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 (lambda em = 450 nm; lambda 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.
Gotti
R
R
Department of Pharmaceutical Sciences, Bologna, Italy.
Andrisano
V
V
Gatti
R
R
Cavrini
V
V
Candeletti
S
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003545:Cysteine / Q000032:analysis; D004229:Dithiothreitol; D005978:Glutathione / Q000032:analysis* / Q000097:blood; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000737:chemistry; D010084:Oxidation-Reduction; D051381:Rats; D013154:Spleen / Q000737:chemistry; D013438:Sulfhydryl Compounds
D013438:Sulfhydryl Compounds; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol