Development and validation of oxygen radical absorbance capacity assay for lipophilic antioxidants using randomly methylated beta-cyclodextrin as the solubility enhancer.

We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORAC(FL) assay is limited in hydrophilic antioxidant due to the aqueous environment of the assay. Lipophilic antioxidants mainly include the vitamin E family and carotenoids, which play a critical role in biological defense systems. In this paper, we expanded the current ORAC(FL) assay to lipophilic antioxidants. Randomly methylated beta-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone-H(2)O mixture was found to sufficiently solubilize vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). This newly developed ORAC assay (abbbreviated ORAC(FL-LIPO)) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORAC(FL-LIPO) assay is reliable and robust. For the first time, by using 6-hydroxy-2,5,7,8-tetramethyl-2-carboxylic acid as a standard (1.0), the ORAC values of alpha-tocopherol, (+)-gamma-tocopherol, (+)-delta-tocopherol, alpha-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and gamma-oryzanol were determined to be 0.5 +/- 0.02, 0.74 +/- 0.03, 1.36 +/- 0.14, 0.00, 0.91 +/- 0.04, 0.16 +/- 0.01, and 3.00 +/- 0.26, respectively. The structural information of oxidized alpha-tocopherol obtained by liquid chromatography/mass spectrometry reveals that the mechanism for the reaction between the vitamin E and the peroxyl radical follows the hydrogen atom transfer mechanism, which is in agreement with the notion that vitamin E is the chain-breaking antioxidant.