Carbon isotope ratios of endogenous steroids in Belgian Blue and Holstein cattle: Method development, reference population studies and application to steroid misuse control

Rationale: The misuse of growth promoters in livestock and breeding animals is prohibited according to the laws of the European Union. Among these growth promoters, the detection of endogenous steroids like testosterone, estradiol or progesterone remains especially challenging as concentration-based urinary thresholds may not provide conclusive results due to large inter-individual variations. In addition to the detection of intact steroid esters in blood or hair, carbon isotope ratio (CIR) determination of urinary steroids has commonly been the method of choice. Methods: A comprehensive sample clean-up procedure was developed and validated, which enables for the first time simultaneous CIR measurements of testosterone metabolites (17 alpha-hydroxyandrost-4-en-3-one, 3 alpha-hydroxy-5 beta-androstan-17-one and 5 alpha-androstane-3 beta,17 alpha-diol), the estradiol metabolite 17 alpha-estradiol (ESTR) and the progesterone metabolite 5 beta-pregnane-3 alpha,20 alpha-diol (PD) from a single urine specimen. As endogenous reference compounds 3 beta-hydroxyandrost-5-en-17-one and 5-androstene-3 beta,17 alpha-diol (5EN) were chosen. The method was validated by means of linear mixing models and a reference population encompassing n = 53 Belgian Blue and Holstein cattle was investigated to enable the calculation of population-based Delta C-13 thresholds. Results: The combined measurement uncertainty determined for the Delta C-13-values of all steroids under investigation was found to be < 0.8 parts per thousand. Within the reference population studies, 5EN was demonstrated to be the most promising endogenous reference compound resulting in comparably low Delta-values and accompanying thresholds. For PD, a surprisingly high number of samples (n = 9) yielded significantly C-13-depleted values and ESTR was only detectable in n = 13 samples. Proof-of-concept was accomplished by investigating two post-administration samples. Conclusions: This first comprehensive investigation on the CIRs of endogenous urinary steroids demonstrated once more the potential of isotope ratios in aiding discrimination between endogenously produced and exogenously administered steroids. By means of the reference population-derived CIRs, it is possible to apply cattle-specific thresholds to differentiate between treated and non-treated animals