Optimization of a Miniaturized and Rapid Sample Preparation Method for HPLC Quantification of Flavonoids in Honey by Response Surface Methodology

Abstract The quantification of flavonoids in honey is often performed as they are considered to be markers of botanical origin and are associated with many health benefits of honey. Several extraction and sample preparation methods have been proposed for the recovery of flavonoids from honey, as it is a prerequisite for their chromatographic analysis. The main objective of this study was to optimize a miniaturized and rapid sample preparation method for the solubilization of honey flavonoids in a low volume of methanol using ultrasound irradiation. A Box–Behnken design has been conducted to study the effect of (i) temperature (40–80 °C), (ii) solvent-sample ratio (5–25 mL g −1 ), and (iii) solubilization time (5–35 min) on catechin, quercetin, and naringenin contents. The desirability index demonstrated that the maximum solubilization of honey flavonoids in methanol can be achieved by ultrasonication of honey at 40 °C for 5 min using methanol at a solvent-sample ratio of 10 mL g −1 . The confirmation test showed the closeness of the predicted results with those of experimental values. Finally, the optimized sample preparation method was applied successfully to the analysis of Cypriot multi-floral honeys. Overall, the novelty of this miniaturized sample preparation method is the direct ultrasound-assisted solubilization of honey flavonoids in a short time (5 min), using a low volume of methanol (3 mL) and a small size of sample (0.3 g) in comparison with existing methods.