A rapid HPLC method to simultaneously quantify therapeutic thiols and monitor their disulfide exchange reactions with cystine.

This study introduces a rapid and environmentally sustainable High Performance Liquid Chromatography method to simultaneously quantify free thiols and monitor their disulfide exchange kinetics with cystine. This method is based on the derivatisation of thiols with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent). The method utilises a mobile phase consisting of 0.1% v/v Trifluoroacetic acid in water and methanol, with Ultraviolet detection at 248 nm. The method exhibited high sensitivity (Limit of Quantification 0.78 µM; Limit of Detection 0.39 µM) and selectivity for biologically relevant thiols (cysteamine, cysteine, tiopronin, glutathione, captopril and penicillamine) in an aqueous environment. Simultaneous quantification of these thiols was successfully achieved in phosphate-buffered saline as well as in biological samples such as human saliva and urine (from a vegetarian subject). Furthermore, it facilitated kinetic analysis of thiol-related reactions, with potential applications in both clinical and pharmacological research.