A novel LC-MS/MS multi-group method for simultaneous determination of antimicrobial residues in legume-based alternative proteins

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.