Determination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies

A sensitive, accurate, and reproducible ultra-high performance liquid chromatography–tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10 mM Tris–HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid–liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1 mm × 100 mm, 1.9 μm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4 mL/min and the total run time was 5 min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100–10,000 ng/mL for warfarin, and 0.5–250 ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.