2.5.2. UHPLC-MS/MS Conditions

The separation and quantification of the four amphenicols and metabolites were performed on a Thermo Scientific Vanquish ultra-high-performance liquid chromatography instrument coupled with a Thermo Scientific TSQ Quantis mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A Waters Acquity UPLC HSS C18 (2.1 mm × 50 mm, 1.8 μm) was used as the analytical column, with a Waters Acquity UPLC HSS C18 VanGuard precolumn (2.1 mm × 5 mm, 1.8 μm) attached to the front end. The column temperature was 40 ◦C, and the injection volume was 2.0 μL. Gradient elution, with water and ACN as mobile phases, was carried out at a constant flow rate of 0.3 mL/min. The starting mobile phase composition was 4:96 (ACN/water) at 0 min. It was switched to 96:4 after 4 min and held for 2 min, returning to the initial conditions at 10 min.

The MS/MS was equipped with an ESI source and scanned in positive ion (PI) and negative ion (NI) mode switching. CAP, TAP, FF and CAP-D5 were analyzed in NI mode, while FFA and FFA-D3 were analyzed in PI mode. The detection mode was selective reaction monitoring. The ESI source was operated with the following capillary voltages: 3.5 kV in PI mode, 2.5 kV in NI mode; sheath gas: 50 Arb; auxiliary gas: 10 Arb; ion transfer tube temperature: 325 ◦C; and evaporator temperature: 350 ◦C. The specific mass spectrometry parameters of amphenicols and metabolites are shown in Table 3. Under these conditions, the limits of detection and limits of quantification for all four analytes were below 1.5 μg/kg and 5.0 μg/kg, respectively, with good precision (RSD < 9.0%) and accuracy (recovery > 72.0%). This means that the extraction and purification procedures used in this experiment are effective and that the UHPLC-MS/MS conditions are applicable to detecting residues of amphenicols and metabolites in livestock and poultry meat samples. The linear working ranges of the current quantitative method were 5.0–50.0 μg/kg for CAP, 1–100 μg/kg for TAP and 5–300 μg/kg for FF and FFA, at which concentration ranges the changes in the peak area were proportional to the changes in the drug concentrations (R2 > 0.9990 for CAP, TAP, FF and FFA).


**Table 3.** Mass spectrometry parameters of amphenicols and metabolites.

\*: quantitative ion; −: negative ion reaction mode; +: positive ion reaction mode.
