Normal-phase liquid chromatographic separation of stratum corneum ceramides with detection by evaporative light scattering and atmospheric pressure chemical ionization mass spectrometry Original Research Article

A new normal-phase HPLC method for the separation of stratum corneum ceramides is presented. Separation was achieved in less than 15 min using a binary gradient consisting of chloroform, n-propanol and acetic acid. Since UV and fluorescence detection are not possible without derivatization and electrospray MS is not compatible to normal-phase LC, we have investigated evaporative light scattering detection (ELSD) and atmospheric pressure chemical ionization mass spectrometry (APCI-MS). When comparing both detection methods, it can be stated that APCI-MS was more sensitive and provides more structural information. However, ELSD can be an economic alternative for routine analysis. The method was applied to human stratum corneum ceramides obtained by lipid extraction. It was shown that the peaks found in LC correspond to the bands described for TLC. The conversion of TLC procedures to HPLC enables an online coupling to mass spectrometry, which is particularly useful in the investigation of changed lipid patterns caused by skin diseases such as psoriasis and atopic dermatitis.