Role of alkoxyl radicals on the fluorescein-based ORAC (Oxygen Radical Absorbance Capacity) assay.

During the last decades the ORAC (Oxygen Radical Absorbance Capacity) assay has been widely employed to evaluate the in vitro antioxidant capacity of polyphenol-rich fruits, vegetables and beverages. The method employs fluorescein (FLH) as target molecule and AAPH (2,2'-azo-bis(2-amidinopropane)dihydrochloride) as the source of peroxyl radicals (ROO•). The protection of FLH, afforded by antioxidants (XH), is often characterized by kinetic profiles with clear lag times (LT), which are directly associated with the stoichiometry (n) of the XH-ROO• reaction. However, even for simple phenolic compounds, the LT measured imply large n values (defined as the number of ROO• moles trapped by each antioxidant molecule) which cannot be explained by a simple reaction mechanism. Nonetheless, they can be explained when considering the formation of alkoxyl radicals (RO•) from the recombination of two AAPH-derived ROO•. In the present work, we provide kinetic data showing that, in the zero order kinetic limit of FLH consumption, there is a low reaction rate incompatible with total trapping of ROO•. Thus, the consumption of FLH should be mostly related to its reaction with RO•. In addition, we present data regarding the assumption that in competitive measurements, the LT is due to efficient trapping of the ROO• by the added phenols, leading to high n values (1.7 to 23) for mono and polyphenols. These values are not in agreement with kinetic studies of the antioxidant consumption mediated by the presence of AAPH carried out by HPLC-DAD technique, which imply a competition by RO•. The results suggest that the use of FLH as probe at low concentrations give, for several antioxidants, ORAC values mainly related to their reaction towards RO• radicals instead of primary ROO•radicals.

Mechanism of pyrogallol red oxidation induced by free radicals and reactive oxidant species. A kinetic and spectroelectrochemistry study.

Pyrogallol red (PGR) presents high reactivity toward reactive (radical and nonradical) species (RS). This property of PGR, together with its characteristic spectroscopic absorption in the visible region, has allowed developing methodologies aimed at evaluating the antioxidant capacity of foods, beverages, and human fluids. These methods are based on the evaluation of the consumption of PGR induced by RS and its inhibition by antioxidants. However, at present, there are no reports regarding the degradation mechanism of PGR, limiting the extrapolation to how antioxidants behave in different systems comprising different RS. In the present study, we evaluate the kinetics of PGR consumption promoted by different RS (peroxyl radicals, peroxynitrite, nitrogen dioxide, and hypochlorite) using spectroscopic techniques and detection of product by HPLC mass spectrometry. The same pattern of oxidation and spectroscopic properties of the products is observed, independently of the RS employed. Mass analysis indicates the formation of only one product identified as a quinone derivative, excluding the formation of peroxides or hydroperoxides and/or chlorinated compounds, in agreement with FOX's assays and oxygen consumption experiments. Cyclic voltammetry, carried out at different pH's, shows an irreversible oxidation of PGR, indicating the initial formation of a phenoxy radical and a second charge transfer reaction generating an ortho-quinone derivative. Spectroelectrochemical oxidation of PGR shows oxidation products with identical UV-visible absorption properties to those observed in RS-induced oxidation.