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. 2023 Jun 16;67(2):275-287.
doi: 10.2478/jvetres-2023-0030. eCollection 2023 Jun.

Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6β-hydroxy-stanozolol in animal urine

Iwona Matraszek-Źuchowska  1 , Alicja Kłopot  1 , Katarzyna Sielska  1 , Beata Korycińska  1 , Sebastian Witek  1 , Paulina Zdonek  1 , Piotr Jedziniak  1
Affiliations

Liquid chromatographytandem mass spectrometry methods for determination of stanozolol and l6β-hydroxy-stanozolol in animal urine

Iwona Matraszek-Źuchowska et al. J Vet Res. .

Abstract

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 16β-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 16β-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 CCβ 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.

Keywords: LC-MS/MS; hormone residue analysis; stanozolol; urine.

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Conflict of interest statement

Conflict of Interests Statement: The authors declare that there is no conflict of interests regarding the publication of this article.

Figures

Fig. 1.
Fig. 1.
Chemical structures, molecular formulas and weights of molecules of hormones tested
Fig. 2.
Fig. 2.
Liquid chromatography-tandem ion trap mass spectrometry selected reaction monitoring (SRM) chromatograms of bovine urine samples spiked with 16β-OH-stanozolol (16β-OH-STAN) and stanozolol (STAN) at 2 μg L−1
Fig. 3.
Fig. 3.
Liquid chromatography-tandem triple quadrupole mass spectrometry multiple reaction monitoring chromatograms of A - a pig blank urine sample; B - a pig urine sample spiked with stanozolol (STAN) and 16β-OH-stanozolol (16β-OH-STAN) at 0.5 μg L−1; C - a pig urine sample spiked with STAN and 16β-OH-STAN at 2 μg L−1
See this image and copyright information in PMC

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