Identification And Quantification of Drugs, Metabolites And Metabolizing Enzymes by Lc-ms

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ISBN: 9780444517104 | 0444517103
Cover: Hardcover
Copyright: 12/29/2005

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As new techniques of transferring from liquid to gas phase and measuring masses of drug molecules and metabolites become more prevalent, so do the technical challenges of putting these techniques into proper use, as well as the task of consolidating emerging applications. Identification and Quantification of Drugs, Metabolites and Metabolizing Enzymes by LC-MS, Volume 6 fills the gap in the lack of presently available literature by providing a critical review in the current use of liquid chromatography-mass spectrometry (LC-MS) in drug discovery and development. With chapters written by experts with a wide range of practical experience from the pharmaceutical industry, emphasis is placed on techniques and applications. The book also includes chapters on how to utilize LC-MS instrumentation for current drug metabolism problems. This book is intended for those beginning to use LC-MS for drug metabolism studies as well as for those considered advanced practitioners.

Preface

List of contributors

1. LC—MS and drug metabolism: a journey back in time, present trends and future directions

K.B. Alton

1.1. Introduction

(4)

References

(2)

2. Strategies for increasing throughput for PK samples in a drug discovery environment

W.A. Korfmacher

2.1. Introduction

(2)

2.2. Strategies for early PK assays

(20)

2.2.1. Enhanced mass resolution — a new analytical tool for PK assays

(4)

2.2.2. CARRS — an example of an integrated PK assay strategy

(7)

2.3. Conclusions

(1)

References

(5)

3. Commonly encountered analytical problems and their solutions in liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods used in drug development

T.K. Majumdar

3.1. Introduction

(1)

3.2. Commonly encountered analytical problems and solutions

(25)

3.2.1. Problems with sample collection and analyte stability

(1)

3.2.2. Problem with positive controls

(1)

3.2.3. Carryover problem

(3)

3.2.4. Problem with retention time shift in HPLC

(2)

3.2.5. Problems related to the mobile phase composition and ionization of analytes

(1)

3.2.6. Problems with ion suppression and matrix effect in the API interface

(4)

3.2.7. Problems with stable isotope labeled IS

(2)

3.2.8. Problem of matrix interference due to the presence of metabolites or other endogenous compounds

(5)

3.2.9. The use of gas-phase cluster ions for method sensitivity problem

(2)

3.2.10. Problem with post-column ion source-induced changes of pro-drugs and metabolites

(1)

3.2.11. Problems with both mass spectrometric and chromatographic resolutions in accurate quantification of parent compound and metabolites

(1)

3.2.12. Problem with quantifying an unstable and light-sensitive compound at ultra-trace level concentrations

(3)

3.3. Conclusions

(1)

Acknowledgments

(1)

References

(4)

4. Pitfalls in quantitative LC–MS/MS: metabolite contribution to measured drug concentration

M. Jemal

4.1. Introduction

(1)

4.2. Analytical pitfalls due to metabolite in-source conversion

(9)

4.3. Analytical pitfalls due to the simultaneous formation of [M+H]+ and [M+NH4]+ ions from a drug as well as its metabolite

(7)

4.4. Analytical pitfalls due to conversion of metabolite to the parent drug during sample collection, handling, extraction, and analysis

(1)

4.5. Bioanalytical method design for drug quantification in the presence of a metabolite that may degrade to generate the drug

(9)

4.6. Strategy of using post-dose samples to establish validity of bioanalytical LC–MS/MS methods

(8)

4.7. Summary and conclusions

(1)

References

100

(5)

5. In vitro DMPK screening in drug discovery, role of LC–MS/MS

I. Chu and A.A. Nomeir

5.1. Introduction

105

(1)

5.2. CYP inhibition screening

105

(6)

5.2.1. Background

105

(1)

5.2.2. Types of inhibition

106

(1)

5.2.3. Experimental procedure

107

(1)

5.2.4. Sample plate set-up

108

(1)

5.2.5. Quantification with LC–MS/MS

109

(1)

5.2.6. IC5„ determination

109

(1)

5.2.7. Throughput

110

(1)

5.3. Caco-2 permeability

111

(5)

5.3.1. Background

111

(1)

5.3.2. Mechanisms of absorption

112

(1)

5.3.3. Caco-2 screening assay

112

(1)

5.3.4. Efflux evaluation

113

(1)

5.3.5. LC–MS or LC–MS/MS?

113

(1)

5.3.6. Multiplexed ion source (MUX®)

114

(1)

5.3.7. Determination of apparent permeability

115

(1)

5.3.8. Assay precision

116

(1)

5.3.9. Throughput

116

(1)

5.4. Blood–Brain barrier

116

(4)

5.4.1. Background

116

(1)

5.4.2. Cell culture

117

(1)

5.4.3. Transport studies

117

(1)

5.4.4. Assay precision

118

(1)

5.4.5. In vivo–in vitro correlation

118

(2)

5.5. Conclusion

120

(1)

Acknowledgment

120

(1)

References

120

(3)

6. Metabolite identification by LC–MS: applications in drug discovery and development

C.E.C.A. Hop and C. Prakash

6.1. Introduction

123

(2)

6.2. Metabolite identification

125

(1)

6.3. Mass spectrometers for metabolite identification

126

(4)

6.3.1. Single and triple quadrupole mass spectrometer

127

(1)

6.3.2. 3-Dimensional ion trap mass spectrometer

127

(1)

6.3.3. Q-TOF mass spectrometer:

128

(1)

6.3.4. Common biotransformations

129

(1)

6.4. Additional tools for metabolite identification

130

(3)

6.4.1. Chemical derivatization

130

(1)

6.4.2. Radiolabeled compounds

131

(1)

6.4.3. Isotope patterns

131

(1)

6.4.4. LC–NMR–MS

132

(1)

6.4.5. H/D exchange

132

(1)

6.5. Applications

133

(20)

6.5.1. Metabolism of a potent neurokinin 1 receptor antagonist

133

(6)

6.5.2. Metabolism of a potent PPARγ agonist

139

(3)

6.5.3. Metabolism of an antipsychotic drug ziprasidone

142

(1)

6.5.3.1. Use of two different radiolabels

143

(1)

6.5.3.2. Precursor ion spectrum

145

(1)

6.5.3.3. Differentiation of isobaric metabolites

145

(1)

6.5.4. Biotransformation of an anxiolytic drug candidate, CP-93,393

146

(7)

6.6. Future trends

153

(1)

Acknowledgments

154

(1)

References

154

(5)

7. The utility of in vitro screening for the assessment of electrophilic metabolite formation early in drug discovery using HPLC—MS/MS

D.E. Grotz, N.A. Clarke, and K.A. Cox

7.1. Introduction

159

(8)

7.1.1. Metabolite characterization

160

(1)

7.1.2. In vitro metabolic screening methodology

161

(2)

7.1.3. The significance of electrophilic metabolites

163

(1)

7.1.3.1. Glutathiones

164

(1)

7.1.3.2. Acylglucuronides

165

(1)

7.1.3.3. Acylglucosides and N-acetylglucosaminides

167

(1)

7.2. Current approaches

167

(5)

7.2.1. Background

167

(2)

7.2.2. Strategy

169

(1)

7.2.2.1. Materials and methods

169

(1)

7.2.2.2. Glutathione conjugation incubations

169

(1)

7.2.2.3. Acylglucuronide conjugation incubations

170

(1)

7.2.2.4. Acylglucoside and N-acetylglucosamini de conjugation incubations

170

(1)

7.2.2.5. Screening method details

171

(1)

7.2.2.6. High performance liquid chromatographic (HPLC) conditions

171

(1)

7.2.2.7. Mass spectrometric analysis

171

(1)

7.3. Results/discussion

172

(5)

7.4. Conclusions

177

(2)

7.5. Future

179

(1)

Acknowledgements

179

(1)

References

179

(4)

8. Liquid chromatography tandem mass spectrometry-based metabolite identification strategies in pharmaceutical research

M.R. Anari, P.R. Tiller, and T.A. Baillie

8.1. Introduction

183

(1)

8.2. Metabolite identification in drug discovery

184

(1)

8.3. Classical mass spectrometry-based metabolite identification approach in drug discovery

185

(1)

8.4. Parallel mass spectrometry-based metabolite identification approach in drug discovery

186

(1)

8.5. Integrated list-dependent fast LC/MSn in identifying metabolic soft spots

187

(1)

8.6. Metabolite identification in preclinical development

188

(1)

8.7. Identification of unanticipated metabolites using data-dependent precursor ion and constant neutral loss scans

188

(1)

8.8. Detailed characterization of metabolites using radiochromatographic techniques coupled to LC/MS

189

(1)

8.9. Metabolite identification in clinical drug development

190

(1)

8.10. Detailed characterization of metabolites in humans

190

(1)

8.11. Quantitative similarity of the circulating metabolites in human versus preclinical species

190

(1)

References

191

(4)

9. Quantification and structural elucidation of low quantities of radiolabeled metabolites using microplate scintillation counting (MSC) techniques in conjunction with LC–MS

M. Zhu, D. Zhang, and G.L. Skiles

9.1. Introduction

195

(1)

9.2. The HPLC–MSC technique

196

(3)

9.3. Analytical characteristics of HPLC–MSC

199

(6)

9.3.1. Sensitivity

199

(2)

9.3.2. Precision and accuracy

201

(1)

9.3.3. Matrix effect of biological samples

201

(3)

9.3.4. Radioactivity recovery determination

204

(1)

9.4. Application of MSC to quantitative analysis of drugs and metabolites

205

(5)

9.4.1. Detection and profiling of low level metabolites

205

(3)

9.4.2. Analysis of drug and metabolite concentrations

208

(1)

9.4.3. Enzymology studies

209

(1)

9.5. Application of a combined of MSC and LC–MS to metabolite identification

210

(10)

9.5.1. A general LC–MSC–MS approach

210

(5)

9.5.2. Use of high resolution LC–MS to enhance analytical selectivity

215

(2)

9.5.3. Use of micro flow LC–MS to enhance analytical sensitivity

217

(3)

9.6. Conclusions

220

(1)

References

220

(5)

10. Oxidative metabolites of drugs and xenobiotics: LC–MS methods to identify and characterize in biological matrices

R. Ramanathan, S.K. Chowdhury, and K.B. Alton

10.1. Introduction

225

(1)

10.2. Dealkylation

226

(16)

10.2.1. Mono-demethylation

227

(1)

10.2.1.1. Verapamil (CalanlVerelan)

227

(1)

10.2.1.2. Diazepam (Valium)

229

(1)

10.2.1.3. Omeprazole (Prilosec®)

230

(1)

10.2.2. Didemethylation and deethylation

231

(1)

10.2.2.1. Dextromethorphan

231

(1)

10.2.2.2. Lidocaine

231

(1)

10.2.2.3. Phenacetin

232

(1)

10.2.2.4. Reboxetine (Edronax®)

232

(1)

10.2.2.5. Sildenafil (Viagra®)

233

(1)

10.2.2.6. Linezolid (Zyvox®)

234

(2)

10.2.3. Depropyl or deisopropylation

236

(1)

10.2.3.1. Selegiline (Sd Deprenyl®/Eldepryl®)

237

(1)

10.2.3.2. Buprenorphine (Subutex®)

238

(1)

10.2.3.3. Fluvastatin (Lescol®)

239

(1)

10.2.4. Dealkylation of larger moieties

240

(1)

10.2.4.1. Ritonavir (Norvir®)

240

(1)

10.2.4.2. Donepezil (Aricept®)

241

(1)

10.2.4.3. Tamoxifen (Nolvadex®)

241

(1)

10.2.4.4. Terfenadine (Seldane®)

242

(1)

10.3. Oxidative deamination

242

(2)

10.3.1. Amphetamine

242

(1)

10.3.2. Benzphetamine (Didrex)

243

(1)

10.3.3. Terbinafine (Lamisil®)

243

(1)

10.4. Oxidation of carbon

244

(12)

10.4.1. Hydroxylation

244

(1)

10.4.1.1. Loratadine (Claritin®)

245

(1)

10.4.1.2. Diclofenac (Cataflam/Voltaren)

247

(1)

10.4.1.3. Debrisoquine

249

(1)

10.4.1.4. Midazolam

250

(1)

10.4.1.5. Bupropion (Wellbutrin®/Zyban®)

252

(1)

10.4.1.6. Terfenadine (Seldane®)

252

(1)

10.4.2. Epoxidation

253

(1)

10.4.2.1. Protryptiline (Vivactil)

253

(1)

10.4.2.2. Carbamazepine (Atretol/Carbatrol/Epitol/Tegretol)

254

(2)

10.5. Oxidation of nitrogen

256

(7)

10.5.1. N-Oxides

256

(1)

10.5.1.1. Loratadine

257

(1)

10.5.1.2. Clozapine (Clozaril®)

259

(1)

10.5.2. Hydroxylamines

260

(1)

10.5.2.1. Dapsone (Avlosulfon)

261

(1)

10.5.2.2. Debrisoquine

262

(1)

10.6. Oxidation of sulfur

263

(5)

10.6.1. S-Oxidation/sulfoxides

263

(1)

10.6.1.1. Cimetidine (Tagamet®)

263

(1)

10.6.1.2. Clindamycin (Cleocin®)

263

(1)

10.6.1.3. Omapatrilat (Vanlev)

264

(2)

10.6.2. Sulfones

266

(1)

10.6.2.1. Chlorpromazine (Megaphen)

266

(1)

10.6.2.2. Ziprasidone (Geodon®)

267

(1)

10.7. β-Oxidation

268

(1)

10.8. Dehalogenation

269

(2)

10.8.1. Halothane

270

(1)

10.8.2. Tolfenamic acid

270

(1)

10.9. Future directions of metabolite characterization/identification

271

(1)

References

272

(5)

11. 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

S.K. Chowdhury, V.S. Gopaul, N. Blumenkrantz, R. Zhong, K.M. Kulmatycki, and K.B. Alton

11.1. Introduction

277

(2)

11.2. Experimental

279

(4)

11.2.1. Materials

279

(1)

11.2.2. Preparation of stock and working solutions

279

(1)

11.2.3. Instrumentation

279

(1)

11.2.4. Chromatographic conditions

280

(1)

11.2.5. Mass spectrometry conditions

281

(1)

11.2.6. Drug administration and specimen collection

281

(1)

11.2.7. Sample preparation for LC–MS analysis

282

(1)

11.2.7.1. Urine

282

(1)

11.2.7.2. Plasma

282

(1)

11.3. Results

283

(6)

11.3.1. Plasma metabolites

285

(3)

11.3.2 Urinary metabolites

288

(1)

11.4. Discussion

289

(3)

11.5. Conclusions

292

(1)

Acknowledgment

293

(1)

References

293

(2)

12. Cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes: role in drug metabolism, polymorphism, and identification of their involvement in drug metabolism

A. Ghosal, R. Ramanathan, N.S. Kishnani, S.K. Chowdhury, and K.B. Alton

12.1. Introduction

295

(1)

12.2. Cytochrome P450 (CYP) and subfamilies

296

(7)

12.2.1. CYP1 family

297

(3)

12.2.2. CYP2 family

300

(1)

12.2.3. CYP3 family

301

(2)

12.2.4. CYP4 family

303

(1)

12.3. Role of CYPs in drug metabolism

303

(1)

12.4. Polymorphism

304

(2)

12.5. Identification of P450 involvement in drug metabolism (phenotyping)

306

(10)

12.5.1. Typical incubation with human liver microsomes

307

(1)

12.5.2. Screening enzymes responsible for the metabolism of NCEs using recombinant human P450 enzymes

307

(1)

12.5.3. Correlation analysis

308

(1)

12.5.4. Inhibition by antibodies

309

(1)

12.5.5. Inhibition by chemicals

309

(1)

12.5.6. Sample analysis

310

(1)

12.5.7. Characterization of enzymology for the metabolism of loratadine: a representative example

310

(6)

12.6. High-throughput screening for inhibition of CYP enzymes

316

(1)

12.7. UDP-glucuronosyltransferase (UGT)

317

(3)

12.8. Identification of UGT enzyme(s) involved in the metabolism

320

(6)

12.8.1. Incubation with pooled human liver microsomes

322

(1)

12.8.2. Screening of recombinant human UGT enzymes

322

(1)

12.8.3. Correlation study

323

(1)

12.8.4. Inhibition study with chemical inhibitors of UGTs

323

(3)

12.9. UGT and polymorphism

326

(1)

12.10. Future trends

326

(1)

Acknowledgment

327

(1)

References

327

(10)

Subject Index

337

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Identification And Quantification of Drugs, Metabolites And Metabolizing Enzymes by Lc-ms

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Table of Contents

Supplemental Materials

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Identification And Quantification of Drugs, Metabolites And Metabolizing Enzymes by Lc-ms

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Table of Contents

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