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Journal of Lipid Research, Vol. 44, 2209-2216, November 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology

Methods

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

Bernd Lieser¹, Gerhard Liebisch¹, Wolfgang Drobnik and Gerd Schmitz²

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

² To whom correspondence should be addressed. e-mail: 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 (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted 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 were 8% and 13% for SPH and SPA, respectively.

The developed HPLC-tandem mass spectrometry 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.

Abbreviations: CER, ceramide; C17-SPH, 17-carbon chain sphingosine; CV, coefficient of variation; ESI, electrospray ionization; FB1, fumonisin B1; IS, internal standard; MS/MS, tandem mass spectrometry; OPA, ortho-phthaldialdehyde; SPA, sphinganine; SPH, sphingosine; SPP, sphingosine-1-phosphate

Supplementary key words high-performance liquid chromatography • sphingolipids • metabolism • high throughput

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