Validation of a liquid chromatography tandem mass spectrometry method to measure oxidized and reduced forms of glutathione in whole blood and verification in a mouse model as an indicator of oxidative stress

• New method for reduced (GSH) and oxidized glutathione (GSSG) was developed.
• We validated analytical performance of the method including precision and recovery.
• Clinical usefulness of the GSH/GSSG ratio was validated using a mouse model.
• Previously well-established mouse model of chronic oxidative stress was employed.
• The GSH/GSSG ratios decreased with increased induction of oxidative stress.

As a possible marker of oxidative stress, many studies have measured whole blood reduced glutathione (GSH) and oxidized glutathione (GSSG). However, large differences in GSH and GSSG levels reported in different studies, calls for a reliable standardized method. In this study, we validate not only analytical performance of new measurement method for GSH and GSSG, but also the clinical utility of these markers in a mouse model with chronic oxidative stress. Twenty mice were randomized into four treatment groups according to iron burden: 0 mg, 5 mg, 10 mg, or 15 mg of iron were injected into the peritoneum per day over 4 weeks. To prevent artifactual GSH auto-oxidation, we pretreated the sample with N-ethylmaleimide (NEM) immediately after sample collection. After protein precipitation using sulfosalicylic acid, GSSG and GSH-NEM were measured using liquid chromatography-tandem mass spectrometry (LC–MS/MS). The mean GSH/GSSG ratios of the mouse model were 163.1, 31.0, 27.9, and 12.8 for control, 5 mg, 10 mg, and 15 mg injection groups, respectively, showing a decrease in the GSH/GSSG ratios according to the amount of oxidative stress induced. Inter-assay coefficients of variation were 4.1% for GSH-NEM and 7.3% for GSSG. Recoveries were 98.0–105.9% for GSH-NEM and 98.0–107.3% for GSSG. No ion suppression was observed at the retention time for GSH-NEM and GSSG. This study suggests an accurate method that can be used for glutathione measurement using LC–MS/MS, and showed that GSH/GSSG ratio could provide an assessment of the degree of oxidative stress.