Analysis of sphingolipids from biological sources using on-line high performance liquid chromatography with detection by atmospheric pressure chemical ionization and electrospray ionization mass spectrometry

Sphingolipids (SPLs) are important structural components of membranes in some types of cells and are involved in numerous signaling processes. Sphingomyelin (SPM) and dihydrosphingomyelin (DHS) are the two major SPLs in membranes. Very little is known about the molecular species and role of DHS in biological systems. In this work, we employed high performance liquid chromatography with detection by atmospheric pressure chemical ionization and electrospray ionization (ESI) mass spectrometry to elucidate the SPL composition in biological extracts. No common dietary source of DHS is known to exist. A novel analytical method developed to analyze complex SPL mixtures was used to show that bovine milk contained substantial amounts of DHS. Also, the human lens is the only known system in which DHS is the most abundant SPL. The molecular species of DHS in cataractous lenses has never been reported. It was shown that there was a preference for monounsaturated species of DHS and SPM in all ages and in cataractous lenses. It was also discovered that SPLs were the primary PLs remaining in cataractous lenses. Finally, the formation of sodium adducts and dimers in the ESI source of the ion trap mass spectrometer prevented the accurate quantitative analysis of PLs. A new method was developed to eliminate these undesirable ions.