Hyper gold c18

Ultra performance liquid chromatography (UPLC) with time-of-flight mass spectrometry (ESI-TOF-MS) was performed at our outsourcing facility by a well-established method [58 (link),59 (link)]. In brief, the sample separation was performed using UltiMate 3000LC combined with Q Exactive mass spectrometry (Thermo) followed by screening with ESI-MS. The LC system is a combination of a two-system unit with Thermo hyper gold C18 (100×2.1mm 1.9 μm) combined with the UltiMate 3000LC system. The mobile phase comprises two solvents—solvent A and solvent B. Solvent A comprises 0.1% formic acid, 5% acetonitrile, and water, and solvent B is a mixture of 0.1% formic acid and acetonitrile with a gradient elution of 0–1.5 min, 0–20% B; 1.5–9.5 min, 20–100% B; 9.5–14.5 min, 100% B; 14.5–14.6 min, 100–0% B; 14.6–18.0 min, 0% B. The flow rate for the mobile phase was fixed at 0.3 mL/min. The column temperature was maintained at 40°C, and the sample manager temperature was set at 4 °C. Mass spectrometry parameters in ESI+ and ESI- modes were set. For positive ion mode (ESI+), the experimental parameters were the following: heater temperature set at 300 °C, flow rate of the sheath gas set at 45 arb, auxiliary gas flow rate set at 15 arb, sweep gas flow rate set at 1 arb with a spray voltage of 3.0 kV, capillary temperature set at 350 °C, and the S-Lens RF level adjusted to 30%.

Analyte separation was performed by liquid chromatography in an Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with a ESI–MS (targeted MS/MS mode). The LC system is composed of a Thermo Hyper gold C18 (100 × 2.1 mm 1.9 μm) with an Ultimate 3000LC. The mobile phase is composed of solvent A (0.1% formic acid–5% acetonitrile–water) and solvent B (0.1% formic acid–acetonitrile) with a gradient elution (0–1.5 min, 100–80% A; 1.5–9.5 min, 80–0% A; 9.5–14.5 min, 0% A; 14.5–14.6 min, 0–100% A; and 14.6–18 min, 100% A)^{27 (link)}. The flow rate of the mobile phase was 0.3 mL min⁻¹. The column temperature was 40 °C, and the sample manager temperature was set at 4 °C^{26 (link)}. The mass spectrometry parameters in ESI+ and ESI− mode are listed as follows: ESI+: Heater Temp 300 °C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; Capillary Temp, 350 °C; S-Lens RF Level, 30%. ESI-: Heater Temp 300 °C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; Capillary Temp, 350 °C; and S-Lens RF Level, 60%^{26 (link),27 (link)}.
At the beginning of the sequence, we run four QC samples to avoid small changes in both chromatographic retention time and signal intensity. The QC samples are also injected at regular intervals (every ten samples) throughout the analytical run.

Chromatographic separation was completed on Thermo Ultimate 3000 system which was equipped with a Hyper gold C₁₈ (100 × 2.1 mm, 1.9 µm, and Thermo) column. Gradient elution of analyses was conducted with 0.1% formic acid in 5% acetonitrile(C) and 0.1% formic acid in acetonitrile (D) for positive ion mode. While, 0.05% acetic acid in 5% acetonitrile (A) and 0.05% acetic acid in acetonitrile (B) for negative mode. An incremental linear gradient of solvent B or D (v/v) was used as follows: 0–1.5 min, 0–20% B/D; 1.5–9.5 min, 20–100% B/D; 9.5–14.5 min, 100% B/D; 14.5–14.6 min, 100–5% B/D; 14.6–18 min, 5% B/D. The column flow rate and temperature were respectively set at 0.3 ml/min and 45°C. 3 μl of each sample was injected into UPLC-Q-Exactive MS/MS for analysis.
The experiments of ESI-MSⁿ were conducted on the Thermo Q Exactive mass spectrometer in positive and negative ion modes, respectively. Mass spectrometry parameters were set as follows: spray voltage was 3.0 kV in positive mode and −3.2 kV in negative mode. Sheath gas was kept at 45 arbitrary units, while auxiliary gas was 15 arbitrary units. The capillary temperature was maintained at 350°C. Full mass scan (m/z 70–1050) and HCD MS/MS spectra were recorded at a resolution of 70,000.