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

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 microg/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 microg/mL in human plasma. The method was successfully applied to determine WAR enantiomeric ratio in patients' plasma undergoing warfarin therapy.