Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe

J Agric Food Chem. 2001 Oct;49(10):4619-26. doi: 10.1021/jf010586o.


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.

MeSH terms

  • Antioxidants / analysis*
  • Antioxidants / chemistry
  • Chromans / chemistry
  • Chromatography, High Pressure Liquid
  • Fluorescein*
  • Fluorescent Dyes*
  • Food Analysis / methods*
  • Fruit / chemistry
  • Mass Spectrometry
  • Oxidation-Reduction
  • Peroxides / chemistry
  • Phycoerythrin
  • Reactive Oxygen Species / chemistry*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Tea / chemistry


  • Antioxidants
  • Chromans
  • Fluorescent Dyes
  • Peroxides
  • Reactive Oxygen Species
  • Tea
  • Phycoerythrin
  • perhydroxyl radical
  • 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
  • Fluorescein