Methenamine, a urinary antiseptic with antimicrobial properties, decomposes into toxic formaldehyde under acidic conditions. Its use is prohibited in dairy cattle in Korea to prevent harmful residues in milk. This study was designed to develop and validate a sensitive and reliable LC–MS/MS method for determining methenamine in raw milk and bovine muscle in compliance with the Positive List System (PLS) regulations. Samples were extracted with acetonitrile (ACN)–methanol (MeOH) (7:3, v/v) containing ammonia water, followed by defatting with n-hexane and purification with primary secondary amine (PSA). Chromatographic separation was performed on a hydrophilic interaction liquid chromatography (HILIC) column, and quantification was conducted using matrix-matched calibration to minimize matrix effects. The method showed excellent linearity (R² > 0.999), low limits of quantification (LOQ) (0.49 μg/kg for raw milk; 0.64 μg/kg for bovine muscle), and acceptable recoveries (78.1–102.8%) with precision (CV ≤ 8.75%), meeting Codex CAC/GL 71-2009 criteria. Stability studies demonstrated that methenamine remained stable in stock solutions, working standards and processed extracts under the storage and handling conditions used. Application to incurred samples resulted in the detection of methenamine in 2 of 32 raw milk samples (0.65 and 1.14 μg/kg) but in none of the 25 bovine muscle samples, with all detected levels below the Korean PLS limit. These findings confirm that the developed method is accurate, sensitive, and applicable for routine surveillance of methenamine residues to ensure consumer safety.

This study evaluated the fitness for purpose of official Korean Food Code LC-MS/MS methods for five prohibited veterinary drugs and one major metabolite—pyrimethamine, dapsone, monoacetyldapsone, roxarsone, chloramphenicol, and vancomycin—in bovine, porcine, and chicken muscle and applied the evaluated methods to nationwide monitoring of production-stage samples. In 2024, dapsone, monoacetyldapsone, pyrimethamine, and roxarsone were analyzed in 202 muscle samples, whereas in 2025, chloramphenicol and vancomycin were analyzed in 300 muscle samples (total n = 502). Analytical performance was assessed for selectivity, linearity, limits of detection (LOD) and quantification (LOQ), recovery, repeatability, inter-day precision, and between-analyst performance; all analyte–matrix combinations met the predefined acceptance criteria. For chloramphenicol, an LOQ of 0.15 μg/kg was achieved in all three matrices, equal to the European Union reference point for action. An external proficiency test for chloramphenicol in bovine muscle yielded a satisfactory z-score of −0.30. No sample met the regulatory reporting criterion for any target analyte included in the respective annual survey. These findings support the fitness for purpose of the evaluated official methods and provide baseline analytical and occurrence data for surveillance of selected prohibited substances in production-stage meat in Korea.