Samples were chemically derivatized using a methyl chloroformate (MCF) method to extract metabolites according to the protocol published by Smart et al 20 . Briefly, 200 µL of sodium hydroxide (1 M) was added to the dried samples, and 167 µL of methanol and 34 µL of pyridine were added. Then, 20 µL of MCF was added with 30s of vortexing, and the addition of another 20 µL of MCF was followed by 30s of vortexing. Then, 400 µL of chloroform and 400 µL of sodium bicarbonate (50 mM) were added and vortexed for 10s. The lower chloroform phase was used for GC-MS analysis.
The samples were analyzed in a GC7890 system with an MSD5975 mass selective detector (Agilent, California, USA). The MSD5975 mass selective detector (Agilent) was a ZB-1701 GC capillary column (30 m x 250 μm id x 0.15 μm with 5-m guard column, Phenomenex, California, USA). One microliter of sample was injected into the GC inlet. Helium gas was the carrier gas flow at 1 mL/min. The auxiliary temperature, MS quadrupole, and MS source were 250°C, 230°C, and 150°C, respectively, and the scan speed was 1.562 u/s.
The samples were analyzed in a GC7890 system with an MSD5975 mass selective detector (Agilent, California, USA). The MSD5975 mass selective detector (Agilent) was a ZB-1701 GC capillary column (30 m x 250 μm id x 0.15 μm with 5-m guard column, Phenomenex, California, USA). One microliter of sample was injected into the GC inlet. Helium gas was the carrier gas flow at 1 mL/min. The auxiliary temperature, MS quadrupole, and MS source were 250°C, 230°C, and 150°C, respectively, and the scan speed was 1.562 u/s.