Analysis of natural and synthetic sphingomyelins using high-performance thin-layer chromatography

The chromatographic behaviour of molecular species of sphingomyelin on HPTLC was investigated. Sphingomyelin gave a double band pattern on HPTLC plates developed using chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) or chloroform/methanol/water (25 : 10 : 1.1, v/v). HPTLC analysis of acyl chain-defined sphingomyelins showed that the Ri values increased linearly with the length of the N-linked acyl chain. A double-banded pattern was therefore seen for natural sphingomyelins with a bimodal fatty acid composition. Racemic sphingomyelins also gave a double band pattern on HPTLC, where the lower band represented the D-erythro and the upper band the L-threo isomer. We also showed that D-erythro-N-16:0-dihydrosphingomyelin migrated faster on HPTLC than D-erythro-N-16:0-sphingomyelin. The upper and lower band sphingomyelins from two different cell lines (human skin fibroblasts and baby hamster kidney cells) were separately scraped off the HPTLC plates and the fatty acid and long-chain base profiles were studied using GC-MS. The lower bands contained short-chain fatty acids and most of the fatty acids in the upper bands were long. The predominant long-chain base was sphingosine, which was found in both upper and lower bands, but sphinganine was found only in the upper bands. To conclude, there are at least three possible reasons for the sphingomyelin double bands on HPTLC; acyl chain length, long-chain base composition and stereochemistry. These reasons might sometimes overlap and, therefore, HPTLC alone is insufficient for complete analysis of the molecular species of sphingomyelin.