LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Thomas Hankemeier,Joke A. Bouwstra,Rob van der Heijden,Louise Hoppel,Jeroen van Smeden,Rob J. Vreeken

doi:10.1194/jlr.m014456

Thomas Hankemeier, Joke A. Bouwstra + Show 4 more

Open Access

https://doi.org/10.1194/jlr.m014456

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Abstract

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.

Highlights

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function
ESI is mainly used for the analysis of CERs, the atmospheric pressure chemical ionization (APCI) mode is used frequently
The limit of detection (LOD) and limit of quantification (LOQ) were defined by the signalto-noise ratio (S/N), being 3 and 10, respectively

Summary

Introduction

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. Important information about CER composition in human SC has been obtained using high performance thin layer chromatography (HPTLC) in conjunction with NMR This resulted in a gradual increase in the number of identified subclasses. The application of LC/MS results in a much more detailed profile of each individual subclass using only small quantities of material [19,20,21,22,23,24,25,26,27,28] Besides their important role in human skin, CERs are key molecules with respect to cell signaling, growth, differentiation, and apoptosis [29,30,31,32,33,34,35,36,37,38].

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