HPLC DETERMINATION OF BIOLOGICALLY-ACTIVE THIOLS USING PRECOLUMN DERIVATIZATION WITH 5,5'-DITHIO-(BIS-2-NITROBENZOIC ACID)

5,5'-Dithio-(bis-2-nitrobenzoic acid), Ellmans reagent (ESSE), is used as a pre-column derivatisation reagent for the determination of biolo gically active thiols by HPLC. D-penicillamine, N-acetyl-D-penicillami ne, N-acetylcysteine, cysteine, captopril and thiomalic acid all give well resolved derivatives. The calibration graph and reproducibility ( %R.S.D. +/- 1.3%) for the analysis of glutathione indicates that the m ethod could be used for quantitative analysis. ESSE is widely used as a reagent in thiol determinations by electronic spectroscopy via the d etection of the Ellmans anion (ES-) generated without any prior separa tion procedures. However, there are considerable reservations over its use for the spectrophotometric determination of thiols because of the possibility of side reactions which generate another Ellmans based sp ecies (ESO2-). The assay described determines the thiol as a derivatis ed mixed disulphide (ESSR) and since speciation between the anion ES- and the oxidation product ESO2- occurs it enables the process of oxida tion to be monitored simultaneously. (C) 1997 Elsevier Science B.V.