Triple tof 5600 1

A Shimadzu LC30-AD HPLC system (Shimadzu, Kyoto, Japan) combined with a quadrupole time-of-flight mass spectrometer (Triple TOF 5600-1, AB SCIEX, Redwood City, CA, USA) was used. An Agilent Zorbax SB-C18 column (4.6 mm × 50 mm, 1.8 μm, Agilent Technologies, USA) with a column temperature maintained at 30 °C was employed. The mobile phase consisted of 0.1% formic acid (A) and acetonitrile (B), using a gradient elution of 10–20% B at 0–2 min, 20–22% B at 2–9 min, 22–40% B at 9–16 min, 40–70% B at 16–17 min, 70–100% B at 17–18 min, and 100% B at 18–19 min with an equilibrium for 4 min. The flow rate was set at 0.4 mL min⁻¹ with an injection volume of 2 μL.
MS detection was performed using QTOF/MS in negative ionization mode with a DuoSpray ion source. The QTOF/MS was calibrated in high sensitivity mode and the automated calibration device system (CDS) was set to perform an external calibration every four samples using a calibration solution. The source parameters were optimized: collision voltage (CE), 50 eV; ion spray voltage floating (ISVF), 4500 V; temperature, 500 °C; nebulizing gas (GS1), 60 psi; heater gas (GS2), 60 psi; curtain gas, 35 psi. The MS was operated in full-scan TOF/MS (100–2000 amu) and MS/MS mode (100–2000 amu) through data-independent acquisition (DIA) in a single-run analysis.^{16 (link)}