Quantitative analysis of the glutathione pathway cellular metabolites by targeted liquid chromatography - tandem mass spectrometry.

Glutathione, its biosynthesis intermediates and other thiol metabolites are of central relevance for the redox homeostasis of cells. Their analysis is critical due to the facile interconversion of redox pairs during sampling, sample preparation, and data acquisition, in particular in the electrospray ionization interface. In this work we propose a fast targeted LC-MS/MS method to accurately analyze 14 metabolites from the glutathione pathway. N-Ethylmaleimide reagent is added with the extraction solvent and instantly stabilizes the thiol-redox state by derivatization. Liquid chromatographic separation of the analytes was performed on a sub-2μm superficially porous HILIC column with sulfobetaine chemistry. Tandem MS with triple-quadrupole mass spectrometry in multiple-reaction monitoring acquisition mode allowed sensitive detection of the targeted metabolites with LOQs in the range of 5-25 nM. Run times of 3 min enable a high throughput analysis of cellular samples. For calibration a 13 C-labelled cell extract was used as internal standard. The method was validated and the concentrations of glutathione and its biosynthesis intermediates determined in HeLa cells. This article is protected by copyright. All rights reserved.