Acquity system

Polyphenolics were analyzed using the Waters ACQUITY system (Waters, Milford, MA, USA), consisting of a binary pump manager, sample manager, column manager, PDA detector, and tandem quadrupole mass spectrometer (TQD) with electrospray ionization (ESI). The separation was carried out using a BEH C18 column (100 × 2.1 mm i.d., 1.7 µm, Waters) kept at 50 °C. The following solvent system was applied: mobile phase A (0.1% formic acid in water v/v) and mobile phase B (0.1% formic acid in 40% ACN in water v/v). The gradient program was set as follows: 0 min 5% B, from 0 to 8 min linear to 100% B, and from 8 to 9.5 min for washing and back to initial conditions. The injection volume of the samples was 5 µL (partial loop with needle overfill) and the flow rate was 0.35 mL/min. The following parameters were used for TQD: capillary voltage 3.5 kV; con voltage 30 V in negative mode, the source was kept at 250 °C and the desolvation temperature was 350 °C; con gas flow 100 L/h; and desolvation gas flow 800 L/h. Argon was used as collision gas at a flow rate of 0.3 mL/min. The polyphenolics detection and identification being based on specific PDA spectra, mass-to-charge ratio and fragment ions obtained after collision induced dissociation (CID). All determinations were performed in duplicate. Waters MassLynx software v.4.1 was used for data acquisition and processing.

Metabolomics was applied using 1,290 ultra-high-performance liquid chromatography (Agilent, CA, USA) coupled with Q Exactive focus MS/MS (Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separations were performed using a Waters ACQUITY system equipped with an ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 × 100 mm). The mobile phase consisted of 0.1% formic acid water (A) and 0.1% formic acid acetonitrile (B) at the flow rate of 0.5 ml/min, and the injection volume was 4 ml. The gradient program was as follows: 85–25% B (0–11.0 min), 25–2% B (11.0–12.0 min), 2–2% B (12.0–14.0 min), 2–85% B (14.0–14.1 min), 85–85% B (14.1–15.0 min), and 85–85% B (15.0–16.0 min). The ESI source was applied to analyze the chemical composition in both positive and negative ion modes with full scan/ddMS2. The MS parameters were set as follows: the scan range was 50–1,000 m/z, the spray voltages were set at 4.0 and 3.6 kV in positive and negative modes, respectively, sheath gas was 35 arb, auxiliary gas was 10 arb, the capillary temperature was 400°C, the maximum injection time for MS1 and ddMS2 was 100 and 45 ms, respectively, and the resolutions for MS1 and ddMS2 were 70,000 and 17,500, respectively; putative molecules of interest were fragmented using three different collision energies (10, 20, and 40 eV).

Ceftiofur was quantified by ultra-performance liquid chromatography (UPLC) using an Acquity system (Waters, Milford, MA, USA) equipped with a binary solvent delivery pump, an autosampler, and a tunable UV detector. The chromatographic separation was performed in an Acquity BEH C18 column with 50 mm length x 2.1 mm diameter, with 1.7 μm particle size (Waters, Milford, MA, USA). The mobile phase consisted of a ratio of 78:22 (v/v) of 20 mM of sodium phosphate buffer at pH 6.0, adjusted with 85% orthophosphoric acid, to acetonitrile at a flow rate of 0.6 mL/min. A wavelength of 292 nm was used to obtain the maximum area under the curve (AUC), and the injection volume was 0.5 μL. A calibration curve was prepared using standard solutions of ceftiofur diluted in the mobile phase at 0.25, 50, 100, and 150 μg/mL.