LC-MS analysis
For LC-MS analysis sample preparation, the same approach was employed as described in GC-MS but extracted with 100 μL 80% v/v methanol.
LC analysis was performed on a Waters Acquity UPLC-I-Class system (Waters Corp., Milford, MA, US) with an Acquity HSS T3 column (100 × 2.1 mm, 1.8 μm) for chromatographic separation (Shan et al., 2014). The column temperature was set at 25 ℃, the flow rate at 0.3 mL/min and the injection volume was 5 μL for each sample. The mobile phase consisted of 0.1% formic acid aqueous solution (A) and acetonitrile containing 0.1% formic acid (B). The gradient elution program was as follows: 0~3 min, 2%~8% B; 3~5 min, 8%~15% B; 5~11 min, 15%~35% B; 11~13 min, 35%~65% B; 13~15.5 min, 65%~100% B; 15.5~16 min 100%~2% B. After UPLC, the effluent was alternatively connected to a Xevo G2-SQ-TOF MS system (Waters Corp., Milford, MA, US). The data acquisition modes were MSE continuum. The experiment was performed in both the ESI (+) and ESI (–) ionization modes. The parameters were set as follows: source and desolvation temperature were 100℃ and 350℃ respectively; desolvation gas flow rate, 900 L/h; capillary voltage, 0.5 KV for ESI (+) and 2 KV for ESI (–); cone voltage, 40 V; collision energy, 6 eV (trap) for low-energy scans, 20-30 eV and 45-70 eV for ESI (–) and ESI (+) in high-energy scans respectively; data acquisition range, 50-1500 Da. The mass accuracy was maintained using a lock spray with leucine enkephalin (200 pg/L, 10 L/min) as the reference.