Glutathione measurement by high-performance liquid chromatography separation and fluorometric detection of the glutathione-orthophthalaldehyde adduct

Glutathione reacts with orthophthalaldehyde to form a stable, highly fluorescent tricyclic derivative which is easily separated and quantitated by high-performance liquid chromatography. Separation of the glutathione adduct is achieved by isocratic elution over a reverse-phase column with 7.5% methanol/92.5% 0.15 M sodium acetate, pH 7.00. The adduct is detected fluorometrically and quantitated by integration of peak area. Detection of 0.1 to 200 pmol glutathione produces a linear response and the recovery of reduced and oxidized glutathione from rat liver homogenate, bile, and plasma is quantitative. The chemical identity of the adduct was confirmed by mass spectrometry.