**1. Introduction**

The use of hormones as growth promoters for fattening purposes in livestock has been banned in the European Union since 1988 by Council Directive 96/22/EC. However, the banned substances are still reported as positive in the European residue monitoring plans [1]. One of these banned substances is the anabolic and androgenic steroid testosterone, which naturally occurs in animal organisms. Testosterone is endogenously secreted by Leydig cells (testes) and is able to accelerate muscle growth (anabolic effect) and improve the development of male characteristics (androgenic e ffect). Testosterone is then secreted into the bloodstream where it primarily (98%) binds to a specific protein beta-globulin termed sex hormone binding globulin (SHBG) and to a lesser extent to albumin. By this binding, testosterone is biologically protected from inactivation in the liver, and is subsequently transported to the target tissues via the bloodstream. A small amount of circulating testosterone is converted to estradiol, but the greater part of free testosterone is converted to 17-ketosteroids, particularly androsterone and its isomer etio-cholanolone (androsterone metabolites) [2]. In some target tissues, testosterone is reduced to 5 α-dihydrotestosterone (DHT) by the cytochrome P450 enzyme 5 α-reductase, an enzyme

highly expressed in male sex organs, skin, and hair follicles. The inactivation and degradation of testosterone and its metabolites in cattle and pigs occurs mainly in the liver and, to a lesser extent, in the kidneys. These mechanisms of inactivation and degradation of testosterone occur with the participation of specific enzymes involved in the catalytic action of the partially transformed steroid molecule. Inactivation and degradation include the following: addition of two hydrogens (reduction) to a double bond or ketone group; removal of two hydrogens (oxidation) from a hydroxyl group; addition of a hydroxyl group (hydroxylation) to a carbon in the steroid molecule; and conjugation of testosterone by reaction of sulfuric acid or glucuronic acid with a hydroxyl group on the steroid molecule, forming testosterone sulphates and glucuronides, respectively. The sulfated or glucuronide conjugated form of testosterone is then excreted in the urine [3].

Testosterone (a natural steroid) is illegally administered to animals in the form of synthetic steroid esters, but these are rapidly hydrolysed to a natural steroid in vivo. For example, after oral administration of testosterone undecanoate, unchanged ester was found in athletes' plasma for only 6 h [4]. In analytical practice, it is di fficult to distinguish between metabolites of natural endogenous testosterone, which is always present in body fluids (plasma, urine), and metabolites of identical exogenous testosterone derived from hydrolysed synthetically prepared esters [5]. In human doping control, this problem is usually solved by determining the urinary ratio of 17β-testosterone/17 α-testosterone levels (T/EpT ratio) or by using gas chromatography with isotopic mass spectrometry (GC-IRMS) and application of the 13 C/12C isotope ratio [6]. The World Anti-Doping Agency (WADA) has established a decision limit if a T/E ratio is equal to or greater than 4, or an epitestosterone (17 α-testosterone) concentration is greater than 200 ng mL−<sup>1</sup> which would require a testing procedure to confirm doping [7,8]. Although important in humans, these analytical parameters have failed in animals because of di fferences in their metabolism [9]. In food safety practices, relatively high or low levels of 17β-testosterone and 17 α-testosterone in urine are often ignored due to a lack of statistically valid reference data on naturally occurring endogenous background levels in animals. However, the EU Community Reference Laboratories (CRLs) for analytical methods recommended in the national monitoring control plans to limit concentrations for plasma (CCα for confirmatory methods) to 0.5 μg L−<sup>1</sup> for heifers 18 months old, 10 μg L−<sup>1</sup> for bullocks six months old, and 30 μg L−<sup>1</sup> for bulls 6–18 months old [10]. For other animals, no such recommendations exist for 17β-testosterone in plasma or urine.

Over time, a number of targeted analytical methods for the determination of testosterone in various biological samples (plasma, urine, muscles and hair) have been developed and described in the literature. In the 1990s, testosterone measurements were often performed by radioimmunoassay (RIA) [11] and immunoassays (ELISA) [12]. Immunological methods are fast, easy-to-perform, cheap, and have a short time to result for a large number of samples, so today they are preferably used in many laboratories primarily for screening. However, cross-reactivity and sensitivity in these assays is a common problem, so these methods are no longer good enough for the detection and quantitative determination of testosterone.

Gas chromatography (GC) and liquid chromatography (LC) are other alternatives for the targeted analysis of testosterone and its esters. GC methods coupled with mass spectrometry (MS) are usually applied for the determination of anabolic steroid levels ranging from micrograms to nanograms in biological samples [13]. The detection of testosterone esters at 1 ng mL−<sup>1</sup> in human plasma by GC/MS has been reported [14]. However, GC-MS methods require a complicated, time-consuming, and expensive step of sample derivatization for steroid analysis. In general, these derivatives are unstable and are susceptible to thermal degradation during analysis, which, in particular, significantly a ffects the reproducibility of the method [15]. In contrast, LC-MS is a good solution for quantitative analysis of steroids because the included sample preparation step is easy, fast, economical and requires no further derivatization step. The high performance liquid chromatography with mass spectrometry (HPLC-MS) used for the analysis of steroid esters in plasma showed greater sensitivity than GC-MS [16]. HPLC is also a commonly used separation technique for the determination of testosterone and its esters in body fluids due to its su fficient sensitivity, good resolution, robustness and short analysis time [17]. Ultra-high performance liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) or today with high-resolution (HR) mass spectrometry is another powerful approach to significantly improve peak resolution, selectivity, sensitivity and speed of the analysis [18–20]. It should be noted that an interesting alternative to inconclusive urine analyses (endogenous testosterone vs. synthetic testosterone) at veterinary inspection may be the analysis of intact natural steroid esters in the hair by UHPLC-MS/MS [21,22] or DESI-MS (desorption ionizing mass spectrometry) [5].

Recently, new synthetic xenobiotic growth promoters have been designed and new ways of application employed, such as the administration of low dose cocktails. However, metabolomics approaches to non-targeted screening for the detection of anabolic practices with natural steroid hormones might change this situation in the future [9,23]. Indeed, several scientific studies have demonstrated the e fficiency of mass spectrometry with high resolution based on urinary fingerprinting to discriminate anabolised animals from control ones. Rijk et al. [24] in their work showed the use of a novel untargeted metabolomics based strategy for the measurement of the anabolic steroid DHEA (dehydroepiandrosterone) and pregnenolone in bovine urine with liquid chromatography coupled with time-of-flight mass spectrometry (LCT Premier). In the same year, Kieken et al. [25] presented a metabolomics strategy involving the characterization of global metabolomic fingerprints in urine samples of non-treated and reGH (recombinant equine growth hormone)-treated horses by LC-HRMS (LTQ-Orbitrap) as a new screening tool for growth hormone abuse in horseracing.

Anizan et al. [26] presented in their study a metabolomics approach to 4-androstenedione (AED) detection after its administration to heifers. Using untargeted profiling by GC-MS, they identified 5α-androst-2-en-17-one in urine as a new biomarker of anabolic AED abuse. From 2011 to the present, several studies have been conducted in cattle in relation to the administration of banned substances for fattening, which have confirmed the correctness of the research focus on non-targeted analyses based on metabolomic approaches [27–32]. However, all these studies were in all cases carried out only in cattle, although in many European countries, for example, pork was consumed significantly more than beef. The only metabolomic study published so far for another animal species was conducted in 2017 in pigs to which a banned beta-agonist substance, ractopamine, was administered [33].

The present study aimed to investigate the e fficacy of metabolomic profiling of pig plasma and urine samples by high resolution mass spectrometry (HRMS) to discriminate between the testosterone ester group and the control group. The experiment was performed in two independent groups of pigs, where individual animals were assigned to groups based on randomization. Plasma and urine samples were continuously collected at specified time intervals, prepared and subsequently measured on a high-resolution hybrid tandem mass spectrometer (QExactive). The obtained metabolomic fingerprints were processed and statistically analyzed using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) multivariate methods. Furthermore, the results of the non-targeted metabolomic analysis obtained in this way were compared with the results of the targeted determination of 17β-testosterone in the same plasma and urine samples. All pigs in the experiment were weighed at weekly intervals and the anabolic e ffect of testosterone was studied based on the body weight gain.
