Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Abstract

A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min−1. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×104 M−1 and 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.