Development of an HPLC-MS/MS method for the quantitative determination of chloramphenicol in honey from Kazakhstan

Farida Baktybayeva, Assiya Serikbayeva, Maxat Toishimanov, Maksat Serikov, Ulzhan Nuraliyeva
Received: 10.10.2024 Revised: 19.03.2025 Accepted: 30.04.2025
Retrieved from Vol. 28, No. 5, 2025 Pages: 54-64
Download article Read article

Abstract

Addressing the issue of antibiotics, particularly chloramphenicol, in bee products is an urgent concern within the trade of these items due to potential risks to human health. Developing highly sensitive techniques for detecting trace amounts of antibiotic residues is crucial. This study aimed to establish an analytical method using HPLC-MS/MS (High-Performance Liquid Chromatography – Tandem Mass Spectrometry) for the identification and quantification of residual chloramphenicol in honey samples sourced from Kazakhstan. The developed method involved ultrasonic extraction followed by HPLC-MS/MS with electrospray ionisation in negative mode. Multiple reaction monitoring was utilised in configuring the parameters of the tandem mass spectrometric detector. The proposed procedure offered notable advantages, such as the simplicity and speed of the extraction process, as well as the sensitivity and accuracy of the detection method. The limit of detection achieved is 0.09 µg/kg, with a linear calibration curve spanning the concentration range of 0.1-1.0 µg/kg and a correlation coefficient of 0.9985. The extraction rate ranged between 97% and 101%. Importantly, this HPLC-MS/MS method for chloramphenicol determination in honey surpassed the maximum residue limit requirements (0.3 µg/kg) in terms of sensitivity, while maintaining methodological precision and a high extraction percentage. This compliance aligned with the standards outlined in Commission Decision 2002/657/EC. The method was applied to 14 honey samples from Kazakhstan, all of which were found to have chloramphenicol levels below the method’s detection limit. However, when testing imported honey samples, two out of seven exceeded the detection limit for chloramphenicol. The developed HPLC-MS/MS method for detecting chloramphenicol residues in honey samples can serve as a valuable tool for routine laboratory analysis and for scrutinising potentially suspicious or questionable samples

Keywords

liquid chromatography; mass spectrometry; analysis; antibiotic; honey

  1. Abera, S., Yaya, E.E., & Chandravanshi, B.S. (2025). Development of HPLC-DAD method for the separation and simultaneous determination of ten antibiotic residues in food and environmental samples using surface floating organic droplet-based air-assisted liquid-liquid micro-extraction. Journal of Chromatography A, 1748, article number 465817. doi: 10.1016/j.chroma.2025.465817.
  2. Akter Mou, S., Islam, R., Shoeb, M., & Nahar, N. (2021). Determination of chloramphenicol in meat samples using liquid chromatography–tandem mass spectrometry. Food Science & Nutrition, 9(10), 5670-5675. doi: 10.1002/ fsn3.2530.
  3. Bonerba, E., Panseri, S., Arioli, F., Nobile, M., Terio, V., Di Cesare, F., Tantillo, G., & Chiesa, L.M. (2021). Determination of antibiotic residues in honey in relation to different potential sources and relevance for food inspection. Food Chemistry, 334, article number 127575. doi: 10.1016/j.foodchem.2020.127575.
  4. Cilia, G., Fratini, F., Marchi, M., Sagona, S., Turchi, B., Adamchuk, L., Felicioli, A., & Kačániová, M. (2020). Antibacterial activity of honey samples from Ukraine. Veterinary Sciences, 7(4), article number 181. doi: 10.3390/ vetsci7040181.
  5. Commission decision 2002/657/EC implementing council directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. (2002). Retrieved from https://cutt.ly/tJzMpi4.
  6. Dar, S.A., Bin Farook, U., Rasool, K., & Ahad, S. (2024). Honey: Classification, composition, safety, quality issues and health benefits. In G.A. Nayik, J. Uddin & V. Nanda (Eds.), Advanced techniques of honey analysis: Characterization, authentication, and adulteration, (pp. 1-37). Cambridge: Academic Press. doi: 10.1016/B978-0443-13175-2.00012-X.
  7. Dvykaliuk, R., Adamchuk, L., Antoniv, A., & Bal-Prylypko, L. (2023). Development of safety and quality of propolis as a food raw material. Animal Science and Food Technology, 14(1), 26-48. doi: 10.31548/ animal.1.2023.26.
  8. European Council. (2022). Retrieved from https://www.consilium.europa.eu/en/.
  9. Garibli, A.S., Suleymanov, T.A., Ollivier, E., & Herbette, G. (2021). Flavonoids from Medicago falcata from the flora of Azerbaijan. Chemistry of Natural Compounds, 57(1), 150-151. doi: 10.1007/s10600-02103302-4.
  10. Honey in Kazakhstan. (2020). Retrieved from https://oec.world/en/profile/bilateral-product/honey/ reporter/kaz.
  11. Hutchings, M., Truman, A., & Wilkinson, B. (2019). Antibiotics: Past, present and future. Current Opinion in Microbiology, 51, 72-80. doi: 10.1016/j.mib.2019.10.008.
  12. Ilderbayeva, G., Rakhyzhanova, S., Utegenova, A., Salkhozhayeva, G., & Ilderbayev, O. (2024). Combined effect of gamma radiation and heavy metals on some living organisms. Biological Trace Element Research, 203, 1764-1775. doi: 10.1007/s12011-024-04272-8.
  13. Jung, H.-N., Park, D.-H., Choi, Y.-J., Kang, S.-H., Cho, H.-J., Choi, J.-M., Shim, J.-H., Zaky, A.A, Abd El-Aty, A.M., & Shin, H.-C. (2022). Simultaneous quantification of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in animal and aquaculture products using liquid chromatography-tandem mass spectrometry. Frontiers in Nutrition, 8, article number 812803. doi: 10.3389/fnut.2021.812803.
  14. Khalifa, H.O., Shikoray, L., Mohamed, M.-Y.I., Habib, I., & Matsumoto, T. (2024). Veterinary drug residues in the food chain as an emerging public health threat: Sources, analytical methods, health impacts, and preventive measures. Foods, 13(11), article number 1629. doi: 10.3390/foods13111629.
  15. Kivrak, Ş., & Kivrak, I. (2021). Ultrasonic-assisted extraction method of phenolic compounds in Extra-Virgin Olive Oils (EVOOs) by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC–MS/MS). Separation Science and Technology, 56(2), 322-329. doi: 10.1080/01496395.2020.1713811.
  16. Kobysh, A., Taran, T., Yakubchak, O., Omelchun, Y., & Romanko, M. (2025). Study of the botanical origin of bee honey of Ukraine. One Health Journal, 3(I), 46-59. doi: 10.31073/onehealthjournal2025-I-05.
  17. Kołacz, R., Iakubchak, O., Taran, T., & Hryb, J. (2023). Safety and quality indicators of rapeseed and sunflower honey from different regions of Ukraine. Ukrainian Journal of Veterinary Sciences, 14(1), 39-56. doi: 10.31548/ veterinary1.2023.39.
  18. Li, Y., Li, J., Huang, H., Jian, D., Shan, Y., Wang, S., & Liu, F. (2021). Rapid quantitative detection for multiple antibiotics in honey using a quantum dot microsphere immunochromatographic strip. Food control, 130, article number 108256. doi: 10.1016/j.foodcont.2021.108256.
  19. Lima, C.M.G., Nora, F.M.D., Seraglio, S.K.T., Silva, J.M.D., Marzoque, H.J., Santana, R.F., Verruck, S., & Scussel, V.M. (2020). Antibiotic residues in honey: A public health issue. Research, Society and Development, 9(11), article number e1739119604. doi: 10.33448/rsd-v9i11.9604.
  20. Manimekalai, M., Sheshadri, A., Kumar, K.S., & Rawson, A. (2019). Ultrasound assisted method development for the determination of selected sulfonamides in honey using liquid chromatography-tandem mass spectrometry. Biosciences Biotechnology Research Asia, 16(2), 289-295. doi: 10.13005/bbra/2745.
  21. Phipps, R. (2025). International honey market report. Retrieved from https://apiculture.com/files/17/InternationalHoney-Market/1354/International-Honey-Market-report-March-2025.pdf.
  22. Pratiwi, R., Ramadhanti, S.P., Amatulloh, A., Megantara, S., & Subra, L. (2023). Recent advances in the determination of veterinary drug residues in food. Foods, 12(18), article number 3422. doi: 10.3390/foods12183422.
  23. Rimkus, G.G., Huth, T., & Harms, D. (2020). Screening of stereoisomeric chloramphenicol residues in honey by ELISA and CHARM ® II test – the potential risk of systematically false-compliant (false negative) results. Food Additives and Contaminants: Part A, 37(1), 94-103. doi: 10.1080/19440049.2019.1682685.
  24. Rivera-Mondragón, A., Broeckx, G., Bijttebier, S., Naessens, T., Fransen, E., Kiekens, F., Caballero-George, C., Heyden, Y.V., Apers, S., Pieters, L., & Foubert, K. (2019). Ultrasound-assisted extraction optimization and validation of an HPLC-DAD method for the quantification of polyphenols in leaf extracts of Cecropia species. Science Reports, 9, article number 2028. doi: 10.1038/s41598-018-37607-2.
  25. Rizzo, S., Russo, M., Labra, M., Campone, L., & Rastrelli, L. (2020). Determination of chloramphenicol in honey using salting-out assisted liquid-liquid extraction coupled with liquid chromatography-tandem mass spectrometry and validation according to 2002/657 European commission decision. Molecules, 25(15), article number 3481. doi: 10.3390/molecules25153481.
  26. Rydchuk, M., Plotycya, S., Yanovych, D., Zasadna, Z., Zayarnyuk, A., & Pazderska, O. (2019). The application of new UPLC-MS/MS multimethod of antimicrobials residues determination for the control of honey safety. Scientific and Technical Bulletin оf State Scientific Research Control Institute of Veterinary Medical Products and Fodder Additives аnd Institute of Animal Biology, 20(2), 375-389. doi: 10.36359/scivp.2019-20-2.49.
  27. Saad, S., Fatthalla, M., Abd-Ellah, H., Hagag, E.S., Taha, S.M., Mahrous, A., & Shahba, M.A. (2024). Antibiotics, pesticides, and heavy metals contaminants of honey as affected by antibiotics usage and agricultural practices in different Egyptian environments. Egyptian Pharmaceutical Journal, 23(3), 555-564. doi: 10.4103/ epj.epj_5_24.
  28. Sniegocki, T., Sell, B., Giergiel, M., & Posyniak, A. (2019). QuEChERS and HPLC-MS/MS combination for the determination of chloramphenicol in twenty-two different matrices. Molecules, 24(3), article number 384. doi: 10.3390/molecules24030384.
  29. Streit, B., Czabany, T., Weingart, G., Marchetti-Deschmann, M., & Prasad, S. (2022). Toolbox for the extraction and quantification of Ochratoxin A and Ochratoxin Alpha applicable for different pig and poultry matrices. Toxins, 14(7), article number 432. doi: 10.3390/toxins14070432.
  30. Sukor, N., Jusoh, R., Rahim, S.A., & Kamarudin, N. (2018). Ultrasound assisted methods for enhanced extraction of phenolic acids from Quercus Infectoria galls. Materials Today: Proceedings, 5(10), 21990-21999. doi: 10.1016/j. matpr.2018.07.060.
  31. Veiga-del-Baño, J.M., Andreo-Martínez, P., Pérez-Lucas, G., & Navarro, S. (2024). Overview of the evolution and trends of the QuEChERS sample preparation procedure. Reviews of Environmental Contamination and Toxicology, 262, article number 22. doi: 10.1007/s44169-024-00073-1.
  32. Vuran, B., Ulusoy, H.I., Sarp, G., Yilmaz, E., Morgül, U., Kabir, A., Tartaglia, A., Locatelli, M., & Soylak, M. (2021). Determination of chloramphenicol and tetracycline residues in milk samples by means of nanofiber coated magnetic particles prior to high-performance liquid chromatography-diode array detection. Talanta, 230, article number 122307. doi: 10.1016/j.talanta.2021.122307.
  33. Yakubchak, O.M., Laposha, O.A., Midyk, S.V., Taran, T.V., & Zabarna, I.V. (2018). Assessment of the conformity of the methods for aflatoxin B1 and deoxynivalenol determination in grain and feeds by method of highperformance liquid chromatography. Methods and Objects of Chemical Analysis, 13(3), 121-130. doi: 10.17721/ moca.2018.121-130.
  34. Yang, Y., Lin, G., Liu, L., & Lin, T. (2022). Rapid determination of multi-antibiotic residues in honey based on modified QuEChERS method coupled with UPLC–MS/MS. Food Chemistry, 374, article number 131733. doi: 10.1016/j.foodchem.2021.131733.
  35. Yanovych, D., Berendsen, B., Zasadna, Z., Rydchuk, M., & Czymai, T. (2018). A study of the origin of chloramphenicol isomers in honey. Drug Testing and Analysis, 10(2), 416-422. doi: 10.1002/dta.2234.
  36. Ye, H., Li, S., Xi, Y., Shi, Y., Shang, X., & Huang, D. (2022). Highly sensitive determination of antibiotic residues in aquatic products by high-performance liquid chromatography-tandem mass spectrometry. Antibiotics, 11(10), article number 1427. doi: 10.3390/antibiotics11101427.
  37. Zhang, Y., Li, X.Q., Li, H.M., Zhang, Q.H., Gao, Y., & Li, X.J. (2019). Antibiotic residues in honey: A review on analytical methods by liquid chromatography tandem mass spectrometry. TrAC Trends in Analytical Chemistry, 110, 344-356. doi: 10.1016/j.trac.2018.11.015.
Baktybayeva, F., Serikbayeva, A., Toishimanov, M., Serikov, M., & Nuraliyeva, U. (2025). Development of an HPLC-MS/MS method for the quantitative determination of chloramphenicol in honey from Kazakhstan. Scientific Horizons, 28(5), 54-64. https://doi.org/10.48077/scihor5.2025.54