Metabolites of 4-chlorotestosterone acetate in cattle urine as diagnostic markers for its illegal use

Seven metabolites of 4-chlorotestosterone acetate were identified in urine of cattle that received a single injection of the drug. The steroids were isolated by means of a series of clean-up steps carried out before and after enzymatic hydrolysis. The obtained extract was fractionated by high-performance liquid chromatography and each fraction was examined both by high-performance thin-layer chromatography and by capillary gas chromatography-mass spectrometry of the m-ethoxime-trimethylsilyl derivatives. The metabolites were tentatively identified by studying the mass spectra of selected peaks not found in blank samples. The structures of two metabolites, viz. 4-chloroandrost-4-ene-3,17-dione and 4-chloroandrost-4-ene-3 alpha,17 beta-diol were confirmed by chemical synthesis. The synthesized metabolites and 4-chloro-17 alpha-testosterone, a third metabolite which was identified tentatively, were located on the thin-layer chromatograms obtained. This study led to the conclusion that the illegal use of 4-chlorotestosterone acetate can be detected by identifying one or more of its metabolites in urine.