2.3.5. Determination of Free Sugars

A total of 1g of bee pollen was mixed with 20 mL of methanol and filled to 50 mL with water. The solution was centrifuged for 10 min at 5000 rpm; the supernatant was transferred into a 50 mL flask and filled with water. The solution was filtered using a 0.45 μm filter prior analysis. The separation of the free sugars (fructose, glucose, sucrose, melesitose and raffinose) was made on Schimadzu HPLC instrument (Kyoto, Japan) with RID (refractive index detector). A Phenomenex Luna® Omega 3 μm SUGAR 100 Å HPLC Column 150 × 4.6 mm (Torrance, CA, USA) was used for the separation. The mobile phases were acetonitrile and water in a mixture of 80:20 (*v*/*v*). The flow rate was set at 1.3 mL·min−1; column and detector temperature was 30 ◦C and the sample volume injection was 10 μL. The free sugars were expressed as % reported to dry mater. All the measurements were carried out in triplicate.
