Measurement of MDA by high-performance liquid chromatography (HPLC)

Briefly, 250 µL thiobarbituric acid (42 mM), 750 µL H₃PO₄ (0.44 M), and 450 µL distilled water were added to the 50-µL RBC suspension in a test tube and the mixture was incubated for 60 min in a boiling water bath (total volume: 1.5 mL). After cooling in ice water, the same volume of alkaline methanol (50 mL methanol + 4.5 mL of 1 M NaOH) (1/1 v/v) was added to the test tube and centrifuged at 3000 × g for 3 min. The supernatant (200 µL) was transferred to a vial and injected into an HPLC apparatus (Agilent, Boblingen, Germany). An RP-18 column (150 ×4.6 mm) and 5 -μm particle size were used. The mobile phase consisted of a mixture of 400 mL of 50-mM phosphate buffer (pH 6.8) and 600 mL of methanol (total volume 1 L). The flow rate of the mobile phase and the injection volume of samples were set to 0.8 mL/min and 20 µL, respectively. Measurements were made against standard samples of different concentrations at an excitation wavelength of 527 nm and an emission wavelength of 551 nm.²¹ MDA levels are expressed as nmol/gHb.