Development and Validation of the Multi-Residue Method for Identification and Quantitation of Six Macroide Antiparasitic Drugs  
[PERSON]  
1 College of Veterinary Medicine, China Agriculture University, Beijing 100193, China; [EMAIL_ADDRESS] (C.C.); [EMAIL_ADDRESS] (Y.C.); [EMAIL_ADDRESS] (X.L.); [EMAIL_ADDRESS] (Y.L.); [EMAIL_ADDRESS] (Q.Q.) 1536  
derivatives include avermectin (AVM), ivermectin (IVM), doramectin (DOR), and environmectin (EPR, also known as acetyl avermectin) ([PERSON] et al., 2015) [2]. These compounds have gained widespread application in animal husbandry and agricultural production due to their novel chemical structures, unique mode of action, high safety profile, exceptional efficacy, and low toxicity. Similarly, milbemycins represent another critical class of antiparasitic agents used in livestock production and cultivation. The primary members of this group include milbemycin oxime (MIL) and moxidectin (MOX). While both avermectins and milbemycins share some functional similarities, they exhibit distinct differences in their chemical structures and physical properties, which influence their specific applications and efficacy. The chemical structures of these six compounds are illustrated in Figure 1.  
Macrolides, while effective against harmful insects and parasites, demonstrate mammalian toxicity ([PERSON] & [PERSON], 2012) [3]. According to World Health Organization (WHO) regulations, avermectins (AVMs) are classified as highly hazardous compounds with significant neurotoxic potential. Animals with elevated AVM concentrations may exhibit central nervous system (CNS) toxicity symptoms, including dyskinesia, respiratory depression, and tremors. The specific toxicological manifestations show slight interspecies variations ([PERSON] & [PERSON], 2016) [4].  
The irrational use of veterinary drugs may lead to drug accumulation in livestock and residues in animal-derived food products, posing direct and indirect risks to human health. In the year 2020, China's Ministry of Agriculture and Rural Affairs issued Announcement No. 194, which prohibited the direct use of antibiotics in animal feed. This regulation aims to reduce the negative impacts of antibiotic misuse and ensure the safety of animal-derived foods and public health. Macrolide drugs are widely used in animal husbandry and agriculture. However, repeated or improper application, including off-label use, may result in excessive drug levels in feed. Livestock can be exposed to macrolide antiparasitic agents through multiple pathways, such as residues in plant-based feed, feed additives, and veterinary drug administration. Such exposure can lead to bioaccumulation in animal tissues, ultimately entering the human food chain and posing health risks.  
This study aimed to develop a robust and sensitive LC-MS/MS method for the simultaneous determination of six macrolide antiparasitic drugs (EPR, AVM, IVM, DOR, MOX, and MIL) in various feed matrices, including premix, concentrate, and complete feed, commonly used in aquaculture. The performance was comprehensively validated by  
Figure 1: Molecular structures of six macrolides drugs.  
evaluating key parameters such as specificity, linearity, recovery, precision, and sensitivity to ensure compliance with regulatory and analytical standards. This validated method provides a reliable approach for monitoring drug residues in feed samples, offering technical support for promoting safe aquaculture practices and protecting public health.