Extraction Strategies for Simultaneous Determination of Florfenicol and Florfenicol Amine in Tilapia (Oreochromis niloticus) Muscle: Quantification by LC-MS/MS

  • Letícia Sayuri Shiroma
  • Sonia C. N. Queiroz
  • Claudio Martin Jonsson
  • Carla Beatriz Grespan BottoliEmail author


A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of the veterinary drug florfenicol (FF) and its major metabolite, florfenicol amine (FFA), in tilapia muscles (Oreochromis niloticus). Three different sample preparation procedures (DLLME, sub-zero, and modified QuEChERS) were tested. The best extraction results were obtained by using the modified QuEChERS. The quantification was made by using a LC-MS/MS analysis, with a Lichrocart Cartridge Purospher Star C8 HPLC column (250 mm×4.6 mm, 5 μm particle size). Analytes were separated with a mobile phase consisting of Milli-Q water to acetonitrile 40:60 (v/v), both with 0.1% formic acid. The validation parameters were recovery of 70 to 79% and 62 to 69%, limit of detection of 0.0625 μg g−1 and 0.125 μg g−1, and limit of quantification of 0.125 μg g−1 and 0.25 μg g−1, for FF and FFA, respectively. CCα was 1183 μg kg−1 and CCβ was 1365 μg kg−1 for FF, intraday and interday precision has CV ≤20%, and linear range was 0.625 to 5.00 μg g−1. This method was shown to be simple and rapid when compared to other, more conventional methods. Also, it has low reagent and solvent consumption, with low waste generation, which is in line with the principles of green chemistry. The method was successfully applied for the analyzes of tilapia exposed to the antibiotic.


Veterinary drugs Green chemistry Residues Antibiotics Aquaculture 



Limit of decision


Detection capacity


European Commission




Florfenicol amine


Ministry of Agriculture, Livestock and Food Supply - Brazil


Molecularly imprinted solid-phase extraction


Matrix solid-phase dispersion extraction


Quick, Easy, Cheap, Effective, Rugged and Safe


Ultrasound-assisted dispersive liquid-liquid microextraction



The authors wish to thank Prof. Carol Collins for language assistance.

Funding Information

This research was supported by BNDES (0117020010606007), FAPESP (2014/50867-3), CNPq (311671/2015-2; 465389/2014-7) and INCT Bioanalítica. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil, Finance Code 001.

Compliance with Ethical Standards

Conflict of Interest

Letícia Sayuri Shiroma, Sonia Claudia do Nascimento de Queiroz, Claudio Martin Jonsson and Carla Beatriz Grespan Bottoli declare no conflict of interest.

Ethical Approval

This article contains studies with animals approved by Ethical Commission for the Use of Animals (CEUA) of the Embrapa Environment (Registration 007/17) (Jonsson et al. 2017).


  1. Alechaga E, Moyano E, Galceran MT (2012) Ultra-high performance liquid chromatography-tandem mass spectrometry for the analysis of phenicol drugs and florfenicol-amine in foods. Analyst 137:2486–2494CrossRefGoogle Scholar
  2. Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. J Assoc Off Anal Chem 86:412–431Google Scholar
  3. Barreto F, Ribeiro C, Barcellos Hoff R, Dalla Costa T (2016) Determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in poultry, swine, bovine and fish by liquid chromatography-tandem mass spectrometry. J Chromatogr A 1449:48–53CrossRefGoogle Scholar
  4. Branco LCC (2016) Farmacocinética e Depleção de resíduos do florfenicol em tambaqui (Colossoma macropomun). University of Campinas, Brazil, DissertationGoogle Scholar
  5. Carraschi SP, Cruz C, Machado Neto JG, Castro MP, Bortoluzzi NL, Girio ACF (2011) Eficácia do florfenicol e da oxitetraciclina no controle de Aeromonas hydrophila em pacu (Piaractus mesopotamicus). Arq Bras Med Vet Zootec 63:579–583CrossRefGoogle Scholar
  6. EC (2002) European Comission. Commission Regulation n° 657/2002 of 12 August 2002Google Scholar
  7. EC (2009) European Comission. Commission Regulation n° 37/2010 of 22 December 2009 on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin, . Acessed 30 October 2018
  8. FAO (2018) Food and Agriculture Organization of the United Nations; the state of world fisheries and aquaculture, Acessed 30 October 2018
  9. Food Ingredients Brasil (2009) Proteínas do peixe, 8:23–32Google Scholar
  10. Gaikowski MP, Mushtaq M, Cassidy P, Meinertz JR, Schleis SM, Sweeney D, Endris RG (2010) Depletion of florfenicol amine, marker residue of florfenicol, from the edible fillet of tilapia (Oreochromis niloticus×O. niloticus and O. niloticus×O. aureus) following florfenicol administration in feed. Aquaculture 301:1–6CrossRefGoogle Scholar
  11. Hu J, Li Y, Zhang W, Wang H, Huang C, Zhang M, Wang X (2009) Dispersive liquid-liquid microextraction followed by gas chromatography–electron capture detection for determination of polychlorinated biphenyls in fish. J Sep Sci 32:2103–2108CrossRefGoogle Scholar
  12. Jonsson CM, Hisano H, Paraiba LC (2017) Comissão de ética de uso de animais. Acúmulo do florfenicol e oxitetraciclina em filé de tambaquis, tilápias e pacus através da água e da ração medicadaGoogle Scholar
  13. Lombardo-Agüí M, García-Campaña AM, Cruces-Blanco C, Gámiz-Gracia L (2015) Determination of quinolones in fish by ultra-high performance liquid chromatography with fluorescence detection using QuEChERS as sample treatment. Food Control 50:864–868CrossRefGoogle Scholar
  14. MPA (2011) Ministério da Pesca e Aquicultura, Boletim estatístico da pesca e aquicultura. Acessed 30 October 2018
  15. Plumb DC (2004) Veterinary Drug Handbook, fifth edn. Iowa State PressGoogle Scholar
  16. MAPA (2011a) Ministério da Agricultura, Pecuária e Abastecimento. Instrução Normativa n° 24 de 01 de Junho de 2011. Acessed 30 October 2018Google Scholar
  17. MAPA (2011b) Ministério da Agricultura, Pecuária e Abastecimento. Guia de Validação e Controle de Qualidade Analítica para Fármacos em Produtos para Alimentação Animal e Medicamentos Veterinários, Acessed 30 October 2018
  18. Marques TV, Paschoal JAR, Barone RSC, Cyrino JEP, Rath S (2018) Depletion study and estimation of withdrawal periods for florfenicol and florfenicol amine in pacu (Piaractus mesopotamicus). Aquac Res 49:111–119CrossRefGoogle Scholar
  19. Martins ML, Primel EG, Caldas SS, Prestes OD, Adaime MB, Zanella R (2012) Microextração líquido-líquido dispersiva (DLLME): fundamentos e aplicações. Sci Chromatogr 4:35–51Google Scholar
  20. Nunes KSD, Vallim JH, Assalin MR, Queiroz SCN, Paraíba LC, Jonsson CM, Reyes FGR (2018) Depletion study, withdrawal period calculation and bioaccumulation of sulfamethazine in tilapia (Oreochromis niloticus) treated with medicated feed. Chemosphere 197:89–95CrossRefGoogle Scholar
  21. Orlando EA, Roque AGC, Losekann ME, Simionato AVC (2016) UPLC–MS/MS determination of florfenicol and florfenicol amine antimicrobial residues in tilapia muscle. J Chromatogr B 1035:8–15CrossRefGoogle Scholar
  22. Pan XD, Wu PG, Jiang W, Ma BJ (2015) Determination of chloramphenicol, thiamphenicol, and florfenicol in fish muscle by matrix solid-phase dispersion extraction (MSPD) and ultra-high pressure liquid chromatography tandem mass spectrometry. Food Control 52:34–38CrossRefGoogle Scholar
  23. Phu TM, Scippo ML, Phuong NT, Tien CTK, Son CH, Dalsgaard A (2015) Withdrawal time for sulfamethoxazole and trimethoprim following treatment of striped catfish (Pangasianodon hypophthalmus) and hybrid red tilapia (Oreochromis mossambicus × Oreochromis niloticus). Aquacult 437:256–262CrossRefGoogle Scholar
  24. Rezaee M, Assadi Y, Milani Hosseini MR, Aghaee E, Ahmadi F, Berijani S (2006) Determination of organic compounds in water using dispersive liquid–liquid microextraction. J Chromatogr A 1116:1–9CrossRefGoogle Scholar
  25. Rezk MR, Riad SM, Khattab FI, Marzouk HM (2015) Multi-residues determination of antimicrobials in fish tissues by HPLC–ESI-MS/MS method. J Chromatogr B 978-979:103–110CrossRefGoogle Scholar
  26. Sadeghi S, Jahani M (2013) Selective solid-phase extraction using molecular imprinted polymer sorbent for the analysis of florfenicol in food samples. Food Chem 141(2):1242–1251CrossRefGoogle Scholar
  27. Tao Y, Zhu F, Chen D, Wei H, Pan Y, Wang X, Liu Z, Huang L, Wang Y, Yuan Z (2014) Evaluation of matrix solid-phase dispersion (MSPD) extraction for multi-fenicols determination in shrimp and fish by liquid chromatography–electrospray ionisation tandem mass spectrometry. Food Chem 150:500–506CrossRefGoogle Scholar
  28. Tsai WH, Chuang HY, Chen HH, Huang JJ, Chen HC, Cheng SH, Huang TC (2009) Application of dispersive liquid–liquid microextraction and dispersive micro-solid-phase extraction for the determination of quinolones in swine muscle by high-performance liquid chromatography with diode-array detection. Anal Chim Acta 656:56–62CrossRefGoogle Scholar
  29. Zhang S, Liu Z, Guo X, Cheng L, Wang Z, Shen J (2008) Simultaneous determination and confirmation of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine in chicken muscle by liquid chromatography–tandem mass spectrometry. J Chromatogr B 875:399–404CrossRefGoogle Scholar
  30. Zhao HY, Zhang GH, Bai L, Zhu S, Shan Q, Zeng DP, Sun YX (2011) Pharmacokinetics of florfenicol in crucian carp (Carassius auratus cuvieri) after a single intramuscular or oral administration. J Vet Pharmacol Therap 34:460–463CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute of ChemistryUniversity of CampinasCampinasBrazil
  2. 2.Laboratory of Residues and Contaminants, Embrapa Meio AmbienteJaguariunaBrazil

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