Determination of reduced and oxidized glutathione in biological samples using liquid chromatography with fluorimetric detection

Abstract

A HPLC method for determination of both reduced(GSH) and oxidized(GSSG) glutathione in plasma,whole blood and rat hepatocytes has been developed and evaluated.Reduced glutathione reacts with orthophthalaldehyde(OPA) to form a stable,highly fluorescent tricyclic derivate at pH 8,while GSSG reacts with OPA at pH 12.For the separation,reverse phase column Discovery C(18),was used.The mixture of methanol and 25 mM sodium hydrogenphosphate,pH 6.0, was used as mobile phase.The intra-assay coefficients of variation were 1.8 and 2.1% for whole blood, 2.0 and 1.9% for rat hepatocytes,4.3 and 5.2% for plasma. The inter-assay coefficient of variation were 5.8 and 6.2% for whole blood,6.6 and 7.1% for rat hepatocytes,6.9 and 7.8% for plasma.The limit of detection was 14.0 and 5.6 fmol,respectively.