Metabolites were extracted from plasma samples. Untargeted metabolomics analysis was conducted by using ultra-high-performance liquid chromatography (UHPLC, 1290 Infinity LC, Agilent Technologies, Palo Alto, CA, USA) and a quadrupole time-of-flight mass spectrometer (TripleTOF 6600; AB Sciex, Framingham, MA, USA). The separation was performed using a 2.1 mm × 100 mm ACQUITY UPLC BEH 1.7 μm column (Waters, Wexford, Ireland). The mobile phase consisted of A. 25 mM ammonium acetate with 25 mM ammonium hydroxide; B. acetonitrile. Gradient elution was performed as follows: 95% B for 0.5 min, and was reduced linearly to 65% in 7 min, next, the gradient was reduced to 40% in 2 min, increased to 95% in 0.1 min, then with a re-equilibration period employed for 3 min. The flow rate was set to 0.3 mL min−1, column temperature at 25 °C and injection volume of 2 µL. The ESI conditions were as follows: Ion Source Gas1(Gas1): 40 psi; Ion Source Gas2 (Gas2): 80 psi; curtain gas (CUR): 30 psi; source temperature: 650℃; IonSpray Voltage Floating (ISVF) ± 5500 V. The raw data were converted to MzXML by MSconventer (ProteoWizard, Palo Alto, CA, USA), and imported into XCMS software (Scripps Research Institute, La Jolla, CA, USA) for alignment, feature detection, retention time correction, and data filtering.
Acquity uplc beh 1.7 μm column
Manufactured by Waters Corporation
Sourced in Ireland
The ACQUITY UPLC BEH 1.7 μm column is a high-performance liquid chromatography (HPLC) column designed for use with ultra-performance liquid chromatography (UPLC) systems. The column features a 1.7 μm particle size and a proprietary bridged ethylene hybrid (BEH) packing material, which provide high efficiency and resolution for a wide range of analytes.
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4 protocols using acquity uplc beh 1.7 μm column
1
Untargeted Metabolomics Analysis of Plasma
2
UHPLC-QTOF-MS/MS for Metabolite Profiling
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Analysis was performed using a UHPLC (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (AB Sciex TripleTOF 6600) at Shanghai Applied Protein Technology Co., Ltd. To achieve HILIC separation, we used a 2.1 × 100 mm ACQUITY UPLC BEH 1.7 μm column (Waters, Ireland). The mobile phases for both ESI+ and ESI− modes consisted of A, which was 25 mmol/L ammonium acetate and 25 mmol/L ammonium hydroxide in water, and B, which was acetonitrile. To obtain RPLC separation, we used a 2.1 × 100 mm ACQUITY UPLC HSS T3 1.8-μm column (Waters, Ireland). For ESI+ mode, the mobile phases included A, which was water with 0.1% formic acid, and B, which was acetonitrile with 0.1% formic acid. On the other hand, for ESI− mode, the mobile phases comprised A, which was 0.5 mmol/L ammonium fluoride in water, and B, which was acetonitrile. During the MS-only acquisition, the instrument was set to capture data within the m/z range of 60–1,000 Da, with an accumulation time of 0.20 s/spectra for the TOF MS scan. Meanwhile, in auto MS-MS acquisition, the instrument captured data within the m/z range of 25–1,000 Da, with an accumulation time of 0.05 s/spectra for the product ion scan. To conduct the product ion scan, we used information-dependent acquisition, specifically selecting the high-sensitivity mode (11 (link)).
3
UHPLC-MS/MS Protocol for Metabolite Analysis
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A UHPLC system (1290 Infinity LC, Agilent Technologies, Santa Clara, CA, USA), with the addition of a quadrupole time-of-flight mass spectrometer (Triple TOF 6600, AB Sciex, Foster City, CA, USA) with an electrospray ionization source (ESI), was used for the analysis. The samples were separated on an ACQUITY UPLC BEH 1.7 μm column (2.1 mm × 100 mm, Waters, Ireland). The mobile phase, which contained A (containing 25 nM ammonium acetate and 25 nM ammonium hydroxide in water) and B (acetonitrile), was used for both of the positive and negative ion modes. The gradient was set as follows: 95% B (1 min), which decreased linearly to 65% (13 min) and then decreased linearly to 40% (2 min); and 40% B (3 min), which then increased to 95% (0.1 min). The ESI parameters were as follows: ion source gas 1, 60; ion source gas 2, 60; and ion spray voltage floating, ± 5500 V. With respect to the MS-only acquisition, apparatus were set to a m/z range of 60–1000 Da and the scanning of TOF MS was 0.2 s per spectrum. For the auto MS/MS acquisition, apparatus were set to a m/z range of 25–1000 Da and the with scanning of product ion was 0.05 s per spectra. For the product ion scan acquisition: fixed collision energy, 35 V ± 15 eV; declustering potential, 60 V (+) and −60 V (−); and exclusion of isotopes within 4 Da.
4
Metabolite Profiling by UHPLC-QTOF-MS
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Analysis was performed by an ultra-high-pressure liquid chromatography (1290 Infinity LC, Agilent Technologies) coupled to a quadrupole time-of-flight (Triple TOF 6600, AB SCIEX). For hydrophilic interaction liquid chromatography separation, a 2.1 mm × 100 mm ACQUITY UPLC BEH 1.7 μm column (Waters, Ireland) was used. The mobile phase contained A = 25 mM of ammonium acetate and 25 mM of ammonium hydroxide in water and B = acetonitrile. The gradient was: 95% B (0-0.5 min), 95–65% B (0.5-7 min), 65–40% B (7–8 min), 40% B (8–9 min), 40–95% (9-9.1 min), and 95% B (9.1–12 min).
After separation, the samples were detected by Triple TOF 6600 through electrospray ionization (ESI) positive and negative modes. The apparatus was set up for MS-only collection with a m/z range of 60–1,000 Da and a TOF MS scan accumulation time of 0.20 s/spectrum. For automatic MS/MS collection, a m/z range of 25–1,000 Da and a product ion scan accumulation time of 0.05 s/spectra were used. Information-dependent acquisition was applied to gather the product ion scan in the selected high-sensitivity mode with the following conditions: the collision energy = 35 ± 15 eV and declustering potential = ± 60 V. Isotopes within 4 Da were excluded. Ten candidate ions were monitored during each cycle.
After separation, the samples were detected by Triple TOF 6600 through electrospray ionization (ESI) positive and negative modes. The apparatus was set up for MS-only collection with a m/z range of 60–1,000 Da and a TOF MS scan accumulation time of 0.20 s/spectrum. For automatic MS/MS collection, a m/z range of 25–1,000 Da and a product ion scan accumulation time of 0.05 s/spectra were used. Information-dependent acquisition was applied to gather the product ion scan in the selected high-sensitivity mode with the following conditions: the collision energy = 35 ± 15 eV and declustering potential = ± 60 V. Isotopes within 4 Da were excluded. Ten candidate ions were monitored during each cycle.
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