Bile acids (cholic acid CA, chenodeoxycholic acid CDCA, deoxycholic acid DCA, ursodeoxycholic acid UDCA, lithocholic acid LCA) including their glycine- and taurine derivatives were analyzed by UPLC-MS/MS. The system consisted of an Acquity UPLC-H Class (Waters, UK) coupled to a Xevo-TQS tandem mass spectrometer (Waters, UK) which is equipped with an ESI source operating in the negative ion mode. Quantitative data were conducted in the multiple reaction monitoring (MRM) mode. The chromatographic separation was performed on Waters UPLC BEH C18 column (100 mm, 2.1 mm ID, 1.7 µm; Waters, UK) using acetonitrile and acidic water (0.1% formic acid) as mobile phases. Analytes were separated by a gradient elution. The injection volume was 5 µL and the column was maintained at 40 °C.
Waters uplc beh c18 column
Manufactured by Waters Corporation
Sourced in United States
The Waters UPLC BEH C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. The column features a bonded phase of ethylene-bridged hybrid (BEH) particles, which provide excellent peak shape, resolution, and low backpressure. The C18 stationary phase is suitable for the analysis of a variety of polar and non-polar compounds.
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Lab products found in correlation
Acquity uplc h class, by Waters Corporation (1 mentions)
Xevo tq s tandem mass spectrometer, by Waters Corporation (1 mentions)
Agilent 6460, by Agilent Technologies (1 mentions)
1260 series, by Agilent Technologies (1 mentions)
Waters 600 pump system, by Waters Corporation (1 mentions)
Vion ims qtof mass spectrometer, by Waters Corporation (1 mentions)
Sepa 300 high sensitive polarimeter, by Horiba (1 mentions)
Uv 1900i uv vis spectrophotometer, by Shimadzu (1 mentions)
Analyst 1, by AB Sciex (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Agilent 6520 q tof, by Agilent Technologies (1 mentions)
Milli q purification system, by Merck Group (1 mentions)
Chromatographic grade methanol, by Thermo Fisher Scientific (1 mentions)
Agilent 1290 infinity uplc, by Agilent Technologies (1 mentions)
Chlorogenic acid, by Yuanye Bio-Technology (1 mentions)
Oleanolic acid, by Yuanye Bio-Technology (1 mentions)
Ursolic acid, by Yuanye Bio-Technology (1 mentions)
Waters acquity uplc system, by Waters Corporation (1 mentions)
6 protocols using waters uplc beh c18 column
1
Quantitative Bile Acid Analysis by UPLC-MS/MS
2
Quantifying O-sulfotyrosine in Serum
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Serum levels of O-sulfotyrosine were analyzed using a high-performance liquid chromatography system (1260 series, Agilent Technologies; Palo Alto, CA, USA) and mass spectrometer (Agilent 6460, Agilent Technologies; Palo Alto, CA, USA) as previously described [21 (link), 22 (link)]. Patient and mouse serum were collected, and 1.5 mL of chloroform/methanol (2:1, v/v) was added. The solution was vortexed for one minute and then centrifuged for 10 min at 3000 g. Next, an 800 μL organic phase adding into a cleaning tube, dried with nitrogen. Mass spectrometry analysis was conducted by adding 200 μL of isopropanol/methanol solution (1:1, v:v), and the supernatant was transferred to HPLC vials for LC-MS analysis. A Waters UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm, Waters, USA) was used for chromatographic separation. Multiple reaction monitoring mode was used to detect O-sulfotyrosine. The analysis was performed by Shanghai Applied Protein Technology Inc.
3
Analytical Characterization of Chemical Compounds
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All solvents and reagents were of analytical grade and purchased from commercial sources. The UV spectra and optical rotations were recorded using a UV-1900i UV-Vis spectrophotometer (Shimadzu Corporation, Kyoto, Japan) and a HORIBA SEPA-300 high-sensitive polarimeter (HORIBA, Kyoto, Japan). The IR spectra were recorded using an FT-720 spectrometer equipped with a DuraSampl IR II ATR instrument (HORIBA). MPLC was performed using the Teledyne ISCO CombiFlash Companion (Teledyne ISCO, Lincoln, NE, USA). Preparative HPLC was conducted using a Waters 600 pump system (Waters, Milford, MA, USA) equipped with a Cosmosil MS-II C18 column (5 μm, 10 mm i.d. × 250 mm; Nacalai, Kyoto, Japan). DAD-LC-MS analyses were conducted using a Waters UPLC H-class system with a Waters UPLC BEH C18-column (1.7 μm, 2.1 mm i.d. × 50 mm; Waters) connected to an AB Sciex API3200 MS/MS system (AB Sciex, Framingham, MA, USA) equipped with an electrospray ionization (ESI) probe. HR–ESI–TOFMS was measured using a Vion IMS QTOF Mass Spectrometer (Waters). NMR spectra were recorded using a JEOL ECA500 FT-NMR spectrometer (JEOL, Tokyo, Japan) at 500 MHz for 1H NMR and 125 MHz for 13C NMR. The chemical shifts were referenced to the corresponding solvent signals (δH 7.24 and δC 77.23 in CDCl3).
4
Quantitative Analysis of URM and UNO in Rat Plasma
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Plasma levels of URM and UNO in rat plasma were determined on a SCIEX Triple QuadTM 5500 mass spectrometer with Turbo VTM source (AB SCIEX, Concord, Ontario, Canada) coupled with Waters AcquityTM I-class system (Waters Corp, Milford, MA, USA). The chromatographic separation was performed on a Waters UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm; Waters Corp), and the column was maintained at 45°C. The mobile phase consisting of (A) 0.1% formic acid with 5 mM ammonium acetate in water and (B) 0.1% formic acid in acetonitrile/methanol (9/1, v/v). Analysis was performed using the following gradient elution at a flow rate of 0.5 mL/min: 0–0.2 min, 10% B; 0.2–1.0 min, 10% B to 60% B; 1.0–1.1 min, 60% to 95% B; 1.1–1.5 min, maintained at 95% B; 1.5–2.0 min, re-equilibration with initial condition. The injection volume was 1 μL.
The MS was operated in positive ionization mode with electrospray voltage 5,500 V, source temperature of 550°C and collision activation dissociation of 7 psi. The detection was performed in the multiple reaction monitoring (MRM) mode at m/z 352.1→120.0 (collision energy (CE) 37 eV) for URM, 368.1→120.0 (CE 42 eV) for UNO and 336.1→120.1 (CE 36 eV) for SCN (IS), respectively, and declustering potential was 150 V for the analytes and IS. Data were acquired and processed in Analyst 1.6.3 software (AB SCIEX, Concord, Ontario, Canada).
The MS was operated in positive ionization mode with electrospray voltage 5,500 V, source temperature of 550°C and collision activation dissociation of 7 psi. The detection was performed in the multiple reaction monitoring (MRM) mode at m/z 352.1→120.0 (collision energy (CE) 37 eV) for URM, 368.1→120.0 (CE 42 eV) for UNO and 336.1→120.1 (CE 36 eV) for SCN (IS), respectively, and declustering potential was 150 V for the analytes and IS. Data were acquired and processed in Analyst 1.6.3 software (AB SCIEX, Concord, Ontario, Canada).
5
Identification of Bioactive Compounds in Plant Extracts
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Periplocin (≥98%), periplocymarin (≥98%), periplogenin (≥98%), and scopoletin (≥98%) were purchased from Chengdu Durst Biotechnology Co., Ltd. (Chengdu, China). Isovanillin (≥98%), isovanillic acid (≥98%), chlorogenic acid (≥98%), ursolic acid (≥98%), and oleanolic acid (≥98%) were obtained from Shanghai Yuanye biotechnology Co., Ltd. (Shanghai, China). Chromatographic grade methanol and acetonitrile were purchased from Fisher company (Thermo Fisher Scientific (China) Co. Ltd., Shanghai, China). Anhydrous ethanol (chromatographically pure) was obtained from Concord technologies Ltd. (Tianjin, China). Water was obtained from a Milli-Q purification system (Millipore, Bedford, MA, USA). Nylon membranes were purchased from Tianjin Bojin Technology Co., Ltd. (Tianjin, China).
The UPLC-Q-TOF-MS/MS system is comprised of an Agilent 1290 Infinity UPLC (Agilent Technologies Inc., Palo Alto, CA, USA) and an Agilent 6520 QTOF. The UPLC system includes an Agilent 4220 binary pump combined with a degasser, an Agilent 4212 diode array detector, an Agilent 4226 well plate sampler, and an Agilent 1316 thermostatic column compartment. Chromatographic separation was performed on a Waters UPLC®BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA).
The UPLC-Q-TOF-MS/MS system is comprised of an Agilent 1290 Infinity UPLC (Agilent Technologies Inc., Palo Alto, CA, USA) and an Agilent 6520 QTOF. The UPLC system includes an Agilent 4220 binary pump combined with a degasser, an Agilent 4212 diode array detector, an Agilent 4226 well plate sampler, and an Agilent 1316 thermostatic column compartment. Chromatographic separation was performed on a Waters UPLC®BEH C18 column (2.1 × 100 mm, 1.7 µm, Waters, Milford, MA, USA).
6
UPLC Quantification of Glycyrrhizic Acid in Hairy Roots
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The UPLC method for determination of the GA contents in G. uralensis hairy roots established in our previous study was adopted (Wang et al. 2021 (link)). The standard compound GA with a purity of 99.45% was purchased from China National Institutes for Food and Drug Control. The stock solution of GA was prepared with 50% methanol at a concentration of 0.0904 mg mL -1 . The linear curve was evaluated at seven points by gradient dilution of the GA stock solution to 0.09040, 0.07232, 0.05424, 0.04520, 0.01808, 0.00904, and 0.00452 mg mL -1 . 100 mg of each hairy root powder sample was extracted with 50 mL 50% methanol using ultrasonic for 30 min (40 kHz, 500 W), and ltered with 0.45 μm lter membranes. The chromatographic conditions were as follows: chromatographic instrument: Waters Acquity UPLC system (Waters Corporation, Milford, Massachusetts, USA), chromatographic column: Waters UPLC BEH C 18 column (2.1 mm × 100 mm, 1.7 μm), UV detection wavelength: 250 nm, column temperature: 40 °C, ow rate: 0.3 mL•min -1 , and injection volume: 1 μL. The mobile phase A was acetonitrile and B was 0.05% phosphoric acid. The UPLC gradient elution program was shown in Table S6.
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