Detection of 20-hydroxyecdysone in calf urine by comparative liquid chromatography/high-resolution mass spectrometry and liquid chromatography/tandem mass spectrometry measurements: application to the control of the potential misuse of ecdysteroids in cattle.

Ecdysteroids, which are steroid hormones in invertebrates, but which are also present in plants, could potentially be used as anabolic agents in food-producing animals. The control of ecdysteroid misuse in cattle relies on the development of an efficient method for their detection in biological matrices at trace levels (microg L(-1)). In order to propose an analytical procedure dedicated to the identification of excreted 20-hydroxyecdysone (20E) in urine and faecal samples of breeding animals, a comparative study of the spectrometric behaviour of these compounds was carried out both by LC/(ESI-)/HRMS(n) (hybrid linear ion trap - orbital trap) and by LC/(ESI+)/MS/MS (triple quadrupole). This study revealed the formation of a large number of product ions both in positive and negative ion mode, corresponding to losses of water molecules and specific cleavages on the side chain. The sample preparation consisted of sequential purification on two solid-phase extraction cartridges (SPE octadecylsilyl and SPE silica). The detection limits were around 0.5 microg L(-1) in the selected reaction monitoring (SRM) mode and recoveries above 60% were obtained. The method was successfully applied to the analysis of real samples collected from calves treated with 60 mg 20E over 4 days. Analysis of the samples allowed the investigation of the kinetics of elimination of 20E in calf urine and determination of the time-frame for the control of potential abuse.