Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Sphingosine-1-Phosphate

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Lahiri S, Futerman AH (2007) The metabolism and function of sphingolipids and glycosphingolipids. Cell Mol Life Sci 64:2270–2284

    Article  PubMed  CAS  Google Scholar 

  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 147:545–558

    Article  PubMed  CAS  Google Scholar 

  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 14:597–605

    Article  PubMed  CAS  Google Scholar 

  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 264:743–750

    Article  PubMed  CAS  Google Scholar 

  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 296:346–349

    Article  PubMed  CAS  Google Scholar 

  6. Pyne NJ, Pyne S (2010) Sphingosine 1-phosphate and cancer. Nat Rev Cancer 10:489–503

    Article  PubMed  CAS  Google Scholar 

  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 207:465–474

    Article  PubMed  CAS  Google Scholar 

  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 31:81–85

    Article  PubMed  CAS  Google Scholar 

  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 477:149–152

    Article  PubMed  CAS  Google Scholar 

  10. Edsall LC, Spiegel S (1999) Enzymatic measurement of sphingosine 1-phosphate. Anal Biochem 272:80–86

    Article  PubMed  CAS  Google Scholar 

  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 230:315–320

    Article  PubMed  CAS  Google Scholar 

  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 363:358–363

    Article  PubMed  CAS  Google Scholar 

  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 358:239–246

    Article  PubMed  Google Scholar 

  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 877:983–990

    Article  PubMed  CAS  Google Scholar 

  15. Murata N, Sato K, Kon J, Tomura H, Okajima F (2000) Quantitative measurement of sphingosine 1-phosphate by radioreceptor-binding assay. Anal Biochem 282:115–120

    Article  PubMed  CAS  Google Scholar 

  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 309:1735–1739

    Article  PubMed  CAS  Google Scholar 

  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 81:162–170

    Article  PubMed  CAS  Google Scholar 

  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):pl1

    Article  PubMed  CAS  Google Scholar 

  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 226:497–509

    PubMed  CAS  Google Scholar 

  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 71:5386–5390

    Article  PubMed  CAS  Google Scholar 

  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 339:129–136

    Article  PubMed  CAS  Google Scholar 

  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 51:641–651

    Article  PubMed  CAS  Google Scholar 

  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 14:1290–1294

    Article  PubMed  CAS  Google Scholar 

  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 2:1011–1021

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Cancer Research Centre, Charité University Medical School, Berlin, Germany

    Constantin Bode & Markus H. Gräler

Authors
  1. Constantin Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Markus H. Gräler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Markus H. Gräler .

Editor information

Editors and Affiliations

  1. University of Melbourne, Parkville, Victoria, Australia

    Alice Pébay

  2. Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Smyth Road 501, Ottawa, K1Y 8L6, Canada

    Kursad Turksen

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Bode, C., Gräler, M.H. (2012). Quantification of Sphingosine-1-Phosphate and Related Sphingolipids by Liquid Chromatography Coupled to Tandem Mass Spectrometry. In: Pébay, A., Turksen, K. (eds) Sphingosine-1-Phosphate. Methods in Molecular Biology, vol 874. Humana Press. https://doi.org/10.1007/978-1-61779-800-9_3

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-800-9_3

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-799-6

  • Online ISBN: 978-1-61779-800-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation