Agilent 7890b 5977a system

The surpernatant was recovered by centrifugation (14,000 × g, 15 min, 4 °C). Surpernatant (100 μl) was lyophilized, and resuspended in 80 μl of methoxyamine solution (20 mg/ml in pyridine), and then was placed in water bath at 37°C for 1.5 h. Subsequently, 60 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) was used for silylation for 1 h. The reaction was stopped by addition of 10 μl hexane. The analysis was performed using the platform based on a DB-5MS column (0.25 μm, 0.25 mm × 30 m, Agilent Technologies, Inc., Santa Clara, CA) and Agilent 7890B-5977A system (Agilent Technologies, Inc.). The temperatures of inlet and ion source were set at 280 and 230°C, respectively. The oven temperature program was set as following: initially kept at 60°C for 3 min, increased to 170°C at the rate of 5 °C/min, 4 °C/min to 234°C, 5°C/min to 280°C, and held for 5 min. The voltage of detector was set at 0.93 kV, and the EI ionization voltage of metabolites was 70 eV. Full scan mode (m/z 33–500) was applied to acquire mass signals.