Xcalibur software 3

All reagents were supplied commercially and they were used without further purification. The melting points (°C) were determined using an electronic melting point apparatus (Electrothermal, Essex, UK) and they were uncorrected. IR spectra were recorded on the Perkin Elmer FT-IR system, spectrum BX, as wave numbers (cm⁻¹) using KBr discs. The NMR analyses were performed in deuterated dimethyl sulfoxide (DMSO-d₆) and they were recorded on different Spectrometers (Tokyo, Japan) including, an Eclipse 300 FT NMR Spectrometer at 300 MHz and on a JNM-Ecx500II FT NMR system spectrometer at 500 MHz for the ¹HNMR analysis, whereas the ¹³CNMR on the Agilent NMR-600, was working at 150 MHz. Chemical shifts (δ) were expressed in ppm relative to tetramethylsilane (TMS) as the internal standard; the coupling constant values (J) were expressed in Hz and the splitting patterns of ¹HNMR were designated as s (singlet), d (doublet), t (triplet), dd (double-doublet), app. (apparent), m (multiplet) and q (quartet). Mass spectra were carried out on a direct probe controller inlet part of a single quadrupole mass analyzer in the Thermo Scientific GCMS model (ISO Lt) using Thermo X-Calibur Software 3.0.

Ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI–MS/MS) was performed using an electrostatic field orbitrap mass spectrometer (QE) coupled with an Ultimate 3000 UPLC System (Thermo Fisher, CA, USA). Instrument control, data acquisition and analysis were performed using Xcalibur software 3.0 (Thermo Fisher, CA, USA). The sample vials were maintained at 10 °C in a thermostatic auto sampler. Chromatographic separation was achieved on a Halo-C18 column (2.1 × 100 mm, 2.7 μm; AMT, USA) with the column temperature set to 45 °C. A total of 5 μL of each sample was injected into the column. The mobile phase consisted of solvent A (0.05% formic acid in water) and solvent B (0.05% formic acid in acetonitrile). The linear gradient programme was as follows: 0–1 min, 2% B; 3 min, 40% B; 15 min, 98% B; 17 min, 98% B; 17.1 min, 2% B; and 3.0 min of equilibration. The flow rate was 0.3 mL·min⁻¹. Before running the samples, 6 QC samples were run to balance the instrument, and then 1 QC sample was run after every 6 samples. Meanwhile, the mass spectrometry detections were performed as follows: capillary temperature, 350 °C and spray voltage, 3.5 kV and 3.0 kV for positive ion mode and negative ion mode, respectively. The mass scan range was from 80 to 1200 Da. The mass resolution was set to 70,000.

Each experiment was repeated at least three times, and the experimental data were expressed as the mean ± standard deviation. The RSE data were analyzed using Design-Expert 11 software. Analysis of variance (ANOVA) was performed using IBM SPSS statistics 22.0 software. HPLC–MS analysis was performed using Xcalibur software 3.0 (Thermo Fisher Scientific Inc., Waltham, MA, USA).