*2.4. Chromatography and Mass Spectrometry Parameters*

A Symmetry C18 (100 mm × 2.1 mm i.d., 3.5 μm) was used. The mobile phases were composed of phase A (0.1% formic acid solution in water) and phase B (acetonitrile, ACN). A flow rate of 0.3 mL/min was maintained with a gradient elution procedure, as presented in Table 1.



The mass spectrometer was operated in positive ion mode (ESI+), utilizing a multiple reaction monitoring (MRM) scan mode. The primary operating parameters are presented in Table 2.

**Table 2.** Operating parameters for mass spectrometer.


The standard stock solution from Section 2.3 was diluted to 1 μg/mL with ACN and injected directly into the spectrometer for mass spectrometric optimization. Molecular ion peaks of the target analytes and the internal standard were identified by full-scan mass spectrometry in positive ion mode: m/z was 285.2 for PMZ, 301.3 for PMZSO, 271.3 for Nor1PMZ, and 291.3 for PMZ-d6. Each precursor ion underwent MS/MS scanning to determine and evaluate monitored ions for each analyte as a quantitative ion and a qualitative ion. The operation parameters for each ion were optimized using the mass spectrometric scan mode of multiple reaction monitoring (MRM). Consequently, the m/z of 86.2 and 198.1 were established as the quantitative and qualitative ions for PMZ, 198.2 and 239.1 for PMZSO, 197.9 and 240.3 for Nor1PMZ, and 92 and 240.3 for PMZ-d6. The quantitative and qualitative ion pairs, declustering potential, and collision energy for each target compound are listed in Table 3. For quantification, PMZ and Nor1PMZ utilized PMZ-d6 as the internal standard, while PMZSO employed an external standard method.

#### *2.5. Sample Preparation*

The blank matrix used in this study came from the muscles, liver, kidneys, and fat of several different pigs and was not mixed during the processing.

Approximately 500 g of muscle, liver, and kidney samples had connective tissue, blood vessels, and fat removed before being chopped into a uniform slurry using a homogenizer. About 5.0 g ± 0.1 g of this slurry was weighed into a 50 mL centrifuge tube, mixed with

100 μL of PMZ-d6 internal standard working solution (1 μg/mL), vortexed for 30 s, and left to stand for 30 min. After adding 10 mL of 0.1% formic acid in acetonitrile, the mixture was vortexed for 1 min and shaken for 10 min at 100% speed on a platform shaker before being centrifuged at 10,000 rpm for 10 min. The supernatant was transferred to a pear-shaped bottle. Another 10 mL of 0.1% formic acid acetonitrile was added to the residue in the centrifuge tube, and the above steps were repeated for a second extraction. Both extraction liquids were collected in a pear-shaped bottle for purification and concentration.

**Table 3.** Qualitative and quantitative ion pairs, declustering voltage, collision energy, and retention time for analytes and internal standard.


Note: The sub ions marked with "\*" are the quantification ions.

Around 500 g of subcutaneous fat from pig, free from muscle and connective tissue, was homogenized to produce a uniform slurry. About 5.0 ± 0.1 g of this fat slurry was weighed into a 50 mL centrifuge tube, into which 100 μL of PMZ-d6 internal standard working solution (1 μg/mL) were added, before being vortexed for 30 s and left to stand for 30 min. Then, 10 mL of acetonitrile saturated n-hexane was added, vortexed until the fat was completely dissolved, and left to stand for 30 min. After adding 10 mL of 0.1% formic acid in acetonitrile, the fat mixture was vortexed for 1 min and shaken for 10 min at 100% speed on a platform shaker, before being centrifuged at 10,000 rpm for 10 min. The upper hexane layer was discarded, and the lower extraction liquid was transferred to a new 50 mL centrifuge tube for purification.

The extraction liquids of muscle, liver, kidney, and fat were added to 10 mL of acetonitrile-saturated n-hexane and vortexed for about 30 s to mix. After settling, the upper hexane layer was discarded and the lower extraction liquid was added to 10 mL of anhydrous ethanol. This was then reduced in volume by using a rotary evaporator at 45 ◦C, then 5 mL of 0.1% formic acid water–acetonitrile was added and vortexed for 30 s to dissolve the residue completely. After this, 5 mL of n-hexane-saturated acetonitrile was added to the solution and vortexed to mix, then left to stand for layering. Approximately 1 mL of the lower solution was transferred to a 1.5 mL centrifuge tube and centrifuged at 14,000 r/min, 0 ◦C, for 10 min. The clarified middle liquid was filtered using 0.22 μm nylon syringe filters, sealed in an autosampler vial, and stored at 4 ◦C for analysis.
