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Analytical Tools and Approaches for Metabolite Identification in Early Drug Discovery

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Abstract

Determination of the chemical structures of metabolites is a critical part of the early pharmaceutical discovery process. Understanding the structures of metabolites is useful both for optimizing the metabolic stability of a drug as well as rationalizing the drug safety profile. This review describes the current state of the art in this endeavor. The likely outcome of metabolism is first predicted by comparison to the literature. Then metabolites are synthesized in a variety of in vitro systems. The various approaches to LC/UV/MS are applied to learn information about these metabolites and structure hypotheses are made. Structures are confirmed by synthesis or NMR. The special topic of reactive metabolite structure determination is briefly addressed.

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Abbreviations

LC:

liquid chromatography

MS:

mass spectrometry

NMR:

nuclear magnetic resonance

UV:

ultraviolet

References

  1. J. L. Walgren, M. D. Mitchell, and D. C. Thompson. Role of metabolism in drug-induced idiosyncratic hepatotoxicity. Crit. Rev. Toxicol. 35:325–361 (2005).

    Article  PubMed  CAS  Google Scholar 

  2. K. Cox. Special requirements for metabolite characterization. In W. A. Korfmacher (ed.), Using Mass Spectrometry for Drug Metabolism Studies, CRC, Boca Raton, FL, 2005, pp. 229–252.

    Google Scholar 

  3. C. E. C. A. Hop. Applications of quadrupole-time-of-flight mass spectrometry to facilitate metabolite identification. Amer. Pharm. Rev. 7:76–79 (2004).

    CAS  Google Scholar 

  4. G. Hopfgartner and M. Zell. Q Trap MS: a new tool for metabolite identification. In W. A. Korfmacher (ed.), Using Mass Spectrometry for Drug Metabolism Studies, CRC, Boca Raton, Florida, 2005, pp. 277–304.

    Google Scholar 

  5. D. B. Kassel. High throughput strategies for in vitro ADME assays: how fast we go? In W. A. Korfmacher (ed.), Using Mass Spectrometry for Drug Metabolism Studies, CRC, Boca Raton, FL, 2005, pp. 83–102.

    Google Scholar 

  6. J. Kingston, D. O’Connor, T. Sparey, and S. Thomas. Hyphenated techniques in drug discovery: purity assessment, purification, quantitative analysis and metabolite identification. In J. M. Rosenfeld (ed.), Sample Preparation for Hyphenated Analytical Techniques, CRC, Boca Raton, FL, 2004, pp. 114–149.

    Chapter  Google Scholar 

  7. D. Q. Liu and C. E. C. A. Hop. Strategies for characterization of drug metabolites using liquid chromatography-tandem mass spectrometry in conjunction with chemical derivatization and on-line H/D exchange approaches. J. Pharm. Biomed. Anal. 37:1–18 (2005).

    Article  PubMed  Google Scholar 

  8. A. E. Nassar and R. E. Talaat. Strategies for dealing with metabolite elucidation in drug discovery and development. Drug Discov. Today 9:317–327 (2004).

    Article  PubMed  CAS  Google Scholar 

  9. A. Nassar, A. Kamel, and C. Clarimont. Improving the decision-making process in the structural modification of drug candidates: enhancing metabolic stability. Drug Discov. Today 9:1020–1028 (2004).

    Article  PubMed  CAS  Google Scholar 

  10. B. Testa, A. L. Balmat, and A. Long. Predicting drug metabolism: concepts and challenges. Pure Appl. Chem. 76:907–914 (2004).

    Article  CAS  Google Scholar 

  11. B. Testa, A. L. Balmat, A. Long, and P. Judson. Predicting drug metabolism—an evaluation of the expert system METEOR. Chem. Biodiv. 2:872–885 (2005).

    Article  CAS  Google Scholar 

  12. S. O. Jonsdottir, F. S. Jorgensen, and S. Brunak. Prediction methods and databases within chemoinformatics: emphasis on drugs and drug candidates. Bioinformatics 21:2145–2160 (2005).

    Article  PubMed  CAS  Google Scholar 

  13. M. R. Anari, R. I. Sanchez, R. Bakhtiar, R. B. Franklin, and T. A. Baillie. Integration of knowledge-based metabolic predictions with liquid chromatography data-dependent tandem mass spectrometry for drug metabolism studies: application to studies on the biotransformation of indinavir. Anal. Chem. 76:823–832 (2004).

    Article  PubMed  CAS  Google Scholar 

  14. M. R. Anari and T. A. Baillie. Bridging cheminformatic metabolite prediction and tandem mass spectrometry. Drug Discov. Today 10:711–717 (2005).

    Article  PubMed  CAS  Google Scholar 

  15. Y. Borodina, A. Rudik, D. Filimonov, N. Kharchevnikova, A. Dmitriev, V. Blinova, and V. Poroikov. A new statistical approach to predicting aromatic hydroxylation sites. Comparison with model-based approaches. J. Chem. Inf. Comput. Sci. 44:1998–2009 (2004).

    PubMed  CAS  Google Scholar 

  16. R. J. Mortishire-Smith, D. O’Connor, J. M. Castro-Perez, and J. Kirby. Accelerated throughput metabolic route screening in early drug discovery using high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry and automated data analysis. Rapid Commun. Mass Spectrom. 19:2659–2670 (2005).

    Article  PubMed  CAS  Google Scholar 

  17. A. Tevell, U. Bondesson, K. Toerneke, and M. Hedeland. Identification of some new clemastine metabolites in dog, horse, and human urine with liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 18:2267–2272 (2004).

    Article  PubMed  CAS  Google Scholar 

  18. G. Cruciani, E. Carosati, B. De Boeck, K. Ethirajulu, C. Mackie, T. Howe, and R. Vianello. MetaSite: understanding metabolism in human cytochromes from the perspective of the chemist. J. Med. Chem. 48:6970–6979 (2005).

    Article  PubMed  CAS  Google Scholar 

  19. M. Y. Lee, C. B. Park, J. S. Dordick, and D. S. Clark. Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proc. Natl. Acad. Sci. U. S. A. 102:983–987 (2005).

    Article  PubMed  CAS  Google Scholar 

  20. O. Pelkonen, M. Turpeinen, J. Uusitalo, A. Rautio, and H. Raunio. Prediction of drug metabolism and interactions on the basis of in vitro investigations. Basic Clin. Pharmacol. Toxicol. 96:167–175 (2005).

    Article  PubMed  CAS  Google Scholar 

  21. D. C. Ackley, K. T. Rockich, and T. R. Baker. Metabolic stability assessed by liver microsomes and hepatocytes. In Z. Yan and G. W. Caldwell (eds.), Optimization Drug Discovery, Humana, Totowa, New Jersey, 2004, pp. 151–162.

    Chapter  Google Scholar 

  22. E. Kantharaj, P. B. Ehmer, A. Tuytelaars, A. Van Vlaslaer, C. Mackie, and R. A. H. J. Gilissen. Simultaneous measurement of metabolic stability and metabolite identification of 7-methoxymethylthiazolo [3,2-a]pyrimidin-5-one derivatives in human liver microsomes using liquid chromatography/ion-trap mass spectrometry. Rapid Commun. Mass Spectrom. 19:1069–1074 (2005).

    Article  PubMed  CAS  Google Scholar 

  23. A. C. Li, D. Alton, M. S. Bryant, and W. Z. Shou. Simultaneously quantifying parent drugs and screening for metabolites in plasma pharmacokinetic samples using selected reaction monitoring information-dependent acquisition on a QTrap instrument. Rapid Commun. Mass Spectrom. 19:1943–1950 (2005).

    Article  PubMed  CAS  Google Scholar 

  24. B. V. Karanam, C. E. C. A. Hop, D. Q. Liu, M. Wallace, D. Dean, H. Satoh, M. Komuro, K. Awano, and S. H. Vincent. In vitro metabolism of MK−0767 [(+−)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl) phenyl]methyl]benzamide], a peroxisome proliferator-activated receptor a/g agonist. I. Role of cytochrome P450, methyltransferases, flavin monooxygenases, and esterases. Drug Metab. Dispos. 32:1015–1022 (2004).

    Article  PubMed  CAS  Google Scholar 

  25. Z. Miao, A. Kamel, and C. Prakash. Characterization of a novel metabolite intermediate of ziprasidone in hepatic cytosolic fractions of rat, dog, and human by ESI-MS/MS, hydrogen/deuterium exchange, and chemical derivatization. Drug Metab. Dispos. 33:879–883 (2005).

    Article  PubMed  CAS  Google Scholar 

  26. M. J. Gomez-Lechon, M. T. Donato, J. V. Castell, and R. Jover. Human hepatocytes in primary culture: the choice to investigate drug metabolism in man. Curr. Drug Metab. 5:443–462 (2004).

    Article  PubMed  CAS  Google Scholar 

  27. C. A. Evans, H. E. Fries, and K. W. Ward. In vitro metabolic fate of a novel structural class: evidence for the formation of a reactive intermediate on a benzothiophene moiety. Chem.-Biol. Interact. 152:25–36 (2005).

    Article  PubMed  CAS  Google Scholar 

  28. R. Gebhardt, J. G. Hengstler, D. Mueller, R. Gloeckner, P. Buenning, B. Laube, E. Schmelzer, M. Ullrich, D. Utesch, N. Hewitt, M. Ringel, B. R. Hilz, A. Bader, A. Langsch, T. Koose, H. J. Burger, J. Maas, and F. Oesch. New hepatocyte in vitro systems for drug metabolism: metabolic capacity and recommendations for application in basic research and drug development, standard operation procedures. Drug Metab. Rev. 35:145–213 (2003).

    Article  PubMed  CAS  Google Scholar 

  29. J. C. Zguris, L. J. Itle, D. Hayes, and M. V. Pishko. Microreactor microfluidic systems with human microsomes and hepatocytes for use in metabolite studies. Biomed. Microdev. 7:117–125 (2005).

    Article  CAS  Google Scholar 

  30. L. Vignati, E. Turlizzi, S. Monaci, P. Grossi, R. de Kanter, and M. Monshouwer. An in vitro approach to detect metabolite toxicity due to CYP3A4-dependent bioactivation of xenobiotics. Toxicology 216:154–167 (2005).

    Article  PubMed  CAS  Google Scholar 

  31. E. F. A. Brandon, T. M. Bosch, M. J. Deenen, R. Levink, E. van der Wal, J. B. M. van Meerveld, M. Bijl, J. H. Beijnen, J. H. M. Schellens, and I. Meijerman. Validation of in vitro cell models used in drug metabolism and transport studies; genotyping of cytochrome P450, Phase II enzymes and drug transporter polymorphisms in the human hepatoma (HepG2), ovarian carcinoma (IGROV-1) and colon carcinoma (CaCo-2, LS180) cell lines. Toxicol. Appl. Pharmacol. 211:1–10 (2006).

    Article  PubMed  CAS  Google Scholar 

  32. R. A. Kemper and D. L. Nabb. In vitro studies in microsomes from rat and human liver, kidney, and intestine suggest that perfluorooctanoic acid is not a substrate for microsomal UDP_glucuronosyltransferases. Drug Chem. Toxicol. 28:281–287 (2005).

    Article  PubMed  CAS  Google Scholar 

  33. B. Blankert, H. Hayen, S. M. van Leeuwen, U. Karst, E. Bodoki, S. Lotrean, R. Sandulescu, N. M. Diez, O. Dominguez, J. Arcos, and J. M. Kauffmann. Electrochemical, chemical and enzymatic oxidations of phenothiazines. Electroanalysis 17:1501–1510 (2005).

    Article  CAS  Google Scholar 

  34. I. Beattie, K. Joncour, and K. Lawson. Ultra performance liquid chromatography coupled to orthogonal quadrupole TOF-MS(MS) for metabolite identification. LC-GC Europe 24:19–21 (2005).

    Google Scholar 

  35. J. Castro-Perez, R. Plumb, J. H. Granger, I. Beattie, K. Joncour, and A. Wright. Increasing throughput and information content for in vitro drug metabolism experiments using ultra-performance liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer. Rapid Commun. Mass Spectrom. 19:843–848 (2005).

    Article  PubMed  CAS  Google Scholar 

  36. M. I. Churchwell, N. C. Twaddle, L. R. Meeker, and D. R. Doerge. Improving LC-MS sensitivity through increases in chromatographic performance: Comparisons of UPLC-ES/MS/MS to HPLC-ES/MS/MS. J. Chromatog. B 825:134–143 (2005).

    Article  CAS  Google Scholar 

  37. C. Giroud, K. Michaud, F. Sporkert, C. Eap, M. Augsburger, P. Cardinal, and P. Mangin. A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography–mass spectrometry (HILIC–MS). J. Anal. Toxicol. 28:464–474 (2004).

    PubMed  CAS  Google Scholar 

  38. A. Baldacci, J. Caslavska, A. B. Wey, and W. Thormann. Identification of new oxycodone metabolites in human urine by capillary electrophoresi-multiple-stage ion-trap mass spectrometry. J. Chromatogr. A 1051:273–282 (2004).

    PubMed  CAS  Google Scholar 

  39. F. Peters, M. Meyer, G. Fritschi, and H. Maurer. Studies on the metabolism and toxicological detection of the new designer drug 4′-methyl-alpha-pyrrolidinobutyrophenone MPBP in rat urine using gas chromatography-mass spectrometry. J. Chromatogr. B 824:81–91 (2005).

    Article  CAS  Google Scholar 

  40. A. Ewald, F. Peters, M. Weise, and H. Maurer. Studies on the metabolism and toxicological detection of the designer drug 4-methylthioamphetamine 4-MTA in human urine using gas chromatography_mass spectrometry. J. Chromatogr. B 824:123–131 (2005).

    Article  CAS  Google Scholar 

  41. W. L. Fitch, M. McGregor, A. R. Katritzky, A. Lomaka, R. Petrukhin, and M. Karelson. Prediction of ultraviolet spectral absorbance using quantitative structure-property relationships. J. Chem. Inf. Comput. Sci. 42:830–840 (2002).

    PubMed  CAS  Google Scholar 

  42. Y. Cai, D. Kingery, O. McConnell, and A. C. Bach II. Advantages of atmospheric pressure photoionization mass spectrometry in support of drug discovery. Rapid Commun. Mass Spectrom. 19:1717–1724 (2005).

    Article  PubMed  CAS  Google Scholar 

  43. S. Ma, S. K. Chowdhury, and K. B. Alton. Thermally induced N-to-O rearrangement of tert-N-oxides in atmospheric pressure chemical ionization and atmospheric pressure photoionization mass spectrometry: differentiation of N-Oxidation from hydroxylation and potential determination of N-Oxidation site. Anal. Chem. 77:3676–3682 (2005).

    Article  PubMed  CAS  Google Scholar 

  44. W. L. Fitch, P. W. Berry, Y. Tu, A. Tabatabaei, L. Lowrie, F. Lopez-Tapia, Y. Liu, D. Nitzan, M. R. Masjedizadeh, and A. Varadarajan. Identification of glutathione-derived metabolites from an IP receptor antagonist. Drug Metab. Dispos. 32:1482–1490 (2004).

    Article  PubMed  CAS  Google Scholar 

  45. J. Olsen, C. Li, I. Bjornsdottir, U. Sidenius, S. H. Hansen, and L. Z. Benet. In vitro and in vivo studies on acyl-coenzyme A-dependent bioactivation of zomepirac in rats. Chem. Res. Toxicol. 18:1729–1736 (2005).

    Article  PubMed  CAS  Google Scholar 

  46. P. H. Stahl and C. G. Wermuth (eds.). Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wiley VCH, Zurich, 2002, pp. 25.

  47. D. Q. Liu, B. V. Karanam, G. A. Doss, R. R. Sidler, S. H. Vincent, and C. E. C. A. Hop. In vitro metabolism of MK-0767 [(+-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)-phenyl] methyl]benzamide], a peroxisome proliferator-activated receptor a/g agonist. II. Identification of metabolites by liquid chromatography_tandem mass spectrometry. Drug Metab. Dispos. 32:1023–1031 (2004).

    Article  PubMed  CAS  Google Scholar 

  48. P. Eichhorn, P. L. Ferguson, S. Perez, and D. S. Aga. Application of ion Trap-MS with H/D exchange and QqTOF-MS in the identification of microbial degradates of trimethoprim in nitrifying activated sludge. Anal. Chem. 77:4176–4184 (2005).

    Article  PubMed  CAS  Google Scholar 

  49. L. Zhou, D. R. Thakker, R. D. Voyksner, M. Anbazhagan, D. W. Boykin, J. E. Hall, and R. R. Tidwell. Metabolites of an orally active antimicrobial prodrug, 2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime, identified by liquid chromatography/tandem mass spectrometry. J. Mass Spectrom. 39:351–360 (2004).

    Article  PubMed  CAS  Google Scholar 

  50. G. Hopfgartner, E. Varesio, V. Tschaeppaet, C. Grivet, E. Bourgogne, and L. A. Leuthold. Triple quadrupole linear ion trap mass spectrometer for the analysis of small molecules and macromolecules. J. Mass Spectrom. 39:845–855 (2004).

    Article  PubMed  CAS  Google Scholar 

  51. T. Mauriala, N. Chauret, R. Oballa, D. A. Nicoll-Griffith, and K. P. Bateman. A strategy for identification of drug metabolites from dried blood spots using triple-quadrupole/linear ion trap hybrid mass spectrometry. Rapid Commun. Mass Spectrom. 19:1984–1992 (2005).

    Article  PubMed  CAS  Google Scholar 

  52. J. P. Qiao, Z. Abliz, F. M. Chu, P. L. Hou, F. Liang, Y. Chang, and Z. R. Guo. Application of a novel quadrupole linear ion trap mass spectrometer to study the metabolism of 6-aminobutylphthalide in rat brains. Rapid Commun. Mass Spectrom. 18:3142–3147 (2004).

    Article  PubMed  CAS  Google Scholar 

  53. M. Wrona, T. Mauriala, K. P. Bateman, R. J. Mortishire-Smith, and D. O’Connor. ‘All-in-One’ analysis for metabolite identification using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry with collision energy switching. Rapid Commun. Mass Spectrom. 19:2597–2602 (2005).

    Article  PubMed  CAS  Google Scholar 

  54. L. Leclercq, C. Delatour, I. Hoes, F. Brunelle, X. Labrique, and J. Castro-Perez. Use of a five-channel multiplexed electrospray quadrupole time-of-flight hybrid mass spectrometer for metabolite identification. Rapid Commun. Mass Spectrom. 19:1611–1618 (2005).

    Article  PubMed  CAS  Google Scholar 

  55. A.-E. F. Nassar, Y. Parmentier, M. Martinet, and D. Y. Lee. Liquid chromatography-accurate radioisotope counting and microplate scintillation counter technologies in drug metabolism studies. J. Chromatogr. Sci. 42:348–353 (2004).

    PubMed  Google Scholar 

  56. D. J. Foltz, J. Castro-Perez, P. Riley, J. R. Entwisle, and T. R. Baker. Narrow-bore sample trapping and chromatography combined with quadrupole/time-of-flight mass spectrometry for ultra-sensitive identification of in vivo and in vitro metabolites. J. Chromatogr. B 825:144–151 (2005).

    Article  CAS  Google Scholar 

  57. R. F. Staack, E. Varesio, and G. Hopfgartner. The combination of liquid chromatography/tandem mass spectrometry and chip-based infusion for improved screening and characterization of drug metabolites. Rapid Commun. Mass Spectrom. 19:618–626 (2005).

    Article  PubMed  CAS  Google Scholar 

  58. L. Zhang, J. D. Laycock, and K. J. Miller. Quantitative small molecule bioanalysis using chip-based nanoESI–MS/MS. J. Assoc. Lab. Automat. 9:109–114 (2004).

    Article  CAS  Google Scholar 

  59. H. Idborg, P. Edlund, and S. Jacobsson. Multivariate approaches for efficient detection of potential metabolites from liquid chromatography/mass spectrometry data. Rapid Commun. Mass Spectrom. 18:944–954 (2004).

    Article  PubMed  CAS  Google Scholar 

  60. C. A. Smith, E. J. Want, G. O’Maille, R. Abagyan, and G. Siuzdak. XCMS: processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal. Chem. 78:779–787 (2006).

    Article  PubMed  CAS  Google Scholar 

  61. A.-E. F. Nassar and P. E. Adams. Metabolite characterization in drug discovery utilizing robotic liquid-handling, quadruple time-of-flight mass spectrometry and in-silico prediction. Curr. Drug Metab. 4:259–271 (2003).

    Article  PubMed  CAS  Google Scholar 

  62. W. Windig, W. F. Smith, and W. F. Nichols. Fast interpretation of complex LC/MS data using chemometrics. Anal. Chim. Acta 446:467–476 (2001).

    Article  CAS  Google Scholar 

  63. G. Zurek, W. D. Herzog, A. Germanus, O. Raether, V. Krone, A. Ingendoh, and C. Baessmann. Rapid metabolite identification using accurate and MSn mass spectra in combination with smart processing tools. In The Applications Book, LC-GC Europe, Chester, UK, 2004, pp. 24–25.

  64. Z. Yan, N. Maher, R. Torres, G. Caldwell, and N. Huebert. Rapid detection and characterization of minor reactive metabolites using stable-isotope trapping in combination with tandem mass spectrometry. Rapid Commun. Mass Spectrom. 19:3322–3330 (2005).

    Article  PubMed  CAS  Google Scholar 

  65. A. Deroussent, M. Re, H. Hoellinger, E. Vanquelef, O. Duval, M. Sonnier, and T. Cresteil. In vitro metabolism of ethoxidine by human CYP1A1 and rat microsomes: identification of metabolites by high-performance liquid chromatography combined with electrospray tandem mass spectrometry and accurate mass measurements by time-of-flight mass spectrometry. Rapid Commun. Mass Spectrom. 18:474–482 (2004).

    Article  PubMed  CAS  Google Scholar 

  66. T. Pfeifer, J. Tuerk, and R. Fuchs. Structural characterization of sulfadiazine metabolites using H/D exchange combined with various MS/MS experiments. J. Am. Soc. Mass Spectrom. 16:1687–1694 (2005).

    Article  PubMed  CAS  Google Scholar 

  67. M. L. Bandu, K. R. Watkins, M. L. Bretthauer, C. A. Moore, and H. Desaire. Prediction of MS/MS Data. 1. a focus on pharmaceuticals containing carboxylic acids. Anal. Chem. 76:1746–1753 (2004).

    Article  PubMed  CAS  Google Scholar 

  68. L. Yi, M. L. Bandu, and H. Desaire. Identifying lactone hydrolysis in pharmaceuticals. A tool for metabolite structural characterization. Anal. Chem. 77:6655–6663 (2005).

    Article  PubMed  CAS  Google Scholar 

  69. S. Tachibana, M. Tanaka, Y. Fujimaki, W. Suzuki, T. Ookuma, Y. Ohori, K. I. Hayashi, H. Iwata, O. Okazaki, and K. I. Sudo. Metabolism of the calmodulin antagonist DY-9760e in animals and humans. Xenobiotica 35:499–517 (2005).

    Article  PubMed  CAS  Google Scholar 

  70. M. Sandvoss, A. D. Roberts, I. M. Ismail, and S. E. North. Direct on-line hyphenation of capillary liquid chromatography to nuclear magnetic resonance spectroscopy: practical aspects and application to drug metabolite identification. J. Chromatogr. A 1028:259–266 (2004).

    Article  PubMed  CAS  Google Scholar 

  71. M. Sandvoss, B. Bardsley, T. L. Beck, E. Lee-Smith, S. E. North, P. J. Moore, A. J. Edwards, and R. J. Smith. HPLC-SPE-NMR in pharmaceutical development: capabilities and applications. Magn. Reson. Chem. 43:762–770 (2005).

    Article  PubMed  CAS  Google Scholar 

  72. M. Godejohann, L. H. Tseng, U. Braumann, J. Fuchser, and M. Spraul. Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe. J. Chromatogr. A 1058:191–196 (2004).

    PubMed  CAS  Google Scholar 

  73. I. M. Ismail, P. D. Andrew, J. Cholerton, A. D. Roberts, G. J. Dear, S. Taylor, K. M. Koch, and D. A. Saynor. Characterization of the metabolites of alosetron in experimental animals and human. Xenobiotica 35:131–154 (2005).

    Article  PubMed  CAS  Google Scholar 

  74. K. Sohda, T. Minematsu, T. Hashimoto, K. Suzumura, M. Funatsu, K. Suzuki, H. Imai, T. Usui, and H. Kamimura. Application of LC-NMR for characterization of rat urinary metabolites of zonampanel monohydrate YM872. Chem. Pharm. Bull. 52:1322–1325 (2004).

    Article  PubMed  CAS  Google Scholar 

  75. G. Grosa, U. Galli, B. Rolando, R. Fruttero, G. Gervasio, and A. Gasco. Identification of 2,3-diaminophenazine and of o-benzoquinone dioxime as the major in vitro metabolites of benzofuroxan. Xenobiotica 34:345–352 (2004).

    Article  PubMed  CAS  Google Scholar 

  76. R. Martino, V. Gilard, F. Desmoulin, and M. Malet-Martino. Fluorine-19 or phosphorus-31 NMR spectroscopy: a suitable analytical technique for quantitative in vitro metabolic studies of fluorinated or phosphorylated drugs. J. Pharm. Biomed. Anal. 38:871–891 (2005).

    Article  PubMed  CAS  Google Scholar 

  77. H. Orhan, J. N. M. Commandeur, G. Sahin, U. Aypar, A. Sahin, and N. P. E. Vermeulen. Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients. Xenobiotica 34:301–316 (2004).

    PubMed  CAS  Google Scholar 

  78. E. Skordi, I. D. Wilson, J. C. Lindon, and J. K. Nicholson. Characterization and quantification of metabolites of racemic ketoprofen excreted in urine following oral administration to man by 1H-NMR spectroscopy, directly coupled HPLC-MS and HPLC-NMR, and circular dichroism. Xenobiotica 34:1075–1089 (2004).

    Article  PubMed  CAS  Google Scholar 

  79. C. E. C. A. Hop, Y. Chen, and L. J. Yu. Uniformity of ionization response of structurally diverse analytes using a chip-based nanoelectrospray ionization source. Rapid Commun. Mass Spectrom. 19:3139–3142 (2005).

    Article  PubMed  CAS  Google Scholar 

  80. D. C. Liebler and F. P. Guengerich. Elucidating mechanisms of drug-induced toxicity. Nature Rev. Drug Disc. 4:410–420 (2005).

    Article  CAS  Google Scholar 

  81. A. S. Kalgutkar and J. R. Soglia. Minimising the potential for metabolic activation in drug discovery. Exp. Opin. Drug. Metab. 1:91–142 (2005).

    Article  CAS  Google Scholar 

  82. A. S. Kalgutkar, I. Gardner, R. S. Obach, C. L. Shaffer, E. Callegari, K. R. Henne, A. E. Mutlib, D. K. Dalvie, J. S. Lee, Y. Nakai, J. P. O’Donnell, J. Boer, and S. P. Harriman. A comprehensive listing of bioactivation pathways of organic functional groups. Curr. Drug Metab. 6:161–225 (2005).

    Article  PubMed  CAS  Google Scholar 

  83. B. R. Baer, A. E. Rettie, and K. R. Henne. Bioactivation of 4-Ipomeanol by CYP4B1: adduct characterization and evidence for an enedial intermediate. Chem. Res. Toxicol. 18:855–864 (2005).

    Article  PubMed  CAS  Google Scholar 

  84. J. Castro-Perez, R. Plumb, L. Liang, and E. Yang. A high-throughput liquid chromatography/tandem mass spectrometry method for screening glutathione conjugates using exact mass neutral loss acquisition. Rapid Commun. Mass Spectrom. 19:798–804 (2005).

    Article  PubMed  CAS  Google Scholar 

  85. K. Johnson and R. Plumb. Investigating the human metabolism of acetaminophen using UPLC and exact mass oa-TOF MS. J. Pharm. Biomed. Anal. 39:805–810 (2005).

    Article  PubMed  CAS  Google Scholar 

  86. C. M. Dieckhaus, C. L. Fernandez-Metzler, R. King, P. H. Krolikowski, and T. A. Baillie. Negative ion tandem mass spectrometry for the detection of glutathione conjugates. Chem. Res. Toxicol. 18:630–638 (2005).

    Article  PubMed  CAS  Google Scholar 

  87. R. S. John, P. H. Shawn, Z. Sabrina, B. John, J. C. Mark, G. B. James, and G. C. Leonard. The development of a higher throughput reactive intermediate screening assay incorporating micro-bore liquid chromatography-micro-electrospray ionization-tandem mass spectrometry and glutathione ethyl ester as an in vitro conjugating agent. J. Pharm. Biomed. Anal. 36:105–116 (2004).

    Article  Google Scholar 

  88. Z. Yan and G. W. Caldwell. Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature. Anal. Chem. 76:6835–6847 (2004).

    Article  PubMed  CAS  Google Scholar 

  89. T. M. Baughman, R. A. Graham, K. Wells-Knecht, I. S. Silver, L. O. Tyler, M. Wells-Knecht, and Z. Zhao. Metabolic activation of pioglitazone identified from rat and human liver microsomes and freshly isolated hepatocytes. Drug Metab. Dispos. 33:733–738 (2005).

    Article  PubMed  CAS  Google Scholar 

  90. Z. Yan, J. Li, N. Huebert, G. W. Caldwell, Y. Du, and H. Zhong. Detection of a novel reactive metabolite of diclofenac: evidence for CYP2C9-mediated bioactivation via arene oxides. Drug Metab. Dispos. 33:706–713 (2005).

    Article  PubMed  CAS  Google Scholar 

  91. D. Argoti, L. Liang, A. Conteh, L. Chen, D. Bershas, C. P. Yu, P. Vouros, and E. Yang. Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Chem. Res. Toxicol. 18:1537–1544 (2005).

    Article  PubMed  CAS  Google Scholar 

  92. S. Bolze. In vitro screening assay of the reactivity of acyl glucuronides. In Z. Yan and G. W. Caldwell (eds.), Optimization Drug Discovery, Humana, Totowa, New Jersey, 2004, pp. 385_404.

    Chapter  Google Scholar 

  93. J. Q. Dong, J. Liu, and P. C. Smith. Role of benoxaprofen and flunoxaprofen acyl glucuronides in covalent binding to rat plasma and liver proteins in vivo. Biochem. Pharmacol. 70:937–948 (2005).

    Article  PubMed  CAS  Google Scholar 

  94. A. G. Siraki, T. Chevaldina, and P. J. O’Brien. Application of quantitative structure-toxicity relationships for acute NSAID cytotoxicity in rat hepatocytes. Chem.-Biol. Interact. 151:177–191 (2005).

    Article  PubMed  CAS  Google Scholar 

  95. S. J. Vanderhoeven, J. Troke, G. E. Tranter, I. D. Wilson, J. K. Nicholson, and J. C. Lindon. Nuclear magnetic resonance (NMR) and quantitative structure-activity relationship (QSAR) studies on the transacylation reactivity of model 1b-O-acyl glucuronides. II: QSAR modeling of the reaction using both computational and experimental NMR parameters. Xenobiotica 34:889–900 (2004).

    Article  PubMed  CAS  Google Scholar 

  96. S. Wainhaus. Acyl glucuronides: assays and issues. In W. A. Korfmacher (ed.), Using Mass Spectrometry for Drug Metabolism Studies, CRC, Boca Raton, FL, 2005, pp. 175_202.

    Google Scholar 

  97. J. Wang, M. Davis, F. Li, F. Azam, J. Scatina, and R. Talaat. A novel approach for predicting acyl glucuronide reactivity via Schiff Base Formation: development of rapidly formed peptide adducts for LC/MS/MS measurements. Chem. Res. Toxicol. 17:1206–1216 (2004).

    Article  PubMed  CAS  Google Scholar 

  98. S. H. Day, A. Mao, R. White, T. Schulz-Utermoehl, R. Miller, and M. G. Beconi. A semi-automated method for measuring the potential for protein covalent binding in drug discovery. J. Pharmacol. Toxicol. Methods 52:278–285 (2005).

    Article  PubMed  CAS  Google Scholar 

  99. J. Gan, T. W. Harper, M. M. Hsueh, Q. Qu, and W. G. Humphreys. Dansyl glutathione as a trapping agent for the quantitative estimation and identification of reactive metabolites. Chem. Res. Toxicol. 18:896–903 (2005).

    Article  PubMed  CAS  Google Scholar 

  100. J. Gan. Fluorescently labeled thiol-containing trapping agent for the quantitation and identification of reactive metabolites in vitro. (USA). 2005-31292[2005186651], 6-20050825. US. 1-7-2005. Ref Type: Patent.

  101. S. Miao, J. Ma, and R. Cho. Formation of NADP+ adducts as a amajor rat/human hepatic microsomal pathway for a novel class of IKKb inhibitors containing thienopyridine. Drug Metab. Rev. 37:155 (2005).

    Google Scholar 

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Acknowledgment

The authors thank Mike Brandl for his helpful suggestions.

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  1. Drug Metabolism and Pharmacokinetics, Roche Palo Alto, 3431 Hillview Ave., Palo Alto, California, 94304, USA

    Yuan Chen, Mario Monshouwer & William L. Fitch

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  1. Yuan Chen
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  2. Mario Monshouwer
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  3. William L. Fitch
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Correspondence to William L. Fitch.

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Chen, Y., Monshouwer, M. & Fitch, W.L. Analytical Tools and Approaches for Metabolite Identification in Early Drug Discovery. Pharm Res 24, 248–257 (2007). https://doi.org/10.1007/s11095-006-9162-7

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  • DOI: https://doi.org/10.1007/s11095-006-9162-7

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