*4.3. Sample Preparation*

#### 4.3.1. Plasma Samples for Targeted Analysis

Each defrosted plasma sample (500 μL) was transferred to a 15 mL centrifuge tube. After adding a methanol solution of 50 μL of 17β-testosterone-D2 internal standard and 5 mL of ethyl acetate, the samples were shaken vigorously for 3 min on a vortex and then centrifuged at 4000× *g* for 10 min. The supernatant (ca. 4 mL) was subsequently transferred to evaporator tubes. The samples were evaporated to dryness at 25 ◦C using a gentle stream of nitrogen (99.99% N2). The samples were reconstituted with 200 μL in mobile phase solution (methanol: water, 70:30, *v*/*v*) and filtered through a 0.45 μm (Hydrophilic PTFE) membrane centrifuge filter. The resulting samples were transferred to the insert in chromatography vials (250 μL). Then, 10 μL samples were injected directly the LC-MS/MS system.

#### 4.3.2. Samples for Metabolomics Profiling

Plasma samples were defrosted at room temperature, homogenized and normalized by the creatinine concentration (Chemistry Analyzer BS-200, MINDRAY, Nanshen, China). Subsequently, 200 μL of each sample was filtered through centrifugal devices (Vivacon 500, cut-off at 10 kDa, 14,000× *g*, 4 ◦C, 30 min) to remove high molecular weight proteins. Filtrates (120 μL) were mixed with 30 μL of internal standard (testosterone-D2 in methanol at the concentration of 1 ng mL−1). After thorough shaking, 5 μL of each sample was injected into the chromatographic system.

Urine samples were defrosted at room temperature and normalized by specific density adjustment [47]. The specific density of the samples was measured using a refractometer (digital refractometer 30GS, Mettler-Toledo, Prague, Czech Republic) and, if necessary, adjusted to approximately 1.010–1.030 kg m<sup>−</sup><sup>3</sup> by dilution with deionized water. The urine samples (500 μL) were then centrifuged through a centrifugal membrane filter (Vivacon 500, cut-off 10 kDa) at 14,000× *g*, 4 ◦C for 30 min to remove high molecular weight proteins. To the sample filtrate (ca. 450 μL), 50 μL of 17β-testosterone-D2 internal standard was added. The urine samples were transferred to chromatographic vials and subsequently analysed.

QC samples for quality control of metabolomics profiling were prepared by the pool method so that they have the same or very similar (bio)-chemical varieties in the same range as individual samples of the study. The QC sample was prepared as a pool of all individual plasma or urine samples that were included in the study. Each QC sample was generated by mixing 20 μL of the filtrate (cut-off) of each individual sample and was analysed at the same time as the study samples as part of the overall measurement sequence.

#### *4.4. Targeted Quantitative Analytical Method*

## 4.4.1. LC Condition

Plasma samples were injected directly into a Thermo Fisher Scientific, (Waltham, MA, USA) LC system Accela 1200 equipped with an autosampler with a temperature controlled tray and column. Chromatographic separation was performed on Waters C18 XTerra MS analytical columns (150 × 2.1 mm, size 3.5 μm) with a Waters C18 XTerra MS guard column (10 × 2.1 mm, size 3.5 μm). The column and autosampler tray temperatures were set at 35 ◦C. The mobile phase consists of 0.1% formic acid in water: methanol (95:5, *v*/*v*) A and 0.1% formic acid in water: methanol (5:95, *v*/*v*) B, the flow rate was constant 300 μL min−1. Gradient elution of 0–2 min with mobile phase 95% A and 5% B was started, 2.1–20 min linear gradient from 5% to 90% B, 20.1–25 min 10% A and 90% B, 25.1–30 min linear gradient from 5% to 95% A and 30.1–35 min 95% A and 5% B. The runtime of the method was 35 min.

## 4.4.2. MS/MS Parameters

The tandem hybrid mass spectrometer Q Exactive (Thermo Fisher Scientific, (Massachusetts, USA) equipped with a heated electrospray ionisation probe measured in a positive mode (H-ESI+). For targeted quantification analysis, the mass spectrometer worked in the parallel reaction monitoring mode PRM (corresponding to the selected reaction monitoring mode SRM) with high resolution RP = 17,500 (FWHM) at 200 *<sup>m</sup>*/*<sup>z</sup>*. Before the start of each acquisition series, the mass spectrometer was externally calibrated to the mass accuracy with the positive ion calibration solution and the negative ion calibration solution (both Thermo Fisher Scientific). The "lock-mass" calibration was set to the molecular mass of [M+Na]<sup>+</sup> = 64.01577 g mol−<sup>1</sup> and [M2+H]<sup>+</sup> = 83.06037 g mol−<sup>1</sup> for the acetonitrile ion, and was run continuously during the acquisition. Instrument and collision cell (HCD) parameters were optimised by direct syringe infusion of working solutions of 50 ng mL−<sup>1</sup> of each targeted compound with a 5 μL min−<sup>1</sup> flow-rate. The mass spectrometer setting was as follows: sheath gas flow rate 30 (unit), aux gas flow rate 5 (unit), spray voltage 4.0 kV, capillary temperature 320 ◦C, heater temperature 220 ◦C, S-lens RF level 50, AGC target of 5 × 106, collision energy 35 eV, and a maximum injection time of 100 ms.

The precursor ions and the four most intense product ions for each analyte were measured for quantification and identification (confirmation), respectively. The whole LC-(HR) MS system was controlled and the acquired data were stored and processed using Xcalibur 3.1 software, and then evaluated using Mass Frontier v. 7.0 for the identification.

## 4.4.3. Method Validation

The targeted quantitative method for the determination of 17β-testosterone and its esters in plasma has been validated to the extent required by European Directive 657/2002/EC [34] used for the determination of residues of foreign substances in biological matrices and according to the VICH GL49 [35] reference guide for validation method. To determine the validation characteristics of the matrix calibration curve, critical values detection limit (LOD), limit of quantification (LOQ), decision limit (CCα), detection capability (CCβ) and calibration range 0.5–80 ng mL−<sup>1</sup> was used for 17β-testosterone and its esters. Samples of pig plasma (blank) were supplemented with the standards of 17β-testosterone and its esters at concentration corresponding to 0.5, 1, 5, 10, 20, 40, and 80 ng mL−1. The concentrations of the internal standards were constantly 10 ng mL−1. For each concentration level, two model samples were prepared and each sample was measured two times. Linear regression was carried out by plotting the peak area ratios of the analyte against the internal standard (dependent variable Y) versus the analyte concentration (independent variable X). To evaluate the precision of the method, repeatability and within-laboratory reproducibility, standard deviation (SD), and the coefficient of variation (CV, %) were determined. Six model samples of pig plasma (*n* = 6) were

prepared at concentrations of 5 and 80 ng mL−<sup>1</sup> for all standards and measurements which were repeated two times for each sample on three different days (3 × 6, *n* = 18).

## *4.5. Metabolomic Profiling*
