Liquid Chromatography−Mass Spectrometry (LC-MS) Investigation of the Thiobarbituric Acid Reactive Substances (TBARS) Reaction

Abstract

The thiobarbituric acid reactive substances (TBARS) assay is a commonly used method for the detection of lipid peroxidation. Malondialdehyde is formed as a result of lipid peroxidation and reacts with thiobarbituric acid to form a pink pigment that has an absorption maximum at 532 nm. Other compounds also react with thiobarbituric acid to form colored species that can interfere with this assay, but little is known about these interfering species. This is the first investigation using LC-MS and MS-MS to study the structures of the pink adduct as well as a common unstable yellow interference compound, which absorbs at 455 nm. Also, the presence of barbituric acid impurities in the thiobarbituric acid reagent was found to produce 1:1:1 thiobarbituric acid/malondialdehyde/barbituric acid and 2:1 barbituric acid/malondialdehyde adducts that absorbed at 513 and 490 nm, respectively, indicating that thiobarbituric acid should be purified before use.