2.3.3. UPLC-MS/MS Analysis

The prepared samples were analyzed by ultra-performance liquid chromatography. The Nexera X2UPLC-MS/MS (Shimadzu Corp., Kyoto, Japan) contained two LC-30AD pumps, SiL-30AC autosampler, CTO-20AC column oven, CBM-20A communication bus module, and mass spectrometer LC-MS8050 with electro spray ionization with positive and negative ion mode (Table 1). Chromatographic separation was performed using an analytical column Kinetex 2.6 μm, Phenyl-Hexyl 100 Å 4.6 mm, 150 mm (Phenomenex, Torrance, CA, USA). The gradient program consisted of the following: 7-min sequence of linear gradient flows of solvent B (acetonitrile:methanol 1:1 *v*/*v*) balanced with solvent A (water with 0.1% formic acid) at a flow rate of 0.6 mL/min: 50–80% B over 1 min, 80–100% B over 2 min, isocratic 100% B for 3 min, and finally, 100–50% B over 1 min. The injection volume was 1 μL and column temperature was 40 ◦C.

The main parameters used to identify analytes were their retention times and multiple reaction monitoring (MRM) ratio, which were obtained at 0.25 μg/mL working standard solutions for most standards, and 2.5 μg/mL for AMP, IB, SPEC, STREP, SFC, SNA and TET. Chromatographic data processing was carried out using LabSolution® software (Shimadzu Corp., Kyoto, Japan).

**Table 1.** The analysis parameters for the monitored ion transitions and triple quadruple mass spectrometer (MS/MS) operation (CE- collision energy, Q1 and Q3Pre Bias—quadruple pre-rod bias voltage).


#### 2.3.4. Quality Assurance/Quality Control (QA/QC)

Ensuring the quality of the measurements results was carried out by:


The calibration step involves the preparation of calibration curves and calculation of response factor (RF) for internal standards. Eleven working standard solutions have been prepared. Limit of determination (LOD) has been calculated from the parameters of a calibration curve constructed on the basis of the three lowest concentrations of working standards solutions. The limit of determination has been calculated according to the formula:

$$LOD = \frac{3.3 \times s}{a} \tag{1}$$

where: a—slope of the calibration curve; s—the standard deviation.

For the calculation of the validation parameters, residual standard deviation (sxy) and standard deviation of the intercept (sa) were taken. Based on LOD(sxy) and LOD(sa) the mean values (LOD) were calculated (see Equation (1)). The coefficients of variation (CV) and uncertainty (U) were calculated based on standard deviations and the average area of the chromatographic peaks obtained during separation of standard mixtures.

To check the matrix effect, two samples were prepared using real soil samples with addition of standards, and using the procedure described in Section 2.3.2. Sample Pretreatment and additionally using solide phase extraction (SPE) cleaning. Based on peak areas, the recovery (%) for standard solutions was calculated.

#### *2.4. Antibiotic Resistance in Microorganisms Isolated from Soil*

The soil samples (1–6A; 1B and 1C, Figure 1) were plated on different growth media for evaluation of microbial growth (Luria Bertani Agar—LA, MacConkey Agar, Chapman Agar, Medium with cetrimide, Merck, Darmstadt, Germany). Briefly, 10 g of the sample was mixed with 90 mL of 0.85% NaCl and shaken for 0.5 h, and then serially diluted to 10−4. In addition, 100 μL of each dilution was plated on LA, while solely the dilution 10−<sup>1</sup> was spread on MacConkey Agar, Chapman Agar and Medium with cetrimide. The plates were incubated at 30 ◦C for 24–72 h to obtain sufficient growth of the colonies. From each sample, several differently-looking colonies were picked and grown to pure cultures on LA, and then stored frozen with 20% glycerol at −40 ◦C.

The isolated bacteria were then subjected to antibiotic resistance disc diffusion test with different antibiotics (TMP 5 μg, SMZ 20 μg, TET 10 μg, SPEC 25 μg, and CIP 10 μg). TET, SPEC, and CIP discs were purchased from Oxoid, UK, while trimethoprim and sulfametazine discs were prepared in the laboratory. Briefly, the cotton discs were cut from MN85/220 paper filters (Macherey-Nagel, Germany) and autoclaved. The appropriate amount of the antibiotic stock solution was poured onto the paper disk with automatic pipette and let dry. For the antibiotic resistance disc test, the bacteria were cultured in LB (Luria Bertani boullion, Btl, Łód´z, Poland) with 150 rpm shaking at 30 ◦C, overnight. Then, the OD600 measurements of the bacterial cultures were taken with Spectroquant Prove 600 (Merck, Darmstadt, Germany) spectrophotometer and diluted in LB to achieve OD600 equal to 0.1. The dilution accurateness was verified with repeated spectrophotometer measurements. In addition, 100 μL of each bacterial suspension was spread on a plate with Muiller-Hinton medium (Graso, Owidz, Poland). Discs with antibiotics were then put on the surface of the plate and the plates were incubated for 24 h at 30 ◦C. Afterwards, the diameter of the growth inhibition zone around each antibiotic disc was measured.
