Sphingolipid Analysis by High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS)

  • Jacek Bielawski
  • Jason S. Pierce
  • Justin Snider
  • Barbara Rembiesa
  • Zdzislaw M. Szulc
  • Alicja Bielawska
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 688)


Sphingolipid (SPL) metabolism (Fig. 1) serves a key role in the complex mechanisms regulating cellular stress responses to environment. Several SPL metabolites, especially ceramide (Cer), sphingosine (Sph) and sphingosine1-phosphate (S1P) act as key bioactive molecules governing cell growth and programmed cell death (Fig. 2). Perturbations in sphingolipids of one type may enhance or interfere with the action of another. To monitor changes in SPL composition therefore, reliable analytical methods are necessary.

Here we present the liquid chromatography tandem mass spectrometry (LC-MS/MS) approach for simultaneous qualitative and quantitative monitoring of SPL components (classes and molecular species) in biological material as an effective tool to study sphingolipid signaling events. The LC-MS/MS methodology is the only available technique that provides high specificity and sensitivity, along with a wealth of structural identification information.
Figure 1.

Sphingolipid biosynthesis and metabolic pathways; metabolomic profiling of sphingolipids. Abbreviations used in the figure: 3-keto-dhSph, 3-keto-dihydrosphingosine; dhSph, dihydrosphingosine; dhS1P, dihydrosphingosine 1-phosphate; dhCer, dihydroceramide; Sph, sphingosine; S1P, sphingosine 1-phosphate; Cer1P, ceramide-1-phosphate; SM, sphingomyelin; lyso-SM, lyso-sphingomyelin; DAG, diacylglycerol; O-Acyl-Sph. O-acyl-sphingosine, O-Acyl-Cer, O-acyl-ceramide; N-Me-Sph, N-methyl-sphingosine; N.N-DMSph, N,N-dimethyl-sphingosine; PA, palmitic acid; EAP, ethanolamine phosphate; HD, hexadecenal; GL, glycerolipids; de-SM, demethylated sphingomyelin; PGLs, phosphoglycerolipids; GlcCer, glucosylceramide; GalCer, galactosylceramide; GSLs, glycosphingolipids.

Figure 2.

Natural sphingolipids are a highly heterogenous system related to the sphingoid bases and derivatization made on the amino- and hydroxy-functions. Structures shown in this figure represent derivatives of 18C-SB (sphingoid bases containing C18-backbone chain) indicating SPLs containing 2-amino-1,3-dihydroxy-octadecene-4E, 2-amino-1,3-dihydroxy-octadecane and 2-amino-1,3,4-trihydroxy-octadecane. General structures, nomenclature and abbreviations for SPLs are cited and described in this presentation. Cn -indicates the chain length of N-acyl part of SPLs.


Molecular Species Multiple Reaction Monitoring Collision Induce Dissociation Sphingoid Base Chemical Noise 
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  1. 1.
    Simons K, Ikonen E, Functional rafts in cell membranes. Nature 1997; 387:569–572.CrossRefPubMedGoogle Scholar
  2. 2.
    Pettus BJ, Chalfant CE, Hannun YA. Ceramide in apoptosis: an overview and current perspectives. Biochim Biophys Acta 2002; 1585:114–125.PubMedGoogle Scholar
  3. 3.
    Cuvillier O. Sphingosine in apoptosis signaling. Biochem Biophys Acta 2002; 1585:153–162.PubMedGoogle Scholar
  4. 4.
    Spiegel S, Kolesnick R. Sphingosine 1-phosphate as a therapeutic agent. Leukemia 2002; 16:1596–1602.CrossRefPubMedGoogle Scholar
  5. 5.
    Lamour N F, Chalfant C E, Ceramide-1-phosphate: The “missing” link in eicosanoid biosynthesis and inflammation. Mol Interv 2005; 5:358–367.CrossRefPubMedGoogle Scholar
  6. 6.
    Sastry PS. Lipids of nervous tissue: composition and metabolism. Prog Lipid Res 1985; 24(2):69–176.CrossRefPubMedGoogle Scholar
  7. 7.
    Vos JP, Lopes-Cardozo M, Gadella BM. Metabolic and functional aspects of sulfogalactolipids. Biochim Biophys Acta 1994; 1211:125–149PubMedGoogle Scholar
  8. 8.
    Merrill AH, Schmelz EM, Dillehay DL et al. Sphingolipids—the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 1997; 142(1):208–225.CrossRefPubMedGoogle Scholar
  9. 9.
    Han XL. Gross RW. Global analyses of cellular lipidomes directly from crude extracts of biological samples by ESI mass spectrometry: a bridge to lipidomics. J Lipid Res 2003; 44:1071–1079.CrossRefPubMedGoogle Scholar
  10. 10.
    Han XL, Yang JY, Cheng H et al. Toward fingerprinting cellular lipidomes directly from biological samples by two-dimensional electrospray ionization mass spectrometry. Anal Biochem 2004; 330:317–331.CrossRefPubMedGoogle Scholar
  11. 11.
    Spener F, Lagarde M, Géloën A et al. What is lipidomics? Eur J Lipid Sci Technol 2003; 105:481–482.CrossRefGoogle Scholar
  12. 12.
    Lagarde M, Geloen A, Record M et al. Lipidomics is emerging. Biochim Biophys Acta 2003; 1634:61–69.PubMedGoogle Scholar
  13. 13.
    Hannun YA. Functional Lipidomics: Lessons and Examples from the Sphingolipids. In: Feng L, Prestwich G eds. Functional Lipidomics. Boca Raton: CRC press, 2005:147–158.CrossRefGoogle Scholar
  14. 14.
    Adams J, Ann Q. Structure determination of sphingolipids by mass spectrometry. Mass Spectrom Rev 1993; 12:51–85.CrossRefGoogle Scholar
  15. 15.
    Adams J, Ann Q. Structure determination of ceramides and neutral glycosphingolipids by collisional activation of (M+Li)+ ions. J Am Soc Mass Spectrom 1992; 3:260–263.CrossRefGoogle Scholar
  16. 16.
    Ann Q, Adams J. Structure-specific collision-induced fragmentation of ceramides cationized with alkali-metal ions. Anal Chem 1993; 22:7–13.CrossRefGoogle Scholar
  17. 17.
    Ann Q, Adams J. Collision-induced decomposition of sphingomyelins for structural elucidation. Biol Mass Spectrom 1993; 22:285–294.CrossRefGoogle Scholar
  18. 18.
    Sullard MC. Sphingolipid Metabolism and Cell Signaling. Methods Enzymol 2000; 312:32–45.CrossRefGoogle Scholar
  19. 19.
    Gu M, Kerwin JL, Watts JD et al. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry. Anal Biochem 1997; 244:347–356CrossRefPubMedGoogle Scholar
  20. 20.
    Van Veldhoven PP, De Ceuster P, Rozenberg R et al. On the presence of phosphorylated sphingoid bases in rat tissues. A mass-spectrometric approach. FEBS Lett 1994; 350(1):91–95.CrossRefPubMedGoogle Scholar
  21. 21.
    Couch LH, Churchwell MI, Doerge DR et al. Identification of ceramides in human cells using liquid chromatography with detection by atmospheric pressure chemical ionization-mass spectrometry. Rapid Commun Mass Spectrom 1997;11:504–512.CrossRefPubMedGoogle Scholar
  22. 22.
    Mano N, Oda Y, Yamada K et al. Simultaneous quantitative determination method for sphingolipid metabolites by liquid chromatography/ion spray ionization tandem mass spectrometry, Anal Biochem 1997; 244:291–300.CrossRefPubMedGoogle Scholar
  23. 23.
    Liebisch G, Derobnik W, Reil M et al. Quantitative measurement of different ceramide spiecies from crude cellular lipid extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Lipid Res 1999; 40:1539–1546.Google Scholar
  24. 24.
    Pettus BJ, Kroesen BJ, Szulc ZM et al. Quantitative measurement of different ceramide species from crude cellular extracts by normal-phase high-performance liquid chromatography coupled to atmospheric pressure ionization mass spectrometry. Rapid Commun Mass Spectrom 2004; 18:577–583.CrossRefPubMedGoogle Scholar
  25. 25.
    Sullard MC, Merrill AH. Analysis of sphingosine-1-phosphate, ceramides and other bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Sci stke 2001; 67: 1–11.Google Scholar
  26. 26.
    Folch J, Lees M, Sloane HS. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1956; 196:497–509.Google Scholar
  27. 27.
    Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification, Can J Biochem and Physiol 1959; 37:911–917.Google Scholar
  28. 28.
    Lin T, Genestier L, Pinkoski MJ. Role of acidic sphingomyelinase in Fas/CD95-mediated cell death. J Biol Chem, 2000: 275(12):8657–8663.CrossRefPubMedGoogle Scholar
  29. 29.
    Bose R, Verheij R, Haimovitz-Friedman A et al. Ceramide synthase mediates daunorubicin-induced apoptosis; an alternative mechanism for generating death signals. Cells 1995; 82:405–414.CrossRefGoogle Scholar
  30. 30.
    Prasad, T, Saini P, Guar NA et al. Functional Analysis of CaIPT1, a Sphingolipid Biosynthetic Gene Involved in Multidrug Resistance and Morphogenesis of Candida albicans. Chemother 2005; 49:3442–3452.Google Scholar
  31. 31.
    Monick MM, Mallampalli RK, Bradford M et al. Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high level of acid ceramidase activity. J Immunology. 2004; 173:123–135.Google Scholar
  32. 32.
    Adams JM, Pratipanawatr T, Berria R et al. Ceramide content is increased in skeletal muscle from obese insulin-resistant humans, Diabetes 2004; 53:25–31.CrossRefPubMedGoogle Scholar
  33. 33.
    Zheng W, Kollmeyer J, Symolon H et al. Ceramides and other bioactive sphingolipids backbone in health and disease: Lipidomic analysis, metabolism and roles in membrane structure, dynamic, signalic and autopaphy, Biochim Biophys Acta 2006; 1758(12):1864–1884.CrossRefPubMedGoogle Scholar
  34. 34.
    Maceyka M, Sankala H, Hait NC et al. SphK1 and SphK2, sphingosine kinase isoenzymes with opposition functions in sphingolipid metabolism. J Biol Chem 2005; 280:37118–37129.CrossRefPubMedGoogle Scholar
  35. 35.
    VanDer Luit AH, Budde M, Zerp S et al. Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin-and cholesterol-deficiency in lipid rafts. Biochem J 2007; 401:541–549.CrossRefPubMedGoogle Scholar
  36. 36.
    Guan XL, He X, Ong WY et al. Non-targeted profiling of lipids during kainate-induced neuronal injury. FASEB J 2006; 20:1152–1161.CrossRefPubMedGoogle Scholar
  37. 37.
    Abe A, Shayman JA, Purification and characterization of 1-O-Acylceramide synthase, a novel phospholipase A2 with transacylase activity. J Biol Chem 1998; 273:8467–8474.CrossRefPubMedGoogle Scholar
  38. 38.
    Brodennec J, Koul O, Aguado I et al. A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges. J Lipid Res 2000; 41:1524–1531.Google Scholar
  39. 39.
    Bodennec J, Brichon G, Zwingelstein G et al. Purification of Sphingoid Classes by Solid-Phase Extraction with Aminopropyl and weak cation Exchange Cartridges. Methods Enzymol 2000; 312:101–114.CrossRefPubMedGoogle Scholar
  40. 40.
    Rizov I, Doulis A. Separation of plant membrane lipids by multiple solid-phase extraction. J Chromatogr 2001; A922:347–354.CrossRefGoogle Scholar
  41. 41.
    Bielawski J, Szulc ZM, Hannun YA et al. Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods 2006; 39:82–91.CrossRefPubMedGoogle Scholar
  42. 42.
    Kralik SF, Du X, Patel C et al. A method for quantitative extraction of sphingosine 1-phosphate into organic solvent. Anal Biochem 2001; 294:190–193.CrossRefPubMedGoogle Scholar
  43. 43.
    McDowall RD. Sample preparation for biomedical analysis. J Chromatogr 1989; 492:3–58.CrossRefPubMedGoogle Scholar
  44. 44.
    Olsson NU, Salem N. Molecular species analysis of phospholipids. J Chromatogr B. 1997; 692:245–256.CrossRefGoogle Scholar
  45. 45.
    Fenn JB, Mann M, Meng CK et al, Electrospray ionization for mass spectrometry of large biomolecules. Science 1989; 246:64–72.CrossRefPubMedGoogle Scholar
  46. 46.
    Fenn JB, Mann M, Meng CK et al. Electrospray ionization-principles and practice. Mass Spectrom 1990; 9:37–70.CrossRefGoogle Scholar
  47. 47.
    Cech NB, Enke CG. Practical implications of some recent studies in electrospray ionization fundamentals. Mass Specrom Rev 2001; 20:362–387.CrossRefGoogle Scholar
  48. 48.
    Gu M, Kerwin JL, Watts JD et al. Ceramide profiling of complex lipid mixtures by electrospray ionization mass spectrometry. Anal Biochem 1997; 244:347–356.CrossRefPubMedGoogle Scholar
  49. 49.
    Vieu C, Chevy, F, Rolland C et al. Coupled assay of sphingomyelin and ceramide molecular species by gas liquid chromatography, J Lipid Res 2002; 43:510–522.PubMedGoogle Scholar
  50. 50.
    Isaac G, Bylund D, Masson JE et al. Analysis of phosphatidylcholine and sphingomyelin molecular species from brain extracts using capillary liquid chromatography electrospray ionization mass spectrometry. J Neurosci Methods. 2003;128(1–2):111–9.CrossRefGoogle Scholar
  51. 51.
    Hoffman ED. Tandem mass spectrometry: a primer. J Mass Spectrom 1996; 31:129–137.CrossRefGoogle Scholar
  52. 52.
    Kuksis A. Myher JJ. Application of tandem mass spectrometry for the analysis of long-chain carboxylic acids. J Chromatogr B 1995; 671:35–70.CrossRefGoogle Scholar
  53. 53.
    Samuelsson K, Samuelsson B. Gas chromatographic and mass spectrometric studies of synthetic and naturally occurring ceramides. Chem Phys Lipids 1970; 5:44–49.CrossRefPubMedGoogle Scholar
  54. 54.
    Ejsing CS, Moehring T, Bahr U et al. Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry. J Mass Spectrom 2006; 41:372–389.CrossRefPubMedGoogle Scholar
  55. 55.
    Junganwala, FB, Hayssen V, Pasquini JM et al. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography. J Lipid Res 1979; 20:579–587.Google Scholar
  56. 56.
    Smith M, Jungalwala FB. Reversed-phase high performance liquid chromatography of phosphatidylcholine: a simple method for determining relative hydrophobic interaction of various molecular species J Lipid Res 1981; 22:697–704.PubMedGoogle Scholar
  57. 57.
    Patton GM, Fasulo JM, Robins SJ. Separation of phospholipids and individual molecular species of phospholipids by high-performance liquid chromatography J Lipid Res 1982; 23:190–196.PubMedGoogle Scholar
  58. 58.
    Abidi SL, Mounts TL. High-performance liquid chromatographic separation of molecular species of neutral phospholipids. J Chromatogr A 1992; 598:209–218.CrossRefGoogle Scholar
  59. 59.
    McHowat J, Jones JH, Creer MH. Quantitation of individual phospholipid molecular species by UV absorption measurements. J Lipid Res. 1996; 37:2450–2460.PubMedGoogle Scholar
  60. 60.
    Christie WW. Separation of molecular species of triacylglycerols by high-performance liquid chromatography with a silver ion column. J Chromatogr 1988; 454:273–284.CrossRefPubMedGoogle Scholar
  61. 61.
    Brouwers JF, Gadella BM, Golde LM et al. Quantitative analysis of phosphatidylcholine molecular species using HPLC and light scattering detection. J Lipid Res 1998; 9(2):344–353.Google Scholar
  62. 62.
    Brouwers JF, Vernooij EA, Tielens AG et al. Rapid separation and identification of phosphatidylethanolamine molecular species. Journal of lipid research. 1999; 40(1):164–169.PubMedGoogle Scholar
  63. 63.
    Christie WW. Detectors for high-performance liquid chromatography of lipids with special reference to evaporative light-scattering detection. In: Christie WW, ed. Advances in Lipid Methodology-One. Bridgewater: Oily Press, 1992:239–271.Google Scholar
  64. 64.
    Karlsson AA, Michelsen P, Odham G. Molecular species of sphingomyelin: Determination by high-performance liquid chromatography mass spectrometry with electrospray and high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization. J Mass Spectrom 1998; 33:1192–1198.CrossRefPubMedGoogle Scholar
  65. 65.
    Cole RB. Electrospray Ionization Mass Spectrometry. New York: John-Willey, 1997.Google Scholar
  66. 66.
    Fox TE. Diabetes alters sphingolipid metabolism in the retina. Diabetes 2006; 55:3573–3580CrossRefPubMedGoogle Scholar
  67. 67.
    Dragusin M, Wehner S, Kelly S et al. Effects of sphingosine-1-phosphate and ceramide-1-phosphate on rat intestinal smooth muscle cells: implications for postoperative ileus. FASEB 2006; 20:1930–1932.CrossRefGoogle Scholar
  68. 68.
    Luberto C, Hannun YA. Sphingomyelin synthase, a potential regulator of intracellular levels of ceramide and diacylglycerol during SV40 transformation-Does sphingomyelin synthase account for the putative, phosphatidylcholine-specific phospholipase. C J Biol Chem 1998; 273:14550–14559.CrossRefGoogle Scholar
  69. 69.
    Bielawska A, Perry DK, Hannun YA. Determination of Ceramides and Diglycerides by the Diglyceride Kinase Assay. Anal Biochem 2001; 298:141–150.CrossRefPubMedGoogle Scholar
  70. 70.
    Sullard MC, Wang E, Peng Q et al. Metabolomic Profiling of Sphingolipids in Human Glioma Cell Lines by Liquid Chromatography Tandem Mass Spectrometry, Cell Mol Biol 2003; 49:789–797.Google Scholar
  71. 71.
    Merrill AH, Jr, Wang E, Mullins RE, Jamison WC, Nimkar S, Liotta DC. Quantitation of free sphingosine in liver by high-performance liquid chromatography. Anal Biochem 1988;171:373–381.CrossRefPubMedGoogle Scholar
  72. 72.
    Edsall LC, Spiegel S. Enzymatic Measurement of Sphingosine 1-Phosphate. Anal Biochem 1999; 272:80–86.CrossRefPubMedGoogle Scholar
  73. 73.
    Caligan TB, Peters K, Ou J, et al. A high-performance liquid chromatographic method to measure sphingosine-1-phosphate and related compounds from sphingosine kinase assays and other biological samples, Anal Biochem 2000; 281:36–44.CrossRefPubMedGoogle Scholar
  74. 74.
    Quanren H, Hirofumi S, Neelesh S, et al. Ceramide Synthase Inhibition by Fumonisin B1 Treatment Activates Sphingolipid-Metabolizing Systems in Mouse. Liver ToxSci Advance Access Toxicol Sci 2006; 94:388–397.Google Scholar
  75. 75.
    Tani M, Kihara A, Igarashi Y. Rescue of cell growth by sphingosine with disruption of lipid microdomain formation in Saccharomyces cerevisiae deficient in sphingolipid biosynthesis Biochem J 2006; 394: 237–242.CrossRefPubMedGoogle Scholar
  76. 76.
    James L. Carroll, J, Diann M. McCoy SE, et al. Pulmonary-specific expression of tumor necrosis factor-alters surfactant lipid metabolism. Am J Physiol Lung Cell Mol Physiol 2002; 282: 735–742.Google Scholar
  77. 77.
    Gaver RC, Sweeley CC. Methods for methanolysis of sphingolipids and direct determination of long-chain bases by gas chromatography. J Am Oil Chem Soc 1965; 42:294–298.CrossRefPubMedGoogle Scholar
  78. 78.
    Samuelsson K, Samuelsson B. Gas chromatographic and mass spectrometric studies of synthetic and naturally occurring ceramides. Chem Phys Lipids 1970; 5:44–79.CrossRefPubMedGoogle Scholar

Copyright information

© Landes Bioscience and Springer Science+Business Media 2010

Authors and Affiliations

  • Jacek Bielawski
    • 1
  • Jason S. Pierce
    • 1
  • Justin Snider
    • 1
  • Barbara Rembiesa
    • 1
  • Zdzislaw M. Szulc
    • 1
  • Alicja Bielawska
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMedical University of South CarolinaCharlestonUSA

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