HPLC DETERMINATION OF GLUTATHIONE AND OTHER THIOLS IN HUMAN MONONUCLEAR BLOOD-CELLS

A simple and sensitive HPLC method is proposed for the determination o f glutathione (GSH) in human mononuclear cells, based on the derivatiz ation of the tripeptide with Ellman's reagent. The mobile phase was co mposed of a mixture of methanol and ammonium formate (10:90 v/v, with a dow rate of 1 mL/min). The stationary phase was a C-18 (4.6 mu m, 25 0 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 calibr ation 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 pre cision (R.S.D.% congruent to 2). The proposed HPLC method allows for t he separation and quantitation of cysteine and N-acetylcysteine, if pr esent in biological samples. Furthermore, the method allows for the de termination of total thiol present in human mononuclear cells. (C) 199 8 John Wiley & Sons, Ltd.