**5. Conclusions**

The present study confirmed the ability of metabolomics approaches and techniques to significantly di fferentiate pigs administered an androgenic anabolic steroid which is on the list of banned substances (17β-testosterone) in EU countries from control pigs. Using metabolomics profiling for plasma and urine samples, it was possible to di fferentiate the used synthetic exogenous testosterone from naturally occurring (endogenous) testosterone based on the results from three statistical models PCA, CA and OPLS-DA. The metabolomics workflow was designed with widely used multivariate statistical methods, analytical techniques, and equipment so that, based on the results of our study, it will be possible to develop validated methodologies for routine screening to prove the illegal use of prohibited substances in pig fattening. Furthermore, the anabolic e ffect of testosterone in pigs was demonstrated by comparing BW gains during the fattening period, and the targeted analysis of plasma testosterone levels provided data for the pharmacokinetic curves.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-1989/10/8/307/s1, Figure S1: Graph of average weekly weight gains in kg, Figure S2: The measured experimental MS1 of the 17β-testosterone standards (up), comparison with the theoretical MS1 (down), Figure S3: The measured experimental MS1 of the 17β-testosterone-D2 standards (up), comparison with the theoretical MS1 (down), Figure S4: Calibration curve of 17β-testosterone in plasma, Figure S5: Hotelling plot for identification of outliers objects in data source matrix X with calculated critical value T2 for plasma, (K—control grup vs. T—treated grup), Figure S6: Hotelling plot for identification of outliers objects in data source matrix X with calculated critical value T2 for urine, Figure S7: The PLS-DA score plots for plasma (A) and urine (B) data matrix demonstrates robust discrimination between the control group of pigs marked with blue color and the group of teated pigs marked with red color (Centering, Statistica), Figure S8: The OPLS-DA permutation tests further confirmed that the proposed statistical models are correct and robust; A—plasma, B—urine, Figure S9: Variable importance in projection (VIP) and S-plots from OPLS-DA were used to determine the most discriminating metabolites between treatments and controls, A—plasma and B—urine, Table S1: The average weekly body weight gains, Table S2: The regression parameters of both linear models of BW growth versus time, Table S3: Identification and confirmation of 17β-testosterone and testosterone esters by mass accuracy (MA) for MS1, MS2 data, Table S4: Linearity of 17β-testosterone in plasma, Table S5: Precision, repeatability and within-laboratory reproducibility, Table S6: Pharmacokinetic of testosterone in plasma.

**Author Contributions:** Conceptualization, K.S., K.P., L.L., M.F. (Milan Franek), P.D. and M.F. (Martin Faldyna); methodology, K.S., K.P., L.L., M.F. (Milan Franek), P.D. and M.F. (Martin Faldyna); validation, K.S., K.P., L.L., M.F. (Milan Franek), P.D. and M.F. (Martin Faldyna); formal Analysis, K.S., K.P., L.L.; investigation, K.S., K.P., L.L., M.F. (Milan Franek), P.D. and M.F. (Martin Faldyna); resources, K.S., K.P., M.F. (Milan Franek), P.D. and M.F. (Martin Faldyna); writing—original draft preparation, K.S., K.P. and M.F. (Milan Franek); All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the Ministry of Agriculture of the Czech (QK1910311 and RO1518) and by the Ministry of Education, Youth and Sports of the Czech Republic (project no. LO1218).

**Conflicts of Interest:** The authors declare no conflict of interest.
