Thermo xcalibur chemstation

HPLC-UV/ESI-MS/MS was carried out to characterize the components in WUE-A4 before and after ultrafiltration by using a Thermo Accela 600 series HPLC connected with a TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). A Waters Symmetry RP-C18 column (4.6 × 250 mm, 5 µm) was used to perform chromatographic analysis at 30 °C, and the mobile phase consisted of H₂O with 0.1% formic acid (A) and ACN (B). The optimized HPLC elution procedures were as follows: 0–15 min, 17% B; 15–40 min, 17–30% B, 40–42 min: 30–56% B. The flow rate was 0.8 mL/min, the injection volume was 10 µL, and the HPLC-UV chromatograms were detected at a wavelength of 254 nm. The negative ion modes were applied to obtained ESI-MS/MS data. Moreover, the parameters of instrument were set as follows: the vaporizer temperature was 350 °C, the capillary temperature was 250 °C, the spray voltage was 3000 V, the cone voltage energy was 40 V, the collision energy was 10 V, the sheath gas pressure was 40 psi, the aux gas pressure was 10 psi, the drying gas flow rate was 6.0 L/min, and the mass range was set from 50 to 1100 (m/z) in the full-scan mode. Finally, the Thermo Xcalibur ChemStation (Thermo Fisher Scientific) was used for data acquisition and analysis.

Thermo Accela 600 series HPLC tandem TSQ Quantum Access MAX mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was used to identify the components in WE-P and the ultrafiltration residues [40 (link)]. Briefly, a Waters Symmetry RP-C18 column (4.6 × 250 mm, 5 µm) was used for chromatographic analysis. The mobile phase consisted of 0.1% FA-H₂O (A) and ACN (B). The optimized gradient HPLC elution procedures were set as follows: 0–28 min, 30% B; 28–32 min, 30–40% B, 32–60 min: 40–45% B. The injection volume was 10 µL, the flow rate was 0.8 mL/min, the column temperature was 30 °C, and the HPLC-UV chromatograms were obtained at a detection wavelength of 230 nm. The ESI-MS/MS parameters were applied as follows: the negative ion mode was set in this system, the spray voltage was set as 3.0 kV, the cone voltage was set as 40 V, the collision energy was set as 10 V according to the results of fragments; the sheath gas (N₂) pressure was set as 40 psi, the aux gas pressure was set as 10 psi, the nebulizing gas flow rate was set as 6.0 L/min, the vaporizer temperature was set as 350 °C, the capillary temperature was set as 250 °C, the mass range from 50 to 1100 (m/z) was set in the full-scan mode. Finally, data acquisition and analysis were carried out by the Thermo Xcalibur ChemStation (Thermo Fisher Scientific, San Jose, CA, USA).