Abstract
An improved method for the measurement of oxygen radical absorbance capacity (ORAC) was developed and validated using fluorescein (3′,6′-dihydroxyspiro[isobenzofuran-1[3H], 9′[9H]-xanthen]-3-one) as a new fluorescence probe (ORACFL). Randomly methylated β-cyclodextrin (RMCD) was introduced as the water-solubility enhancer for lipophilic antioxidants. 7% RMCD (w/v) in 50% acetone–H2O mixture sufficiently solubilized vitamin E compounds and other lipophilic phenolic antioxidants in 75 mM phosphate buffer (pH 7.4). Results indicated that fluorescein shows excellent photostability under the plate reader conditions. This ORACFL was validated through linearity, precision, accuracy, and ruggedness. The validation results demonstrated that the ORACFL assay is reliable and robust. The mean of intraday and interday CVs were <15%; for hydrophilic ORAC, LOD and LOQ are 5 and 6.25 μM, respectively; for lipophilic ORAC, LOD and LOQ are 6.25 and 12.5 μM, respectively. It is concluded that unlike other popular methods, the ORACFL assay provides a direct measure of total antioxidant capacity against the peroxyl radicals.
Method Validation by Interlaboratory Studies of Improved Hydrophilic Oxygen Radical Absorbance Capacity Methods for the Determination of Antioxidant Capacities of Antioxidant Solutions and Food Extracts
