Multi-site binding of fenoprofen to human serum albumin studied by a combined technique of microdialysis with high performance liquid chromatography

A simple and fast method for the determination of the multi-site binding of fenoprofen (FP) to human serum albumin (HSA) has been developed by utilizing microdialysis sampling techniques combined with high performance liquid chromatography (HPLC). The drug and protein were mixed in different molar ratios in 0.067 Mol potassium phosphate buffer, pH 7.4, and incubated at 37 degrees C in a water-bath. Then the microdialysis probe was put in the FP-HSA solution and sampled at the perfusion rate of 1 microL/min. The concentrations of FP in microdialysates were determined by the reversed-phase high performance liquid chromatography. Relative recovery (R) was also determined in vitro on similar condition, R is about 56.03 +/- 1.11% (n = 3). Fenoprofen was found to bind to two classes of sites, the association constant (K1) and the number of the binding sites on primary binding sites of a HSA molecule (n1) for fenoprofen are 3.4 x 10(5)/M and 2.5, respectively, and those for secondary binding are 1.0 x 10(4)/M and 10.0, respectively. The competitive interaction of ibuprofen (IP) and palmitic acid with fenoprofen to HSA were also studied, both compounds significantly decrease the binding degree of fenoprofen to HSA.