flanagan - fundamentals of analytical toxicology (wiley, 2007)

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
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2007 John Wiley & Sons Ltd,
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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.
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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
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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
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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
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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
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CONTENTS ix

8.2 HPLC: general considerations
178
8.2.1 The column
179
8.2.1.1 Column oven
180
8.2.2 The eluent
181
8.2.3 The pump
182
8.2.4 Sample introduction
184
8.2.5 System operation
185
8.3 Detection in HPLC
186
8.3.1 UV/visible absorption detection
188
8.3.2 Fluorescence detection
189
8.3.3 Chemiluminescence detection
189
8.3.4 Electrochemical detection
190
8.3.5 Chemiluminescent nitrogen detection
192
8.3.6 Evaporative light scattering detection
193
8.3.7 Charged aerosol detection
193

8.3.8 Radioactivity detection
194
8.3.9 Chiral detection
195
8.3.10 Post-column modification
195
8.3.11 Immunoassay detection
196
8.4 Columns and column packings
196
8.4.1 Column configuration
197
8.4.2 Column packings
197
8.4.2.1 Chemical modification of silica
198
8.4.2.2 Bonded-phase selection
199
8.4.2.3 Stability of silica packings
200
8.4.2.4 Monolithic columns
200
8.4.2.5 Hybrid particle columns
201
8.5 Modes of HPLC
202
8.5.1 Normal-phase chromatography
202
8.5.2 Reversed-phase chromatography
202

8.5.3 Ion-exchange chromatography
203
8.5.4 Ion-pair chromatography
204
8.5.5 Size-exclusion chromatography
204
8.5.6 Affinity chromatography
205
8.5.7 Semipreparative and preparative chromatography
206
8.6 Chiral separations
207
8.6.1 Chiral stationary phases
208
8.6.1.1 Amylose and cellulose polymers
208
8.6.1.2 Crown ethers
208
8.6.1.3 Cyclodextrins
209
8.6.1.4 Ligand-exchange chromatography
210
8.6.1.5 Macrocyclic glycopeptides
210
8.6.1.6 Pirkle brush-type phases
211
8.6.1.7 Protein-based phases
213
8.6.2 Chiral eluent additives
213

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8.7 Derivatives for HPLC
214
8.7.1 Fluorescent derivatives
214
8.7.2 Electroactive derivatives
215
8.7.3 Chiral derivatives
215
8.8 Use of HPLC in analytical toxicology
216
8.8.1 Acidic and neutral compounds
216
8.8.2 Basic drugs and quaternary ammonium compounds
217
8.8.2.1 Nonaqueous ionic eluent systems
219
8.8.3 Systematic toxicological analysis
222
8.8.4 Chiral analyses
224
8.9 Summary
224
9 Capillary Electrophoretic Techniques 231
9.1 Introduction
231
9.2 Electrophoretic mobility
232
9.3 Efficiency and zone broadening

234
9.3.1 Joule heating
235
9.3.2 Electrodispersion
235
9.3.3 Adsorption of analyte onto the capillary wall
236
9.4 Sample injection
236
9.4.1 Hydrodynamic injection
236
9.4.2 Electrokinetic injection
237
9.4.3 Sample ‘stacking’
237
9.5 Detection
237
9.6 Reproducibility of migration time
239
9.7 Applications of capillary electrophoresis
240
9.8 Micellar electrokinetic capillary chromatography
240
9.9 Other capillary electrokinetic modes
242
9.9.1 Capillary electrochromatography
242
9.9.2 Capillary gel electrophoresis
244
9.9.3 Capillary isoelectric focusing

244
9.10 CE techniques in analytical toxicology
244
9.11 Chiral separations
244
9.12 Summary
246
10 Mass Spectrometry 249
10.1 Introduction
249
10.1.1 Historical development
250
10.2 Instrumentation
251
10.2.1 Sector instruments
252
10.2.2 Quadrupole instruments
253
10.2.3 Quadrupole ion-trap instruments
253
10.2.4 Ion cyclotron resonance
254
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CONTENTS xi
10.2.5 Controlled fragmentation (MS-MS)
254
10.3 Presentation of mass spectral data
255
10.4 Gas chromatography-mass spectrometry
256

10.4.1 Electron ionization
258
10.4.2 Chemical ionization
259
10.4.3 Application in analytical toxicology
260
10.5 Liquid chromatography-mass spectrometry
266
10.5.1 Atmospheric-pressure chemical ionization
268
10.5.2 Atmospheric-pressure photoionization
269
10.5.3 Electrospray or ionspray ionization
269
10.5.4 Flow fast-atom bombardment ionization
271
10.5.5 Particle-beam ionization
271
10.5.6 Thermospray
271
10.5.7 Application in analytical toxicology
272
10.6 Interpretation of mass spectra
274
10.7 Quantitative mass spectrometry
277
10.8 Summary
278
11 Trace Elements and Toxic Metals 281
11.1 Introduction

281
11.1.1 Historical development
281
11.2 Sample collection and storage
282
11.3 Sample preparation
284
11.3.1 Analysis of tissues
285
11.3.2 Analyte enrichment
285
11.4 Atomic spectrometry
286
11.4.1 General principles of AES, AAS and AFS
286
11.4.2 Atomic absorption spectrometry
287
11.4.2.1 Flame atomization
288
11.4.2.2 Electrothermal atomization
289
11.4.2.3 Sources of error
290
11.4.3 Atomic emission and atomic fluorescence spectrometry
292
11.4.3.1 Atomic emission spectrometry
292
11.4.3.2 Atomic fluorescence spectrometry
293
11.4.4 Inductively coupled plasma-mass spectrometry

293
11.4.4.1 Ion sources
294
11.4.4.2 Mass analyzers
294
11.4.4.3 Interferences
294
11.4.5 Vapour generation approaches
295
11.4.5.1 Hydride generation
295
11.4.5.2 Mercury vapour generation
296
11.4.6 X-ray fluorescence
297
11.5 Colorimetry and fluorimetry
298
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xii CONTENTS
11.6 Electrochemical methods
299
11.6.1 Anodic stripping voltammetry
299
11.6.2 Ion-selective electrodes
300
11.7 Catalytic methods
301
11.8 Neutron activation analysis
302
11.9 Chromatographic methods

302
11.9.1 Chromatography
302
11.9.2 Speciation
303
11.10 Quality assurance
303
11.11 Summary
304
12 Immunoassays and Enzyme-Based Assays 309
12.1 Introduction
309
12.1.1 Historical development
309
12.2 Basic principles of competitive binding assays
310
12.2.1 Antibody formation
310
12.2.2 Specificity
311
12.2.3 Performing the assay
313
12.2.3.1 Classical radioimmunoassay
313
12.2.3.2 Modern radioimmunoassay (RIA)
315
12.2.4 Non-isotopic immunoassay
315
12.2.5 Assay sensitivity and selectivity
316

12.2.6 Immunoassay development
317
12.2.7 Radioreceptor assays
318
12.3 Heterogeneous immunoassays
318
12.3.1 Tetramethylbenzidine reporter system
318
12.3.2 Antigen-labelled competitive ELISA
319
12.3.3 Antibody-labelled competitive ELISA
320
12.3.4 Sandwich ELISA
320
12.3.5 Lateral flow competitive ELISA
321
12.3.6 Chemiluminescent immunoassays (CLIA)
321
12.4 Homogenous immunoassays
321
12.4.1 Enzyme-multiplied immunoassay technique (EMIT)
321
12.4.2 Fluorescence polarization immunoassay (FPIA)
323
12.4.3 Cloned enzyme donor immunoassay (CEDIA)
324
12.5 Microparticulate and turbidimetric immunoassays
326
12.5.1 Microparticle enzyme immunoassay (MEIA)
326

12.5.2 Chemiluminescent magnetic immunoassay (CMIA)
327
12.6 Assay calibration, quality control and quality assurance
327
12.6.1 Immunoassay calibration
327
12.6.2 Drug screening
329
12.7 Interferences and assay failures
329
12.7.1 Digoxin
330
12.7.1.1 Digoxin-like immunoreactive substances (DLIS)
330
12.7.1.2 Other digoxin-like immunoreactive substances
331
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CONTENTS xiii
12.7.1.3 Measurement of plasma digoxin after F
ab
antibody fragment
administration
331
12.7.2 Insulin and C-peptide
331
12.8 Enzyme-based assays
332
12.8.1 Paracetamol
332
12.8.2 Ethanol

333
12.8.3 Anticholinesterases
334
12.9 Summary
334
13 Toxicology Testing at the Point of Care 339
13.1 Introduction
339
13.1.1 Historical development
340
13.2 Use of POCT
340
13.2.1 Samples and sample collection
341
13.3 Analytes
343
13.3.1 Ethanol
343
13.3.1.1 Breath ethanol
343
13.3.1.2 Saliva ethanol
343
13.3.2 Drugs of abuse
344
13.3.2.1 Urine testing
345
13.3.2.2 Oral fluid testing
346
13.3.2.3 Sweat testing
346

13.3.3 Paracetamol and salicylates
346
13.3.4 Snake envenomation
347
13.3.5 Therapeutic drug monitoring
347
13.3.5.1 Lithium
348
13.3.5.2 Theophylline
348
13.3.5.3 Anticonvulsants
348
13.4 Interferences and adulterants
348
13.5 Quality assurance
349
13.6 Summary
350
14 Basic Laboratory Operations 353
14.1 Introduction
353
14.1.1 Reagents and standard solutions
354
14.1.2 Reference compounds
354
14.1.3 Preparation and storage of calibration solutions
356
14.2 Aspects of quantitative analysis
358
14.2.1 Analytical error

358
14.2.1.1 Confidence intervals
360
14.2.2 Minimizing random errors
361
14.2.2.1 Preparation of a solution of known concentration
362
14.2.3 Accuracy and Precision
362
14.2.3.1 Assessing precision and accuracy
363
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xiv
CONTENTS
14.2.3.2 Detecting systematic error (fixed bias)
363
14.2.3.3 Identifying sources of variation: analysis of variance
364
14.2.4 Calibration graphs
365
14.2.4.1 Linear regression
366
14.2.4.2 Testing for linearity
368
14.2.4.3 Weighted linear regression
370
14.2.4.4 Nonlinear calibration curves
370
14.2.4.5 Residuals and standardized residuals
372

14.2.4.6 Blank samples and the intercept
372
14.2.4.7 Method of standard additions
373
14.2.4.8 Limits of detection and quantitation
373
14.2.4.9 Curve fitting and choice of equation
374
14.2.4.10 Single point calibration
375
14.2.5 Batch analyses
375
14.3 Use of internal standards
376
14.3.1 Advantages of internal standardization
378
14.3.1.1 Reproducibility of injection volume
378
14.3.1.2 Instability of the detection system
379
14.3.1.3 Pipetting errors and evaporation of extraction solvent
379
14.3.1.4 Extraction efficiency
380
14.3.1.5 Derivatization and nonstoichiometric reactions
381
14.3.2 Internal standard availability
381
14.3.3 Potential disadvantages of internal standardization
382

14.4 Method comparison
382
14.4.1 Bland–Altman plots
383
14.5 Nonparametric statistics
384
14.5.1 Sign Tests
385
14.5.1.1 Wilcoxon signed rank test
386
14.5.2 Runs test
387
14.5.3 Mann–Whitney U-test
387
14.5.4 Spearman rank correlation
387
14.5.5 Nonparametric regression
388
14.6 Quality control and proficiency testing
389
14.6.1 Quality control charts
390
14.6.1.1 Shewhart charts
390
14.6.1.2 Cusum charts
390
14.6.1.3 J-chart
391
14.6.1.4 Westgard rules
392

14.6.2 External quality assurance
392
14.7 Operational considerations
393
14.7.1 Staff training
393
14.7.2 Recording and reporting results
394
14.7.3 Toxicology EQA schemes
395
14.8 Summary
397
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CONTENTS xv
15 Absorption, Distribution, Metabolism and Excretion of Xenobiotic
Compounds 399
15.1 Introduction
339
15.1.1 Historical development
399
15.2 Routes of administration
400
15.2.1 Oral dosage
400
15.2.1.1 P-Glycoprotein
402
15.2.1.2 Presystemic metabolism
403
15.2.2 Intravenous injection
403

15.2.3 Intramuscular and subcutaneous injection
404
15.2.4 Sublingual and rectal administration
404
15.2.5 Intranasal administration
405
15.2.6 Transdermal administration
405
15.2.7 Inhalation
405
15.2.8 Other routes of administration
405
15.3 Absorption
406
15.3.1 Passive diffusion
406
15.3.1.1 Partition coefficient
407
15.3.1.2 Ionization
407
15.3.2 Carrier-mediated absorption
408
15.3.3 Absorption from muscle and subcutaneous tissue
409
15.4 Distribution
409
15.4.1 Ion trapping
410
15.4.2 Binding to macromolecules
411

15.4.2.1 Plasma protein binding
411
15.4.3 Distribution in lipid
412
15.4.4 Active transport
412
15.5 Metabolism
412
15.5.1 Phase 1 metabolism
413
15.5.1.1 The cytochrome P450 family
413
15.5.1.2 Other phase 1 oxidases
414
15.5.1.3 Microsomal reductions
416
15.5.1.4 Hydrolysis
416
15.5.2 Phase 2 reactions
417
15.5.2.1 D-Glucuronidation
417
15.5.2.2 O-sulfation and N-acetylation
418
15.5.2.3 O-, N- and S-methylation
419
15.5.2.4 Conjugation with glutathione
419
15.5.2.5 Amino acid conjugation
419

15.5.3 Metabolic reactions of analytical or toxicological importance
420
15.5.3.1 Oxidative dealkylation
420
15.5.3.2 Hydroxylation
421
15.5.3.3 S- and N-oxidation
422
15.5.3.4 Oxidative dehalogenation
423
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xvi CONTENTS
15.5.3.5 Desulfuration
425
15.5.3.6 Trans-sulfuration and trans-esterification
425
15.6 Excretion
425
15.6.1 The kidney
426
15.6.1.1 Tubular secretion
427
15.6.1.2 Excretion of metabolites
427
15.6.2 Biliary excretion
427
15.6.2.1 Enterohepatic recirculation
427
15.7 Summary
428

16 Pharmacokinetics 431
16.1 Introduction
431
16.1.1 Historical development
431
16.1.2 Symbols and conventions
432
16.2 Fundamental concepts
432
16.2.1 Rates, rate constants and reaction order
432
16.2.1.1 First-order elimination
433
16.2.1.2 Zero-order elimination
434
16.2.2 Dependence of half-life on volume of distribution
and clearance
434
16.2.2.1 Apparent volume of distribution
435
16.2.2.2 Organ clearance
435
16.2.2.3 Whole body clearance
436
16.3 Absorption and elimination
437
16.3.1 First-order absorption
437
16.3.2 Bioavailability
438

16.3.3 Maximum concentration (C
max)
439
16.4 Drug accumulation
439
16.4.1 Intravenous infusion
439
16.4.1.1 Loading doses
440
16.4.2 Multiple dosage
440
16.5 Sustained-release preparations
441
16.5.1 Intramuscular depot injection
442
16.5.2 Other sustained-release preparations
443
16.6 Non-linear pharmacokinetics
443
16.6.1 Ethanol
445
16.7 Multicompartment models
447
16.7.1 Calculation of rate constants
449
16.7.2 Volumes of distribution in a two-compartment model
450
16.8 Model-independent pharmacokinetic parameters
451
16.8.1 Apparent volume of distribution

452
16.8.2 Clearance
453
16.8.3 Model-independent approach
453
16.9 Pharmacokinetics and the interpretation of results
454
16.9.1 Back-calculation of dose or time of dose
454
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CONTENTS xvii
16.9.1.1 How much substance was administered?
455
16.9.1.2 When was the substance administered?
455
16.9.1.3 Practical examples
456
16.9.1.4 Calculation of time of cannabis exposure
456
16.9.2 Toxicokinetics
458
16.10 Summary
461
17 Clinical Interpretation of Analytical Results 463
17.1 Introduction
463
17.2 Pharmacogenetics
463
17.2.1 Acetylator status
465

17.2.1.1 Isoniazid
465
17.2.1.2 Sulfonamides
465
17.2.2 Cytochrome P450 polymorphisms
466
17.2.2.1 CYP2D6 polymorphism
466
17.2.2.2 CYP2C9 and CYP2C19 polymorphisms
467
17.2.2.3 Other CYP polymorphisms
467
17.2.3 Atypical cholinesterase
467
17.2.4 Glucose-6-phosphate dehydrogenase (G6PD)
468
17.2.5 Alcohol dehydrogenase and aldehyde dehydrogenase
468
17.3 Effects of age, sex and disease on drug disposition
468
17.3.1 Age
468
17.3.1.1 Effect of age on renal function
469
17.3.2 Disease
469
17.3.3 Sex
470
17.4 Enzyme induction and inhibition
471

17.4.1 Enzyme induction
471
17.4.2 Enzyme inhibition
472
17.5 Investigation of acute poisoning
472
17.5.1 Selectivity and reliability of analytical methods
474
17.5.2 Route and duration of exposure and mechanism of toxicity
474
17.5.3 Hair analysis
476
17.5.4 Sources of further information
477
17.6 Postmortem toxicology
478
17.6.1 Choice of sample and sample collection site
479
17.6.2 Assay calibration
480
17.6.3 Interpretation of analytical results
481
17.7 Gazetteer
483
17.7.1 Antidepressants
483
17.7.2 Anti-epileptics and antipsychotics
483
17.7.3 Carbon monoxide and cyanide
483

17.7.4 Cannabis
484
17.7.5 Cardioactive drugs
485
17.7.6 Cocaine
485
17.7.7 Drug-facilitated sexual assault
486
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xviii CONTENTS
17.7.8 Ethanol (ethyl alcohol, ‘alcohol’)
487
17.7.9 Heroin/morphine
488
17.7.10 Hypoglycaemic agents
489
17.7.11 Methadone
489
17.7.12 Methylenedioxymetamfetamine and related compounds
490
17.7.13 Volatile substance abuse (VSA)
490
17.8 Summary
490
Index 495
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Preface
The analytical toxicologist may be required to detect, identify, and in many cases measure a
wide variety of compounds in samples from almost any component of the body or in related
materials such as residues in syringes or in soil. Many difficulties may be encountered. The

analytes may include gases such as carbon monoxide, drugs, solvents, pesticides, metal salts, and
naturally occurring toxins. Some samples may be pure chemicals and others complex mixtures.
New drugs, pesticides, and other substances continually present novel challenges in analysis and
in the interpretation of results. The analyte might be an endogenous compound such as acetone,
or an exogenous compound such as a drug and/or metabolite(s) of the drug, whilst the sample
matrix may range from urine to bone.
Many biological samples contain muscle, connective tissue, and so forth, which may have to
be separated or degraded prior to the analysis, as well as a multitude of small and large molecular
weight compounds. The concentration of the analyte to be measured can range from g L
−1
(parts
per thousand) in the case of blood ethanol to ␮gL
−1
(parts per thousand million) in the case of
plasma digoxin. The stability of the analytes also varies considerably, ranging from a few minutes
for protease sensitive peptides and esters such as aspirin and heroin, to several years for some
other drugs and pesticides.
This book aims to give principles and practical information on the analysis of drugs, poisons
and other relevant analytes in biological specimens, particularly clinical and forensic specimens.
The material presented here is intended to cover part of the basic theoretical syllabus of the
Association for Clinical Biochemistry (ACB) pre-registration training course in Clinical Chem-
istry, subspecialty Analytical Toxicology ( />tox.pdf,
accessed 20 April 2006). The book should also prove useful to other analytical toxicol-
ogy trainees and also to those undertaking preregistration training in Clinical Chemistry
( />biochem.pdf). As such, this volume extends the
scope of the World Health Organization (WHO) basic analytical toxicology manual (Flanagan,
et al. Basic Analytical Toxicology. WHO, Geneva: 1995), but is not intended to provide detailed
coverage such as that given in Clarke’s Analysis of Drugs and Poisons (Moffat, et al., eds. Edition
3. Pharmaceutical Press, London: 2004).
It has been assumed that the reader will be familiar with basic analytical laboratory operations.

Many of the topics discussed here (use of clinical specimens, samples and standards, sample pre-
treatment, TLC, UV/visible spectrophotometry, GLC, HPLC, etc.) are the subject of monographs
in the Analytical Chemistry by Open Learning (ACOL) series, and study of these volumes is rec-
ommended for those with a limited background in analytical chemistry. A distance learning course
in analytical chemistry is available ( ac-
cessed 3 August 2007).
A detailed basic text (Laboratory Skills Training Handbook. Bailey and Barwick, 2007) has
been produced under the Valid Analytical Measurement (VAM) programme. The VAM website
( accessed 3 August 2007) details a number of related publica-
tions, many of which can be downloaded free of charge.
xix
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xx PREFACE
The appendices to this book (incorporating a guide to interpreting analytical toxicology re-
sults, CAS registry numbers, a list of volatile compounds & conversion factors, and a glos-
sary of commonly-used terms) are available online, and can be accessed at www.wiley.com/
go/flanagantoxicology.
The authors and publishers would like to remind readers that web addresses cited in the text,
including those in the preface, are current and accurate as we go to press. However, we cannot
guarantee that cited resources or content will remain indefinitely at the exact addresses given.
JWBK176-Flanagan-FM JWBK176-Flanagan December 21, 2007 19:8 Char Count= 0
Health and Safety
Care should be taken to ensure the safe handling of all chemical or biological materials, and par-
ticular attention should be given to the possible occurrence of allergy, infection, fire, explosion, or
poisoning (including transdermal absorption or inhalation of toxic vapours). Readers are expected
to consult local health and safety regulations and adhere to them.
xxi
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Nomenclature, Symbols

and Conventions
We have followed IUPAC nomenclature for chemical names except when Chemical Abstracts
nomenclature or trivial names are more readily understood. With regard to symbols we have
adopted the convention that variables and constants are italicized, but labels and mathe-
matical operators are not (IUPAC Compendium of Chemical Terminology, Edition 2, 1997;
accessed 20 April 2006). Thus, for
example, the acid dissociation constant is written K
a
, a being a label to denote that it is an acid
dissociation constant. The notation for the negative logarithm of K
a
is pK
a
; p is a mathemat-
ical symbol and is not italicized. Where the subscript is a variable then it is italicized, so the
concentration at time t,isC
t
, but the concentration at time 0 is C
0
. Note especially that relative
molecular mass (molecular weight, relative molar mass), ratio of the mass of an atom or molecule
to the unified atomic mass unit (u), is referred to throughout as M
r
. The unified atomic mass unit,
sometimes referred to as the dalton (Da), is defined as 1/12 of the mass of one atom of
12
C.
The symbol amu for atomic mass unit can sometimes be found, particularly in older works. The
unified atomic mass unit is not a Syst`eme International (SI) unit of mass, although it is (only by
that name, and only with the symbol u) accepted for use with SI.

We have adopted the convention whereby square brackets [] used in figure legends, etc. denote
analyte concentration.
As to drugs and pesticides, we have used recommended International Nonproprietary Name
(rINN) or proposed International Nonproprietary Name (pINN) whenever possible. For drugs of
abuse, the most common chemical names or abbreviations have been used. It is worth noting that
for rINNs, it is now general policy to use ‘f’ for ‘ph’ (e.g. in sulfur), ‘t’ for ‘th’ (e.g. chlortalidone
not chlorthalidone) and ‘i’ for ‘y’ (mesilate not mesylate for methanesulfonate, for example).
However, so many subtle changes have been introduced in recent years that it is difficult to ensure
compliance withall suchchanges. Namesthatmay beencountered includeBritishApproved Name
(BAN), British Pharmacopoeia (BP) name, United States Adopted Name (USAN), United States
National Formulary (USNF) name, and United States Pharmacopoeia (USP) name. Where the
rINN is markedly different from common US usage, e.g. acetaminophen rather than paracetamol,
meperidine instead of pethidine, the alternative is given in parentheses at first use and in the index.
A useful source of information on drug and poison nomenclature is the Merck Index (Edition
14. O’Neil et al., eds. (2006) Merck & Co., Whitehouse Station, NJ); it should be noted that the
reference number of a particular compound (Merck Index number) changes with edition. Chem-
ical Abstracts Service (CAS) Registry Numbers (RN) provide a unique identifier for individual
compounds, but it is again important to be clear which numbers refers to salts, hydrates, race-
mates, etc. Similarly, when discussing dosages we have tried to be clear when referring to salts,
etc. and when to free bases or quaternary ammonium compounds.
We emphasize that cross-referral to an appropriate local or national formulary is mandatory
before any patient treatment is initiated or altered. Proprietary names must be approached with
xxiii