Q exactive plus orbitrap high resolution mass spectrometer

GXF formula was analyzed using U3000 UPLC system equipped with Q Exactive Plus Orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific). Chromatographic separation was performed on a ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm, Waters). The column temperature was set at 35 ℃. The flow rate was 0.3 mL/min. The mobile phase consisted of deionized water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The mass spectrometer analysis was conducted in both positive and negative ion modes. The scan mass ratio was within the mass range of m/z 50–1500.
The analysis data were analyzed by Compound Discoverer software 3.2 (Thermo Fisher Scientific). The mass deviations of characteristic peak element matching, molecular formula prediction and isotope distribution matching were all set to within 5 ppm. Chemical identification was based on chromatographic elution behavior, mass spectrometry fragment patterns and mass spectrometry databases (MZ cloud, MZ vault).

The fraction which showed the best inhibitory activity was analyzed using LC-MS/MS. The samples (10 mg) were dissolved in 5 mL methanol and filtered using PTFE membrane filter (PTFE 25 mm; 0.22 μm; pore size, Anpel, China). An amount of 2 μL sample was injected into LC-MS/MS with a flow rate of 0.2 mL/min. LC-MS/MS analysis was performed on a Thermo Scientific Vanquish Flex Binary UHPLC (Thermo Fisher Scientific, USA) connected to a Thermo Scientific Q Exactive Plus Orbitrap High Resolution Mass Spectrometer (Thermo Fisher Scientific, USA). The mass spectrometer was equipped with electrospray ionization (ESI) in the positive mode. The MS/MS spectra were obtained with a mass range of m/z 100-1500. The chromatographic separation of the sample was performed on a C18 column (100 mm × 2.1 mm; 1.5 μm, Accucore C18, Thermo Fisher Scientific, USA). The mobile phase consisted of water containing 0.1% formic acid for solvent A and acetonitrile containing 0.1% formic acid for solvent B. The gradient elution was 0-1 min (5% B), 1-25 min (5-95% B), 25-28 min (95% B), and 28-30 min (5% B).