Measurement of malondialdehyde by high performance liquid chromatography with fluorescence detection

Abstract

Abstract 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.