Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies

Anal Biochem. 2001 Nov 1;298(1):40-9. doi: 10.1006/abio.2001.5383.


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.

Publication types

  • Comparative Study

MeSH terms

  • Calibration / standards
  • Chromatography, Liquid / methods*
  • Cytochrome P-450 Enzyme System / drug effects
  • Cytochrome P-450 Enzyme System / metabolism*
  • Hepatocytes / enzymology
  • Humans
  • Ketoconazole / pharmacology
  • Mass Spectrometry / methods*
  • Microsomes, Liver / enzymology
  • Polychlorinated Dibenzodioxins / pharmacology
  • Stereoisomerism
  • Warfarin / analogs & derivatives*
  • Warfarin / analysis*
  • Warfarin / chemistry*
  • Warfarin / metabolism


  • Polychlorinated Dibenzodioxins
  • Warfarin
  • 10-hydroxywarfarin
  • Cytochrome P-450 Enzyme System
  • Ketoconazole