Comparative investigation of human stratum corneum ceramides

Abstract

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, without further analysis, some uncertainty remains about the molecular composition of lipids represented by every TLC band of an unknown sample. We therefore analyzed each ceramide band further by subjecting the isolated lipids to a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or LC/MS). LC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociation experiments revealed that ceramide band 4 contained not only N-(omega-OH-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(alpha-OH-acyl)-sphingosine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results demonstrate the benefit of LC/MS analysis for selective identification of human SC ceramides. Moreover, the combination of HPTLC for pre-separation and LC/MS for identification of lipids is an even more powerful tool for detailed ceramide analysis.

Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization

“…This ion is of low-abundance in the product-ion spectrum obtained by highenergy CAD and has not been reported [7,8]. The ion was also observed in the tandem mass spectrum obtained by ion-trap mass spectrometry [3][4][5] and has been mistakenly assigned as an amide radical anion by Vietzke et al [5] Ϫ ion of the N-oleoylaminoethanol at m/z 324 is a labile ion, which dissociates to a prominent N-oleoylaminoethylen-1-ol ion at m/z 322 (a 2 ) via a H 2 loss, when subjected to a collision energy of 20 eV (data not shown), which is 15 eV lower than that optimized for ceramides. The loss of H 2 mainly involves the two non-exchangeable hydrogens bonded to the ethanol backbone, as evidenced by the product-ion spectrum of the H-D exchanged analogs of the m/z 325 ion (Figure 6c), which contains a prominent ion of m/z 323, corresponding to a H 2 loss.…”

“…In the negative ion mode, ceramide forms the [M ϩ Cl] Ϫ ion with Cl Ϫ . Ceramide also yields [M Ϫ H] Ϫ ions with less sensitivity, when subjected to ESI [3][4][5].…”

“…The structural information obtained from the product-ion spectra was thought to be complimentary to that arising from the [M ϩ Li] ϩ or [M ϩ H] ϩ ions in positive-ion mode [6 -8]. Recently, Raith and Neubert [3,4] and Vietzke et al [5] reported the application of quadrupole ion-trap tandem mass spectrometric method with ESI to characterize ceramide as the [M Ϫ H] Ϫ ions. However, the studies provide very limited structural information and the evidence of the proposed fragmentation pathways is inadequate.…”

Ceramides and Sphingomyelins with High Proportions of Very Long-chain Polyunsaturated Fatty acids in Mammalian Germ Cells

“…Ceramides were resolved using chloroform/methanol/acetic acid, 95:4.5:0.5, by vol.) (8) or chloroform/methanol/ammonia/ water (90:10:05:0.5, by vol.) as solvents.…”

“…Thus, the steady-state levels of Cer in cells may be modified by a set of enzymes in different locations that form, remove, or modify the lipid. This complexity, and the fact that, with only a few exceptions like the skin (8), Cer is naturally a minor lipid class in most animal tissues and cells, may explain why general information concerning the fatty acids of this lipid is in general scarce. Our interest in the fatty acids of the ceramides of testis and spermatozoa began with the expectation that they should differ from those of other tissues.…”