Optimisation and Validation of a Microscale FRAP Assay with Matrix-Matched (Interaction-Corrected) Blanking for...

This study optimised and validated a microplate Ferric-Reducing Antioxidant Power (FRAP) assay for honey that incorporates a honey-specific matrix-matched blank-a paired absorbance measurement lacking the chromogenic probe 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ)-to separate true ferric reduction from non-reductive Fe³⁺-matrix interactions that otherwise inflate apparent antioxidant capacity. A 30 min incubation at 37 °C in the dark was selected as the optimal compromise between reaction completeness and reagent/matrix stability, using 20 µL sample and 180 µL FRAP reagent per well (absorbance at 620 nm). The assay was validated according to International Council for Harmonisation (ICH) principles, showing linearity for FeSO₄·7H₂O from 200-1200 µM (R² ≥ 0.9988), Limit of detection/limit of quantification (LOD/LOQ) of 0.28/0.94 mmol Fe²⁺ equivalents kg^-1, recoveries of 101-103%, and precision ≤ 5% relative standard deviation (RSD). All tested individual sugars except maltodextrin and the artificial honey matrix produced FRAP responses below the LOQ. Flavonoids, however, showed pronounced, time-dependent, non-reductive Fe³⁺ interactions and were markedly overestimated under conventional water blanking, while phenolic acids were less affected. Applied to 47 Western Australian honeys, the validated assay yielded FRAP activities of 2.80-9.52 mmol Fe²⁺ kg^-1; matrix-matched blanking gave consistently lower, more chemically specific values than conventional water blanking, with corrections ranging up to >30% depending on honey type. Matrix-matched blanking is therefore essential, not optional, for accurate FRAP measurement in honey, and the same principle should apply directly to other phenolic-rich foods like wine, tea, and fruit extracts-wherever iron-polyphenol interactions confound conventional antioxidant assays.