*2.6. Limit of Detection and Limit of Quantification*

To establish the limit of detection (LOD) and limit of quantification (LOQ), a blank tissue sample homogenate (5 ± 0.1 g) was spiked with 100 μL of PMZ-d6 internal standard working solution (1 μg/mL) and 100 μL of mixed standard working solution. Thus, spiked samples at varying concentrations of 0.05 μg/kg, 0.1 μg/kg, 0.5 μg/kg, and 1 μg/kg were prepared. These samples were processed and analyzed by the method described in Sections 2.4 and 2.5. The concentration of the sample with a signal-to-noise ratio (S/N) ≥ 3 was considered the LOD, and the concentration with an S/N ≥ 10 was regarded as the LOQ.

#### *2.7. Calibration Curve and Linearity*

Blank tissue sample slurries (5 ± 0.1 g) were spiked with a 1 μg/mL PMZ-d6 internal standard working solution (100 μL) and a mixed standard working solution (100 μL) to achieve varying concentrations—for PMZ and PMZSO, ranging from 0.1 μg/kg to 50 μg/kg; for Nor1PMZ, ranging from 0.5 μg/kg to 50 μg/kg. These samples were processed and analyzed by the method described in Sections 2.4 and 2.5. The calibration curve and

correlation coefficient (r) were determined using a weighted least-squares method with the ratio of the concentration of PMZ, Nor1PMZ, and PMZ-d6 as the abscissa and the peak area ratio of the quantitative ion pairs of PMZ, Nor1PMZ, and PMZ-d6 as the ordinate, with the weight chosen as 1/X2. The calibration curve and correlation coefficient of PMZSO were obtained using a weighted least-squares method with the concentration of PMZSO as the abscissa and the peak area of the PMZSO quantitative ion pair as the ordinate, with the weight chosen as 1/X2. The experiment was repeated in triplicate.
