Identification and confirmation of the presence of some steroid-like growth promoters in the urine of cattle and swine

English: Anabolic steroids and/or growth promoters are used to improve growth rate and feed conversion efficiency of livestock. The residues of these anabolics, which are present in the meat, may have a pharmacological activity due to oral bioavailability, and pose a risk to the consumer. Certain anabolics can be given legally to farm animals in some countries, but are banned in most others because of their proved or alleged toxic and/or carcinogenic properties. The use of these substances is completely forbidden within the European Community (EC). Before meat products can be exported to any member state of the EC, it is compulsory for the exporting country to have a monitoring programme to test for illegal use of these anabolic substances. The necessity to test for illegal use or to determine residue levels after legal use, has led to a strong interest in developing analytical methods for the identification and confirmation of anabolic agents in biological samples. The objectives of this study were to develop suitable analytical methods with a view to identify residues of some growth-promoting veterinary drugs in the urine of cattle and/or swine, to confirm the presence of these veterinary drugs unequivocally in the urine and to examine the stability of these drugs in urine under different environmental storing conditions. The excretion of clenbuterol, diethylstilbestrol, nandrolone, trenbolone and zeranol from cattle and/or swine were studied. A thorough literature study was done on the published analytical methods as well as the metabolism and pharmacokinetics of these drugs in cattle and/or swine. Reference standards were used to develop a GC-MS screening method for the identification of these drugs and/or their metabolites in the urine of cattle and/or swine. Recoveries of 61-99% and detection limits of 0.9-2.1 ng/ml were obtained for the different analytes with the developed analytical method. Trials were conducted in which these drugs were administered to cattle and/or swine. Urine samples were collected at regular time intervals and stored immediately at -20°C until time of analysis. Reference standards were also used for the development of GC-MS-MS analytical methods to confirm the presence of these drugs and/or their metabolites in the urine of cattle and/or swine. Confirmation of the substances in urine was done by obtaining a MS-MS spectrum of the extract and comparing this with the MS-MS spectrum of a reference standard. The MS-MS spectra was obtained by using the ion-trap technique. Urine samples are often collected at different farms and transported to the laboratory for residue analysis. Although it is standard procedure to freeze samples immediately after collection, it is not always possible. The results of this study show that the analytes contained in urine samples that were stored frozen remained stable for at least 10 days. If urine samples are stored at ambient temperature, concentrations of the analytes can decrease with as much as 30% after 10 days. Epi-nandrolone (metabolite ofnandrolone in cattle) could however not be detected in the urine samples after 4 days. If urine samples are stored in direct sunlight, concentrations of the analytes can decrease with as much as 90% after 10 days. It can be concluded that a method was developed to identify residues of some growthpromoting veterinary drugs and/or their metabolites in the urine of cattle and/or swine, and to confirm the presence of these drugs unequivocally in the urine. It can further be concluded that urine samples should be frozen as soon as possible after collection to prevent false negative results.