Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS to study the redox state and drug-mediated modulation in cells, worms and animal tissue

by Selleckchem | Sep 22 2023

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.

Comments:

The passage describes a study that aims to develop a stable and reliable method for sample preparation and quantification of reduced and oxidized glutathione (GSH/GSSG) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The study focuses on different biological matrices such as lysates from HepG2 cells, C. elegans, and mouse liver tissue.

To prevent the autoxidation of GSH, the researchers treated the samples with the thiol-masking agent N-ethylmaleimide (NEM) and sulfosalicylic acid (SSA) in a single step during sample processing. This step is crucial to maintain the integrity of GSH and obtain accurate measurements. The developed LC-MS/MS method allows for simultaneous determination of GSH and GSSG within a short analysis time of 5 minutes, enabling high sample throughput with high sensitivity.

The study also validates the method using different parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, interday, and intraday variability. Additionally, the researchers verified the method's effectiveness by using menadione and L-buthionine-(S,R)-sulfoximine (BSO), which are known to modulate cellular GSH and GSSG concentrations. Menadione demonstrated reliable results as a positive control, even in C. elegans, suggesting its potential applicability in oxidative and protective substance screening in both in vitro and in vivo models.

Overall, this optimized LC-MS/MS method provides a robust and efficient approach for measuring GSH and GSSG levels, allowing researchers to investigate oxidative stress and disease progression in toxicological research and assess the effects of various substances on GSH/GSSG concentrations.