List of contributors .......................................... xvi
Preface ...................................................... xvii
Physico-chemical properties of drugs and metabolites and
their extraction from biological material ....................... 1
1. HUGH WILTSHIRE
1.1. Introduction ............................................... 1
1.1.1. Metabolite isolation ................................ 1
1.1.2. Bioanalysis ......................................... 1
1.1.3. Enrichment of drugs and metabolites ................. 1
1.1.4. Differences between metabolite isolation and drug
analysis ............................................ 2
1.2. Physico-chemical properties of drugs and solvents .......... 2
1.2.1. Energy changes on solution .......................... 2
1.2.2. Molecular phenomena behind solubility/miscibility ... 3
1.2.3. Water miscibility and water immiscibility ........... 8
1.3. Partition .................................................. 9
1.3.1. Extraction efficiency ............................... 9
1.4. Ionisation and its effect on the extraction of drugs ...... 11
1.4.1. Ionisation, pH and pK .............................. 11
1.4.2. Titration curves ................................... 12
1.4.3. Henderson-Hasselbach equation ...................... 13
1.4.4. Buffers ............................................ 16
1.4.5. Distribution coefficient ........................... 17
1.5. Solvent extraction ........................................ 18
1.5.1. Choice of solvent .................................. 18
1.5.2. Mixed solvents ..................................... 20
1.5.3. Dealing with plasma proteins and emulsions ......... 21
1.5.4. Choice of pH for solvent extraction ................ 21
1.5.5. Artefacts arising during the extraction of drugs
and metabolites .................................... 21
1.5.6. Modification and derivatisation of drugs and
metabolites ........................................ 24
1.5.7. Ion-pair extraction ................................ 26
1.5.8. Recoveries ......................................... 26
1.6. The vfirst law of drug metabolism' ........................ 26
1.7. Bibliography .............................................. 27
2. Solid-phase extraction
CHRIS JAMES
2.1. Introduction .............................................. 28
2.2. General properties of bonded silica sorbents .............. 30
2.3. Sorbent/analyte interactions .............................. 30
2.3.1. Solvation .......................................... 30
2.3.2. Non-polar .......................................... 31
2.3.3. Polar .............................................. 32
2.3.4. Ion exchange ....................................... 32
2.3.5. Covalent ........................................... 33
2.3.6. Mixed-mode interactions ............................ 34
2.3.7. Polymeric sorbents ................................. 35
2.3.8. Miscellaneous ...................................... 36
2.4. Sample. pretreatment of different biological matrices ..... 36
2.4.1. Liquid samples ..................................... 36
2.4.2. Protein binding .................................... 36
2.4.3. Solid samples ...................................... 37
2.5. Developing SPE methods .................................... 37
2.6. Example of an SPE method .................................. 38
2.7. Disc cartridges ........................................... 38
2.7.1. Potential advantages ............................... 39
2.7.2. Disadvantages ...................................... 40
2.8. 96-Well format (e.g. Porvair Microsep™ system) ............ 40
2.9. Direct injection of plasma ................................ 41
2.10.Other new developments .................................... 41
2.10.1.Fines .............................................. 41
2.10.2.A cartridge in a pipette tip? ...................... 41
2.11.Conclusions and future perspectives ....................... 42
2.12.Bibliography .............................................. 42
3. Basic HPLC theory and practice
ANDY GRAY
3.1. Origins ................................................... 44
3.2. Applications .............................................. 44
3.3. Apparatus ................................................. 45
3.3.1. Column ............................................. 45
3.3.2. Plumbing ........................................... 47
3.3.3. Pumps .............................................. 48
3.3.4. Injectors .......................................... 49
3.3.5. Column ovens ....................................... 50
3.3.6. Detectors .......................................... 50
3.4. The chromatographic process ............................... 50
3.4.1. Basic principles ................................... 50
3.4.2. Molecular forces ................................... 51
3.4.3. Distribution ....................................... 51
3.4.4. Theoretical plates ................................. 52
3.5. The chromatogram .......................................... 54
3.5.1. Retention .......................................... 54
3.5.2. Resolution ......................................... 54
3.5.3. Peak shape ......................................... 58
3.5.4. Effect of temperature .............................. 61
3.5.5. Effect of flow rate and linear velocity ............ 62
3.5.6. Effect of sample volume ............................ 64
3.6. Separation mode ........................................... 64
3.6.1. Normal phase ....................................... 64
3.6.2. Reverse phase ...................................... 65
3.6.3. Gradient reverse phase ............................. 68
3.6.4. Ion suppression and ion pairing .................... 69
3.6.5. Ion exchange ....................................... 72
3.6.6. Others ............................................. 72
3.7. Column care ............................................... 73
3.8. Bibliography .............................................. 74
4. HPLC optimisation ........................................... 75
DAVID BAKES
4.1. Objective ................................................. 75
4.2. System parameters ......................................... 75
4.3. Reverse-phase HPLC ........................................ 76
4.4. Ion-pair HPLC ............................................. 82
4.5. Ion-exchange HPLC ......................................... 85
4.6. Normal-phase HPLC ......................................... 87
4.7. Chiral HPLC ............................................... 90
4.7.1. Chiral columns ..................................... 91
4.7.2. Diastereoisomers ................................... 94
4.7.3. Chiral complexing agents ........................... 96
4.7.4. Chiral summary ..................................... 96
4.8. Column switching in HPLC .................................. 97
4.9. Gradient reverse-phase HPLC .............................. 100
4.10.Column conditions ........................................ 101
4.11.Computerised optimisation of HPLC ........................ 103
4.12.Conclusions .............................................. 104
4.13.Glossary ................................................. 104
4.14.References ............................................... 105
5. HPLC detectors ............................................. 106
RICHARD F. VENN
5.1. Introduction ............................................. 106
5.2. Principles of detection .................................. 107
5.2.1. Solute-property detectors ......................... 107
5.2.2. Bulk-property detectors ........................... 108
5.3. Selectivity in detectors ................................. 108
5.4. Detector response ........................................ 109
5.4.1. Linearity ......................................... 109
5.4.2. Time constant ..................................... 110
5.5. Detector types ........................................... 111
5.5.1. UV-visible detectors .............................. 111
5.5.2. Fluorescence detectors ............................ 115
5.5.3. Electrochemical detectors ......................... 120
5.5.4. Multifunctional detectors ......................... 122
5.5.5. Radiochemical detectors ........................... 123
5.5.6. Other detectors ................................... 124
5.6. Sensitivity considerations ............................... 126
5.6.1. Irradiation ....................................... 126
5.6.2. Pre-column derivatisation ......................... 126
5.6.3. Post-column derivatisation ........................ 127
5.7. Selectivity .............................................. 127
5.8. Detector problems ........................................ 128
5.8.1. Noise due to bubbles .............................. 128
5.8.2. Spurious peaks .................................... 128
5.8.3. Baseline instability .............................. 128
5.9. Appendix ................................................. 129
5.9.1. Buying a detector ................................. 129
5.9.2. Which detector to use? ............................ 129
5.10.Bibliography ............................................. 129
6. Gas chromatography: what it is and how we use it ........... 131
PETER ANDREW
6.1. Why gas chromatography works ............................. 131
6.2. Factors that affect the chromatography ................... 132
6.3. Choices in GC ............................................ 133
6.3.1. Stationary phase .................................. 133
6.3.2. Mobile phase ...................................... 135
6.3.3. Column length ..................................... 136
6.3.4. Column diameter ................................... 136
6.3.5. Film thickness .................................... 136
6.3.6. Flow rate ......................................... 137
6.3.7. Temperature ....................................... 137
6.3.8. Some rules of thumb ............................... 138
6.4. GC hardware .............................................. 138
6.4.1. Pneumatics ........................................ 139
6.4.2. Sample introduction ............................... 140
6.4.3. Detectors ......................................... 145
6.5. Derivatisation for GC .................................... 147
6.6. A GC strategy for bioanalysis ............................ 148
6.7. Bibliography ............................................. 148
7. Thin-layer chromatography .................................. 149
HUGH WILTSHIRE
7.1. Introduction ............................................. 149
7.2. Uses of TLC .............................................. 150
7.2.1. Preparative TLC ................................... 150
7.2.2. Metabolic profiling ............................... 151
7.2.3. 'Rules of thumb' .................................. 155
7.3. Some recommended solvent systems ......................... 158
7.4. Detection of compounds on TLC plates ..................... 158
7.5. Bibliography ............................................. 159
8. Capillary electrophoresis: an introduction ................. 160
PETER ANDREW
8.1. Introduction ............................................. 160
8.2. How capillary electrophoresis works ...................... 160
8.3. Why capillary electrophoresis works ...................... 162
8.3.1. Electro-osomotic flow ............................. 162
8.3.2. Free-solution capillary electrophoresis ........... 162
8.3.3. Micellar electrokinetic capillary
chromatography .................................... 164
8.3.4. Electrochromatography (electrically driven
HPLC) ............................................. 165
8.4. CE hardware .............................................. 166
8.4.1. The capillary ..................................... 166
8.4.2. Sample introduction ............................... 166
8.4.3. Detectors in CE ................................... 168
8.4.4. Sensitivity in CE ................................. 169
8.5. Use in bioanalysis ....................................... 169
8.6. Bibliography 170
9. Immunoassay techniques ..................................... 171
RICHARD F. VENN
9.1. Introduction ............................................. 171
9.2. Definitions .............................................. 171
9.3. Theory ................................................... 172
9.3.1. Mass action ....................................... 172
9.3.2. Competitive assays ................................ 173
9.3.3. Non-competitive assays ............................ 174
9.4. Requirements for immunoassay ............................. 175
9.4.1. Antibody .......................................... 175
9.4.2. Label ............................................. 175
9.4.3. Separation ........................................ 177
9.5. Practical aspects ........................................ 178
9.5.1. Preparation of hapten-carrier protein
conjugates ........................................ 178
9.5.2. Immunisation ...................................... 179
9.5.3. Antibody detection ................................ 180
9.5.4. Antibody titres ................................... 180
9.5.5. Calibration curves ................................ 180
9.5.6. Matrix effects .................................... 182
9.6. Data handling ............................................ 182
9.6.1. Standard curves ................................... 182
9.6.2. Fitting ........................................... 182
9.6.3. Precision profile ................................. 182
9.7. Advantages of immunoassay ................................ 184
9.7.1. Sensitivity ....................................... 184
9.7.2. Throughput ........................................ 184
9.7.3. Selectivity ....................................... 184
9.7.4. Ease .............................................. 184
9.7.5. Automation ........................................ 184
9.8. Disadvantages of immunoassays ............................ 185
9.8.1. Time: how long does it take? ...................... 185
9.8.2. Selectivity ....................................... 185
9.8.3. Matrix effects .................................... 185
9.9. What can go wrong? ....................................... 185
9.9.1. Matrix effects .................................... 185
9.9.2. Concentration effects ............................. 187
9.10.Immunoassay strategy ..................................... 187
9.11.Example .................................................. 187
9.11.1.Sampatrilat ....................................... 187
9.12.Affinity chromatography .................................. 187
9.12.1.Immobilisation techniques and media ............... 190
9.12.2.Elution techniques ................................ 191
9.12.3.Re-use/reconditioning ............................. 192
9.12.4.The interface between affinity chromatography
and analysis ...................................... 193
9.13.The future ............................................... 193
9.13.1.Phage libraries for antibodies .................... 193
9.13.2.Monoclonal antibodies ............................. 193
9.13.3.Molecular imprinting .............................. 193
9.13.4.Non-competitive assays for small molecules ........ 194
9.13.5.Use of low-specificity immunoassay for
discovery compounds ............................... 194
9.13.6.Indwelling optical fibre probes ................... 194
9.14.Summary .................................................. 194
9.15.Bibliography ............................................. 194
10.Automation of sample preparation ........................... 196
CHRIS JAMES
10.1.Introduction ............................................. 196
10.2.Approaches to automation ................................. 197
10.2.1.SPE ............................................... 197
10.2.2.Protein precipitation methods ..................... 198
10.2.3.Multi-well plate technology ....................... 198
10.2.4.Liquid-handling procedures ........................ 198
10.2.5.Avoiding evaporation .............................. 199
10.3.Simple automation ........................................ 199
10.4.Column switching ......................................... 200
10.5.Prospekt and Merck OSP-2 ................................. 202
10.6.Benchtop instruments - sequential sample processing ...... 202
10.6.1.Zymark BenchMate .................................. 203
10.6.2.Gilson ASPEC XL ................................... 203
10.6.3.Hamilton MicroLab ................................. 204
10.7.Benchtop instruments - parallel sample processing ........ 205
10.7.1.Zymark RapidTrace ................................. 205
10.7.2.Gilson ASPEC 4 .................................... 205
10.7.3.Multiple probe liquid-handling robots ............. 205
10.8.Gilson ASTED ............................................. 206
10.9.Full robotic systems ..................................... 207
10.10.When to automate? ....................................... 207
10.11.Example methods ......................................... 208
10.12.Conclusions and future perspectives ..................... 208
10.13.Bibliography ............................................ 209
11.Fundamental aspects of mass spectrometry: overview of
tenninology ................................................ 211
MIRA V.DOIG
11.1.Introduction ............................................. 211
11.2.Inlets ................................................... 211
11.2.1.Septum inlet ...................................... 211
11.2.2.Direct probe inlet ................................ 212
11.2.3.GC inlets ......................................... 212
11.2.4.LC inlets ......................................... 213
11.3.Ion sources .............................................. 216
11.3.1.Introduction ...................................... 216
11.3.2.Electron impact ionisation ........................ 216
11.3.3.Chemical ionisation ............................... 218
11.3.4.Atmospheric-pressure chemical ionisation .......... 219
11.3.5.Fast atom bombardment ............................. 220
11.3.6.Thermospray ....................................... 221
11.3.7.Electrospray ...................................... 223
11.3.8.Other desorption techniques ....................... 225
11.4.Analysers ................................................ 226
11.4.1.Single-focusing magnetic instruments .............. 226
11.4.2.Double-focusing instruments ....................... 228
11.4.3.Quadrupole analysers .............................. 229
11.4.4.Time of flight (ToF) analysers .................... 231
11.4.5.Ion-trap mass analysers ........................... 231
11.5.Detectors ................................................ 233
11.5.1.Electron multipliers .............................. 233
11.5.2.Negative-ion detection ............................ 234
11.6.Data acquisition and processing .......................... 234
11.6.1.Instrument control ................................ 234
11.6.2.Data acquisition/preliminary data processing ...... 234
11.6.3.Secondary data processing/data presentation ....... 235
11.7.Bibliography ............................................. 239
12.Applications of mass spectrometry: quantitative mass
spectrometry ............................................... 240
MIRA V.DOIG
12.1.Quantification ........................................... 240
12.1.1.Gas chromatography-mass spectrometry (GC-MS) ...... 240
12.1.2.Liquid chromatography-mass spectrometry (LC-MS) ... 241
12.1.3.Quantitative API LC-MS and its contribution to
the drug development process ...................... 241
12.2.Internal standardisation ................................. 242
12.3.Data acquisition ......................................... 243
12.3.1.Selected ion versus mass chromatogram ............. 243
12.3.2.Mass analysis ..................................... 243
12.3.3.Calculation of the mass of the selected ion ....... 244
12.3.4.Data storage and processing ....................... 245
12.4.Developing a quantitative method ......................... 245
12.5.Analysis of prostanoids by GC-MS ......................... 246
12.6.An example of thermospray LC-MS .......................... 249
12.7.Examples of API LC-MS .................................... 250
12.8.The future ............................................... 253
12.9.Bibliography ............................................. 254
13.Mass spectrometric identification of metabolites ........... 255
JANET OXFORD AND SORAYA MONTE
13.1.Objectives ............................................... 255
13.2.Introduction ............................................. 255
13.3.Tandem mass spectrometry (MS-MS) ......................... 256
13.3.1.Theory ............................................ 256
13.3.2.Instrumentation ................................... 256
13.3.3.MS-MS scans and their application to metabolite
identification .................................... 258
13.4.Isotopically labelled compounds in metabolite
identification ........................................... 265
13.5.Practical aspects for the identification of
metabolites by mass spectrometry ......................... 266
13.5.1.Introduction ...................................... 266
13.5.2.Electron impact ionisation and chemical
ionisation ........................................ 268
13.5.3.Fast atom bombardment ............................. 270
13.5.4.Thermospray LC-MS ................................. 271
13.5.5.Electrospray LC-MS ................................ 273
13.5.6.Ion-trap mass spectrometry coupled to external
atmospheric-pressure ionisation sources ........... 274
13.5.7.Summary ........................................... 275
13.5.8.Overall comments .................................. 276
13.6.Bibliography ............................................. 277
14.Nuclear magnetic resonance in drug metabolism .............. 278
PHIL GILBERT
14.1.Introduction ............................................. 278
14.2.Basic theory of the NMR phenomenon ....................... 278
14.3.Parameters of the NMR spectrum ........................... 280
14.3.1.Chemical shift .................................... 280
14.3.2.Spin-spin coupling ................................ 281
14.3.3.Intensity ......................................... 286
14.4.Practical considerations ................................. 286
14.4.1.Types of spectrometer ............................. 286
14.4.2.Sample preparation ................................ 287
14.5.NMR applications in drug development ..................... 288
14.5.1.No sample preparation ............................. 288
14.5.2.Solid-phase extraction sample preparation ......... 288
14.5.3.HPLC fractions .................................... 291
14.5.4.Fluorinated compounds ............................. 291
14.5.5.Stable isotope labelling .......................... 293
14.6.Plasma metabolites ....................................... 294
14.7.Biochemical changes ...................................... 294
14.8.Summary .................................................. 294
14.9.Appendix: fourier transform and some multi-pulse
techniques ............................................... 294
14.9.1.Why use pulse NMR? ................................ 294
14.9.2.The pulse ......................................... 296
14.9.3.Time and frequency ................................ 296
14.9.4.Multi-pulse experiments ........................... 296
14.9.5.Conclusion ........................................ 301
14.10.Bibliography ............................................ 301
15.Strategy in metabolite isolation and identification ........ 302
HUGH WILTSHIRE
15.1.Stage 1: radiochemical synthesis ......................... 302
15.1.1.Choice of label ................................... 302
15.1.2.Position of 14C label ............................. 303
15.2.Stage 2: animal experiments .............................. 303
15.2.1.Routes of excretion ............................... 304
15.2.2.Formulation and route of administration ........... 304
15.2.3.Collection of urine and bile ...................... 304
15.3.Stage 3: metabolite isolation and characterisation ....... 304
15.3.1.Enrichment ........................................ 304
15.3.2.Analysis .......................................... 306
15.3.3.Separation ........................................ 307
15.3.4.Purification ...................................... 315
15.3.5.Characterisation .................................. 315
15.4.Stage 4: identification of metabolites ................... 321
15.4.1.Mass spectrometry ................................. 323
15.4.2.NMR ............................................... 325
15.4.3.Degradation, derivatisation and comparison with
authentic material ................................ 327
15.4.4.Ambiguities ....................................... 330
15.5.Stage 5: quantitative aspects of metabolism .............. 330
15.5.1.Quantification of excretion balance studies ....... 330
15.5.2.Quantitative aspects of metabolite isolation ...... 331
15.5.3.Quantitative measurement of metabolic profiles .... 331
15.6.In vitro studies ......................................... 333
15.6.1.Isolation of metabolites from in vitro
incubations ....................................... 334
15.6.2.Cross-species comparisons of metabolic profiles ... 336
15.6.3.Mechanistic studies ............................... 337
15.7.Identification of plasma metabolites ..................... 337
15.8.Good laboratory practice ............................ 339
15.9.Conclusions ......................................... 341
16.Strategy for the development of quantitative analytical
procedures ................................................. 342
DAVID BAKES
16.1.Introduction ............................................. 342
16.2.Preliminary requirements ................................. 343
16.3.Detection ................................................ 345
16.4.Separation ............................................... 348
16.5.Sample preparation ....................................... 349
16.6.Solid-phase extraction ................................... 349
16.7.Extraction sequence ...................................... 350
16.8.Liquid/liquid extraction ................................. 352
16.9.Quantification ........................................... 354
16.9.1.Rule of one and two ............................... 354
16.9.2.Standardisation ................................... 354
16.9.3.Peak height and area .............................. 355
16.9.4.Calibration check ................................. 355
16.10.Validation .............................................. 356
16.11.Support work ............................................ 356
16.11.1.Matrix substitution .............................. 356
16.11.2.Stability ........................................ 357
16.11.3.Metabolites ...................................... 358
16.12.Conclusions ............................................. 358
Index ......................................................... 359
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