Reexamination of the ORAC assay: effect of metal ions

Original Paper

DOI: 10.1007/s00216-011-4884-8

Cite this article as:
Nkhili, E. & Brat, P. Anal Bioanal Chem (2011) 400: 1451. doi:10.1007/s00216-011-4884-8


The oxygen radical absorbance capacity (ORAC) assay method has been employed extensively in the field of antioxidant and oxidative stress. It uses fluorescein as probe for oxidation by peroxyl radical. Hundreds of reports have been published on the use of this method to determine antioxidant capacity in food and biological samples. The question is whether the results of all these reports are influenced by antioxidant autoxidation, which occurs during the ORAC test. Indeed, the presence of metal ions in the studied matrix will influence antioxidant stability, thereby leading to the underestimation of their antioxidant properties. Ethylenediaminetetraacetic acid hydrate (EDTA) can be used as a metal complexation agent. This paper examines the effect of the addition of EDTA on the ORAC values of pure compounds (quercetin, ascorbic, and dehydroascorbic acid) and five food juices (kiwi, orange, tomato, red grape, and apple). Metal complexation by EDTA (80 μM) clearly increased the ORAC values, given that the antioxidant was protected against rapid autoxidation incited by trace metal ions within samples and then by free radicals. Our finding also undoubtedly demonstrated that the number of literature values is potentially underestimated.


ORAC Antioxidant activity Phenolic compounds Metal chelator Fruit juices 



Ascorbic acid


2,2′-Azobis(2-methylpropionamidine) dihydrochloride


Dehydroascorbic acid


Ethylenediaminetetraacetic acid hydrate


Oxygen radical absorbance capacity


Phosphate buffer without EDTA


Phosphate buffer with EDTA


Reactive oxygen species


Trolox equivalent

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Département PERSYSTCentre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD)Montpellier Cedex 5France

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