An Acquity UPLC system (Waters Corporation, Milford, MA, USA) combined with a Q-Exactive high-resolution mass spectrometer (Thermo Fisher, Waltham, MA, USA) with an Orbitrap mass analyzer was used. Samples (5 μL) were injected onto an ACQUITY UPLC BEH Shield RP18 column (150 × 2.1 mm, particle size 1.7 μm) (Waters, Manchester, MA, USA), with a flow rate of 0.35 μL min−1 at 50 °C. Mobile phases contained 0.1% (v/v) formic acid in water (A) (LC-MS grade, Merck, Darmstadt, Germany) and acetonitrile (B) (LC–MS grade, Merck). A multi-step linear gradient was as follows: 5% B—1.5 min, 80% B—10.5 min, 98% B—11.5 min, 5% B—13 min.
Mass spectrometry analysis was performed using heated electrospray ionization (H-ESI) in positive and negative modes. A 3.5 kV and 2.5 kV ion spray voltage was applied for positive and negative ionization, respectively. Ion source temperature was 320 °C. Data were acquired in Full MS/data-dependent MS2 mode in the 100–1500 m/z range. The resolution of Full MS was 70,000 and of ddMS2 17,500. Normalized collision energy in the ddMS2 experiment was set to 30%. Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA) was used for system operation, data acquisition, and data analysis [58 (link)].
Mass spectrometry analysis was performed using heated electrospray ionization (H-ESI) in positive and negative modes. A 3.5 kV and 2.5 kV ion spray voltage was applied for positive and negative ionization, respectively. Ion source temperature was 320 °C. Data were acquired in Full MS/data-dependent MS2 mode in the 100–1500 m/z range. The resolution of Full MS was 70,000 and of ddMS2 17,500. Normalized collision energy in the ddMS2 experiment was set to 30%. Xcalibur software (ThermoFisher Scientific, Waltham, MA, USA) was used for system operation, data acquisition, and data analysis [58 (link)].
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