Liquid chromatography-tandem mass spectrometry methods for determination of stanozolol and 16β-hydroxy-stanozolol in animal urine

Introduction: Because of the activities and effects they induce, hormones are prohibited for use for anabolic purposes in farm animals intended for slaughter, which is regulated in the European Union by relevant legal provisions. Therefore, there is an obligation to monitor residues of hormones in animals and food of animal origin to ensure consumer safety. A hormone banned but used formerly for fattening cattle, stanozolol, and its metabolite 16 beta-OH-stanozolol are synthetic compounds that belong to a large group of steroid hormones. This study investigates residues of these compounds in animal urine. Material and Methods: From 2006-2022, 2,995 livestock urine samples were tested for stanozolol residues in Poland as part of the National Residue Monitoring Programme. A liquid chromatography-tandem mass spectrometry method to determine stanozolol and 16 beta-OH-stanozolol in animal urine was developed and validated according to the required criteria. Urine sample analysis was based on enzymatic hydrolysis of hormones potentially present in it to the free form, extraction of them from the sample with a mixture of n-hexane and butyl alcohol, purification of an extract on an NH2 amine column and finally, instrumental detection. Results: The apparent recovery and precision parameters of the developed method were in line with the established criteria, while its decision limits CC alpha and detection capabilities CC beta were lower than the recommended concentration for analytical purposes set at 2 mu g L-1 (valid until December 15, 2022; currently set as 0.5 mu g L-1). Conclusion: All examined samples were compliant with the evaluation criteria.