DETERMINATION OF GLUTATHIONE IN BIOLOGICAL SAMPLES BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

A selective and sensitive high performance liquid chromatographic (HPL C) method has been developed for the determination of reduced glutathi one (GSH) in biological samples (rat liver, spleen and plasma). The me thod 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 sele ctive 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. Fluo rescence detection (lambda(em)=450 nm; lambda(exc)=310 nm) was used an d 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 glut athione in rat plasma after a preliminary reduction with dithiothreito l.