Quantification of Malondialdehyde via HPLC

Homogenate (100 μL) was used for MDA analysis. Briefly, 700 μL of 1% orthophosphoric acid and 200 μL of thiobarbituric acid (42 mM) were added to the sample. Then, the sample was boiled for 60 min in a water bath, and afterwards, it was immediately cooled on ice. Two hundred microliters was transferred to a 2 mL tube containing 200 μL sodium hydroxide/methanol (1:12 v/v). The sample was vortex-mixed for 10 s, and centrifuged for 3 min at 13,000× g. The supernatant (200 μL) was transferred to a 300 μL glass vial and 50 μL injected onto the column. The HPLC was a Shimadzu LC-10AD system (Kyoto, Japan) equipped with a C18 Luna column (5 μm, 150 × 4.60 mm, Phenomenex Inc., Torrance, CA, USA), a Shimadzu RF-535 fluorescence detector (excitation 525 nm, emission 551 nm), and 0.5 mL/min flow of phosphate buffer (KH2PO4 1mM, pH 6.8) [32 (link)]. MDA was quantified by determination of the peak area in the chromatograms relative to a standard curve of known concentrations.