Thermo accela 600 series hplc system

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 HPLC-UV/ESI-MS/MS analysis was performed using a TSQ Quantum Access MAX mass spectrometer in the negative mode, which was connected with a Thermo Accela 600 series HPLC system (Thermo Fisher Scientific, San Jose, CA, USA). The chromatographic separation of samples was carried out using a Waters Symmetry RP-C18 column (250 mm × 4.6 mm, 5 μm) at 25 °C. The mobile phases consisted of 0.1% (v/v) formic acid–water (A) and acetonitrile (B). The HPLC elution procedures were optimized as follows: 0–30 min, 8–30% B; 30–40 min, 30–95% B. The injection volume of all the samples was 10 μL, the flow rate was set at 800 μL/min, and the wavelength was monitored at 280 nm. Moreover, the optimized parameters of the MS instrument were implemented as follows: capillary temperature of 350 °C; vaporizer temperature of 300 °C; spray voltage of 3,000 V; sheath gas (nitrogen, N₂) of 40 psi; auxiliary gas (N₂) of 10 psi; and mass range (m/z) of 100–1,500. The mass spectrum data were obtained in the full-scan and the data-dependent mode and analyzed by the Thermo Xcalibur ChemStation (Thermo Fisher Scientific).