Db 35ms ultra inert column

Seven-day-old M. polymorpha gemmalings (∼50 mg) were collected and immediately frozen in liquid nitrogen prior to storage at −80°C right after HS treatment at 37°C for 0, 1, and 5 h. The samples were then homogenized using a mortar and pestle precooled with liquid nitrogen and extracted in 700-µL methanol, and 60-µL of internal standard (0.2-mg ribitol mL⁻¹ H₂O) was subsequently added as a quantification standard. The extraction, derivatization, standard addition, and sample injection were conducted as described previously (Lisec et al., 2006 (link)). Untargeted GC–MS analysis was performed using a previously described system (Avin-Wittenberg et al., 2015 (link)). In brief, samples were separated using a DB-35ms Ultra Inert column (30 m × 0.25 mm × 0.25 µm, Agilent, USA) with Agilent 7200 Q-TOF GC–MS coupled to Agilent 7890B Gas Chromatograph by Chemical, Molecular, and Materials Analysis Centre (CMMAC), National University of Singapore. Raw MS data were extracted using the MassHunter Profinder suite (B.08.00, Agilent), and resulting peaks were aligned and mapped to the NIST database (2017) using Mass Profiler Professional software (v 8.0, Agilent) with default setting. Student’s t test (P < 0.05) was applied to generate differentially accumulated compounds in HS versus control conditions in at least one pair-wise comparison.

Metabolite extraction, derivatization, and GC-MS condition were performed according to previously described (Lisec et al., 2006 (link)). Root extracts were separated using DB-35ms Ultra Inert column (30 m × 0.32 mm × 0.25 µm, Agilent, Santa Clara, CA, USA) with Agilent 7200 Q-TOF GCMS coupled to Agilent 7890B Gas Chromatograph by Chemical, Molecular and Materials Analysis Centre, National University of Singapore. Peak extraction and compound identification were performed by Mass Hunter Suite with default settings using the NIST 2014 mass spectral database.