High Performance Liquid Chromatography Coupled with Pre-column Derivatization for Determination of Oxidized Glutathione Level in Rats Exposed to Paraquat

Abstract

Glutathione (GSH) is one of the most important antioxidants that plays an essential role in detoxification of reactive oxygen species (ROS) which oxidizes to glutathione disulfide (GSSG). Paraquat (PQ), awidely used herbicide, causes pulmonary injury with the productionof ROS. Excessive ROS accumulation as a consequence of PQ exposure are frequently targeted by GSH thereby oxidative stress leads to depletion of cellular GSH by transforming of GSH to glutathione disulfide (GSSG). A precise method of measuring of GSSG concentration in plasma as indicator of oxidative stress is needed. Some analytical techniques such as high-performance liquid chromatography (HPLC), gas chromatography and capillary electrophoresis have been used for determination of GSSG concentration. In the present study, a new HPLC method with fluorescence detection based on derivatization of the amine group of glutathione with 9-fluorenylmethyl chloroformate (FMOC-Cl) was developed. Male Wistar albino rats exposed to different doses of PQ (20-60 mg/kg) and control group were used and after protein precipitation, their plasma was subjected to derivatization with FMOC in the presence of borate buffer. The derivatized samples were injected to HPLC system with C18 column, mobile phase consisting of methanol and phosphate buffer, λem= 315 nm, λex = 260 nm. Among all experimental groups, the rats which received 60 mg/kg PQ, showed a significant increase in the amount of oxidized glutathione (GSSG) compared to the control group. In this study, the applied derivatization and HPLC method made it possible to measure small amounts of glutathione in plasma using a precise and sensitive technique.