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Simultaneous determination of total homocysteine, cysteine, glutathione, and N ‐acetylcysteine in brain homogenates by HPLC
Author(s) -
Kamińska Adrianna,
Olejarz Patrycja,
Borowczyk Kamila,
Głowacki Rafał,
Chwatko Grażyna
Publication year - 2018
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201800381
Subject(s) - glutathione , chemistry , derivatization , chromatography , cysteine , high performance liquid chromatography , thiol , homocysteine , acetylcysteine , detection limit , quantitative analysis (chemistry) , biochemistry , antioxidant , enzyme
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.