*2.3. Sugar Composition Determination*

An HPLC Dionex UltiMate 3000 Series system (Thermo Scientific, Germering, Germany) supplied with a refractive index detector RefractoMax521 (ERC Inc., Kawaguchi, Saitama, Japan) was used to measure the sugar content (fructose, glucose, and sucrose) at 35 ◦C. After dissolving in 25 mL of 25% methanol, the honey sample was filtered through a 0.22 m nylon filter and then injected (5 μL) into the HPLC. The HPLC column was a Hypersil GOLD Amino 150 × 3 mm (particle size 3 μm) (Thermo Scientific, Germany), equipped with a guard column Hypersil GOLD Amino 10 × 3 mm column with the same particle size. Acetonitrile and water (80:20, *v*/*v*) served as the mobile phase and were filtered using a 0.22 m membrane filter at a flow rate of 1 mL/min. At room temperature, all measurements were made. Thermo Scientific's Chromeleon®7 software (Version 7.1, Dionex, Sunnyvale, CA, USA) was used to control the system. Sugar concentrations in the samples were measured using external calibration curves created by standard solutions. By comparing honey sugars' retention periods and peak areas to those of standard sugar solutions, honey sugars were identified and quantified.
