Ultimate 3000 series uhplc system

Electrospray Mass (ESI-MS) spectra were recorded with an UltiMate^® 3000 Series UHPLC system (Thermo Fisher Scientific, Waltman, MA, USA) connected to an amaZon speed^® ESI-Ion Trap-MS (Bruker, Billerica, MA, USA) mass spectrometer, utilizing a C18 Acquity^® UPLC BEH column (2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA) as stationary phase. HPLC parameters were set as follows: solvent A: H₂O + 0.1% formic acid, solvent B: acetonitrile (ACN) + 0.1% formic acid; gradient 5% B for 0.5 min, increasing to 100% B in 19.5 min, keeping 100% B for further 5 min; flow rate 0.6 mL/min, with diode array (DAD) detection in the range of 200–600 nm.
High Resolution Electrospray Mass (HR-ESI-MS) spectra were obtained with an Agilent 1200 Infinity Series HPLC (Agilent Technologies, Santa Clara, CA, USA) connected to a maXis^® Electrospray Time-of-flight mass spectrometer (ESI-TOF-MS; Bruker). The HPLC conditions were the same as for ESI-MS measurements.
NMR spectra were recorded with an Avance III 500 spectrometer (Bruker, ¹H NMR: 500 MHz, ¹³C NMR: 125 MHz). Optical rotations were taken with a MCP 100 circular polarimeter (Anton Paar, Graz, Austria) and UV/vis spectra with a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). ECD spectra were recorded on a J-815 spectropolarimeter (JASCO, Pfungstadt, Germany).

Chromatographic analyses were performed on a Thermo Scientific Ultimate 3,000 Series UHPLC system (Thermo Fisher Scientific, Bremen, Germany) equipped with a pump, an auto-sampler, a rapid separation-column compartment and a diode array detector. Chromatographic separation was carried on an Agilent Poroshell SB-C18 reverse-phase column (4.6 mm × 150 mm, 2.7 μm, Agilent Technologies Inc., Santa Clara, CA, United States). The column was eluted with a gradient concentration of acetonitrile and 0.1% formic acid aqueous solution at a flow rate of 0.9 ml/ min using the following program. Solvent A: 0.1% formic acid in Milli-Q^® water, solvent B: acetonitrile, 0–5 min, 5% B; 5–10 min, 5–10% B; 10–15 min, 10% B; 15– min, 10–20% B; 25–40 min, 20–30% B; 40–45 min, 30–40% B. The injection volume was set at 5 μL, 240 nm was chosen as the detection wavelength and the column temperature was maintained at 15°C.

Briefly, the UHPLC analysis was performed using the Ultimate 3000 series UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA), whereas the chromatographic separation was performed using the Thermo Hypersil aQ column (100 mm × 2.1 µm × 1.9 µm) maintained at 40 °C. For testing, a sensor wavelength of 206 nm was used. The detector was a photodiode array.
The flow rate used was 0.4 mL/min, and the injection volume of samples was 5 µL with mobile phases comprising 0.1% formic acid in water (solution A) and 0.1% formic acid in acetonitrile (solution B). A device controller and data collector were involved in the operation with Xcalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA, USA). A linear gradient elution was applied as follows: (1) 5% eluent B for 10 min, (2) a gradient from 5% to 65% eluent B over 30 min, (3) holding for 5 min, (4) returning to 5% eluent B in 35 min and (5) holding for 5 min.