Simple and sensitive quantification of glutathione hydropersulfide alkylated using iodoacetamide by high-performance liquid chromatography with post-column derivatization.

A novel analytical method was developed for the quantification of glutathione hydropersulfide (G-SSH) in biological samples by high-performance liquid chromatography (HPLC) with post-column derivatization. G-SSH was treated with iodoacetamide as an alkylating agent for 5 min at 37 °C, and the resultant acetamide-labeled G-SSH (G-SS-acetamide) was subjected to HPLC. After separation on a reversed-phase column, G-SS-acetamide was quantified by detection using a post-column reaction with orthophthalaldehyde under alkaline conditions. The standard G-SS-acetamide was synthesized through the S-S exchange reaction between oxidized glutathione and 2-mercaptoacetamide. It should be noted that some types of alkylating agents, including N-ethylmaleimide and monobromobimane, cleave the polysulfide chains of polysulfides that consist of glutathione, resulting in the production of alkylated G-SSHs. We confirmed that iodoacetamide did not enhance the cleavage of acetamide-labeled glutathione trihydropersulfide (G-SSS-acetamide). The lowest quantification limit was estimated to be 25 nM for G-SS-acetamide. This method can be useful for studying the dynamics of sulfane sulfur in glutathione-containing matrices.