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. 2010 Jul;51(7):2001-11.
doi: 10.1194/jlr.D005322. Epub 2010 Mar 12.

A rapid and quantitative LC-MS/MS method to profile sphingolipids

Max Scherer  1 , Kerstin Leuthäuser-Jaschinski, Josef Ecker, Gerd Schmitz, Gerhard Liebisch
Affiliations

A rapid and quantitative LC-MS/MS method to profile sphingolipids

Max Scherer et al. J Lipid Res. 2010 Jul.

Abstract

Sphingolipids comprise a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signaling molecules. To understand the differential role of sphingolipids in a regulatory network, it is important to use specific and quantitative methods. We developed a novel LC-MS/MS method for the rapid, simultaneous quantification of sphingolipid metabolites, including sphingosine, sphinganine, phyto-sphingosine, di- and trimethyl-sphingosine, sphingosylphosphorylcholine, hexosylceramide, lactosylceramide, ceramide-1-phosphate, and dihydroceramide-1-phosphate. Appropriate internal standards (ISs) were added prior to lipid extraction. In contrast to most published methods based on reversed phase chromatography, we used hydrophilic interaction liquid chromatography and achieved good peak shapes, a short analysis time of 4.5 min, and, most importantly, coelution of analytes and their respective ISs. To avoid an overestimation of species concentrations, peak areas were corrected regarding isotopic overlap where necessary. Quantification was achieved by standard addition of naturally occurring sphingolipid species to the sample matrix. The method showed excellent precision, accuracy, detection limits, and robustness. As an example, sphingolipid species were quantified in fibroblasts treated with myriocin or sphingosine-kinase inhibitor. In summary, this method represents a valuable tool to evaluate the role of sphingolipids in the regulation of cell functions.

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Figures

Fig. 1.
Fig. 1.
Product ion spectrum and proposed fragmentation of DimetSPH (A), TrimetSPH (B), dhCer1P (C), and PhytoSPH (D) in positive ion mode.
Fig. 2.
Fig. 2.
Chromatogram of a sphingolipid standard mixture. Displayed are MS/MS transitions representative of distinct sphingolipid classes and their respective ISs.
Fig. 3.
Fig. 3.
Chromatogram of a fibroblast sample. Displayed is a representative mass chromatogram obtained from a human skin fibroblast lipid extract.
Fig. 4.
Fig. 4.
The effect of myriocin and SKI on intracellular sphingolipids in primary human skin fibroblasts. Cells were treated with increasing concentrations of myriocin (A + B) and SKI (C + D) for 24 h, respectively. SPH (closed circle), SPA (open circle), SPC (closed triangle), S1P (open triangle), HexCer (open triangle), and LacCer (closed square) were quantified by LC-MS/MS; Cer (closed triangle), SM (closed circle), and dhSM (open circle) were quantified by flow injection analysis (ESI-MS/MS). Values represent the mean ± SD of three independent samples.
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