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MB Sample ID: SA236576
| Local Sample ID: | EicoT49 |
| Subject ID: | SU002443 |
| Subject Type: | Mammal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Age Or Age Range: | 26 weeks of age |
| Weight Or Weight Range: | 320-460 grams |
| Gender: | Male |
| Animal Animal Supplier: | RRRC (University of Missouri) |
| Animal Housing: | Center for Comparative Medicine UConn |
| Animal Feed: | Modified AIN-93G diet from Research Diets |
| Animal Water: | ab libitum |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002443 |
| Subject Type: | Mammal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Age Or Age Range: | 26 weeks of age |
| Weight Or Weight Range: | 320-460 grams |
| Gender: | Male |
| Animal Animal Supplier: | RRRC (University of Missouri) |
| Animal Housing: | Center for Comparative Medicine UConn |
| Animal Feed: | Modified AIN-93G diet from Research Diets |
| Animal Water: | ab libitum |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| EicoT49 | SA236576 | FL029291 | F344 Pirc | Genotype |
| EicoT49 | SA236576 | FL029291 | EPA-FFA (2% EPA) | Treatment |
| EicoT49 | SA236576 | FL029291 | Eicosanoid | Metabolite |
| EicoT49 | SA236576 | FL029291 | Tumor-adjacent normal colon | Tissue |
Collection:
| Collection ID: | CO002436 |
| Collection Summary: | Animals were sacrificed by CO2 narcosis, and large intestines were excised, washed with ice-cold PBS, and laid flat on filter paper. Tumor-adjacent full-thickness colon segments were flash-frozen in liquid Nitrogen and stored at -80C until analysis. Blood was collected via cardiac puncture and put in lithium heparin tubes on ice. Samples were centrifuged at 2,000 g for 20 minutes, and supernatant (plasma) was collected and stored at -80C until analysis. |
| Sample Type: | Blood (Plasma) and Large Intestine |
Treatment:
| Treatment ID: | TR002455 |
| Treatment Summary: | Rats were fed two doses of TP-252 equivalent to consuming 1.3 and 2.6 g/kg/day for LD TP-252 and HD TP-252, respectively. Alternatively, rats were also fed a diet containing 2% EPA-FFA in their diet (equivalent to 1.14 g/kg/day of EPA). In groups receiving naproxen, the dose of naproxen was 11.4 mg/kg/day, or roughly 4 mg/rat/day. These treatment groups were carried out for the 20 weeks, and then animals were sacrificed and tissue collected and analyzed. |
Sample Preparation:
| Sampleprep ID: | SP002449 |
| Sampleprep Summary: | Samples (0.85 ml) were spiked with 5 ng each (in 150 μl methanol) of 15(S)-HETE-d8,14(15)-EpETrE-d11, Resolvin D2-d5, Leukotriene B4-d4, and Prostaglandin E1-d4 as internal standards for recovery and quantitation and mixed thoroughly. The samples were then extracted for PUFA metabolites using C18 extraction columns as described earlier [1-4]. Briefly, the internal standard spiked samples were applied to conditioned C18 cartridges, washed with 15% methanol in water followed by hexane and dried under vacuum. The cartridges were eluted with 0.5 ml methanol. The eluate was dried under a gentle stream of nitrogen. The residue was redissolved in 50 μl methanol-25 mM aqueous ammonium acetate (1:1) and subjected to LC-MS analysis. |
| Sampleprep Protocol Filename: | Methods of Eicosanoid Detection.pdf |
Combined analysis:
| Analysis ID | AN003842 | AN003843 | AN003844 | AN003845 |
|---|---|---|---|---|
| Analysis type | MS | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | Reversed phase | Reversed phase |
| Chromatography system | Prominence XR | Prominence XR | Prominence XR | Prominence XR |
| Column | Targa C8, 2x10 mm, 5μ | Luna C18 (150 x 2.1mm,3um) | Targa C8, 2x10 mm, 5μ | Luna C18 (150 x 2.1mm,3um) |
| MS Type | ESI | ESI | ESI | ESI |
| MS instrument type | QTRAP | QTRAP | QTRAP | QTRAP |
| MS instrument name | ABI Sciex 5500 QTrap | ABI Sciex 5500 QTrap | ABI Sciex 5500 QTrap | ABI Sciex 5500 QTrap |
| Ion Mode | NEGATIVE | NEGATIVE | NEGATIVE | NEGATIVE |
| Units | ng/g tissue | mg/mL | ng/mL | mg/g tissue |
Chromatography:
| Chromatography ID: | CH002844 |
| Chromatography Summary: | HPLC was performed on a Prominence XR system (Shimadzu) using Luna C18 (3μ, 2.1x150 mm) column. The mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate. The gradient program with respect to the composition of B was as follows: 0-1 min, 50%; 1-8 min, 50-80%; 8-15 min, 80-95%; and 15-17 min, 95%. The flow rate was 0.2 ml/min. The HPLC eluate was directly introduced to ESI source of QTRAP5500 mass analyzer (ABSCIEX) in the negative ion mode with following conditions: Curtain gas: 35 psi, GS1: 35 psi, GS2: 65 psi, Temperature: 600 ˚C, Ion Spray Voltage: -1500 V, Collision gas: low, Declustering Potential: -60 V, and Entrance Potential: -7 V. The eluate was monitored by Multiple Reaction Monitoring method to detect unique molecular ion – daughter ion combinations for each of the lipid mediators using a scheduled MRM around the expected retention time for each compound. Optimized Collisional Energies (18 – 35 eV) and Collision Cell Exit Potentials (7 – 10 V) were used for each MRM transition. Spectra of each peak detected in the scheduled MRM were recorded using Enhanced Product Ion scan to confirm the structural identity. The data was collected using Analyst 1.6.2 software and the MRM transition chromatograms were quantitated by MultiQuant software (both from ABSCIEX). The internal standard signals in each chromatogram were used for normalization, recovery, as well as relative quantitation of each analyte. |
| Instrument Name: | Prominence XR |
| Column Name: | Targa C8, 2x10 mm, 5μ |
| Flow Rate: | 0.25ml/min |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002845 |
| Chromatography Summary: | HPLC was performed on a Prominence XR system (Shimadzu) using Luna C18 (3μ, 2.1x150 mm) column. The mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate. The gradient program with respect to the composition of B was as follows: 0-1 min, 50%; 1-8 min, 50-80%; 8-15 min, 80-95%; and 15-17 min, 95%. The flow rate was 0.2 ml/min. The HPLC eluate was directly introduced to ESI source of QTRAP5500 mass analyzer (ABSCIEX) in the negative ion mode with following conditions: Curtain gas: 35 psi, GS1: 35 psi, GS2: 65 psi, Temperature: 600 ˚C, Ion Spray Voltage: -1500 V, Collision gas: low, Declustering Potential: -60 V, and Entrance Potential: -7 V. The eluate was monitored by Multiple Reaction Monitoring method to detect unique molecular ion – daughter ion combinations for each of the lipid mediators using a scheduled MRM around the expected retention time for each compound. Optimized Collisional Energies (18 – 35 eV) and Collision Cell Exit Potentials (7 – 10 V) were used for each MRM transition. Spectra of each peak detected in the scheduled MRM were recorded using Enhanced Product Ion scan to confirm the structural identity. The data was collected using Analyst 1.6.2 software and the MRM transition chromatograms were quantitated by MultiQuant software (both from ABSCIEX). The internal standard signals in each chromatogram were used for normalization, recovery, as well as relative quantitation of each analyte. |
| Instrument Name: | Prominence XR |
| Column Name: | Luna C18 (150 x 2.1mm,3um) |
| Flow Rate: | 0.2ml/min |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002846 |
| Chromatography Summary: | HPLC was performed on a Prominence XR system (Shimadzu) using Luna C18 (3μ, 2.1x150 mm) column. The mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate. The gradient program with respect to the composition of B was as follows: 0-1 min, 50%; 1-8 min, 50-80%; 8-15 min, 80-95%; and 15-17 min, 95%. The flow rate was 0.2 ml/min. The HPLC eluate was directly introduced to ESI source of QTRAP5500 mass analyzer (ABSCIEX) in the negative ion mode with following conditions: Curtain gas: 35 psi, GS1: 35 psi, GS2: 65 psi, Temperature: 600 ˚C, Ion Spray Voltage: -1500 V, Collision gas: low, Declustering Potential: -60 V, and Entrance Potential: -7 V. The eluate was monitored by Multiple Reaction Monitoring method to detect unique molecular ion – daughter ion combinations for each of the lipid mediators using a scheduled MRM around the expected retention time for each compound. Optimized Collisional Energies (18 – 35 eV) and Collision Cell Exit Potentials (7 – 10 V) were used for each MRM transition. Spectra of each peak detected in the scheduled MRM were recorded using Enhanced Product Ion scan to confirm the structural identity. The data was collected using Analyst 1.6.2 software and the MRM transition chromatograms were quantitated by MultiQuant software (both from ABSCIEX). The internal standard signals in each chromatogram were used for normalization, recovery, as well as relative quantitation of each analyte. |
| Instrument Name: | Prominence XR |
| Column Name: | Targa C8, 2x10 mm, 5μ |
| Flow Rate: | 0.25ml/min |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH002847 |
| Chromatography Summary: | HPLC was performed on a Prominence XR system (Shimadzu) using Luna C18 (3μ, 2.1x150 mm) column. The mobile phase consisted of a gradient between A: methanol-water-acetonitrile (10:85:5 v/v) and B: methanol-water-acetonitrile (90:5:5 v/v), both containing 0.1% ammonium acetate. The gradient program with respect to the composition of B was as follows: 0-1 min, 50%; 1-8 min, 50-80%; 8-15 min, 80-95%; and 15-17 min, 95%. The flow rate was 0.2 ml/min. The HPLC eluate was directly introduced to ESI source of QTRAP5500 mass analyzer (ABSCIEX) in the negative ion mode with following conditions: Curtain gas: 35 psi, GS1: 35 psi, GS2: 65 psi, Temperature: 600 ˚C, Ion Spray Voltage: -1500 V, Collision gas: low, Declustering Potential: -60 V, and Entrance Potential: -7 V. The eluate was monitored by Multiple Reaction Monitoring method to detect unique molecular ion – daughter ion combinations for each of the lipid mediators using a scheduled MRM around the expected retention time for each compound. Optimized Collisional Energies (18 – 35 eV) and Collision Cell Exit Potentials (7 – 10 V) were used for each MRM transition. Spectra of each peak detected in the scheduled MRM were recorded using Enhanced Product Ion scan to confirm the structural identity. The data was collected using Analyst 1.6.2 software and the MRM transition chromatograms were quantitated by MultiQuant software (both from ABSCIEX). The internal standard signals in each chromatogram were used for normalization, recovery, as well as relative quantitation of each analyte. |
| Instrument Name: | Prominence XR |
| Column Name: | Luna C18 (150 x 2.1mm,3um) |
| Flow Rate: | 0.2ml/min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003584 |
| Analysis ID: | AN003842 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | Mass spectra for each detected lipid metabolite were recorded using the enhanced production feature to verify the identity of the detected peak. Data were collected and quantified using Analyst 1.6.2 (SCIEX) and MultiQuant (SCIEX) software, respectively. Correction for recovery efficiencies and relative quantitation of each analyte were performed using signals from each chromatogram corresponding to the spiked-in internal standards. Under standardized conditions of liquid chromatography-mass spectrometry quantitation, the detection limits for the eicosanoids are 1–2 pg on the column and the limit of quantitation is 5 pg at a signal-to-noise ratio of 3. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003585 |
| Analysis ID: | AN003843 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | Mass spectra for each detected lipid metabolite were recorded using the enhanced production feature to verify the identity of the detected peak. Data were collected and quantified using Analyst 1.6.2 (SCIEX) and MultiQuant (SCIEX) software, respectively. Correction for recovery efficiencies and relative quantitation of each analyte were performed using signals from each chromatogram corresponding to the spiked-in internal standards. Under standardized conditions of liquid chromatography-mass spectrometry quantitation, the detection limits for the eicosanoids are 1–2 pg on the column and the limit of quantitation is 5 pg at a signal-to-noise ratio of 3. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003586 |
| Analysis ID: | AN003844 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | Mass spectra for each detected lipid metabolite were recorded using the enhanced production feature to verify the identity of the detected peak. Data were collected and quantified using Analyst 1.6.2 (SCIEX) and MultiQuant (SCIEX) software, respectively. Correction for recovery efficiencies and relative quantitation of each analyte were performed using signals from each chromatogram corresponding to the spiked-in internal standards. Under standardized conditions of liquid chromatography-mass spectrometry quantitation, the detection limits for the eicosanoids are 1–2 pg on the column and the limit of quantitation is 5 pg at a signal-to-noise ratio of 3. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS003587 |
| Analysis ID: | AN003845 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | QTRAP |
| MS Type: | ESI |
| MS Comments: | Mass spectra for each detected lipid metabolite were recorded using the enhanced production feature to verify the identity of the detected peak. Data were collected and quantified using Analyst 1.6.2 (SCIEX) and MultiQuant (SCIEX) software, respectively. Correction for recovery efficiencies and relative quantitation of each analyte were performed using signals from each chromatogram corresponding to the spiked-in internal standards. Under standardized conditions of liquid chromatography-mass spectrometry quantitation, the detection limits for the eicosanoids are 1–2 pg on the column and the limit of quantitation is 5 pg at a signal-to-noise ratio of 3. |
| Ion Mode: | NEGATIVE |
