Microanalytical systems for separations of stratum corneum ceramides

Abstract

Original PaperMicroanalytical systems for separations of stratum corneum ceramidesThe small amount of lipids from human skin obtained with noninvasive sampling method led us to investigate microanalytical separation techniques. The lipid class analysis was performed with a micro polyvinyl alcohol-silica (PVA-Silm) column. The gradient elution was from heptane to acetone/butanol 90:10 v/v in 4%/min at 78 lL/ min. In addition an evaporative light scattering detector (ELSD) was modified for micro-LC. All solvents contained 0.1% of triethylamine and formic acid in stoichiometric amount, which increased the ELSD response. In these conditions, the cholesterol eluted before free fatty acid, and squalene and triglycerides close to the dead volume. The various ceramide classes eluted following the order of the increased number of hydroxyl groups. The LOD for ceramides was 2.2 ng. The advantages of this method are the use of a normal stationary phase more reliable due to its chemical stability, its surface homogeneity and its development in microchromatography without chlorinated solvents which offers small LOD and the whole profile of lipids present in stratum corneum (SC). A method using a narrow-bore PVA-Sil column was used to collect ceramide fraction. Then the molecular species were analysed with a porous graphitic carbon column in capillary LC using a gradient from CH 3 OH/CHCl 3 70:30 v/v to CHCl 3 at 2%/min with a flow rate at 5 lL/min. The LOD obtained for ceramide was 1 ng. Both methods were assessed with SC samples obtained by rinsing a 5.7 cm 2 area of the forearm with 25 mL of ethanol. IntroductionStratum corneum (SC) consists of corneocytes organized in a matrix of highly ordered multilamellar lipid sheets [1,2]. For the structural and physical properties of this lipid arrangement, SC is considered to play an essential role in the skin barrier function. Ceramides constitute 50% of this lipid barrier. Free fatty acid and cholesterol (Cho) constitute the remaining lipid classes composing this lipid barrier. The ceramide classes are highly heterogeneous in structure due to the sphingoid base and fatty acid moieties constituting these amide molecules. They differ by a different number of hydroxyl groups and carbon double bonds, and the chain length [3]. This structural heterogeneity is an important element to study the lipid barrier property. The nature of the structural difference between molecular species included in each lipid class is apolar. Therefore, apolar stationary phases are required for their discrimination such as octadecyl bonded silica [4][5][6] or porous graphitic carbon (PGC) [7]. This latter column provides original selectivity between molecular species. However, to apply such analytical methods to skin samples, the lipids should be previously extracted and the various lipid classes separated.Several noninvasive methods for extraction of lipids from SC exist, e.g. by stripping [8][9][10], scraping [11], skin rinsing [3,12] or with an extraction chamber [11,13]. Whatever the approach, the three...

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