In modern agricultural production, pesticides are essential for decreasing the prevalence of pests and weeds as well as raising grain yield and income [1], and their residues continue to infiltrate the environment as a result of widespread use, endangering the health of humans and animals, as well as causing extensive damage to soil, water, and other living organisms [2]. The quality and safety of animal products are becoming more and more significant to society as their share in the average diet continually increases. The European Union, the United States, Japan, and other nations and regions have established maximum residue limits (MRLs) for pesticide residues in animal-derived foods in order to prevent pesticide contamination that endangers consumers’ health. The MRLs for 119 pesticides in animal-derived foods, such as eggs, milk, and meat, are specified by the Chinese national standard GB 2763-2021 and range from 0.005 to 2 mg/kg [3]. Nowadays, pesticide residues in animal-derived foods are largely monitored by measuring the amount of residue in animal tissues. However, this method necessitates the slaughter of animals and is unable to monitor pesticide pollution in real time during the breeding process. Animals mostly eliminate pesticides in their urine, and only trace amounts of residue are left in the heart, liver, kidney, muscle, and other tissues [4]. Urine sampling is more ethical and less expensive than tissue sampling because it is easier and less invasive. Therefore, developing analytical techniques for a wide range of pesticide residues in animal urine is critical for tracking pesticide levels in animal-derived foods.

The analysis of multiple pesticide residues in urine is commonly carried out through the coupled technologies of chromatography and mass spectrometry, such as gas chromatography–mass spectrometry (GC–MS) [5,6], liquid chromatography–tandem mass spectrometry (LC–MS/MS) [7], [8], [9], and liquid chromatography–high-resolution mass spectrometry (LC–HRMS) [10,11]. However, GC–MS cannot detect difficult-to-volatilize pesticides. LC–HRMS is relatively expensive and mainly used for screening analytes. LC–MS/MS is widely used for the high throughput and highly selective screening and detection of targeted pesticides. The complexity of urine's composition, which includes high levels of urea, sodium chloride, proteins, and other small molecules resulting from metabolism, may explain why detecting pesticide residues in this matrix is challenging. In contrast to other matrices such as food and environmental water, only a limited number of methods have been developed for analyzing pesticide residues in urine. To establish an efficient analytical method with optimal sensitivity for daily monitoring, the pretreatment of urine samples must take into account the relationship between co-extracts, purification level, and enrichment factors. Although solid-phase extraction (SPE) is the most commonly utilized pretreatment option [7,11,12], it remains challenging to identify a solid-phase material that can be employed for the simultaneous analysis of hundreds of pesticides possessing different chemical properties. Dispersive liquid–liquid microextraction (DLLME) has been utilized for detecting pesticide residues in human urine due to its ease of use and low cost [9,13,14]. However, it is challenging to use this method to analyze hundreds of pesticides because of extractant limitations. The QuEChERS approach, which was initially put forth by Anastassiades and associates, has a number of benefits, including being quick, easy, affordable, reliable, and robust. Based on the aforementioned benefits, QuEChERS has been used to analyze pesticide residues in human urine, even though it lacks a sample concentration step or has a lower concentration coefficient than SPE and DLLME [6,8,10].

The literature focuses on human urine as a research object for developing biomonitoring methods that will provide effective tools for assessing the health risks of pesticides to occupationally and non-occupationally exposed populations. To the best of our knowledge, the detection methods for the simultaneous analysis of hundreds of pesticide residues in livestock urine have not been reported, nor has the exposure assessment of pesticide residues in livestock. Thus, as target analytes, this study chose pesticides that have MRLs in animal-derived foods, are commonly used in agricultural production, and have specific harmful effects on human health. An analytical method for 106 pesticide residues in livestock urine was developed, and the exposure assessment was carried out based on the monitored pesticide residue levels. A modified QuEChERS cleanup combined with liquid chromatography–tandem mass spectrometry was established for the determination of multiple pesticide residues. The method-validation results proved its suitability for the analysis of pesticide residues in livestock urine, which could provide an approach for monitoring pesticide pollution in animal breeding.