化学
谷胱甘肽
甲萘醌
色谱法
氧化应激
检出限
液相色谱-质谱法
串联质谱法
谷胱甘肽二硫化物
体内
氧化磷酸化
样品制备
生物化学
质谱法
酶
生物技术
生物
作者
Alicia Thiel,Ann-Kathrin Weishaupt,Merle M. Nicolai,Kristina Lossow,Anna P. Kipp,Tanja Schwerdtle,Julia Bornhorst
标识
DOI:10.1016/j.jchromb.2023.123742
摘要
Alterations in reduced and oxidized glutathione (GSH/GSSG) levels represent an important marker for oxidative stress and potential disease progression in toxicological research. Since GSH can be oxidized rapidly, using a stable and reliable method for sample preparation and GSH/GSSG quantification is essential to obtain reproducible data. Here we describe an optimised sample processing combined with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, validated for different biological matrices (lysates from HepG2 cells, C. elegans, and mouse liver tissue). To avoid autoxidation of GSH, samples were treated with the thiol-masking agent N–ethylmaleimide (NEM) and sulfosalicylic acid (SSA) in a single step. With an analysis time of 5 min, the developed LC-MS/MS method offers simultaneous determination of GSH and GSSG at high sample throughput with high sensitivity. This is especially interesting with respect of screening for oxidative and protective properties of substances in in vitro and in vivo models, e.g. C. elegans. In addition to method validation parameters (linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, interday, intraday), we verified the method by using menadione and L-buthionine-(S,R)-sulfoximine (BSO) as well established modulators of cellular GSH and GSSG concentrations. Thereby menadione proved to be a reliable positive control also in C. elegans.
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