Novel multi-mode ultra performance liquid chromatography–tandem mass spectrometry assay for profiling enantiomeric hydroxywarfarins and warfarin in human plasma

Coumadin (R/S-warfarin) is a commonly prescribed anticoagulant for over ∼20 million Americans. Although highly efficacious, positive clinical outcomes during warfarin therapy depend on maintaining a narrow therapeutic range for the drug. This goal is challenging due to large inter-individual variability in patient response, which has been attributed to diversity in drug metabolism. Warfarin is given as a racemic mixture and evidence suggest differences of R and S-warfarin in their therapeutic activities and metabolism. Previous investigation of warfarin metabolism has been hampered by the inability to quantify the individual enantiomers. To overcome this limitation a multi-mode LC–MS/MS method is reported. This strategy combines phenyl based reverse phase chromatography with chiral phase chromatography prior to quantitation by liquid chromatography tandem mass spectrometry. This approach was made possible through advances in UPLC technology producing narrow peaks suitable for transferring to a second column. The reported method separated individual R and S enantiomers of hydroxywarfarin and warfarin. All four possible isomers of 10-hydroxywarfarin were resolved to reveal unprecedented insights into the stereo-specific metabolism of warfarin. Characterization of the method demonstrated that it is robust and sensitive with inter-day coefficients of error between <7% and a detection limit of 2 nM in sample or 10 fmol on column for each analyte. Individual metabolites may be suitable surrogate biomarkers or predictive markers that predict warfarin dose, adverse interactions, or other important clinical outcomes during anticoagulant therapy. Consequently, the metabolite profiles obtained through this dual phase UPLC–MS/MS method are expected to increase our understanding of the role warfarin metabolism plays in patient response to therapy and yield new strategies to improve patient outcomes.