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

Dimitrieska Stojkovikj, Elizabeta; Koceva, Dushica; Angjeleska, Aleksandra; Stojković, Goran; Stojanovska Dimzoska, Biljana; Hajrulai-Musliu, Zehra

doi:DOI: 10.20450/mjcce.2025.3166

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

Journal

Macedonian Journal of Chemistry and Chemical Engineering

Date Issued

2025-12-24

Author(s)

Koceva, Dushica

Angjeleska, Aleksandra

Stojković, Goran

DOI

DOI: 10.20450/mjcce.2025.3166

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 methods were validated for parameters in-cluding 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. 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 un-der two temperature conditions. 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.

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