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Protocol for Lipid Identification by Untargeted Tandem Mass Spectrometry Coupled with Ultra-High-Pressure Liquid Chromatography

1 Lipid Extraction

A range of lipid extraction methods have been documented in the scientific literature, including the Folch and Bligh and Dyer methods, as well as MTBE. This protocol outlines a modified version of the Bligh and Dyer method.

To minimize unsaturated lipid oxidation during sample handling, a mixture of inhibitors is typically added prior to lipid extraction. These inhibitors, such as ethylene-diaminotetraacetic acid (EDTA, 2 mM final concentration) and butylated hydroxytoluene (BHT, 100 μM final concentration), act as chelators.

To prevent lipase activity, tissue samples should be flash-frozen in liquid nitrogen and handled as little as possible before lipid extraction, such as during weighing.

When working with cell pellets, they can be extracted directly to avoid further changes in lipid composition, and the resulting dry lipid samples can be stored at −80 °C under argon until analysis.

The lipid sample (e.g., 100 mg flash-frozen tissue homogenate or 1 × 107 cells suspended in 500 μL of buffer) is mixed with an equal volume of methanol and vortexed for a minute. It is then extracted with chloroform (typically at the same volume as the buffer plus methanol) and this process is repeated three times.

As plasticizers from plastic containers can contaminate the lipid samples, HPLC column, and mass spectrometer, only glass tubes or vials should be used, and a Hamilton syringe or glass pipette is preferable.

After each extraction, the mixture is centrifuged at 4 °C for 10 min at 3500 RCF to separate the organic and aqueous phases. The lower chloroform phases are combined and evaporated to dryness, typically using a nitrogen gas evaporator.

The nitrogen is then switched to argon, which is denser than air, to ensure the samples are sealed under an argon blanket. Parafilm can be used to seal the vials. This extraction procedure can be used for cells in suspension, cell pellets, or homogenized tissue.

2 Chromatographic Separation of Lipids: HPLC Method

Owing to their great structural diversity lipids have varied chemical properties and polarities and a single system for lipid separation is challenging. An example of a chromatographic separation method involves either HPLC or UHPLC on a reverse phase C18 column using as buffer system; A—10 mM ammonium acetate or formate solution in 40% acetonitrile in water and as buffer B—10 mM ammonium acetate or formate solution in 10% acetonitrile in isopropanol. A typical 27 min separation method, which achieves lipid class separation and some species separation.

3 Instrument Acquisition Method for Lipid Analysis

A variety of instrumentation can be used for mass spectrometric detection of lipids. However, typically a tandem mass spectrometer is used which can acquire full MS, fragment the most intense lipid ions, and acquire fragment ion information on the measured lipids. The most popular instruments for lipid analysis are Orbitraps (Q-Exactive, Orbitrap Fusion) and Q-TOFs (Agilent, Waters, Sciex, Bruker, etc.). A triple quadrupole instrument is usually used for targeted analyses of specific lipids.

4 Data Analysis

Several software packages are available for the analysis of mass spectrometric data for lipid identification and quantitation such as Lipid Search (Thermo Fisher), Lipidblast, Simlipid (Premier Biosoft). Matching the MS/MS experimental data with spectra from various databases provides the identification. Historically, this used to be done based on accurate mass search due to lack of MS/MS databases. Currently, MS/MS matching against an experimental or in silico database is the preferred identification method used in combination with parent ion accurate mass.


  1. Bhattacharya, S. K. (2017). Lipidomics. Methods in Molecular Biology, 1609.
* For Research Use Only. Not for use in diagnostic procedures.
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