Development and validation of HILIC-MS/MS method for the analysis of glutathione metabolism in cell cultures

Glutathione is a critical intracellular antioxidant that neutralizes reactive oxygen species and participates in detoxification. The ratio of its two forms, the reduced and disulfide, serves as an indicator of cellular oxidative stress associated with both acute and chronic disorders. Monitoring intracellular levels of glutathione and thiols involved in its metabolism is important for the proper characterization of cellular injury. However, current analytical methods often require tedious chemical derivatization, lack adequate retention and selectivity for highly polar analytes, or suffer from severe matrix effects when profiling the broader metabolic pathway. In this study, a robust hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and optimized for the comprehensive analysis of glutathione metabolism in cell samples without chemical derivatization. The method focused on the label-free quantitation of 21 key metabolites, encompassing intact glutathione and other thiols, their oxidized forms, precursor amino acids, and related sulfur-containing compounds. Chromatographic performance was systematically investigated in HILIC mode using sulfobetaine zwitterionic stationary phase. The final method employed 0.05% difluoroacetic acid in the mobile phase and 5% 5-sulfosalicylic acid for sample preparation, ensuring efficient protein precipitation, stabilization of thiols, and compatibility with electrospray ionization. The method demonstrated high analytical performance, with intra- and inter-day precision (≤5%) and accuracy (≤15%) for all target analytes. Application to A549 lung cancer cells incubated with CdCl₂ and cisplatin for 24 and 48 h revealed significant glutathione depletion and multiple metabolic alterations, including elevated γ-glutamylcysteine levels.