Optimal Incubation Time and Use of Random Methylated β-Cyclodextrin (RMCD) to Improve the Solubility of Lipophilic Antioxidants for the Frap Antioxidant Capacity Assay

FRAP is one of the most widely used methods to measure the antioxidant capacity of foods. However, there are some major problems: first, there is no standard time for reading the FRAP value; second, the end point of the FRAP reaction differs among antioxidant compounds and among samples; third, the reaction occurs in aqueous solution, making it difficult to measure the antioxidant capacity of lipophilic compounds. Therefore, we evaluated the linearity of the FRAP values at 593 nm as the reaction progressed through the most commonly reported reading times. Also, we tested random methylated β-cyclodextrin (RMCD), which has been used to improve the solubility and resolution of the lipophilic fraction in other antioxidant assays. The FRAP values of pure antioxidant compounds commonly found in tomatoes (ascorbic acid, gallic acid, lycopene, β-carotene, and lutein) and Trolox (the accepted standard), were determined at seven different concentrations; and also in red, orange, and yellow tomato cultivars with or without lycopene added. The hydrophilic and lipophilic fractions of pure antioxidants and fruit samples were measured under standard conditions and using RMCD. The reaction was monitored every 1 minute for 60 minutes. The linearity of the FRAP value versus antioxidant concentration was evaluated at 1, 4, 8, 15, 30, and 60 minutes by comparing the linear regression parameters R² and slope. Also, we compared the FRAPsubTE values of the tomato samples at selected reading times. We found that the FRAP reaction increased for every antioxidant compound except for Trolox, which tended to decrease. Time significantly increased the FRAP sensitivity for ascorbic acid (P=0.0002), gallic acid (P<0.0001), lutein (P=0.0101), β-carotene (P=0.0549), and lycopene (P=0.0073), whereas the sensitivity for Trolox decreased (P=0.0002). Only in the case of β-carotene did time positively affect precision (P=0.0173). Furthermore, the sensitivity for lipophilic antioxidants was greatly increased by RMCD only in the case of lutein (P<0.0001), while RMCD had strong limitations in keeping highly lipophilic compounds like β-carotene and lycopene in solution. Even though standard conditions produced higher sensitivity for β-carotene (P<0.0001) and lycopene (P<0.0001), RMCD increased the precision in the case of lutein and β-carotene. Similarly absorbance constantly increased over time for every tomato sample and RMCD produced a positive effect on the absorbance of the lipophilic fraction. In conclusion, the FRAP reaction should be conducted for at least 30 minutes to accurately measure the antioxidant capacity and to avoid losing the Trolox standard resolution.