Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA ) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells an d cultured fibroblasts. OPA derivatives are separated on a reversed-phase H PLC column with an acetonitrile-sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) c oncentration is calculated by subtraction of the GSH level from the GSHt le vel. The assay shows high sensitivity (50 fmol per injection, the lowest re ported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSS G close to 100%, and linearity (r > 0.999). This HPLC technique is very sim ple and rapid. Its wide applicability and high sensitivity make it a conven ient and reliable method for glutathione determination in various biologica l samples. (C) 2001 Elsevier Science B.V. All rights reserved.