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

The chromatographic behaviour of molecular species of sphingomyelin on HPTL C was investigated. Sphingomyelin gave a double band pattern on HPTLC plate s 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 ac yl chain-defined sphingomyelins showed that the Ri values increased linearl y with the length of the N-linked acyl chain. A double-banded pattern was t herefore seen for natural sphingomyelins with a bimodal fatty acid composit ion. 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 is omer. We also showed that D-erythro-N-16:0-dihydrosphingomyelin migrated fa ster on HPTLC than D-erythro-N-16:0-sphingomyelin. The upper and lower band sphingomyelins from two different cell lines (human skin fibroblasts and b aby 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 l ower 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 rea sons for the sphingomyelin double bands on HPTLC; acyl chain length, long-c hain base composition and stereochemistry. These reasons might sometimes ov erlap and, therefore, HPTLC alone is insufficient for complete analysis of the molecular species of sphingomyelin.