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

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


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 


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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Molecular Cancer Research CentreCharité University Medical SchoolBerlinGermany

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