Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry
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 RP 8 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 (A S /A IS , 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 (Cer 16 ), N-stearoyl-D-erythro-sphingosine (Cer 18 ), N-tetracosanoyl-D-erythro-sphingosine (N24 : 0, lignoceric ceramide, Cer 24:0 ), and N-tetracos-15 9-enoyl-D-erythro-sphingosine (N24 : 1, nervonic ceramide, Cer 24 : 1 ), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.