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.

A novel high-throughput image based rapid Folin-Ciocalteau assay for assessment of reducing capacity in foods

“…Similar changes in the brightness intensity were shown with different water volume ( Figure 3C), which might be associated with the retention of incident light of the scanner due to a refraction phenomenon produced by the aqueous solution. On the other hand, the optical pathlength in a microplate is not fixed and it is directly related to the sample volume [5], which explains the increases of the slope of the standard curve using a 315 µL volume because the slope is directly related to the optical pathlength and the chemical nature of chromophore [5].…”

“…In this context, the natural antioxidants contained in foods, fruits, beverages, spices, and supplements have received much attention for the prevention or treatment of diseases [3] and therefore antioxidant capacity is widely assessed in food science and nutrition research [4][5][6][7][8][9][10][11].…”

A FRAP Assay at pH 7 unveils Extra Antioxidant Activity from Green, Black, White and Rooibos Tea but not Apple Tea

“…The TptzFRAP (pH 3.6) regent was produced as described by Benzie and Strain [4] and adapted for microplate format described recently [16].…”

“…A particular limitation relevant to foods is that polyphenols from fruits and vegetables show decreasing antioxidant activity at low pH [10][11][12][13][14][15] which might affect results obtained with the TptzFRAP (pH3.6) method. Our laboratory recently highlighted the benefits of calibrating the microplate TptzFRAP (pH 3.6) assay, using a manual pathlength correction, to achieve results consistent with a 1-cm spectrophotometer but retaining the high throughput characteristics so important for basic research [16].…”

“…The overall aims of this study were, to evaluate the antioxidant activity of tea infusions at pH 7.0 using a ferrozine based FRAP (FzFRAP(pH 7)) assay adapted to the 96-well format and to compare the results with values obtained using the calibrated microplate TptzFRAP(pH 3.6) assay reported recently and which we applied to honey samples not tea [16]. In this investigation the antioxidant activity for several pure antioxidant compounds were also tested as a function of pH.…”