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

Author:
Kamińska, Adrianna, Olejarz, Patrycja, Borowczyk, Kamila, Głowacki, Rafał, Chwatko, Grażyna
Source:
Journal of separation science 2018 v.41 no.16 pp. 3241-3249
ISSN:
1615-9306
Subject:
acetylcysteine, brain, cysteine, derivatization, detection limit, disulfide bonds, glutathione, homocysteine, phosphine, reversed-phase high performance liquid chromatography, statistical analysis, swine, thiols
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 10 min. Linearity was observed in the range of 10–300, 0.7–10, 2–30, and 3–20 μmol/L homogenate with a limit of detection of 3.7, 0.2, 0.8, and 1.2 μmol/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.
Agid:
6095408