Separation and purification of sphingomyelin diastereomers by high-performance liquid chromatography

All naturally occurring sphingomyelins have the d-erythro-(2S,3R) configuration of the sphingoid base. We have developed a normal-phase HPLC method for the separation of this natural stereoisomer from the l-threo-sphingomyelin, which is the other stereoisomer commonly present in semisynthetic preparations of acyl-chain defined sphingomyelins. The chromatographic method was developed by modification of a previously reported method for phospholipid separation on a normal-phase diol column. The separation was accomplished by a binary gradient of solvent mixtures (A) hexane:isopropanol:acetic acid (82:17:1.0 by vol) and (B) isopropanol:water:acetic acid (85:14:1.0 by vol) with 0.08 vol% triethylamine added to both solvent mixtures. The program of gradient elution was optimized for maximal separation of sphingomyelin diastereomers. For detection of the lipids, a light-scattering detector was used. This analytical scale HPLC method was also used for purification of the stereoisomers (up to 0.5 mg of N-oleoyl-sphingomyelin in a single injection). The purified stereoisomers were at least 99% pure according to high-performance thin-layer chromatography and analytical HPLC.