A Sensitive Fluorometric Method for the Determination of Glutathione and Some Thiols in Blood and Mammalian Tissues by High Performance Liquid Chromatography

Abstract

A fluorescent reagent, N-(9-acridinyl)maleimide (NAM), was used for the determination of thiols in biological samples by high performance liquid chromatography. NAM-labeled glutathione (GSH), homocysteine, coenzyme A (CoA) and cysteine (CySH) were separated on a reversed-phase partition (octadecylsililated silica gel) column with the elution conditions of 0.06 M borate buffer pH 8.8: methanol (13:1) at a flow rate of 0.6ml/min within 15 min. In the absence of CoA in the sample, the elution conditions of 0.1 M borate buffer pH 8.8: methanol (15:1) at a flow rate of 0.8ml/min was used for the separations. Calibration curves were held up to 2.5 pmol for GSH and 11 pmol for CySH. About 0.17 μl of rat blood and 0.03mg of rat liver equivalent to 0.1 nmol of GSH were determined. The sensitivity was 100 times higher than that obtained with an automatic amino acid analyzer.