Vietzke J P, Brandt O, Abeck D, Rapp C, Strassner M, Schreiner V, Hintze U
Beiersdorf AG, 20245 Hamburg, Germany.
Lipids. 2001 Mar;36(3):299-304. doi: 10.1007/s11745-001-0721-9.
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.
角质层（SC）需要神经酰胺、胆固醇和脂肪酸来提供皮肤渗透屏障。SC脂质可通过正相高效薄层色谱法（HPTLC）进行分析。然而，在没有进一步分析的情况下，未知样品的每个TLC条带所代表的脂质分子组成仍存在一些不确定性。因此，我们通过将分离出的脂质进行反相高效液相色谱与电喷雾电离质谱直接联用（HPLC/ESI-MS，或LC/MS），对每个神经酰胺条带进行了进一步分析。LC/MS分析以及ESI-MS/MS负离子和碰撞诱导解离实验表明，神经酰胺条带4不仅含有N-（ω-羟基酰基）酰基-6-羟基鞘氨醇，即Cer（EOH），还含有N-（α-羟基酰基）-鞘氨醇。条带5仅含有N-酰基-6-羟基鞘氨醇。我们的结果证明了LC/MS分析在选择性鉴定人SC神经酰胺方面的优势。此外，将用于预分离的HPTLC和用于脂质鉴定的LC/MS相结合，是进行详细神经酰胺分析的更强大工具。
