Comparative investigation of human stratum corneum ceramides

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids t o provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, w ithout further analysis, some uncertainty remains about the molecular compo sition of lipids represented by every TLC band of an unknown sample. We the refore analyzed each ceramide band further by subjecting the isolated lipid s to a direct coupling of reversed-phase high-performance liquid chromatogr aphy and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or LC/MS). LC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociati on experiments revealed that ceramide band 4 contained not only N-(omega -O H-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(alpha -OH-acyl)-sphingo sine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results dem onstrate the benefit of LC/MS analysis for selective identification of huma n SC ceramides. Moreover, the combination of HPTLC for pre-separation and L C/MS for identification of lipids is an even more powerful tool for detaile d ceramide analysis.