Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies.
A sensitive assay using high-performance liquid chromatography tandem mass spectrometry (MS/MS) has been established for the quantitative analysis of cytochrome P450 form-specific activities using warfarin as a probe substrate. Four metabolites, 6-, 7-, 8-, and 10-hydroxywarfarin, were chromatographically resolved within 10 min using gradient mobile phases. The mass spectrometry was operated under negative ionization mode. The MS/MS product ion spectra of warfarin and the metabolites were generated using collision-activated dissociation and interpreted. The abundant product ions of the metabolites were selected for quantification applying multiple reaction monitoring. Quantification was based on a quadratic or power curve of the peak area ratio of the metabolite over the internal standard against the respective concentration of the metabolite. This assay has been validated from 2 to 1000 nM for 10-hydroxywarfarin and from 2 to 5000 nM for 6-, 7-, and 8-hydroxywarfarin and successfully applied to evaluate cytochrome P450-mediated drug-drug interactions in vitro using human hepatocytes and liver microsomal preparations.
Zhang
Z Y
ZY
Department of Pharmacokinetics and Drug Metabolism, Eisai Research Institute, Wilmington, Massachusetts 01887, USA. zhi-yi_zhang@eri.eisai.com
King
B M
BM
Wong
Y N
YN
D002138:Calibration / Q000592:standards; D002853:Chromatography, Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000187:drug effects / Q000378:metabolism*; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans; D007654:Ketoconazole / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000201:enzymology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D013237:Stereoisomerism; D014859:Warfarin / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D000072317:Polychlorinated Dibenzodioxins; D014859:Warfarin; C065719:10-hydroxywarfarin; D003577:Cytochrome P-450 Enzyme System; D007654:Ketoconazole