Robust High-Performance Liquid Chromatography and Liquid Chromatography-Tandem Mass Spectrometry Methods for the Determination of Malondialdehyde in Biological Samples Using Thiobarbituric Acid Derivatization and Column Cleaning with High-Concentration Ammonium Acetate

Malondialdehyde (MDA) is a widely used biomarker of lipid peroxidation and is commonly quantified as a red chromophore formed by the reaction with thiobarbituric acid (TBA). However, we found that previously unrecognized byproducts generated during the TBA reaction gradually accumulated on high-performance liquid chromatography (HPLC) columns, leading to poor elution and unstable retention of the TBA derivative of MDA. To address this issue, we developed a highly sensitive and reproducible HPLC-based method that incorporates a column-cleaning step by the injection of 1 M ammonium acetate during column washing, which effectively removes the retained byproducts and restores consistent chromatographic performance. As a result, MDA could be reliably quantified at concentrations as low as 1 μM by HPLC and 10 nM by liquid chromatography-tandem mass spectrometry, respectively. The method was successfully applied to detect elevated MDA levels in the blood and kidneys of mice after oral administration of fructose at a rate of 2 g per kg body weight.