Development of a potential reference material for evaluating antioxidant activity

Phenolic phytochemical are known to perform several functions ranging from phytoprotectants, to protecting lipids in food products, to antioxidant activity in animals and humans. The need for a common standard mixture containing multiple phenolic phytochemicals is critical for the development of a robust validation assay for accurately quantifying antioxidant activity in various matrixes. Different research groups have used A wide array of single purified reference phenolic compounds in this regard. A 5 compound mixture (caffeic acid, morin hydrate, hesperetin, catechin hydrate, and epigallocatechin gallate) containing phenolic compounds from 4 subgroups (phenolic acid, flavone, flavanone, and flavan-3-ol) was prepared. The mixture was assayed for stability evaluation by high-performance liquid chromatography (HPLC) using a diode array detection procedure for a 3 month time interval. HPLC analysis confirmed that there was no significant interaction between different components of the mixture. The among-sample relative standard deviation (RSD) of all 5 phenolic compounds, as well as the total HPLC area, was < 1%. The RSD due to instrument variation was < 2% and the total RSD among-days was < 5%. These results unambiguously suggest that the sample was stable for a 3 month time interval in an amber vial stored in a refrigerator below 5 degrees C. This mixture is currently being used for the single-laboratory validation study for the assay of total phenolic content by the Folin-Ciocalteu method and the antioxidant capacity by oxygen radical absorbing capacity procedure.