HPLC determination of glutathione and other thiols in human mononuclear blood cells

A simple and sensitive HPLC method is proposed for the determination of glutathione (GSH) in human mononuclear cells, based on the derivatization of the tripeptide with Ellman's reagent. The mobile phase was composed of a mixture of methanol and ammonium formate (10:90 v/v, with a flow rate of 1 mL/min). The stationary phase was a C18 (4.6 microns, 250 x 4 mm) reversed phase column. The detection of GSH was performed at 280 nm, resulting in a neat chromatographic peak at 5.8 min. A calibration curve showed good linearity over the concentration range 3 x 10(-6) - 6 x 10(-5) M, with a satisfactory precision. The method was found to yield a quantitative recovery of glutathione (96%), to be sensitive (down to 30 pmol of glutathione per injection) and to have a high precision (R.S.D.% approximately equal to 2). The proposed HPLC method allows for the separation and quantitation of cysteine and N-acetylcysteine, if present in biological samples. Furthermore, the method allows for the determination of total thiol present in human mononuclear cells.