Skip to main content
SpringerLink
Log in

Metabolite Identification in Drug Discovery

  • Protocol
  • First Online:
Optimization in Drug Discovery

Part of the book series: Methods in Pharmacology and Toxicology ((MIPT))

Abstract

Early knowledge on the structures of metabolites from in vitro and in vivo metabolism studies is very useful for improving the biopharmaceutical, efficacy and safety properties of lead candidates in drug discovery. The recognition of the value in what metabolite identification brings to drug discovery led to its inclusion in ADMET toolbox and recent trend to multiplex assessment of metabolic stability and metabolite identification. In this chapter, we will cover the in vitro and in vivo systems typically used for metabolite identification. Fast LC-MS/MS with capability of data-dependent multiple-stage mass analysis is the instrument of choice and the workhorse for multiplexing assessment of metabolic stability and metabolite identification from a single analysis. Therefore, various LC-MS/MS instruments and techniques used for metabolite identification including software to speed up data-mining along with estimating major metabolites based on UV methods will be discussed. In general, the exact site of biotransformation is difficult to obtain based solely on MS data. As a result, microchemistry will also be discussed to help narrowing down site of modification in metabolites.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cheng KC, Korfmacher WA, White RE, Njoroge FG (2007) Lead optimization in discovery drug metabolism and pharmacokinetics/case study: the hepatitis C virus (HCV) protease inhibitor SCH 503034. Perspect Medicin Chem 1:1–9

    PubMed  Google Scholar 

  2. Kalgutkar AS, Dalvie D, Obach RS, Smith DA (2012) Role of reactive metabolites in drug-induced toxicity—the tale of acetaminophen, halothane, hydralazine, and tienilic acid. In: Reactive drug metabolites. Eds. Mannhold R, Kubinyi H, and Folkers G. Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

    Google Scholar 

  3. Zhang Z, Zhu M, Tang W (2009) Metabolite identification and profiling in drug design: current practice and future directions. Curr Pharm Des 15:2220–2235

    Article  PubMed  CAS  Google Scholar 

  4. Working with hepatocytes in suspension (Video tutorials included 1) Thawing cryosuspension hepatocytes; 2) Cell counting using the Trypan Blue exclusion method and 3) Reconstitution of cryosuspension hepatocytes. http://www.celsisivt.com/working-with-hepatocytes-in-suspension from Celsis/In Vitro Technology

  5. Yan Z, Caldwell GW, Maher N (2008) Unbiased high-throughput screening of reactive metabolites on the linear ion trap mass spectrometer using polarity switch and mass tag triggered data-dependent acquisition. Anal Chem 80:6410–6422

    Google Scholar 

  6. Mutlib A, Lam W, Atherton J, Chen H, Galatsis P, Stolle W (2005) Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites. Rapid Commun Mass Spectrom 19:3482–3492

    Article  PubMed  CAS  Google Scholar 

  7. Bonn B, Leandersson C, Fontaine F, Zamora I (2010) Enhanced metabolite identification with MS(E) and a semi-automated software for structural elucidation. Rapid Commun Mass Spectrom 24:3127–3138

    Article  PubMed  CAS  Google Scholar 

  8. Ruijken MMA (2010) MsXelerator RM: a software platform for reactive metabolite detection using low and high resolution mass spectrometry data. The 58th American Society for Mass Spectrometry (ASMS). Salt Lake City, UT, 23–27 May

    Google Scholar 

  9. Actis-Goretta L, Lévèques A, Giuffrida F, Destaillats F, Nagy K (2012) Identification of O-methyl-(−)-epicatechin-O-sulphate metabolites by mass-spectrometry after O-methylation with trimethylsilyldiazomethane. J Chromatogr A 1245:150–157

    Article  PubMed  CAS  Google Scholar 

  10. Nagy K, Redeuil K, Williamson G, Rezzi S, Dionisi F, Longet K, Destaillats F, Renouf M (2011) First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry. J Chromatogr A 1218(2011):491

    Article  PubMed  CAS  Google Scholar 

  11. Lamoureux G, Aguero C (2009) A comparison of several modern alkylating agents. ARKIVOC i:251–264

    Article  Google Scholar 

  12. Kulanthaivel P, Barbuch RJ, Davidson RS, Yi P, Rener GA, Mattiuz EL, Hadden CE, Goodwin LA, Ehlhardt WJ (2004) Selective reduction of N-oxides to amines: application to drug metabolism. Drug Metab Dispos 32:966–972

    PubMed  CAS  Google Scholar 

  13. Chen G, Daaro I, Pramanik BN, Piwinski JJ (2009) Structural characterization of in vitro rat liver microsomal metabolites of antihistamine desloratadine using LTQ-Orbitrap hybrid mass spectrometer in combination with online hydrogen/deuterium exchange HR-LC/MS. J Mass Spectrom 44:203–213

    Article  PubMed  CAS  Google Scholar 

  14. Lam W, Ramanathan R (2002) In electrospray ionization source hydrogen/deuterium exchange LC–MS and LC–MS/MS for characterization of metabolites. J Am Soc Mass Spectrom 13:345–353

    Article  PubMed  CAS  Google Scholar 

  15. Tolonen A, Turpeinen M, Uusitalo J, Pelkonen O (2005) A simple method for differentiation of monoisotopic drug metabolites with hydrogen–deuterium exchange liquid chromatography/electrospray mass spectrometry. Eur J Pharm Sci 25:155–162

    Article  PubMed  CAS  Google Scholar 

  16. Peiris DM, Lam W, Michael S, Ramanathan R (2004) Distinguishing N-oxide and hydroxyl compounds: impact of heated capillary/heated ion transfer tube in inducing atmospheric pressure ionization source decompositions. J Mass Spectrom 39:600–606

    Article  PubMed  CAS  Google Scholar 

  17. Wang WW, Khetani SR, Krzyzewski S, David Duignan D, Obach RS (2010) Assessment of micropatterned hepatocyte co-culture system to generate metabolites. Drug Metab Dispos 38:1900–1905

    Article  PubMed  CAS  Google Scholar 

  18. Josephs JL, Sanders M (2005) Chapter 13: an integrated LC-MS strategy for preclinical candidate optimization. In: Lee MS (ed) Integrated strategies for drug discovery using mass spectrometry. Wiley, New York, p 379

    Chapter  Google Scholar 

  19. Yang Y, Grubb MF, Luk CE, Humphreys WG, Josephs JL (2011) Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction. Rapid Commun Mass Spectrom 25:3245–3251

    Article  PubMed  CAS  Google Scholar 

  20. Sanders M, Ruzicka J, McHale K, Shipkova P. Thermo application notes # 476: accurate and sensitive all-ions quantitation using ultra-high resolution LC/MS http://planetorbitrap.com/data/uploads/ZFS1327867271789_AN476.pdf

  21. Baillie TA, Davis MR (1993) Mass spectrometry in the analysis of glutathione conjugates. Biol Mass Spectrom 22:319–325

    Article  PubMed  CAS  Google Scholar 

  22. Lafaye A, Junot C, Gall BR, Fritsch P, Ezan E, Tabet J-C (2004) Profiling of sulfoconjugates in urine using precursor ion and neutral loss scans in tandem mass spectrometry. Application to the investigation of heavy metal toxicity in rats. J Mass Spectrom 39:655–664

    Article  PubMed  CAS  Google Scholar 

  23. Xia Y-Q, Miller JD, Bakhtiar R, Franklin RB, Liu DQ (2003) Use of a quadrupole linear ion trap mass spectrometer in metabolite identification and bioanalysis. Rapid Commun Mass Spectrom 17:1137–1145

    Article  PubMed  CAS  Google Scholar 

  24. Liu DQ, Hop CECA (2005) 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

    Article  PubMed  Google Scholar 

  25. Jackson PJ, Brownsill RD, Taylor AR, Walther B (1995) Use of electrospray ionization and neutral loss liquid chromatography/tandem mass spectrometry in drug metabolism studies. J Mass Spectrom 30:446–451

    Article  CAS  Google Scholar 

  26. Clarke NJ, Rindgen D, Korfmacher WA, Cox KA (2001) Systematic LC/MS metabolite identification in drug discovery. Anal Chem 73:430A–439A

    PubMed  CAS  Google Scholar 

  27. Kostiainen R, Kotiaho T, Kuuranne T, Auriola S (2003) Liquid chromatography/atmospheric pressure ionization-mass spectrometry in drug metabolism studies. J Mass Spectrom 38:357–372

    Article  PubMed  CAS  Google Scholar 

  28. Zhang H, Zhang D, Ray K (2003) A software filter to remove interference ions from drug metabolites in accurate mass liquid chromatography/mass spectrometric analyses. J Mass Spectrom 38:1110–1112

    Article  PubMed  CAS  Google Scholar 

  29. Zhang H, Zhang D, Ray K, Zhu M (2009) Mass defect filter technique and its applications to drug metabolite identification by high-resolution mass spectrometry. J Mass Spectrom 44:999–1016

    Article  PubMed  CAS  Google Scholar 

  30. Zhu M, Zhang H, Yao M, Zhang D, Ray K, Skiles GL (2004) Detection of metabolites in plasma and urine using a high resolution LC/MS-based mass defect filter approach: comparison with precursor ion and neutral loss scan analyses. Drug Metab Rev 36(Suppl 1):43

    Google Scholar 

  31. Mortishire-Smith RJ, O’Connor D, Castro-Perez JM, Kirby J (2005) 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

    Article  PubMed  CAS  Google Scholar 

  32. Lim HK, Chen J, Cook K, Sensenhauser C, Silva J, Evans DC (2008) A generic method to detect electrophilic intermediates using isotopic pattern triggered data-dependent high-resolution accurate mass spectrometry. Rapid Commun Mass Spectrom 22:1295–1311

    Article  PubMed  CAS  Google Scholar 

  33. Cuyckens F, Hurkmans R, Castro-Perez JM, Leclercq L, Mortishire-Smith RJ (2009) Extracting metabolite ions out of a matrix background by combined mass defect, neutral loss and isotope filtration. Rapid Commun Mass Spectrom 23:327–332

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The views expressed here are solely those of the author and do not reflect the opinions of Janssen Research & Development, LLC.

Author information

Authors and Affiliations

  1. Drug Safety Sciences, Drug Metabolism and Pharmacokinetics, Janssen Research & Development, LLC, Spring House, PA, USA

    Wing W. Lam, Jie Chen, Rongfang Fran Xu, Jose Silva & Heng-Keang Lim

Authors
  1. Wing W. Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jie Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rongfang Fran Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jose Silva
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heng-Keang Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Gary W. Caldwell

  2. CREATe Core Science Technologies, Janssen Research & Development, LLC, Spring House, Pennsylvania, USA

    Zhengyin Yan

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Lam, W.W., Chen, J., Xu, R.F., Silva, J., Lim, HK. (2014). Metabolite Identification in Drug Discovery. In: Caldwell, G., Yan, Z. (eds) Optimization in Drug Discovery. Methods in Pharmacology and Toxicology. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-742-6_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-742-6_26

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-741-9

  • Online ISBN: 978-1-62703-742-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation