Simultaneous Determination of Endogenous Antioxidants and Malondialdehyde by RP-HPLC Coupled with Electrochemical Detector in Serum Samples

Abstract

A new method has been developed and validated based on high-performance liquid chromatography for simultaneous determination of endogenous aminothiols (cystine, cysteine, homocystiene, methionine, reduced and oxidized glutathione, N-acetylcysteine), ascorbic acid, and malondialdehyde in single elution using electrochemical detector kept at 900 mV electrode potential on Discovery 250 mm C18 RP chromatographic column. The elution time was 20 min, using flow rate of 0.6 mL/min, injecting 5 µL sample for analysis after extraction. The method was validated according to standard guidelines for accuracy, linearity, specificity, reproducibility, limit of detection and limit of quantification and was successfully applied to serum samples of healthy animal model. The analytes were determined using dilution method and values were calculated in mmol/L. This method can be applied to clinical pathologies related to oxidative stress and metabolic syndromes for assessment of severity and facilitate in developing a correlation between the endogenous antioxidant capacities with exogenous antioxidant interventions.