PROTEIN-BINDING HIGH-PERFORMANCE FRONTAL ANALYSIS OF (R)-WARFARIN AND(S)-WARFARIN ON HSA WITH AND WITHOUT PHENYLBUTAZONE

Applicability of high-performance frontal analysis (HPFA) to the stere oselective study of drug-drug interaction upon plasma protein binding has been investigated. Racemic warfarin and phenylbutazone were used a s model drugs. An on-line HPFA/HPLC system consisting of a HPFA column (diol-silica column), an extraction column, and a chiral separation c olumn was developed, and human serum albumin solution containing racem ic warfarin and/or phenylbutazone was injected directly to the HPFA co lumn, When the injection volume das large enough, the binding equilibr ium in the sample solution was reproduced in the column, and consequen tly a plateau region appeared on the chromatogram. This plateau region contains unbound drug(s). A given volume of eluent in the plateau par t was transferred into the extraction column by column-switching. The concentrated drug(s) was then transferred to the chiral separation col umn to determine the unbound concentrations of the enantiomers and/or the competitor. The results agreed with those obtained by a convention al ultrafiltration-HPLC method. The influence of phenylbutazone upon t he protein binding of warfarin is enantioselective. in warfarin and hu man serum albumin mixed solution, the unbound concentration of (R)-war farin was 1.22 times higher than that of the S-isomer. By addition of phenylbutazone, the unbound concentration of (S)-warfarin increased mo re than that of (R)-warfarin, resulting in the reversed enantioselecti vity, i.e., the unbound concentration of (S)-warfarin became 1.19 time s larger than that of(R)-warfarin. The present method was also applica ble to human plasma samples.