Q exactivetm high resolution mass spectrometer

Thermo ScientificTM DionexTM Ultimate 3000 RSLCnano UHPLC (ultra-high-performance liquid chromatography) and Q ExactiveTM High-Resolution Mass Spectrometer (ThermoFisher, MA, USA) were used to screen and discover non-targeted chemical compounds from the fraction showing the best antibiofilm activity. The mobile phase was composed of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The programming for the gradient mode was as follows: at t = 0–15 min, B 5%; at t = 16–20 min, B 90%; at t = 21–25 min, B 90%. The analytical column used was Phenyl-Hexyl 100 × 2.1 mm with a flow of 0.20 mL/min and an injection volume of 5 µl. MS1 was rendered at 70,000 FWHM, whereas MS2 was rendered at 17,500 FWHM. This experiment utilized Heated Electrospray Ionization (H-ESI) in both positive and negative modes. The spray voltage used was 3.8 kV. The flow rates for Sheath gas and Aux gas were 15 and 7, respectively. The capillary temperature was 250 °C. The mass range used was between 50 and 750 m/z. Thermo Scientific ™ Compound Discoverer Software was used for identifying the compounds.

Formic acid (0.1 percent) was used to dissolve the sample. Following that, 5 µL of the sample was injected. Mobile phases A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid) were analyzed using the Thermo Scientific^TM DionexTM Ultimate 3000 RSLC nano UHPLC, paired with the Thermo Scientific^TM Q Exactive^TM High-Resolution Mass Spectrometer. The acquired data were processed using Full MS 70,000 FWHM Resolution. Thermo Scientific^TM Proteome Discoverer 2.2 Software was used to identify proteins, which were then compared to the Master Protein database.

Roots and leaves extracts were subjected to HRMS in order to investigate the formation of T2 and HT2 biotransformation products, for which analytical standards were not available. Chromatographic conditions were the same used for the targeted analysis. LC-HRMS full scan spectra were recorded using Q-ExactiveTM high resolution mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with electrospray ionization (ESI). The Q-Exactive mass analyzer was operated in the full MS/data dependent MS/MS mode (full MS–dd-MS/MS) in positive ionization mode. The following parameters were set: sheath and auxiliary gas flow rates 40 and 10 arbitrary units, respectively; spray voltage 3.5 kV; heater temperature 250 °C; capillary temperature 300 °C. Following parameters were used in full MS mode: resolution 70,000 FWHM (defined for m/z 200), scan range 400 ¬ 900 m/z, automatic gain control (AGC) target 3e6, maximum inject time (IT) 200 ms. Parameters for dd-MS/MS mode: resolution 17,500 FWHM (defined for m/z 200) AGC target 2e5, maximum IT 50 ms, normalized collision energy (NCE) 35% with ±25% step was used.