Effects of reducing agents on the determination of thiolic compounds in the presence of their disulfides using bimane pre-column derivatization

Summary The influence of reducing agents (sodium borohydride, tributylphosphine, dithioerythritol and dithiothreitol) on the conversion of disulfides into their parent thiols, with specific application to cysteine was investigated. Dithioerythritol and dithiothreitol were found to be most suitable for this reaction. A contact time of one hour at room temperature provided quantitative reduction as tested using cystine as a pure disulfide standard. A modified fluorescence labelling procedure with monobromobimane followed by reversed-phase HPLC allows quantitation of the parent thiol and the disulfide content when completing the labelling reaction with and without preliminary treatment with reducing agent. The effects of various bimane reducing agent ratios on the yield of the reaction are discussed. Precautions should be taken when dealing with complex matrices with respect to reagent concentrations and ratios.