Microchim Acta (2008) 160: 413–419 DOI 10.1007/s00604-007-0777-0 Printed in The Netherlands Original Paper Mechanism of antioxidant capacity assays and the CUPRAC (cupric ion reducing antioxidant capacity) assay Res ° at Apak, Kubilay Gu ¨c ° lu ¨, Mustafa O ¨ zyu ¨rek, Saliha Esin C ° elik Department of Chemistry, Faculty of Engineering, Istanbul University, Istanbul, Turkey Received November 29, 2006; accepted March 21, 2007; published online May 21, 2007 # Springer-Verlag 2007 Abstract. We report on the application of a simple and versatile antioxidant capacity assay for dietary polyphenols, vitamin C and vitamin E utilizing the copper(II)-neocuproine (Cu(II)-Nc) reagent as the chromogenic oxidant, which we term the CUPRAC (cupric reducing antioxidant capacity) method. It in- volves mixing the antioxidant solution (directly or after acid hydrolysis) with solutions of CuCl 2 , neocuproine, and ammonium acetate at pH 7, and measuring the absorbance at 450 nm after 30 min. Slowly reacting antioxidants required an incubation at 50  C for 20 min for color development. The flavonoid glycosides were hydrolyzed to their corresponding aglycones by re- fluxing in 1.2 M HCl-containing 50% MeOH for fully exhibiting their antioxidant potencies. Certain compounds also needed incubation after acid hydro- lysis for color development. The CUPRAC absorbances of mixture constituents were additive, indicating lack of chemical deviations from Beer’s law. The CUPRAC antioxidant capacities of a wide range of polyphenolics are reported in this work and compared to those found by ABTS=persulfate and Folin assays. The trolox-equivalent capacities of the antioxidants were linearly correlated (r ¼ 0.8) to those found by ABTS but not to those of Folin. The highest antioxidant capacities in the CUPRAC method were observed for epicatechin gallate, epigallocatechin gallate, quercetin, fisetin, epigallocatechin, catechin, caffeic acid, epicatechin, gallic acid, rutin, and chlorogenic acid in this order, in accordance with theoretical expec- tations. The experiences of other CUPRAC users also are summarized. Keywords: CUPRAC antioxidant capacity; dietary flavonoids; polyphenols; copper(II)-neocuproine; spectrophotometry Reactive oxygen species (ROS) that emerge as a result of the respirative cycle of oxidative phosphorylation may attack biological macromolecules like cellular DNA and proteins. Excessive ROS may give rise to single- and double-strand DNA breaks that may eventually cause cell ageing, cardiovascular diseases, mutagenic changes and cancerous tumor growth [1, 2]. Consumption of foods naturally bearing antioxidant power is the most efficient way of combating such undesired transforma- tions and health risks. Consequently, the opportunity for improving health by improving diet is great [3]. The chemical diversity of antioxidants makes it dif- ficult to separate and quantify individual antioxidants (i.e., parent compounds, glycosides, polymers, and many isomers) from the vegetable matrix. Moreover, the total antioxidant power is often more meaningful to evaluate health beneficial effects because of the coop- erative action of antioxidants. Therefore it is desirable to establish a method that can measure the total anti- Correspondence: Res ° at Apak, Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, TR-34320 Istanbul, Turkey, e-mail: rapak@istanbul.edu.tr