Validation of high-performance liquid chromatography-boron-doped diamond detection for assessing hepatic glutathione...

Glutathione redox status is a commonly used oxidative stress biomarker. High-performance liquid chromatography-ultraviolet (HPLC-UV) and HPLC-electrochemical detection (HPLC-ECD) have been used to assess glutathione status but have potential limitations due to challenging sample preparation procedures or electrochemical signal degradation. Thus, this study aimed to validate an HPLC-ECD approach using boron-doped diamond (BDD), a novel electrode material exhibiting excellent electrochemical stability. Liver homogenates from obese (ob/ob) mice and their lean littermates (n=4/genotype) as well as from rats fed high- or low-fat diets (n=8/treatment) were analyzed in parallel by HPLC-BDD and -UV. HPLC-BDD responses for reduced glutathione (GSH) and oxidized glutathione (GSSG) were linear over more than four orders of magnitude at 1475 mV, the optimal oxidation potential. Within- and between-day precision values of GSH, GSSG, and GSH/GSSG were 2.1% to 7.9%, and accuracy values of GSH and GSSG were 96% and 105%, respectively. Electrochemical responses were stable up to 48 h of continuous system use. Using HPLC-BDD and -UV, hepatic GSH, GSSG, and GSH/GSSG from mice (r=0.64-0.94) and rats (r=0.79-0.92) were well correlated (P<0.05), and no significant differences in thiol levels were observed between detection methods. Collectively, our findings support HPLC-BDD as a relatively simple, accurate, and validated approach for evaluating hepatic glutathione redox status.