Application of the 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) Radical Cation Assay to a Flow Injection System for the Evaluation of Antioxidant Activity of Some Pure Compounds and Beverages NICOLETTA PELLEGRINI,* DANIELE DEL RIO,BARBARA COLOMBI, MARTA BIANCHI, AND FURIO BRIGHENTI Department of Public Health, University of Parma, Via Volturno, 39, 43100 Parma, Italy The 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS •+ ) assay was adapted to a flow injection (FI) system to obtain a sensitive and rapid technique for the monitoring of antioxidant activity of pure compounds and complex matrixes, such as beverages and food extracts. The FI system includes a HPLC pump that flows the mobile phase (a solution of ABTS •+ in ethanol) through a 20 μL loop injector, a single bead string reactor filled with acid-washed silanized beads, a delay coil and a photodiode array UV-visible detector. The technique was very sensitive, with limits of detection and of quantification of 4.14 and 9.29 μmol of Trolox/L, respectively, and demonstrated high repeatability and reproducibility. The proposed technique was then applied to the evaluation of the antioxidant activity of some pure compounds, demonstrating good agreement with published data obtained by the original spectrophotometric ABTS •+ assay. Finally, the total antioxidant activity of 10 beverages was determined by both the proposed and the original method. The values ranged from 0.09 mmol L -1 for cola to 49.24 mmol L -1 for espresso coffee and did not result significantly different from those obtained by the original spectrophotometric ABTS •+ assay (Student’s paired t-test:t ) 1.4074, p ) 0.1929). In conclusion, the proposed FI technique seems suitable for the direct, rapid and reliable monitoring of total antioxidant activity of pure compounds and beverages and, due to the ability to operate in continuous, it allows the analysis of about 30 samples h -1 making the assay particularly suitable for large screening of total antioxidant activity in food samples. KEYWORDS: Antioxidant activity; ABTS radical cation assay; antioxidant compounds; beverages; method INTRODUCTION Due to their chemical reactivity, free radicals and other reactive oxygen species, derived either from normal metabolic processes or from external sources, can damage all types of cellular macromolecules. These effects have been implicated in the causation of some degenerative diseases, such as cataracts, atherosclerosis, and certain types of cancer (1-4). The consumption of antioxidant-rich foods might play an important role in the maintenance of health and in disease prevention (5). Epidemiological studies have demonstrated an inverse association between the intake of antioxidants from fruits and vegetables and the morbidity and mortality from coronary heart diseases (6, 7) and cancer (8-10). Nevertheless, many clinical studies have not shown direct beneficial effects of individual antioxidant molecules, such as vitamin E and -carotene, on various chronic diseases (11-13) suggesting that the functionality of dietary antioxidants might be strongly linked to cooperative mechanisms among different antioxidant mol- ecules present in the matrix. On the basis of these observations, a number of assays have been introduced in the past decade for determining the total antioxidant activity (intended as the cumulative capacity of food components to scavenge free radicals) of food extracts and beverages (14-16). These assays diverge in that they relate to the generation of different radicals, often acting through different mechanisms and/or target mol- ecules, and the measures are made on a range of different end- points (17). In general, two types of approach have been taken: (1) inhibition assays, for which the extent of the scavenging of a preformed free radical by hydrogen or electron donation is the marker of antioxidant activity; (2) assays involving the presence of antioxidant systems during the generation of the radical, for which the activity is measured on the rate of oxidation of a target molecule. The quenching of the 2,2′-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid) radical cation (ABTS •+ ) forms the basis of a spectrophotometric method that, thanks to a recent improvement in its chemistry, allows the evaluation of both water-soluble and lipid-soluble antioxidants (18). The method is a decolori- zation assay that, after the addition of an antioxidant, measures * To whom correspondence should be addressed. Tel. +39 0521 903835. Fax: + 39 0521 903832. E-mail: mailnico@nemo.unipr.it. 260 J. Agric. Food Chem. 2003, 51, 260-264 10.1021/jf020657z CCC: $25.00 © 2003 American Chemical Society Published on Web 11/23/2002