An Agilent UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was used to characterize the CER profiles. The mobile phase was composed of solvent A (water with 20 mM ammonium formate pH 5) and solvent B (methanol). Initially, 70% of B was held isocratically for 1 min, followed by a linear increase to 100% of B within 75 min, and return to initial conditions in 5 min. The CERs were separated on an RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) with a flow rate of 0.5 mL/min. The QTOF mass spectrometer was operated in positive ion mode (electrospray voltage 3.5 kV) with a capillary temperature of 300 °C and a sheath gas flow rate of 8 L/min. The data was collected in DDA mode. Identification of CER species was based on the presence of the [M + H]+ molecular ion, retention time and characteristic fragmentation patterns observed in MS/MS spectra, which was previously described in detail19 (link). Cermide internal standards, N-lignoceroyl-d-erythro-sphingosine and N-lignoceroyl-d-erythro-sphinganine (Avanti Polar Lipids, Alabaster, AL, USA) were used for quantification of CERs.
Acquity beh shield 2.1 100 mm 1 7 μm
Manufactured by Waters Corporation
Sourced in United States
The Acquity BEH Shield 2.1 × 100 mm; 1.7 μm is a high-performance liquid chromatography column produced by Waters Corporation. The column features a 2.1 mm internal diameter, a length of 100 mm, and a particle size of 1.7 micrometers. This column is designed for use in analytical applications that require high-resolution separation and sensitivity.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 6540, by Agilent Technologies (4 mentions)
Agilent 1290, by Agilent Technologies (4 mentions)
N lignoceroyl d erythro sphingosine, by Avanti Polar Lipids (1 mentions)
N lignoceroyl d erythro sphinganine, by Avanti Polar Lipids (1 mentions)
Uplc esi qtof ms system, by Agilent Technologies (1 mentions)
4 protocols using acquity beh shield 2.1 100 mm 1 7 μm
1
UPLC-ESI-QTOF-MS Characterization of Ceramide Profiles
2
RP-LC-MS/MS for Ceramide Profiling
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The RP-LC-MS/MS was applied to obtain the CER profiles. The same Agilent UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was applied for analysis. Chromatographic separation of CERs was performed on RP-C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) in gradient elution with the mixture of water (20 mM ammonium formate, pH 5) and methanol. The QTOF mass spectrometer was operated in positive (electrospray voltage 3.5 kV) ion mode with a capillary temperature of 300 °C and a sheath gas flow of 8 L/min, as previously described in details [27 (link)].
3
UPLC-ESI-QTOF-MS Ceramide Profiling
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An Agilent UPLC–ESI–QTOF–MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was used to characterize the CER profiles. The mobile phase was composed of solvent A (water with 20 mM ammonium formate pH 5) and solvent B (methanol). Initially, 70% of B was held isocratically for 1 min, followed by a linear increase to 100% of B within 75 min, and return to initial conditions in 5 min. The ceramides were separated on an RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA) with a flow rate of 0.5 mL/min. The QTOF mass spectrometer was operated in positive ion mode (electrospray voltage 3.5 kV) with a capillary temperature of 300 °C and a sheath gas flow rate of 8 L/min. The data was collected in DDA mode. Identification of ceramide species was based on the presence of the [M+H]+ molecular ion, retention time, and characteristic fragmentation patterns observed in MS/MS spectra, which was previously described in detail [71 (link)].
4
Lipidomic Analysis of Ceramide Profiles
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Reversed-phase (RP) chromatography LC-MS/MS was utilized to characterize ceramide (CER) profiles. The same UPLC-ESI-QTOF-MS system (Agilent 1290; Agilent 6540; Agilent Technologies, Santa Clara, CA, USA) was used for the analysis. The separation of ceramides was carried out on an RP C18 column (Acquity BEH Shield 2.1 × 100 mm; 1.7 μm; Waters, Milford, MA, USA). The mobile phase consisted of water with 20 mM ammonium formate at pH 5 (A) or methanol (B). The solvent gradient started at 70% eluent B held for 1 min, linearly increasing to 100% within 75 min, and returning to initial composition over a final 5 min period. Flow rate was 0.5 mL/min. The MS analysis was performed in positive-ion mode. Electrospray voltage set to 3.5 kV; the drying and sheath gas temperatures set to 300 °C, and the drying and sheath gas flow rates set to 6 and 8 L/min, respectively, were the typical ESI conditions. Data was acquired in DDA mode. Ceramides were identified according to the presence of the [M + H]+ molecular ion, retention time, and characteristic fragmentation patterns, as described previously [33 (link)].
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