Three primary approaches have been used to quantify bioactive lipids in cells and biological samples: ELISA, GC-MS, and LC-MS/MS. The basics of each technique are described below along with some of the advantages and pitfalls of each method. The choice of which method one should use will depend on many factors including availability of assays/instrumentation, cost of analysis, scope of project, analytical performance requirements, etc.

ELISA 

Enzyme-Linked Immunosorbent Assay (ELISA) is a common technique that has been successfully used for many years to measure target analytes in cell cultures and biological matrices.

Advantages of ELISA

1. Well-characterized antibodies for a large variety of lipids

2. Cost-effective option for quantification of single lipid species

3. Very sensitive method for low abundance lipids (e.g., leukotrienes)

Disadvantages of ELISA

1. Single analyte quantitation - multiple analytes require multiple kits
   

2. Assay development is slow
   

3. Antibody may cross react with similar lipids

4. Antibodies among vendors are not equivalent 

GC-MS

Gas Chromatography-Mass Spectrometry (GC-MS) has been used successfully for many years to detect and quantify bioactive lipids. Many analytes may be quantified in a single run and the resolution and sensitivity provided by GC-MS overcomes some of the analytical challenges with the analysis of bioactive lipids. However, for successful analysis of oxidized lipids by GC-MS several derivatization steps are required, including silylation of alcohol functional groups, preparation of esters of acid functional groups, and derivatization of ketone functional groups.

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Advantages of GC-MS

1. Multiple analytes can be quantified in a single experiment

2. High throughput and cost effectiveness improve with scale

3. Method development time is much less than antibody development

4. Greater specificity and accuracy compared to ELISA

Disadvantages of GC-MS

1. Derivatization step(s) are time consuming and can introduce errors

2. Not as sensitive as ELISA

3. Not universal – large, polar lipids and temperature-sensitive compounds are not amenable to GC-MS

4. Instrumentation is expensive and requires training to operate
   

LC-MS/MS

A more recently developed and increasingly used tool for the quantification of bioactive lipids is LC-MS/MS. Improvements in chromatographic resolution and mass spectrometer sensitivity in combination with its high specificity and multiplexing capability have made this an ideal technique for the analysis of lipids.

Advantages of LC-MS/MS

1. Many analytes (hundreds) can be quantified in a single analysis

2. Sensitivity is comparable to ELISA

3. High specificity – analytes are separated and detected by unique transitions

4. Throughput advantage scales with analyte number and sample number

Disadvantages of LC-MS/MS

1. Instrumentation is expensive
   

2. Highly trained staff are required to operate and maintain equipment

3. Sensitivity is analyte specific and can be poor for some compound classes

4. Significant sample cleanup is required to reduce matrix interference effects

List of Common MRM Transitions for Eicosanoids and Docosanoids (Negative ESI)

Sources

Murphy, R.C. Tandem mass spectrometry of lipids: Molecular analysis of complex lipids. Cambridge, UK: Royal Society of Chemistry (2015).

Zhang, X., Yang, N., Ai, D., et al. Systematic metabolomic analysis of eicosanoids after omega-3 polyunsaturated fatty acid supplementation by a highly specific liquid chromatography–tandem mass spectrometry-based method. J. Proteome Res. 14(4), 1843-1853 (2015). [PMID: 25736083 ]

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