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

UHPLC-MS/MS analysis of antimicrobials in feed

Authors

  • Elizabeta Dimitrieska Stojkovikj Ss. Cyril and Methodius University in Skopje, Faculty of Veterinary Medicine - Skopje, Lazar Pop Trajkov 5-7, 1001 Skopje, North Macedonia https://orcid.org/0000-0001-8315-5267
  • Dushica Koceva Ss. Cyril and Methodius University in Skopje, Faculty of Veterinary Medicine - Skopje, Lazar Pop Trajkov 5-7, 1001 Skopje, North Macedonia https://orcid.org/0009-0004-5777-9593
  • Aleksandra Angjeleska Ss. Cyril and Methodius University in Skopje, Faculty of Veterinary Medicine - Skopje, Lazar Pop Trajkov 5-7, 1001 Skopje, North Macedonia https://orcid.org/0000-0002-7109-2681
  • Goran Stojković Ss. Cyril and Methodius University in Skopje, Faculty of Natural Sciences and Mathematics, Institute of Chemistry, Arhimedova 5, 1001 Skopje, North Macedonia https://orcid.org/0009-0004-9207-0985
  • Biljana Stojanovska Dimzoska Ss. Cyril and Methodius University in Skopje, Faculty of Veterinary Medicine - Skopje, Lazar Pop Trajkov 5-7, 1001 Skopje, North Macedonia https://orcid.org/0000-0003-1688-6179
  • Zehra Hajrulai-Musliu Ss. Cyril and Methodius University in Skopje, Faculty of Veterinary Medicine - Skopje, Lazar Pop Trajkov 5-7, 1001 Skopje, North Macedonia https://orcid.org/0000-0003-4999-9022

DOI:

https://doi.org/10.20450/mjcce.2025.3166

Keywords:

Antimicrobial substances, poultry feed, multi-class UHPLC-MS/MS method, matrix effect, stability study.

Abstract

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.

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2025-12-22 — Updated on 2025-12-24

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Dimitrieska Stojkovikj, E., Koceva, D., Angjeleska, A., Stojković, G. ., Stojanovska Dimzoska, B., & Hajrulai-Musliu, Z. (2025). Multi-class analysis of antimicrobial substances in poultry feed at cross-contamination levels by UHPLC-MS/MS – Method establishment, validation, and application: UHPLC-MS/MS analysis of antimicrobials in feed. Macedonian Journal of Chemistry and Chemical Engineering, 44(2), 195–213. https://doi.org/10.20450/mjcce.2025.3166 (Original work published December 22, 2025)

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Vol. 44 No. 2 (2025)

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Analytical Chemistry

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Copyright (c) 2025 Elizabeta Dimitrieska Stojkovikj, Dushica Koceva, Aleksandra Angjeleska, Goran Stojković, Biljana Stojanovska Dimzoska, Zehra Hajrulai-Musliu

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