A new approach was developed in order to control testosterone abuse in animal production. A gas chromatographic-combustion-isotope ratio mass spectrometric (GC-C-IRMS) method was used to distinguish the exogenous character from the endogenous character of the main metabolites of testosterone (epitestosterone and etiocholanolone) in cattle urine. This method is based on a comparison between the carbon isotope ratio (13C/12C) of testosterone metabolites and those of testosterone endogenous precursors. After urinary steroid purification, extracts were acetylated with acetic anhydride and injected into the GC-C-IRMS system. In order to validate the method, testosterone enanthate was administered to a 4 year old cow. The 13C/12C isotope ratios of testosterone exogenous metabolites appeared to be significantly different to the 13C/12C precursor ratios and were detected until 3 weeks after the anabolic administration. These preliminary results appear to be promising for the difficult control of natural hormones in livestock.

The optimum conditions for hydrolysing conjugated metabolites of steroid hormones in bovine urine were performed with Helix pomatia juice, beta-glucuronidase from bovine liver and preparations of limpets and abalone entrails using response surface methodology. The experimental design and empirical modelling used allowed us to assess the main effects of factors (time, temperature, pH and enzyme quantity) and to predict the optimum conditions for each enzyme preparation. Confirmatory experiments were applied to check the predicted values and to validate the model. The comparison of the enzyme preparation efficiency for various conjugate steroids and the study of possible by-product synthesis led us to select abalone entrails to hydrolyse natural dehydroepiandrosterone, etiocholanolone, epitestosterone; 17 alpha-estradiol and estrone in bovine urine. The optimum conditions were found to be 20 h at 42 degrees C with the pH adjusted to 5.2 and using 12,000 units of enzyme preparation.

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The detection of exogenous testosterone in bovine urine was investigated by using gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). The carbon isotopic ratio measurement of epitestosterone, etiocholanolone (testosterone metabolite) and DHEA (testosterone precursor) in female bovine urines after testosterone enanthate administration was carried out. An important modification in the 13C/12C ratio of testosterone metabolites was observed, such that significant differences between precursor and metabolites of testosterone occurred until three weeks after intramuscular administration of testosterone enanthate. The factors influencing the 13C/12C of endogenous steroids were studied especially through cattle feeding and age. The DHEA mean delta13C value was found to vary between -25 and -26/1000 when hay and concentrate diet were used for fattening. On the other hand the delta13C value observed when maize silage was used increased to -20/1000. Testosterone metabolites showed the same delta13C increase as their precursor. Moreover, we observed a clear relationship between age and efficiency of misuse determination. Indeed, because of the lower concentration of natural hormones in young animals, the contribution of exogenous molecules increases significantly compared with older subjects. Consequently, demonstration of administration is easier to achieve in calves than in mature animals.

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