A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans

Rapid Commun Mass Spectrom. 2012 May 15;26(9):1134-40. doi: 10.1002/rcm.6216.


Rationale: Sphingolipids are important components of cell membranes that serve as cell signaling molecules; ceramide plays a central role in sphingolipid metabolism. De novo ceramide biosynthesis depends on fatty acid availability, but whether muscle uses circulating free fatty acids or pre-existing intracellular stores is unknown. Our goal was to develop a method to detect the incorporation of intravenously infused [U-(13)C]palmitate into intramyocellular ceramides.

Methods: We used liquid chromatography/tandem mass spectrometry (LC/MS/MS) to measure the concentrations of different sphingolipid species and (13)C-isotopic enrichment of 16:0-ceramide. Chromatographic separation was performed using ultra-performance liquid chromatography. The analysis was performed on a triple quadrupole mass spectrometer using a positive ion electrospray ionization source with selected reaction monitoring (SRM).

Results: The sphingolipids ions, except enriched ceramide, were monitored as [M+2+H](+). The [(13)C(16)]16:0-ceramide was monitored as [M+16+H](+). By monitoring two different transitions of the [(13)C(16)]16:0-ceramide (554/536 and 554/264) we could indirectly measure enrichment of the palmitate that is not a part of the sphingoid base. Concentration and enrichment could be measured using 20 mg of muscle obtained from volunteers receiving a low dose [U-(13)C]palmitate infusion.

Conclusions: LC/MS/MS can be used to detect the incorporation of plasma palmitate into muscle ceramides in humans, in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes
  • Ceramides / analysis
  • Ceramides / chemistry
  • Ceramides / metabolism*
  • Chromatography, High Pressure Liquid / methods*
  • Humans
  • Linear Models
  • Muscle, Skeletal / chemistry
  • Muscle, Skeletal / metabolism*
  • Palmitates / administration & dosage
  • Palmitates / blood
  • Palmitates / chemistry
  • Palmitates / metabolism*
  • Tandem Mass Spectrometry / methods*


  • Carbon Isotopes
  • Ceramides
  • Palmitates