Multi-class analysis of antimicrobial substances in poultry feed at cross-contamination levels by UHPLC-MS/MS – Method establishment, validation, and application

ABSTRACT The Commission Delegated Regulation (EU) 2024/1229 establishes maximum limits (MLs) for the cross-contamination of antimicrobial substances in feed intended for food-producing animals. This study presents the development and in-house validation of two multi-class analytical methods for detecting antimicrobial substances in poultry feed at cross-contamination levels, using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Methods with and without solid-phase extraction (SPE) purification were developed for 26 antimicrobial substances across eight classes, covering a concentration range of 5–300 µg kg–1. The established methods were validated for parameters including selectivity, limit of quantification, within- and between-day precision, accuracy, decision limit, matrix effect, and short-term analyte stability in the corresponding extracts. Both methods met the required performance criteria for detecting antimicrobial substances at cross-contamination levels, except for reduced sensitivity to lincomycin in the SPE method. The between-day precision was below 18 % and 19 %, with recoveries ranging from 95 to 103 %, for the method without SPE, and from 88 % to 101 % for the method with SPE, respectively. The analytes remained stable after two days of storage in the dark under two temperature regimes. These methods were applied to 39 poultry feed samples, revealing that approximately 41 % contained one or more antimicrobial substances, some of which exceeded the cross-contamination MLs. This study underscores the importance of simple, rapid, sensitive, and reliable analytical methods for controlling the presence of antimicrobials at cross-contamination levels. Such methods enable stringent control to prevent uncontrolled antimicrobial use in poultry production systems, thereby mitigating the development of antimicrobial resistance.