Determination of drug–protein interactions by microdialysis coupled with liquid chromatography and electrochemical detection based on a nickel hexacyanoferrate modified electrode

Abstract

Microdialysis sampling coupled with liquid chromatography–electrochemical detection (LC-ED) was developed and applied to determine the interaction of thiopurine (TP) with bovine serum albumin (BSA). A nickel hexacyanoferrate (NiHCNFe) modified electrode was fabricated and used as the working electrode in LC-ED for the determination of TP. Cyclic voltammetric experiments demonstrate that this chemically modified electrode (CME) can effectively catalyze the electrooxidation of TP. The mechanism of the catalytic oxidation of TP at the CME involves an ‘EC’ process. In the LC-ED, the CME also shows good stability and reproducibility for the determination of TP. The limit of detection is 5.0 × 10⁻⁷ mol l⁻¹ for 6-TP with a signal-to-noise ratio of 3. The utility of microdialysis as a quantitative sampling technique for in vitro studies of drug–protein interactions was studied. The microdialysis experiments were performed in phosphate buffer solution (pH 7.4) containing different molar ratios of the drug and protein at 37 °C. The collected microdialysis sample with unbound TP was analyzed at the NiHCNFe CME in the LC-ED. The relative recovery of TP determined in vitro is 18.3% at a perfusion rate of 1.0 μl min⁻¹ and the RSD is about 2.1%. The association constant and the number of binding sites on a BSA molecule calculated with the Scatchard equation are 3.72 × 10⁻³ (l mol⁻¹) and 1.51, respectively. This method provides a fast, sensitive and simple technique for the study of drug–protein interactions.