2.2. Extraction and Chromatographic determination of glucose, fructose, sucrose, myo-inositol, threhalose, FOS and RFOS.
For carbohydrates analyses, 2 g of frozen strawberry powder (wet) were extracted with 5 ml of distilled water, homogenized and centrifuged at 10000 rpm for 30 min at 4 ºC, and determined by UPAEC-PAD with a Carb2-250 (250 x 4mm) column, as previously described (Blanch et al., 2015b). Samples were analysed on Bioscan module (817 IC Metrohm, Herisau, Switzerland) equipped with a pulsed amperometric detector, (IC Pump 812) and coupled degasser (IC-837). Isocratic elution was carried out with 100 mM NaOH- 10 mM NaOAc, and the flow rate through the column was 0.5 mL/min, leading to sampling times of 60 min. For FOS and RFOS analyses, 3 g of frozen strawberry powder (wet) were homogenized in 4 ml of ultra-pure water and the mixture was boiled under reflux in a water bath for 15 min. After cooling, the samples were sonicated for 10 min at 40 ºC and the pH of each sample was adjusted to 7.5 with 10% NH4OH. The samples were then centrifuged at 14700g for 15 min at 4 ºC and the supernatants were filtered thought a 0.45 µm pore size membrane. The analyses of extracts and calibration standards were performed using an Agilent 1200 liquid chromatography coupled to an Agilent Triple Quadrupole MS detector G6410B (HPLC-QqQ-SIM, Agilent Technologies, Palo Alto, CA, USA). The chromatographic separation was achieved on an HYPERCARB (100 mm X 2.1μm X 5μm) column, with a mobile phase consisting of water containing 5 mM ammonium formate (A) and performing an elution using acetonitrile (B) in gradient as follows: %B: 0 min, 5%; 5 min, 10%; 10 min, 10%; 20 min, 50%; 30 min, 5%; 40, min, 5% at 25 ºC with an injection volume of 5 μL.