Monitoring monohydroperoxides in docosahexaenoic acid using high-performance liquid chromatography

The oxidation of free DHA has been investigated with respect to monohydroperoxides and polyhydroperoxides, which were analyzed with a novel HPLC method. The temperature and physical system, i.e., bulk and liposome, were varied. We have also studied the effects of antioxidants such as alpha-tocopherol, ascorbic acid, and juice from sea buckthorn on DHA. The HPLC method, which was performed isocratically, eluted eight peaks, each containing one or two isomers of monohydroperoxy-DHA. This method showed that the double bond farthest from the carboxyl group was easily oxidized, as shown by the rapid increase in the amount of C20-monohydroperoxy-DHA, which always provided the largest contribution to the total amount of monohydroperoxides. The monohydroperoxy-DHA containing the hydroperoxy group located on the double bond nearest the carboxyl group also was shown to increase considerably during an increase in the total amount of monohydroperoxides. This demonstrates that the double bonds located nearest and farthest from the carboxyl group were the most prone to hydroperoxide formation. DHA was more stable when stored in liposomes than as bulk. Addition of alpha-tocopherol to the DHA-containing liposomes reduced the oxidation of these double bonds. The antioxidant effect of alpha-tocopherol was prolonged when combined with ascorbic acid, since alpha-tocopherol was regenerated.