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 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 (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 (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.
Camera
Emanuela
E
Istituto Dermatologico S. Gallicano (IRCCS), Via S. Gallicano 25/A, 00153 Roma, Italy.
Picardo
Mauro
M
Presutti
Carla
C
Catarcini
Paolo
P
Fanali
Salvatore
S
D002518:Ceramides / Q000737:chemistry*; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods; D000078404:Epidermal Cells; D006801:Humans; D007477:Ions; D015603:Keratinocytes / Q000378:metabolism; D008055:Lipids / Q000032:analysis / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D012867:Skin / Q000166:cytology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013110:Sphingosine / Q000737:chemistry; D013997:Time Factors
D002518:Ceramides; D007477:Ions; D008055:Lipids; D013110:Sphingosine