A Chiral HPLC-MS/MS Method for Simultaneous Quantification of Warfarin Enantiomers and its Major Hydroxylation Metabolites of CYP2C9 and CYP3A4 in Human Plasma

Warfarin is an oral anticoagulant that requires frequent therapeutic drug monitoring due to a narrow therapeutic window, considerable inters individual variability in drug response, and susceptibility to drug-drug and drug-diet interactions. Enantiomeric separation and quantification of warfarin enantiomers and clinically important major hydroxylation metabolites are essential for drug interaction studies and phenotypic characterization of CYP2C9 and CYP3A4, the major Cytochrome P450 (CYP) enzymes involved in warfarin metabolism. Here, we describe the development and validation of a chiral high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS)-based quantification of R-warfarin, S-warfarin, S-7-hydroxywarfarin (the major CYP2C9metabolite) and (9R; 10S)-10-hydroxywarfarin (the CYP3A4 metabolite) in human plasma. Simple protein precipitation-based extraction showed good recovery of analyses (82.9 - 96.9%). The developed method exhibited satisfactory intra-day and inter-day accuracy and precision. The lower limits of detection were 0.25nM (or ~0.08 ng/ml) for the war far in enantiomers and 0.1nM (or ~0.04 ng/mL) for S-7-hydroxywarfarinand (9R; 10S)-10- hydroxywarfarin using only 50μL plasma during extraction. The validated method was successfully applied to analyze plasma samples obtained from a healthy human subject who enrolled in a clinical drug interaction study involving warfarin.