*2.3. Determination of Sugar Composition*

Determination of sugars (fructose, glucose, and sucrose) was carried out by High Performance Liquid Chromatography (Agilent Infinity 1260 II, Agilent, Santa Clara, CA, USA), using a refractive index detector following the AOAC official method 977.20-1977. Sample preparation was carried out by dissolving 2 mg honey in 25 mL deionized water. Standard solutions of 1% fructose, 1% glucose, and 0.5% sucrose (Sigma-Aldrich, St. Louis, MO, USA) were prepared in distilled water. Mixed standards were prepared at 1.0% for fructose and glucose and 0.5% for sucrose. The mixed standard was diluted to yield 0.2, 0.4, 0.6 and 0.8%. The chromatographic separation of sugars was achieved using an Agilent Zorbax carbohydrate column maintained at 35 ◦C with acetonitrile/water (75:25, *v*/*v*), which was used as the mobile phase. Then 10 μL of sample was injected at a flow rate of 1.0 mL/min. The temperature of the column was maintained at 27 ◦C during the entire run.
