Stopped-flow method for assessment of pH and timing effect on the ABTS total antioxidant capacity assay

The pH and timing effect on the relative capacity of antioxidant compounds to scavenge the ABTS [2,2ʹ-azinobis(3-ethylbenzothiazoline-6-sulphonic acid)] radical cation (ABTS++) expressed as the trolox equivalent antioxidant capacity (TEAC) is assessed for a wide range of antioxidants using a modification of the original ABTS assay at different pHs (4.6, 5.4 and 7.4). To study both fast and slow reacting antioxidants, a stopped-flow method based on a low cost laboratory-made system was used. For most of the tested antioxidants, total antioxidant capacity values were found to be a function of pH and time allowed for the reaction. Structurally similar compounds have the same time and pH-dependent behaviour even if they have significant differences in total antioxidant capacity values. The ABTS + scavenging reaction rate was found to depend strongly on pH for p-coumaric acid, glutathione, BHT and albumin. Quercetin, gallic acid, (+)-catechin and (-)-epicatechin showed the higher activities at all pHs. The stopped-flow method can be utilized for screening of antioxidant compounds of unknown kinetics towards ABTS+ at different pHs and the results can be used to predict the total antioxidant capacity of structurally related compounds.