Comparison of conventional antioxidants assays for evaluating potencies of natural antioxidants as food additives by collaborative study

In recent years, there has been a need for a new quality criterion for antioxidants based on its antioxidative activity in Japan. In particular, novel official methods of evaluating the antioxidative activities of food additives are required. In order to investigate the possibility of developing official methods, the reproducibilities of three simple conventional antioxidant assays (DPPH assay, ABTS assay, and WST-1 assay) based on spectrophotometric detection were evaluated by collaborative study between three laboratories. DPPH and ABTS assays were performed to evaluate the antioxidant activity (TEAC, Trolox equivalent activity) of all nine antioxidants used in this study, whereas the WST-1 assay was suitable for evaluating the superoxide dismutase (SOD)-equivalent activity of seven antioxidants (excepting D-α-tocopherol and D-δ-tocopherol). The HorRat values from the collaborative study revealed that the DPPH and ABTS assays showed higher reproducibility than the WST-1 assay. It was assumed that the lower reproducibility would be due to the measuring principle, because the WST-1 assay, unlike the DPPH and the ABTS assays, is a competitive enzyme assay. Based on the results, we concluded that the DPPH and ABTS assays would be candidates in validation studies of methods for the purpose of developing an official methodology.