A flow-injection ultrafiltration sampling chemiluminescence system for on-line determination of drug–protein interaction

A flow-injection ultrafiltration sampling chemiluminescence system for on-line determination of cimetidine–bovine serum albumin (BSA) interaction is proposed in this paper. Cimetidine can be oxidized by N-bromosuccinimide (NBS) and sensitized by fluorescein to produce high chemiluminescence emission in basic media. The concentration of cimetidine is linear with the CL intensity in the range 3×10−7–1×10−4 mol L−1 with a detection limit of 1×10−7 mol L−1 (3σ). The drug and protein were mixed in different molar ratios in 0.067 mol L−1 phosphate buffer, pH 7.4, and incubated at 37 °C in a water bath. The ultrafiltration probe was utilized to sample the mixed solution at a flow rate of 5 µL min−1. The data obtained by the proposed ultrafiltration flow-injection chemiluminescence method was analyzed with Scrathard analysis and a Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scrathard analysis and Klotz plot were 3.15×104 L mol−1 and 0.95, 3.25×104 L mol−1 and 0.92, respectively. The proposed system proved that flow-injection chemiluminescence analysis coupled with on-line ultrafiltration sampling is a simple and reliable technique for the study of drug–protein interaction.