Thermo high resolution q exactive focus mass spectrometer

A Thermo Scientific Dionex Ultimate 3000 UHPLC system, hyphenated with a Thermo high resolution Q-Exactive focus mass spectrometer (Thermo, Bremen, Germany) was used for the analysis. The chromatographic system was coupled to the MS with a heated electrospray ionization source II (HESI II). Nitrogen (purity > 99.999%) obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA) was employed to produce MS fragmentation. Mass calibration for the Orbitrap spectrometer and HESI parameters were explained in detail and precisely in previous reports [5 (link),24 (link)].
Solvent delivery was performed at 1 mL/min using ultrapure water supplemented with 1% formic acid (A) and acetonitrile with 1% acid formic (B) and a program starting with 5% B at zero time, then maintained 5% B for 5 min, then changing to 30% B within 10 min, then maintaining 30% B for 15 min, then going to 70% B for 5 min, then maintaining 70% B for 10 min, and finally returning to 5% B in 10 min and keeping this condition for twelve additional minutes to achieve column stabilization before the next injection of 20 µL [5 (link),24 (link)].

Optical rotation was measured with a Jasco DIP-370 digital polarimeter (Tokyo, Japan). Ultraviolet (UV) spectra were recorded on a Shimadzu UV-2401PC spectrophotometer (Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra were measured on an Avance III-600 spectrometer (Bruker Biospin, Rheinstetten, Germany). Electrospray ionization mass spectrometry (ESI–MS) spectra were recorded on a Thermo high-resolution Q Exactive Focus mass spectrometer (Thermo, Bremen, Germany). Column chromatography was performed on silica gel G (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Piscataway, NJ, USA) columns.

Equipment: A Thermo Scientific Dionex Ultimate 3000 UHPLC system operated by Chromeleon 7.2 Software (Thermo Fisher Scientific, Bremen, Germany) hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo, Bremen, Germany) were employed for analysis. Nitrogen obtained from a generator (Ciantecnologica, Seville, Spain) was used as both the collision and damping gas. All calibration and equipment parameters were set as reported (Simirgiotis et al., 2016 (link)).
LC parameters: The column used was a UHPLC C18 column (Acclaim, 150 mm × 4.6 mm ID, 2.5 µm, Restek Corporation, Bellefonte PA, USA) operated at 25 °C. The detection was set at 320, 254, 280, and 440 nm, and 800–200 nm in the PDA was recorded. Mobile phases were 1% formic aqueous solution (A) and acetonitrile with 1% formic aqueous solution (B). The gradient program was: (0.00, min, 5% B); (5.00 min, 5% B); (10.00 min, 30% B); (15.00 min, 30% B); (20.00 min, 70% B); (25.00 min, 70% B); (35.00 min, 5%B); and 12 minutes before each injection for equilibration. The flow rate was set at 1.00 mL min⁻¹, and the injection volume was 10 µL.
MS parameters: The HESI II and other parameters were optimized as previously reported (Simirgiotis et al., 2016 (link)).