Substrate and TBARS variability in a multi‐phase oxidation system

The thiobarbituric acid reactive substances (TBARS) assay is widely used to measure antioxidant activity in the presence of lipid substrates such as linoleic acid (LA). In one version of this assay, LA is oxidized with and without antioxidant in a multi‐phase system in which LA lies over the surface of the aqueous layer of catalyst and antioxidant solution, with dissolved and atmospheric oxygen. Results from this assay showed low precision and therefore we conducted a systematic study to identify possible sources of variability including: order of addition of reactants; pre‐formed peroxides in LA (as precursors of TBARS); and the effect of different concentrations of added antioxidant, Trolox. These investigations showed that all contributed to variability of both formation of oxidation products and the antioxidant activity of Trolox. Furthermore, through the use of LA with pre‐formed peroxides removed, it was discovered that antioxidant activities reported in other studies using this system were most likely not due to the protective effect of antioxidant against formation of primary oxidation products. Rather, the antioxidant is likely to interfere with the radical scission of pre‐formed peroxides, resulting in lower TBARS. Practical applications: The results obtained from the TBARS assay have been used to interpret the potential antioxidant value of compounds added to lipid‐containing food products, such as in oil and meat. Alternatively, the TBARS assay has been used to assess multiple compounds or extracts to rank their effectiveness as antioxidants, thus selecting the most active ones for further study. In both cases, pre‐formed peroxides in the system may not be routinely measured. The results from this study suggest that pre‐formed peroxides may have a large impact on the results of antioxidant studies when TBARS are the measure of oxidation. Therefore, further research in other systems is necessary to ensure that robust antioxidant measurements are being performed. TBARS variability: absorbance values at 532 nm for different batches of linoleic acid. Absorbance values show TBARS variability in a multi‐phase oxidation system in measuring antioxidant activity of 250 and 1000 µM Trolox.