**4. Discussion**

Thiophene compounds encompass amino groups, which, when dissociated in water, exhibit alkalinity. These compounds may be adsorbed by residual silicon hydroxyl groups present on the surface of the stationary phase of a chromatographic column. To address this issue, the selection of fully end-capped C18, phenyl, and C8 chromatographic columns is recommended. The Symmetry C18 column (100 mm × 2.1 mm i.d., 3.5 μm, Waters, Milford, MA, USA) was utilized for separation in this investigation. In LC–MS/MS analysis, the ESI+ mode is favorable for alkaline PMZ and its metabolites, while acidic mobile phase systems tend to form [M + H] <sup>+</sup> ions. Acetonitrile and water, which are frequently used as mobile phases, can be proportioned according to specific requirements. Formic acid or acetic acid serve as typical protonation reagents in the LC–MS mobile phase. This study examined the effects of introducing different ratios of formic acid or acetic acid into the mobile phase. It was discovered that acetic acid increased the baseline of the Nor1PMZ representative ion chromatogram, rendering it unsuitable. The most optimal retention time and representative ion chromatograms for the analytes were achieved by adding 0.1% (by volume) formic acid to the aqueous phase.

Matrix effects from animal tissue samples can interfere with the accuracy of drug content analysis in tissues. The internal standard method is routinely employed to mitigate matrix effects and significantly enhance the accuracy and precision of the analysis. Numerous studies have reported the use of the internal standard method in determining PMZ and its metabolite content. Metronidazole served as the internal standard for estimating PMZ and PMZSO in rat plasma and various tissues [36]. PMZ-d6 and PMZSO-d6 were utilized as internal standards to detect the content of PMZ and PMZSO in pig muscle, liver, and kidney [43]. Donepezil was used to detect drugs, include PMZ, in human plasma and urine [31]. Haloperidol was reported to be used as internal standard to quantify chlorpromazine and PMZ in pig kidneys [21], and loratadine was used as internal standard when

studying PMZ and ephedrine mixture [38]. The PMZ-d6, a deuterated isotope of PMZ, was employed as the internal standard for quantification in this study.

In the research work, it was found that PMZ, PMZSO, Nor1-PMZ, and PMZ-d6 stock solutions and working solutions were stable long-term at −20 ◦C and 4 ◦C, and were stable at room temperature and during the sample preparation process. However, after evaporating the solvent of PMZ-d6 working solution, the response value of PMZ-d6 detected by LC–MS/MS significantly decreased after one week at room temperature and exposed to the air. Therefore, we sealed and stored the solution containing PMZ-d6 in the refrigerator. After the solvent of the sample solution containing PMZ-d6 is evaporated by a rotary evaporator, it should be immediately re-dissolved, sealed, and stored at 4 ◦C.

It was found that the recovery of PMZSO was generally significantly high (>120%) while quantified by PMZ-d6 with internal standard method, though the recovery of PMZ and Nor1PMZ was in the range of 80–120%. After investigations, it was found that, in spiked samples, the actual extraction recovery of PMZ, PMZ-d6, and Nor1PMZ were all between 60% and 70%, which were very close. However, the actual extraction recovery of PMZSO was above 85%, which was significantly different from the internal standard and other analytes, as shown in Figure 3. As such, PMZ-d6 is unsuitable for quantification analysis of PMZSO. As a metabolite, PMZSO shows stronger polarity than PMZ, with its chemical properties differing from those of PMZ, PMZ-d6, and Nor1PMZ. Finally, the internal standard method was used for quantifying PMZ and Nor1PMZ, and the external standard method was used for quantifying PMZSO.

Based on the physicochemical characteristics of the target analytes in this study, along with evidence from previous studies [33,41,46], several extraction solvents, including ACN, 0.1% formic acid in ACN, ethyl acetate—ACN (20:80, *v*/*v*), and 1% ammoniated ACN, were investigated for their extraction recovery efficacy in pig tissues. Results indicated that formic acid–acetonitrile combination exhibited the most efficient extraction recovery across all analytes, as depicted in Figure 3. Considering the efficiency of extraction for PMZ, PMZSO, Nor1PMZ, and PMZ-d6 across four tissue samples, 0.1% formic acid in ACN was utilized as the extraction solvent for this study. It was observed that the extraction efficiency could be boosted by adding a slight amount of acid. However, with an increasing increment in formic acid volume, the extraction liquid for liver and kidney became darker, harboring more impurities, and, thus, posing interference in instrument detection. Consequently, an optimal extractant ratio of 0.1% formic acid in ACN was established.

Fat tissue is a significant animal source food and one of the target tissues for monitoring drug residues. However, fat samples pose challenges in sample preparation and detection procedures due to their high lipophilic impurity content. The extraction recovery of analytes in fat is generally low. In this study, various procedures were explored to enhance extraction and purification efficacy in fat samples. It was determined that complete dissolution of the fat sample slurry in n-hexane prior to analyte extraction improved extraction recovery. During the sample concentration and purification process, the lipid-rich impurity content in the sample solvent could be discarded through extraction with n-hexane, both before and after the extraction solvent was removed using a rotary evaporator. Centrifuging the sample solution at 0 ◦C or lower facilitated the upward migration of lipid-interfering substances. Finally, the parameters of the fat tissue detection method complied with the requirements of technical guiding principles.

#### **5. Conclusions**

We have, for the first time, developed and validated an LC–MS/MS method for the determination of promethazine and its two metabolites across all edible tissues of swine, in accordance with the Ministry of Agriculture and Rural Affairs of China's technical guiding principles. In this method, 0.1% formic acid in acetonitrile served as the extraction solvent, and LC–MS/MS was used for analyte detection. The limit of quantification ranged between 0.1 μg/kg and 0.5 μg/kg, demonstrating a sensitivity equal to or surpassing previous reports. This study included swine fat as a research subject for the first time and Nor1-PMZ

as one of the target analytes, thereby presenting an accurate and reliable detection method for monitoring PMZ residues and its metabolites in swine edible tissues.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/foods12112180/s1, Table S1. Reports of analysis methods on PMZ and its metabolites in edible animal tissues [21,33,34,41,43,47–52]; Table S2. Original Data of Recovery and precision of spiked blank samples.

**Author Contributions:** Investigation, D.W., R.S., H.H., R.C., Y.Z. and H.C.; methodology, H.C., D.W. and R.L.; validation, D.W., R.S., H.H., R.C. and Y.Z.; writing—original draft preparation, D.W. and R.C.; writing—review and editing, H.C.; project administration, H.C.; funding acquisition, H.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Agriculture and Rural Affairs of China's FORMULATION AND REVISION OF NATIONAL STANDARDS FOR VETERINARY DRUGS, grant number: Formulation and revision of agricultural industry standards 125C0701.

**Data Availability Statement:** There were no publicly archived datasets created during the study.

**Acknowledgments:** The instrument parameters presented in this article were inspired by the Master's thesis of Ting Peng: Pharmacokinetic and Residue Elimination Study of Promethazine in Sheep.

**Conflicts of Interest:** The authors declare no conflict of interest.
