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

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 Fe2+ 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 Fe2+ per 100 g honey) were strongly correlated with UMF ratings (R2 = 0.977) and total phenols content (R2 = 0.982)whilst the UMF rating was correlated with the total phenols (R2 = 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.