A Simplified High-Performance Liquid Chromatographic Method for Direct Determination of Warfarin Enantiomers and Their Protein Binding in Stroke Patients

Abstract

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