A SIMPLIFIED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC METHOD FOR DIRECT DETERMINATION OF WARFARIN ENANTIOMERS AND THEIR PROTEIN-BINDING IN STROKE PATIENTS

A simplified method for direct determination of warfarin enantiomers b y high-pressure liquid chromatography with fluorescence detection has been developed. This method involves solid phase extraction of warfari n in plasma, precolumn derivatization to form diastereoisomeric esters , and postcolumn reaction to discriminate each enantiomer separately. Ultrafiltration was employed in the separation of unbound warfarin ena ntiomers. Twelve plasma samples from six stroke patients taking warfar in regularly were analyzed. The average concentration of total warfari n was 0.47 +/- 0.17 mg/L for the S-isomer and 0.69 +/- 0.18 mg/L for t he R-isomer. The average protein binding was 99.67 +/- 0.33% for S-war farin and 99.44 +/- 0.33% for R-warfarin. This methodology provides a quick and reliable technique for determining enantiomeric protein bind ing of warfarin in clinical settings.