Development and Validation of Oxygen Radical Absorbance Capacity Assay for Lipophilic Antioxidants Using Randomly Methylated β-Cyclodextrin as the Solubility Enhancer

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Abstract
We recently reported the improved oxygen radical absorbance capacity (ORAC) assay using fluorescein (FL) as the fluorescent probe. The current ORACFL 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 ORACFL assay to lipophilic antioxidants. Randomly methylated β-cyclodextrin (RMCD) was introduced as the water solubility enhancer for lipophilic antioxidants. Seven percent RMCD (w/v) in a 50% acetone−H2O 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 ORACFL-LIPO) was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrate that the ORACFL-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 α-tocopherol, (+)-γ-tocopherol, (+)-δ-tocopherol, α-tocopherol acetate, tocotrienols, 2,6-di-tert-butyl-4-methylphenol, and γ-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 α-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. Keywords: ORAC; cyclodextrin; LC/MS; lipophilic antioxidants; vitamin E; chain breaking; hydrogen atom transfer