Tracefinder 4.1 clinical

The sample was digested with 2 M HCl at 80 °C for 3 h. An aliquot (200 μL) of the supernatant was evaporated under a gentle stream of N₂. For UHPLC-ESI-MS/MS analysis, the samples were dissolved in 1 mL of 30 % methanol (vol/vol) and transferred to insert-equipped vials. The sample extracts were analysed using an UPLC-Orbitrap-MS system (UPLC, Vanquish; MS, QE). The analytical conditions were as follows, UPLC: column, Waters ACQUITY UPLC HSS T3 (1.8 μm, 2.1 mm × 50 mm); column temperature, 40℃; flow rate, 0.3 mL/min; injection volume, 2 μL; solvent system, water (0.1 % formic acid): acetonitrile (0.1 % formic acid); gradient program, 90:10 V/V at 0 min, 90:10 V/V at 1.0 min, 65:35 V/V at 6 min, 5:95 V/V at 8 min, 5:95 V/V at 10 min, 90:10 V/V at 10 min, 90:10 V/V at 12 min. HRMS data were recorded on a Q Exactive hybrid Q-Orbitrap mass spectrometer equipped with a heated ESI source (Thermo Fisher Scientific) using SIM MS acquisition methods. The ESI source parameters were set as follows: spray voltage, −2.8 kV; sheath gas pressure, 40 arb; aux gas pressure, 10 arb; sweep gas pressure, 0 arb; capillary temperature, 320 ℃; and aux gas heater temperature, 350 ℃. Data were acquired on Q-Exactive by using Xcalibur 4.1 (Thermo Scientific), processed using TraceFinder™4.1 Clinical (Thermo Scientific). Quantified data were output into Excel format.