We use cookies to improve your experience. By continuing to browse this site, you accept our cookie policy.×
Research Article

An improved LC–MS/MS method for simultaneous evaluation of CYP2C9, CYP2C19, CYP2D6 and CYP3A4 activity

    Santosh Kumar Sreevatsav Adiraju

    Centre for Integrated Preclinical Drug Development, School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia

    ,
    Kiran Shekar

    Critical Care Research Group, The Prince Charles Hospital & The University of Queensland, Brisbane, Queensland, Australia

    ,
    John F Fraser

    Critical Care Research Group, The Prince Charles Hospital & The University of Queensland, Brisbane, Queensland, Australia

    ,
    Maree T Smith

    Centre for Integrated Preclinical Drug Development, School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia

    School of Pharmacy, Faculty of Health & Behavioural Sciences, The University of Queensland, Brisbane, Queensland, Australia

    &
    Sussan Ghassabian

    *Author for correspondence: Tel.: +61 0 7 3346 5194, Fax: +61 0 7 3365 5444;

    E-mail Address: susan.ghassabian@gmail.com

    Centre for Integrated Preclinical Drug Development, School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia

    Published Online:https://doi.org/10.4155/bio-2018-0102

    Aim: To develop an LC–MS/MS assay to quantitate well-tolerated substrates; midazolam (CYP3A), omeprazole (CYP2C19), dextromethorphan (CYP2D6), losartan (CYP2C9) and their respective metabolites’ concentrations in plasma samples. Patients & methods: A solid-phase extraction method was optimized to extract analytes of interest simultaneously from human plasma samples. The assay analyzed plasma samples collected from patients who received equal or lower than therapeutic doses of CYP substrates. Results: This assay was validated based on the European Medicines Agency guideline for bioanalytical method validation and was sensitive, linear, accurate and precise with acceptable recovery and matrix effects. Conclusion: Small sample volume and dose of cytochrome P450 substrates, short-run time, using stable isotope internal standards and being cost effective are the major advantages of the assay.

    Papers of special note have been highlighted as: • of interest; •• of considerable interest

    References

    • 1 Fuhr U, Jetter A, Kirchheiner J. Appropriate phenotyping procedures for drug metabolizing enzymes and transporters in humans and their simultaneous use in the ‘cocktail’ approach. Clin. Pharmacol. Ther. 81(2), 270–283 (2007). • Discusses the basics and requirements of cytochrome P450 (CYP) phenotyping and different substrates for each of the CYP isoenzymes. It also discusses the advantages and problems of the cocktail approach and a summary of all CYP phenotyping cocktails that have been published until 2007.
    • 2 Christensen M, Andersson K, Dalen P et al. The Karolinska cocktail for phenotyping of five human cytochrome P450 enzymes. Clin. Pharmacol. Ther. 73(6), 517–528 (2003).
    • 3 Ghassabian S, Chetty M, Tattam BN et al. A high-throughput assay using liquid chromatography–tandem mass spectrometry for simultaneous in vivo phenotyping of 5 major cytochrome p450 enzymes in patients. Ther. Drug Monit. 31(2), 239–246 (2009).
    • 4 de Andres F, Sosa-Macias M, Llerena A. A rapid and simple LC–MS/MS method for the simultaneous evaluation of CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 hydroxylation capacity. Bioanalysis 6(5), 683–696 (2014).
    • 5 Bosilkovska M, Samer CF, Deglon J et al. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots. Clin. Pharmacol. Ther. 96(3), 349–359 (2014).
    • 6 Ryu JY, Song IS, Sumwoo YE et al. Development of the ‘Inje cocktail’ for high-throughput evaluation of five human cytochrome P450 isoforms in vivo. Clin. Pharmacol. Ther. 82(5), 531–540 (2007). •• Used the five substrates for the major CYP isoenzymes for the first time and they showed that there is no interaction among those substrates, and the substrates possessed produce low adverse effects and are specific for each of the CYP enzymes of interest.
    • 7 Tanaka S, Uchida S, Inui N et al. 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(1), 18–25 (2014).
    • 8 Oh KS, Park SJ, Shinde DD et al. High-sensitivity liquid chromatography–tandem mass spectrometry for the simultaneous determination of five drugs and their cytochrome P450-specific probe metabolites in human plasma. J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 895–896, 56–64 (2012).
    • 9 Puris E, Pasanen M, Gynther M et al. A liquid chromatography–tandem mass spectrometry analysis of nine cytochrome P450 probe drugs and their corresponding metabolites in human serum and urine. Anal. Bioanal. Chem. 409(1), 251–268 (2017).
    • 10 Tanaka E, Kurata N, Yasuhara H. How useful is the ‘cocktail approach’ for evaluating human hepatic drug metabolizing capacity using cytochrome P450 phenotyping probes in vivo? J. Clin. Pharm. Ther. 28(3), 157–6165 (2003).
    • 11 de Andres F, Teran S, Bovera M et al. Multiplex phenotyping for systems medicine: a one-point optimized practical sampling strategy for simultaneous estimation of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 activities using a cocktail approach. Integr. Biol. 20(2), 88–96 (2016). • Promoted the use of a one-point sampling strategy for CYP phenotyping that will be ideal.
    • 12 Donzelli M, Derungs A, Serratore MG et al. The basel cocktail for simultaneous phenotyping of human cytochrome P450 isoforms in plasma, saliva and dried blood spots. Clin. Pharmacokinet. 53(3), 271–282 (2014).
    • 13 EMA. Guidance on Bioanalytical Method Validation (2011). http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf.
    • 14 Petsalo A, Turpeinen M, Pelkonen O et al. Analysis of nine drugs and their cytochrome P450-specific probe metabolites from urine by liquid chromatography–tandem mass spectrometry utilizing sub 2 microm particle size column. J. Chromatogr. A 1215(1-2), 107–115 (2008).
    • 15 Chen R, Zheng X, Hu P. CYP2D6 phenotyping uisng urine, plasma, and saliva metabolic ratios to assess the impact of CYP2D6*10 on interindividual variation in a chineses population. Front Pharmacol. 8, 239 (2017). •• The authors found a significant correlation between urinary magnetic resonance and plasma magnetic resonance (Spearman r = 0.78). This finding makes using plasma instead of the more cumbersome urine samples justified.
    • 16 Hennion MC. Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography. J. Chromatogr. A. 856(1–2), 3–54 (1999).
    • 17 Wang S, Cyronak M, Yang E. Does a stable isotopically labeled internal standard always correct analyte response? A matrix effect study on a LC/MS/MS method for the determination of carvedilol enantiomers in human plasma. J. Pharm. Biomed. Anal. 43(2), 701–707 (2007).
    • 18 Kumar A, Mann HJ, Remmel RP. Simultaneous analysis of cytochrome P450 probes-dextromethorphan, flurbiprofen and midazolam and their major metabolites by HPLC-mass-spectrometry/fluorescence after single-step extraction from plasma. J. Chromatogr. B. Analyt. Technol. Biomed. Life. Sci. 853(1–2), 287–293 (2007).
    • 19 Yin OQ, Lam SS, Lo CM et al. 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. Commun. Mass. Spectrom. 18(23), 2921–2933 (2004).
    • 20 Chainuvati S, Nafziger AN, Leeder JS et al. Combined phenotypic assessment of cytochrome p450 1A2, 2C9, 2C19, 2D6, and 3A, N-acetyltransferase-2, and xanthine oxidase activities with the ‘Cooperstown 5 + 1 cocktail’. Clin. Pharmacol. Ther. 74(5), 437–447 (2003). • Addresses the basics of the CYP phenotyping cocktail approach that was been used by many other researchers.