GC-TOF-MS Metabolomic Analysis Protocol

Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) analysis was performed as previously described by Jung et al. [50 (link)]. For analysis, all dried samples were oximated with 50 μL of methoxyamine hydrochloride (20 mg/mL in pyridine) for 90 min at 30 °C and silylated with 50 μL of N-methyl-N-(trimethylsilyl) trifluoroacetamide for 30 min at 37 °C. The derivatized samples were analyzed on an Agilent 7890A GC system (Santa Clara, CA, USA) coupled with an Agilent 7693 auto-sampler and Pegasus^® HT TOF MS (LECO Corp., St. Joseph, MI, USA). An Rtx-5MS column (30 m × 0.25 mm, 0.25-μm particle size, Restek Corp., St. Joseph, MI, USA) was used at a constant flow of 1.5 mL/min with helium used as the carrier gas. Next, 1 μL of the derivatized samples were injected into the GC in a splitless mode. The GC oven temperature was asset to 75 °C for 2 min, and then increased by 15 °C/min to 300 °C with a 3-min hold time as the final temperature. The mass data collection rate was set to 10 scans/s over a scan range of 50–1000 m/z followed by −70 eV of an electron ionization mode. The front inlet and transfer line temperatures were set to 250 °C and 240 °C, respectively.