JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 FUNDAMENTALS OF ANALYTICAL TOXICOLOGY Robert J Flanagan Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London, UK Andrew Taylor Royal Surrey County Hospital, Guildford, Surrey, UK Ian D Watson Department of Clinical Biochemistry, University Hospital Aintree Liverpool, UK Robin Whelpton School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 FUNDAMENTALS OF ANALYTICAL TOXICOLOGY JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 FUNDAMENTALS OF ANALYTICAL TOXICOLOGY Robert J Flanagan Department of Clinical Biochemistry, King’s College Hospital NHS Foundation Trust, London, UK Andrew Taylor Royal Surrey County Hospital, Guildford, Surrey, UK Ian D Watson Department of Clinical Biochemistry, University Hospital Aintree Liverpool, UK Robin Whelpton School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 Copyright C  2007 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): cs-books@wiley.co.uk Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. 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Anniversary Logo Design: Richard J. Pacifico Library of Congress Cataloging-in-Publication Data Fundamentals of analytical toxicology / Robert J. Flanagan [et al.]. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-31934-5 (hb : alk. paper) ISBN 978-0-470-31935-2 (pbk. : alk. paper) 1. Analytical toxicology. I. Flanagan, Robert James. [DNLM: 1. Toxicology—methods. 2. Chemistry, Clinical—methods. 3. Specimen Handling—methods. QV 602 F9805 2007] RA1221.F86 2007 615.9  07—dc22 2007013704 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library. ISBN 9780470319345 (HB) 9780470319352 (PB) Typeset by Aptara, New Delhi, India. Printed and bound in Great Britain by Antony Rowe Ltd, Chippenham, Wiltshire. This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production. JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 Contents Preface xix Health and Safety xxi Nomenclature, Symbols and Conventions xxiii Amount Concentration and Mass Concentration xxv Acknowledgements xxvii List of Abbreviations xxix 1 Analytical Toxicology: Overview 1 1.1 Introduction 1 1.1.1 Historical development 1 1.2 Modern analytical toxicology 2 1.2.1 Drugs and pesticides 4 1.2.2 Ethanol and other volatile substances 6 1.2.3 Trace elements and toxic metals 7 1.3 Provision of analytical toxicology services 8 1.3.1 Samples and sampling 8 1.3.2 Choice of analytical method 8 1.3.3 Method implementation and validation 9 1.3.4 Quality control and quality assurance 11 1.4 Applications of analytical toxicology 13 1.4.1 Clinical toxicology 13 1.4.2 Forensic toxicology 14 1.4.3 Drug abuse screening 15 1.4.4 Therapeutic drug monitoring (TDM) 16 1.4.5 Occupational and environmental toxicology 17 1.5 Summary 18 2 Sample Collection, Transport, and Storage 21 2.1 Introduction 21 2.2 Clinical samples and sampling 21 2.2.1 Health and safety 21 2.2.2 Clinical sample types 23 2.2.2.1 Arterial blood 23 2.2.2.2 Venous blood 23 2.2.2.3 Serum 26 2.2.2.4 Plasma 26 2.2.2.5 Blood cells 27 2.2.2.6 Urine 28 v JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 vi CONTENTS 2.2.2.7 Stomach contents 28 2.2.2.8 Faeces 28 2.2.2.9 Tissues 29 2.3 Guidelines for sample collection for analytical toxicology 29 2.3.1 Sample collection and preservation 32 2.3.2 Blood (for quantitative work) 32 2.3.3 Blood (for qualitative analysis) 33 2.3.4 Urine 33 2.3.5 Stomach contents 35 2.3.6 Saliva/oral fluids 36 2.3.6.1 Collection devices for saliva/oral fluids 37 2.3.7 Sweat 38 2.3.8 Exhaled air 38 2.3.9 Cerebrospinal fluid 38 2.3.10 Vitreous humour 38 2.3.11 Synovial fluid 39 2.3.12 Liver 39 2.3.13 Other tissues 39 2.3.14 Insect larvae 39 2.3.15 Keratinaceous tissues (hair and nail) 40 2.3.16 Bone and bone marrow 41 2.3.17 Injection sites 41 2.3.18 ‘Scene residues’ 41 2.4 Sample transport and storage 42 2.5 Common interferences 44 2.6 Summary 45 3 Sample Preparation 49 3.1 Introduction 49 3.2 Modes of sample preparation 51 3.2.1 Direct analysis/on-line sample preparation 51 3.2.2 Protein preciptation 52 3.2.3 Microdiffusion 54 3.2.4 Headspace and ‘purge-and-trap’ analysis 55 3.2.5 Liquid–liquid extraction 57 3.2.5.1 Theory of pH-controlled liquid–liquid extraction 63 3.2.5.2 Ion-pair extraction 66 3.2.5.3 Liquid–liquid extraction columns 67 3.2.6 Solid-phase extraction 67 3.2.7 Solid-phase microextraction 73 3.2.8 Liquid-phase microextraction 76 3.2.9 Supercritical fluid extraction 77 3.2.10 Accelerated solvent extraction 78 3.3 Measurement of nonbound plasma concentrations 79 3.3.1 Ultrafiltration 80 3.3.2 Equilibrium dialysis 81 JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 CONTENTS vii 3.4 Hydrolysis of conjugated metabolites 82 3.5 Extraction of drugs from tissues 84 3.5.1 Hair analysis for drugs and organic poisons 85 3.6 Derivatization 87 3.7 Summary 88 4 Colour Tests, and Spectrophotometric and Luminescence Techniques 95 4.1 Introduction 95 4.1.1 Historical development 95 4.2 Colour tests 96 4.3 UV/visible spectrophotometry 97 4.3.1 The Beer–Lambert law 98 4.3.2 Instrumentation 100 4.3.2.1 Derivative spectrophotometry 102 4.3.3 Spectrophotometric assays 104 4.3.3.1 Salicylates in plasma or urine 106 4.3.3.2 Carboxyhaemoglobin (COHb) in whole blood 106 4.3.3.3 Cyanide in whole blood by microdiffusion 107 4.3.3.4 Colorimetric measurement of sulfonamides 108 4.4 Luminescence 108 4.4.1 Fluorescence and phosphorescence 108 4.4.1.1 Intensity of fluorescence and quantum yield 109 4.4.1.2 Instrumentation 110 4.4.1.3 Fluorescence assays 111 4.4.2 Chemiluminescence 112 4.4.2.1 Instrumentation 114 4.4.2.2 Chemiluminescence assays 114 4.5 Summary 115 5 Introduction to Chromatography and Capillary Electrophoresis 117 5.1 General introduction 117 5.1.1 Historical development 117 5.2 Theoretical aspects of chromatography 119 5.2.1 Analyte phase distribution 119 5.2.2 Column efficiency 121 5.2.3 Zone broadening 122 5.2.3.1 Multiple path and eddy diffusion 122 5.2.3.2 Longitudinal diffusion 123 5.2.3.3 Resistance to mass transfer 123 5.2.4 Extra-column contributions to zone broadening 125 5.2.5 Temperature programming and gradient elution 125 5.2.6 Selectivity 126 5.2.7 Peak asymmetry 127 5.3 Measurement of analyte retention 128 5.4 Summary 129 JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0 viii CONTENTS 6 Thin-Layer Chromatography 131 6.1 Introduction 131 6.2 Preparation of thin-layer plates 132 6.3 Sample application 133 6.4 Developing the chromatogram 133 6.5 Visualizing the chromatogram 135 6.6 Retention factor (R f ) 137 6.7 Toxi-Lab 140 6.8 High-performance thin-layer chromatography 140 6.8.1 Forced-flow planar chromatography 141 6.9 Quantitative thin-layer chromatography 141 6.10 Summary 142 7 Gas Chromatography 145 7.1 Introduction 145 7.2 Instrumentation 146 7.2.1 Injectors and injection technique 147 7.2.1.1 Cryofocusing/thermal desorption 148 7.2.2 Detectors for GC 149 7.2.2.1 Thermal-conductivity detection 150 7.2.2.2 Flame-ionization detection 150 7.2.2.3 Nitrogen–phosphorus detection 151 7.2.2.4 Electron capture detection 152 7.2.2.5 Pulsed-discharge detection 154 7.2.2.6 Flame-photometric detection 155 7.2.2.7 Atomic-emission detection 155 7.2.2.8 Fourier-transform infrared detection 156 7.3 Columns and column packings 156 7.3.1 Packed columns 157 7.3.2 Capillary columns 160 7.3.3 Multidimensional GC 163 7.4 Derivatization for GC 164 7.4.1 Electron-capturing derivatives 165 7.5 Chiral separations 166 7.6 Applications of gas chromatography in analytical toxicology 167 7.6.1 Systematic toxicological analysis 167 7.6.2 Quantitative analysis of drugs and other poisons 179 7.6.2.1 Measurement of carbon monoxide and cyanide 170 7.6.2.2 Measurement of ethanol and other volatiles 170 7.7 Summary 173 8 High-Performance Liquid Chromatography 177 8.1 Introduction 177 [...]... spectrometry/spectrometric N -methyl-N -trimetylsilyl-trifluoroacetamide methyl tert-butyl ether neutron activation analysis National Academy of Clinical Biochemistry (US) N -acetyl-L-cysteine nonaqueous capillary electrophoresis nicotine adenine dinucleotide N -acetyl- p-benzoquinoneimine 2,3-naphthalenedialdehyde nicotine adenine dinucleotide phosphate N -naphthylethylenediamine 7-nitrobenz-2-oxa-1,3-diazole National... ionization monoamine oxidase inhibitor 6-monoacetylmorphine 3,4-methylenedioxy-N -methylbutanamine 2,2 -Dichloro-4,4 -methylene dianiline N -Methyl-bis(trifluoroacetamide) microcrystalline triacetate 4,4 -methylenedianiline (occupational context); 3,4-methylenedioxyamfetamine (drug of abuse) 3,4-methylenedioxyethylamfetamine multidimensional gas chromatography 3,4-methylenedioxymetamfetamine multidrug... C(Z)E capillary (zone) electrophoresis CEA chiral eluent additive CEC capillary electrochromatography CECF 1-chloroethyl chloroformate CEDIA cloned enzyme donor immunoassay CHAPS 3-[ (3-cholamidopropyl)dimethylammonio ]-1 -propanesulfonate CHAPSO 3-( [3-cholamidopropyl]dimethylammonio )-2 -hydroxy-1-propanesulfonate CI chemical ionization (mass spectrometry), confidence interval (statistics) CIEF capillary... fatty acid phase flow-injection analysis flame ionization detector fluorescein isothiocyanate flavin-containing monooxygenase flame photometric detector ferric/perchloric/nitric (acids) fluorescence polarisation immunoassay 3-( 2-furoyl)quinoline-2-carboxaldehyde free solution capillary electrophoresis xxxi xxxii LIST OF ABBREVIATIONS FSH FTICR-MS FTIR FRAT GC GCE GFR GHB GI GLC G-6-P GPC G-6-PDH GPR GSC GSH... FITC FMO FPD FPN FPIA FQ FSCE deferoxamine (desferrioxamine) drug facilitated sexual assault digoxin-like immunoreactive substance(s) dimethyldichlorosilane dinitro-ortho-cresol (4,6-dinitro-2-methylphenol) dansyl chloride drugs of abuse 3-( 3,4-dihydroxyphenyl)alanine dodecyltrimethylammonium bromide 5-( 4,6-dichlorotriazinyl)aminofluorescein Drug Treatment and Testing Order driving under the influence enzyme... department (medicine) 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (methadone metabolite I) ethylenediaminetetra-acetic acid (or sodium salt) energy dispersive X-ray fluorescence electron ionization enzyme immunoassay enzyme-linked immunosorbent assay evaporative light scattering detector extensive metabolizer ecgonine methyl ester enzyme-multiplied immunoassay technique electro-osmotic flow Environmental... (University of Sheffield), Prof H Maurer (Saarland University, Homburg), Dr G Mills (University of Portsmouth), Mr P Morgan (Guy’s and St Thomas’ Hospital), Prof P Myers (University of York), Dr I Ojanper¨ (University of Helsinki), Prof D Perrett (Queen a Mary, University of London), Prof F Pragst (University Hospital Charit´ , Berlin), Miss J Smith e (Selly Oak Hospital), Dr J Wilson (University of Cardiff),... chromatography glucose-6-phosphate gel permeation chromatography glucose-6-phosphate dehydrogenase general purpose reagent gas–solid chromatography reduced glutathione oxidized glutathione glutathione S-transferase glyceryl nitrate (trinitrin) glycated haemoglobin human chorionic gonadotropin 2-hydroxyethyl mercapturic acid [N -acetyl-S-(2-hydroxyethyl)-L-cysteine] height equivalent to a theoretical plate heptafluorobutanoic... 15.5.1.4 Hydrolysis 15.5.2 Phase 2 reactions 15.5.2.1 D-Glucuronidation 15.5.2.2 O-sulfation and N -acetylation 15.5.2.3 O-, N - and S-methylation 15.5.2.4 Conjugation with glutathione 15.5.2.5 Amino acid conjugation 15.5.3 Metabolic reactions of analytical or toxicological importance 15.5.3.1 Oxidative dealkylation 15.5.3.2 Hydroxylation 15.5.3.3 S- and N -oxidation 15.5.3.4 Oxidative dehalogenation xv 399... isobutyl ketone (4-methyl-2-pentanone) molecularly imprinted polymer Michaelis–Menten xxxiv LIST OF ABBREVIATIONS MMDA MMP MOCA Mr MR MS MSTFA MTBE NAA NACB NAC NACE NAD NAPQI NDA NADP NED NBD NIH NIMH NIST NLM NMR NPD NPT NSD OATP OCT ODS OIA OP OPA OPLC OPLC OR OTC PAN PBA PC PCA PCP PCT PDD PDMS PDVB 3-methoxy-4,5-methylenedioxyamfetamine mixed-mode (stationary) phase 4,4 -methylenebis(2-chloroaniline) . JWBK176 -Flanagan December 21, 2007 19:8 Char Count= 0 JWBK176 -Flanagan- FM JWBK176 -Flanagan December 21, 2007 19:8 Char Count= 0 FUNDAMENTALS OF ANALYTICAL TOXICOLOGY JWBK176 -Flanagan- FM JWBK176 -Flanagan. Pacifico Library of Congress Cataloging-in-Publication Data Fundamentals of analytical toxicology / Robert J. Flanagan [et al.]. p. ; cm. Includes bibliographical references and index. ISBN 97 8-0 -4 7 0-3 193 4-5 . 2007 19:8 Char Count= 0 JWBK176 -Flanagan- FM JWBK176 -Flanagan December 21, 2007 19:8 Char Count= 0 FUNDAMENTALS OF ANALYTICAL TOXICOLOGY Robert J Flanagan Department of Clinical Biochemistry, King’s