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Pesticides and veterinary pharmaceuticals are used for effective crop production and prevention of livestock diseases; these chemicals are released into the environment via various pathways. Although the chemicals are typically present in trace amounts post-release, they could disturb aquatic ecosystems and public health through resistance development toward drugs or diseases, e.g., reproductive disorders. Thus, the residues of pesticides and veterinary pharmaceuticals in the environment must be managed and monitored. To that end, we developed a simultaneous analysis method for 41 target chemicals in environmental water samples using ultra-high-performance liquid chromatography (UHPLC)–quadrupole-orbitrap high-resolution mass spectrometry (HRMS) coupled with an on-line solid-phase extraction system. Calibration curves for determining linearity were constructed for 10–750 ng∙L−1, and the coefficient of determination for each chemical exceeded 0.99. The method’s detection and quantitation limits were 0.32–1.72 ng∙L−1 and 1.02–5.47 ng∙L−1, respectively. The on-line solid-phase extraction system exhibited excellent method reproducibility and reduced experimental error. As the proposed method is applicable to the monitoring of pesticides and veterinary pharmaceuticals in surface water and groundwater samples acquired near agricultural areas, it allows for the management of chemicals released into the environment.

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