Screening and identification of the analyte and metabolite profiling using LC-QTOF/MS analysis
SEPARATION
For qualitative determination of untargeted compounds, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was performed on an Agilent 6540 QTOF/MS (Agilent Technologies, USA) equipped with an Agilent 1260 infinity liquid chromatography system. The stationary phase of the chromatographic separation was performed on a 150 mm × 4.6 mm, 5.0 µm Phenomenex Luna C18 column (Phenomenex, USA) using a mobile phase consisting of 0.1% formic acid in water pH 2.5–2.7 (A) and 100% acetonitrile (B) at a constant flow rate of 0.5 mL/min with a controlled oven temperature at 35 °C, (and 10 µL) of injection volume. The elution gradient was started with 30%B, and was increased to 90%B within 20 min, then maintained at 95%B during 25–30 min followed by a post-run for 5 min.Mass spectrometric analysis was performed using an ESI negative and positive modes with varying collision energies (10, 20, and 40 V) over a mass range of m/z 100–1200 Da. The MS conditions were as follows: capillary voltage = 3500 V; flow rate of drying gas (N