Summary of Study ST002354

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001511. The data can be accessed directly via it's Project DOI: 10.21228/M8CQ6T This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002354
Study TitleCombination of TP-252 and Naproxen elicit tumor protective Eicosanoid changes.
Study SummaryColorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds to thousands of polyps that unless removed by prophylactic colectomy will progress to CRC at an early age. Non-steroidal anti-inflammatory drugs (NSAIDs) and -3 marine polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA), have been evaluated for their chemopreventive potential in delaying the onset of CRC in high-risk patients. In this study, we determined whether the NSAID, naproxen, alone or in combination with a chemically-stable form of EPA (TP-252), affects tumor formation in the ApcPirc rat model. When compared to control diet, animals fed naproxen or HD TP-252 had 66%, and 82% fewer tumors respectively. However, when fed a combination of naproxen and HD TP-252, animals exhibited a 95% reduction in tumor formation and a 98% reduction in tumor volume, respectively. To elucidate potential mechanisms of tumor protection, a comprehensive, targeted lipidomic analysis was performed on colonic mucosa to determine changes in eicosanoid metabolism. Animals receiving TP-252 alone or in combination with naproxen had significantly reduced mucosal levels of pro-inflammatory -6 eicosanoids (PGE2, 5-HETE, and 14,15-DiHETrE), along with a simultaneous increase in anti-inflammatory EPA-derived -3 eicosanoids. Our colonic mucosal lipidomic analysis also uncovered several potential pharmacodynamic (PD) lipid biomarkers, including resolvin E2, 9-HEPE, 12-HEPE and 18-HEPE, that were increased in both the tissue and plasma of rats receiving TP-252 and were significantly correlated with tumor protection. Further studies with this drug combination should be focused on dose optimization and the role of EPA-derived lipid mediators in CRC initiation and progression.
Institute
UConn Health
Last NameBeach
First NameRyan
Address263 Farmington Avenue, Farmington CT 06030
Emailrbeach@uchc.edu
Phone860-679-8703
Submit Date2022-11-21
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailLC-MS
Release Date2022-12-22
Release Version1
Ryan Beach Ryan Beach
https://dx.doi.org/10.21228/M8CQ6T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001511
Project DOI:doi: 10.21228/M8CQ6T
Project Title:Combination of TP-252 and Naproxen elicit tumor protective Eicosanoid changes.
Project Summary:Comprehensive analysis of the lipidomic changes resulting from feeding with a over EPA analogue alone, and in combination with the NSAID naproxen.
Institute:UConn Health
Department:Center for Molecular Oncology
Laboratory:Rosenberg Laboratory
Last Name:Beach
First Name:Ryan
Address:263 Farmington Avenue, Farmington, Connecticut, 06030, USA
Email:rbeach@uchc.edu
Phone:860-679-8703
Funding Source:NCI PREVENT funding

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:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype Treatment Metabolite Tissue
SA236512EicoP8F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236513EicoP9F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236514EicoP10F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236515EicoP11F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236516EicoP14F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236517EicoP13F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236518EicoP12F344 Pirc 200 ppm Naproxen Eicosanoid Plasma
SA236519EicoT13F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236520EicoT14F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236521EicoT12F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236522EicoT11F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236523EicoT8F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236524EicoT9F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236525EicoT10F344 Pirc 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236526TFA-P11F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236527TFA-P12F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236528TFA-P10F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236529TFA-P8F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236530TFA-P14F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236531TFA-P9F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236532TFA-P13F344 Pirc 200 ppm Naproxen Fatty Acid Plasma
SA236533TFA-T11F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236534TFA-T14F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236535TFA-T10F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236536TFA-T12F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236537TFA-T13F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236538TFA-T9F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236539TFA-T8F344 Pirc 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236540EicoP1F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236541EicoP6F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236542EicoP2F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236543EicoP3F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236544EicoP4F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236545EicoP5F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236546EicoP7F344 Pirc AIN-93G (control) Eicosanoid Plasma
SA236547EicoT4F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236548EicoT2F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236549EicoT1F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236550EicoT3F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236551EicoT7F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236552EicoT5F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236553EicoT6F344 Pirc AIN-93G (control) Eicosanoid Tumor-adjacent normal colon
SA236554TFA-P2F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236555TFA-P7F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236556TFA-P6F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236557TFA-P5F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236558TFA-P4F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236559TFA-P3F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236560TFA-P1F344 Pirc AIN-93G (control) Fatty Acid Plasma
SA236561TFA-T1F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236562TFA-T2F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236563TFA-T3F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236564TFA-T4F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236565TFA-T6F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236566TFA-T7F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236567TFA-T5F344 Pirc AIN-93G (control) Fatty Acid Tumor-adjacent normal colon
SA236596EicoP50F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236597EicoP56F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236598EicoP52F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236599EicoP51F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236600EicoP53F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236601EicoP55F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236602EicoP54F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Plasma
SA236603EicoT56F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236604EicoT55F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236605EicoT53F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236606EicoT50F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236607EicoT54F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236608EicoT52F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236609EicoT51F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Eicosanoid Tumor-adjacent normal colon
SA236610TFA-P51F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236611TFA-P50F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236612TFA-P52F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236613TFA-P53F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236614TFA-P54F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236615TFA-P55F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236616TFA-P56F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Plasma
SA236617TFA-T50F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236618TFA-T52F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236619TFA-T51F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236620TFA-T56F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236621TFA-T53F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236622TFA-T55F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236623TFA-T54F344 Pirc EPA-FFA (2%) + 200 ppm Naproxen Fatty Acid Tumor-adjacent normal colon
SA236568EicoP46F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236569EicoP45F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236570EicoP47F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236571EicoP43F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236572EicoP44F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236573EicoP49F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236574EicoP48F344 Pirc EPA-FFA (2% EPA) Eicosanoid Plasma
SA236575EicoT48F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236576EicoT49F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236577EicoT46F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236578EicoT45F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236579EicoT44F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236580EicoT43F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236581EicoT47F344 Pirc EPA-FFA (2% EPA) Eicosanoid Tumor-adjacent normal colon
SA236582TFA-P48F344 Pirc EPA-FFA (2% EPA) Fatty Acid Plasma
SA236583TFA-P44F344 Pirc EPA-FFA (2% EPA) Fatty Acid Plasma
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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 Triple quadrupole Triple quadrupole Triple quadrupole Triple quadrupole
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:Triple quadrupole
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:Triple quadrupole
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:Triple quadrupole
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:Triple quadrupole
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
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