This review summarizes the multifaceted aspects of antioxidants and the basic kinetic models of inhibited autoxidation and analyzes the chemical principles of antioxidant capacity assays. Depending upon the reactions involved, these assays can roughly be classified into two types: assays based on hydrogen atom transfer (HAT) reactions and assays based on electron transfer (ET). The majority of HAT-based assays apply a competitive reaction scheme, in which antioxidant and substrate compete for thermally generated peroxyl radicals through the decomposition of azo compounds. These assays include inhibition of induced low-density lipoprotein autoxidation, oxygen radical absorbance capacity (ORAC), total radical trapping antioxidant parameter (TRAP), and crocin bleaching assays. ET-based assays measure the capacity of an antioxidant in the reduction of an oxidant, which changes color when reduced. The degree of color change is correlated with the sample's antioxidant concentrations. ET-based assays include the total phenols assay by Folin-Ciocalteu reagent (FCR), Trolox equivalence antioxidant capacity (TEAC), ferric ion reducing antioxidant power (FRAP), "total antioxidant potential" assay using a Cu(II) complex as an oxidant, and DPPH. In addition, other assays intended to measure a sample's scavenging capacity of biologically relevant oxidants such as singlet oxygen, superoxide anion, peroxynitrite, and hydroxyl radical are also summarized. On the basis of this analysis, it is suggested that the total phenols assay by FCR be used to quantify an antioxidant's reducing capacity and the ORAC assay to quantify peroxyl radical scavenging capacity. To comprehensively study different aspects of antioxidants, validated and specific assays are needed in addition to these two commonly accepted assays.

An improved method of oxygen radical absorbance capacity (ORAC) assay has been developed and validated using fluorescein (3',6'-dihydroxyspiro[isobenzofuran-1[3H],9'[9H]-xanthen]-3-one) as the fluorescent probe. Our results demonstrate that fluorescein (FL) is superior to B-phycoerythrin. The oxidized FL products induced by peroxyl radical were identified by LC/MS, and the reaction mechanism was determined to follow a classic hydrogen atom transfer mechanism. In addition, methodological and mechanistic comparison of ORAC(FL) with other widely used methods was discussed. It is concluded that, unlike other popular methods, the improved ORAC(FL) assay provides a direct measure of hydrophilic chain-breaking antioxidant capacity against peroxyl radical.

The oxygen radical absorbance capacity (ORAC) assay has been widely accepted as a standard tool to measure the antioxidant activity in the nutraceutical, pharmaceutical, and food industries. However, the ORAC assay has been criticized for a lack of accessibility due to the unavailability of the COBAS FARA II analyzer, an instrument discontinued by the manufacturer. In addition, the manual sample preparation is time-consuming and labor-intensive. The objective of this study was to develop a high-throughput instrument platform that can fully automate the ORAC assay procedure. The new instrument platform consists of a robotic eight-channel liquid handling system and a microplate fluorescence reader. By using the high-throughput platform, the efficiency of the assay is improved with at least a 10-fold increase in sample throughput over the current procedure. The mean of intra- and interday CVs was

Methods are described for the extraction and analysis of hydrophilic and lipophilic antioxidants, using modifications of the oxygen radical absorbing capacity (ORAC(FL)) procedure. These methods provide, for the first time, the ability to obtain a measure of "total antioxidant capacity" in the protein free plasma, using the same peroxyl radical generator for both lipophilic and hydrophilic antioxidants. Separation of the lipophilic and hydrophilic antioxidant fractions from plasma was accomplished by extracting with hexane after adding water and ethanol to the plasma (hexane/plasma/ethanol/water, 4:1:2:1, v/v). Lipophilic and hydrophilic antioxidants were efficiently partitioned between hexane and aqueous solvents. Conditions for controlling temperature effects and decreasing assay variability using fluorescein as the fluorescent probe were validated in different laboratories. Incubation (37 degrees C for at least 30 min) of the buffer to which AAPH was dissolved was critical in decreasing assay variability. Lipophilic antioxidants represented 33.1 +/- 1.5 and 38.2 +/- 1.9% of the total antioxidant capacity of the protein free plasma in two independent studies of 6 and 10 subjects, respectively. Methods are described for application of the assay techniques to other types of biological and food samples.