https://doi.org/10.1016/j.fochx.2022.100440
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2.3. UHPLC−HRMS analysis
The chromatographic separation was performed on a UHPLC system (Waters, Milford, MA, USA), with a BEH C8 column (2.1 mm × 100 mm, 1.7 μm, Waters, Milford, USA) at 50 °C. Mobile phase A and mobile phase B consisted of 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in ACN, respectively. The flow rate was 0.35 mL/min. A gradient started with 5% B and held for 1 min, then increased to 100% B at 23 min and maintained for 4 min. Lastly, the gradient was back to 5% B in 0.1 min and held for 1.9 min. The volume for injection was 1 µL and the sample manager temperature was 4 °C.
The MS data acquisition was carried out on a Q Exactive HF (Thermo Fisher Scientific, Rockford, IL, USA) system. All spectra were obtained in positive ion mode, the resolutions were 120,000 (full width at half-maximum, FWHM) in full scan mode and 30,000 in data-dependent MS/MS (ddMS2) mode. The automatic gain control target of the full scan was 3 × 10 6 and the maximum injection time of the full scan was set as 100 ms. For ddMS2, the automatic gain control target and maximum injection time were 1 × 105 and 50 ms, respectively. Normalized collision energy (NCE) was set as 15%, 30%, and 45%. The top N was 10, spray voltage was 3.5 kV. The aux gas heater temperature and capillary temperatures were 350 °C and 300 °C, respectively. Aux gas flow rate was 10 arbitrary units and sheath gas was 45 arbitrary units. The S-lens RF level was 50 arbitrary units.
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