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Preparative steps necessary for the accurate measurement of malondialdehyde by high-performance liquid chromatography

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
197
Issue
2
Identifiers
DOI: 10.1016/0003-2697(91)90392-7
Disciplines
  • Biology

Abstract

Abstract The need for a more specific, reliable, and reproducible technique for the measurement of malondialdehyde (MDA) has prompted modifications of currently available methods based on the formation and recovery of the complex between MDA and thiobarbituric acid (TBA). To 500 μl of plasma or to 300 mg of liver homogenate, 2 ml of H 2O and 500 μl of 0.5% butylated hydroxytoluene in methanol were added to prevent further formation of MDA. Precipitation of proteins carried out with 200 μl of 0.66 n H 2SO 4 and 150 μl of 10% Na 2WO 4 (w/v) led to complete recovery of the MDA standard. Maximum formation of the MDA-TBA complex was obtained by adjusting the pH between 2.5 and 4.5 and heating the MDA-TBA mixture at 100°C for 60 min. Extraction of the MDA-TBA complex was a critical step and proved complete with n-butanol at pH < 0.75. It was then evaporated at 37°C under nitrogen. The MDA-TBA complex solubilized in H 2O was shown to be stable for at least 7 days. These preparative steps led to the detection of a single peak that on spectral analysis was identified as pure MDA-TBA. This procedure offers several advantages in terms of specificity, recovery, and reproducibility.

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