Quantification of Soluble Sugars by HPAEC-PAD

Total soluble sugars were extracted six times from 10 mg pulverized dry mass samples with 1.5 mL of 80% ethanol at 80°C for 20 min. The Alcohol Insoluble Residue (AIR) was dried at 45°C overnight and subsequently used for starch evaluation. After each extraction, the supernatant was recovered by centrifugation. The supernatant pool was vacuum concentrated (ThermoScientific^® Savant SC 250 EXP) and resuspended in 1 mL of water and 1 mL of chloroform. The recovered water-soluble sugars (sucrose, fructose, glucose, and raffinose) were analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) in a Dionex system (ICS 5,000) using a CarboPac PA1 column and eluted with 150 μM NaOH on an isocratic run of 27 min (Pagliuso et al., 2018 (link)).