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Quantification of Sphingosine-1-Phosphate and Related Sphingolipids by Liquid Chromatography Coupled to Tandem Mass Spectrometry

  • Constantin Bode
  • Markus H. GrälerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 874)

Abstract

Liquid chromatography coupled to tandem mass spectrometry has evolved as the method of choice for the detection of sphingolipid metabolites due to its high sensitivity and superior specificity compared to other methodological approaches. Here, we describe a simple and robust method for the detection and quantification of sphingosine-1-phosphate (S1P) and related sphingolipids in biological samples. Tissue homogenates, cells, supernatant, plasma, and whole blood are spiked with an internal standard to account for loss of material during sample handling. After chloroform extraction of lipids under acidified conditions, the solvent is evaporated, and the remaining lipid extracts are dissolved in 20% CHCl3 and 80% methanol. Following reversed-phase high-performance liquid chromatography step-gradient separation of sphingolipids and positive electrospray ionization, detection is carried out with the AB Sciex QTrap triple-quadrupole mass spectrometer operating in multiple reaction monitoring. Characteristic fragment ions of S1P and related sphingolipids are monitored and subsequently analyzed relative to known standard concentrations of the pure compounds. Known problems of S1P quantification, such as carryover and insufficient HPLC separation, are discussed.

Key words

Sphingosine-1-phosphate Sphingolipids Electrospray ionization Mass spectrometry Liquid chromatography Reversed phase Multiple reaction monitoring 

References

  1. 1.
    Lahiri S, Futerman AH (2007) The metabolism and function of sphingolipids and glycosphingolipids. Cell Mol Life Sci 64:2270–2284PubMedCrossRefGoogle Scholar
  2. 2.
    Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, Spiegel S (1999) Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 147:545–558PubMedCrossRefGoogle Scholar
  3. 3.
    Yu H, Okada T, Kobayashi M, Abo-Elmatty DM, Jahangeer S, Nakamura S (2009) Roles of extracellular and intracellular sphingosine 1-phosphate in cell migration. Genes Cells 14:597–605PubMedCrossRefGoogle Scholar
  4. 4.
    Lee OH, Kim YM, Lee YM, Moon EJ, Lee DJ, Kim JH, Kim KW, Kwon YG (1999) Sphingosine 1-phosphate induces angiogenesis: its angiogenic action and signaling mechanism in human umbilical vein endothelial cells. Biochem Biophys Res Commun 264:743–750PubMedCrossRefGoogle Scholar
  5. 5.
    Mandala S, Hajdu R, Bergstrom J, Quackenbush E, Xie J, Milligan J, Thornton R, Shei GJ, Card D, Keohane C, Rosenbach M, Hale J, Lynch CL, Rupprecht K, Parsons W, Rosen H (2002) Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 296:346–349PubMedCrossRefGoogle Scholar
  6. 6.
    Pyne NJ, Pyne S (2010) Sphingosine 1-phosphate and cancer. Nat Rev Cancer 10:489–503PubMedCrossRefGoogle Scholar
  7. 7.
    Oskeritzian CA, Price MM, Hait NC, Kapitonov D, Falanga YT, Morales JK, Ryan JJ, Milstien S, Spiegel S (2010) Essential roles of sphingosine-1-phosphate receptor 2 in human mast cell activation, anaphylaxis, and pulmonary edema. J Exp Med 207:465–474PubMedCrossRefGoogle Scholar
  8. 8.
    Skoura A, Michaud J, Im DS, Thangada S, Xiong Y, Smith JD, Hla T (2011) Sphingosine-1-phosphate receptor-2 function in myeloid cells regulates vascular inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol 31:81–85PubMedCrossRefGoogle Scholar
  9. 9.
    Kulakowska A, Zendzian-Piotrowska M, Baranowski M, Kononczuk T, Drozdowski W, Gorski J, Bucki R (2010) Intrathecal increase of sphingosine 1-phosphate at early stage multiple sclerosis. Neurosci Lett 477:149–152PubMedCrossRefGoogle Scholar
  10. 10.
    Edsall LC, Spiegel S (1999) Enzymatic measurement of sphingosine 1-phosphate. Anal Biochem 272:80–86PubMedCrossRefGoogle Scholar
  11. 11.
    Yatomi Y, Ruan F, Ohta J, Welch RJ, Hakomori S, Igarashi Y (1995) Quantitative measurement of sphingosine 1-phosphate in biological samples by acylation with radioactive acetic anhydride. Anal Biochem 230:315–320PubMedCrossRefGoogle Scholar
  12. 12.
    Ruwisch L, Schafer-Korting M, Kleuser B (2001) An improved high-performance liquid chromatographic method for the determination of sphingosine-1-phosphate in complex biological materials. Naunyn Schmiedebergs Arch Pharmacol 363:358–363PubMedCrossRefGoogle Scholar
  13. 13.
    Andréani P, Gräler MH (2006) Comparative quantification of sphingolipids and analogs in biological samples by high-performance liquid chromatography after chloroform extraction. Anal Biochem 358:239–246PubMedCrossRefGoogle Scholar
  14. 14.
    He X, Huang CL, Schuchman EH (2009) Quantitative analysis of sphingosine-1-­phosphate by HPLC after napthalene-2,3-­dicarboxaldehyde (NDA) derivatization. J Chromatogr B Analyt Technol Biomed Life Sci 877:983–990PubMedCrossRefGoogle Scholar
  15. 15.
    Murata N, Sato K, Kon J, Tomura H, Okajima F (2000) Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay. Anal Biochem 282:115–120PubMedCrossRefGoogle Scholar
  16. 16.
    Schwab SR, Pereira JP, Matloubian M, Xu Y, Huang Y, Cyster JG (2005) Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science 309:1735–1739PubMedCrossRefGoogle Scholar
  17. 17.
    Schmidt H, Schmidt R, Geisslinger G (2006) LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples. Prostaglandins Other Lipid Mediat 81:162–170PubMedCrossRefGoogle Scholar
  18. 18.
    Sullards MC, Merrill AH Jr (2001) Analysis of sphingosine 1-phosphate, ceramides, and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Sci STKE 2001(67):pl1PubMedCrossRefGoogle Scholar
  19. 19.
    Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509PubMedGoogle Scholar
  20. 20.
    Yamaguchi J, Ohmichi M, Jingu S, Ogawa N, Higuchi S (1999) Utility of postcolumn addition of 2-(2-methoxyethoxy)ethanol, a signal-enhancing modifier, for metabolite screening with liquid chromatography and negative ion electrospray ionization mass spectrometry. Anal Chem 71:5386–5390PubMedCrossRefGoogle Scholar
  21. 21.
    Berdyshev EV, Gorshkova IA, Garcia JG, Natarajan V, Hubbard WC (2005) Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography-tandem mass spectrometry. Anal Biochem 339:129–136PubMedCrossRefGoogle Scholar
  22. 22.
    Wijesinghe DS, Allegood JC, Gentile LB, Fox TE, Kester M, Chalfant CE (2010) Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels. J Lipid Res 51:641–651PubMedCrossRefGoogle Scholar
  23. 23.
    Dams R, Huestis MA, Lambert WE, Murphy CM (2003) Matrix effect in bio-analysis of illicit drugs with LC-MS/MS: influence of ionization type, sample preparation, and biofluid. J Am Soc Mass Spectrom 14:1290–1294PubMedCrossRefGoogle Scholar
  24. 24.
    Lahaie M, Mess JN, Furtado M, Garofolo F (2010) Elimination of LC-MS/MS matrix effect due to phospholipids using specific solid-phase extraction elution conditions. Bioanalysis 2:1011–1021PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Molecular Cancer Research CentreCharité University Medical SchoolBerlinGermany

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