5 m duraguard column

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)}.

All samples were ground finely before freeze drying. Then, total fatty acids were extracted from approximately 30 mg dry power as detailed by Guo et al. (2017) (link) and quantified as methyl esters through a gas chromatography-flame ionization detector (GC-FID). Heptadecanoic acid (C17:0) as the internal standard was added to the samples prior to extraction. Helium was used as the carrier gas at a constant velocity of 1.0 mL/min. The injector and FID temperatures were set at 240 and 250°C, respectively.
Soluble sugars of 15 mg dry powder were extracted and derivatized sequentially with methoxyamine hydrochloride and N-methyl-N-trimethylsilyl-trifluoroacetamide, as detailed by Zhu et al. (2021) (link). Metabolites were quantified with an Agilent 7890 A GC/5975C MS system (Agilent Technology) on a DB-5MS capillary column (20 m × 0.18 mm × 0.18 µm) with a 5 m Duraguard column (Agilent Technology).