Uhplc esi qtof ms

The phenolic profiles of fresh Moringa oleifera seedlings were assessed using ultra-high-performance liquid chromatography instrumentation connected to a PDA and combined with electrospray ionization quadruple-pole time-of-flight mass spectrometry equipment from Agilent Technologies (UHPLC/ESI/QTOF-MS, Micromass, Newcastle, UK) [47 (link)]. To acquire maximum signals for polyphenols, the optimized TQD tuning parameters were as follows: capillary voltage, 2.0 kV; cone voltage, 36 V; source temperature, 120 °C; desolvating temperature, 500 °C; source desolvating gas flow, 1000 L/h; and cone gas flow, 50 L/h. The flow rate was 0.25 mL/min and the injection volume was 5 μL. Mass spectrometry data were obtained using electrospray ionization in positive ionization mode. The range of the entire scan mass was from m/z 100 to 1200. High-purity nitrogen was utilized as a desolvation, cone, and collision gas. Finally, 1 µL of the solution was injected into the UPLC-ESI-MS/MS system for analysis. Data were obtained using MassLynx software (version 4.1) and processed using the TargetLynx program.

We selected phenols (gastrodin), coumarins (esculin), isoflavones (daidzin), and flavonoids (baicalin) as the transformation substrates of TtBgl3 to analyze the transformation ability of the recombinant TtBgl3 to different phenolic glycosides. The reaction mixture (400 μL) consisted of 200 mM sodium phosphate buffer (pH 6.0), 1 mg/mL substrate and 80 U/mL recombinant TtBgl3. After incubating the reaction mixture for 12 h at 37°C, the reaction was stopped using 400 μL of methanol. The mixture was centrifuged at 10,000g for 30 min at 4°C. The supernatant was analyzed by UHPLC-ESI-Q-TOF-MS (Agilent Technologies, United States) to confirm the identity of the transformation products. The optimal dosage of the recombinant TtBgl3 was determined by adding 1, 5, 10, 20, 40, 60, and 80 U/mL of the recombinant TtBgl3 to the reaction system for 12 h at 37°C. The optimal transformation time of the recombinant TtBgl3 was determined by incubating the transformation system after adding 10 U/mL recombinant TtBgl3 at 37°C for 0.5, 1, 2, 4, 8, and 12 h. All samples were analyzed by high-performance liquid chromatography (HPLC).