Q exactive plus orbitrap ftms mass spectrometer

An UltiMate 3000 U-HPLC system (Dionex) was used to create a 45 min linear gradient of 5%-35% (v/v) acetonitrile in 0.1% (v/v) FA in water at a flow rate of 0.19 ml min -1 . Five microlitres of each extract was injected and compounds were separated on a Luna C18 column (2.0 × 150 mm, 3 µm; Phenomenex) maintained at 40°C (De Vos et al., 2007) . The detection of compounds eluting from the column was performed with a Q-Exactive Plus Orbitrap FTMS mass spectrometer (Thermo Scientific). Full scan MS data were generated with electrospray in switching positive/negative ionization mode at a mass resolution of 35 000 (FWHM at m/z 200) in a range of m/z 95-1350. Subsequent MS/MS experiments for the identification of selected metabolites were performed with separate positive or negative electrospray ionization at a normalized collision energy of 27 and a mass resolution of 17 500. The ionization voltage was optimized at 3.5 kV for positive mode and 2.5 kV for negative mode; the capillary temperature was set at 250°C; the auxiliary gas heater temperature was set to 220°C; sheath gas, auxiliary gas and the sweep gas flow were optimized at 36, 10 and 1 arbitrary units, respectively. Automatic gain control was set at 3 e 6 and the injection time at 100 ms. External mass calibration with FA clusters was performed in both positive and negative ionization modes before each sample series.

SFE and different fractions of SFE were diluted in DMSO to a concentration of 10 mg mL -1 . Next a 100× dilution was prepared in the assay buffer. The diluted samples were mixed with methanol containing formic acid (FA, 0.1%) in a 3 : 7 ratio. An UltiMate 3000 U-HPLC system (Dionex, Sunnyvale, CA, USA) was used to create a 45 min linear gradient of 5-75% acetonitrile (v/v) in 0.1% FA in water at a flow rate of 0.19 ml min -1 , which was followed by 15 min of washing and equilibration. Of each extract, 5 µL was injected and compounds were separated on a Luna C18 column (2.0 × 150 mm, 3 µm; Phenomenex) at 40 °C. A Q Exactive plus-Orbitrap FTMS mass spectrometer (Thermo Fisher Scientific), operating at a resolution of 35 000 with positive electrospray ionization (ESI) mode over the m/z range 90-1350, was used to detect eluting compounds. The compounds were identified based on the accurate mass and where possible by comparison with commercial standards (lactucin, lactucopicrin, 11β-13-dihydrolactucin and 11β-13-dihydrolactucopicrin). 8-Deoxylactucin and 11β-13dihydro-8-deoxylactucin were identified tentatively based on the accurate mass, retention time and MSn analysis.