4.5.1. LC Separation

Plasma and urine samples prepared for non-targeted analysis (fingerprinting) were injected directly into the Accela 1200 LC system with a mass spectrometer. Chromatographic separation was performed on a C18 Hypersil GOLD (50 × 2.1 mm, 1.9 μm size) analytical column equipped with a C18 Hypersil GOLD (10 × 2.1 mm, 1.9 μm size) guard column, both from Thermo Fisher Scientific. The temperatures of the column and the autosampler tray were set at 35 ◦C and 20 ◦C, respectively. The flow rate through the column was constantly set at 200 μL min−1. The injection volume of the sample was 5 μL. 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 μLmin−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.

#### 4.5.2. Non-Targeted Mass Spectrometry

For non-targeted metabolomic analysis, the tandem hybrid mass spectrometer Q Exactive worked in the positive full scan mode with resolving power (PR) = 70,000 (FWHM) at 200 *m*/*z* in the range 50 to 750 *m*/*z* and in the centroid mode. Before the start of each acquisition series, the mass spectrometer was externally calibrated to the mass accuracy with a positive ion calibration solution and a negative ion calibration solution (both Thermo Fisher Scientific (Massachusetts, USA). 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. The mass spectrometer setting was as follows: sheath gas flow rate 30 (unit), aux gas flow rate 5 (unit), spray voltage 4.5 kV, capillary temperature 320 ◦C, heater temperature 180 ◦C, S-lens RF level 50, AGC target of 6 × 10<sup>6</sup> and a maximum injection time of 200 ms. The whole LC-(HR)MS system was controlled and the obtained data were stored and processed using Xcalibur 3.1 software.
