Analytical and Bioanalytical Chemistry

, Volume 408, Issue 13, pp 3475–3483 | Cite as

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

  • Dimple Kauhanen
  • Marko Sysi-Aho
  • Kaisa M. Koistinen
  • Reijo Laaksonen
  • Juha Sinisalo
  • Kim Ekroos
Research Paper


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.

Graphical Abstract

Workflow of molecular ceramide analysis


Mass spectrometry Chromatography Ceramides Cardiovascular Clinical High throughput 



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.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

216_2016_9425_MOESM1_ESM.pdf (13 kb)
ESM 1 (PDF 12 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Dimple Kauhanen
    • 1
  • Marko Sysi-Aho
    • 1
  • Kaisa M. Koistinen
    • 1
  • Reijo Laaksonen
    • 1
  • Juha Sinisalo
    • 2
  • Kim Ekroos
    • 1
  1. 1.Zora BiosciencesEspooFinland
  2. 2.Heart and Lung CenterHelsinki University HospitalHelsinkiFinland

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