Metabolites were analyzed using a Dionex UltiMate 3000 UHPLC system coupled with an Orbitrap Fusion Lumos system (Thermo Fisher Scientific, Milford, MA, USA). Each sample was run twice with technical repetitions. Metabolite samples were injected into an Agilent Poroshell 120 SB-C18 column (2.1 × 100 mm, 2.7 μm) using an autosampler. Chromatographic separation was achieved with two mobile phases: mobile phase A [5 % (v/v) acetonitrile and 0.1 % (v/v) formic acid] and mobile phase B [0.1 % (v/v) formic acid in acetonitrile]. The flow rate was set at 0.2 mL/minutes. The elution program was structured as follows: 0–2 mL/minutes with 100 % mobile phase A, followed by a gradient from 100 % to 0 % A over the next 10 minutes, and then a 4 mL/minutes maintenance period.
The mass spectrometer parameters for this experiment were as follows: electrospray voltage in positive mode was 3.5 kV; the temperature of the electrospray ionization source was 350 °C; the resolution of the initial full scan was 70,000; the scan range extended from 100 to 1,000 m/z; the resolution of the secondary data dependency scan was 35,000; and the automated gain control target was set to 1e6. To ensure the stability of the analysis, the QC was conducted by injecting a sample once every 14 samples throughout the analysis to monitor the consistency of sample preparation and instrument performance.
The mass spectrometer parameters for this experiment were as follows: electrospray voltage in positive mode was 3.5 kV; the temperature of the electrospray ionization source was 350 °C; the resolution of the initial full scan was 70,000; the scan range extended from 100 to 1,000 m/z; the resolution of the secondary data dependency scan was 35,000; and the automated gain control target was set to 1e6. To ensure the stability of the analysis, the QC was conducted by injecting a sample once every 14 samples throughout the analysis to monitor the consistency of sample preparation and instrument performance.