Improved method for plasma malondialdehyde measurement by high-performance liquid chromatography using methyl...

Measurement of malondialdehyde (MDA) is an important contribution to the assessment of oxidative stress. We report a sensitive and reproducible high-performance liquid chromatography (HPLC) method for measurement of plasma MDA in the assessment of lipid peroxidation. Using methyl malondialdehyde (Me-MDA) as an internal standard with reversed-phase HPLC and UV detection and derivatisation with 2,4 dinitrophenylhydrazine (DNPH), we obtained maximum MDA values with 60-min incubation of 10% plasma with 1 M NaOH at 60 degrees C. The dilution of the plasma and a longer incubation time in the alkaline hydrolysis step greatly improved recovery of MDA from its bound form. Ratios of peak height of MDA/Me-MDA were linear over a range of 0-100 microM with correlation coefficients >0.99. The recovery was 88.5%. Within and between run variations were <4 and <7%, respectively. The mean MDA value measured in 20 healthy volunteers was 13.8 microM (+/-1.32).