HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY WITH THERMOSPRAY IONIZATION OF FREE ECDYSTEROIDS

High-performance liquid chromatography/mass spectrometry (HPLC/MS) wit h thermospray ionization has been applied to the analysis of free ecdy steroids (polyhydroxylated steroids that serve as arthropod moulting h ormones). Of a range of analytical conditions that were tested, optimu m results were obtained by the use of reversed-phase HPLC using a meth anol + water mixture containing 0. 1 m ammonium acetate. The positive- ion spectra of ecdysteroids showed pseudo-molecular ions, [M + H]+, an d fragment ions corresponding to the sequential losses of hydroxyl gro ups. The [M + H - H2O]+ and [M + H - 2H2O]+ ions were the most abundan t for all the compounds tested. Ecdysteroids bearing a C-20 hydroxyl g roup (e.g. 20-hydroxyecdysone) showed ions characteristic of cleavage of the side-chain from the steroid nucleus. This mode of fragmentation was much less obvious in the case of ecdysteroids lacking C-20 hydrox ylation (e.g. ecdysone). Full-scan mass spectra were readily obtained from 1 mug of free ecdysteroids, while a limit of detection of 5 ng (s ignal-to-noise ratio 3) was attained in the selected-ion monitoring mo de. The application of HPLC/MS to the study of ecdysteroids in biologi cal materials is demonstrated by the analysis of extracts of newly lai d eggs of the desert locust (Schistocerca gregaria) and pupae of the c otton leafworm (Spodoptera litoralis).