Quantitative study of competitive binding of drugs to protein by microdialysis/high-performance liquid chromatography.

A displacement equation describing competitive binding of drugs to protein in solution is derived and examined with four nonsteroidal anti-inflammatory drugs and human serum albumin as model drugs and protein, respectively. Microdialysis/high-performance liquid chromatography was adopted to determine simultaneously the unbound solute and displacing agent in drug-protein solutions. The method is able to locate the binding site and determine affinity constants even up to 10(7) L/mol accurately. A comparison of association constants determined by this method and from capacity factors on HSA-CSP is given.