Classical vs Modified Folin–Ciocalteu Assay for Total Phenolic Content Determination in Honey

The total phenolic content (TPC), determined by Folin–Ciocalteu (FC) assay in an alkaline medium (pH 10–12), is an important honey quality indicator. To avoid the interference of honey main components (fructose and glucose), FC analysis in a weakly alkaline (pH 8) medium was recently proposed. Here a systematic study of polyphenols and sugars reactivity in FC assay at pH 8–10 is presented. Particularly, lowering the pH causes a noticeable decrease in the reactivity of flavonoids, which are important components of honey polyphenols. Consequently, at pH 8 the TPC is significantly underestimated, as the flavonoids are missed. Furthermore, the presence of sugars resulted in an overestimation of TPC at both pH 8 and 10, caused primarily by a coupled reaction where polyphenols induce additional FC reagent reduction by sugars, rather than a simple additive effect. Thus, the original pH 10 conditions ensure more uniform polyphenol recovery and better metrological comparability of honey analysis results.