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Optimization of Gradient Hydrophilic Interaction High-Performance Liquid Chromatography and Mass Spectrometry Method for Analysis of Glutathione Metabolism
Rok: 2023
Druh publikace: ostatní - přednáška nebo poster
Strana od-do: nestránkováno
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Jazyk Název Abstrakt Klíčová slova
eng Optimization of Gradient Hydrophilic Interaction High-Performance Liquid Chromatography and Mass Spectrometry Method for Analysis of Glutathione Metabolism Glutathione (GSH) is the major low-molecular thiol antioxidant synthesised in the cytosol. GSH performs many vital functions, such as antioxidant defence, detoxifying toxic electrophiles, and many more. GSH also occurs in the form of glutathione disulfide (GSSG). The ratio between GSH and GSSG is an important indicator of cellular oxidative stress and a biomarker of many diseases, e. g., cataracts, cancer, neurodegenerative diseases, kidney failure, pneumonia, cystic fibrosis, liver infection, or heavy metal poisoning. To date, many methods employing high-performance liquid chromatography (HPLC) have been reported for the assessment of GSH or GSSG, lesser number of methods have been aimed at GSH metabolomics. However, many of these methods employ derivatisation, which may be complicated and time-consuming. The aim of this work was to develop and optimise an HPLC method for the determination of GSH metabolism without derivatisation of the compounds. The goal was achieved using hydrophilic interaction chromatography (HILIC) separation on the ZIC-HILIC column coupled with mass spectrometry (MS) detection. This method was optimised for the separation and determination of glutathione (GSH), glutathione disulfide (GSSG), γ-glutamyl-cysteine (gGC), cysteine (Cys), glycine (Gly), glutamic acid (Glu), cystine (CysS) and homocystine (HCysS) i.e. the gradient profile was proposed using retention modelling and window diagram approach. The properties of the method were demonstrated by analysis of GSH and related metabolites in various cell lines.

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