*2.5. Validation of ic-ELISA Analysis*

Tissue samples of pork, swine liver, swine kidney, chicken, and chicken liver were purchased from local supermarkets. The homogenized samples (1 ± 0.05 g) were weighed, and 12 mL of extractant (ethyl acetate: acetonitrile = 1:1) was added to them. The mixture was mixed thoroughly for 3 min and then centrifuged at 4000× *g* for 10 min at room temperature. The organic phase was pipetted, and 2 mL of the aqueous NaOH solution (0.5 mol/L) was added. After shaking for 5 min, all organic phases were blown dry under nitrogen at 40 ◦C in a water bath. Then, the residue was resuspended with 1 mL of the compound solution (PBS: methanol = 9:1) and diluted 16 times with PBS to be ready for ic-ELISA analysis.

The sensitivity of the ic-ELISA analysis for practical applications is determined by the detection of the actual samples. Twenty different blank samples (pork, swine liver, swine kidney, chicken, and chicken liver) that were confirmed to be free of drug residues by LC–MS/MS were performed to ic-ELISA analysis. The mean (C) of the concentrations and the standard deviation (SD) were calculated for all blank samples. The limit of detection (LOD) and limit of quantification (LOQ) of this ic-ELISA method were calculated according to LOD = C + 3 × SD and LOQ = C + 10 × SD.

To demonstrate the accuracy and precision of the ic-ELISA analysis, the recovery rate and coefficient of variation (CV) were evaluated as indicators. DMEQ standard solutions were added to different types of homogenized samples at final concentrations of 1 × LOQ, 2 × LOQ, and 4 × LOQ, with 5 replicates of each concentration. The samples were mixed well by vortexing and left to stand for 1 h to allow full absorption of the drug, followed by sample pretreatment and ic-ELSIA analysis. The calculating equation is as follows:

recovery = (actual concentration/spiked concentration) × 100%. CV = (SD/C) × 100%

To confirm the reliability of the developed ic-ELISA method, four spiked chicken samples were selected for comparison using ic-ELISA analysis and LC–MS/MS. The detailed procedures for LC–MS/MS analysis and sample preparation were performed according to the previous description [20]. Briefly, 10 mL of methanol-water (*v*/*v*, 5:95) and 20 mg of N-propyl ethylenediamine were added to the homogenized samples. They were vortexed

for 1 min and then centrifuged at 15,000× *g* for 10 min, and the supernatant was collected and filtered through a 0.22 μm membrane. A Thermo Hypersil Gold column (150 × 2.1 mm i.d., 5.0 μm) was used for LC–MS/MS analysis, and water-formic acid (*v*/*v*, 100:0.1) and acetonitrile were used for the mobile phases. For the analysis of spiked chicken samples by ic-ELISA and LC–MS/MS, correlation fitting curves were drawn to assess the correlation between the two methods.
