Comparison of enzyme kinetics of warfarin analyzed by LC–MS/MS QTrap and differential mobility spectrometry

•This work is the first to separate all the six hydroxylated metabolites of warfarin including 3′,4′,6,7,8,10-hydroxy warfarin, and the parent in single run.•This manuscript reports low limit of quantitation of 1nM for all analytes, previously reported LLOQs were significantly higher.•This manuscript reports enzyme kinetics for the formation of 3′,4′,6,7,8,10-hydroxy warfarin from racemic warfarin, previously kinetics of only major metabolites was reported.•This manuscript reports use of differential mobility spectrometry for increasing specificity, reducing run time to separate similar compounds using ion mobility.•This manuscript compares the choice between specificity and sensitivity using different technologies available, i.e., QTrap and SelexIon and how different technologies are compound specific. Warfarin is an anticoagulant used in the treatment of thrombosis and thromboembolism. It is given as a racemic mixture of R and S enantiomers. These two enantiomers show differences in metabolism by CYPs: S-warfarin undergoes 7 hydroxylation by CYP2C9 and R-warfarin by CYP3A4 to form 10 hydroxy warfarin. In addition, warfarin is acted upon by different CYPs to form the minor metabolites 3′-hydroxy, 4′-hydroxy, 6-hydroxy, and 8-hydroxy warfarin. For analysis, separation of these metabolites is necessary since all have the same m/z ratio and similar fragmentation pattern. Enzyme kinetics for the formation of all of the six hydroxylated metabolites of warfarin from human liver microsomes were determined using an LC–MS/MS QTrap and LC–MS/MS with a differential mobility spectrometry (DMS) (SelexION™) interface to compare the kinetic parameters. These two methods were chosen to compare their selectivity and sensitivity. Substrate curves for 3′-OH, 4′-OH, 6-OH, 7-OH, 8-OH and 10-OH warfarin formation were generated to determine the kinetic parameters (Km and Vmax) in human liver microsomal preparations. The limit of quantitation (LOQ) for all the six hydroxylated metabolites of warfarin were in the range of 1–3nM using an LC–MS/MS QTrap method which had a run time of 22min. In contrast, the LOQ for all the six hydroxylated metabolites using DMS interface technology was 100nM with a run time of 2.8min. We compare these two MS methods and discuss the kinetics of metabolite formation for the metabolites generated from racemic warfarin. In addition, we show inhibition of major metabolic pathways of warfarin by sulfaphenazole and ketoconazole which are known specific i