2.2.4. Free Sugars

The lyophilized samples were extracted using a methodology previously described by the authors [23]. The separation of compounds was performed using a HPLC system (Knauer, Smartline system 1000, Berlin, Germany) coupled with a refraction index detector (Knauer Smartline 2300). The mobile phase consisted of an acetonitrile:water mixture (70:30 *v*/*v*, acetonitrile HPLC-grade,

Lab-Scan, Lisbon, Portugal), and separation was achieved using a Eurospher 100-5 NH2 column (4.6 × 250 mm, 5 μm, Knauer, Berlin, Germany). After separation, the compounds were identified by comparison with standards and quantification was performed by the IS method (melezitose). Results were processed using the Clarity 2.4 software (DataApex, Prague, Czech Republic) and expressed in g per 100 g of fw.
