A method to measure antioxidant activity in organic media: Application to lipophilic vitamins

Abstract

The 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS.+) can be generated by the enzymatic system formed by hydrogen peroxide and horseradish peroxidase in an organic medium. The ABTS radical is easily generated in acidified ethanol medium in about 100 s with a stability of 1.7 x 10-3 (-Δabs/min) monitored at 730 nm. Other organic solvents, such as methanol or acetone, have lower radical generation times but the radical is less stable. The addition of Trolox or a lipophilic antioxidant such as α-tocopherol or β-carotene produces a decrease in absorbance that can be used to estimate antioxidant capacity. Using a spectrophotometric end-point method and microplate-reader equipment, we have developed a method that estimates the antioxidant activity of different lipophilic vitamins. The use of Trolox as an antioxidant standard led to a limit of detection of 0.08 nmoles and limit of quantitation of 0.28 nmoles, while similar values were obtained for α-tocopherol and β-carotene. The relative antioxidant activity values obtained by different antioxidants showed that α-tocopherol has a similar antioxidant potential to Trolox and that β-carotene has 2.6 times the antioxidant potential of Trolox. In our opinion, this method can be useful for estimating the antioxidant activity in lipophilic samples and as a complement to other methods that measure antioxidant activity in aqueous media.