Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

Emanuela Camera, Mauro Picardo, Carla Presutti, Paolo Catarcini, Salvatore Fanali

Research output: Contribution to journalArticlepeer-review

Abstract

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (AS/AIS, 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15′-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.

Original languageEnglish
Pages (from-to)971-976
Number of pages6
JournalJournal of Separation Science
Volume27
Issue number12
DOIs
Publication statusPublished - Aug 2004

Keywords

  • Ceramides
  • HPLC
  • Keratinocytes
  • Mass-spectrometry
  • Skin lipids

ASJC Scopus subject areas

  • Molecular Medicine
  • Clinical Biochemistry
  • Chemical Engineering(all)
  • Filtration and Separation

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