Abstract
A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous analysis of three different classes of veterinary drugs—antibiotics, antifungals, and antiparasitics—in legume-based alternative protein samples, e.g. beans, peas, and nuts. The sample preparation process utilized a modified dilute-and-shoot (DnS) technique, achieving recoveries ranging from 72.08 to 108.11% for 18 of the 26 target analytes. The method demonstrated excellent repeatability (n = 12) with relative standard deviations (RSD) between 1.39 and 9.26%, and intermediate precision (over three days, n = 18) ranging from 5.76 to 19.94%. Limits of detection (LOD) and quantification (LOQ) ranged from 0.04 to 15.64 ng.g−1 and 0.10 and 47.40 ng.g−1, respectively, with good linearity. The optimized method was applied to 97 legume samples (primary and processed products) originating from domestic and international markets. Occurrence analysis revealed that all analytes monitored were below the detection limits, suggesting that antimicrobial contamination in legumes-based alternative protein products is low within the Association of Southeast Asian Nations (ASEAN) region.
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The authors declare that all data supporting the findings of this study are available in the paper.
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Acknowledgements
This research has received funding support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation (PMU-B) [grant number B13F660129], Thailand Science Research and Innovation (TSRI) Fundamental Fund, fiscal year 2026, and the National Science, Research and Innovation Fund, Thailand Science Research and innovation (TSRI) (Grant No.: FFB670076/0337). This research has received funding support from the European Union’s Horizon Europe research and innovation program under the Marie Skłodowska-Curie grant agreement No 101131125 - Mycobeans. Additionally, this study was financially supported by Bualuang Chair Professor Fund (contract number TUBC 08/2022). The authors wish to express their gratitude to the National Center for Genetic Engineering and Biotechnology (BIOTEC) at National Science and Technology Development Agency (NSTDA), Thailand for providing the facilities for the project. The authors also wish to thank the master's student, Miss Warissara Kasikonsunthonchai, for her assistance in quantifying certain studies.
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C.B.: Development and analysis, Validation methodology, Data curation, Writing—original draft, Figures Preparation. U.W.: Investigation methodology, Conceptualization, Writing—review and editing. N.W.: Analysis, Software, Data curation. K.S.: Analysis, Data curation. B.G., N.K., R.P., and D.B. Writing—review and editing. C.T.E.: Writing—review and editing, Resources, Supervision. A.P.: Writing—review and editing, Resources, Funding acquisition, Project administration. All authors have read and agreed to the published version of the manuscript.
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Professor Chris Elliott, one of the co-authors, serves as Co-Editor-in-Chief of npj Science of Food. He was not involved in the peer review process for this manuscript. The remaining authors declare no competing interests.
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Boonkanon, C., Uawisetwathana, U., Waesoh, N. et al. A novel LC-MS/MS multi-group method for simultaneous determination of antimicrobial residues in legume-based alternative proteins. npj Sci Food (2026). https://doi.org/10.1038/s41538-025-00678-3
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DOI: https://doi.org/10.1038/s41538-025-00678-3
