The coloring reaction of the thiobarbituric acid test for hydroperoxides was completely inhibited by the addition of EDTA. Therefore, it was necessary to add a metal salt to the reaction mixture to complete the reaction and also to add an antioxidant to prevent autoxidation when unoxidized unsaturated fatty acids co‐exist. The optimal pH of the reaction was found at 3.6 using glycine‐hydrochloric acid buffer.

In order to understand the effect of α-tocopherol on the autoxidation mechanism of edible oil under storage conditions, methyl linoleate was allowed to autoxidize at 50 C in bulk phase without any radical initiator. The reaction was monitored by determining the production of four isomeric hydroperoxides (13-cis,trans; 13-trans,trans; 9-cis,trans; 9-trans,trans) by high performance liquid chromatographic analysis after reduction. In the absence of α-tocopherol, the rate of autoxidation depended on the sample size, and the duration of the induction period was affected by the initial level of hydroperoxides. However, the distribution of c-t and t-t hydroperoxide isomers remained constant during the propagation period regardless of the sample size. The addition of α-tocopherol at 0.1 and 1.0% caused a linear increase in the amount of hydroperoxides and elevated the distribution of the c-t isomers. The rate of hydroperoxidation appeared to be governed by the initial concentration of α-tocopherol rather than the sample size or the initial hydroperoxide level. This peroxidizing effect of α-tocopherol was suppressed by the presence of ascorbyl palmitate. A mechanism in which chromanoxy radical participates is proposed for the effect of α-tocopherol on lipid autoxidation in bulk phase. It is therefore suggested that α-tocopherol at high concentrations influences the mechanism of autoxidation of edible oil.

The thiobarbituric acid (TBA) test has been used in the field of medical science in recent years to detect lipid peroxides. In this case, it is necessary for hydroperoxides to be decomposed to secondary products during the reaction. When purified methyl linoleate and methyl linolenate monohydroperoxides were used as the sample for the TBA test, they did not decompose entirely to secondary products, but did so completely when an iron catalyst (ferrous sulfate) was added. However, the iron catalyst also accelerated the autoxidation of coexisting unsaturated fatty acids. Therefore, the addition of antioxidants was required. Fifteen min of heating was sufficient to complete the reaction. With additions of catalyst and antioxidant to the TBA test, it may be possible to make useful distinctions between hydroperoxides and secondary products of lipid oxidation.