Binding of sulfamethoxazole to human serum albumin studied by a combined technique of microdialysis with liquid chromatography

A new, simple and fast method for the determination of the interaction parameters of sulfamethoxazole (SMZ) to human serum albumin (HSA) has been developed by utilizing a microdialysis sampling technique combined with liquid chromatography (LC). The drug and protein 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. Then the microdialysis probe was put in the SMZ-HSA solution and sampled at a perfusion rate of 1 μl/min. The concentration of SMZ in the microdialysates was determined by reversed-phase liquid chromatography. The recovery (R) was also determined in vitro under similar conditions, R is about 41.8% with an RSD of about 2.3%. The association constant (K) and the number of the binding sites on one HSA molecule (n) are calculated by three equations, the values of nK estimated by three methods are quite similar. The values found for K and n are 3.24 × 103 M−1 and 3.04, respectively.