High-performance liquid chromatographic determination of plasma malondialdehyde level without a solvent extraction procedure.

A highly sensitive and simple HPLC method for measuring malondialdehyde (MA) in plasma was developed. For sample preparation, an aliquot of plasma (10 microliters) was mixed with 2.0% thiobarbituric acid (TBA) in 2 M sodium acetate buffer (containing 1 mM DTPA and 0.05% BHT), pH 3.5. After incubating at 95 degrees C for 45 min, the reaction solution was passed through a 0.2-micron membrane filter. An aliquot of 10 microliters filtrate was injected into HPLC system without any extraction procedures. The thiobarbituric acid-malondialdehyde (TBA-MA) complex was separated on reversed-phase HPLC and quantitated with a fluorescence detector. The mobile phase was mixture of acetonitrile:water (7:3, v/v) under isocratic conditions at ambient temperature, and a single analysis was completed in ca. 2.5 min. The minimum detection level for MA was 0.01 pmol. A very close correlation was observed between this method and the Yagi method, which is widely used for clinical diagnosis. The simple and inert mobile phase along with a very simple sample preparation makes this method applicable to numerous clinical samples without any difficulties.