*4.4. Sample Preparation and Extraction*

To detect sulfonamide residues, we used TFDA's directions for multiresidue analysis of residues of veterinary drugs in foods, which required the cleaning and homogenization of fish samples first [46]. In brief, 5 g of the homogenate and 25 mL of ACN in 5% MeOH were mixed on a vortex mixer for 3 min. Then, 10 g of ASS was added to the homogenate and mixed for 10 min; subsequently, centrifugation was executed at 3500 g at 4 ◦C for 10 min, after which only the supernatant layer was retained. The remaining tissue pellets were re-extracted using 25 mL of ACN in 5% MeOH, followed by centrifugation. The first extract was combined with the previously separated ACN layer. The resulting mixture was subjected to liquid–liquid extraction in a separating funnel. The filtrate was then added to 30 mL of ACN-saturated n-hexane and mixed on a vortex mixer for 10 min. The ACN-extracted layer was dried at 40 ◦C in a nitrogen evaporator. The evaporation residue was dissolved in 1 mL of 50% MeOH and filtered through a 0.2-μm polyvinylidene fluoride filter (Whatman, Maidstone, UK). Next, the derived filtrate was transferred to an autosampler vial before injection into a chromatograph.

For analyzing residues of insecticides in fish samples, we used the European Committee for Standardization-developed QuEChERS extraction procedure [6,7,47]. In brief, 10 g of homogenized fish sample and 10 mL of ACN were mixed vigorously in a 50-mL centrifuge tube for 1 min; then, QuEChERS extraction salt was added, and the mixture was mixed on a vortex for 1 min and then subjected to a 5-min centrifugation process executed at 3000 g. Thereafter, crude ACN extract (~6 mL) was transferred into QuEChERS cleanup tubes. The ACN layer was mixed vigorously for 2 min and centrifuged at 3000 g for 5 min. Next, 1 mL of the extract was filtered through a 0.2-μm filter membrane and transferred into an autosampler vial for LC-MS/MS. Another 1 mL of extracted solution was near-completely dried in the nitrogen evaporator at 40 ◦C. Finally, the residue was redissolved with 1 mL of a 1:1 (v/v) n-hexane and acetone mixture, filtered through a 0.2-μm filter membrane, and introduced into an autosampler vial for GC-MS/MS.

#### *4.5. LC-MS*/*MS Parameters*

The injection volume used for detecting the sulfonamide and organophosphorus insecticide residues was 10 μL. The mobile phase was binary, comprising eluents A (0.1% FA) and B (0.1% FA in MeOH), and the gradient of the mobile phase was developed as follows: 5% eluent B from 0 to 2 min (flow rate, 0.3 mL/min); followed by a step increase of eluent B to 20% from 2 to 3 min, 25% from 3 to 6 min, 27% from 6 to 8.6 min, and 37% from 8.6 to 14.5 min; then linear increase to 100% eluent B from 14.5 to 14.7 min; and finally decrease to 4% eluent B at 18.7 min, which was maintained from 18.7 to 20 min. MS was determined in positive ESI modes with monitoring of the two most abundant MS/MS (precursor/product) ion transitions by using an MRM program for each analyte. The MS parameter settings are outlined as follows: collision gas argon pressure, 0.12 mL/min; desolvation flow, 1000 L/h; source temperature, 150 ◦C; desolvation temperature, 500 ◦C; dwell time for every MRM transition, 5 ms; cone gas flow, 50 L/h; capillary voltage, 3 kV. Table S1 lists the precursor and corresponding product ions with optimum collision energy obtained through the MRM detection for 12 sulfonamides and LC-amenable 6 organophosphorus insecticides.

#### *4.6. GC-MS*/*MS Parameters*

GC-MS/MS analysis was executed in positive and negative electron-impact ionization interface modes. The carrier gas, namely helium, was applied at a constant flow rate of 1 mL/min. The temperatures of injector were 280 ◦C. In addition, the oven temperature was set at 60 ◦C—it was initially maintained isothermal for 1 min, next raised to 170 ◦C at 40 ◦C/min, and finally maintained at 310 ◦C for 8 min. The set source and transfer-line temperatures were 300 and 250 ◦C, respectively. In the splitless mode, the injection volume was determined to be 10.0 μL. In the collision chamber (second quadrupole), these ions were collision-activated with argon at 4.4 mTorr. Table S2 lists the precursor and corresponding product ions with optimum collision energy obtained through the MRM detection for GC-amenable 12 organophosphorus insecticides.

#### *4.7. Quality Assurance and Validation*

To validate our described method, recovery, repeatability, linearity, and LOQ were estimated [2,4,6]. For repeatability and recovery estimation, we spiked blank samples (in triplicate) with a standard mixture of the analytes at two concentrations 5 (low) and 25 (high) ng/g for analysis of sulfonamides and 10 (low) and 50 (high) ng/g for analysis of LC- and GC-amenable organophosphorus insecticides. The recovery was then calculated through the comparison of the noted concentrations of samples spiked before extraction with blanks spiked at the same concentration after extraction.

The derived reproducibility is presented herein as RSD (%); moreover, the LOQs were derived as the analyte concentration that generated a peak signal 3–10 times higher than the background noise in the chromatogram. For evaluation of linearity, matrix-matched calibration was executed by using blank sample extracts and then adding the corresponding amount of the working target compound solution at concentrations of 1–500 ng/mL. Linearity of the calibration curves was detected by fitting least-squares regression analysis in a linear mode (R2 <sup>≥</sup> 0.990) in the studied concentration range. All sample concentrations lower than their corresponding LOQs were regarded as undetectable [4,7,47].
