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The Folin-Ciocalteu assay is a widely used method for measuring the total phenolic content (TPC) in honey, but it can be affected by the presence of reducing sugars in honey, which can lead to interference and an over-estimation of its TPC. To optimize the Folin-Ciocalteu assay for honey analysis, the effect of pH on the assay was investigated. A number of pH scenarios were tested using different concentrations of Na2CO3 (0.00%, 0.75%, 0.94%, and 7.50%) in order to minimize reducing sugar interference and maximize the reaction of phenolics in the assay. The modified TPC method was then validated in accordance with current International Council on Harmonisation (ICH) guidelines. The findings of this study demonstrate that the traditional Folin-Ciocalteu assay (using 7.50% aqueous Na2CO3 solution, pH 10.8) leads to a significant overestimation of the TPC of honey due to the interference of reducing sugars. However, a pH of 7.9, achieved by using a 0.75% aqueous Na2CO3 solution, provides suitable conditions to account for most of the phenolic compounds without interference from reducing sugars. This finding was further confirmed by testing various sugar solutions and artificial honey which yielded TPC values below the established limit of detection and quantification of the assay. However, a slight increase in pH, even by a moderate deviation (pH 8.9), leads to significant discrepancies in absorbance readings, indicating that pH control is crucial for the accurate analysis of TPC in honey.
Lawag et al. (Tue,) studied this question.