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.

Fast analysis of 19 anabolic steroids in bovine tissues by high performance liquid chromatography with tandem mass spectrometry.

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients   > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.

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.