Identification and Quantification of Drugs, Metabolites and Metabolizing Enzymes by LC-MS (eBook)

Cover 1
Identification and Quantification of Drugs, Metabolites and Metabolizing Enzymes by LC–MS 4
Preface 6
Contents 8
List of contributors 16
LC–MS and Drug Metabolism: A Journey Back in Time, Present Trends and Future Directions 18
Introduction 18
References 22
Strategies for Increasing Throughput for PK Samples in a Drug Discovery Environment 24
Introduction 24
Strategies for Early PK Assays 26
Enhanced Mass Resolution – A New Analytical Tool for PK Assays 35
CARRS – An Example of an Integrated PK Assay Strategy 39
Conclusions 46
References 47
Commonly Encountered Analytical Problems and their Solutions in Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) Methods used in Drug Development 52
Introduction 52
Commonly Encountered Analytical Problems and Solutions 53
Problems with Sample Collection and Analyte Stability 53
Problem with Positive Controls 54
Carryover Problem 55
Problem with Retention Time Shift in HPLC 58
Problems Related to the Mobile Phase Composition and Ionization of Analytes 60
Problems with Ion Suppression and Matrix Effect in the API Interface 60
Problems with Stable Isotope Labeled IS 64
Problem of Matrix Interference due to the Presence of Metabolites or Other Endogenous Compounds 66
The Use of Gas-Phase Cluster Ions for Method Sensitivity Problem 71
Problem with Post-Column Ion Source-Induced Changes of Pro-Drugs and Metabolites 73
Problems with Both Mass Spectrometric and Chromatographic Resolutions in Accurate Quantification of Parent Compound and Metab 74
Problem with Quantifying an Unstable and Light-Sensitive Compound at Ultra-Trace Level Concentrations 75
Conclusions 78
Acknowledgments 78
References 78
Pitfalls in Quantitative LC–MS/MS: Metabolite Contribution to Measured Drug Concentration 82
Introduction 82
Analytical Pitfalls Due to Metabolite In-Source Conversion 82
Analytical Pitfalls Due to the Simultaneous Formation of [M+H]+ and [M+NH4]+ Ions from a Drug as Well as Its Metabolite 91
Analytical Pitfalls Due to Conversion of Metabolite to the Parent Drug During Sample Collection, Handling, Extraction, and An 98
Bioanalytical Method Design for Drug Quantification in the Presence of a Metabolite that may Degrade to Generate the Drug 99
Strategy of Using Post-Dose Samples to Establish Validity of Bioanalytical LC–MS/MS Methods 108
Summary and Conclusions 116
References 117
In Vitro DMPK Screening in Drug Discovery, Role of LC–MS/MS 122
Introduction 122
CYP Inhibition Screening 122
Background 122
Types of Inhibition 123
Experimental Procedure 124
Sample Plate Set-up 125
Quantification with LC–MS/MS 126
IC50 Determination 126
Throughput 127
Caco-2 Permeability 128
Background 128
Mechanisms of Absorption 129
Caco-2 Screening Assay 129
Efflux Evaluation 130
LC–MS or LC–MS/MS? 130
Multiplexed Ion Source (MUX®) 131
Determination of Apparent Permeability 132
Assay Precision 133
Throughput 133
Blood–Brain barrier 133
Background 133
Cell Culture 134
Transport Studies 134
Assay Precision 135
In Vivo–In Vitro Correlation 135
Conclusion 137
Acknowledgment 137
References 137
Metabolite Identification by LC–MS: Applications in Drug Discovery and Development 140
Introduction 140
Metabolite Identification 142
Mass Spectrometers for Metabolite Identification 143
Single and Triple Quadrupole Mass Spectrometer 144
3-Dimensional Ion Trap Mass Spectrometer 144
Q-TOF Mass Spectrometer: 145
Common Biotransformations 146
Additional Tools for Metabolite Identification 147
Chemical Derivatization 147
Radiolabeled Compounds 148
Isotope Patterns 148
LC–NMR–MS 149
H/D Exchange 149
Applications 150
Metabolism of a Potent Neurokinin 1 Receptor Antagonist 150
Metabolism of a Potent PPAR. Agonist 156
Metabolism of an Antipsychotic Drug Ziprasidone 159
Use of Two Different Radiolabels 160
Precursor Ion Spectrum 162
Differentiation of Isobaric Metabolites 162
Biotransformation of an Anxiolytic Drug Candidate, CP-93,393 163
Future Trends 170
Acknowledgments 171
References 171
The Utility of in Vitro Screening for the Assessment of Electrophilic Metabolite Formation Early in Drug Discovery Using HPLC–MS/MS 176
Introduction 176
Metabolite Characterization 177
In Vitro Metabolic Screening Methodology 178
The Significance of Electrophilic Metabolites 180
Glutathiones 181
Acylglucuronides 182
Acylglucosides and N-Acetylglucosaminides 184
Current Approaches 184
Background 184
Strategy 186
Materials and Methods 186
Glutathione Conjugation Incubations 186
Acylglucuronide Conjugation Incubations 187
Acylglucoside and N-Acetylglucosaminide Conjugation Incubations 187
Screening Method Details 188
High Performance Liquid Chromatographic (HPLC) Conditions 188
Mass Spectrometric Analysis 188
Results/Discussion 189
Conclusions 194
Future 196
Acknowledgements 196
References 196
Liquid Chromatography Tandem Mass Spectrometry-Based Metabolite Identification Strategies in Pharmaceutical Research 200
Introduction 200
Metabolite Identification in Drug Discovery 201
Classical Mass Spectrometry-Based Metabolite Identification Approach in Drug Discovery 202
Parallel Mass Spectrometry-Based Metabolite Identification Approach in Drug Discovery 203
Integrated List-Dependent Fast LC/MSn in Identifying Metabolic Soft Spots 204
Metabolite Identification in Preclinical Development 205
Identification of Unanticipated Metabolites using Data-Dependent Precursor Ion and Constant Neutral Loss Scans 205
Detailed Characterization of Metabolites using Radiochromatographic Techniques Coupled to LC/MS 206
Metabolite Identification in Clinical Drug Development 207
Detailed Characterization of Metabolites in Humans 207
Quantitative Similarity of the Circulating Metabolites in Human versus Preclinical Species 207
References 208
Quantification and Structural Elucidation of Low Quantities of Radiolabeled Metabolites using Microplate Scintillation Counting (MSC) Techniques in Conjunction with LC–MS 212
Introduction 212
The HPLC–MSC Technique 213
Analytical Characteristics of HPLC–MSC 216
Sensitivity 216
Precision and Accuracy 218
Matrix Effect of Biological Samples 218
Radioactivity Recovery Determination 221
Application of MSC to Quantitative Analysis of Drugs and Metabolites 222
Detection and Profiling of Low Level Metabolites 222
Analysis of Drug and Metabolite Concentrations 225
Enzymology Studies 226
Application of a Combined of MSC and LC–MS to Metabolite Identification 227
A general LC–MSC–MS Approach 227
Use of High Resolution LC–MS to Enhance Analytical Selectivity 232
Use of Micro Flow LC–MS to Enhance Analytical Sensitivity 234
Conclusions 237
References 237
Oxidative Metabolites of Drugs and Xenobiotics: LC–MS Methods to Identify and Characterize in Biological Matrices 242
Introduction 242
Dealkylation 243
Mono-Demethylation 244
Verapamil (Calan/Verelan) 244
Diazepam (Valium) 246
Omeprazole (Prilosec®) 247
Didemethylation and Deethylation 248
Dextromethorphan 248
Lidocaine 248
Phenacetin 249
Reboxetine (Edronax®) 249
Sildenafil (Viagra®) 250
Linezolid (Zyvox®) 251
Depropyl or Deisopropylation 253
Selegiline (Sd Deprenyl®/Eldepryl®) 254
Buprenorphine (Subutex®) 255
Fluvastatin (Lescol®) 256
Dealkylation of Larger Moieties 257
Ritonavir (Norvir®) 257
Donepezil (Aricept®) 258
Tamoxifen (Nolvadex®) 258
Terfenadine (Seldane®) 259
Oxidative Deamination 259
Amphetamine 259
Benzphetamine (Didrex) 260
Terbinafine (Lamisil®) 260
Oxidation of Carbon 261
Hydroxylation 261
Loratadine (Claritin®) 262
Diclofenac (Cataflam/Voltaren) 264
Debrisoquine 266
Midazolam 267
Bupropion (Wellbutrin®/Zyban®) 269
Terfenadine (Seldane®) 269
Epoxidation 270
Protryptiline (Vivactil) 270
Carbamazepine (Atretol/Carbatrol/Epitol/Tegretol) 271
Oxidation of Nitrogen 273
N-Oxides 273
Loratadine 274
Clozapine (Clozaril®) 276
Hydroxylamines 277
Dapsone (Avlosulfon) 278
Debrisoquine 279
Oxidation of Sulfur 280
S-Oxidation/Sulfoxides 280
Cimetidine (Tagamet®) 280
Clindamycin (Cleocin®) 280
Omapatrilat (Vanlev) 281
Sulfones 283
Chlorpromazine (Megaphen) 283
Ziprasidone (Geodon®) 284
ß-Oxidation 285
Dehalogenation 286
Halothane 287
Tolfenamic Acid 287
Future Directions of Metabolite Characterization/Identification 288
References 289
Detection and Characterization of Highly Polar Metabolites by LC–MS: Proper Selection of LC Column and use of Stable Isotope-Labeled Drug to Study Metabolism of Ribavirin in Rats 294
Introduction 294
Experimental 296
Materials 296
Preparation of Stock and Working solutions 296
Instrumentation 296
Chromatographic Conditions 297
Mass Spectrometry Conditions 298
Drug Administration and Specimen Collection 298
Sample Preparation for LC–MS Analysis 299
Urine 299
Plasma 299
Results 300
Plasma Metabolites 302
Urinary Metabolites 305
Discussion 306
Conclusions 309
Acknowledgment 310
References 310
Cytochrome P450 (CYP) and UDP-Glucuronosyltransferase (UGT) Enzymes: Role in Drug Metabolism, Polymorphism, and Identification of their Involvement in Drug Metabolism 312
Introduction 312
Cytochrome P450 (CYP) and Subfamilies 313
CYP1 Family 314
CYP2 Family 317
CYP3 Family 318
CYP4 Family 320
Role of CYPs in Drug Metabolism 320
Polymorphism 321
Identification of P450 Involvement in Drug Metabolism (Phenotyping) 323
Typical Incubation with Human Liver Microsomes 324
Screening Enzymes Responsible for the Metabolism of NCEs Using Recombinant Human P450 Enzymes 324
Correlation Analysis 325
Inhibition by Antibodies 326
Inhibition by Chemicals 326
Sample Analysis 327
Characterization of Enzymology for the Metabolism of Loratadine: A Representative Example 327
High-Throughput Screening for Inhibition of CYP Enzymes 333
UDP-glucuronosyltransferase (UGT) 334
Identification of UGT Enzyme(s) Involved in the Metabolism 337
Incubation with Pooled Human Liver Microsomes 339
Screening of Recombinant Human UGT Enzymes 339
Correlation Study 340
Inhibition Study with Chemical Inhibitors of UGTs 340
UGT and Polymorphism 343
Future trends 343
Acknowledgment 344
References 344
Subject Index 354