2.7. Malondialdehyde Determination

MDA was quantified according to the Templar et al. [47] protocol with some modifications. Briefly, human plasma was deproteinized using 5% TCA and supernatant was treated with fresh 0.6% TBA and incubated for 45 min at 90°C. The mixture was cooled at room temperature (25°C) and 120 μL was injected in the HPLC. The MDA concentration was determined by interpolating the area of the peak corresponding to the adduct MDA-TBA₂ of the sample into a MDA calibration curve prepared through acid hydrolysis of 1,1,3,3-tetraethoxypropane. The HPLC measurements were performed using a reverse-phase HPLC Merck-Hitachi 7000 series (Merck-Hitachi, Darmstadt, Germany) equipped with an autosampler device. The separation was reached using an Inertsil ODS-3 column (GL Sciences, Tokyo, Japan) and a mobile phase composed of a 50 mM sodium phosphate buffer, pH 7 (65%) and methanol (35%). The detection was conducted using a UV-visible photodiode array detector and a fluorescence detector (λ_EXC = 515 nm; λ_EM = 550 nm). The fluorescence chromatograms were only used for MDA analysis.