Yeo H C, Helbock H J, Chyu D W, Ames B N
Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Anal Biochem. 1994 Aug 1;220(2):391-6. doi: 10.1006/abio.1994.1355.
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.
通过气相色谱 - 质谱联用仪（GC-MS）对生物样品中的丙二醛（MDA）进行飞摩尔量的测定。作为席夫碱捕获在蛋白质中的MDA通过硫酸释放，使用钨酸钠沉淀蛋白质，并用五氟苯肼将MDA衍生化以形成稳定的加合物N - 五氟苯基吡唑。负化学电离（NCI）能力允许在柱上以5 nM的水平灵敏检测生物样品中的这种MDA加合物。使用稳定同位素标记的MDA，[2H2]MDA作为定量内标。血浆中MDA的回收率为76％。该测定提供了两种形式的分析物确认，保留时间和质量离子，从而最大限度地减少了由于干扰化合物引起的误差。常用的用于MDA的硫代巴比妥酸测定法将MDA水平高估了10倍以上，这可能是由于与其他醛的交叉反应以及100℃温度条件下的人为氧化所致。在我们的测定中，所有步骤均在室温下进行，从而抑制了样品的人为氧化。我们已成功将该测定应用于包括血浆、组织匀浆和精子在内的生物样品。
