Measurement of Malondialdehyde Levels in Food by High-Performance Liquid Chromatography with Fluorometric Detection

A sensitive and reproducible HPLC assay with fluorometric detection was used to measure the malondialdehyde (MDA) concentration in food (butter, margarine, oil, fish, and meat tissue). Samples were homogenized in water supplemented with butylated hydroxytoluene. Proteins were precipitated with ice-cold 5% trichloroacetic acid and removed by centrifugation. The supernatant was incubated in a 0.28% thiobarbituric acid (TBA) mixture from which the oxygen was depleted. Optimal incubation time and temperature, for the TBA treatment, were found to be 30 min and 90 °C, respectively. The MDA-TBA adduct was fractionated by reverse phase HPLC and detected by fluorescence (?EX = 515 nm; ?EM = 543 nm). Elution was performed at 1 mL/min flow rate with a mixture of acetonitrile and sodium phosphate at pH 7 (15:85 v/v). The described sample preparation procedure minimizes the lipid oxidation and provides high sensitivity (0.01 pmol of MDA), reproducibility, and specificity.