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Eicosanoid Analysis Service for Targeted UPLC–MS/MS Lipid Mediator Quantification

Creative Proteomics provides high-sensitivity, targeted LC-MS/MS quantification to solve the critical challenges of low abundance, structural isomers, and matrix interference in eicosanoid research. We translate unstable lipid mediators into actionable, publication-ready pathway insights, ensuring your metabolic and inflammatory data is reliable and reproducible.

Absolute Quantification: Anchored by 26+ isotope-labeled internal standards to eliminate matrix effects and ensure accuracy.
Isomer Resolution: Baseline separation of structural isomers (e.g., 5-HETE vs 12-HETE) using optimized Waters ACQUITY UPLC.
Comprehensive Coverage: Targeted profiling across the entire COX, LOX, and CYP enzymatic branches.
Stability-First Workflow: Specialized sample handling protocols to prevent ex vivo degradation and artifact formation.

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Service Scope
Analytes Panel
Advantages
Workflow
Methods
Deliverables
Sample Submission
FAQ

What Are Eicosanoids (Lipid Mediators)?

Eicosanoids are potent lipid signals made from polyunsaturated fatty acids. They include prostaglandins, thromboxanes, leukotrienes, lipoxins, and many hydroxylated or epoxidized products.

Teams measure them as direct pathway readouts. They can shift quickly after stimulation, treatment, or stress. They also appear at low levels in many matrices.

Eicosanoid quantification is challenging because:

Many compounds share the same mass and fragment patterns, so separation matters.
Matrix effects can distort response, making internal-standard correction important.
Sample handling can change oxygenated lipids, so pre-analytics must be controlled.

Eicosanoid Analysis Service Options

Targeted quant panels (core eicosanoids)

A focused panel for eicosanoid pathway profiling across COX/LOX/CYP outputs. It fits inhibitor studies, perturbation screens, and time-course designs.

Expanded eicosanoid coverage (metabolites and pathway branches)

Adds commonly requested downstream metabolites and pathway branch markers when they are feasible in your matrix.

Custom targets and add-on pathways

Add specific analytes tied to your mechanism or model, subject to standard availability and chromatographic feasibility.

Absolute quantification (isotope dilution, optional)

Stable isotope–labeled internal standards and calibration curves can be applied to report absolute concentrations for targets with suitable standards and validated transitions.

If your study extends beyond arachidonic acid–derived eicosanoids—such as DHA/EPA lipid mediators—or you need broader pathway coverage, see our Oxylipin Quantification and Pathway Profiling for expanded panels, including specialized pro-resolving mediators and other oxidized PUFA products.

Complete Eicosanoid Target List for LC–MS/MS Quantification

Prostaglandins
Thromboxanes
Leukotrienes
Lipoxins
EETs
Substrates

Prostaglandins and related metabolites (COX)

Subgroup	Analytes
Series prostaglandins	PGE2, PGD2, PGF2α, PGE1, PGD1, PGF1α, PGE3, PGD3, PGF3α
Prostacyclin pathway markers	6-keto-PGF1α, 6-keto-PGE1
Dehydration / cyclopentenones	PGA2, PGB2, PGJ2, 15-deoxy-PGJ2, 15-deoxy-PGD2, 15-deoxy-PGA2
Oxidation / breakdown markers	15-keto-PGE2, 15-keto-PGD2, 15-keto-PGF2α, 15-keto-PGF1α, PGEM, PGFM, bicyclo-PGE2
Hydroxy derivatives	11β-PGF2α, 11β-PGE2, 19-hydroxy-PGE2, 20-hydroxy-PGE2

Thromboxanes and pathway markers (COX)

Subgroup	Analytes
Thromboxane readouts	TXB2, TXB1, TXB3

Leukotrienes and related LOX products

Subgroup	Analytes
Leukotriene series	LTB4, 20-hydroxy-LTB4, 20-carboxy-LTB4, LTC4, LTD4, LTE4
Hydroxy and oxo products	5-HETE, 12-HETE, 15-HETE, 20-HETE, 11-HETE, 5-oxo-ETE

Lipoxins and pro-resolving–adjacent eicosanoid family members

Subgroup	Analytes
Lipoxins	LXA4, LXB4

CYP epoxygenase products (AA-derived)

Subgroup	Analytes
Epoxides	8,9-EET, 11,12-EET, 14,15-EET

Optional substrate readouts

Subgroup	Analytes
Pathway substrate	Arachidonic acid

Why Choose Our Eicosanoid LC–MS/MS Assay

Quantitation with isotope-labeled internal standards
Eicosanoids are highly sensitive to matrix effects. Public LC–MS/MS workflows describe using 26 deuterated internal standards for isotope-dilution quantification. We follow this approach and verify internal-standard performance in QC.
Broad coverage with targeted control
Targeted LC–MS/MS can scale from focused panels to expanded pathway coverage. Peer-reviewed methods report panels of up to184 eicosanoid targets under a single setup, showing what is feasible with optimized separation and transitions. We scope coverage based on your matrix and target feasibility.
Scheduled MRM built for run-to-run stability
Published methods commonly use scheduled windows (for example, 30 seconds per transition) to tolerate minor retention shifts while maintaining panel breadth. We use scheduled acquisition to balance sensitivity and coverage.
Clear LOD/LOQ rules for consistent reporting
We apply defined criteria for detection and quantitation. A commonly used framework sets LOD >3:1 and LOQ >7:1 by signal-to-noise. Results below quant thresholds are flagged.

Workflow: From Study Intake to Quantified Results

Schematic of the targeted UPLC–MS/MS workflow for eicosanoid quantification.

Methods and Key Instrument Parameters for Eicosanoid Quantification

Eicosanoids are low-abundance lipid mediators with many structural isomers. To support confident quantification in research matrices, we use targeted UPLC–MS/MS (scheduled MRM) on a triple quadrupole–class platform.

Instrument Platform

Component	Model	Purpose in eicosanoid quantification
UPLC	Waters ACQUITY UPLC	Fast, stable separations to support isomer handling and reproducible retention time
MS/MS	SCIEX QTRAP 6500	Targeted MRM quantification with high selectivity for low-level lipid mediators
Ion source	Turbo V ESI (QTRAP 6500 source)	Stable ionization for acidic eicosanoids in complex matrices

Chromatography (Key Parameters)

We tune chromatography to separate closely related mediators and reduce interference.

Parameter	Setting
Column	ACQUITY UPLC BEH Shield RP18, 2.1 × 100 mm, 1.7 μm
Column temperature	40 °C
Flow rate	0.5 mL/min
Injection volume	10 μL
Mobile phase A	ACN/water/acetic acid (60/40/0.02, v/v)
Mobile phase B	ACN/isopropanol (50/50, v/v)
Gradient	0–4.0 min: 0.1–55% B; 4.0–4.5 min: 55–99% B; 4.5–5.0 min: 99% B

MS/MS Acquisition

We use scheduled MRM to maintain sensitivity while supporting multi-analyte panels.

Parameter	Setting
Ionization mode	ESI negative
Acquisition mode	Scheduled MRM
Scheduling window	30 s per transition
Curtain gas (CUR)	10 psi
Gas 1 (GS1)	30 psi
Gas 2 (GS2)	30 psi
Ion spray voltage	−4.5 kV
Source temperature	525 °C
Collision gas	Nitrogen (CID optimized per analyte)

Waters ACQUITY UPLC System (Figure from Waters)

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Deliverables: What You Receive From Eicosanoid Analysis

Standard Deliverables

Quantified results table for the agreed eicosanoid targets (per sample, with defined units/normalization).
QC summary covering internal standard performance, calibration checks, and key run-level flags.
Representative extracted-ion chromatograms for critical targets and any flagged calls.
Method appendix with the instrument setup, acquisition mode, and processing notes used for your batch.

LC-MS/MS eicosanoid EIC with baseline isomer resolution.

Representative EICs and isomer resolution. Traces show analyte/IS co-elution (PGE2, LTB4) and baseline separation of 12/15-HETE isomers, ensuring high analytical specificity.

Eicosanoid calibration curve and residual plot (0.1–1000 pg/mL).

Isotope dilution delivers linear calibration (R² ≥ 0.998) across 0.1–1000 pg/mL, with residuals held within ±15% to support robust quantitative precision.

Eicosanoid heatmap grouped by COX, LOX, and CYP pathways.

Log2 fold-changes grouped by COX, LOX, and CYP branches reveal global shifts in lipid mediator flux across experimental groups.

Eicosanoid volcano plot highlighting significant metabolic hits.

Volcano plot of differential mediators. Statistically significant hits are color-coded by metabolic branch (COX/LOX/CYP) for rapid identification of key bioactive lipids.

Advanced Data Analysis (Optional)

COX/LOX/CYP pathway grouping with pathway-level summaries and selected ratios (project-defined).
Exploratory statistics such as PCA, clustering, and trend plots for time-course or multi-group studies.
Differential analysis outputs including fold-change tables and comparison summaries aligned to your design.
Interpretation notes highlighting consistent pathway shifts and data-quality considerations.

Data Formats

.xlsx and .csv result tables.
.pdf report.
Raw vendor files (on request) and/or .mzML for archiving and re-analysis.

Explore our Lipidomics Solutions brochure to learn more about our comprehensive lipidomics analysis platform.

Download Brochure

Research Applications of Eicosanoid Profiling

Inflammatory stimulation kinetics

Track rapid eicosanoid synthesis changes across COX/LOX/CYP after LPS/cytokine or stress perturbations.

Mechanism-of-action and PD readouts

Quantify pathway outputs to confirm target engagement or pathway rerouting in inhibitor/agonist studies.

Tumor microenvironment research

Profile prostaglandins/leukotrienes/HETEs to connect lipid mediators with immune and inflammatory phenotypes in models.

Metabolism and nutrition interventions

Measure how omega-3/omega-6 inputs reshape downstream mediator patterns in controlled designs.

Oxidative stress models

Separate enzymatic eicosanoid changes from oxidation-linked signatures under defined stress conditions.

Genetics and pathway engineering

Compare eicosanoid pathway outputs across KO/KD/overexpression systems to validate branch dependencies.

Sample Requirements and Submission Guide

Matrix	Typical minimum	Container and handling notes
Plasma / serum	200–500 μL	Use pre-chilled polypropylene; keep cold; avoid repeated freeze–thaw
Urine	1–2 mL	Clarify, aliquot, and freeze; normalization strategy can be discussed
CSF	~200 μL	Use low-bind tubes when possible; freeze quickly
Tissue	30–100 mg	Snap-freeze; record wet weight; avoid thaw cycles
Cell pellets	≥1×106 cells	Cold washes; snap-freeze; provide cell count metadata
Culture media	1–2 mL	Clarify and freeze; note serum supplementation

Collection, Storage, and Shipping

Keep samples cold during processing and aliquoting.
Minimize time at room temperature and avoid repeated freeze–thaw cycles.
Use polypropylene or low-bind tubes, and label vials clearly.
Snap-freeze tissue and cell pellets promptly; record wet weight or cell count.
Store at −80 °C when possible and ship on dry ice with an inventory sheet

FAQs for Eicosanoid Pathway Analysis Service

How do you separate eicosanoid isomers (e.g., 5-HETE vs 12-HETE)?

We use Waters ACQUITY UPLC with BEH Shield RP18 chemistry to improve chromatographic resolution for structural isomers with similar MS/MS behavior. Separation is confirmed by retention time (RT) and qualifier/quantifier consistency before quantitation.

Why use isotope dilution for eicosanoid quantification?

Eicosanoids are prone to ESI matrix effects. We apply isotope dilution with 26+ deuterated internal standards spiked pre-extraction to correct recovery loss and ionization variability, improving quantitative reliability in complex matrices.

How do you reduce ex vivo oxidation and artifacts?

We follow a stability-focused handling plan: rapid processing, cold-chain control, and project-appropriate stabilization steps to minimize enzymatic activity and non-enzymatic oxidation during collection and storage.

Can you report results by COX/LOX/CYP pathways?

Yes. Outputs can be organized into COX, LOX, and CYP sub-panels to support pathway-level interpretation and mechanism studies.

Why use LC–MS/MS instead of ELISA for eicosanoids?

LC–MS/MS improves chemical specificity and supports multiplex measurement, reducing cross-reactivity risk that can affect antibody-based assays.

What's the difference between eicosanoids and oxylipins?

Oxylipins are a broad class of oxidized polyunsaturated fatty acid mediators. Eicosanoids are a major subset of oxylipins, typically derived from 20-carbon fatty acids such as arachidonic acid, and include prostaglandins, thromboxanes, leukotrienes, and related products.

Can you normalize eicosanoids in cell culture supernatants?

Yes. We can report results normalized to cell count, total protein, or other study-defined metadata you provide.

Are eicosanoids hormones?

Eicosanoids are often described as hormone-like local mediators (autacoids). They act near their site of production and change rapidly with pathway activity, which is why targeted LC–MS/MS is commonly used to quantify them in research models.

Publications

Anti-inflammatory activity of black soldier fly oil associated with modulation of tlr signaling: A metabolomic approach. International Journal of Molecular Sciences, 2023. https://doi.org/10.3390/ijms241310634
Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion. PLoS Pathogens, 2023. https://doi.org/10.1371/journal.ppat.1011232
Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation. The FASEB Journal, 2023. https://doi.org/10.1096/fj.202201088R
Evidence for phosphate-dependent control of symbiont cell division in the model anemone Exaiptasia diaphana. mBio, 2024. https://doi.org/10.1128/mbio.01059-24
Lipid Membrane Engineering for Biotechnology. Doctoral dissertation, Aston University, 2023. https://doi.org/10.48780/publications.aston.ac.uk.00046663

* Our services can only be used for research purposes and Not for clinical use.

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