A universally calibrated microplate ferric reducing antioxidant power (FRAP) assay for foods and applications to Manuka honey

Abstract

The ferric reducing antioxidant power (FRAP) assay was recently adapted to a microplate format. However, microplate-based FRAP (mFRAP) assays are affected by sample volume and composition. This work describes a calibration process for mFRAP assays which yields data free of volume effects. From the results, the molar absorptivity (ε) for the mFRAP assay was 141,698 M(-1) cm(-1) for gallic acid, 49,328 M(-1) cm(-1) for ascorbic acid, and 21,606 M(-1) cm(-1) for ammonium ferrous sulphate. The significance of ε (M(-1) cm(-1)) is discussed in relation to mFRAP assay sensitivity, minimum detectable concentration, and the dimensionless FRAP-value. Gallic acid showed 6.6 mol of Fe(2+) equivalents compared to 2.3 mol of Fe(+2) equivalents for ascorbic acid. Application of the mFRAP assay to Manuka honey samples (rated 5+, 10+, 15+, and 18+ Unique Manuka Factor; UMF) showed that FRAP values (0.54-0.76 mmol Fe(2+) per 100g honey) were strongly correlated with UMF ratings (R(2)=0.977) and total phenols content (R(2) = 0.982)whilst the UMF rating was correlated with the total phenols (R(2) = 0.999). In conclusion, mFRAP assay results were successfully standardised to yield data corresponding to 1-cm spectrophotometer which is useful for quality assurance purposes. The antioxidant capacity of Manuka honey was found to be directly related to the UMF rating.

Keywords: 2,4,6-Tripyridyl-s-Triazine (PubChem CID: 77258); Ammonium ferrous sulphate (PubChem CID: 197097); Antioxidant capacity; Ascorbic acid (PubChem CID: 54670067); FRAP; Ferric reducing antioxidant power; Gallic acid (PubChem CID: 370); Manuka honey; Microplate assay.