3.3.1. Elution Conditions

Accucore aQ-MS column (2.1 × 100 mm, 2.6 μm, Thermo Fisher Scientific, USA), was employed to perform sample separation with a thermostat at 30 ◦C. The binary mobile phases (MP) were 0.1% FA in water (containing 5 mM Ammonium Formate, A) and 0.1% FA in MeOH (containing 5 mM Ammonium Formate, B). Their gradient elution was started with 2% B, linearly increasing to 20% in 4 min and continuously ramped to 40% within 1.5 min. Subsequentially, B was increased to 98% in the subsequent 5 min, and kept for 2.4 min. Then, B was restored to the initial conditions 2%B in the following 2.1 min, and kept for 5 min to re-equilibrate for the next injection. The whole elution process for one injection analysis took 20 min. The flow rate was kept at 0.3 mL·min−1. The injection volume for analysis was 10 μL for each sample.

#### 3.3.2. Mass Spectrometer Condition

Parameters for electrospray were as following: spray voltage, 3200 V (positive mode), 2800 V (negative mode), sheath gas flow rate at 40 <sup>L</sup>·min−1, auxiliary gas flow rate at 10 <sup>L</sup>·min−1, sweep gas flow rate at 1.0 <sup>L</sup>·min−1, auxiliary gas temperature at 350 ◦C, capillary temperature at 325 ◦C and S-lens RF level at 60 V. The scan mode for high-resolution mass spectrometry acquisition was Full MS/dd-MS2 (with inclusion list) mode. Recorded mass range for full mass record was between *m*/*z* 100–1000 (positive mode) and 150–1000 (negative mode), at resolution of 70,000. The Full MS/dd-MS2 (with inclusion list) mode can simultaneously record the precursor mass and the MS/MS (fragmentation) spectra for selected precursors. The MS/MS acquisition for fragment scanning of the selected ions was carried out at the isolation window of 2.0 *m*/*z* and the resolution of 15,000. For each round of fragmentation acquisition, the top 2 (TopN, 2, loop count 1) abundant precursors above the threshold 5 × 10<sup>5</sup> were sequentially transferred into the C-Trap (AGC, 5 × 105, Max IT, 100 ms) for collision at normalized energies (NCE, 20, 50, 80) in HCD multipole and pumped to Orbitrap for MS/MS acquisition.

All the units of UHPLC-ESI-Q-Orbitrap HRMS system were controlled through the Tracefinder software.




