Trolox Equivalent Antioxidant Capacity (TEAC) Assay

The Trolocks equivalent antioxidant capacity (TEAC) assay measures the antioxidant capacity of a given substance, as compared to the standard, Trolox. Most commonly, antioxidant capacity is measured using the ABTS Decolorization Assay. Other antioxidant capacity assays which use Trolox as a standard include the diphenylpicrylhydrazyl (DPPH), oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP) assays. The TEAC assay is often used to measure the antioxidant capacity of foods, beverages and nutritional supplements.^[1]

The trolox equivalent antioxidant capacity (TEAC) assay was first developed as a simple and convenient method for total antioxidant capacity (TAC) determination (Miller et al., 1993). The assay measures the ability of antioxidants to scavenge the stable radical cation ABTS+">ABTS+ (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)), a blue-green chromophore with maximum absorption at 734 nm that decreases in its intensity in the presence of antioxidants. Antioxidants can neutralize the radical cation ABTS+">ABTS+, generated from ABTS, by either direct reduction via electron donation or by radical quenching via hydrogen atom donation, and the balance of these two mechanisms is generally determined by antioxidant structure and pH of the medium (Prior et al., 2005). Hence, although TEAC assay is usually classified as an ET-based method, the HAT mechanism also applies.

The extent of discoloration of the blue-green color, quantified as a decrease in absorbance at 734 nm, depends on the duration of reaction, the intrinsic antioxidant activity, and concentration in the sample. Similar to other radical scavenging assays discussed above, the TEAC assay can either monitor the absorbance decay with time or the drop at a certain end point. When operated as an end point assay, the TEAC method requires reliable determination of an end point of the reaction for quantitative evaluation of antioxidant activity (Huang et al., 2005). Results obtained by TEAC assay are reported as trolox equivalents.

In the original TEAC assay, metmyoglobin and hydrogen peroxide were used to generate an intermediate ferrylmyoglobin radical, which then reacted with ABTS to produce ABTS+">ABTS+. The oxidizing agent was later replaced by peroxide or persulfate. Potassium persulfate is the most commonly used oxidant for ABTS+">ABTS+ generation.

Modifications of the original assay have been proposed (Prior et al., 2005). Durmaz (2012) prepared a ready-to-use radical powder of ABTS+">ABTS+ by incubating ABTS with potassium peroxodisulfate followed by freeze-drying of the resultant radical solution. This freeze-dried radical powder was then used as a replacement for the freshly prepared radical solution, and its linearity and accuracy of the modified assay was demonstrated by the authors. In another version of the assay, the radical anion, ABTS-">ABTS-, was used as the target radical instead of the cation. ABTS⋅−">ABTS⋅− was generated via oxidation of ABTS2− by potassium persulfate, and the radical anion ABTS-">ABTS- was reduced back by antioxidants to the colorless product ABTS2-">ABTS2- (Huang et al., 2005).

The TEAC assay has been used to measure the total antioxidant activity of pure substances, body fluids, and plant materials. TEAC assay, similar to other radical scavenging methods, can be automated and adapted to microplates and flow injection techniques (Milardovic, Kerekovic, & Rumenjak, 2007). It may also be coupled with HPLC by including a post-column reaction with ABTS radical cation to facilitate the search of individual antioxidants from a complex mixture. HPLC-TEAC provides an efficient and rapid method for separation and identification of bioactive compounds from the source material (Gong, Plander, Xu, Simandi, & Gao, 2011;

Raudonis, Bumblauskiene, Jakstas, Pukalskas, & Janulis, 2010). Better sensitivity and precision of the TEAC assay can be achieved when coupled with other detection techniques, such as amperometric (Chan-Eam, Teerasong, Damwan, Nacapricha, & Chaisuksant, 2011; Milardovic et al., 2007) and FTIR (Cerretani et al., 2010), among others. Many of these modified TEAC assays employ online enzymatic generation of ABTS">ABTS. For example, Milardovic et al. (2007) reported generation of ABTS+">ABTS+ by glucose oxidase and peroxidase separately immobilized in tubular flow-through reactors for flow injection analysis of alcoholic beverages for their TEAC values.