Determination of glutathione in human plasma using high-performance liquid chromatography with electrochemical detection with a carbon-epoxy resin composite electrode chemically modified with cobalt phthalocyanine

Abstract

High-performance liquid chromatography with electrochemical detection (LCEC), incorporating a novel carbon-epoxy resin working electrode modified with cobalt phthalocyanine, has been employed for preliminary studies directed towards the determination of normal circulating levels of reduced glutathione (GSH) in human plasma. The mobile phase consisted of 0.05 M phosphate buffer (pH 3) containing 0.1%m/m ethylenediaminetetraacetic acid (EDTA); the calibration graph was linear in the range 0.24–30.7 ng of GSH injected. The mean recovery of GSH added to a control serum over the physiological concentration range (0.38–3.07 ng ml^–1) was 99%; this was achieved following a simple sample pre-treatment method, prior to LCEC, involving chelation of divalent cations with EDTA and subsequent acidification with orthophosphoric acid. Using the LCEC method, the mean circulating level of GSH in plasma, found in three normal subjects, was 2.69 µM, which is in agreement with previously reported values. The limit of detection for GSH in plasma was calculated to be 55 ng ml^–1, which is equivalent to a plasma concentration of 0.17 µM GSH; this indicates that the method might be applicable to the determination of depressed circulating levels of GSH.