Thermo fisher q exactive plus orbitrap mass spectrometer

UPLC-MS analysis was conducted with a Thermo-Fisher Q-Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, MA, USA) coupled to an Acquity UPLC system (Waters Corporation, Milford, MA, USA) using a reversed-phase column (BEH C18, 1.7 μm, 2.1 × 50 mm, Waters Corporation, Milford, MA, USA). Each sample was analyzed in triplicate at 0.1 mg mL⁻¹ in methanol with a 3μL injection. A binary gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) began at 10% B for 0.5 min and increased 100% B from 0.5–8.0 min, then held for 30 s. From 8.5–9.0 min, starting conditions were re-established, and the composition remained at 10% B until 10.0 min. Analysis was conducted in positive and negative ion modes with 150–1500 m/z, scan time 200 ms, capillary temperature 256°C, S-lens RF 50.00, spray voltage 3.50 kV, sheath gas flow 47.50, and auxiliary gas flow 11.25. A data-dependent method that fragmented the top four ions with HCD of 35.0 was employed.

UPLC-MS analysis was completed using a Thermo-Fisher Q-Exactive Plus Orbitrap mass spectrometer (ThermoFisher Scientific, MA, USA) connected to an Acquirity UPLC system (Waters, Milford, MA, USA) with reversed phase UPLC column (BEH C18, 1.7 μm, 2.1 × 50 mm, Waters Corporation, Milford, MA, USA). All fractions were analyzed at 0.1 mg mL⁻¹ in methanol (mass of sample per volume of solvent), with 3 μL injections. The gradient was comprised of solvent A (water with 0.1% formic acid) and solvent B (acetonitrile with 0.1% formic acid). The gradient began with 90:10 (A:B) from 0–0.5 min, and increased to 0:100 (A:B) from 0.5–8.0 min after which it was held at 100% B for 0.5 min, before returning to starting conditions over 0.5 min and held from 9.0–10.0 min. Analysis was performed in both positive and negative modes over a m/z range of 150–1500 with the: capillary voltage at −0.7 V, capillary temperature at 310°C, S-lens RF level at 80.00, spray voltage at 3.7 kV, sheath gas flow at 50.15, and auxiliary gas flow at 15.16. The four most intense ions were fragmented with HCD of 65.0. Flash chromatography was conducted with a CombiFlash RF system (Teledyne-Isco) with a photo-diode array (PDA) detector. All chemicals were acquired through Sigma-Aldrich and were spectroscopic or microbiological grade.