HPLC determination of malondialdehyde in ECV304 cell culture medium for measuring the antioxidant effect of vitexin-4″-O-glucoside

摘要

To investigate the antioxidant effect of vitexin-4″-O-glucoside, a flavone glycoside, isolated from the leaves of Crataegus pinnatifida Bge. var. major, we developed a simple and sensitive high-performance liquid chromatography (HPLC) method to determine levels of malondialdehyde (MDA) in ECV304 cell culture medium after induction by tert-butyl-hydroperoxide (TBHP). The preparation of analyzed samples involved a one-step derivatization with thiobarbituric acid (TBA). HPLC analysis was performed on a Synergi™ Hydro-RP, a polar end-capped C₁₈ column (250×4.6 mm, 4 μm), using an acetonitrile-ammonium acetate aqueous solution (10 mM, pH 6.8) as the mobile phase under linear gradient conditions with UV detection at 532 nm. The calibration curve was linear over 0.0125–1.25 μM MDA (r = 0.9951). Relative standard deviations (RSDs) of intra-day and inter-day precision were less than 6.1% and 5.0%, respectively. The mean recovery was 96.9 ± 1.6%. The lower limit of quantification (LLOQ) of MDA was 0.0125 μM. This chromatographic method was successfully applied to investigating the in vitro antioxidant effect of vitexin-4″-O-glucoside. Vitexin-4″-O-glucoside (120 M) protected ECV304 cells from peroxidation induced by TBHP.