PRINCIPLES AND PRACTICE OF BIOANALYSIS

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

2 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 Metabolite isolation Bioanalysis Enrichment of drugs and metabolites Differences between metabolite isolation and drug analysis Physico-chemical properties of drugs and solvents Energy changes on solution Molecular phenomena behind solubility/miscibility Water miscibility and water immiscibility Partition Extraction efficiency Ionisation and its effect on the extraction of drugs Ionisation, ph and pk Titration curves Henderson-Hasselbach equation Buffers Distribution coefficient Solvent extraction Choice of solvent Mixed solvents Dealing with plasma proteins and emulsions Choice of ph for solvent extraction Artefacts arising during the extraction of drugs and metabolites Modification and derivatisation of drugs and metabolites 24

3 1.5.7 Ion-pair extraction Recoveries The 'first law of drug metabolism' Bibliography 27 2 Solid-phase extraction 28 CHRIS JAMES 2.1 Introduction General properties of bonded silica sorbents Sorbent/analyte interactions Solvation Non-polar Polar Ion exchange Covalent Mixed-mode interactions Polymeric sorbents Miscellaneous Sample pretreatment of different biological matrices Liquid samples Protein binding Solid samples Developing SPE methods Example of an SPE method Disc cartridges Potential advantages Disadvantages Well format (e.g. Porvair Microsep system) Direct injection of plasma Other new developments Fines A cartridge in a pipette tip? Conclusions and future perspectives Bibliography 42 3 Basic HPLC theory and practice 44 ANDY GRAY 3.1 Origins Applications Apparatus Column Plumbing 47 VI

4 3.3.3 Pumps Injectors Column ovens Detectors The chromatographic process Basic principles Molecular forces Distribution Theoretical plates The chromatogram Retention Resolution Peak shape Effect of temperature Effect of flow rate and linear velocity Effect of sample volume Separation mode Normal phase Reverse phase Gradient reverse phase Ion suppression and ion pairing Ion exchange Others Column care Bibliography 74 4 HPLC optimisation 75 DAVID BAKES 4.1 Objective System parameters Reverse-phase HPLC Ion-pair HPLC Ion-exchange HPLC Normal-phase HPLC Chiral HPLC Chiral columns Diastereoisomers Chiral complexing agents Chiral summary Column switching in HPLC Gradient reverse-phase HPLC Column conditions Computerised optimisation of HPLC 103 vn

5 4.12 Conclusions Glossary References HPLC detectors RICHARD F. VENN Introduction 106 Principles of detection Solute-property detectors Bulk-property detectors 108 Selectivity in detectors 108 Detector response Linearity Time constant 110 Detector types UV-visible detectors Fluorescence detectors Electrochemical detectors Multifunctional detectors Radiochemical detectors Other detectors 124 Sensitivity considerations Irradiation Pre-column derivatisation Post-column derivatisation 127 Selectivity 127 Detector problems Noise due to bubbles Spurious peaks Baseline instability 128 Appendix Buying a detector Which detector to use? 129 Bibliography Gas chromatography: what it is and how we use it 131 PETER ANDREW 6.1 Why gas chromatography works Factors that affect the chromatography Choices in GC Stationary phase Mobile phase 135 Vlll

6 6.3.3 Column length Column diameter Film thickness Flow rate Temperature Some rules of thumb GC hardware Pneumatics Sample introduction Detectors Derivatisation for GC A GC strategy for bioanalysis Bibliography Thin-layer chromatography 149 HUGH WILTSHIRE 7.1 Introduction UsesofTLC Preparative TLC Metabolic profiling 'Rules of thumb' Some recommended solvent systems Detection of compounds on TLC plates Bibliography Capillary electrophoresis: an introduction 160 PETER ANDREW 8.1 Introduction How capillary electrophoresis works Why capillary electrophoresis works Electro-osomotic flow Free-solution capillary electrophoresis Micellar electrokinetic capillary chromatography Electrochromatography (electrically driven HPLC) CE hardware The capillary Sample introduction Detectors in CE Sensitivity in CE Use in bioanalysis Bibliography 170 IX

7 Immunoassay techniques 171 RICHARD F. VENN 9.1 Introduction Definitions Theory Mass action Competitive assays Non-competitive assays Requirements for immunoassay Antibody Label Separation Practical aspects Preparation of hapten-carrier protein conjugates Immunisation Antibody detection Antibody titres Calibration curves Matrix effects Data handling Standard curves Fitting Precision profile Advantages of immunoassay Sensitivity Throughput Selectivity Ease Automation Disadvantages of immunoassays Time: how long does it take? Selectivity Matrix effects What can go wrong? Matrix effects Concentration effects Immunoassay strategy Example Sampatrilat Affinity chromatography Immobilisation techniques and media Elution techniques Re-use/reconditioning 192

8 The interface between affinity chromatography and analysis The future Phage libraries for antibodies Monoclonal antibodies Molecular imprinting Non-competitive assays for small molecules Use of low-specificity immunoassay for discovery compounds Indwelling optical fibre probes Summary Bibliography Automation of sample preparation 196 CHRIS JAMES 10.1 Introduction Approaches to automation SPE Protein precipitation methods Multi-well plate technology Liquid-handling procedures Avoiding evaporation Simple automation Column switching Prospekt and Merck OSP Benchtop instruments - sequential sample processing Zymark BenchMate Gilson ASPEC XL Hamilton MicroLab Benchtop instruments - parallel sample processing Zymark RapidTrace Gilson ASPEC Multiple probe liquid-handling robots Gilson ASTED Full robotic systems When to automate? Example methods Conclusions and future perspectives Bibliography 209 XI

9 11 Fundamental aspects of mass spectrometry: overview of terminology 211 MIRA V. DOIG 11.1 Introduction Inlets Septum inlet Direct probe inlet GC inlets LC inlets Ion sources Introduction Electron impact ionisation Chemical ionisation Atmospheric-pressure chemical ionisation Fast atom bombardment Thermospray Electrospray Other desorption techniques Analysers Single-focusing magnetic instruments Double-focusing instruments Quadrupole analysers Time of flight (ToF) analysers Ion-trap mass analysers Detectors Electron multipliers Negative-ion detection Data acquisition and processing Instrument control Data acquisition/preliminary data processing Secondary data processing/data presentation Bibliography Applications of mass spectrometry: quantitative mass spectrometry 240 MIRA V. DOIG 12.1 Quantification Gas chromatography-mass spectrometry (GC-MS) Liquid chromatography-mass spectrometry (LC-MS) Quantitative API LC-MS and its contribution to the drug development process Internal standardisation Data acquisition Selected ion versus mass chromatogram Mass analysis 243 xn

10 Calculation of the mass of the selected ion Data storage and processing Developing a quantitative method Analysis of prostanoids by GC-MS An example of thermospray LC-MS Examples of API LC-MS The future Bibliography Mass spectrometric identification of metabolites 255 JANET OXFORD AND SORAYA MONTE 13.1 Objectives Introduction Tandem mass spectrometry (MS-MS) Theory Instrumentation MS-MS scans and their application to metabolite identification Isotopically labelled compounds in metabolite identification Practical aspects for the identification of metabolites by mass spectrometry Introduction Electron impact ionisation and chemical ionisation Fast atom bombardment Thermospray LC-MS Electrospray LC-MS Ion-trap mass spectrometry coupled to external atmosphericpressure ionisation sources Summary Overall comments Bibliography Nuclear magnetic resonance in drug metabolism 278 PHIL GILBERT 14.1 Introduction Basic theory of the NMR phenomenon Parameters of the NMR spectrum Chemical shift Spin-spin coupling Intensity Practical considerations Types of spectrometer Sample preparation 287 xiu

11 14.5 NMR applications in drug development No sample preparation Solid-phase extraction sample preparation HPLC fractions Fluorinated compounds Stable isotope labelling Plasma metabolites Biochemical changes Summary Appendix: fourier transform and some multi-pulse techniques Why use pulse NMR? The pulse Time and frequency Multi-pulse experiments Conclusion Bibliography Strategy in metabolite isolation and identification 302 HUGH WILTSHIRE 15.1 Stage 1: radiochemical synthesis Choice of label Position of 14 C label Stage 2: animal experiments Routes of excretion Formulation and route of administration Collection of urine and bile Stage 3: metabolite isolation and characterisation Enrichment Analysis Separation Purification Characterisation Stage 4: identification of metabolites Mass spectrometry NMR Degradation, derivatisation and comparison with authentic material Ambiguities Stage 5: quantitative aspects of metabolism Quantification of excretion balance studies Quantitative aspects of metabolite isolation Quantitative measurement of metabolic profiles 331 xiv

12 xv CONTENTS 15.6 In vitro studies Isolation of metabolites from in vitro incubations Cross-species comparisons of metabolic profiles Mechanistic studies Identification of plasma metabolites Good laboratory practice Conclusions Strategy for the development of quantitative analytical procedures 342 DAVID BAKES 16.1 Introduction Preliminary requirements Detection Separation Sample preparation Solid-phase extraction Extraction sequence Liquid/liquid extraction Quantification Rule of one and two Standardisation Peak height and area Calibration check Validation Support work Matrix substitution Stability Metabolites Conclusions 358 Index 359

Introduction to Pharmaceutical Chemical Analysis

Introduction to Pharmaceutical Chemical Analysis Hansen, Steen ISBN-13: 9780470661222 Table of Contents Preface xv 1 Introduction to Pharmaceutical Analysis 1 1.1 Applications and Definitions 1 1.2 The