A simple, rapid, highly sensitive and reproducible quantification method for plasma malondialdehyde by high-performance liquid chromatography.

A simple, rapid, highly sensitive and reproducible quantification method for plasma malondialdehyde (MDA) was developed using high-performance liquid chromatography (HPLC). The present method is based on the thiobarbituric acid (TBA) reaction and reversed-phase HPLC separation with fluorescence detection. For sample preparation, an aliquot of 5 microL plasma was mixed with 0.2% (w/v) TBA in 0.1 M sodium acetate buffer, pH3.5. After heating at 95 degrees C for 60 min, the reaction solution was centrifuged and an aliquot of 10 microL supernatant was injected into the HPLC system without neutralization or extraction procedures. The TBA-MDA adduct was separated on a reversed-phase column and quantified by a fluorescence detection (lambda ex = 515 nm; lambda em = 553 nm). The mobile phase was a mixture of acetonitrile: water (7:3, v/v) under isocratic conditions at ambient temperature. These procedures gave good results with regard to sensitivity (detection limit S/N = 3, 0.5 fmol per injection), linearity (0.5-500 fmol per injection), precision (between-assay C.V = 3.1%, within-assay C.V = 1.2%) and recovery (98.6%). The simple sample procedure, the highly sensitive detection limit and the stability of the TBA-MDA adduct make the present method applicable to numerous clinical samples. As many as 250-300 samples per day can be assayed using an autosampler.