Abstract: This study addresses critical food safety risks arising from disinfectant residues and their highly toxic transformation by-products in milk powder processing. As a staple nutrient source for all age groups, especially infants and young children, milk powder requires rigorous microbial control throughout the production chain; intensive, high-frequency sanitation practices, including clean-in-place (CIP) cleaning, equipment surface disinfection, and environmental hygiene management, are indispensable to ensure production safety, yet can leave residual chemicals on equipment, transfer pipelines, and process water systems, while subsequent concentration, evaporation, and spray drying steps further enrich trace contaminants in the final product, significantly elevating potential dietary exposure risks for consumers. Quaternary ammonium compounds (QACs), a class of cationic surfactants with broad-spectrum antimicrobial efficacy, are ubiquitously used in cleaning and disinfection procedures within the global dairy industry, whereas halobenzoquinones (HBQs) are a group of emerging highly toxic disinfection by-products, with well-documented cytotoxicity and genotoxicity over 1β000 times higher than traditional regulated disinfection by-products such as trihalomethanes and haloacetic acids. However, HBQs remain largely unincorporated into existing global food safety regulatory frameworks. Both compound classes are closely linked to a range of adverse health outcomes, including oxidative stress induction, cellular antioxidant system disruption, DNA adduct formation, and chromosomal aberrations in mammalian cells. Current analytical methods for these target contaminants are severely limited by narrow target coverage, insufficient detection sensitivity, long analytical cycles, and pronounced matrix interference from the complex composition of milk powder, which is characterized by high levels of proteins, lipids, and other co-extracted matrix components. Notably, most reported methods only target a small number of common QACs, and no validated analytical method for simultaneous determination of QACs and HBQs in milk powder has been reported to date, creating an urgent need for a robust, sensitive, high-throughput multi-residue analytical platform to support systematic exposure assessment and regulatory monitoring. Here, we developed and fully validated a comprehensive analytical method for simultaneous quantification of 31 target analytes (18 QACs, 2 halogenated disinfectants, and 11 HBQs) in milk powder, which integrates a modified QuEChERS sample preparation workflow with ultra performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry (UPLC-Orbitrap HRMS), a platform that offers superior mass accuracy, anti-interference capability, and qualitative confirmation performance for trace contaminants in complex matrices; milk powder samples were reconstituted with ultrapure water, followed by extraction with 1% formic acid in acetonitrile via vortex mixing and ultrasonic-assisted extraction, and after low-temperature centrifugation, one supernatant aliquot was directly analyzed for QACs, while the other was purified using an optimized PSA/C₁₈/GCB mixed sorbent system, with sorbent ratios optimized via the Box-Behnken response surface methodology to maximize analyte recoveries and minimize matrix effects, while chromatographic separation was achieved on a Waters HSS T3 column, with an upstream trap column to eliminate system background contamination, and optimized mobile phase systems and gradient elution ensured baseline resolution of four pairs of critical HBQ isomers, with full-scan/data-dependent MS/MS acquisition performed in positive (for QACs) and negative (for HBQs) electrospray ionization modes, and accurate quantification achieved via matrix-matched calibration to offset ionization interference. Full method validation demonstrated excellent analytical performance: all 31 analytes exhibited strong linearity (R²>0.999) over their tested concentration ranges, with matrix-matched recoveries of 68.4%‒105.4% and acceptable intra- and inter-day relative standard deviations (RSD<7.8%), while limits of detection and quantification fully met the requirements for trace-level contaminant analysis in national food safety monitoring; application to 64 batches of commercial milk powder revealed no detectable halogenated disinfectants or HBQs, while QACs were detected in 79.7% of samples, with 8 individual QACs identified and a maximum BAC-C₁₂ concentration of 68.1 μg/kg, and notably, 3 detected QACs are not included in current national supplementary testing methods for dairy products, highlighting critical gaps in existing monitoring frameworks. This sensitive, robust, high-throughput method fills key technical gaps in dairy contaminant analysis, providing a reliable technical platform for systematic contamination assessment, dietary exposure evaluation, and evidence-based development of food safety standards and regulatory supervision practices.

Key words: QuEChERS, ultra performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry (UPLC-Orbitrap HRMS), halobenzoquinones (HBQs), quaternary ammonium compounds (QACs), disinfection by-products (DBPs), milk powder