Measurement of malondialdehyde by high performance liquid chromatography with fluorescence detection

A method has been developed to measure malondial-dehyde (MDA) in biological systems. MDA was reacted with 2-thiobarbituric acid (TBA) in the presence of butylated hydroxytoluene (BHT) to minimize formation of artifacts. Initial separation of the TBA-MDA adduct was accomplished by isobutanol extraction. Further elimination and separation of interfering substances was achieved by high performance liquid chromatography. The mobile phase consisted of a 1∶1 (v/v) mixture of methanol and water with 0.05% (w/v) tetrabutyl ammonium dihydrogen phosphate added as an ion pairing reagent. At a flow rate of 1 ml/min, the TBA-MDA adduct was eluted from a 15-cm, c-18, reversed phase column in approximately 4.9 min. The TBA-MDA adduct was quantitated with a fluorescence detector set at 515 nm excitation and 550 nm emission. Using this method, picomole quantities of MDA can be easily detected in plasma and liver samples.