Tsq quantum access max mass spectrometer

The qualitative and quantitative analysis of the potential ligands in CIFE were carried out with the Thermo Accela 600 series HPLC system, which was connected with a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, United States). For the HPLC analysis, a Waters Sunfire RP-C18 column (4.6 mm × 150 mm, 5 μm) was employed. 0.1% formic acid—H₂O (0.1% FA-H₂O, A) and acetonitrile (ACN, B) were used as the mobile phases. The HPLC elution sequences were: 0–2 min, 5%B; 2–30 min, 5%–45% B; 30–35 min, 45%–95% B; 35–40 min, 95%–5%. The flow rate, injection volume, column temperature and UV detection wavelength were set as 800 μL/min, 10 μL, 30°C and 320 nm, respectively. For the ESI-MS/MS analysis, the optimized MS instrument parameters in negative ion mode were: capillary temperature, 350°C; vaporizer temperature, 300°C; spray voltage, 3.0 KV; sheath gas (N₂), 40 psi; mass range, m/z 100–1000. The full-scan and data-dependent mode was employed to obtain the mass spectrum data, which was further analyzed with the Thermo Xcalibur ChemStation.

The reaction products were analyzed by an LC-20AT HPLC system (Shimadzu, Kyoto, Japan), using an Inertsil ODS-SP reverse phase column (250 mm × 4.6 mm, 5 μm, Shimadzu) at 25°C. Solvent A was 0.1% formic acid in Milli-Q water, and solvent B was HPLC-grade acetonitrile. The system was equilibrated at 14% B for 10 min, and samples were separated on the column at a flow rate of 0.8 ml/min using a water-acetonitrile gradient in the mobile phase (14–50% B for 35 min, 50–70% B for 2 min, and 70–14% B for 1 min). The assays were monitored at 260 nm for detection of isoflavones and their glucosides, and 280 nm for flavones and their respective glucosides.
Liquid chromatography–mass spectrometry analysis was performed on an Accela LC system coupled with TSQ Quantum Access Max mass spectrometer (Thermo Scientific, USA). The column and analysis method were same with the HPLC analyses as described above. The MS data were recorded with ranges of m/z 100–800. Other parameters were set according to the previous report (Li et al., 2014 (link)).

A Thermo Accela 1250 HPLC equipped with an auto-sampler and a UV-visible detector (Thermo Fisher Scientific, San Jose, CA, USA) was employed for the analysis of crude alkaloids. A 10 μL sample was analyzed on a Phenomenex Kinetex column (2.6 μm, C18, 100 × 2.1 mm) at 25 °C. The flow rate was 0.2 mL/min and the chromatograms were recorded at the wavelength of 280 nm. 0.5% formic acid solution (adjusted to pH = 4.5 by ammonium, A) and acetonitrile (B) were selected as mobile phase, and the gradient was set as follows: 0–25 min, 5–20% (B); 25–40 min, 20% (B); 40–55 min, 20–35% (B); 55–65 min, 35–80% (B).
For ESI-MS/MS analysis, a Thermo Accela 600 HPLC system with both UV detector and TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used for the LC-MS analysis in the positive mode. The mass condition was set as follows: mass range from 200 to 800 Da; Spray Voltage, 3.0 kV; Capillary temperature, 350 °C; Sheath gas pressure, 40 psi; Aux gar pressure, 10 psi.

For HPLC analyses, samples were detected by an LC-20AT instrument (Shimadzu, Kyoto, Japan) using an inertsil ODS-SP reverse phase column (250 mm × 4.6 mm, 5 μm) at 30°C. The Milli-Q water containing 0.4% phosphoric acid (solvent A) and HPLC-grade methanol (solvent B) were used as the mobile phase. The samples from the reactions with quercetin, kaempferol, and luteolin were separated using 65% B for 45 min at a flow rate of 0.8 ml/min. For other analyses, 0.1% (v/v) formic acid (A) and acetonitrile (B) were used as the mobile phase and samples were separated as follows: 0–30 min, 25–90% B; 30–35 min, 90–25% B; 35–40 min, 25% B and the flow rate was 0.8 ml min^-1. The detection wave length was set at 260 nm for isoflavones, 280 nm for liquiritigenin, 350 nm for apigenin and luteolin, and 370 nm for quercetin, kaempferol, and isoliquiritigenin. A calibration standard curve was made from different concentrations (5–100 μg ml^-1) of each chemical standard. LC–MS/MS analysis was acquired using Accela LC system with TSQ Quantum Access Max mass spectrometer (Thermo Scientific, Waltham, MA, USA) and electrospray ionization source. The analysis method was same as the HPLC analyses described above. The MS data was recorded in a positive ion mode with the ranges of m/z 50–500. Other parameters were set according to those described previously (Li et al., 2014 (link)).

¹H-, ¹³C-, and 2D-NMR spectra were carried out in CDCl₃ using a Bruker AM-400 MHz spectrometer (Karlsruhe, Baden-Wuerttemberg, Germany) with TMS as internal standard (IS); δ in ppm and J in Hz. ESI-MS was obtained with a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Column chromatography was performed using silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), RP-C18 silica gel (150–200 mesh, Merck, Darmstadt, Hesse, Germany) and Sephadex LH-20 (20–100 μm, Sigma, St Louis, MO, USA). FBS (fetal bovine serum) and DMEM (Dulbecco’s Modified Eagle Medium) were purchased from Gibco (Life Technologies, Grand Island, NY, USA). Doxorubicin was provided by Sigma-Aldrich (St Louis, MO, USA). All other chemicals and solvents were of analytical grade.