Small-scale analysis of phytoecdysteroids in seeds by HPLC-DAD-MS for the identification and quantification of specific analogues, dereplication and chemotaxonomy

Introduction Phytoecdysteroids are analogues of arthropod steroids occurring in plants. They contribute to invertebrate deterrence. A wide diversity of ecdysteroids occurs in phytoecdysteroid-containing plant species, sometimes in high amounts. Ecdysteroids demonstrate potentially useful pharmaceutical actions in mammals. Objectives Establish reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC-MS/MS) and RP-HPLC-DAD-MS (diode array detector mass spectrometry) methods for the separation, identification and quantification of ecdysteroids to screen for species containing significant amounts of 20-hydroxyecdysone (20E) and other useful ecdysteroids. Materials and methods Micro-extracts of seed samples (ca. 30 mg) in 50% ethanol were subjected to RP-SPE (solid-phase extraction) purification prior to analysis by RP-HPLC-MS/MS and RP-HPLC-DAD-MS. The method was initially applied to genera (Amaranthus,Centaurea,Lychnis,Ourisia,Serratula,SileneandTrollius) where high-accumulating species had been previously encountered. Seeds of 160 randomly selected species, many of which have not previously been assessed, were then analysed. HPLC-MS/MS with a short analysis time initially identifies ecdysteroid-positive extracts and quantifies 20E. The positive extracts (20 ng 20E) are then analysed by HPLC-MS/MS with a longer analysis time to identify and quantify 17 common phytoecdysteroids and, finally, HPLC-DAD-MS (0.1-0.25 mu g 20E) is used to obtain UV- and MS-spectra to confirm identifications or as a basis for characterisation of partially identified or novel analogues. Results Lychnis coronaria,Silene fimbriataandSilene hookeriecdysteroids are characterised for the first time and those ofCucubalus bacciferandIpheion uniflorumare more extensively characterised. Conclusions The procedure provides a rapid/sensitive method for screening small plant samples for the presence, quantification and identification of ecdysteroids. It permits ready dereplication of samples, identifying extracts containing large amounts or novel analogues.