College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan, ROC.
Anal Biochem. 2010 Jun 1;401(1):107-13. doi: 10.1016/j.ab.2010.02.009. Epub 2010 Feb 12.
A sensitive and accurate normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) method for determining the standard ceramide [NS] (Cer[NS]) was developed and validated so as to improve the traditional thin-layer chromatography (TLC) technique and LC-electrospray ionization (ESI)-MS method to profile and quantify ceramides in nude mouse skin. Normal-phase LC-APCI-MS was optimized to separate the nine classes of ceramides presented in the stratum corneum (SC) of nude mouse skin. A normal-phase silica column eluted with the gradient system from heptane:acetone/butanol (90:10, v/v) of 75:25 to 100% acetone/butanol (90:10, v/v) (with each solvent containing 0.1% [v/v] triethylamine and 0.1% [v/v] formic acid) at a flow rate of 0.8 ml/min was found to be optimal for analyzing standard Cer[NS]. The analysis of Cer[NS] was validated and employed as the standard for constructing a calibration curve to quantitate all classes of ceramides. This method was applied to profile the classes and contents of ceramides in the SC of nude mouse skin and proved to be workable. It was concluded that this improved method can be used to directly detect and quantify all classes of ceramides in the SC of nude mouse skin and that it is more convenient and labor-saving than the traditional TLC method.
建立并验证了一种灵敏、准确的正相液相色谱-大气压化学电离质谱（LC-APCI-MS）方法，用于测定标准神经酰胺 [NS]（Cer[NS]），以改进传统的薄层色谱（TLC）技术和 LC-电喷雾电离（ESI）-MS 方法，用于对裸鼠皮肤中的神经酰胺进行谱分析和定量。对正相 LC-APCI-MS 进行了优化，以分离裸鼠皮肤角质层（SC）中存在的九类神经酰胺。正相硅胶柱以庚烷：丙酮/丁醇（90:10，v/v）75:25 至 100%丙酮/丁醇（90:10，v/v）（每种溶剂均含有 0.1%[v/v]三乙胺和 0.1%[v/v]甲酸）的梯度系统洗脱，流速为 0.8 ml/min，是分析标准 Cer[NS]的最佳条件。对 Cer[NS]的分析进行了验证，并用作构建校准曲线以定量所有类神经酰胺的标准。该方法应用于分析裸鼠皮肤 SC 中神经酰胺的种类和含量，并证明是可行的。结论是，该改进的方法可用于直接检测和定量裸鼠皮肤 SC 中的所有类神经酰胺，比传统的 TLC 方法更方便、省力。
