Abstract
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 liquid chromatography-tandem mass spectrometry 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 hydrophilic interaction liquid chromatography column with sulfobetaine chemistry. Tandem mass spectrometry with triple-quadrupole mass spectrometry in multiple-reaction monitoring acquisition mode allowed sensitive detection of the targeted metabolites with limits of quantification in the range of 5-25nM. Run times of 3min enable a high throughput analysis of cellular samples. For calibration, a 13 C-labelled cell extract was used as an internal standard. The method was validated and the concentrations of glutathione and its biosynthesis intermediates were determined in HeLa cells.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
