Flow injection on-line oxidizing fluorometry coupled to dialysis sampling for the study of carbamazepine–protein binding

Abstract

The mechanism of binding of carbamazepine (CBZ) with bovine serum albumin (BSA) has been investigated in vitro based on a new flow injection fluorometry coupled to the technique of dialysis sampling. The CBZ and BSA were mixed in different molar ratios in 0.050 mol L −1 phosphate buffer (containing 0.9% NaCl), pH 7.4, and incubated at 37 ± 0.5 °C in a water bath. The dialysis sampler was utilized to sample free CBZ from mixed solution with a relative dialytic efficiency of 7.6%. Then the CBZ in dialysis solution was injected into carrier and on-line oxidized by lead dioxide solid-phase reactor into fluorescent product with a maximum excitation wavelength of 355 nm and a maximum emission wavelength of 478 nm. The fluorescence intensity measured was linear proportional with the concentration of free CBZ in mixed solution over the range of 1 × 10 −5 to 2 × 10 −4 mol L −1 with the detection limit of 6 × 10 −6 mol L −1. According to the fluorescence measurement results from mixed solution, the association constant ( K) estimated for CBZ–BSA binding and the number of the binding site ( n) with Scatchard analysis were 1.08 × 10 4 L mol −1 and 0.94, respectively. Stern–Volmer plots indicated the presence of dynamic component in the quenching mechanism. The acting force was suggested to be mainly hydrophobic and the distance between the acceptor and donor was 3.12 nm. The estimated binding parameters agreed well with literature values.