Simultaneous quantitation of oxidised and reduced glutathione in equine biological fluids by reversed-phase high-performance liquid chromatography using electrochemical detection.

A change in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) can be used to indicate oxidative stress in vivo. A rapid and highly sensitive isocratic reversed-phase high-performance liquid chromatographic method using coulometric electrochemical detection (LCEC) has been developed to simultaneously detect GSH and GSSG in equine biological fluids. Perchloric acid was used to extract GSH and GSSG from equine plasma and haemolysates, and methanol was used to deproteinise bronchoalveolar lavage fluid samples. Injection of extracts onto a Hypersil ODS HPLC column produced well resolved peaks corresponding to GSH and GSSG. The concentrations of GSH and GSSG found in equine haemolysates were similar to those previously found in humans and laboratory animals, although, to the authors' knowledge, previous attempts to measure GSH and GSSG in bronchoalveolar lavage fluid using LCEC have been unsuccessful. This method can be used to measure the GSH redox ratio in biological fluids during physiological conditions that may induce oxidative stress, such as exercise and disease.