Plasma kynurenine levels in treated and control naive hemophilia mice were determined at the indicated time points using High Pressure Liquid Chromatography-Mass Spectrometry (HPLC-MS) by an adapted method from University of Washington Nutrition Obesity Research Center (NORC) Metabolomics Subcore. The LC system was composed of a Agilent 1260 binary pump, an Agilent 1260 auto-sampler, and Agilent 1290 column compartment (Agilent Technologies, Santa Clara, CA). Injection volume was 2 μL. Chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on SeQuant ZIC-cHILIC column (Merck KGaA, Darmstadt, Germany) with a flow rate at 0.3 mL/min. The identities of L-kynurenine and L-tyrosine were confirmed by spiking the pooled serum sample (QC sample) with a mixture of standard L-kynurenine and L-tyrosine. After the chromatographic separation, MS ionization and data acquisition were performed using an AB Sciex QTrap 5500 mass spectrometer (AB Sciex, Toronto, ON, Canada) equipped with an electrospray ionization (ESI) source working in positive ionization mode.
Sequant zic chilic column
Manufactured by Merck Group
Sourced in Germany, United States
The SeQuant ZIC-cHILIC column is a liquid chromatography column designed for the separation and analysis of polar and hydrophilic compounds. It utilizes zwitterionic ion chromatography principles to achieve efficient separation of a wide range of analytes.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 1290 column compartment, by Agilent Technologies (2 mentions)
Qtrap 5500 mass spectrometer, by AB Sciex (2 mentions)
Agilent 1260, by Agilent Technologies (1 mentions)
Phenol red, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Cambridge Isotopes (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Thermo q exactive plus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Dmem without glucose, by Thermo Fisher Scientific (1 mentions)
13c6 glucose, by Cambridge Isotopes (1 mentions)
Xcalibur software, by Thermo Fisher Scientific (1 mentions)
Vacuum centrifuge, by Labconco (1 mentions)
Oasis wax cartridge, by Waters Corporation (1 mentions)
Aquity uplc system, by Waters Corporation (1 mentions)
Agilent 1260 binary pump, by Agilent Technologies (1 mentions)
Column switching valve, by Agilent Technologies (1 mentions)
Agilent 1260 autosampler, by Agilent Technologies (1 mentions)
Quantitative analysis, by Agilent Technologies (1 mentions)
Masshunter qualitative analysis, by Agilent Technologies (1 mentions)
9 protocols using sequant zic chilic column
1
Quantification of Plasma Kynurenine Levels
2
Metabolomic Profiling by LC-MS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Metabolites were analysed using liquid chromatography followed by mass spectrometry. For the analysis, an Agilent 1200 ultra-high-pressure liquid chromatography system (Agilent technologies, Santa Clara, CA, USA) coupled to a SCIEX TripleTOF 5600 quadrupole-time-of-flight mass spectrometer (Framingham, MA, USA) was used. Samples were kept at 10 °C in the autosampler and 5 µL of sample was injected on the analytical column. The chromatographic separation was established using a SeQuant ZIC-cHILIC column (PEEK 100 × 2.1 mm, 3.0 µm particle size; Merck KGaA, Darmstadt, Germany), which was kept at 15 °C using a column thermostat (560-CIL, Cleuzeau Info Labo, France). The flow rate was 0.2 mL/min. The mobile phases where composed of (A) 90/10 acetonitrile/H2O with 5 mM ammonium acetate at pH 6.8 and (B) 10/90 acetonitrile/H2O with 5 mM ammonium acetate at pH 6.8, respectively. Metabolites were separated using a gradient composed of 100% A for 3 min; ramping 3–20 min to 36% A; ramping 28–28.5 to 100% A and re-equilibrated from 28.5 to 36 min with 100% A. The MS data was acquired at full scan range 50–800 amu at a scan rate of 10 scans/s in negative ionisation mode and the source temperature was kept at 400 °C.
3
Doxycycline Impacts Metabolic Flexibility
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To study whether doxycycline alters metabolic flexibility and mitochondrial metabolism [34 (link),49 (link)], we performed 13C-fluxomics as previously [53 (link)]. In short, the incubation was performed for 2 h in 500.000 cells/well, in DMEM without glucose, pyruvate, glutamine and phenol red (Life technologies, Bleiswijk, The Netherlands) supplemented with 50 μM oleic acid in 0.2% BSA, 1 mM glutamine, 50 μM carnitine, and 5 mM [13C6]-glucose (Cambridge Isotope Laboratories, Tewksbury, MA, USA). Every condition was performed in duplicate.
Cells were washed in saline twice, collected in ice-cold methanol-water, chloroform (1:1:2 v/v) and centrifuged at 10,000× g for 10 min. The aqueous phase of the extractions was collected and evaporated. The metabolite pellet was dissolved in 100 µL methanol-water (3:2 v/v) and analyzed by ultra-high-pressure liquid chromatography (Thermo Scientific, Waltman, MA, USA) with a SeQuant ZIC-cHILIC column (PEEK 100 × 2.1 mm, 3.0 µm particle size, Merck, Darmstadt, Germany) at 15 °C coupled to a Thermo Q Exactive (Plus) Orbitrap mass spectrometer (Thermo Scientific, Waltman, MA, USA). Data were acquired in negative-scan mode. Data analysis was performed in Xcalibur software (Thermo Scientific, Waltman, MA, USA). [13C]-label enrichment was calculated and corrected based on mass distribution isotopomer analysis [54 (link)].
Cells were washed in saline twice, collected in ice-cold methanol-water, chloroform (1:1:2 v/v) and centrifuged at 10,000× g for 10 min. The aqueous phase of the extractions was collected and evaporated. The metabolite pellet was dissolved in 100 µL methanol-water (3:2 v/v) and analyzed by ultra-high-pressure liquid chromatography (Thermo Scientific, Waltman, MA, USA) with a SeQuant ZIC-cHILIC column (PEEK 100 × 2.1 mm, 3.0 µm particle size, Merck, Darmstadt, Germany) at 15 °C coupled to a Thermo Q Exactive (Plus) Orbitrap mass spectrometer (Thermo Scientific, Waltman, MA, USA). Data were acquired in negative-scan mode. Data analysis was performed in Xcalibur software (Thermo Scientific, Waltman, MA, USA). [13C]-label enrichment was calculated and corrected based on mass distribution isotopomer analysis [54 (link)].
4
HILIC-based Metabolite Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen cell extracts were defrosted by 1 to 2 min of incubation in a 37°C water bath and sonicated for 10 min in a water-ice slurry. After 10 min of centrifugation at 20,000 × g, samples were loaded on a 1 ml/30 mg Oasis Wax cartridge (Waters) preconditioned with 1 ml of MeOH and 1 ml 50 mM ammonium acetate buffer (pH 4.5). After washing with 1 ml ammonium acetate buffer was performed, analytes were eluted with 200 μl of 2.8% ammonium hydroxide–MeOH/ACN/H2O 50:30:20 (vol/vol/vol). After addition of 10 μl of 5% trehalose and brief vortex mixing, the samples were dried in a vacuum centrifuge (Labconco). Dried trehalose-stabilized extracts were redissolved in 20 μl of MeOH/ACN/H2O (50:30:20 [vol/vol/vol]) and moved to an autosampler vial for analysis. Separation was performed on an iHilic column (Hilicon) (2.1 mm by 100 mm, 3.5-μm pore size) or a SeQuant Zic-cHILIC column (Merck) (2.1 mm by 100 mm, 3-μm pore size) at 0.3 ml/min using the following binary gradient: 100% B, ramp to 85% B in 1.5 min followed by 10 min of isocratic hold at 85% B and a linear decrease to 30% B in 3 min with a 2-min hold at 30% B and 8 min reequilibration under the initial conditions (A, 3.75 mM ammonium acetate–1.25 mM acetic acid–2 mM acetylacetone–Milli-Q water, B, 11.25 mM ammonium acetate–3.75 mM acetic acid–2 mM acetylacetone–80% ACN). The injection volume was 2 μl.
5
UPLC-MS/MS Analysis of Metabolites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The column used in UPLC was a SeQuant ZIC-cHILIC column (2.1 mm × 150 mm, 3 μm, Merck, USA) with mobile phase A was an aqueous solution containing 0.1 % formic acid; and B was acetonitrile. The elution program was set as 0–8 min, 95 %–85 % B; 8–22 min, 85 %–60 % B; 22–25 min, 60 %–95 % B; 25–35 min, 95 % B; with the column temperature 30 °C, injection volume 10 µL, and flow rate 0.25 mL/min.
Mass spectrometry was set to ESI ionization mode, with detection in negative ion mode. The parameters were set to scanning mode: Full Scan /dd-MS2; the acquisition range was 80–1200 m/z; the spray voltage was 3.5 kV for positive and 2.5 kV for negative; the sheath gas flow rate was 35 arb; the auxiliary gas flow rate was 10 arb; the capillary temperature was 320 °C; and the resolution: MS Full Scan 35 000 FWHM, and MS/MS 17 500 FWHM.
Mass spectrometry was set to ESI ionization mode, with detection in negative ion mode. The parameters were set to scanning mode: Full Scan /dd-MS2; the acquisition range was 80–1200 m/z; the spray voltage was 3.5 kV for positive and 2.5 kV for negative; the sheath gas flow rate was 35 arb; the auxiliary gas flow rate was 10 arb; the capillary temperature was 320 °C; and the resolution: MS Full Scan 35 000 FWHM, and MS/MS 17 500 FWHM.
6
Metabolomic Analysis of Freeze-Dried Muscle Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used 2–4 mg of freeze-dried muscle tissue for a metabolomics analysis, as previously described (20 (link)). Briefly, the metabolite analysis was carried out in an Aquity UPLC system (Waters) coupled to an Impact II Ultra-High Resolution Qq-Time-Of-Flight MS (Bruker). Chromatographic separation of the compounds was achieved using a SeQuant ZIC-cHILIC column (PEEK 100 × 2.1 mm; 3 μm particle size; Merck) at 30°C. The LC method consisted of a gradient running at 0.25 mL/min from 100% mobile phase B (9:1 acetonitrile: water with 5 mM ammonium acetate pH 6.8) to 100% mobile phase A (1:9 acetonitrile: water with 5 mM ammonium acetate pH 6.8) in 28 minutes, followed by a pre-equilibration step at 100% B of 5 minutes. MS data were acquired both in negative and positive ionization modes in full scan mode over the range of m/z 50–1200.
7
Serum Metabolite Profiling via LC-MS/MS
8
HILIC-MS Analysis of Metabolites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LC system was composed of two Agilent 1260 binary pumps, an Agilent 1260 auto-sampler and Agilent 1290 column compartment containing a column-switching valve (Agilent Technologies, Santa Clara, CA). Each sample was injected twice, at 10 µl for analysis using the negative-ionization mode and 2 µl for analysis using the positive-ionization mode. Both chromatographic separations were performed in the HILIC mode on two parallel SeQuant ZIC-cHILIC columns (150 by 2.1 mm, 3.0-µm particle size) (Merck KGaA, Darmstadt, Germany). While one column was performing the separation, the other column was reconditioning in preparation for the next injection. The flow rate was 0.300 ml/min, the autosampler temperature was kept at 4°C, the column compartment was set at 40°C, and the total separation time for each ionization mode was 18 min. The mobile phase was composed of solvents A and B. The gradient conditions for both separations were identical and are as follows: 0 to 2 min, 25% solvent A and 75% solvent B; 2 to 5 min, 25 to 70% solvent A, linear gradient; 5 to 9 min, 70% solvent A; 9 to 11 min, 70 to 25% solvent A, linear gradient; 11 to 18 min, 25% solvent A.
9
Optimized HILIC-RP LC Strategy for Metabolomics
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!