Separation and determination of warfarin enantiomers in human plasma using a novel polymeric surfactant for micellar electrokinetic chromatography–mass spectrometry

Abstract

Warfarin is a widely used oral anticoagulant which is mostly administrated as a racemic mixture containing equal amount of R- and S-enantiomers. The two enantiomers are shown to exhibit significant differences in pharmacokinetics and pharmacodynamics. In this study, a new chiral micellar electrokinetic chromatography–mass spectrometry (MEKC–MS) method has been developed using a polymeric chiral surfactant, polysodium N-undecenoyl- l,l-leucyl-valinate (poly- l,l-SULV), as a pseudostationary phase for the chiral separation of (±)-warfarin (WAR) and (±)-coumachlor (COU, internal standard). Under optimum MEKC–MS conditions, the enantio-separation of both (±)-WAR and (±)-COU was achieved within 23 min. Calibration curves were linear ( R = 0.995 for ( R)-WAR and R = 0.989 for ( S)-WAR) over the concentration range 0.25–5.0 μg/mL. The MS detection was found to be superior over the commonly used UV detection in terms of selectivity and sensitivity with LOD as low as 0.1 μg/mL in human plasma. The method was successfully applied to determine WAR enantiomeric ratio in patients’ plasma undergoing warfarin therapy.