이전 사이트 이동

Sample preparation was achieved by protein precipitation. A 400 µL of extraction solution (MeOH:ACN = 1:1) was added to 100 µL of each plasma sample and vortexed for 5 minutes at room temperature. We centrifuged samples at 14,000 rpm at 4 ℃ for 10 minutes. The supernatants were transferred into new tubes. 4 µL of each sample was injected into the Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany).

An ACQUITY UPLC HSS T3, 1.8 µm x 100 mm column (Waters Corporation, Milford, MA, USA) was connected to the UPLC system for metabolite separation. For the mobile phase, we used water containing 0.1% formic acid (mobile phase A) and MeOH with 0.1% formic acid (mobile phase B) under gradient conditions. The gradient elution started with 5% B at 1 minute, increased to 95% B at 6 minutes, and 98% B at 12 minutes. It was maintained up to 22 minutes and reduced to 5% B at 25 minutes. The flow rate was 0.4 mL/min, and the column temperature was maintained at 50 ℃.

We used the ultra-high-performance liquid chromatography (UPLC) system equipped with a Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) for metabolomics analysis. Sheath Gas, 3 Arb; Aux Gas, 2 Arb; Sweep Gas, 0 Arb; Ion Transfer Tube Temperature, 320 °C; Vaporizer Temperature, 0 °C; Resolution, 120000; Scan range, 100-1000 m/z.