Abstract

A novel, highly specific, simple and rapid method for the determination of malondialdehyde (MDA), the routinely used marker for free radical generation in body fluids has been developed and evaluated. Serum samples from 30 healthy volunteers in heparin and 1,4-dithiothreitol-containing tubes stored at −80 °C were analyzed. The MDA-thiobarbituric acid complex was separated from interfering substances using HPLC. For the separation, reverse phase column MAC (4 × 250 mm, Biospher SI 120 PSI C₁₈, particle size 7 μm) was used. The mixture of methanol and 8.3 mmol/l phosphate buffer, pH=7.2, (35:65, v/v) was used as mobile phase. The volume of serum samples injected on the column was 50 μl. The analyte was detected at 532 nm. Retention time of MDA-thiobarbituric acid complex was 4.9±0.1 min at the flow rate 0.7 ml/min. Excellent linearity was achieved. The intra- and interassay coefficient of variation was 7.3% and 8.8%, respectively. The recovery was 95.6% and the detection limit was 0.1 μmol/l. The validity of this method was proved by comparison with the spectrophotometric determination of MDA-thiobarbituric acid complex by the method of Yagi at three different wavelengths (485, 532 and 560 nm) with Allen's correction.