An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant’s scavenging capacity against AAPH-derived free radicals

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC–FL) is an indirect method that monitors the antioxidant’s ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC–EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC–FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC–EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC–FL, but absolute values showed significant discrepancies. ORAC–EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.