Quantification of adenine nucleotides in VSMCs was done as previously described [41 ]. Briefly, serum-deprived VSMCs were exposed to PIO (30 μM, 3hr) or vehicle control. VSMCs were collected in ice-cold PBS, lysed in ice-cold 5% perchloric acid, and then centrifuged at 10,000 × g for 5 min at 4°C to remove the acid-insoluble material. Perchloric acid in the collected supernatant was extracted by three washes with 10% excess volume of a 1:1 mixture of tri-n-octylamine and 1,1,2-trichlorotrifluroethane. Adenine nucleotides in the aqueous phase were analyzed using liquid chromatography/tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) on a 4000 QTRAP LC/MS/MS system (Applied Biosystems, Carlsbad, CA). The samples and standards were run on a Amide XBridge HPLC column (Cat # 186004860; 3.5 μM particle size; 2.1 mm inner diameter × 100 mm length, Waters, Milford, MA) using buffer A (20 mM ammonium hydroxide and 20 mM ammonium acetate in 5% acetonitrile, pH 9.0) and buffer B (100% acetonitrile) at a flow rate of 0.3 ml/min for 10 min. The mobile phase consists of isocratic elusion with 20% buffer B. The concentrations of AMP, ADP and ATP were calculated from standard curves of a serial dilution of a standard consisting of known concentrations of AMP, ADP and ATP that were run in parallel with the samples in the same session.
Amide xbridge hplc column
Manufactured by Waters Corporation
Sourced in United States
The Amide XBridge HPLC column is a high-performance liquid chromatography column designed for the separation and analysis of a wide range of polar compounds. The column features a versatile amide stationary phase that provides excellent retention and selectivity for analytes such as carbohydrates, amino acids, and other polar molecules.
Automatically generated - may contain errors
Lab products found in correlation
4000 qtrap lc ms ms system, by Thermo Fisher Scientific (1 mentions)
Agilent 6460, by Agilent Technologies (1 mentions)
Hplc system, by Agilent Technologies (1 mentions)
Guard column, by Waters Corporation (1 mentions)
A955 4, by Thermo Fisher Scientific (1 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (1 mentions)
4 protocols using amide xbridge hplc column
1
Quantification of Adenine Nucleotides in VSMCs
2
HPLC-QqQ MS Targeted Metabolomics
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The pseudotargeted metabolite analysis was conducted by HPLC-QqQ MS (Agilent 6460 coupled with an Agilent 1260 HPLC system, QqQ MS; Agilent, Los Angeles, CA). Dried samples were resuspended with 60 μL of water and then injected into the HPLC system (Agilent, CA, USA) through an Amide XBridge HPLC column (4.6 × 100 mm, 3.5 µm) with a guard column (Waters, Milford, MA, USA) for the separation. The gradient conditions and solvents (A: 95:5 (v/v) water:acetonitrile with ammonium acetate (20 mM) and ammonium hydroxide (20 mM) and B: 100% acetonitrile) were adopted and modified [29 (link)]. The data were acquired by electrospray ionization (+)/(−) ion-switching targeted metabolomics
3
Faecal Metabolite Profiling by LC-MS
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To measure metabolites in faecal samples, 50 mg of fresh faeces was suspended in 1.5 mL of PBS and passed through a 40 μm cell strainer. 100 mL of sample was collected following centrifugation at 7200×g for 10 min. Targeted metabolomic analysis was performed on a liquid chromatography–tandem mass spectrometry (LC–MS/MS) system consisting of an Agilent 1260 Infinity LC system (Santa Clara, CA, USA) coupled to a QTRAP 5500 mass spectrometer (AB Sciex, Foster City, CA, USA). Hydrophilic interaction chromatography, with an Amide XBridge HPLC column (Waters, Millford, MA; #186004860; 4.6 mm × 100 mm, 3.5 μm) was used to separate and analyse 65 polar metabolites, including amino acids; nucleotide and nucleoside phosphates; high-energy intermediates; organic acids; Krebs cycle intermediates and glycolytic intermediates56 (link). Mobile Phase A consisted of HPLC grade acetonitrile:water (5:95% v/v) (Fisher Chemical, #FSBW6-4) with 20 mM ammonium acetate (Sigma-Aldrich, #73594-25G-F) and 20 mM ammonium hydroxide (Fluka, #44273), while Mobile Phase B consisted of 100% acetonitrile (Fisher Chemical, #A955-4). A full-scan analysis over 70–800m/z at 70,000 resolution and a 3-Hz data acquisition rate was used.
4
Targeted Metabolite Profiling of Mouse Liver
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Mice liver tissue was homogenized in ultrapure water (1:10 w/v). Liver homogenate (100 μL) was extracted by adding 500 μL methanol containing IS (13C‐gutamine). The samples were then centrifuged, dried and reconstituted prior to analysis on liquid chromatography mass spectrometry quadrupole time‐of‐flight (LCMS‐Q‐TOF). Finally, 20 μL aliquot was injected into LCMS‐Q‐TOF for analysis. HPLC separation was conducted by a Waters Amide XBridge HPLC column (4.6 × 100 mm, 3.5 μm; Waters) maintained at 30°C. The mass spectrometer was detected in negative electrospray ionization (ESI) mode by a TripleTOF system. More detailed sample preparation and LCMS‐Q‐TOF setup is shown in Appendix S1 .
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