Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC-electrospray ionization tandem mass spectrometry

Bernd Lieser, Gerhard Liebisch, Wolfgang Drobnik, and Gerd Schmitz

Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg 93042

Corresponding Author: gerd.schmitz{at}klinik.uni-regensburg.de

Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation and apoptosis. The currently available methods for the quantification of SPH are in part complicated, time consuming, insensitive or unselective. Therefore a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (ESI-MS/MS). Quantitation is achieved by use of a constant concentration of a non-naturally occurring internal standard C17-SPH, together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA co-eluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation (CV) were 8 % and 13 % for SPH and SPA, respectively. The developed LC-MS/MS methodology with an analysis time of 3.5 min, simple sample preparation and automated data analysis allows high throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.

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