Vanquish flex binary uhplc

Aromatic waters were diluted in MeOH (1:1, v/v), centrifuged (4000 rpm) and injected (5 μL injection volume) into the LC system composed by a Vanquish Flex Binary UHPLC coupled with a Vanquish DAD and a Q Exactive Plus mass spectrometer, Orbitrap-based FT-MS system, equipped by an ESI source (Thermo Fischer Scientific Inc., Bremem, Germany). Elution was performed on a Kinetex^® Biphenyl column (100 × 2.1 mm, 2.6 μm) provided of a SecurityGuard^TM Ultra Cartridges (Phenomenex, Bologna, Italy), using formic acid in MeOH 0.1% v/v (solvent A) and formic acid in H₂O 0.1% v/v (solvent B) as eluent and developing a linear solvent gradient of increasing 5 to 55% A within 15 min, at a flow rate 0.3 mL/min. During analysis, autosampler and column oven temperatures were maintained at 4 and 35 °C, respectively. UV data were registered using 254, 280 and 325 nm as preferential channels. A positive ion mode was used for ESI interface in a scan range of m/z 150–1200 and spectra were acquired both in full (70,000 resolution, 220 ms maximum injection time) and data dependent-MS/MS scan (17,500 resolution, 60 ms maximum injection time). The following ionization parameters were used: spray voltage 3500 V, capillary temperature 300 °C, sheath gas (N₂) 20 arbitrary unit, auxiliary gas (N₂) 3 arbitrary unit, collisionally activated dissociation (HCD) 18 eV. Data were elaborated with Xcalibur software.

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).