*3.1. Optimization of HPLC–MS/MS Conditions*

The Symmetry C18 column (100 mm × 2.1 mm i.d., 3.5 μm), supplied by Waters, USA, was chosen for separation in this study. Several mobile phase combinations were tested, including 0.1% formic acid water–acetonitrile, 0.1% acetic acid water–acetonitrile, 0.2% formic acid water–acetonitrile, and a blend of 0.1% formic acid and 0.1% acetonitrile. The results indicated that the 0.1% formic acid water–acetonitrile mobile phase system provided the optimum response value and retention time. Figure 1 depicts the characteristic ion mass spectrometry of a mixed standard working solution of 0.005 μg/mL, with mobile phase of 0.1% formic acid in water and acetonitrile.

**Figure 1.** The characteristic ion mass spectrometry of the mixed standard working solution of 0.005 μg/mL.

After the optimization of operational parameters, the molecular ions and the product ions of PMZ, PMZSO, Nor1PMZ, and PMZ-d6 in standard working solutions were scanned under suitable conditions, as illustrated in Figure 2.

#### *3.2. Selection of Extraction Reagents*

The actual absolute recoveries of four analytes in muscle, liver, kidney, and fat tissue were compared using four extraction reagents: acetonitrile, 0.1% formic acid in acetonitrile, a blend of ethyl acetate and acetonitrile (20/80, *v*/*v*), and 1% ammoniated acetonitrile, as depicted in Figure 3. In Figure 3, the bar represents the average absolute recovery rate of each analyte in four types of tissues, extracted using different extraction reagents, and the error bar represents the standard deviation. The extraction efficiency of 0.1% formic acid in acetonitrile was superior to the others. Consequently, it was chosen as the extraction reagent for the four analytes.

**Figure 2.** Representative MRM chromatograms of the precursor ions and primary product ions of the analytes and internal standard in the standard working solution: (**a**) PMZ, (**b**) PMZ-d6, (**c**) PMZSO, and (**d**) Nor1PMZ.

**Figure 3.** Average absolute recoveries of four analytes in four types of tissues in different extraction reagents.

#### *3.3. Methodological Validation*

Selectivity was evaluated by comparing the chromatograms derived from spiked tissue samples and blank tissue samples, processed and detected following the method outlined in Sections 2.4 and 2.5. It was demonstrated that no endogenous peaks from blank samples were present, and no interfering signals were observed at the retention times of each monitored ion of the analytes. As such, the method developed in this study allowed for accurate qualitative and quantitative analysis of PMZ and its metabolites, PMZSO and Nor1PMZ.

The limit of detection (LOD), limit of quantification (LOQ), linear range, and linearity were assessed using spiked samples. After processing and detecting the samples in accordance with Section 2.6, the LOD and LOQ for PMZ and PMZSO were determined to be 0.05 μg/kg and 0.1 μg/kg, respectively, in muscle, liver, and kidney samples; for Nor1PMZ, the LOD and LOQ were 0.1 μg/kg and 0.5 μg/kg, respectively. For spiked fat samples, the LOD and LOQ for all three analytes were found to be 0.05 μg/kg and 0.1 μg/kg, respectively. Employing the method described in Section 2.7, PMZ and PMZSO displayed good linear relationships in the range of 0.1 μg/kg to 50 μg/kg across the four tissue types. Nor1PMZ also exhibited a strong linear relationship in the range of 0.5 μg/kg to 50 μg/kg, with correlation coefficients (r) exceeding 0.99. Refer to Table 4 for additional details.

**Table 4.** Linear equations, correlation coefficient (r), limit of detection (LOD), and limit of quantification (LOQ) of PMZ and its two metabolites.


\* Y: peak area of analyte, x: concentration of analyte.

Recovery and precision were evaluated using spiked samples, following the methodology presented in Section 2.8. As can be seen in Table 5 (original data are shown in Table S2), average recoveries for PMZ, PMZSO, and Nor1PMZ in muscle, liver, kidney and fat ranged from 77% to 111%. The intra-day and inter-day precision for all tissues remained less than 15%, thereby meeting the Ministry of Agriculture and Rural Affairs of China's technical guiding principles for residue analysis methods.

After processing and analyzing the samples as described in Section 2.9, the matrix effects were determined, as presented in Table 6. The matrix effects for the four types of tissue were predominantly negative, signifying a suppression effect on the signal of the compounds. The matrix effect on the three target compounds in pig fat tissue suggested weak matrix interference. In contrast, the matrix effect on the three target compounds in pig muscle and kidney tissues indicated moderate matrix interference. In pig liver tissue, the matrix effect on the three target compounds signified strong matrix interference. These findings underscore the necessity for thoughtful consideration of tissue matrix types when analyzing analytes, as varying matrices can influence the accuracy of the results.


**Table 5.** Recovery and precision of spiked blank samples.

The stability of the samples was assessed following the methodology outlined in Section 2.10. As detailed in Table 7, the relative standard deviations (RSDs) for each analyte concentration within tissue samples, subjected to conditions such as ambient temperature and light exposure for 24 h, refrigeration at 4 ◦C for 48 h, a three-cycle freeze–thaw process, and prolonged storage for a month, typically hovered around ±15%. Hence, the structural and compositional stability of PMZ, PMZSO, and Nor1PMZ in tissue samples proved to be fairly robust under a range of conditions.

**Table 6.** Matrix effects (%) of PMZ and its metabolites in four tissue types (n = 3).



**Table 7.** Stability investigation of target compounds in various tissues, RSDs (%, n = 3).
