Identification of lipids of the stratum corneum by high performance thin layer chromatography and mass spectrometry

The stratum corneum, the outermost layer of the epidermis, is the most important skin barrier against exogenous physical and chemical effects, in addition to protecting against dehydration. Ceramides are integral parts of the intercellular lipid lamellae of the stratum corneum and play an important role in the barrier function of mammalian skin. Ceramides are sphingolipids consisting of sphingoid bases linked to fatty acids by an amide bond. Typical sphingoid bases in the skin are composed of dihydrosphingosine, sphingosine, phytosphingosine, and 6-hydroxysphingosine, and the fatty acid acyl chains are composed of non-hydroxy fatty acid, α-hydroxy fatty acid, ω-hydroxy fatty acid, and esterified ω-hydroxy fatty acid. Analytical methods, such as gas chromatography/mass spectrometry, high performance thin layer chromatography with UV detection, and liquid chromatography/mass spectrometry, have been developed for the identification and quantification of ceramides in the stratum corneum. However, only a few publications relate to the mass fragmentation patterns specific to ceramide types to determine the structure of skin ceramides. Moreover, these studies provide very limited structural information and only for some ceramides. Therefore, the aim of our study was to develop a quick and easy method of quantification of ceramides, cholesterol, and free fatty acids by high performance thin layer chromatography with ultraviolet detection. High performance thin layer chromatography with ultraviolet detection was also coupled with mass spectrometry using negative ionization by electrospray and tandem mass spectrometry (MS/MS) for identification of ceramides’ structure