2.8. Solid-phase extraction of sugars

Extraction of sucrose, glucose and fructose started with the extraction assisted by ultrasound according to the method reported by Gil et al., (2019b) [33]. First, 20 mg of raw and fermented cotyledons (moisture >10 %) or 10 mg of dried fermented cotyledons (moisture <10 %) were diluted into 600 μL of MilliQ H2O in a microcentrifuge tube of 2 mL. Second, after dilution, the suspension was mixed for 5 min × 3000 rpm. Third, each microcentrifuge tube was carried out in an ultrasound bath (15 min, 25 Hz, 25 °C, 99 %), followed by centrifugation at 10000 rpm during 15 min.

Solid-phase extraction (SPE) cartridges were used to clean-up the samples before UHPLC-C-CAD analysis (UltiMateTM 3000 Dionex, Thermo Scientific, Sunnyvale, CA, USA). 80 μL of supernatant were passed through a cartridge (SePak C18, 1 cc Vac of 50 mg, Waters, USA), previously activated with 1mL methanol and 1 mL of MilliQ water. The extract of sugars was eluted with 720 μL of MilliQ H2O. The eluate was collected and 400 μL were mixed with 200 μL of acetonitrile prior to analysis.

The chromatographic analysis was performed using a micro-HPLC Ultimate 3000 (Thermo Scientific™ Dionex™ Corona™ Veo™). Chromatographic separation was carried out with a Shodex Asahipak NH2 P-50 4E column (5 μm, 150 mm × 4.6 mm) at 30 °C and 10 μL of sample were defined as injection volume. The mobile phase composition was acetonitrile: water 75:25 (v/v) and the flow rate was fixed at 1 mL min⁻¹ during 19 min for each sample.

The corona-charged aerosol detector (C-CAD) was set as follows: the nitrogen generator (Peak Scientific, USA) was selected as nebulizing gas, a pressure of 60 ± 7 psi (purity gas ≥99 %), and collection velocity 10 Hz were also fixed for all analyses. The chromatographic data analysis was carried out using a Chromeleon 7.0 software.