HPLC simultaneous analysis of thiols and disulfides: on-line reduction and indirect fluorescence detection without derivatization

Abstract

A liquid chromatography method with indirect fluorescence detection has been developed for simultaneous detection of cysteine, cystine, homocysteine, homocystine, glutathione and glutathione disulfide. After separation in their native forms, a post-column solution of tris(2-carboxyethyl)phosphine (TCEP) at 32 mM, pH 8 is added to reduce the disulfides on-line to the corresponding thiol. The effluent is then merged with a second post-column solution of the highly fluorescent complex Cd(HQS)₂²⁻. The cadmium is complexed by the eluting thiols, effectively quenching the fluorescence. Optimization of the separation, the on-line reduction and the indirect fluorescence detection are discussed. Detection limits from 0.3–4.3 µM (0.04 to 2.6 ppm) are achieved for the six analytes in a 20 min separation.