**1. Introduction**

Promethazine (PMZ) is a first-generation antihistamine drug known for its anti-allergic properties. It exhibits additional central inhibitory effects on the subcortical regions of the brain, resulting in significant central sedation, hypnotic, antiemetic, and antipyretic effects, making it commonly used for sedation and sleep [1–3]. In China, PMZ is approved for treating allergic reactions in animals such as sheep and pigs, including urticaria and serum

**Citation:** Wen, D.; Shi, R.; He, H.; Chen, R.; Zhang, Y.; Liu, R.; Chen, H. Development and Validation of a High-Performance Liquid Chromatography–Tandem Mass Spectrometry Method to Determine Promethazine and Its Metabolites in Edible Tissues of Swine. *Foods* **2023**, *12*, 2180. https://doi.org/10.3390/ foods12112180

Academic Editor: Rosaria Saletti

Received: 23 April 2023 Revised: 25 May 2023 Accepted: 27 May 2023 Published: 29 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

sickness. Occasionally, a small number of farmers illegally use it in the breeding process of food animals in order to reduce animal movement, speed up weight gain, or reduce stress reactions during transportation [4].

There have been reports of adverse reactions due to PMZ abuse in humans, including drug-induced mental disorders and cardiovascular diseases in certain individuals [5–7]. However, the illegal use of PMZ in animal feed and breeding can also pose health hazards to consumers through drug residues in animal-derived foods and result in environmental pollution and other risks [8,9]. Chinese Ministry of Agriculture Announcements No. 176 and No. 2583 prohibit the use of promethazine hydrochloride in animal feed and drinking water. In March 2010, the Chinese Ministry of Health published the fourth batch of "non-food substances that may be illegally added to food and food additives that are easily abused" list, which included promethazine. Regulations in Japan, the United States, and the European Union also prohibit the residuals of thiazine tranquilizers and their metabolites in animal-derived foods. Furthermore, the use of PMZ formulations in food animals has not been approved in the European Union, the United States, and other countries and regions. The Ministry of Agriculture and Rural Affairs of China, in order to ensure the safety of animal-derived food and regulate the use of veterinary drugs, has arranged research projects which include PMZ residue studies. We were fortunate to participate in these research projects, to establish a detection method for PMZ and its metabolites in accordance with the Ministry of Agriculture and Rural Affairs of China's No. 326 Announcement "Guiding Principles for Veterinary Drug Residue Elimination Tests" and the "Technical Guiding Principles for Quantitative Analysis Method Validation of Biological Samples", released on 20 June 2022. In these technical guiding principles, experimental approaches, standards, parameters, and reference threshold values for detection method comply with the current international norms, such as COMMISSION IMPLEMENTING REGULATION (EU) 2021/808 of 22 March 2021.

According to previous reports, PMZ is primarily metabolized by CYP450 enzymes in animals [10–12]. Studies on PMZ metabolism in pig tissues seem to be scarce; no literature on PMZ metabolism in pigs was found. However, from the existing literature (see Table S1), PMZ metabolizes into five to eight metabolites, including PMZSO and Nor1PMZ in humans, rats, and mice. PMZSO and Nor1PMZ appear to be stable when present and account for a high proportion of metabolites which can be found in humans, rats, and mice. If drugs metabolize in mammals through CYP450 enzymes, there is a certain similarity in the metabolic pathways. Hence, we initially attempted to establish an LC–MS/MS analytical method for PMZ, PMZSO, and Nor1PMZ in pig plasma and tissues, then carried out a dosing trial in three experimental pigs. After a single intramuscular injection of PMZ, PMZ and its metabolites PMZSO and Nor1PMZ were found in the plasma of all three pigs. Ten days after the injection, PMZ, PMZSO, and Nor1PMZ were still present in plasma and tissue above the limit of quantification. Therefore, we eventually chose PMZ and its metabolites PMZSO and Nor1PMZ as the target analytes. Nor1PMZ was chosen as a target compound of analysis on drug residues in edible tissues for the first time in this study.

Various methods have been employed to detect PMZ, including enzyme-linked immunosorbent assay [13,14], spectroscopy [15–17], capillary electrophoresis [18,19], high performance liquid chromatography [20,21], gas chromatography–mass spectrometry [22–29], and liquid chromatography–tandem mass spectrometry (LC–MS/MS) [30–43]. However, most of the reported detection methods are used for PMZ formulations or detecting illegally added PMZ in animal feed [32,35,44,45]. Only a handful of methods have been developed to detect PMZ residues, or PMZ along with one of its metabolites, PMZSO, in animalderived foods [14,21,33,37,41,43,44]. These methods are applicable to only certain edible tissues such as muscle, liver, and kidney. Notably, previous studies have not included fat tissue, which is an important animal source food. Therefore, in this study, fat tissue was included as a research object for the first time, considering its significance as an animal source food and as one of the target tissues for monitoring drug residues in food. The objective of this study was to establish a sample preparation and LC–MS/MS method for

detecting PMZ and two of its metabolites in all edible tissues of swine, in order to provide technical support for monitoring PMZ and its metabolites in swine edible tissues, ensuring food safety.
