Determination of reduced-form glutathione and total glutathione in blood and plasma by high performance liquid chromatography with on-column fluorescence derivatization

摘要

An on-column fluorometric derivatization method has been developed for the determination of reduced-form glutathione and total glutathione in blood and plasma by high-performance liquid chromatography. Blood hemolysate or diluted plasma was deproteinized with perchloric acid. For total glutathione determination, any oxidized forms of glutathione in samples were converted to reduced-form glutathione with dithiothreitol prior to protein precipitation. Deproteinized samples were injected without pretreatment. Glutathione was fluorescence-derivatized and separated on a C18-bonded vinyl alcohol polymer column with ortho-phthalaldehyde in sodium berate buffer-acetonitrile, as mobile phase: the ortho-pathalaldehyde-glutathione, fluorescent isoindole adduct is measured by its fluorescence response. Due to the high sensitivity, selectivity reproducibility and the simple HPLC system, the method is particularly suitable for routine assay of numerous or small amounts of samples. [References: 38]