PRINCIPLES AND PRACTICE OF BIOANALYSIS

PRINCIPLES AND PRACTICE OF BIOANALYSIS Richard F. Venn Pfizer Central Research, Sandwich, UK London and New York

List of contributors Preface xvi xvii 1 Physico-chemical properties of drugs and metabolites and their extraction from biological material 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 'first law of drug metabolism' 26 1.7 Bibliography 27 2 Solid-phase extraction 28 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 44 ANDY GRAY 3.1 Origins 44 3.2 Applications 44 3.3 Apparatus 45 3.3.1 Column 45 3.3.2 Plumbing 47 VI

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 vn

4.12 Conclusions 104 4.13 Glossary 104 4.14 References 105 5 HPLC detectors RICHARD F. VENN 106 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 Introduction 106 Principles of detection 107 5.2.1 Solute-property detectors 107 5.2.2 Bulk-property detectors 108 Selectivity in detectors 108 Detector response 109 5.4.1 Linearity 109 5.4.2 Time constant 110 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 Sensitivity considerations 126 5.6.1 Irradiation 126 5.6.2 Pre-column derivatisation 126 5.6.3 Post-column derivatisation 127 Selectivity 127 Detector problems 128 5.8.1 Noise due to bubbles 128 5.8.2 Spurious peaks 128 5.8.3 Baseline instability 128 Appendix 129 5.9.1 Buying a detector 129 5.9.2 Which detector to use? 129 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 Vlll

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 UsesofTLC 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 IX

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 XI

11 Fundamental aspects of mass spectrometry: overview of terminology 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 xn

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 atmosphericpressure 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 xiu

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 14 C 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 xiv

xv CONTENTS 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