Accumulation of the xenobiotic anabolic steroids ethinyloestradiol and methyltestosterone in calf hair

Abstract

An analytical method to detect the illegal application of anabolic steroids in cattle by hair analysis was developed. The time course of incorporation of the orally active xenobiotic steroids ethinyl‐oestradiol (EE2) and methyltestosterone (MT) into growing hair, the duration of their detection by hair analysis and the influence of hair pigmentation on steroid concentrations were investigated in detail. Female veal calves of different coat colour were fed with 3.5 μg of EE2 kg‐1 of body weight and 35 μg of MT kg‐1 of body weight, twice daily, for 10 days. Before, during and after treatment, hair samples were obtained and analyzed for EE2 and MT residues. An appropriate method was developed to extract EE2 and MT from hair. The extraction procedure consisted of liquid‐liquid and solid‐phase extraction and was followed by an essential high‐pressure liquid chromatography step for further purification of the extracts. The final quantification was carried out with specific enzyme immunoassays. EE2 and MT residues could be detected in hair from day 4 (approximately 1 ng of EE2 g‐1 of hair and 5 ng of MT g‐1) of the experiment up to day 38 (approximately 0.3 ng of EE2 g‐1) and day 94 (approximately 0.5 ng of MT g‐1) respectively. The plasma concentrations of MT during the treatment were about 10‐fold higher than the plasma concentrations of EE2. Steroid accumulation in hair seemed to depend on the plasma levels of the steroids applied because the accumulation of MT in hair was higher (approximately 7 ng g‐1) than the accumulation of EE2 (approximately 1 ng g‐1). In addition, the tracking of MT by hair analysis was possible for a much longer duration (up to day 94 of the experiment) than the detection of EE2 (day 38). There was no influence of pigmentation on the steroid concentrations in hair. These results suggest that hair analysis is a powerful means with which to detect and track the illegal use of anabolic steroids in animal production, horse‐racing and sports.