2.3.4. Determination of Organic Acids

A total of 0.5 g of bee pollen was mixed with 2.5 mL 4% metaphosphoric acid (*w*/*v*), after homogenization the solution was centrifuged for 10 min at 5000 rpm. The supernatant was filtered using a 0.45 μm filter prior analysis. The determination was carried out on a high performance liquid chromatograph Shimadzu (Kyoto, Japan) with diode array detector. The separation of the organic acids (gluconic acid, formic acid, butyric acid propionic acid, lactic acid and acetic acid, respectively) was made using a Phenomenex Kinetex® <sup>5</sup> <sup>μ</sup>m C18 100 Å HPLC Column 250 × 4.6 mm [35]. The mobile phase consisted of a mixture of 0.5% metaphosphoric acid and acetonitrile (50/50, *v*/*v*), and the flow rate was set at of 0.8 mL·min−1. The injection volume was set at 10 <sup>μ</sup>L. The detector was set at 210 nm. The organic acids were expressed as mg/kg dry mater. All the measurements were carried out in triplicate.
