Separation and characterization 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 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-159-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.