Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6ß-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 16ß-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 16ß-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 CCα and detection capabilities CCß were lower than the recommended concentration for analytical purposes set at 2 µg L-1 (valid until December 15, 2022; currently set as 0.5 µg L-1). Conclusion: All examined samples were compliant with the evaluation criteria.

Determination of selected testosterone esters in blood serum of slaughter animals by liquid chromatography with tandem mass spectrometry.

Anabolic hormones, which cause muscle growth, have been banned for anabolic purposes in animal husbandry in Europe since the 1980s. Control of hormones from the list of Annex I to Directive 96/23/EC is mandatory in the European Union. The presence of hormones in samples of animal origin may be due to their endogeneous nature or illegal use. One way to distinguish their origin is to study hormones, particularly steroids in the form of ester derivatives. In the body synthetic hormone esters could be only exogenous therefore their detection in animal tissues is the undisputed evidence of illegal administration. The analytical procedure involves the extraction of esters from serum with organic solvents, derivatisation with methoxyamine and detection by liquid chromatography tandem mass spectrometry. The method was approved in accordance with the applicable legislative criteria and its effectiveness was verified in the proficiency test. The research material consisted of bovine serum samples officially taken. During the validation good apparent recovery, precision, decision limits and detection capabilities in the range 0.006-0.012 µg L-1 and 0.010-0.020 µg L-1 respectively were obtained. The developed method met the criteria for confirmation set out in Commission Decision 2002/657/EC. Since the inclusion of serum in 2018 for testing for testosterone esters in the National Residue Control Program, 130 samples have been examined. In none of the serum samples, esters above the decision limits were found. The control of animals and food of animal origin for hormone esters will be continued to ensure the health and safety of consumers.

Determination of Steroid Esters in Hair of Slaughter Animals by Liquid Chromatography with Tandem Mass Spectrometry.

INTRODUCTION: The use of growth promoters in animal husbandry to increase weight gain and efficiency of feed conversion into muscle has been banned in the European Union since 1988, and under Directive 96/23/EC, surveillance for anabolic steroid hormones is obligatory. The hormones present in animal tissues may be of endogenous origin or may result from illegal administration. Steps have been taken to determine selected steroids in the form of esters in the alternative matrix of animal hair. Their detection in biological material is direct proof of the illegal use of anabolics. MATERIAL AND METHODS: The procedure for the determination of steroid esters in animal hair, based on digestion, extraction, purification, and liquid chromatography with tandem mass spectrometry was validated under the current regulations. In total, 348 samples of animal hair were examined using this method. RESULTS: Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCß) were in the ranges of 2.57-4.18 µg kg-1 and 4.38-7.12 µg kg-1, respectively. The method met the criteria for confirmation techniques with respect to Commission Decision 2002/657/EC. CONCLUSION: Testing for steroid esters in animal hair was introduced into the National Residue Control Programme in 2017. Steroid esters were not found in any hair samples above the CCα, which indicates that illegal use of anabolics was not confirmed.

Control of Residues of Thyreostats in Slaughter Animals in Poland in 2011-2017.

INTRODUCTION: In the European Union, the use of thyreostatic drugs for fattening slaughter animals has been banned since 1981 under Council Directive 81/602/EEC. For protection of consumer health against unwanted residues and in compliance with Directive 96/23, each EU country must monitor thyreostats in samples of animal origin. This paper presents the results of research on thyreostatic residues carried out in Poland in 2011-2017. MATERIAL AND METHODS: The material for testing was urine (n = 3,491), drinking water (n = 127), and muscle samples (n = 349) officially collected by Veterinary Sanitary Inspectors in slaughterhouses and farms throughout the country in accordance with the national residue control plan. The samples were examined for the presence of tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil using liquid chromatography tandem mass spectrometry through an accredited method. RESULTS: In four bovine and three porcine urine samples, the permissible thiouracil concentration was exceeded. In one sample of porcine urine, methyl- and propylthiouracil were found. The presence of thiouracil and its derivatives in urine samples is most likely due to feeding animals diet containing cruciferous plants. CONCLUSIONS: The results of research indicate that thyreostats are not used for anabolic purposes in slaughter animals in Poland.

Liquid chromatography tandem mass spectrometry with ion trap and triple quadrupole analyzers for determination of thyreostatic drugs in urine and muscle tissue.

Liquid chromatography tandem mass spectrometry methods were developed and validated to screen for and confirm residues of the thyreostatic drugs: tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil in bovine and porcine urine and muscle tissues using dimethylthiouracil as internal standard. Thyreostats were extracted from urine samples with diethyl ether after derivatisation with 3-iodobenzylbromide in basic medium (pH 8.0) and analyzed by gradient elution on a Nucleosil C18 column with ion trap mass spectrometry detection using an electrospray source and triple quadrupole MS detection with turbo spray source. Thyreostats were extracted from muscle tissue with methanol, the denaturation of matrix protein was performed and then the same steps as for the urine samples were carried out. The methods were validated in accordance with the Commission Decision 2002/657/EC. Good thyreostats recoveries were obtained (from 82% to 117%) as well as acceptable within-lab reproducibility. The values of the decision limit CCα and the detection capability CCß of five thyreostatic drugs are found to be below the recommended concentration set at 10 µg L(-1) (kg(-1)). The results of the validation demonstrate that liquid chromatography mass spectrometry with ion trap detection does not meet the criteria for confirmation for some thyreostats and therefore was applied for screening purpose only.