Determination of glutathione and glutathione disulfide in human whole blood using HPLC with coulometric detection: A comparison with fluorescence detection

• Published in: Collection of Czechoslovak chemical communications Vol. 76; no. 4; pp. 277 - 294
• Main Authors: Kanďár, Roman, Žáková, Pavla, Marková, Miroslava, Lotková, Halka, Kučera, Otto, Červinková, Zuzana
• Format: Journal Article
• Language: English
• Published: Prague Blackwell Publishing Ltd 01-01-2011
• Subjects: Analytical chemistry; Chemistry; Chromatography; Methods; Molecular weight; Oxidative stress
• ISSN: 1212-6950; 1212-6950; 2192-6506
• DOI: 10.1135/cccc2010038

We describe a relatively simple method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human whole blood. We have used an HPLC with coulometric electrochemical detection for the simultaneous measurement of GSH and GSSG. Diluted and filtered trichloroacetic acid extracts were injected directly into the HPLC system and were eluted isocratically on a Polaris 5u C18-A, 250 × 4.6 mm analytical column. Glutathione in samples extracted with trichloroacetic acid and diluted with 1.0 m
M
hydrochloric acid was stable at 4 °C for at least 8 h. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked whole blood samples were at intervals 91.6–97.6% for GSH and 85.0–104.4% for GSSG. The linear range is 5.0–2000.0 μmol/l, with a detection limit of 2.1 μmol/l (signal-to-noise ratio = 3) for GSH and 2.0–250.0 μmol/l, with a detection limit of 0.9 μmol/l for GSSG.