HPLC determination of biologically active thiols using pre-column derivatisation with 5,5'-dithio-(bis-2-nitrobenzoic...

5,5'-Dithio-(bis-2-nitrobenzoic acid), Ellmans reagent (ESSE), is used as a pre-column derivatisation reagent for the determination of biologically active thiols by HPLC. D-penicillamine, N-acetyl-d-penicillamine, 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 method could be used for quantitative analysis. ESSE is widely used as a reagent in thiol determinations by electronic spectroscopy via the detection of the Ellmans anion (ES-) generated without any prior separation 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 species (ESO2-). The assay described determines the thiol as a derivatised mixed disulphide (ESSR) and since speciation between the anion ES- and the oxidation product ESO2- occurs it enables the process of oxidation to be monitored simultaneously.