Determination of γ-glutamylglutathione and other low-molecular-mass biological thiol compounds by isocratic high-performance liquid chromatography with fluorimetric detection

Abstract

A method was developed for the simultaneous determination of γ-glutamylglutathione (γ-GluGSH) and other low-molecular-mass thiol compounds (cysteine, cysteamine, homocysteine, cysteinylglycine, γ-glutamylcysteine, glutathione and N-acetylcysteine) using high-performance liquid chromatography combined with precolumn fluorescence labeling with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate (SBD-F). These SBD-labeled thiol compounds were separated within 35 min on a Cosmosil 5C-18AR column with isocratic elution using 75 m M sodium citrate buffer (pH 2.90)–methanol (98:2) and detected fluorimetrically (ex. 386 nm, em. 516 nm). The calibration graphs using 2-mercaptoethanol as an internal standard showed good linearity in the range from 20 pmol to 10 nmol for all thiol compounds examined. The application of this method for the quantitative determination of thiol compounds in the urine from γ-glutamyl transpeptidase-deficient mice was also demonstrated. This method is sufficiently simple, rapid and sensitive for the determination of γ-GluGSH and other low-molecular-mass thiol compounds in biological samples.