Thin-layer chromatography of ecdysone acyl esters and their 2,3-acetonide derivatives

Abstract

Thin-layer chromatography (TLC) has been investigated as a means of separating ecdysone 22-acyl esters from each other and from other classes of ecdysteroid. Various types of high-performance TLC (HPTLC) plate have been evaluated with a range of solvent systems of differing polarity. Adsorption HPTLC on silica gives minimal separation of the various ecdysone 22-long-chain fatty acyl esters, either after a single development or after multiple development. Silica HPTLC does, however, clearly resolve the ecdysone 22-long-chain fatty acyl esters from their 2,3-acetonide derivatives and from ecdysone 2-stearate, ecdysone 2,22-distearate, ecdysone 2,3,22-tristearate or from the various ecdysone acetates. Reversed-phase HPTLC of the ecdysone 22-acyl esters and of their 2,3-acetonide derivatives has been extensively investigated on C 8-plates and the results compared with those obtained on C 2−, C 18− and CN-bonded phases. All types of reversed-phase HPTLC plate give good separation of the saturated long-chain fatty acyl esters of ecdysone or their 2,3-acetonide derivatives when a mobile phase of appropriate polarity is used, mobility decreasing with increasing fatty acid carbon chain length and increasing hydrocarbon chain length of the bonded phase. However, the different hydrocarbon chain lengths of the three bonded phases show different selectivities for the unsaturated long-chain fatty acyl esters, relative to the saturated acyl esters. In all systems increased unsaturation results in greater mobility but the magnitude of the increase in mobility is dependent on the hydrocarbon chain length of the bonded phase, being greatest with C 18-plates and least with CN-plates. HPTLC plates with a CN-bonded phase also provide suitable separations in the normal-phase mode, giving slightly better resolution of the various ecdysone 22-long-chain fatty acyl esters than is achieved on silica. The applicability of HPTLC coupled with radioscanning as a means of rapidly detecting the presence of and partially identifying apolar ecdysteroid conjugates in biological studies involving the metabolism of [ 3H]ecdysone is demonstrated and discussed.