a measurable flow of liquid inside the capillary (typically tens of nanolitres per minute), termed ‘electroosmotic flow’ (EOF). Capillaries made of materials other than silica (e.g. Teflon), or with the wall coated with neutral or charged coatings (negative or positive), will display the same phenomenon, depending on the degree of ionisation of the wall surface. It is

Clarke's Analysis of Drugs and Poisons Chapter No. 42 Dated: 10/3/2011 At Time: 16:24:6

Instrumental hardware

v ¼ ðmi þ mEOF Þ  E

ð42:4Þ

The set-up of a capillary electropherograph is usually with the detector close to the end of the capillary towards which the EOF is directed, and liquid flow will drag towards the detector all the solutes contained in the injected sample, excluding those with an electrophoretic counter-migration velocity higher than the EOF itself (small ions with high charge-tomass ratio). Inside this flow of solvent, the ionic species migrate according to the respective mi, whereas neutral solutes (with mi ¼ 0) migrate all together at the velocity of the EOF. In short, with the usual instrumental arrangement, having the injector at the anode end and the detector close to the cathode end of an uncoated fused-silica capillary (negatively charged at the inner wall), the cations with the highest mobility will arrive at the detector first, followed by the cations with progressively lower mobility (lower charge-to-mass ratio); subsequently, the bulk of the neutrals will appear at the detector, followed by the anions in reversed order of mobility (slow anions first, fast anions last). Only the fastest anions with electrophoretic mobility higher than that of the EOF will not be detected, because they will escape from the opposite end of the separation capillary. The EOF, being driven by a force generated close to the capillary wall, has a peculiar ‘piston-like’ flow profile, which is particularly beneficial for molecular separations. In fact, it minimises band broadening, which is a typical drawback of capillary liquid chromatography, where the pressure-driven flow of the mobile phase, hindered by shear forces at the wall, yields a parabolic flow profile. The coexistence of both electrophoretic and electrokinetic phenomena, which can be tuned separately and in combination to achieve molecular separation, is a unique feature of CE that can be exploited to perform a great variety of separations modes. In addition, interactions taking place between analytes and other molecules (e.g. complexforming molecules, organic solvents, micelles, polymer gels) present in the medium in which separation occurs further contribute to differentiation of the migration velocities of different chemical species according to non-electrophoretic principles. For a detailed explanation of the theoretical principles of CE, readers are referred to publications listed in the ‘Further reading’ section of this chapter, and particularly to the book by Ahuja and Jimidar (2008). Finally, although quantification methods used in CE are similar to those applied in other forms of chromatography, in that they mainly use internal standardisation, two important differences must be emphasised. First, in CE most separations have an ‘on-column’ detection step. In this process, the velocity of the peaks crossing the detection window differs according to the respective ion mobilities of the analytes. As a result, the ‘apparent peak width’ of zones moving at different velocities will differ, even if the ‘real band width’ is the same. Consequently, two equal bands differing only in mobility will show different ‘peak areas’, because of the difference in their residence times inside the detector cell. To overcome this problem, for quantitative computations the ratio of peak area to migration time can be used instead of peak areas. An alternative is to use peak heights. Second, since only minute amounts of sample (of the order of a few tens of nanolitres) are injected onto the separation capillary, it is difficult to handle these volumes with precision using current technology. This seriously restricts the use of external standardisation for quantitative methods. Thus, as for example with gas chromatography (GC), internal standardisation is much preferred.

Instrumental hardware The outstanding feature of CE instrumentation is its basic simplicity. A schematic representation of a capillary electropherograph is given in Figure 42.1. In brief, as in other chromatographic techniques, a CE analysis is based on three major steps: sample injection, separation and detection. As a consequence, a capillary electropherograph can be described as an assembly of the following components: n n n n

An injection system A high-voltage power supply A separation capillary (generally located in a thermostatted compartment) A detector.

In CE, only a minute amount of sample can be introduced into the capillary, in order to maintain the separation efficiency. Overloading of samples causes peak broadening and peak distortion. Sample loading is limited by capillary dimensions. Typically, the length of the sample plug should not exceed 1–2% of the total capillary length and this corresponds to volumes of between 10 and 100 nL, for typical CE capillaries, which have an inner volume ranging between 1 and 4 mL. Sample injection is achieved by replacing the inlet buffer reservoir with the sample vial for a few seconds. The forces driving sample introduction into the separation capillary may be brought about by applying pressure, voltage or both. The most common modes are hydrodynamic and electrokinetic injection. Hydrodynamic injection is the most widely used and is carried out by the application of a positive pressure at the inlet end of the capillary, by the application of a vacuum at the terminal end or by exploiting siphoning between the two capillary ends. In this way, the amount of sample loaded is almost independent of the sample composition and is representative of the whole sample solution. Hydrodynamic injection is described by Equation (42.5): V¼

DPd4 pt 128hL

ðHagen
Poiseuille equationÞ

ð42:5Þ

where V ¼ sample volume, DP ¼ applied pressure, d ¼ capillary internal diameter, t ¼ injection time, L ¼ capillary length and h ¼ buffer viscosity. The typical applied pressure range is between 5 and 30 psi (1 psi ¼ 69 mbar ¼ 6.9 kPa) and injection times are between 5 and 20 seconds, although these parameters depend strongly on method optimisation. The advantages of this injection mode are reproducibility and suitability for quantitative analysis. However, it is subject to limited sensitivity owing to the tiny volume of sample introduced into the capillary and hence the limited mass of analytes that can be introduced into the capillary.

PC control

Power supply (0–30 kV)

AU

important to point out that even ‘neutral’ capillaries may display a charged surface because of the adsorption on the wall of molecules present in the solution. Electrophoretic migration and electroosmotic flow may have either the same or the opposite direction. Thus, they sum as vectors and induce ionised species to migrate with an apparent velocity (v) resulting from the sum of their intrinsic mobility (mi) and the mobility of the EOF (mEOF) (Equation 42.4):

759

Time (min)

Capillary

Detector Electrodes Inlet reservoir

Outlet reservoir

Figure 42.1 Schematic representation of a capillary electropherograph.

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Electrokinetic injection is obtained by applying a voltage for a few seconds while the injection end of the capillary is dipped into the sample vial. In this way, analytes enter the separation capillary by both electrophoretic migration and the pumping effect of the EOF. The amount of sample introduced is given by Equation (42.6): Q¼

ðmi þmEOF ÞVpr 2 Ct L

ð42:6Þ

where Q ¼ grams or moles of injected amount, mi ¼ analyte electrophoretic mobility, mEOF ¼ EOF mobility, V ¼applied voltage, r ¼ capillary radius, t ¼ injection time, L ¼ capillary length and C ¼ analyte concentration. It can be seen that, in electrokinetic injection, discrimination occurs on the basis of the electrophoretic mobility of the sample components, so that ionic species with mobilities towards the detector will be preferentially introduced with respect to neutral species and ions with the opposite charge. This results in selectivity and sensitivity enhancements for the analytes that are preferentially loaded, but also in delicate reproducibility, strongly affected by the sample composition. Electrokinetic injection is the injection mode exploited to realise ‘sample stacking’ procedures that allow large increases in analytical sensitivity, as discussed below with regard to sample pretreatment and sample enrichment. Separation capillary Fused silica is by far the most widely used material for CE capillaries, since it is chemically and electrically inert, physically resistant, UV transparent (for the needs of in-capillary UV detection) and displays good thermal conductivity (in order to ensure high dissipation of the joule heat). To achieve the necessary mechanical resistance, capillaries are externally coated with a protective polyimide layer, in a similar way to GC capillaries. This external layer is optically opaque and must be removed in the region where an optical detector is to be placed. This can easily be achieved by burning or scraping off the material in a capillary segment corresponding to the detection window. In-capillary optical detection has the advantage of avoiding any post-separation added volumes and consequent band broadening, but has disadvantages in terms of optical path length and cell geometry. Typical ranges for inner and outer diameters of CE capillaries are 25–100 mm and 350–400 mm, respectively. Capillary length typically rages between 20 and 100 cm. Fused-silica capillaries can be internally ‘uncoated’ (naked) or ‘coated’ with thin layers of polymers. The most common internal coatings include amines, polyacrylamide, cellulose, poly(vinyl alcohol)s, amino acids, surfactants, aryl pentafluoro compounds, poly (vinylpyrrolidinone) and poly(ethyleneamine). Also, liquid chromatography (C2, C8, C18) or GC (poly(ethylene glycol) and phenyl methyl silicone) stationary phases can be used as capillary wall coatings. An inherent problem in CE separations is reproducibility, since the EOF and consequently migration times of analytes are affected by the inner surface conditions. A common practice adopted to refresh the inner surface of uncoated silica capillaries and remove adsorbed materials is that of flushing with strong bases, which dissolve a thin layer of the silica surface. Usually, 1 mol/L sodium hydroxide solution is flushed through the capillary, followed by 0.1 mol/L sodium hydroxide solution and then by the separation buffer for final conditioning. The frequency of base conditioning during the experimental work depends on the nature of the samples, the buffer employed, the working pH, the background electrolyte (BGE) concentration and the nature of the capillary surface. Acid washing and flushing with solvents are less popular but effective practices, particularly when coated capillaries are used that would suffer from exposure to basic solutions. Thermostatting is another key point to ensure reproducibility of capillary separations. Temperature variations due to joule heating should be minimised in CE analyses so as to minimise viscosity changes in both separation and injection. Thermostatting is better achieved by using a refrigerant fluid, but air streams are also effective when separations generate low currents.

Power supply A high-voltage power supply is a fundamental part of a CE instrument. It should be capable of delivering up to 30 kV, with currents up to 200–300 mA. Because of the typical direction of the EOF (towards the cathode, in naked fused-silica capillaries), the standard polarity configuration has the anode (positive electrode) placed at the injection end of the capillary and the cathode (negative electrode) at the opposite end, close to the detector (a configuration known as ‘normal polarity’). However, in many cases (e.g. when the EOF is directed towards the anode, because the capillary wall is coated with a positively charged wall modifier) the application of a ‘reversed polarity’ configuration is needed in order to allow the analytes to migrate to the detector. For this reason, dual-polarity power supplies that are capable of rapidly switching the electrode polarity are used in modern CE instrumentation. CE separations are usually carried out at a constant potential, but separations at constant current can sometimes be needed. An advantage of constant-current mode is the possibility of compensating by automatic adjustments of the voltage for viscosity changes caused by inadequate temperature control. Voltage programming, i.e. applying gradients of voltage or current during the analysis, has also been reported to improve separation efficiency in the case of complex samples, but the advantages of gradient application are not as important as in GC and high performance liquid chromatography (HPLC), where temperature or buffer composition gradients are the most popular mode for tuning separation. Detectors Although it might be expected that the detection of analytes directly inside the capillary through its transparent silica wall would be relatively simple, this is not the case. Inherently, the main problem related to detection is sensitivity, not in terms of detectable mass (picogram amounts of analytes are easily detectable inside the capillary with simple UV detectors), but in terms of concentration of analyte. Since only minute volumes (nanolitres) of samples can be introduced into the tiny separation compartment (i.e. the capillary, with a typical volume 1 mL), only very small amounts of analytes will reach the detector. On this basis, taking into account the high efficiency of CE (i.e. the ability to produce separations without generating dilution of the electrophoretic zones), it is mandatory to avoid zone broadening in order to achieve an acceptable sensitivity, either ‘in-capillary’ or ‘off-capillary’. The way to achieve this is by limiting any ‘dead volumes’ in the system, particularly at the detection side, and increasing the response time of the detectors. In the next subsections the most common CE detectors are described and their relative advantages and drawbacks discussed. Optical methods

UV is the most commonly employed means of detection in CE instruments. Absorbance detection is usually performed ‘in-capillary’, with the optical beam focused directly into the capillary, crossing a transparent window made in it by removing the protective polyimide external coating. The limited optical path inside the capillary and the poor optical shape of the capillary section are the major factors negatively affecting sensitivity. There are also other features peculiar to CE separation that have to be taken into account in quantitative analysis. As mentioned previously, detection in CE occurs when separation is still taking place, in contrast to chromatographic techniques where all analytes move at the same velocity in front of the detector, which is located after the separation column. In CE, the migration velocity differs from one analyte to another during detection such that their residence time in the detector is affected by their different velocities, and this affects the respective peak areas. To correct this artefact in quantitative analysis, peak areas in CE should be divided by their migration times. Moreover, in CE the dynamic linear range of optical detection is less extended than in HPLC, particularly with UV detection, since deviations from the Beer–Lambert Law occur owing to the small size and curvature of the capillary cell.

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Modes of separation Despite these limitations, UV detection is generally applicable, easy to perform and acceptably sensitive for most applications. Capillary design and optical cell design may be optimised to improve sensitivity. Two examples are Z-shaped cells and bubble cells. In Z-cells, a double right angle, in the capillary region where detection takes place, results in an increased optical path length. The light beam runs axially for the length included between the two right angles. In bubble cells, solutes pass through an expanded region (‘bubble’) inside the capillary, where the analyte zone expands radially and, consequently, contracts longitudinally, increasing the path length without sacrificing separation. The availability of multichannel, dispersive optical detectors (diode array detectors, DADs) for commercial CE instruments has greatly impacted CE instrumentation, allowing for enhanced information content at each analytical run and easier method development. Fluorescence detection, mainly based on laser-induced fluorescence (LIF), has been proficiently applied both to fluorescent analytes and to molecules that can be made fluorescent after chemical derivatisation. In this case, sensitivity can be as high as 10
12 mol/L. In the case of non-UV-absorbing ionised molecules, ‘indirect detection’ can be performed by introducing a detectable (UV-absorbing) additive, with the same charge as the analyte of interest and similar mobility to the running buffer. Displacement of the additive by the nonabsorbing analyte will occur in the capillary in the zone of residence of the analyte and consequently a negative peak will be recorded by the detector (Beckers, Bocek, 2003; Johns et al. 2003). Indirect detection, for instance, makes possible the determination of small organic and inorganic ions, most of which do not absorb UV light, using the UV detectors present in all commercial CE instruments. Electrochemical detectors

Electrochemical (EC) detectors, particularly amperometric and conductimetric detectors, can be successfully coupled with CE. Electrochemical detection can be performed either ‘in-capillary’ or ‘off-capillary’. Amperometric detectors record the electronic current generated by an electrochemical reaction involving the analyte molecules, thus displaying, for oxidisable (or reducible) analytes, high sensitivity and good selectivity. This technique is mass sensitive and therefore independent of the cross-sectional pathlength of the capillary. Conductimetric detectors are simpler, although less selective, than amperometric detectors. Their application to CE allows direct detection of small ions, making possible ion analysis in a configuration resembling ion chromatography. A general problem with EC detection in CE is the isolation of the separation circuit from the detection circuit to avoid interferences. A further problem of the EC detection mode is the necessity of using miniaturised electrodes, compatible with capillary dimensions. These devices are not commercially available so far, making EC detection (particularly amperometric) still reliant on home-made components. Mass spectrometry

(See also Chapters 37 and 41.) Hyphenation with mass spectrometry (CE-MS) is the latest major innovation in CE. In recent years the application of CE-MS has grown substantially, and now accounts for a large number of publications in the pharmaceutical, biotechnological, environmental and toxicological science literature (Schmitt-Kopplin, Frommberger 2003; SchmittKopplin, Engelmann 2005). The key issue when coupling CE with MS is the efficient transfer of ions from the CE capillary to the mass analyser without sacrifice of separation performance and sensitivity (Smith et al. 1988). The electrospray ionisation (ESI) source has proved to be the most suitable for CE-MS coupling. This produces gas-phase ions from ions in solution, which are introduced into the mass spectrometer after being sprayed in a plume of droplets, as a result of a voltage applied on a holed metal tip. The ESI source is compatible with the CE-limited flows, since it is concentration sensitive and not mass sensitive. Other ion sources can be coupled with CE, for example atmospheric pressure chemical ionisation (APCI; Tanaka et al. 2003) and atmospheric pressure photoionisation (APPI; Mol et al. 2005), but the recent successful results of CE-MS interfacing are due to the development of the CE-ESI hyphenation.

761

Coupling CE with ESI involves joining a high-voltage-driven separation technique with a high-voltage-based molecule ionisation technique. Thus, it is necessary to ensure electrical continuity between the two systems while at the same time keeping the respective circuits independent. Second, a stable flow of ionised molecules must be constantly produced during analysis and therefore a BGE compatible with the ion source must be chosen. CE can be interfaced with ESI with essentially two possible alternatives, as well described by Cai and Henion (1995): the ‘sheath liquid’ interface and the ‘sheathless’ interface. In the former case the electrical continuity is brought about by an additional aqueous/organic flow (the sheath liquid) of the order of magnitude of mL/min, which dilutes the liquid stream coming out of the separation capillary (nL/min). This accessory flow also ensures the onset of a stable ion spray. The diluting effect on the sample may be seen as a drawback in terms of sensitivity, but, on the other hand, it allows for the use of non-volatile BGEs or small amounts of additives or coating agents in the separation buffer (van Wijk et al. 2007). In a sheathless interface, the electrical contact between the CE buffer and ion source is established by making the terminal end of the separation capillary conductive via several devices, such as stainless-steel connections, microelectrodes, conductive polymer coatings, and metal coatings of gold, silver, nickel or chromium. Unfortunately, the lack of reproducibility and robustness, due to delicate fabrication and manipulation of miniaturised components, limits its use on a large scale despite its advantages in terms of sensitivity (Smith 1990). Novel sheathless configurations have recently been designed, exploiting the results of the developments in nanospray and other miniaturised technology, with promising results (Janini et al. 2003; Kele et al. 2005). Fast mass analysers with a high sampling rate along the electropherogram are required to follow the tiny, fast and closely moving CE peaks, which reflect the intrinsic high efficiency of this separation technique. CE has been hyphenated with almost all types of MS detector: magnetic sectors (Perkins, Tomer 1994), single and triple quadrupoles (Baidoo et al. 2003), ion traps (IT; McClean et al. 2000; Wey et al. 2000; Wey, Thormann 2001a; Iio et al. 2003), Fourier transform ion cyclotron resonance (FTICR; Hofstadler et al. 1994, 1996) and time-offlight (TOF) mass spectrometers (Lazar et al. 1998a, 1998b; Ullsten 2004). At present, as in HPLC, ion traps remain the most widely employed detectors for CE because of their relative speed of scanning, relatively low cost and the possibility of performing multistep fragmentation experiments (MSn). The hyphenation of CE with TOF MS is seen as particularly promising, because of the high scan rate (10–200 ms per spectrum) and high sampling frequency of this detector (Lazar et al. 1998a, 1998b; 1999). TOF shows a particular advantage for toxicological analyses owing to the possibility of identifying molecules by determining their accurate mass and not requiring fragmentation databases. Microchip CE A specific advantage of CE is that instrumentation can easily be miniaturised in a microchip format, since a precise control of fluidics and other separation conditions can be obtained simply by changing the voltage and without the need of mechanical pumps, valves, etc. as in HPLC. CE microchips can also provide custom design, versatility, reduced consumption of reagents and sample, low waste generation, and increased analysis speed and portability. Because of the minimal amounts of sample injected, the most commonly used detection method is LIF, which offers the highest sensitivity. Microchip CE is mostly suitable for portable devices and fully automated analysers. Instrumentation based on this technique is now commercially available. Table 42.1, taken from the third edition of this work, gives an indication of the relative sensitivities of CE detectors towards various analytes in terms of mass and concentration limit of detection (LOD).

Modes of separation A particular advantage of CE is the possibility of using the same hardware to perform different separation modes by simply adjusting a few

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Table 42.1 Relative sensitivity of CE detection systems towards appropriate compounds Detection mode

LOD (moles injected)
13

–10


16

LOD (mol/L) 10


3


8

–10

Commercial availability

Comments

Yes; DAD supplied with the major instruments

At 3) of about 16 ng/mL in spiked urine using the sweeping-MEKC method with fluorescence detection (lex ¼ 320 nm; lem ¼ 390 nm), and a LOD of about 1 ng/mL with CSEI-sweep-MEKC. The sweeping-MEKC was also used for the online concentration and analysis of LSD in clandestine tablets (Fang et al. 2003). LIF detection, using a He–Cd laser at 325 nm wavelength, has also been applied to the determination of LSD in specimens of human blood (Frost, Koehler 1998). The CE method used a citrate–acetate system as running buffer. The use of DAD as a CE detector in routine methods has attracted particular attention in drug analysis. As expected from a multichannel detector, recording an entire signal spectrum enhances the capability of correct identification and determination of analytes and the possibility of studying the peak purity. An example of a CE-DAD application can be found in Nieddu et al. (2005). This rapid method realises the screening of a class of amfetamine designer drugs, comprising 10 methylenedioxy derivatives of amfetamines and phenethylamine in human blood. The detection limits and recoveries from blood samples were between 10 and 30 ng/mL and 81 and 90%, respectively. CSEI was used by Meng et al. (2006) as an on-line concentration method for the high-sensitivity analysis of illicit amfetamines. Using this approach (CSEI with micellar sweeping), a LOD lower than 50 pg/mL was achieved, whereas using normal MEKC it was about 10 ng/mL. The quantitative reproducibility of CSEI-micellar sweeping for the analysis of amfetamine, metamfetamine and MDMA using a benzylamine internal standard was satisfactory (standard deviation around 10%). The method was also tested on hair samples. Among multichannel detectors, mass-selective detectors have rapidly become the gold standard in toxicological analysis for both generic and specific determinations. Recently CE-MS coupling has begun to find applications in the field of drug analysis (Smyth 2006). Benzodiazepines are a challenging class of drugs to assay in biological fluids and represent a typical example of the proficient use of MS detection based on its outstanding identification power and selectivity. A double-dynamic coating CZE method with IT-MS detection was developed by Vanhoenacker et al. (2004a) for the separation of six benzodiazepines in spiked urine, and MS2 experiments were performed for confirmation. McClean et al. (2000) optimised a CZE-ESI-IT-MS method for the determination of selected 1,4-benzodiazepines, identified by sequential product ion fragmentation. MS3 fragmentation was also exploited by Wey et al. (2000) to unambiguously identify codeine, morphine, dihydrocodeine and their glucuronides in urine samples previously screened using opiate immunoassay. The simultaneous detection and quantification of a number of abused drugs in human hair (namely 6-monoacetylmorphine, morphine, amfetamine, metamfetamine (MA), methylenedioxyamfetamine (MDA), methylenedioxymetamfetamine (MDMA), benzoylecgonine, ephedrine and cocaine) was obtained by Gottardo et al. (2007a) with a rapid CZE-ESI-IT-MS method (Figure 42.3). Ammonium formate (25 mmol/L), pH 9.5 and 15 kV separation were employed for the separation using a bare fused-silica capillary. Under field-amplified sample stacking conditions, LODs were below 0.1 ng/mg for all drugs in hair matrix and good linearity was achieved in the concentration range 0.025–5 ng for each analyte per mg of sample. The same group investigated the application of these separation conditions when coupling CEESI with a TOF detector (Gottardo et al. 2007b), in order to exploit the advantages of high mass accuracy and fast scanning capability of this technique. Drugs and metabolites were identified in hair samples, after a single extraction procedure, by exact mass and isotopic pattern matching (Figure 42.4). Analytical precision in real matrices proved acceptable in both within-day and day-to-day tests.

Intensity × 106

766

MA + EPH A MDMA + MDA

3

COC

2 MOR IS + 6-MAM

1 BE

0 13

14

15

2

16 17 Time (min)

18

19

1

0 2

4

6

8

10

12

14

16

18

Time (min)

Figure 42.3 Toxicological analysis of drugs of abuse in hair using CZE-ion trap MS. Peak identification: A, amfetamine; MA, metamfetamine; MDA, methylenedioxyamfetamine; MDMA, methylenedioxymetamfetamine; EPH, ephedrine; COC, cocaine; BE, benzoylecgonine; MOR, morphine; 6-MAM, 6-acethylmorphine; IS, internal standard (folcodine). For analytical details, see text. (From Gottardo et al. 2007a, with permission.)

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Applications of CE to forensic and clinical drug analyses

767

2.5

Intensity × 106

2.0

1.5

1.0

0.5

(a)

0.0

8

10

12

14

16

18

20

Time (min) 2.0 6

Intensity × 106

1.5

1.0

2 5

0.5

IS

1 10 9

4 8

3

(b) 0.0

8

10

12

14

16

18

20

Time (min)

Figure 42.4 Toxicological analysis of drugs of abuse in hair using CZE-TOF MS. Peak identification: 1, MA, metamfetamine; 2, MDMA, methylenedioxymetamfetamine; 3, amfetamine; 4, MDA, methylenedioxyamfetamine; 5, ephedrine; 6, cocaine; 7, codeine; 8, benzoylecgonine; 9,6-acethylmorphine; 10, morphine, at a concentration of 0.2 ng/mg each drug; IS, internal standard (folcodine). For analytical details, see text. (From Gottardo et al. 2007b, with permission.)

the capillary, with resulting enantiomeric separation. When MS detection is used, the anionic chiral selector does not enter the MS ion source, because it is driven in the opposite direction by the applied voltage. Iio et al. (2003) used the complete filling technique in which the chiral selector, a diluted mixture of 3 mmol/L b-cyclodextrin and 10 mmol/L heptakis(2,6-di-O-methyl)-b-cyclodextrin, was instead added directly to the BGE. This method achieved the enantioseparation of metamfetamine and its metabolites in human urine samples with detection limits (with MS) in the range 0.03–0.05 mg/mL. Amfetamine, metamfetamine, MDA, MDMA and MDEA were also used as test compounds in a study by Souverain et al. (2006) on the different strategies for rapid chiral analysis using CE. The adoption of

hydroxypropyl-b-cyclodextrin as the chiral selector was investigated using different approaches (short-end injection, high electric fields, external pressure application, dynamic coating of the capillary) with a view to decreasing analysis time and increasing sensitivity. A general paper on the optimisation of chiral separations using dual neutral cyclodextrins (b-CD and dimethyl-b-CD) was published by Nhujak et al. (2005), showing the potential advantages of adding two chiral selectors to the running buffer. As previously discussed, non-aqueous capillary electrophoresis (NACE) is particularly eligible for CE-MS coupling, because of the high volatility of most organic solvents, which is beneficial for ionisation. Moreover, the observed change in selectivity in comparison with

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Capillary Electrophoresis

separations in aqueous buffer may offer additional practical advantages in resolution. A complete review on NACE-MS has been published by Scriba (2007), collecting all the published NACE-MS applications up to November 2006. The low number of papers reviewed is probably due to the difficulties in adopting a separation technique whose physical–chemical mechanisms are still scarcely understood. Peri-Okonny et al. (2003) exploited NACE-MS for the separation of nine basic drugs, including tricyclic antidepressant and bronchodilator drugs, with 80 mmol/L acetate buffer dissolved in methanol–acetonitrile (80 : 20 v/v). The results showed improved efficiency compared with HPLC separation. In a paper by Steiner and Hassel (2005), a few organic solvents (namely methanol, acetonitrile, DMSO, formamide, N-methylformamide and N,N-dimethylformamide) were compared against water for the preparation of ammonium acetate separation buffer. Selectivity, peak efficiency and average plate counts were evaluated in the separation of basic drugs. In the seven different solvents (including water), the shortest run time was obtained with acetonitrile, the best peak resolution with the amphiprotic solvents (especially methanol), best peak efficiency with methanol and formamide, and the most sensitive ESIMS detection with acetonitrile and methanol, but with only a slight advantage compared with water. Anderson et al. (2004) developed a NACE-MS method for the determination of lidocaine and two of its metabolites in human plasma. The effects of sheath liquid composition, drying gas temperature and nebulising gas pressure on separation efficiency were evaluated. Fluoxetine and related compounds were analysed with NACE-MS by Cherkaoui and Veuthey (2002), using 25 mmol/L ammonium acetate–1 mol/L acetic acid in acetonitrile as the running buffer and a 30 kV separation voltage. A significant increase in sensitivity was obtained compared with UV detection. NACE-MS was also reported by Geiser et al. (2000) for separation of amfetamine derivatives in spiked urine, after LLE. Although proposed as an attractive alternative to chromatography and supported by positive results, CE traditionally lacks sensitivity compared with chromatographic methods. However, on-line and offline sample enrichment and stacking injection techniques have been employed successfully to enhance sensitivity. As previously mentioned, CSEI was used by Meng et al. (2006) for the high-sensitivity analysis of illicit amfetamines and achieved a LOD of 15 mg/L, limit of detection, 80% of the dose is excreted in urine, 40 to 45% of which remains unchanged. The drug is distributed into central spinal fluid. Therapeutic Concentration Six healthy men, aged between 21 and 31 years, were administered an oral dose of 10 and 20 mg/kg body weight eflornithine, and a 10 mg/kg IV dose. Peak plasma concentrations of 31.4 to 51.0 nmol/L were reached, within 2 to 6 h, for the 10 mg/kg oral dose and 27.4 to 137.5 nmol/L for the 20 mg/kg dose (1.5 to 4 h) [Haegele et al. 1981]. Toxicity Ototoxicity can occur and loss of hearing has been observed with a cumulative dose. Low toxicity via ingestion and IP routes. Bioavailability Oral bioavailability, 55%. Half-life 3 to 4 h. Volume of Distribution 0.33 to 0.37 L/kg. Clearance Total body clearance, 1.17 mL/min/kg. Dose A dose of 100 mg/kg body weight is administered every 6 h, IV, for at least 14 days, which may be followed by a 300 mg/kg body weight daily dose (orally) for 3 to 4 weeks. Dose is decreased for patients with impaired renal function. Cohen JL et al. (1989). High-pressure liquid chromatographic analysis of eflornithine in serum. J Pharm Sci 78(2): 114–116. Gunaratna PC et al. (1994). Pharmacokinetic studies of alpha-difluoromethylornithine in rabbits using an enzyme-linked immunosorbent assay. J Pharm Biomed Anal 12(10): 1249–1257. Haegele KD et al. (1981). Kinetics of alpha-difluoromethylornithine: an irreversible inhibitor of ornithine decarboxylase. Clin Pharmacol Ther 30: 210–215.

Quantification Serum HPLC Column: Lichrosorb C18 (240  4.0 mm i.d.). Mobile phase: acetonitrile : water : methanol (35 : 35 : 30), flow rate 1 mL/min. UV detection (l ¼ 245 nm). Retention time: 9 min. Limit of quantification and limit of detection not reported [Heikinheimo et al. 1994]. Disposition in the Body Elcometrine is poorly absorbed by the oral route and rapidly metabolised during hepatic first-pass metabolism. It has a very short halflife because of its inability to bind to sex-hormone-binding globulin or to cortisolbinding globulin, transport proteins that prolong the biological half-life of many steroid hormones. Therapeutic Concentration A group of 25 postpartum nursing women were administered an implant containing 50 mg elcometrine. Mean serum concentrations were reported as 171.5 ng/L (463 pmol/L) on day 15 and 141.2 ng/L (381 pmol/L) on day 75. Elcometrine mean serum concentrations were also measured on day 75 in breast milk and in serum from babies and were reported as 373 pmol/L and 19.3 pmol/L, respectively [Coutinho et al. 1999]. After oral administration of 100 mg of elcometrine in fasting women, the mean peak plasma concentration reached 156.7 ng/L (423 pmol/L) after 10 min. This level was reduced by 50% at 60 min [Noe et al. 1993]. Six healthy postpartum women were administered 20 mg elcometrine parenterally. After 2 weeks, mean plasma and breast milk concentrations were measured by radioimmunoassay and reported as 62 and 38 mg/L, respectively [Lahteenmaki et al. 1990]. Bioavailability Approximately 12%. Half-life Biphasic, 0.13 h and 14.6 h. Volume of Distribution Approximately 263 L. Clearance Approximately 126 L/h. Coutinho EM et al. (1999). Use of a single implant of elcometrine (ST-1435), a nonorally active progestin, as a long acting contraceptive for postpartum nursing women. Contraception 59: 115–122. Heikinheimo O et al. (1994). The progestin ST 1435—rapid metabolism in man. Contraception 50: 275–289. Lahteenmaki PL et al. (1990). Milk and plasma concentrations of the progestin ST-1435 in women treated parenterally with ST-1435. Contraception 42: 555–562. Noe G et al. (1993). Pharmacokinetics and bioavailability of ST 1435 administered by different routes. Contraception 48: 548–556.

Eletriptan 5-HT1 Receptor Agonist, Antimigraine C22H26N2O2S = 382.5 CAS—143322-58-1 IUPAC Name 3-[[(2R)-1-Methylpyrrolidin-2-yl]methyl]-5-(2-phenylsulfonylethyl)-1H-indole Synonyms 5-[2-(Benzenesulfonyl)ethyl]3-(1-methylpyrrolidin-2(R)-ylmethyl)1H-indole; 3-[[(2R)-1-methyl-2-pyrrolidinyl]methyl]-5-[2-(phenylsulfonyl)ethyl]1H-indole; 3-[(1-methylpyrrolidin-2-yl)methyl]-5-(2-phenylsulfonylethyl)-1Hindole; UK-116044.

Elcometrine Contraceptive, Progestogen, Progestational Steroid C23H30O4 = 370.5 CAS—7759-35-5 IUPAC Name [(8R,9S,10R,13S,14S,17R)-17-Acetyl-13-methyl-16-methylidene3-oxo-2,6,7,8,9,10,11,12,14,15-decahydro-1H-cyclopenta[a]phenanthren-17-yl] acetate Synonyms 17-(Acetyloxy)-16-methylene-19-norpregn-4-ene-3,20-dione; 16methylene-17a-acetoxy-19-norpregn-4-ene-3,20-dione; 16-methylene-17a-acetoxy-19 norprogesterone; ST-1435. Proprietary Names Elmetrine; Nestorone.

Chemical Properties Crystals. Mp 178
to 179
.

Chemical Properties pKa 9.2 [Cooper et al. 1999]. Log P (octanol/water), 3.58 [Wishart 2006]. Eletriptan Hydrobromide C22H26N2O2S,HBr = 463.4 CAS—177834-92-3 Proprietary Name Relpax Chemical Properties Awhite to light pale coloured powder. Readily soluble in water.

Quantification Plasma HPLC Column (100  4.6 mm i.d., 5 mm). Mobile phase: acetonitrile : 500 mmol/L potassium phosphate buffer (pH 3.5) : water (30 : 6 : 64), flow rate 1.0 mL/min. UV detection (l ¼ 225 nm). Limit of quantification, 0.5 mg/L [Cooper et al. 1999]. Oral Fluid HPLC Column (100  4.6 mm i.d., 5 mm). Mobile phase: acetonitrile : 500 mmol/L potassium phosphate buffer (pH 3.5) : water (30 : 6 : 64), flow rate 1.0 mL/min. UV detection (l ¼ 225 nm). Limit of quantification, 0.5 mg/L [Cooper et al. 1999]. Disposition in the Body Readily absorbed after oral administration, with peak plasma concentrations reached 1.5 h after dosing (2 h in patients with migraine). It is primarily metabolised by CYP3A4, with a small contribution (