A simultaneous liquid chromatography/mass spectrometric assay of glutathione, cysteine, homocysteine and their disulfides in biological samples

Abstract

A liquid chromatography/mass spectrometric (LC/MS) method was developed for simultaneous detection and quantitation of glutathione (GSH), glutathione disulfide (GSSG), cysteine (CysSH), homocysteine (HCysSH) and homocystine in biological samples (rat brain, lung, liver, heart, kidneys, erythrocytes and plasma). Thiols were derivatized with a large excess of Ellman's reagent, a thiol-specific reagent, to ensure an instantaneous and complete derivatization. The derivatization blocked the oxidation of the thiols to disulfides, preventing errors caused by thiol oxidation. The samples were then analyzed by LC/MS. The method provides a highly selective and sensitive assay for these endogenous thiols and their corresponding disulfides. The detection limits for GSH, GSSG, CysSH, HCysSH and homocystine were 3.3, 3.3, 16.5, 29.6 and 14.9 pmol, respectively. An attempt for cystine analysis was unsuccessful due to earlier elution of the compound and strong interferences caused by other endogenous compounds. This method will be a useful tool in the investigation of the roles of these important thiol-containing compounds and their corresponding disulfides in physiological and pathological processes.