*Article* **Profiling of Metabolomic Changes in Plasma and Urine of Pigs Caused by Illegal Administration of Testosterone Esters**

#### **Kamil Stastny 1,\*, Kristina Putecova 1, Lenka Leva 1, Milan Franek 1, Petr Dvorak 2 and Martin Faldyna 1**


Received: 3 June 2020; Accepted: 6 July 2020; Published: 27 July 2020

**Abstract:** The use of anabolic steroid hormones as growth promoters in feed for farm animals has been banned in the European Union since 1988 on the basis of Council Directive 96/22/EC. However, there is still ongoing monitoring and reporting of positive findings of these banned substances in EU countries. The aim of this work was to investigate the e fficacy and discriminatory ability of metabolic fingerprinting after the administration of 17β-testosterone esters to pigs. Plasma and urine samples were chromatographically separated on a Hypersil Gold C18 column. High resolution mass spectrometry metabolomic fingerprints were analysed on a hybrid mass spectrometer Q-Exactive. Three independent multivariate statistical methods, namely principal component analysis, clustre analysis, and orthogonal partial least squares discriminant analysis showed significant di fferences between the treated and control groups of pigs even 14 days after the administration of the hormonal drug. Plasma samples were also analysed by a conventional quantitative analysis using liquid chromatography with tandem mass spectrometry and a pharmacokinetic curve was constructed based on the results. In this case, no testosterone residue was detected 14 days after the administration. The results clearly showed that a metabolomics approach can be a useful and e ffective tool for the detection and monitoring of banned anabolic steroids used illegally in pig fattening.

**Keywords:** metabolomic; anabolic practices; testosterone; plasma; urine; pigs
