A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their g rowing popularity is in part due to their operational simplicity and cost r eduction in solvents, and partly because they are easier to automate, Sphin golipids are implicated in various cellular events such as growth, differen tiation, and apoptosis, However, their separation by small SPE cartridges h as attracted limited attention. Here we describe an SPE procedure on aminop ropyl cartridges that by sequential elution allows the separation elf a lip id mixture into free ceramides, neutral glycosphingolipids, neutral phospho lipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides, Indivi dual components are obtained in high yield and purity. We applied the proce dure to obtain data on separation of [H-3]myristic acid-labeled sphingolipi ds from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-per formance thin-layer chromatography (HPTLC) systems. The chromatographic beh avior of free sphingoid bases is also reported.