Separation and Purification of Sphingomyelin Diastereomers by High-Performance Liquid Chromatography

✍ Scribed by Bodil Ramstedt; J.Peter Slotte

• Publisher: Elsevier Science
• Year: 2000
• Tongue: English
• Weight: 73 KB
• Volume: 282
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2000.4612

✦ Synopsis

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 Noleoyl-sphingomyelin in a single injection). The purified stereoisomers were at least 99% pure according to highperformance thin-layer chromatography and analytical HPLC.