Principles and Practice of Bioanalysis, Second Edition

Principles and Practice of Bioanalysis provides a guide to the methods available and the techniques currently used in this field. It provides up to the minute information and guidance on the methods and strategy used in developing and running ultra-trace analyses for drugs, metabolites and other substances. The authors writes in an informal and didactic style, offering a logical path through the problems of small molecule (bio)analysis and enables readers to choose appropriate methods of analysis for their needs. Principles and Practice of Bioanalysis provides an overview of analytical methods for analytical scientists within the pharmaceutical industry, research and development, the agrochemical industry, and scientists in the health service, biology and biochemistry. It also gives postgraduate students a useful reference for their research methods. Doody Review Services Reviewer: Eugene A Davidson, PhD(Georgetown University School of Medicine) Description: This is an introduction to practical, analytical methods used in the pharmacology industry. Purpose: This book is intended to serve as a text for a course in analysis developed by the pharmaceutical industry. This goal is well reflected in the nature of the material covered and the emphases. Audience: Students needing an appreciation of the analytical challenges faced by pharmaceutical manufacturing will profit from this text. Features: An introductory chapter covers a set of drugs and their metabolites - the focus is mainly on ionization and solubility properties and does not cover modes of drug metabolism. Examples are provided but the relationship of chemical or physical properties to a given separation or analytical method is missing. A detailed discussion of HPLC follows that includes theory and detectors. Similar coverage of thin layer and gas-liquid chromatography and capillary electrophoresis completes the discussion of separation methods. The remainder is devoted to analytical methods, including immunoassays, mass spectrometry and NMR. Since no protocols are included, it can be assumed that the companion course does not include practical exercises. Assessment: It should be realized that for most of the technologies discussed, only superficial treatment is provided. Conversely, if one needs mass spectral data in an industrial setting, the company will have experts available for that purpose. Book Cover......Page 1 Half-Title......Page 2 Title......Page 3 Copyright......Page 4 Contents......Page 5 Contributors......Page 19 Preface......Page 20 1.1.3 Enrichment of drugs and metabolites......Page 22 1.2.1 Energy changes on solution......Page 23 Ionic bonds and ‘salting-out’......Page 24 Hydrogen bonds......Page 26 Van der Waals forces......Page 27 1.2.3 Water miscibility and water immiscibility......Page 29 1.3.1 Extraction efficiency......Page 30 1.4.1 Ionisation, pH and pK......Page 31 1.4.2 Titration curves......Page 33 1.4.3 Henderson-Hasselbalch equation......Page 34 1.4.4 Buffers......Page 36 1.4.5 Distribution coefficient......Page 37 1.5.1 Choice of solvent......Page 38 1.5.2 Mixed solvents......Page 40 1.5.5 Artefacts arising during the extraction of drugs and metabolites......Page 41 1.5.6 Modification and derivatisation of drugs and metabolites......Page 44 1.6 The ‘first law of drug metabolism’......Page 46 1.7 Bibliography......Page 47 2.1 Introduction......Page 48 2.3.1 Solvation......Page 50 2.3.2 Non-polar......Page 51 2.3.4 Ion exchange......Page 52 2.3.5 Covalent......Page 53 2.3.6 Mixed-mode interactions......Page 54 2.3.7 Polymeric sorbents......Page 55 2.4.3 Solid samples......Page 56 2.5 Developing SPE methods......Page 57 2.7 Disc cartridges......Page 58 2.7.2 Disadvantages......Page 59 2.9 Direct injection of plasma......Page 60 2.11 Conclusions and future perspectives......Page 61 2.12 Bibliography......Page 62 3.2 Applications......Page 64 3.3.1 Column......Page 65 3.3.2 Plumbing......Page 67 3.3.3 Pumps......Page 68 3.3.4 Injectors......Page 69 3.4.1 Basic principles......Page 70 3.4.3 Distribution......Page 71 3.4.4 Theoretical plates......Page 72 3.5.1 Retention......Page 73 3.5.2 Resolution......Page 74 Skewed peaks......Page 77 Band broadening......Page 79 3.5.4 Effect of temperature......Page 80 3.5.5 Effect of flow rate and linear velocity......Page 81 3.5.6 Effect of sample volume......Page 82 3.6.1 Normal phase......Page 83 3.6.2 Reverse phase......Page 84 3.6.3 Gradient reverse phase......Page 87 3.6.4 Ion suppression and ion pairing......Page 88 3.6.6 Others......Page 91 3.7 Column care......Page 92 3.8 Bibliography......Page 93 4.2 System parameters......Page 94 4.3 Reverse-phase HPLC......Page 95 4.4 Ion-pair HPLC......Page 100 4.5 Ion-exchange HPLC......Page 104 4.6 Normal-phase HPLC......Page 106 4.7.1 Chiral columns......Page 109 4.7.2 Diastereoisomers......Page 112 4.7.4 Chiral summary......Page 114 4.8 Column switching in HPLC......Page 115 4.9 Gradient reverse-phase HPLC......Page 118 4.10 Column conditions......Page 119 4.13 Glossary......Page 121 References......Page 122 5.1 Introduction......Page 124 5.2.1 Solute-property detectors......Page 125 5.3 Selectivity in detectors......Page 126 5.4.1 Linearity......Page 127 5.4.2 Time constant......Page 128 Principle of operation......Page 129 Design......Page 130 Advantages of UV-visible detectors......Page 132 Principles......Page 133 Design and use......Page 134 Advantages......Page 136 Other considerations......Page 137 Dynamic detectors......Page 138 5.5.4 Multifunctional detectors......Page 139 Principles......Page 140 Optical activity......Page 141 Nitrogen detectors......Page 142 5.6.2 Pre-column derivatisation......Page 143 5.7 Selectivity......Page 144 5.8.3 Baseline instability......Page 145 5.10 Bibliography......Page 146 6.1 Why gas chromatography works......Page 148 6.2 Factors that affect the chromatography......Page 149 6.3.1 Stationary phase......Page 150 6.3.3 Column length......Page 152 6.3.5 Film thickness......Page 153 6.3.8 Some rules of thumb......Page 154 6.4 GC hardware......Page 155 6.4.2 Sample introduction......Page 156 Splitless injection......Page 157 Direct on-column injection......Page 158 Thermionic detector (TID)......Page 161 Electron capture detector......Page 162 6.5 Derivatisation for GC......Page 163 6.7 Bibliography......Page 164 7.1 Introduction......Page 165 7.2.1 Preparative TLC......Page 166 7.2.2 Metabolic profiling......Page 167 Conjugates are relatively polar on TLC.......Page 171 7.4 Detection of compounds on TLC plates......Page 174 7.5 Bibliography......Page 175 8.2 How capillary electrophoresis works......Page 176 8.3.2 Free-solution capillary electrophoresis......Page 178 Choice of buffer and ionic strength......Page 179 8.3.3 Micellar electrokinetic capillary chromatography......Page 180 8.3.4 Electrochromatography (electrically driven HPLC)......Page 181 Gravimetric (syphonic)......Page 182 Differential pressure......Page 183 Electrochemical detection......Page 184 8.5 Use in bioanalysis......Page 185 8.6 Bibliography......Page 186 9.2 Definitions......Page 187 9.3.1 Mass action......Page 188 9.3.2 Competitive assays......Page 189 9.3.3 Non-competitive assays......Page 190 9.4.2 Label......Page 191 Enzymes......Page 192 Dextran-coated charcoal......Page 193 Immobilisation......Page 194 Typical immunisation protocol......Page 195 Radioimmunoassays......Page 196 DELFIA......Page 197 9.6.2 Fitting......Page 198 9.6.3 Precision profile......Page 199 9.7.4 Ease......Page 200 9.8.3 Matrix effects......Page 201 9.10 Immunoassay strategy......Page 202 9.12 Affinity chromatography......Page 204 Soft gel supports......Page 206 Rigid supports......Page 207 Biospecific desorption......Page 208 9.13.1 Phage libraries for antibodies......Page 209 9.14 Summary......Page 210 9.15 Bibliography......Page 211 10.1 Introduction......Page 213 10.2.1 SPE......Page 214 10.2.4 Liquid-handling procedures......Page 215 10.3 Simple automation......Page 216 10.4 Column switching......Page 217 10.6 Benchtop instruments—sequential sample processing......Page 219 10.6.2 Gilson ASPEC XLTM......Page 220 10.6.3 Hamilton MicroLabTM......Page 221 10.7.3 Multiple probe liquid-handling robots......Page 222 10.9 Full robotic systems......Page 224 10.11 Example methods......Page 225 10.13 Bibliography......Page 226 11.2.1 Septum inlet......Page 229 11.2.3 GC inlets......Page 230 11.2.4 LC inlets......Page 231 Particle-beam inlet......Page 232 Other inlets......Page 233 11.3.1 Introduction......Page 234 11.3.2 Electron impact ionisation......Page 235 11.3.3 Chemical ionisation......Page 236 11.3.4 Atmospheric-pressure chemical ionisation......Page 237 11.3.5 Fast atom bombardment......Page 238 Ion evaporation......Page 239 11.3.7 Electrospray......Page 241 11.3.8 Other desorption techniques......Page 243 11.4.1 Single-focusing magnetic instruments......Page 244 11.4.3 Quadrupole analysers......Page 246 11.4.4 Time of flight (ToF) analysers......Page 248 11.4.5 Ion-trap mass analysers......Page 249 11.5 Detectors......Page 250 11.5.2 Negative-ion detection......Page 251 11.6.2 Data acquisition/preliminary data processing......Page 252 Total-ion current (TIC) chromatogram......Page 253 Mass chromatography......Page 254 11.7 Bibliography......Page 256 12.1.1 Gas chromatography-mass spectrometry (GC-MS)......Page 258 Research/discovery......Page 259 12.2 Internal standardisation......Page 260 12.3.2 Mass analysis......Page 261 12.3.3 Calculation of the mass of the selected ion......Page 262 12.4 Developing a quantitative method......Page 263 12.5 Analysis of prostanoids by GC-MS......Page 264 12.6 An example of thermospray LC-MS......Page 267 12.7 Examples of API LC-MS......Page 268 12.9 Bibliography......Page 271 13.2 Introduction......Page 273 13.3.2 Instrumentation......Page 274 Product ion mass spectrum......Page 276 Precursor ion mass spectrum......Page 280 Neutral loss mass spectrum......Page 281 13.4 Isotopically labelled compounds in metabolite identification......Page 283 13.5.1 Introduction......Page 284 Direct-probe analysis......Page 286 Particle-beam liquid chromatography-mass spectrometry (LC-MS)......Page 287 13.5.3 Fast atom bombardment......Page 288 13.5.4 Thermospray liquid chromatography-mass sectrometry (TSP LC-MS)......Page 289 13.5.5 Electrospray liquid chromatography-mass spectrometry......Page 291 13.5.6 Ion-trap mass spectrometry coupled to external atmospheric-pressure ionisation sources......Page 292 13.5.7 Summary......Page 293 13.6 Bibliography......Page 294 14.2 Basic theory of the NMR phenomenon......Page 296 14.3.1 Chemical shift......Page 298 14.3.2 Spin-spin coupling......Page 299 14.4.1 Types of spectrometer......Page 304 14.4.2 Sample preparation......Page 305 14.5.2 Solid-phase extraction sample preparation......Page 306 14.5.4 Fluorinated compounds......Page 309 14.6 Plasma metabolites......Page 311 14.9.1 Why use pulse NMR?......Page 312 Spectral editing......Page 314 Homonuclear 2-dimensional correlation spectroscopy (COSY)......Page 315 14.9.5 Conclusion......Page 318 14.10 Bibliography......Page 319 15.1.1 Choice of label......Page 320 15.2 Stage 2: animal experiments......Page 321 15.3 Stage 3: metabolite isolation and characterisation......Page 322 15.3.1 Enrichment......Page 323 15.3.2 Analysis......Page 325 15.3.3 Separation......Page 326 Changing the mobile phase......Page 328 Changing the stationary phase......Page 330 Changing the pH......Page 332 15.3.5 Characterisation......Page 333 15.4 Stage 4: identification of metabolites......Page 339 15.4.1 Mass spectrometry......Page 341 15.4.2 NMR......Page 344 15.4.3 Degradation, derivatisation and comparison with authentic material......Page 346 15.5.3 Quantitative measurement of metabolic profiles......Page 349 15.6 In vitro studies......Page 352 15.6.1 Isolation of metabolites from in vitro incubations......Page 353 15.6.2 Cross-species comparisons of metabolic profiles......Page 355 15.7 Identification of plasma metabolites......Page 356 15.8 Good laboratory practice......Page 358 15.9 Conclusions......Page 359 16.1 Introduction......Page 360 16.2 Preliminary requirements......Page 361 16.3 Detection......Page 363 16.4 Separation......Page 365 16.5 Sample preparation......Page 366 16.7 Extraction sequence......Page 367 16.8 Liquid/liquid extraction......Page 369 16.9.2 Standardisation......Page 371 16.9.4 Calibration check......Page 372 16.10 Validation......Page 373 Stability of extracts......Page 374 16.12 Conclusions......Page 375 Index......Page 377 Analysis of chemicals in the complex environments in which they occur is carried out by a vast range of institutions for a variety of purposes, from pharmaceutical and agrochemical companies to hospital biochemistry labs and university departments, from environmental monitoring to safety and toxicity testing of new drugs. The range of compounds for analysis is enormous, from naturally-occurring compounds such as vitamins to man-made chemicals from the pharmaceutical and agrochemical industries. This book offers an integrated, readable reference text describing the full range of analytical techniques available for such small molecules in an up-to-date manner and should appeal to all involved in the rapidly growing field of analytical sciences