Detection and identification of 20-hydroxyecdysone metabolites in calf urine by liquid chromatography-high resolution or tandem mass spectrometry measurements and establishment of their kinetics of elimination after 20-hydroxyecdysone administration

Ecdysteroids, which are steroid hormones in invertebrates, but are also present in plants, could be potentially used as anabolic agents in food-producing animals because of their growth-promoting properties. In this context, the metabolism of 20-hydroxyecdysone (20E) has been investigated in cattle in order to efficiently control its potential misuse. The analytical procedure involves purification on two solid-phase extraction cartridges (SPE octadecylsilyl and SPE silica) prior to detection based on liquid chromatography coupled to high resolution mass spectrometry in negative electrospray ionization mode (LC-(ESI−)-HRMS). Each new signal appearing on full-scan HRMS (30,000) during the analysis was investigated by tandem mass spectrometry (MS/MS). Comparison of the mass spectra pattern between 20E and potential metabolites has given informations on the chemical structures of the metabolites. This targeted approach, combining HRMS and MSn measurements on a linear trap in tandem with an orbital trap, allowed us to elucidate the structure of several 20E metabolites in calf urine: 14-deoxy-20-hydroxyecdysone, 20,26-dihydroxyecdysone and 14-deoxy-20,26-dihydroxyecdysone, the last of which had never previously been reported in bovine. The kinetics of elimination of these metabolites were investigated, and two of them were monitored by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) measurements over 6 days after 20E administration to calves, thus increasing the time-window for detection after 20E administration and thereby allowing for more efficient control of its misuse.