Assay of malondialdehyde in biological fluids by gas chromatography-mass spectrometry

Malondialdehyde (MDA) is assayed in femtomole quantities in biological samples by gas chromatography-mass spectrometry (GC-MS). The MDA trapped in protein as a Schiff base is released by H2SO4, the protein precipitated using Na2WO4, and the MDA derivatized with pentafluorophenylhydrazine to form the stable adduct, N-pentafluorophenylpyrazole. Negative chemical ionization (NCI) capability allows the sensitive detection of this MDA adduct in biological samples at a level of 5 nM on-column. A stable-isotope-labeled MDA, [2H2]MDA, was used as an internal standard for quantitation. MDA recovery from plasma was 76%. This assay provides two forms of confirmation of the analyte, retention time and mass ion, thus minimizing error due to interfering compounds. The commonly used thiobarbituric acid assay for MDA overestimates the MDA levels by over 10-fold, possibly resulting from cross-reactivity with other aldehydes and artifactual oxidation due to 100 degrees C temperature conditions. In our assay, all steps were performed at room temperature thereby suppressing artifactual oxidation of the sample. We have successfully applied this assay to biological samples including plasma, tissue homogenates, and sperm.