Skip to main content
SpringerLink
Log in

LC–MS–MS Method to Simultaneously Determine Six Probe Drugs for CYP450 Isozymes in Human Liver Microsomes

  • Original
  • Published:
Chromatographia Aims and scope Submit manuscript

    We’re sorry, something doesn't seem to be working properly.

    Please try refreshing the page. If that doesn't work, please contact support so we can address the problem.

Abstract

Here, we report a rapid and specific method based on high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) capable of quantifying six CYP450-specific probe substrates in human liver microsomal incubation mixtures simultaneously. These analytes were prepared by single-step extraction and detected in one run by switching polarity of electrospray ionization mode three times. Following optimization of the chromatographic conditions, the peaks were well separated, and retention times ranged between 2.0 and 8.4 min. The total run time for a single injection was within 9 min. This method was fully validated over linear range of 18.8–3,000.0 ng mL−1 for diclofenac, 0.8–3,000.0 ng mL−1 for dapson, 1.5–3,000.0 ng mL−1 for dextromethorphan, 2.0–4,000.0 ng mL−1 for omeprazole, 75.0–3,000.0 ng mL−1 for chlorzoxazone and 0.8–3,000.0 ng mL−1 for phenacetin using diazepam as internal standard. Samples were prepared by protein precipitation and analyzed on the LC–MS–MS equipped with ESI interface. For each analyte, inter- and intra-day precision (RSD%) were <15 % and accuracy was within 85–115 %. The specificity, precision, accuracy, stabilities and matrix effect were evaluated.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Lewis DF (2004) 57 Varieties: the human cytochromes P450. Pharmacogenomics 5:305–318

    Article  CAS  Google Scholar 

  2. Wrighton SA, Vardonbradon M, Ring BJ (1996) The human drug metabolizing cytochromes P450. J Pharmacokinet Biopharm 26:461–473

    Article  Google Scholar 

  3. Venkatakrishnan K, Von Moltke LL, Greenblatt DJ (2001) Human drug metabolism and the cytochromes P450: application and relevance of in vitro models. J Clin Pharmacol 41:1149–1179

    Article  CAS  Google Scholar 

  4. Zhang TY, Zhu YX, Gunaratna C (2003) Simultaneous determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography with UV detection. Curr Sep 20:87–91

    Google Scholar 

  5. Yin H, Racha J, Li SY, Olejnik N, Satoh H, Moore D (2000) Automated high throughput human CYP isoform activity assay using SPE-LC/MS method: application in CYP inhibition evaluation. Xenobiotica 30:141–154

    Article  CAS  Google Scholar 

  6. Bu HZ, Magis L, Knuth K, Teitelbaum P (2001) High-throughput cytochrome P450 (CYP) inhibition screening via a cassette probe-dosing strategy. VI. Simultaneous evaluation of inhibition potential of drugs on human hepatic isozymes CYP2A6, 3A4, 2C9, 2D6 and 2E1. Rapid Commun Mass Spectrom 15:741–748

    Article  CAS  Google Scholar 

  7. Bhoopathy S, Xin B, Unger SE, Karnes HT (2005) A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes. J Pharm Biomed Anal 37:739–749

    Article  CAS  Google Scholar 

  8. Bendriss EK, Markoglou N, Wainer IW (2001) High-performance liquid chromatography assay for simultaneous determination of dextromethorphan and its main metabolites in urine and in microsomal preparations. J Chromatogr B Biomed Sci Appl 754:209–215

    Article  CAS  Google Scholar 

  9. Scott RJ, Palmer J, Lewis IA, Pleasance S (1999) Determination of a ‘GW cocktail’ of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 13:2305–2319

    Article  CAS  Google Scholar 

  10. Tanaka S, Uchida S, Inui N, Takeuchi K, Watanabe H, Namiki N (2014) Simultaneous LC–MS/MS Analysis of the Plasma Concentrations of a Cocktail of 5 Cytochrome P450 Substrate Drugs and Their Metabolites. Biol Pharm Bull 37:18–25

    Article  CAS  Google Scholar 

  11. Zhang T, Zhu Y, Gunaratna C (2002) Rapid and quantitative determination of metabolites from multiple cytochrome P450 probe substrates by gradient liquid chromatography-electrospray ionization-ion trap mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 780:371–379

    Article  CAS  Google Scholar 

  12. Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H, Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450 s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29:23–29

    CAS  Google Scholar 

  13. Ayrton J, Plumb R, Leavens WJ, Mallett D, Dickins M, Dear GJ (1998) Application of a generic fast gradient liquid chromatography tandem mass spectrometry method for the analysis of cytochrome P450 probe substrates. Rapid Commun Mass Spectrom 12:217–224

    Article  CAS  Google Scholar 

  14. Bu HZ, Magis L, Knuth K, Teitelbaum P (2000) High-throughput cytochrome P450 (CYP) inhibition screening via cassette probe-dosing strategy. I. Development of direct injection/on-line guard cartridge extraction/tandem mass spectrometry for the simultaneous detection of CYP probe substrates and their metabolites. Rapid Commun Mass Spectrom 14:1619–1624

    Article  CAS  Google Scholar 

  15. Pillai VC, Strom SC, Caritis SN, Venkataramanan R (2013) A sensitive and specific CYP cocktail assay for the simultaneous assessment of human cytochrome P450 activities in primary cultures of human hepatocytes using LC–MS/MS. J Pharm Biomed Anal 74:126–132

    Article  CAS  Google Scholar 

  16. Yin OQ, Lam SS, Lo CM, Chow MS (2004) Rapid determination of five probe drugs and their metabolites in human plasma and urine by liquid chromatography/tandem mass spectrometry: application to cytochrome P450 phenotyping studies. Rapid Commu Mass Spectrom 18:2921–2933

    Article  CAS  Google Scholar 

  17. Raucy JL, Lasker JM (1991) Methods in enzymology. Academic Press, San Diego

    Google Scholar 

  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  19. Zeng S (2004) Drug Metabolism. Zhejiang University Publishing House, China

    Google Scholar 

  20. Tang J, Usmani KA, Hodgson E, Rose RL (2004) In vitro metabolism of fipronil by human and rat cytochrome P450 and its interactions with testosterone and diazepam. Chem Biol Interact 147:319–329

    Article  CAS  Google Scholar 

  21. Agrawala AK, Hopa CECA, Panga J, Desaib RC, Leunga KH, Franklina RB (2005) In vitro metabolism of a new oxazolidinedione hypoglycemic agent utilizing liver microsomes and recombinant human cytochrome P450 enzymes. J Pharm Biomed Anal 37:351–358

    Article  Google Scholar 

  22. Sams C, Loizou GD, Cocker J, Lennard MS (2004) Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP). Toxicol Lett 147:253–260

    Article  CAS  Google Scholar 

  23. Morin S, Loriot MA, Poirier JM, Tenneze L, Beaune PH, Funck-Brentano C, Jaillon P, Becquemont L (2001) Is diclofenac a valuable CYP2C9 probe in humans? Eur J Clin Pharmacol 56:793–797

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors deeply appreciated the contributions of the study team during the conduct of this study. This work was supported by a grant from the National Program on Key Research Project of New Drug Innovation (No. 2008ZX09312-016).

Author information

Authors and Affiliations

  1. Clinical Pharmacology Research Centre, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, China

    Qian Zhao, Xin Zheng, Ji Jiang & Pei Hu

  2. Pharmacology Department, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, China

    Yan Li & Jinping Hu

Authors
  1. Qian Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinping Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xin Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ji Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Pei Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Pei Hu.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, Q., Li, Y., Hu, J. et al. LC–MS–MS Method to Simultaneously Determine Six Probe Drugs for CYP450 Isozymes in Human Liver Microsomes. Chromatographia 77, 913–922 (2014). https://doi.org/10.1007/s10337-014-2695-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10337-014-2695-8

Keywords

Search

Navigation