Development of clenbuterol reference materials: Lyophilized bovine eye samples free of clenbuterol (CRM 673) and containing clenbuterol (CRM 674). Part 1. Preparation, homogeneity and stability

Within the EU Standards, Measurement and Testing Program (SMT) two clenbuterol reference materials (RMs) were developed. Since clenbuterol readily accumulates and is slowly depleted from pigmented tissues such as the retina, homogenized eye liquid content is the most sensitive tissue for the detection of clenbuterol misuse. Therefore, both of the RMs were produced from bovine eye matrix: a negative control - RM 673 eye reference material, clenbuterol free (<0.50 μg/kg eye matrix) and a positive - RM 674 eye reference material containing clenbuterol (∼10 μg/kg eye matrix). Eyes were sampled from 103 German Simmental cattle and the inner liquid content was homogenized to a wet homogenized liquid content (HLC). This clenbuterol negative pool was divided into two sub-pools, one of which was spiked with clenbuterol to a final concentration of 10 μg clenbuterol/kg HLC. Of each pool exactly 2.0±0.01 g (±0.5%) portions were weighed into 790 containers. Lyophilization of the 1580 containers was performed in one batch. Parameters for the filling of containers, dry matter content, and residual moisture were in accordance with EU requirements. A three-year stability study and two homogeneity studies at various storage temperatures (-60°C, -20°C, +4°C, +20°C, and +37°C) were performed. Low variation was observed within all of the homogeneity studies, proving that each of the RMs were homogeneous and that this was independent of storage temperature and storage time. In the stability studies, measured clenbuterol concentrations remained constant for RM 673 under the detection limit at 0.15±0.01 μg clenbuterol equivalent/kg HLC (n=110) and were also constant for RM 674 at 11.21±0.15 μg clenbuterol/kg HLC (n=150; measured as duplicates). These studies demonstrate that clenbuterol-containing and clenbuterol-free RMs in bovine eye matrix can be successfully produced. Based on the results described above, it is concluded that both RMs may be suitable as candidates for certification.