Uplc beh c18 column

Determination of organosulfur compounds was carried out using a UPLC equipped with a binary pump, column and sample manager, photodiode array detector (PDA), and tandem quadrupole mass spectrometer (TQD) with electrospray ionization (ESI) source working in negative mode (Waters, Milford, MA, USA), as described elsewhere [92 (link)]. Separation was performed using the UPLC BEH C18 column (1.7 µm, 100 mm × 2.1 mm, Waters) at 50 °C, at a flow rate of 0.35 mL/min. The injection volume of the samples was 5 µL. The mobile phase consisted of water (solvent A) and 40% acetonitrile in water, v/v (solvent B). The following parameters were used: capillary voltage of 3500 V; con voltage of 30 V; con gas flow of 100 L/h; source temperature of 120 °C; desolvation temperature of 350 °C; and desolvation gas flow rate of 800 L/h. Polyphenolic identification and quantitative analyses were performed on the basis of the mass-to-charge ratio, retention time, specific PDA spectra, fragment ions, and comparison of data obtained with commercial standards and literature findings. The analyses were performed in two replications.

Harmaline and harmine concentrations were simultaneous quantitative determined on a Waters-ACQUITY™ UPLC system (Waters Corp., Milford, MA, USA) using an ACQUITY UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm particle size). Mass spectrometric detection was performed using a triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA) equipped with electrospray ionization in positive ionization mode, and all other instrumental parameters were set according to our previous study (Li et al., 2016 (link)). The UPLC-ESI-MS/MS method was well-validated (data not shown) and the analytical method was successfully applied to determine the concentration of harmine and harmaline in the HBSS buffers. Figure S2 presents the representative of typical MRM chromatograms of blank HBSS, blank HBSS spiked with harmine, harmaline and IS, and IS-spiked HBSS sample collected at 30 min after administration of harmine and harmaline.

Sample extraction was performed as described by Farneti et al. [32 (link)]. Ursolic, oleanolic, and betulinic acids were identified and quantified using the ACQUITY Ultra Performance LC system with a binary solvent manager (Waters Corp., Milford, MA, USA), a UPLC BEH C18 column (1.7 μm, 2.1 mm × 150 mm, Waters Corp., Milford, MA, USA), and a Q-TOF mass spectrometer (Waters, Manchester, UK) equipped with an electrospray ionization (ESI) source, operating in the negative mode. The elution solvent was methanol-acetonitrile (15:85, v/v), at a flow rate of 0.1 mL min⁻¹. The m/z for betulinic acid was 455.3452, for oleanolic acid 455.3496, and for ursolic acid 455.3365, and the retention times were 6.80, 7.50, and 8.85 min, respectively. The compounds were monitored at 210 nm. All data were obtained in triplicate. The results were expressed as mg/100 g DW.

LC-MS/MS analysis was performed on an Agilent ultra-high performance liquid chromatography 1290 UPLC system with a Waters UPLC BEH C18 column (1.7 μm 2.1∗100 mm). The flow rate was set at 0.4 mL/min, and the sample injection volume was set at 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% in acetonitrile (B). The multistep linear elution gradient program was as follows: 0–3.5 min, 95–85% A. 3.5-min, 85–70%A. 6–6.5, 70–70% A. 6.5–12 min, 70–30% A. 12–12.5 min, 30–30% A. 12.5–18 min, 30–0% A. 18–25 min, 0–0% A. 25–26 min, 0–95% A. 26–30 min, 95–95% A.
A Q Exactive focus mass spectrometer coupled with an Xcalibur software was employed to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was from 100 to 1500, the top three of every cycle were screened, and the corresponding MS/MS data were further acquired. Sheath gas flow rate: 45 Arb, Aux gas flow rate: 15 Arb, capillary temperature: 400°C, full ms resolution: 70000, MS/MS resolution: 17500, collision energy: 15/30/45 in NCE mode, spray voltage: 4.0 kV (positive) or −3.6 kV (negative).

Metabolomics was applied using 1,290 ultra-high-performance liquid chromatography (Agilent, CA, USA) coupled with Q Exactive focus MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separations were performed using a Waters ACQUITY system equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) at the flow rate of 0.5 ml/min, and the injection volume was 4 ml. The gradient program was as follows: 85–25% B (0–11.0 min), 25–2% B (11.0–12.0 min), 2–2% B (12.0–14.0 min), 2–85% B (14.0–14.1 min), 85–85% B (14.1–15.0 min), and 85–85% B (15.0–16.0 min). The ESI source was applied to analyze the chemical composition in both positive and negative ion modes with full scan/ddMS2. The MS parameters were set as follows: the scan range was 50–1,000 m/z, the spray voltages were set at 4.0 and 3.6 kV in positive and negative modes, respectively, sheath gas was 35 arb, auxiliary gas was 10 arb, the capillary temperature was 400°C, the maximum injection time for MS1 and ddMS2 was 100 and 45 ms, respectively, and the resolutions for MS1 and ddMS2 were 70,000 and 17,500, respectively; putative molecules of interest were fragmented using three different collision energies (10, 20, and 40 eV).

We used UHPLC-QE-MS to detect and analyze the potential active components of YS to exert its medicinal effect and further clarify the components of YS. LC-MS/MS analysis was performed on a UHPLC system (Vanquish, Thermo Fisher Scientific) with a Waters UPLC BEH C18 column (1.7 μm 2.1 ^*100 mm). The flow rate was set at 0.4 mL/min, and the sample injection volume was set at 5 μL. The mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). The multi-step linear elution gradient program was as follows: 0–3.5 min, 95–85% A; 3.5–6 min, 85–70% A; 6–6.5 min, 70–70% A; 6.5–12 min, 70–30% A; 12–12.5 min, 30–30% A; 12.5–18 min, 30–0% A; 18–25 min, 0–0% A; 25–26 min, 0–95% A; 26–30 min, 95–95% A.
An Orbitrap Exploris 120 mass spectrometer coupled with an Xcalibur software was employed to obtain the MS and MS/MS data based on the IDA acquisition mode. During each acquisition cycle, the mass range was from 100 to 1,500, and the top four of every cycle were screened. The corresponding MS/MS data were further acquired. Sheath gas flow rate: 30 Arb, Aux gas flow rate: 10 Arb, Ion Transfer Tube Temp: 350°C, Vaporizer Temp: 350°C, Full ms resolution: 60,000, MS/MS resolution: 15,000, Collision energy: 16/38/42 in NCE mode, Spray Voltage: 5.5 kV (positive) or−4 kV (negative).