Determination of the molar extinction coefficient for the ferric reducing/antioxidant power assay

The FRAP reagent contains 2,4,6-tris(2-pyridyl)-s-triazine, which forms a blue–violet complex ion in thepresence of ferrous ions. Although the FRAP (ferric reducing/antioxidant power) assay is popular and hasbeen in use for many years, the correct molar extinction coefficient of this complex ion under FRAP assayconditions has never been published, casting doubt on the validity of previous calibrations. A previouslyreported value of 19,800 is an underestimate. We determined that the molar extinction coefficient was21,140. The value of the molar extinction coefficient was also shown to depend on the type of assayand was found to be 22,230 under iron assay conditions, in good agreement with published data. Redoxtitration indicated that the ferrous sulfate heptahydrate calibrator recommended by Benzie and Strain,the FRAP assay inventors, is prone to efflorescence and, therefore, is unreliable. Ferrous ammonium sulfatehexahydrate in dilute sulfuric acid was a more stable alternative. Few authors publish their calibrationdata, and this makes comparative analyses impossible. A critical examination of the limited numberof examples of calibration data in the published literature reveals only that Benzie and Strain obtained asatisfactory calibration using their method