Compositional Analysis of Phospholipids by Mass Spectrometry and Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy

  • M. Cecilia YappertEmail author
Part of the Methods in Pharmacology and Toxicology book series (MIPT)


As the roles of phospholipids continue to be unraveled in an expansive list of biological processes, the availability of fast, accurate, and precise analytical approaches becomes of utmost relevance. Traditional methods rely on the separation of phospholipid classes, each with a different polar head group, by either thin-layer or liquid chromatography. The length and degree of unsaturation of the hydrophobic chains are subsequently determined by derivatization and gas chromatography. Although these methods are well developed, they are time-consuming and do not allow for in situ analysis.

The combination of P-31 nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) provides a powerful alternative for the analysis of phospholipids in complex mixtures. With the use of P-31 NMR spectroscopy, phospholipids can be analyzed quantitatively in either organic solvents or aqueous media containing a detergent. Matrix-assisted laser desorption/ionization (MALDI) and other forms of ionization such as electron-spray ionization (ESI) coupled with mass spectrometry (MS) allow the analysis of intact lipids both in vitro and in situ.

This chapter describes conventional approaches for phospholipid analysis first. The criteria and current options in the choice of matrices for MALDI MS are addressed. The ability of MALDI MS to image phospholipids is then highlighted as well as the remaining challenges. Regarding P-31 NMR, current studies on the measurement of temperature coefficients and pH dependences to improve the accuracy in the assignment of P-31 resonances are described. Finally, the complementary nature of P-31 NMR and MALDI MS is illustrated in the reevaluation of the unusual composition of phospholipids in human lens membranes.

Key words

Phospholipid analysis mass spectrometry MALDI matrices mass spectral imaging 31P NMR spectroscopy, bile salts, temperature coefficient 



The ongoing, 25-year collaboration with Professor Douglass Borchman is most deeply appreciated. The discussions and generous donation of animal ocular tissue by the former Director of the Louisville Zoo, Dr. William Foster, and current veterinarians, Dr. Roy Burns and Dr. Zoole Gymesi are thankfully acknowledged. Professor Donald DuPré’s theoretical predictions of P-31 chemical shifts have been most helpful for the interpretation of experimental trends. Without the funding provided by the National Eye Institute many of the studies cited in this chapter would not have been completed. Finally, to the many students in our graduate program that carried out these studies, a heartfelt thank-you.



2-(2-Aminoethylamino)-5-nitropyridine; a basic organic matrix that promotes the generation of anions.


Atmospheric pressure chemical ionization.


2,5-Dihydroxybenzoic acid, one of the matrices most commonly used for the MALDI-MS analysis of small molecules, including phospholipids. Its acidic character facilitates the formation of cations.


Mild ionization technique involving nebularization and a high electric field at a metallic nozzle. Compatible with online chromatography as well as direct infusion, and results primarily in molecular ions.


Matrix-assisted laser desorption/ionization, a commonly used means of ionizing and vaporizing an analyte for introduction into a mass spectrometer.


Phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, common phospholipids (PL) differing at the head group moiety.


Ether lipids where the first position of glycerol bears a vinyl residue in an ether linkage rather than an ester. The double bond next to the ether bond.


Para-nitroaniline, a nearly neutral matrix used in the MALDI-MS analysis of phospholipids in both positive and negative modes. It also enables imaging of phospholipids in tissues.


Class of lipids based on sphingosine, which has a serine backbone. Different derivatives on the serine hydroxyl give different family members such as ceramide and sphingomyelin (SM).

Temperature coefficient

Variation of, for example, chemical shift with temperature. Often linear, and then defined as dδ/dT. The sign and magnitude may provide clues about interactions, such as hydrogen bonding.


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Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of LouisvilleLouisvilleUSA

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