Production of clenbuterol, diethylstilbestrol and trenbolone mass standards in lyophilised bovine urine Original Research Article

Clenbuterol (CLEN), diethylstilbestrol (DES) and trenbolone (TbOH) are xenobiotic growth promoters and have been illegally applied as repartitioning agents in meat-producing animals. In the EU, proper detection methods and control systems are established for many hormones and anabolic substances, and therefore there is a need to improve the accuracy and repeatability of quantification. In order to define a common basis for accurate measurement and quantification, standard materials were produced as lyophilised bovine urine: ‘negative’ drug free controls, and ‘positive’ standards (CLEN ∼3 μg kg−1; DES ∼2 μg kg−1, TbOH ∼3 μg kg−1). Two milligram of CLEN or DES were applied orally in milk replacer to the calves. Two gram of TbOH-acetate was given as an implant. ‘Positive’ urine was sampled frequently between 12 and 24 h after application. ‘Negative’ urine was sampled from untreated calves only fed with milk replacer. Urines were pooled separately and diluted with ‘negative’ urine to a suitable level of final concentrations. Exactly 5.0 g portions of each standard were weighed into 750 brown glass containers and lyophilised in one batch. Container filling, dry matter and residual water content were determined and optimized. A 3-year stability and homogeneity study at various storage temperatures (−20 °C, +4 °C and +20 °C and +37 °C) indicate that all standards are stable and homogeneous. Reconstituted urines were quantified via ELISA and findings after reconstitution remained constant in ‘positive’ (+) standards and ‘negative’ (−) controls: CLEN+ (2.75±0.62 μg kg−1; n=133), CLEN− (0.023 μg kg−1 equivalents; n=130), DES+ (1.75±0.52 μg kg−1; n=133), DES− (0.034 μg kg−1 equivalents; n=133), TbOH+ (2.78±0.47 μg kg−1; n=133), TbOH− (0.022 μg kg−1 equivalents; n=133). Additionally, concentrations and identities of the drugs were confirmed by two independent accredited laboratories via HPLC–ELISA, LC–MS and GC–MS.