Journal
JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
Volume 852, Issue 1-2, Pages 554-561Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jchromb.2007.02.024
Keywords
aminothiols; homocysteine; cysteine; cysteinylglycine; glutathione; oxidative stress
Funding
- NHLBI NIH HHS [R01 HL070938, R01 HL070938-03, R01 HL 070938] Funding Source: Medline
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A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobeDzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of ammothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients (r) > 0.99. The intra- and inter-day precision and accuracy were within 10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of antinothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease. (c) 2007 Elsevier B.V. All rights reserved.
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