Skip to main content
SpringerLink
Log in

Development and validation of a high-throughput LC–MS/MS assay for routine measurement of molecular ceramides

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Monitoring the levels of the ceramides (Cer) d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1 and ratios thereof in human plasma empowers the prediction of fatal outcome of coronary artery disease (CAD). We describe a validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) methodology for clinical-scaled measurement of the four distinct ceramides. Rapid plasma precipitation was accomplished in 96-well format. Excellent extraction recoveries in the range of 98–109 % were achieved for each ceramide. Addition of corresponding D7-labeled ceramide standards facilitated precise quantification of each plasma ceramide species utilizing a novel short 5-min LC–MS/MS method. Neither matrix interference nor carryover was observed. Robust intra- and inter-assay accuracy and precision <15 % at five different concentrations were obtained. Linear calibration lines with regressions, R 2 > 0.99, were achieved for all analytes. Short-term bench top, long-term plasma, and extract stability demonstrated that the distinct ceramides were stable in the conditions evaluated. The validity of the methodology was demonstrated by determining the precise ceramide concentrations in a small CAD case–control study. Thus, our LC–MS/MS methodology features simple sample preparation and short analysis time for accurate quantification of Cer d18:1/16:0, Cer d18:1/18:0, Cer d18:1/24:0, and Cer d18:1/24:1, designed for routine analysis.

Workflow of molecular ceramide analysis

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Maceyka M, Spiegel S. Sphingolipid metabolites in inflammatory disease. Nature. 2014;510:58–67.

    Article  CAS  Google Scholar 

  2. Tarasov K, Ekroos K, Suoniemi M, Kauhanen D, Sylvänne T, Hurme R, et al. Molecular lipids identify cardiovascular risk and are efficiently lowered by simvastatin and PCSK9 deficiency. J Clin Endocrinol Metab. 2014;99:E45–52.

    Article  Google Scholar 

  3. Haynes CA, Allegood JC, Park H, Sullards MC. Sphingolipidomics: methods for the comprehensive analysis of sphingolipids. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877:2696–708.

    Article  CAS  Google Scholar 

  4. Mullen TD, Hannun YA, Obeid LM. Ceramide synthases at the centre of sphingolipid metabolism and biology. Biochem J. 2012;441:789–802.

    Article  CAS  Google Scholar 

  5. Merrill AH. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem Rev. 2011;111:6387–422.

    Article  CAS  Google Scholar 

  6. Dobrzyń A, Górski J. Ceramides and sphingomyelins in skeletal muscles of the rat: content and composition. Effect of prolonged exercise. Am J Physiol Endocrinol Metab. 2002;282:E277–85.

    Article  Google Scholar 

  7. Previati M, Bertolaso L, Tramarin M, Bertagnolo V, Capitani S. Low nanogram range quantitation of diglycerides and ceramide by high-performance liquid chromatography. Anal Biochem. 1996;233:108–14.

    Article  CAS  Google Scholar 

  8. Vielhaber G, Brade L, Lindner B, Pfeiffer S, Wepf R, Hintze U, et al. Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide. Glycobiology. 2001;11:451–7.

    Article  CAS  Google Scholar 

  9. Samuelsson K, Sameulsson B. Gas chromatographic and mass spectrometric studies of synthetic and naturally occurring ceramides. Chem Phys Lipids. 1970;5:44–79.

    Article  CAS  Google Scholar 

  10. Liebisch G, Drobnik W, Reil M, Trümbach B, Arnecke R, Olgemöller B, et al. Quantitative measurement of different ceramide species from crude cellular extracts by electrospray ionization tandem mass spectrometry (ESI-MS/MS). J Lipid Res. 1999;40:1539–46.

    CAS  Google Scholar 

  11. Simons B, Kauhanen D, Sylvänne T, Tarasov K, Duchoslav E, Ekroos K. Shotgun lipidomics by sequential precursor Ion fragmentation on a hybrid quadrupole time-of-flight mass spectrometer. Metabolites. 2012;2:195–213.

    Article  CAS  Google Scholar 

  12. Breslow DK, Collins SR, Bodenmiller B, Aebersold R, Simons K, Shevchenko A, et al. Orm family proteins mediate sphingolipid homeostasis. Nature. 2010;463:1048–53.

    Article  CAS  Google Scholar 

  13. Han X. Characterization and direct quantitation of ceramide molecular species from lipid extracts of biological samples by electrospray ionization tandem mass spectrometry. Anal Biochem. 2002;302:199–212.

    Article  CAS  Google Scholar 

  14. Merrill AH, Sullards MC, Allegood JC, Kelly S, Wang E. Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry. Methods. 2005;36:207–24.

    Article  CAS  Google Scholar 

  15. Bielawski J, Szulc ZM, Hannun YA, Bielawska A. Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry. Methods. 2006;39:82–91.

    Article  CAS  Google Scholar 

  16. Jiang H, Hsu F-F, Farmer MS, Peterson LR, Schaffer JE, Ory DS, et al. Development and validation of LC-MS/MS method for determination of very long acyl chain (C22:0 and C24:0) ceramides in human plasma. Anal Bioanal Chem. 2013;405:7357–65.

    Article  CAS  Google Scholar 

  17. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine (2001) Guidance for Industry: Bioanalytical method validation. http://www.fda.gov/downloads/Drugs/Guidance/ucm070107.pdf

  18. Vaara S, Nieminen MS, Lokki M-L, Perola M, Pussinen PJ, Allonen J, et al. Cohort profile: the corogene study. Int J Epidemiol. 2012;41:1265–71.

    Article  Google Scholar 

  19. Ann Q, Adams J. Structure determination of ceramides and neutral glycosphingolipids by collisional activation of [M + Li]+ ions. J Am Soc Mass Spectrom. 1992;3:260–3.

    Article  CAS  Google Scholar 

  20. Hsu F-F, Turk J. Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization. J Am Soc Mass Spectrom. 2002;13:558–70.

    Article  CAS  Google Scholar 

  21. Hsu F-F, Turk J, Stewart ME, Downing DT. Structural studies on ceramides as lithiated adducts by low energy collisional-activated dissociation tandem mass spectrometry with electrospray ionization. J Am Soc Mass Spectrom. 2002;13:680–95.

    Article  CAS  Google Scholar 

  22. Schiller J, Arnhold J, Glander HJ, Arnold K. Lipid analysis of human spermatozoa and seminal plasma by MALDI-TOF mass spectrometry and NMR spectroscopy—effects of freezing and thawing. Chem Phys Lipids. 2000;106:145–56.

    Article  CAS  Google Scholar 

  23. Li W, Tang Y, Guo J, Shang E, Qian Y, Wang L, et al. Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach. J Chromatogr A. 2014;1346:49–56.

    Article  CAS  Google Scholar 

  24. Sonomura K, Kudoh S, Sato TA, Matsuda F. Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. J Sep Sci. 2015;38:2033–7.

    Article  CAS  Google Scholar 

  25. Krishnamurthy K, Dasgupta S, Bieberich E. Development and characterization of a novel anti-ceramide antibody. J Lipid Res. 2007;48:968–75.

    Article  CAS  Google Scholar 

  26. Scherer M, Schmitz G, Liebisch G. High-throughput analysis of sphingosine 1-phosphate, sphinganine 1-phosphate, and lysophosphatidic acid in plasma samples by liquid chromatography-tandem mass spectrometry. Clin Chem. 2009;55:1218–22.

    Article  CAS  Google Scholar 

  27. Taylor RL, Grebe SK, Singh RJ. Quantitative, highly sensitive liquid chromatography-tandem mass spectrometry method for detection of synthetic corticosteroids. Clin Chem. 2004;50:2345–52.

    Article  CAS  Google Scholar 

  28. Gold H, Mirzaian M, Dekker N, Ferraz MJ, Lugtenburg J, Codée JDC, et al. Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry. Clin Chem. 2013;59:547–56.

    Article  CAS  Google Scholar 

  29. Müller MJ, Volmer DA. Mass spectrometric profiling of vitamin D metabolites beyond 25-hydroxyvitamin D. Clin Chem. 2015;61:1033–48.

    Article  Google Scholar 

  30. Ng TWK, Ooi EMM, Watts GF, Chan DC, Meikle PJ, Barrett PHR. Association of plasma ceramides and sphingomyelin with VLDL apoB-100 fractional catabolic rate before and after rosuvastatin treatment. J Clin Endocrinol Metab. 2015;100:2497–501.

    Article  CAS  Google Scholar 

  31. Xia JY, Holland WL, Kusminski CM, Sun K, Sharma AX, Pearson MJ, et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic steatosis. Cell Metab. 2015;22:266–78.

    Article  CAS  Google Scholar 

  32. Perman JC, Boström P, Lindbom M, Lidberg U, StÅhlman M, Hägg D, et al. The VLDL receptor promotes lipotoxicity and increases mortality in mice following an acute myocardial infarction. J Clin Invest. 2011;121:2625–40.

    Article  CAS  Google Scholar 

  33. Sampaio JL, Gerl MJ, Klose C, Ejsing CS, Beug H, Simons K, et al. Membrane lipidome of an epithelial cell line. Proc Natl Acad Sci U S A. 2011;108:1903–7.

    Article  CAS  Google Scholar 

  34. Han X, Rozen S, Boyle SH, Hellegers C, Cheng H, Burke JR, et al. Metabolomics in early Alzheimer’s disease: identification of altered plasma sphingolipidome using shotgun lipidomics. PLoS One. 2011;6:e21643.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Walt Shaw and Lisa Connell at Avanti Polar Lipids for their great support. We thank all members of our laboratory for valuable discussions and suggestions. The research leading to these results has received funding from the European Union’s Seventh Framework Programme FP7/2007-2013 under grant agreement no. 305739.

Author information

Authors and Affiliations

  1. Zora Biosciences, Biologinkuja 1, 02150, Espoo, Finland

    Dimple Kauhanen, Marko Sysi-Aho, Kaisa M. Koistinen, Reijo Laaksonen & Kim Ekroos

  2. Heart and Lung Center, Helsinki University Hospital, 00029 HUS, Helsinki, Finland

    Juha Sinisalo

Authors
  1. Dimple Kauhanen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marko Sysi-Aho
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kaisa M. Koistinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Reijo Laaksonen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Juha Sinisalo
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kim Ekroos
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kim Ekroos.

Ethics declarations

Conflict of interest

The authors declare that there is no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 12 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kauhanen, D., Sysi-Aho, M., Koistinen, K.M. et al. Development and validation of a high-throughput LC–MS/MS assay for routine measurement of molecular ceramides. Anal Bioanal Chem 408, 3475–3483 (2016). https://doi.org/10.1007/s00216-016-9425-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9425-z

Keywords

Search

Navigation