Optimization of a miniaturized and green ultrasound-assisted sample preparation method for HPLC quantification of phenolic acid and flavonoids in honey

Honey is one of the most appreciated and valued natural products; it is used not only as a nutritional product but also in traditional medicine for the treatment of several clinical conditions [ 1 ].Polyphenols are linked with diverse health effects and considered as markers of botanical and geographical origin [ 2 ] [ 3 ]. The extraction of polyphenols from honey requires complicated and time-consuming extraction techniques due their low contents and matrix particularity [ 4 ]. The objective of this study was to develop and optimize the direct ultrasound-assisted solubilization of phenolic acids and flavonoids in honey samples before chromatographic separations. Thus, Box-Behnken designs have been conducted to study the effect of: (i) percentage of alcohol, (ii) temperature, (iii) solvent-honey ratio and (iv) sonication time on the contents of phenolic acids and flavonoids. The desirability index demonstrated that the maximum solubilization of honey flavonoids in pure methanol can be achieved by ultrasonication of honey at 40°C for 5 min using methanol at solvent-sample ratio of 10 mL x g. Regarding to phenolic acids, the use of 60.0% (v/v) ethanol at a solvent-honey ratio of 10 mL x g −1 for a period of 17.8 min at a temperature of 54.6 o C resulted in their maximum solubilization. Furthermore, the optimized sample preparation methods were applied successfully for the analysis of multifloral honey produced in different districts of Cyprus island. In conclusion, the present work demonstrates novel, miniaturized sample preparation methods for the direct ultrasound-assisted solubilization of honey phenolic compounds at short time, using low volume of alcohols and small size of sample in comparison with existing methods. Publication History Article published online: 05 March 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany