Development and Validation of an Improved Oxygen Radical Absorbance Capacity Assay Using Fluorescein as the Fluorescent Probe

TLDR

An improved ORAC assay was developed and validated using fluorescein as the fluorescent probe, and its methodology and mechanism were compared with other widely used methods. The assay showed that fluorescein is superior to B‑phycoerythrin, its oxidized products were identified by LC/MS, the reaction follows a classic hydrogen‑atom transfer mechanism, and it provides a direct measure of hydrophilic chain‑breaking antioxidant capacity against peroxyl radicals.

Abstract

An improved method of oxygen radical absorbance capacity (ORAC) assay has been developed and validated using fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one) as the fluorescent probe. Our results demonstrate that fluorescein (FL) is superior to B-phycoerythrin. The oxidized FL products induced by peroxyl radical were identified by LC/MS, and the reaction mechanism was determined to follow a classic hydrogen atom transfer mechanism. In addition, methodological and mechanistic comparison of ORAC(FL) with other widely used methods was discussed. It is concluded that, unlike other popular methods, the improved ORAC(FL) assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radical.