Multi-residue method for the determination of residues of nitroimidazoles, tetracyclines, sulfonamides and trimethoprim in honey by liquid chromatography–tandem mass spectrometry

Kamila Mitrowska, Department of Pharmacology and Toxicology, National Veterinary Research Institute (PIWet), Pulawy, Poland (kamila.mitrowska@piwet.pulawy.pl)

Nitroimidazoles, tetracyclines, sulfonamides and trimethoprim are a group of antibacterial compounds that have been applied in apiculture to prevent and control bacterial and protozoan diseases such as American foulbrood, European foulbrood and nosemosis. However, the use of antimicrobial substances in commercial beekeeping is not allowed in the European Union because there are no Maximum Residue Limits (MRLs) for these drugs in honey. Thus, illegal use of antibacterials in beekeeping could result in an accumulation of their residues in bee products, including honey, and pose a threat to human health. Therefore, to carry out official control, we have developed a multi-residue method for the determination of 12 nitroimidazoles (metronidazole, hydroxymetronidazole, dimetridazole, ronidazole, hydroxydimetridazole, ipronidazole, hydroxyipronidazole, carnidazole, ornidazole, secnidazole, ternidazole, tinidazole), 7 tetracyclines (oxytetracycline, tetracycline, chlortetracycline, doxycycline, 4-epi-oxytetracycline, 4-epi-tetracycline, 4-epi-chlortetracycline), 16 sulfonamides (sulfaguanidine, sulfacetamide, sulfathiazole, sulfadiazine, sulfapyridine, sulfamerazine, sulfamethazine, sulfamethoxypyridazine, sulfameter, sulfamonomethoxine, sulfachloropyridazine, sulfadoxine, sulfamethoxazole, sulfaquinoxaline, sulfadimethoxine, sulfaphenazole) and trimethoprim in honey.

The extraction of the analytes from honey included an acid hydrolysis step with 1 M hydrochloric acid to release the sugar-bound sulfonamides followed by adjustment to pH 4 and clean-up on a Strata X cartridge using solid phase extraction (SPE). The obtained extract was evaporated to dryness, reconstituted in 0.1% formic acid and analysed by a liquid chromatography-tandem mass spectrometer (LC-MS/MS) system with positive electrospray ionization operated in MRM mode. The separation of analytes was performed on a Luna 3µ, C18(2) 100A, 150 x 2 mm analytical column using 0.1% formic acid and 0.1% formic acid in acetonitrile as mobile phases with gradient elution. The proposed multi-residue method was successfully validated according to the EU Commission Decision 2002/657/EC requirements and all validation criteria were in the required ranges. The decision limits (CCα) and detection capabilities (CCβ) for all tested analytes ranged from 2.12 to 2.98 μg/kg and from 2.13 to 3.47 μg/kg, respectively.

Acknowledgements

The project was funded by the National Science Centre allocated on the basis of the decision number DEC-2015/19/B/NZ7/02356.

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Abstract Reference & Short Personal Biography of Presenting Author

Dr. Kamila Mitrowska graduated in medical analytics from the Jagiellonian University Medical College in Krakow in 2002. Since then, she has been working at the National Veterinary Research Institute (PIWet) in Pulawy, where she obtained her Ph.D. degree in veterinary sciences in 2006 and was promoted to associate professor in 2015. She was a post-doc fellow at the Institute for Reference Materials and Measurements (IRMM) in Geel from 2008 to 2010. Currently, Dr. Mitrowska is the First Deputy Head of the Department of Pharmacology and Toxicology of PIWet in Pulawy which acts as the National Reference Laboratory (NRL) for veterinary drug residues and chemical contaminants in food of animal origin. Her research is mainly focused on applications of liquid chromatography coupled to mass spectrometry especially on food safety control and pharmacokinetics of veterinary drugs.