Collect. Czech. Chem. Commun.
2011, 76, 277-294
https://doi.org/10.1135/cccc2010038
Published online 2011-03-15 10:59:19
Determination of glutathione and glutathione disulfide in human whole blood using HPLC with coulometric detection: A comparison with fluorescence detection
Roman Kanďára,*, Pavla Žákováa, Miroslava Markováa, Halka Lotkováb, Otto Kučerab and Zuzana Červinkováb
a Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, 532 10 Pardubice, Czech Republic
b Department of Physiology, Faculty of Medicine in Hradec Králové, Charles University in Prague, 500 38 Hradec Králové, Czech Republic
Abstract
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 mM 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.
Keywords: Glutathione; Glutathione disulfide; Glutathione stability; Liquid chromatography; Coulometric electrochemical and fluorescence detection.
References: 43 live references.
