Quantitation of glutathione and its oxidation products in erythrocytes by multiple-label stable-isotope dilution

A multiple-label stable isotope dilution assay for quantifying glutathione (GSH), glutathione disulfide (GSSG), and glutathione sulfonic acid in erythrocytes was developed. As the internal standards, [¹³C ₃,¹⁵N]glutathione, [¹³C₄, ¹⁵N₂]glutathione disulfide, and [¹³C ₃,¹⁵N]glutathione sulfonic acid were used. Analytes and internal standards were detected by LC-MS/MS after derivatization of GSH with iodoacetic acid and dansylation of all compounds under study. The calibration functions for all analytes relative to their respective isotopologic standards revealed slopes close to 1.0 and negligible intercepts. As various labelings of the standards for GSH and GSSG were used, their simultaneous quantitation was possible, although GSH was partly oxidized to its disulfide during analysis. The degree of this artifact formation of GSSG was calculated from the abundance of the mixed disulfide formed from unlabeled GSH and its respective standard. Thus, the detected GSSG amount could be corrected for the artifact amount. In this way, the amount of GSSG in erythrocytes was found to be less than 0.5% of the GSH concentration. Similar to GSSG, the detected amount of glutathione sulfonic acid was found to be formed at least in part during the analytical process, but the degree could not be quantified.