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Free Fatty Acids Analysis Service

Creative Proteomics specializes in high-precision free fatty acids (FFA) analysis using advanced LC-MS/MS and GC-MS technologies. We provide comprehensive lipid profiling, absolute quantification, and pathway-specific analysis to support research in nutrition, metabolism, agriculture, and environmental science. Our services help identify lipid imbalances, optimize bio-based production, and ensure food quality with accurate, high-sensitivity data. With rapid turnaround times and customizable solutions, we deliver reliable insights for complex lipidomics research.

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Service We Provide
List of Free Fatty Acids
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Platform
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Sample Requirements
FAQ

What are Free Fatty Acids?

Free fatty acids (FFAs) are a crucial class of lipid molecules that exist unbound in biological systems. Structurally, FFAs consist of a hydrocarbon chain terminating in a carboxyl functional group, which determines their chemical properties and metabolic roles. These fatty acids play a central role in cellular energy metabolism, serving as vital fuel sources. Additionally, they contribute to various physiological processes, including membrane biosynthesis, signaling pathways, and lipid homeostasis.

Free Fatty Acid Analysis in Creative Proteomics

Full-Spectrum Free Fatty Acid Profiling

Simultaneous detection of 50+ FFAs (C6–C24) across biological and food samples using LC-MS/MS and GC-MS platforms

Free Fatty Acid Absolute Quantification

Trace-level quantification (detection limit: 0.6 μM) with isotope-labeled internal standards for precision

Customized Free Fatty Acid Panels

Targeted analysis of specific FFAs (e.g., palmitic, oleic, linoleic acids) or pathway-focused panels (e.g., β-oxidation intermediates)

Multi-Omics Integration Analysis

Combine FFA data with proteomics or metabolomics for holistic insights into lipid metabolism

Free Fatty Acid Classification We Can Identify

Saturated
Monounsaturated
Polyunsaturated
Hydroxylated
Branched-Chain

Saturated Fatty Acids (SFAs)

Saturated fatty acids contain no double bonds in their hydrocarbon chains. They play critical roles in energy storage and membrane structure.

Compound	Chain Length	Related Metabolites	Metabolic Pathway
Palmitic Acid (C16:0)	16	Palmitoyl-CoA, Acetyl-CoA	β-Oxidation, Lipogenesis
Stearic Acid (C18:0)	18	Stearoyl-CoA, Acetyl-CoA	β-Oxidation, Lipogenesis
Myristic Acid (C14:0)	14	Myristoyl-CoA	β-Oxidation, Protein Modification
Lauric Acid (C12:0)	12	Dodecanoyl-CoA	β-Oxidation, Lipogenesis

Monounsaturated Fatty Acids (MUFAs)

Monounsaturated fatty acids contain one double bond, contributing to membrane fluidity and cellular signaling.

Compound	Chain Length	Related Metabolites	Metabolic Pathway
Oleic Acid (C18:1)	18	Oleoyl-CoA, Hydroxyoleic Acid	Lipid Signaling, Lipogenesis
Palmitoleic Acid (C16:1)	16	Palmitoleoyl-CoA, Acetyl-CoA	β-Oxidation, Fatty Acid Desaturation
Vaccenic Acid (C18:1)	18	Vaccenoyl-CoA	Lipid Signaling, Energy Metabolism

Polyunsaturated Fatty Acids (PUFAs)

Polyunsaturated fatty acids contain multiple double bonds and are essential for inflammatory response, brain function, and hormone production.

Compound	Chain Length	Related Metabolites	Metabolic Pathway
Linoleic Acid (C18:2)	18	Arachidonic Acid, Eicosanoids	Eicosanoid Synthesis, Lipogenesis
Arachidonic Acid (C20:4)	20	Prostaglandins, Leukotrienes	Inflammatory Response, Cell Signaling
Docosahexaenoic Acid (C22:6)	22	DHA-Derived Mediators	Neuroprotection, Lipid Signaling
Eicosapentaenoic Acid (C20:5)	20	Resolvins, Protectins	Anti-Inflammatory Pathways

Hydroxylated Fatty Acids

Hydroxylated fatty acids have hydroxyl (-OH) groups, contributing to lipid signaling and metabolic regulation.

Compound	Chain Length	Related Metabolites	Metabolic Pathway
3-Hydroxybutyric Acid (C4:0)	4	Acetoacetate, Acetyl-CoA	Ketogenesis, Energy Metabolism
12-Hydroxystearic Acid (C18:0)	18	Hydroxyoctadecanoic Acid	Lipid Signaling, Inflammatory Response

Branched-Chain Fatty Acids (BCFAs)

Branched-chain fatty acids have methyl branches, often derived from microbial metabolism and are detected in dairy and meat products.

Compound	Chain Length	Related Metabolites	Metabolic Pathway
Phytanic Acid (C20:0)	20	Pristanic Acid, Acetyl-CoA	α-Oxidation, Peroxisomal Metabolism
Pristanic Acid (C19:0)	19	Acyl-CoA, Propionyl-CoA	β-Oxidation, Energy Metabolism

Why Choose Our Free Fatty Acids Services?

High Sensitivity and Accuracy – Detection limits as low as 1 pmol with LC-MS/MS and GC-MS.
Comprehensive Lipid Coverage – Capable of identifying and quantifying over 500 FFA species.
Fast Turnaround Time – Standard results delivered within 4 to 6 weeks, expedited service available in 2 weeks.
Accurate Quantification – Internal standards ensure precision across a 10 nM to 100 µM dynamic range.

In-Depth Data Analysis – Statistical and pathway analysis with significance levels of p < 0.05.
Flexible Sample Compatibility – Requires only 200 µL of plasma or 50 mg of tissue for analysis.
Customizable Analysis – Targeted and untargeted lipidomics tailored to research needs.

What Methods are Used for Our Free Fatty Acids Analysis?

Gas Chromatography-Mass Spectrometry (GC-MS)

Instrument: Agilent 7890B GC System coupled with Agilent 5977B MSD

Ideal for volatile and derivatized fatty acid analysis.
High separation efficiency and sensitivity.
Suitable for analyzing short-chain and medium-chain fatty acids.
Derivatization using methylation or silylation enhances detection.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS)

Instrument: Thermo Fisher Scientific Q Exactive™ Plus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer

Provides high-resolution, accurate mass (HRAM) data.
Excellent for detecting long-chain and polyunsaturated fatty acids (PUFAs).
Suitable for targeted and untargeted lipidomics studies.

High-Performance Liquid Chromatography (HPLC)

Instrument: Agilent 1260 Infinity II HPLC System

Effective for separation and quantification of free fatty acids without derivatization.
Suitable for large-scale lipid screening and quality control applications.
Can be used with UV or fluorescence detectors for non-MS analysis.

Agilent 7890A GC System (Figure from Agilent)

Thermo Fisher Q Exactive (Figure from Thermo Fisher)

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Demo Results of Free Fatty Acids Analysis

Free Fatty Acids (FFA) Quantification Report

Results we provide:

Total FFA concentration (e.g., µmol/L) to assess overall lipid content.
Individual fatty acid concentrations (e.g., Palmitic Acid C16:0, Oleic Acid C18:1, Arachidonic Acid C20:4) for detailed profiling.
Statistical analysis to identify significant differences between experimental groups.

Format:

Tabular data displaying concentrations of FFAs and relevant biomarkers.
Graphical representation using bar graphs and pie charts for visual comparison across samples.
Summary report with key findings and interpretations.

GC–MS chromatogram of analysis of free fatty acids extracted from phospholipid of H. parasuis SC096 (Feng, Saixiang, et al., 2017).

Extracted ion chromatograms (EICs) of fatty acids in a standard solution (500 ng/mL) (Feng, Saixiang, et al., 2017).

Lipid Profile Report

Contents:

Relative abundance of fatty acid classes to differentiate the proportions of SFAs, MUFAs, and PUFAs.
Key biomarkers identification for monitoring lipid metabolism, including essential fatty acids and bioactive lipid intermediates.
Unsaturation index to evaluate the degree of lipid unsaturation.

Format:

Bar graphs for comparing the abundance of fatty acid categories across sample groups.
Pie charts representing the proportional distribution of lipid classes.
Heatmaps for visualizing changes in lipid composition across multiple samples.

Comparative Analysis Report

Contents:

Statistical comparisons using appropriate tests (e.g., t-tests, ANOVA) to evaluate FFA differences between experimental groups.
Significant biomarker Identification with fold changes and p-values to highlight metabolically relevant FFAs.
Pathway analysis to interpret lipidomic alterations and identify potential disruptions in metabolic pathways.

Format:

Summary table displaying FFA species, mean concentrations, standard deviations, fold changes, and p-values.
Volcano plots to visualize significantly altered fatty acids.
Cluster analysis for exploring sample similarities and grouping based on FFA profiles.
Principal component analysis (PCA) to show sample distribution and lipidomic variations.

Lipidome Data Analysis Workflow Workflow of Lipidome Data Analysis

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

Download Brochure

What Our Free Fatty Acids Analysis Used For

Metabolic Research

Investigate the role of free fatty acids in cellular energy metabolism, lipid biosynthesis, and biochemical pathway regulation.

Biotechnology Development

Optimize lipid production in microbial strains for biofuel, bioplastic, and other bio-based material applications.

Agricultural Studies

Analyze plant lipids, soil fatty acids, and microbial communities to monitor soil health, crop quality, and agricultural productivity.

Environmental Monitoring

Track changes in fatty acid profiles as biomarkers for pollution exposure, microbial activity, and ecosystem health assessments.

Nutritional Research

Evaluate the fatty acid composition of foods, oils, and dietary supplements to assess nutritional value and lipid quality.

Food Quality and Safety

Monitor fatty acid composition, detect lipid oxidation, and ensure product stability during food processing, storage, and distribution.

Sample Requirements for Free Fatty Acids Analysis Solutions

Sample Type	Required Amount	Storage Conditions	Notes
Plasma	≥ 200 µL	-80°C	Avoid hemolysis; use EDTA or heparin tubes.
Serum	≥ 200 µL	-80°C	Allow complete clotting before centrifugation; remove lipemic samples if possible.
Tissue	≥ 50 mg	-80°C	Flash freeze in liquid nitrogen; store in cryotubes.
Cell Culture	≥ 1 × 10⁶ cells	-80°C	Wash cells with PBS, pellet cells, and freeze immediately.
Urine	≥ 500 µL	-80°C	Collect midstream urine and store without preservatives.
Feces	≥ 100 mg	-80°C	Ensure minimal exposure to air; freeze promptly.
Plant Samples	≥ 100 mg	-80°C	Freeze immediately and store in sealed containers to prevent lipid oxidation.
Food and Oil Samples	≥ 1 g or 1 mL	4°C or -20°C	Store in dark, airtight containers to avoid oxidation.
Microbial Cultures	≥ 1 × 10⁹ cells or equivalent	-80°C	Pellet cultures by centrifugation and wash with PBS before freezing.

How Does Free Fatty Acids Analysis Work

Workflow of free fatty acids analysis service.

FAQs for Free Fatty Acids Analysis Service

How should biological samples (e.g., plasma, tissues) be collected to ensure FFA stability?

Critical Steps:

Immediate Cooling: Collect samples on ice to inhibit lipase activity, which rapidly hydrolyzes triglycerides into FFAs. Serum/plasma must be separated from cells within 4 hours post-collection to avoid artifactual FFA elevation .
Anticoagulants: Use EDTA (1 mM) to stabilize blood samples and prevent enzymatic degradation .
Storage: Freeze at -80°C immediately after separation. Avoid repeated freeze-thaw cycles, as this accelerates lipid oxidation.

What are the best practices for handling lipid-rich samples (e.g., oils, dairy products)?

Contamination Control:

Use methanol-washed glass tubes instead of plastic containers to reduce exogenous FFA contamination (e.g., palmitic acid levels reduced by 73%) .
For oils, extract FFAs using chloroform-hexane to isolate them from triglycerides, achieving ≥99% recovery .

Oxidation Prevention: Add antioxidants (e.g., 0.01% BHT) during extraction and perform steps under nitrogen/argon atmosphere .

How do I optimize FFA extraction for complex matrices like sewage sludge or microbial cultures?

Specialized Protocols:

Magnetic Nanoparticle-Based Extraction: For environmental samples (e.g., sludge), superhydrophobic Fe3O4 nanoparticles functionalized with FFAs enable efficient PAH enrichment with 82.8–116.6% recovery .
Microbial FFA Extraction: Centrifuge cultures at 10,000 ×g for 10 min, lyophilize biomass, and use MTBE-based solvent systems to enhance yield .

What are the QA/QC measures to ensure FFA quantification accuracy?

Internal Standards: Use deuterated FFAs (e.g., d3-palmitic acid) to correct matrix effects and validate recovery rates (intra-day CV ≤5%) .
Cross-Platform Validation: Combine LC-MS/MS (sensitivity: 0.01 ng/mL) with GC-MS or NMR for isomer resolution (e.g., ω-3 vs. ω-6 FFAs) .

Can I analyze FFAs in small-volume or non-invasive samples (e.g., dried blood spots)?

Dried Blood Spots (DBS): Validated LC-MS/MS methods allow quantification of PUFA-FFAs (e.g., EPA, DHA) from a single 3.2 mm punch, with linearity up to 6.14% and precision ≤16% .
Skin Sebum: Extract FFAs from Sebutape patches using chloroform-methanol (2:1), followed by FTIR or GC-MS profiling .

How do I design experiments for metabolic flux or lipid oxidation studies?

Tracer Studies: Use 13C-glucose or 13C-palmitate to track FFA β-oxidation rates in cell/tissue models. Data analyzed via INCA software for kinetic modeling .
Oxidation Markers: Pair FFA analysis with lipid peroxidation products (e.g., malondialdehyde) via LC-MS to assess oxidative stress.

What are the industry-specific considerations for FFA analysis?

Food Industry: Monitor FFA levels in oils to detect rancidity (correlates with peroxide value). FT-Raman spectroscopy enables reagent-free, rapid screening (RMSEP: 0.29%) .
Biofuel Research: Optimize FFA esterification in waste cooking oil using ion-exchange resins (e.g., Amberlyst-15 achieves 60.2% conversion at 65°C) .

Publications

White matter lipid alterations during aging in the rhesus monkey brain. GeroScience, 2024. https://doi.org/10.1007/s11357-024-01353-3
Characterization of Dnajc12 knockout mice, a model of hypodopaminergia. bioRxiv, 2024. https://doi.org/10.1101/2024.07.06.602343
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
Lipid Membrane Engineering for Biotechnology (Doctoral dissertation, Aston University). Lipid Membrane Engineering for Biotechnology, 2023. https://doi.org/10.48780/publications.aston.ac.uk.00046663
Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction. EMBO Journal, 2019. https://doi.org/10.15252/embj.201899558

References

Feng, Saixiang, et al. "Either fadD1 or fadD2, which encode acyl-CoA synthetase, is essential for the survival of Haemophilus parasuis SC096." Frontiers in Cellular and Infection Microbiology 7 (2017): 72. https://doi.org/10.3389/fcimb.2017.00072
Kokotou, Maroula G., Christiana Mantzourani, and George Kokotos. "Development of a liquid chromatography–high resolution mass spectrometry method for the determination of free fatty acids in milk." Molecules 25.7 (2020): 1548. https://doi.org/10.3390/molecules25071548

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

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