Total Antioxidant Capacity Assay Using Optimized Ferricyanide/Prussian Blue Method

The existing ferricyanide/Prussian blue assay of reducing capacity measurement was optimized so as to obtain a more reproducible, linear and additive response from antioxidants. The modification involved the simultaneous use of ferricyanide and iron(III) to regulate more favorable redox conditions for a greater variety of antioxidants. Prussian blue precipitation was hindered with the addition of sodium dodecyl sulfate, and the optimal pH was adjusted to 1.7 to maintain the redox activity of ferric ion while preventing its hydrolysis. Incubation of the reaction mixture at room temperature for 30 min enabled more complete oxidations than observed in the conventional ferricyanide method. The order of trolox equivalent antioxidant capacities was quercetin > rosmarinic acid > gallic acid > ferulic acid ≥ catechin > caffeic acid ≥ rutin ≥ ascorbic acid ≈ trolox. Synthetic antioxidant mixtures gave the theoretically expected total antioxidant capacities conforming to Beer’s law. The assay was nonresponsive to simple sugars and citric acid (which are not true antioxidants) but responsive to biologically important thiols which are not oxidized by other Fe(III)-based assays. The assay was used in real sample solutions by using the method of standard additions to green tea, nettle, and sage, and validated against other similar antioxidant assays.