HPLC method for the analysis of acetonides of ecdysteroids providing structural information on different vicinal diols
The polyhydroxy structure of ecdysteroids provides a basis for the formation of various derivatives including the acetonides that have been frequently prepared mainly in preparative work. Acetonides can be formed between vicinal diols in cis position such as positions C-2/C-3 and C-20/C-22 in 20-hydroxyecdysone, for example. We have modified an established procedure for the preparation of acetonides, employing a higher concentration of phosphomolybdic acid as a catalyst, and developed a reverse-phase HPLC method to analyze the products on a micro scale. The derivatization method is fast, taking minutes or less, and does not produce artifacts. Thirteen structurally diverse ecdysteroids were studied in detail concerning speed of acetonide formation, and the number and type of intermediate and end products. Mass spectrometry coupled to HPLC was employed to confirm and/or identify ecdysteroids and their acetonide derivatives. Analysis of the chromatographic profiles revealed that detailed information on vicinal diols of ecdysteroids can be derived from the differences in retention times (Deltar.t.)1 of products relative to the parent steroids on reverse-phase HPLC using a linear water/methanol gradient for elution. The specific Deltar.t. values characterize a product as a monoacetonide, a diacetonide, or a triacetonide. Furthermore, this chromatographic shift upon acetonide formation is significantly different among the various monoacetonides and, thus, specific for the site on the ecdysteroid molecule. The same seems to hold for diacetonides. As variation of the Deltar.t. values for a specific type of acetonide was rather small among the tested ecdysteroids, the results should provide reliable structural information. Muristerone A did not completely fit this common pattern. However, this may specify its uncommon ecdysteroid structure. The method has recently been successfully employed to solve the structures of new ecdysteroid metabolites. Arch. Copyright 1999 Wiley-Liss, Inc.
Novartis Crop Protection AG, Research Biochemistry, Basel, Switzerland.
H
Kayser
P
Eilinger