*2.3. Sample Preparation*

Stock solutions were prepared in methanol at 1 μg/mL for ENR and ENR-*d5*, and 100 μg/mL for ENR-*d3*. Further dilution was applied for recovery and calibration solutions. The blank samples were prepared using the muscle of grass carp obtained from a local supermarket in Shanghai (China). All blank samples were firstly screened to ensure that they were free of the antibiotics of interest. Two calibration curves (Curve 1 and 2) were established using a standard solution of different concentrations prepared with a blank matrix solution; the curves were fitted by linear regression with ENR-*d5* and ENR-*d3*, respectively. For the plot of calibration Curve 1, the standard solutions were at 1, 3, 9, 27, 81, and 243 ng/mL of ENR, with 5 ng/mL of ENR-*d5* as the isotope standard for each point. For the plot of calibration Curve 2, the standard solutions were 27, 81, 243, 729, 2187, and 6561 ng/mL of ENR with 100 ng/mL of ENR-*d3*. Considering that a high amount of ENR can saturate the MS system detector, the standard solutions of Curve 2 can be diluted 20 times to obtain good linearity for the subsequent quantification of the correspondingly

diluted sample solution. Finally, the actual concentrations of Curve 2 were 1.35, 4.05, 12.2, 36.4, 109, and 328 ng/mL of ENR with 5 ng/mL of ENR-*d3* for determination by LC–MS/MS.

The analyte was extracted following the optimized method in [29]. Briefly, a 5 g homogenized sample was weighed in a 50 mL centrifuge tube and added to 50 ng of ENR-*d5* (50 μL of solution of 1 μg/mL ENR-*d5* in methanol) and 1000 ng of ENR-*d3* (100 μL of solution of 10 μg/mL ENR-*d5* in methanol), respectively. An amount of 10 mL of acetonitrile–water solution (85:15, containing 1 mL/100 mL acetic acid) was then added, and the sample was vortex–mixed for 10 min. Afterward, the sample was extracted by ultrasonication for 10 min, and centrifuged (5000× *g*) for 5 min at 4 ◦C. Following this, a 1 mL aliquot was filtered through a 0.22 μm hydrophobic polytetrafluoroethylene (PTFE) membrane as solution 1 for analysis. Meanwhile, 50 μL of solution 1 was diluted with 950 μL of extraction solvent, and the obtained solution (solution 2) was used for analysis.
