DETERMINATION OF DRUG-PROTEIN INTERACTIONS BY COMBINED MICRODIALYSIS AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A simple and rapid method for determination of the parameters of the i nteraction between drugs and protein, including the association consta nt and the number of binding sites, has been developed by use of a mic rodialysis sampling technique combined with highperformance liquid chr omatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazep ine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as exa mples) were mixed in different molar ratios in 0.067 M potassium phosp hate buffer, pH 7.4, and incubated at 37 degrees C in a water-bath. Th e microdialysis probe was then used to sample the mixed CBZ-HSA soluti on at a perfusion rate of 1 mu L min(-1). The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance l iquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; the RSD of R was approxi mately 1.85%. The estimated association constant (K) and the number of the binding sites, n, on one molecule of HSA were 1.06x10(4) M(-1) an d 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.