HPLC and TLC characterisation of ecdysteroid alkyl ethers

Semi-synthetic ecdysteroid alkyl ethers have increased potential over natural ecdysteroids as actuators of ligand-inducible gene-expression systems based on the ecdysteroid receptor for in vivo applications. However, a scalable synthesis of these compounds has yet to be developed. We report a set of reversed-phase (RP; C18 and C6) and normal-phase (NP; diol) HPLC systems which can be used to analyse and separate ecdysteroid ethers with single or multiple O-methyl substitutions at the 2α-, 3β-, 14α-, 22- and 25-positions. The elution order of methyl ether analogues of the prototypical ecdysteroid 20-hydroxyecdysone (20E) was 3-methyl < 2-methyl < 14-methyl < 25-methyl < 22-methyl with both C18- and C6-RP-HPLC, when eluted with methanol/water mixtures. Further, the elution order of 20E 22-O-alkyl ethers was methyl < ethyl < allyl < n-propyl < benzyl < n-butyl with both C18- and C6-RP-HPLC. Moreover, the ecdysteroid alkyl ethers can also be adequately resolved by NP-HPLC and silica HPTLC. On the latter, detection of ecdysteroid O-alkyl ethers with the p-anisaldehyde/sulphuric acid reagent distinguishes 22-O-alkyl ethers from non-22-O-alkyl ether analogues by the colour of the resulting spot.