Validation of high-performance liquid chromatography–boron-doped diamond detection for assessing hepatic glutathione redox status

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.