Determination of glutathione in biological samples by high performance liquid chromatography with fluorescence detection

A selective and sensitive high performance liquid chromatographic (HPLC) method has been developed for the determination of reduced glutathione (GSH) in biological samples (rat liver, spleen and plasma). The method involved a prechromatographic thiol derivatization with methyl 4‐(6‐methoxynaphthalen‐2‐yl)‐4‐oxo‐2‐butenoate; the reaction was rapid (5 min) under mild conditions (pH 7.5 and ambient temperature) and selective for the sulphydryl group. The thiol adducts were separated on a reversed‐phase C‐18 column using acetonitrile: 0.05 M triethylammonium (TEA) phosphate (pH 4) solution 32:68 (v/v) as the mobile phase. Fluorescence detection (λ em = 450 nm; λ exc = 310 nm) was used and the detection limit (S/N = 3) was about 0.5 pmole of the injected GSH adduct. The method was also applied to the determination of total glutathione in rat plasma after a preliminary reduction with dithiothreitol.