Evaluation of a Selective Approach for the Determination of 5-Nitroimidazoles in Aquaculture Products by Capillary Liquid Chromatography Using Molecularly Imprinted Solid-Phase Extraction


Capillary liquid chromatography with UV detection is proposed for the determination of 5-nitroimidazole residues in aquaculture products. The separation was carried out in a C18 column at 20 °C, using a mobile phase consisting of water and acetonitrile, at 7 μL/min and 320 nm as detection wavelength. Furthermore, full loop injection mode (8 μL) was selected and water was considered as injection solvent. The optimized method was applied to the monitoring of nine 5-nitroimidazoles, including three metabolites, in crab, salmon, prawn, and velvet swimming crab. Molecularly imprinted solid-phase extraction has been evaluated for sample cleanup. The method was characterized in all the matrices in terms of linearity (R 2 ≥ 0.9964), precision (repeatability, RSD ≤ 7.9%, and reproducibility, RSD ≤ 11.1%) and trueness (recoveries between 80.4 and 108.7%). Decision limits, CCα, ranging from 0.2 to 1.5 μg/kg and detection capabilities, CCβ, from 0.2 to 1.8 μg/kg, were obtained.

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Molecularly imprinted solid-phase extraction






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  1. Antignac J, Marchand P, Bizec BL (2002) Identification of ractopamine residues in tissue and urine samples at ultra-trace level using liquid chromatography-positive electrospray tandem mass spectrometry. J Chromatogr B 774:59–66

    CAS  Article  Google Scholar 

  2. Bendesky A, Menéndez D, Ostrosky-Wegman P (2002) Is metronidazole carcinogenic? Mutat Res 511(2):133–144

    CAS  Article  Google Scholar 

  3. Cháfer-Pericás C, Maquieira Á, Puchades R (2010) Fast screening methods to detect antibiotic residues in food samples. TrAC Trends Anal Chem 29(9):1038–1049

    Article  Google Scholar 

  4. Cronly M, Behan P, Foley B, Malone E, Regan L (2009) Development and validation of a rapid method for the determination and confirmation of 10 nitroimidazoles in animal plasma using liquid chromatography tandem mass spectrometry. J Chromatogr B 877(14–15):1494–1500

    CAS  Article  Google Scholar 

  5. Dolan JW (2012) When should an internal standard be used? LCGC N Am 30:474–480

    CAS  Google Scholar 

  6. European Commission (2002) Commission Decision (2002/657/EC) of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results. Off J Eur Communities L221:8–36

    Google Scholar 

  7. European Union Reference Laboratories (2007) CRL guidance paper (7 December 2007). CRLs view on state of the art analytical methods for national residue control plans. http://www.rivm.nl/bibliotheek/digitaaldepot/crlguidance2007.pdf. Accessed on 16th October 2016

  8. Fanali C, Dugo L, Dugo P, Mondello L (2013) Capillary-liquid chromatography (CLC) and nano-LC in food analysis. TrAC Trends Anal Chem 52:226–238

    CAS  Article  Google Scholar 

  9. Granja RHMM, Nino AMM, Reche KVG, Giannotti FM, de Lima AC, Wanschel ACBA, Salerno AG (2013) Determination and confirmation of metronidazole, dimetridazole, ronidazole and their metabolites in bovine muscle by LC-MS/MS. Food Additives & Contaminants. Part A 30(6):970–976

  10. Hernández-Mesa M, García-Campaña AM, Cruces-Blanco C (2014) Novel solid phase extraction method for the analysis of 5-nitroimidazoles and metabolites in milk samples by capillary electrophoresis. Food Chem 145:161–167

    Article  Google Scholar 

  11. Hernández-Mesa M, Lara FJ, Cruces-Blanco C, García-Campaña AM (2015) Determination of 5-nitroimidazole residues in milk by capillary electrochromatography with packed C18 silica beds. Talanta 144:542–550

    Article  Google Scholar 

  12. Leiros H-KS, Kozielski-Stuhrmann S, Kapp U, Terradot L, Leonard GA, McSweeney SM (2004) Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans. J Biol Chem 279(53):55840–55849

    CAS  Article  Google Scholar 

  13. Mahugo-Santana C, Sosa-Ferrera Z, Torres-Padrón ME, Santana-Rodríguez JJ (2010) Analytical methodologies for the determination of nitroimidazole residues in biological and environmental liquid samples: a review. Anal Chim Acta 30:113–122

    Article  Google Scholar 

  14. Martín-Esteban A (2013) Molecularly-imprinted polymers as a versatile, highly selective tool in sample preparation. TrAC Trends Anal Chem 45:169–181

    Article  Google Scholar 

  15. Mitrowska K, Posyniak A, Zmudzki J (2014) Selective determination of fourteen nitroimidazoles in honey by high-performance liquid chromatography–tandem mass spectrometry. Anal Lett 47(10):1634–1649

    CAS  Article  Google Scholar 

  16. Mohamed R, Mottier P, Treguier L, Richoz-Payot J, Yilmaz E, Tabet J-C, Guy PA (2008) Use of molecularly imprinted solid-phase extraction sorbent for the determination of four 5-nitroimidazoles and three of their metabolites from egg-based samples before tandem LC-ESIMS/MS analysis. J Agric Food Chem 56(10):3500–3508

    CAS  Article  Google Scholar 

  17. Płotka J, Tobiszewski M, Sulej AM, Kupska M, Górecki T, Namieśnik J (2013) Green chromatography. J Chromatogr A 1307:1–20

    Article  Google Scholar 

  18. Rodríguez Ferreiro G, Cancino Badías L, Lopez-Nigro M, Palermo A, Mudry M, González Elio P, Carballo MA (2002) DNA single strand breaks in peripheral blood lymphocytes induced by three nitroimidazole derivatives. Toxicol Lett 132(2):109–115

    Article  Google Scholar 

  19. Saito Y, Jinno K, Greibrokk T (2004) Capillary columns in liquid chromatography: between conventional columns and microchips. J Sep Sci 27(17–18):1379–1390

    CAS  Article  Google Scholar 

  20. Sams MJ, Strutt PR, Barnes KA, Damant AP, Rose MD (1998) Determination of dimetridazole, ronidazole and their common metabolite in poultry muscle and eggs by high performance liquid chromatography with UV detection and confirmatory analysis by atmospheric pressure chemical ionisation mass spectrometry. Analyst 123(12):2545–2549

    CAS  Article  Google Scholar 

  21. Shimelis O, Wihlborg A, Rudolfsson M, Boyd B, Trinh A (2009) Extraction of nitroimidazoles from milk and egg samples using SupelMIP SPE Nitroimidazoles and LC-MS-MS (Technical Note—Sigma-Aldrich®)

  22. Sorensen LK, Hansen H (2000) Determination of metronidazole and hydroxymetronidazole in trout by a high-performance liquid chromatographic method. Food Additives & Contaminants 17(3):197–203

    CAS  Article  Google Scholar 

  23. Sun H-W, Wang F-C, Ai L-F (2007) Simultaneous determination of seven nitroimidazole residues in meat by using HPLC-UV detection with solid-phase extraction. J Chromatogr B 857(2):296–300

    CAS  Article  Google Scholar 

  24. The European Commission (2010) Commission Regulation (EU) No. 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off J Eur Union L15(2377):1–72

    Google Scholar 

  25. Tölgyesi A, Sharma VK, Fekete S, Fekete J, Simon A, Farkas S (2012) Development of a rapid method for the determination and confirmation of nitroimidazoles in six matrices by fast liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 64-65:40–48

    Article  Google Scholar 

  26. Turiel E, Martín-Esteban A (2010) Molecularly imprinted polymers for sample preparation: a review. Anal Chim Acta 668(2):87–99

    CAS  Article  Google Scholar 

  27. Xia X, Li X, Zhang S, Ding S, Jiang H, Li J, Shen J (2008) Simultaneous determination of 5-nitroimidazoles and nitrofurans in pork by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1208(1–2):101–108

    CAS  Article  Google Scholar 

  28. Xia X, Li X, Ding S, Zhang S, Jiang H, Li J, Shen J (2009) Determination of 5-nitroimidazoles and corresponding hydroxy metabolites in swine kidney by ultra-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Anal Chim Acta 637(1–2):79–86

    CAS  Article  Google Scholar 

  29. Zelnícková H, Rejtharová M (2013) Determination of 5-nitroimidazoles in various types of matrices using molecular imprinted polymer purification. Food Additives & Contaminants, Part A 30(6):1123–1127

    Article  Google Scholar 

  30. Zhou J, Shen J, Xue X, Zhao J, Li Y, Zhang J, Zhang S (2007) Simultaneous determination of nitroimidazole residues in honey samples by high-performance liquid chromatography with ultraviolet detection. J AOAC Int 90(3):872–878

    CAS  Google Scholar 

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Corresponding author

Correspondence to Ana M. García-Campaña.

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This study was funded by the Andalusian Government (Junta de Andalucía) for Excellence Project Ref: P12-AGR-1647. M. Hernández-Mesa thanks the “Plan Propio” of University of Granada for a pre-doctoral fellowship. D. Moreno-González acknowledges the program to strengthen research groups of the University of Granada for his postdoctoral contract.

Conflict of Interest

M. Hernández-Mesa declares that he has no conflict of interest. D. Moreno-González declares that he has no conflict of interest. C. Cruces-Blanco declares that she has no conflict of interest. A.M. García-Campaña declares that she has no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Hernández-Mesa, M., Moreno-González, D., Cruces-Blanco, C. et al. Evaluation of a Selective Approach for the Determination of 5-Nitroimidazoles in Aquaculture Products by Capillary Liquid Chromatography Using Molecularly Imprinted Solid-Phase Extraction. Food Anal. Methods 10, 3647–3657 (2017). https://doi.org/10.1007/s12161-017-0928-9

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  • 5-Nitroimidazoles
  • Aquaculture products
  • Capillary liquid chromatography
  • Molecularly imprinted solid-phase extraction