A new approach to assess the total antioxidant capacity using the TEAC assay Mariken J.T.J. Arts * , J. Sebastiaan Dallinga, Hans-Peter Voss, Guido R.M.M. Haenen, Aalt Bast Department of Pharmacology and Toxicology, Faculty of Medicine, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands Received 1 August 2003; received in revised form 5 February 2004; accepted 5 February 2004 Abstract The trolox equivalent antioxidant capacity (TEAC) assay is a popular method for assessing the capacity of a compound to scavenge ABTS radicals (ABTS Å ). Under the conditions in which the assay is performed, the reaction between most antioxidants and ABTS Å does not reach completion within the time span applied. This leads to an underestimation of the TEAC of these antioxidants. In the present study, incubations with different concentrations of ABTS Å and a fixed concentration of antioxidant were performed. The decrease in ABTS Å concentration in 6 min was plotted against the initial concentration of ABTS Å and fitted by an exponential function. Extrapolation of the fit to an infinite excess of ABTS Å gives the maximal concentration of ABTS Å that can be scavenged by the antioxidant at the concentration employed. This can be used to determine the actual TEAC of antioxidants, i.e. the total an- tioxidant capacity. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: TEAC; Antioxidant capacity; ABTS radical 1. Introduction The trolox equivalent antioxidant capacity (TEAC) assay (Miller, Rice-Evans, Davies, Gopinathan, & Mil- ner, 1993) is widely applied to assess the amount of radicals that can be scavenged by an antioxidant, i.e. the antioxidant capacity (Lien, Ren, Bui, & Wang, 1999; Plumb, Price, & Williamson, 1999; Rice-Evans, Miller, & Paganga, 1996). In the most recent version of this assay, an antioxidant is added to a pre-formed ABTS radical (ABTS Å ) solution and, after a fixed time period, the remaining ABTS Å is quantified spectrophotometri- cally (Berg van den, Haenen, Berg van den, & Bast, 1999; Re et al., 1999; Williamson, Plumb, & Garcia- Conesa, 1999). The reduction in ABTS Å concentration, induced by a certain concentration of antioxidant, is related to that of trolox and gives the TEAC value of that antioxidant. The assay is rapid, easy and correlates with the biological activity of antioxidants (Berg van den, Haenen, Berg van den, Vijgh van der, & Bast, 2000; Rezk, Haenen, Vijgh van der, & Bast, 2003). A major problem associated with the TEAC assay is that substantial differences in reported TEAC values of an antioxidant are observed, e.g. the TEAC of quercetin varies from 3.1 (Re et al., 1999) to 6.4 (Berg van den et al., 1999). The variation cannot only be ascribed to differences in procedure. Also, with the same procedure, the TEAC of a compound may vary. With the same method, the TEAC of quercetin at a concentration of 1.5 or 1.0 lM is reported to be 5.6 or 6.4, respectively (Berg van den et al., 1999). The aim of this study is to develop a procedure to determine the true total antiox- idant capacity of a compound that is independent of the concentration of the antioxidant. 2. Materials and methods 2.1. Chemicals 2,2 0 -Azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2, 5,7,8-tetramethylchroman-2- * Corresponding author. Tel.: +31-43-3881340; fax: +31-43-3884149. E-mail address: mariken.arts@farmaco.unimaas.nl (M.J.T.J. Arts). 0308-8146/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2004.02.008 Food Chemistry 88 (2004) 567–570 www.elsevier.com/locate/foodchem Food Chemistry