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

Autor: Serafimov K; Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Tübingen, Germany., Aydin Y; Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Tübingen, Germany., Lämmerhofer M; Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Tübingen, Germany.
Jazyk: angličtina
Zdroj: Journal of separation science [J Sep Sci] 2024 Jan; Vol. 47 (1), pp. e2300780. Date of Electronic Publication: 2023 Nov 14.
DOI: 10.1002/jssc.202300780
Abstrakt: 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-2 μm 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 5-25 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 an internal standard. The method was validated and the concentrations of glutathione and its biosynthesis intermediates were determined in HeLa cells.
(© 2023 The Authors. Journal of Separation Science published by Wiley-VCH GmbH.)
Databáze: MEDLINE
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