Concurrent quantification of oxidized and reduced glutathione and cysteine by targeted mass spectrometry:...

Glutathione (GSH) and cysteine (Cys) are crucial low-molecular-weight thiol antioxidants that protect ocular tissues from oxidative stress, a key driver of age-related eye diseases. The balance between their reduced (GSH/Cys) and oxidized (GSSG/CySS) forms reflect tissue oxidative status. Despite their importance, existing analytical methods typically quantify these analytes separately, requiring multiple assays. In this work, a robust liquid chromatography-tandem mass spectrometry method for the concurrent quantification of GSH, GSSG, Cys, and CySS in ocular tissues has been developed and validated. The workflow uses derivatization of reduced thiols with monobromobimane, followed by solid-phase extraction and separation using reversed-phase high-performance liquid chromatography with an 8-min gradient elution of acetonitrile and heptafluorobutyric acid. Analytes were detected by positive-ion mode multiple reaction monitoring on a triple quadrupole mass spectrometer and quantified using extracted ion chromatograms. The method demonstrated excellent linearity, precision, and accuracy, with all quality control samples meeting acceptance criteria. Method sensitivity and reproducibility were also validated. Application to human, rat, bovine, and rabbit lenses, as well as rat retina, cornea, aqueous humor, and vitreous humor revealed distinct tissue- and species-specific redox profiles. Rat ocular tissues were found to be in a predominantly reduced state, with reduced thiols at higher concentrations than their oxidized counterparts. Rabbit lenses exhibited the highest GSH concentrations, while human lenses showed lower levels, a finding potentially related to donor age. This method has broad applicability in studies investigating the role of oxidative stress in ocular health and the development of novel treatments to prevent ocular disease.