Optimum Conditions for Derivatization of Glutathione,Cysteine and Cysteinylglycine in Human Plasma with Ammonium 7-Fluorobenzo-2-Oxa-1,3-Diazole-4-Sulfonate for Accurate Quantitation by High-Performance Liquid Chromatography

For the accurate quantitation of glutathione (GSH), cysteine (Cys) and cysteinylglycine (CysGly) in human plasma by detection of fluorescence after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F), a thiol-specific fluorogenic agent and high-performance liquid chromatography (HPLC), we attempted to identify optimum conditions for the derivatization and treatment of samples. The extent of derivatization with SBD-F of endogenous thiol compounds in human plasma was affected by several factors, such as the concentration of SBD-F and the reaction time. Acidification and deproteinization of plasma immediately after the separation of plasma from whole blood were essential and prevented the determination of inappropriately low values. The method developed in the present study allowed recovery that was closed to 100% for each compound. The mean values determined for 61 healthy individuals were as follows: Cys, 50.1 μM; CysGly, 12.2 μM; and GSH, 5.3 μM. INTRODUCTION Glutathione (GSH) is a tripeptide, gammaglutamylcysteinylglycine, that is widely distributed in animal tissues and plasma 1). GSH plays several essential roles in the maintenance of the integrity of thiol moieties of proteins and other compounds;the activity of some enzymes;and the protection of cells against oxidative stress, free radicals and the toxicity of certain drugs and other chemicals 1-3).