2.7 HPLC procedures and PK analysis of tylosin

Frozen plasma samples were thawed at room temperature, and 245 uL of plasma was transferred to new prechilled centrifuge tubes combined with 5 uL of 0.025 μg/mL as the internal standard. For drug extraction, plasma sample aliquots were mixed with methanol (2 mL). After vortexing (15 min) and centrifugation (12,000 × g, 10 min), the supernatant was separated and filtered through a 0.22-μm nylon syringe filter, and dried in a water bath using nitrogen at 50°C. The residue was dissolved in 100-uL methanol, agitated (1 min), and centrifuged (12,000 rpm, 10 min). Drug levels in the final 70 uL were determined using the liquid chromatography-tandem mass spectrometry method (Agilent 1200 HPLC system; API 4000 triple quadrupole mass spectrometer, CA, United States). The mass spectrometer was set up with an electrospray positive ionization mode (ESI+) using a capillary voltage of 3,500 V and had optimal ESI–MS parameters, including a drying gas temperature of 350°C, a drying gas flow of 5 L/min, and a nebulizing gas pressure of 45 psi. Separations were accomplished using an Eclipse plus C18 column (2.1 × 100 mm, 3.5 μm) (Agilent Technologies, CA, United States). The mobile phase consisted of a mixture of 0.1% formic acid in water (Eluent A) and 0.1% formic acid in acetonitrile solution (Eluent B) with a ratio of 30:70 (v/v) and a concentration of 1 mM. The flow rate was 0.4 mL/min, and the sample injection volume was 3 μL. The column temperature was maintained at 40°C. The monitored precursor ion for tylosin was 916.3 m/z. The validation of the assay was performed by spiked plasma samples at five different levels. The limit of detection was the concentration at which the signal-to-noise ratio was greater than three with a value of 0.017 μg/mL, whereas the limit of quantification was the concentration at which the signal-to-noise ratio was ten with a value of 0.053 μg/mL. The correlation coefficient (R) was above 0.98 in the linear range of 0.025–4 μg/mL. Inter- and intra-assay precision was determined to be all <10% and the accuracy of the assay was 101.38% ± 34.24% (Huang et al., 2018).

Tylosin time–concentration data in the plasma of individual pigs were analyzed using WinNonlin Version 8.3 software (Certara, NJ, United States) employing non-compartmental modeling. The maximal drug concentration (Cmax) was directly determined from the data with T_max defined as the time of the first occurrence of C_max. To calculate the AUC, the linear trapezoidal rule was used. Additional PK parameters, including terminal half-life (T_1/2λ_z) and mean residence time (MRT), were also determined.