Metabolite Profiling by GC-TOF-MS

GC–TOF–MS analysis was performed using an Agilent 7890A GC system (Agilent Technologies, Palo Alto, CA, USA) equipped with an L-PAL3 autosampler and Pegasus^® HT TOF-MS system (LECO Corp., St. Joseph, MI, USA). Metabolites were separated using an RTX-5MS column (30 m length × 0.25 mm inner diameter × 0.25 μm particle size, Restek Corp., St. Joseph, MI, USA) with a constant flow of helium (1.5 mL) as the carrier gas. 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 (1 μL) were injected into the GC column in splitless mode. The analytical program for sample analysis was adopted from our previous study [22 (link)]. Moreover, metabolite analysis was performed in a random manner to reduce bias and systematic errors.