Prepared samples were analysed on a LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer (Thermo Scientific). A Sequant ZIC-HILIC column (4.6mm x 150mm, 3.5μm) (Merck) was used to separate the metabolites with the mobile phase mixed by A=0.1% (v/v) formic acid in water and B=0.1% (v/v) formic acid in acetonitrile. A gradient program starting at 20% of A and linearly increasing to 80% at 30 min was used followed by washing (92% of A for 5 mins) and re-equilibration (20% of A for 10min) steps. The total run time of the method was 45 min. The LC stream was desolvated and ionised in the HESI probe. The Exactive mass spectrometer was operated in full scan mode over a mass range of 70–1,200 m/z at a resolution of 50,000 with polarity switching. The LCMS raw data was converted into mzML files by using ProteoWizard and imported to MZMine 2.10 for peak extraction and sample alignment. An in-house database including all possible 13C and 15N isotopic m/z values of the relevant metabolites was used for the assignment of LCMS signals. Finally the peak areas were used for comparative quantification.
Accela 600 lc system
Manufactured by Thermo Fisher Scientific
The Accela 600 LC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. It features a flow rate range of 0.001 to 10 mL/min and can operate at pressures up to 600 bar. The system includes a degasser, a quaternary pump, an autosampler, a column compartment, and a variety of detection options, such as UV-Vis and Diode Array detectors.
Automatically generated - may contain errors
Lab products found in correlation
Exactive mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Sequant zic philic column, by Merck Group (3 mentions)
Sequant zic hilic column, by Merck Group (2 mentions)
Quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Exactive, by Thermo Fisher Scientific (1 mentions)
6 protocols using accela 600 lc system
1
Quantitative HILIC-LCMS Metabolomics
2
Lipid Profiling using HPLC-Orbitrap MS
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Lipid samples were loaded onto a high‐performance liquid chromatograph (HPLC) Ascentis® Express C18 Column (15 cm, 2.1 mm, 2.7 µm) with a C18 (octadecyl) phase as matrix active group on a fused‐core particle platform and 90 Å pore size. For reversed‐phase chromatography, an HPLC Thermo Scientific Accela 600 LC System instrument was used to elute the lipids onto Q‐Exactive Orbitrap mass spectrometer, which is a hybrid quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific), with a resolving power up to 140 000 m/z 200, and internal mass accuracy of <1 ppm RMS and external <5 ppm RMS. The lipid samples were kept at −80°C until use and then resuspended in chloroform:methanol (1:1, v/v). Solvent A was a mixture of methanol:water:formic acid 0.2% in a 10 mmol L−1 ammonium acetate solution, and solvent B was a mixture of methanol:chloroform:formic acid 0.2% in a 10 mmol L−1 ammonium acetate solution. The lipids were gradient eluted following a 15 minutes 35%‐100% of solvent B gradient at a 260 μL/min flow rate. The mass spectrometer was operated both in positive and negative modes, within a range of collision energies.
3
LCMS-Based Metabolite Profiling Protocol
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Prepared samples were analysed on a LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer (Thermo Scientific). A Sequant ZIC-pHILIC column (4.6 mm × 150 mm, 5 μm) (Merck) was used to separate the metabolites with the mobile phase mixed by A= 20 mM ammonium carbonate in water and B= acetonitrile. A gradient program starting at 20% of A and linearly increasing to 80% at 30 min was used followed by washing (92% of A for 5 min) and re-equilibration (20% of A for 10 min) steps. The total run time of the method was 45 min. The LC stream was desolvated and ionised in the HESI probe. The Exactive mass spectrometer was operated in full scan mode over a mass range of 70–1,200 m/z at a resolution of 50,000 with polarity switching. The LCMS raw data was converted into mzML files by using
ProteoWizard (v3.0.4472, 64-bit) and imported to
MZMine (2.10) for peak extraction and sample alignment. A house-made database including all possible
13C isotopic m/z values of the relevant metabolites was used for the assignment of LCMS signals. Finally the peak areas were used for comparative quantification.
13C isotopic m/z values of the relevant metabolites was used for the assignment of LCMS signals. Finally the peak areas were used for comparative quantification.
4
LC-HRMS Metabolite Profiling Protocol
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LC-HRMS was performed in an Accela 600 LC system (Thermo Fisher Scientific) coupled to an Exactive (Orbitrap) mass spectrometer (Thermo Fisher Scientific). Metabolite separation was done using a SeQuant ZIC-pHILIC column (4.6 mm × 150 mm, 5 μm) (Merck). The mobile phase consisted of an aqueous solution of ammonium carbonate (20 mM, pH 9.2) (A) and acetonitrile (B) and the following gradient profile was applied: linear increase of A from 20 to 80% at 0 to 30 min, 92% A 31–37 min and linear decrease of A to 20% at 37–46 min. Total injection volume was 20 µL and samples were maintained at 4 °C throughout analysis. The spectrometer was operated in both positive and negative electrospray ionisation (ESI) modes, full-scan mode over a mass range of m/z 70–1200 at a resolution of 50,000. The capillary temperature was 320 °C and the seath and auxiliary gas flow rates were 50 and 17 units, respectively. Thermo raw files were converted to mzML files using ProteoWizard, separated into ESI positive and negative modes, and imported to MZMine 2.53 for peak processing. Metabolite identification was performed using an in-house standard library and peak areas were exported for analysis. The (peak area) energy charge ratio (ECR) was calculated from the formula ECR = ([ATP] + 0.5 [ADP])/([ATP] + [ADP] + [AMP]), where [metabolite] is the metabolite’s peak area [19 (link)].
5
Quantitative HILIC-LCMS Metabolomics
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Prepared samples were analysed on a LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer (Thermo Scientific). A Sequant ZIC-HILIC column (4.6mm x 150mm, 3.5μm) (Merck) was used to separate the metabolites with the mobile phase mixed by A=0.1% (v/v) formic acid in water and B=0.1% (v/v) formic acid in acetonitrile. A gradient program starting at 20% of A and linearly increasing to 80% at 30 min was used followed by washing (92% of A for 5 mins) and re-equilibration (20% of A for 10min) steps. The total run time of the method was 45 min. The LC stream was desolvated and ionised in the HESI probe. The Exactive mass spectrometer was operated in full scan mode over a mass range of 70–1,200 m/z at a resolution of 50,000 with polarity switching. The LCMS raw data was converted into mzML files by using ProteoWizard and imported to MZMine 2.10 for peak extraction and sample alignment. An in-house database including all possible 13C and 15N isotopic m/z values of the relevant metabolites was used for the assignment of LCMS signals. Finally the peak areas were used for comparative quantification.
6
LCMS Analysis of Metabolites
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LCMS analysis was performed as described previously 56 . Prepared samples were analysed on a LCMS platform consisting of an Accela 600 LC system and an Exactive mass spectrometer (Thermo Fisher Scientific). A Sequant ZIC-pHILIC column (4.6mm x 150mm, 5μm) (Merck) was used to separate the metabolites with the mobile phase mixed by A=20mM ammonium acetate in water and B=acetonitrile. A gradient program starting at 20% of A and linearly increasing to 80% at 30 min was used followed by washing (92% of A for 5 mins) and re-equilibration (20% of A for 10min) steps. The total run time of the method was 45 min. The LC stream was desolvated and ionised in the HESI probe. The Exactive mass spectrometer was operated in full scan mode over a mass range of 70-1,200 m/z at a resolution of 50,000 with polarity switching.
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