Simultaneous determination of total homocysteine, cysteine, glutathione, and N-acetylcysteine in brain homogenates by HPLC.

Abstract

We have developed a simple, fast, accurate, and cheap method for the simultaneous determination of total cysteine, homocysteine, glutathione, and N-acetylcysteine in brain homogenates based on the reduction of disulfide bonds by tris(2-carboxyethyl) phosphine, pre-column derivatization of free thiol groups with 2-chloro-1-methylquinolinium tetrafluoroborate followed by ion-pair reversed-phase high-performance liquid chromatography separation with ultraviolet detection. The separation of thiol derivatives was achieved in 10min. Linearity was observed in the range of 10-300, 0.7-10, 2-30, and 3-20μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2μmol/L homogenate for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The precision, calculated as relative standard deviation, was in the range of 1.21-4.77, 1.53-14.35, 0.47-1.92, and 1.61-8.95% for cysteine, homocysteine, glutathione, and N-acetylcysteine, respectively. The presented method was successfully applied to the selective determination of total amino thiols in pig brain tissue samples.