GC-MS Profiling of Soluble Sugars

As described previously^{3 (link),45 (link)}, soluble sugars and hexose phosphates were extracted in 75% methanol with ribitol added as an internal standard and then derivatized sequentially with methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide. After derivatization, the metabolites were analyzed with an Agilent 7890 A GC/5975C MS system (Agilent Technology, Palo Alto, CA, USA) on a DB-5MS capillary column (20 m × 0.18 mm × 0.18 µm) with a 5 m Duraguard column (Agilent Technology). The tissue residue after 75% methanol extraction for gas chromatography–mass spectrometry analysis was re-extracted three times with 80% (v/v) ethanol at 80 °C, and the pellet was retained for enzymatic determination of starch as glucose equivalents^{3 (link)}.