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Normal phase liquid chromatography coupled to quadrupole time of flight atmospheric pressure chemical ionization mass spectrometry for separation, detection and mass spectrometric profiling of neutral sphingolipids and cholesterol.

Abstract
Many lipidomic approaches focus on investigating aspects of sphingolipid metabolism. Special emphasis is put on neutral sphingolipids and cholesterol and their interaction. Such an interest is attributed to the fact that those lipids are altered in a series of serious disorders including various sphingolipidoses. High performance thin-layer chromatography (HPTLC) has become a widely used technique for lipid analysis. However, mass spectrometric profiling is irreplaceable for gaining an overview about the various molecular species within a lipid class. In this work we have developed a sensitive method based on a gradient normal phase high performance liquid chromatography (HPLC) coupled to quadrupole time of flight (QTOF) atmospheric pressure chemical ionization mass spectrometry (APCI-MS) in positive mode, which for the first time enables separation, on-line detection, and mass spectrometric profiling of multiple neutral sphingolipids including ceramide, glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, sphingomyelin as well as cholesterol within less than 15min. An important advantage of the presented HPLC/APCI-MS approach is that the separation pattern emulates the one obtained by an optimized HPTLC method with a multiple stage development. Thus, the lipid classes previously separated and quantified by HPTLC can be easily screened regarding their mass spectrometric profiles by HPLC/APCI-MS. In addition, the selected ionization conditions enable in-source fragmentation providing useful structural information. The methods (HPLC/APCI-MS and the optimized HPTLC) were applied for the analysis of the mentioned lipids in human fibroblasts. This approach is aimed basically at investigators who perform studies based on genetic modifications or treatment with pharmacological agents leading to changes in the biochemical pathways of neutral sphingolipids and cholesterol. In addition, it can be of interest for research on disorders related to impairments of sphingolipid metabolism.
AuthorsHany Farwanah, Jennifer Wirtz, Thomas Kolter, Klaus Raith, Reinhard H H Neubert, Konrad Sandhoff
JournalJournal of chromatography. B, Analytical technologies in the biomedical and life sciences (J Chromatogr B Analyt Technol Biomed Life Sci) Vol. 877 Issue 27 Pg. 2976-82 (Oct 01 2009) ISSN: 1873-376X [Electronic] Netherlands
PMID19646933 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Neutral Glycosphingolipids
  • Cholesterol
Topics
  • Cells, Cultured
  • Cholesterol (analysis, isolation & purification)
  • Chromatography, High Pressure Liquid (methods)
  • Chromatography, Thin Layer (methods)
  • Fibroblasts (metabolism)
  • Humans
  • Mass Spectrometry (methods)
  • Neutral Glycosphingolipids (analysis, isolation & purification)
  • Reproducibility of Results
  • Sensitivity and Specificity

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