Quantitation of ceramides in nude mouse skin by normal-phase liquid chromatography and atmospheric pressure chemical ionization mass spectrometry

Abstract

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.