Quantitative Liquid Chromatography/Mass Spectrometry/Mass Spectrometry Warfarin Assay for in Vitro Cytochrome P450 Studies

✍ Scribed by Zhi-Yi Zhang; Belinda M. King; Y.Nancy Wong

• Publisher: Elsevier Science
• Year: 2001
• Tongue: English
• Weight: 120 KB
• Volume: 298
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5383

✦ Synopsis

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 collisionactivated 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.