Generation of Glutathione Persulfide and Optimization of Its Mass-Spectrometric Determination

Glutathione persulfide (GSSH) is an important cellular metabolite involved in redox regulation and a potential therapeutic agent. Due to its instability and the lack of commercial GSSH standards, the development of procedures for its generation in situ and quantitative determination in various test systems is a problem of considerable current interest. In this work, conditions for the generation of GSSH in a reaction of oxidized glutathione with sodium sulfide with the fluorescent monitoring of released hydrogen sulfide were optimized. To derivatize the generated GSSH and reduced glutathione (GSH) with the formation of both derivatives in similar amounts, the reaction was carried out in the presence of an excess of N-ethylmaleimide. A procedure for the determination of GSSH based on the level of GSH in a model reaction using HPLC–mass spectrometry in the multiple reaction monitoring mode was described. The contribution of the GSH impurity in a solution of oxidized glutathione to the determined amount of GSSH and the detection limit of GSSH in the reaction mixture were found. The results are of interest for the preparation and mass spectrometric analysis of biologically relevant persulfides using various derivatizing agents.