5600 q tof mass spectrometer

The separation equipment for this assay was Sciex Exion LC, and the chromatographic column was Waters Acquity HSS T3 (2.1 × 150 mm, 1.7 μm). The temperature was set at 35°C, and the flow rate was 0.3 ml/min. The mobile phases were 0.1% formic acid in water (A) and acetonitrile (B), with the optimized gradient as follows: 0–5 min from 3% B to 8% B, 5–11 min from 8% B to 30% B, 11–20 min from 30% B to 80% B, 20–21 min from 80% B to 95% B, 21–25 min was maintained at 95% B, and then back to the initial ratio and re-equilibration for 7 min.
The 5600 QTOF mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with an ESI ion source was operated in positive and negative modes, and the mass range was m/z of 100–1250. The details of mass spectrometry conditions were summarized as follows: gas 1 and gas 2, 45 psi; curtain gas, 35 psi; heat block temperature, 500°C; ion spray voltage, −4.5 kV in negative mode and 5.5 kV in positive; declustering potential, 50V; collision energy, ±35 V; and the collision energy spread (CES), ±15 V. Sciex OS 1.6.1 was the basal data processing platform, and MetabolitePilot 2.0.4 software was applied for further metabolite fishing.

To provide high-resolution detection, a 5600 Q-TOF mass spectrometer (AB Sciex, Foster City, CA, USA) equipped with an electrospray ionization source (Turbo Ionspray) was applied. MS detection was implemented both in negative and positive ion mode with the mass rang at m/z 100–1,250. The parameters of the mass spectrometer were summarized as follows: gas 1 and gas 2, 45 psi; curtain gas, 35 psi. Heat block temperature, 550°C; ion spray voltage, −4.5kV in negative mode and 5.5 kV in positive; declustering potential, 50 V; collision energy, ±35 V; and the collision energy spread (CES) was ±15 V. To monitor the reproducibility and stability of the acquisition system, QC samples were prepared by pooling small aliquots of each sample. The QC specimens were analyzed every five samples throughout the whole analysis procedure.

In the current study, a TOF-MS/MS method was developed to detect flavonoid constituents and their metabolites in db/db mice. A Shimadzu LC-30A system (Kyoto, Japan) was used in this analysis. The mobile phase consisting of mobile phase A (0.1% formic acid-water) and mobile phase B (acetonitrile) was used for separation on an XSelect HSS T3 column (2.1 × 150 mm, 3.5 μm) with a flow rate of 0.3 mL/min. The following gradient elution program was used: 0–2 min, 5% B; 2–7 min, 9%–40% B; 7–12 min, 40%–65% B; 12–15 min, 65% B; 15–17 min, 65%–90% B; 17–20 min, 90% B; 20–22 min, 90%–5% B and 22–25 min, 5% B. The injection volume was 10 μL.
Mass spectrum data were acquired on AB Sciex 5600 Q-TOF mass spectrometer (Framingham, MA, United States) using negative information-dependent acquisition (IDA) modes. The source parameters were optimized and set as follows: MS and MS/MS scan mass range, m/z 100–1200 Da; gas 1, 60 Arb; gas 2, 65 Arb; curtain gas, 35 Arb; ion spray voltage, −4500 V; temperature, 550 °C; DP, −100 V; CE, −10 eV for MS scan, and 55 ± 25 eV for MS/MS scan. Real-time calibration was achieved by injecting APCI calibration solutions for every 5 samples. Analyst TF software (AB SCIEX) was used to acquire MS data.

The AlyDS44 band was excised from the gel, washed, reduced/alkylated, and digested with Trypsin, as described previously [28 (link)]. Alginate lyase peptides were then sequenced on a 5600+ qTOF mass spectrometer (AB Sciex, Framingham, MA, USA) fitted with an Ekspert nano LC 415 high performance liquid chromatography (HPLC) (Eksigent, AB Sciex, Dublin, CA, USA) Peptides were loaded onto a C18 trap and eluted onto a Nikkyo Technos (Tokyo, Japan) 15-cm capillary spray column containing 5 micron C18 beads using an acetonitrile gradient [49 (link)]. The spectra were analyzed by Pilot Protein software (version 4.5) (www.sciex.com, accessed on 6 October 2021) using protein mode against the Streptomyces amino acid database (UniProt).

The contents of chemicals were determined as described in the literature (Tao et al., 2019 (link)) with minor modifications. The analytical facility contained an UPLC system (Shimadzu, Japan) and a Q-TOF 5600⁺ mass spectrometer provided with Turbo V sources (AB sciex, USA). The chromatographic conditions were set as below: Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); sample injection volume, 5 μL; temperature of column oven, 35°C; flowrate, 0.4 mL/min; mobile phases, water with 0.1% formic acid (solvent A) and acetonitrile (solvent B). A gradient programmer was employed as follows: 5% B (0 - 2 min), 5-30% B (2.0 - 8.0 min), 30-45% B (8.0 - 9.0 min), 45-60% B (9.0 - 10.0 min), 60-80% B (10.0 - 16.0 min), 80-95% B (16.0 - 21.0 min), 95-100% B (21.0 - 22.0 min). The operating parameters for Q-TOF-MS were set as below: full-scan data acquisition was performed from m/z 100 to 1,500 in the negative mode; ion spray voltage, - 4.5 kV; collision energy, - 35 eV.

HPLC analysis was obtained by Agilent 1260 Infinity HPLC system (Agilent Technology, Santa Clara, CA, USA) equipped with a ZORBAX SB-C18 column (4.6 mm*250 mm, 5 µm) at 35 °C. Acetonitrile (solvent A) and 0.05% phosphate (solvent B) were used as the mobile phases. Gradient elution was performed by the followings: solvent A and solvent B from 23:77 to 40:60 (v:v) for 20 min, 40:60 to 75:25 for 10 min, 75:25 to 90:10 for 2 min, and keeping it until 45 min. The flow rate of the mobile phase was 1.0 mL/min, and it was monitored at 203 nm. MS analysis was obtained by Q-TOF 5600⁺ mass spectrometer provided with turbo V sources (AB sciex, USA). The operating parameters of Q-TOF–MS were set as below: full-scan data acquisition from m/z 100 to 1500 in negative mode, ion spray voltage (− 4.5 kV), and collision energy (− 35 eV).
The products prepared by the semi-preparative liquid phase were dried and determined by TLC and HPLC. The samples were then taken and dissolved in pyridine-d5 for NMR analysis.