Uhplc q orbitrap ms

The qualitative analysis was performed on a UHPLC/Q-Orbitrap-MS (Thermo Fisher Scientific, San Jose, CA, USA) with chromatographic separation achieved using an ACQUITY UPLC® BEH C18 (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA) at 40°C. The mobile phase system was composed of 0.1% formic acid aqueous solution (v/v) (A) and methanol (B) at 0.3 mL min⁻¹, which was performed according to following optimized gradient program: 0–5 min, 3%–9% B; 5–9 min, 9%–15% B; 9–11 min, 15%–17% B; 11–15 min, 17%–27% B; 15–18 min, 27%–28% B; 18–26 min, 28%–50% B; 26–31 min, 50%–66% B; 31–35 min, 66%–74% B; and 35–36 min, 74%–3% B. The mass spectrometer was carried out in both positive and negative ion modes with centroided MS and MS² spectra recorded from 100 to 1500 m/z in full MS and dd-MS² (TopN) modes at a resolution of 70000 and 17500, respectively. The maximum injection time was set at 50 and 100 ms for MS¹ and MS², respectively. The automatic gain control (AGC) target of MS¹ and MS² was 3e⁶ and 1e⁵, respectively. The optimal MS parameters were as follows: spray voltage, −2.5 kV in negative ion mode and 3.5 kV in positive ion mode; sheath gas flow rate, 35 Arb; aux gas flow rate, 10 Arb; capillary temperature, 350°C; aux gas heater temperature, 350°C; collision energy at 20, 40, and 60 eV. All the data were acquired and processed by the Thermo Scientific™ Xcalibur™ system.