Development and Validation of a HPLC-HESI-MS/MS Method for Simultaneous Determination of Robenidine Hydrochloride and Its Metabolites in Fish and Exploration of Their Kinetic Regularities in Grass Carp

  • Yongtao LiuEmail author
  • Yi Song
  • Bo Cheng
  • Jing Dong
  • Ning Xu
  • Shun Zhou
  • Qiuhong Yang
  • Xiaohui AiEmail author


An original method for the simultaneous determination of robenidine hydrochloride (ROBH) and its main metabolites 4-chlorohippuric acid (PCHA) and 4-chlorobenzoic acid (PCBA) in fish plasma and muscle was established by high-performance liquid chromatography coupled with heat electrospray ionization tandem mass spectrometry (HPLC-HESI-MS/MS) using predefined time segments in the alternating positive/negative mode. Fish muscle samples were prepared using a modified QuEChERS procedure, and plasma samples were prepared by a liquid–liquid extraction (LLE) procedure. The entire procedure was validated according to the guidelines defined by the US Food and Drug Administration. Matrix-matched calibration curves for plasma and muscle of fish showed good linearity with correlation coefficients (R2) ≥ 0.9985. The accuracy exhibited a relative error (RE) ranging from −14.2–8.2%, and intra- and inter-day precisions of analytes expressed as relative standard deviation (RSD) were within 12.4%. Limits of detection (LODs) and limits of quantitation (LOQs) were lower than 2.5 μg L−1 and 5 μg L−1 for target compounds in plasma and not more than 2.5 μg kg−1 and 5 μg kg−1 for analytes in muscle, respectively. The present method was successfully applied to explore the kinetic profiles of ROBH and its metabolites in grass carp (Ctenopharyngodon idella), and it demonstrated that PCBA is the major metabolite of ROBH in grass carp plasma and muscle. The elimination half-lives (t1/2β) of ROBH and PCBA in grass carp muscle were calculated to be 17.31 h and 138.53 h, respectively.


Robenidine hydrochloride Metabolites Kinetic regularity Fish HPLC-HESI-MS/MS 


Funding Information

This study was supported financially by the Central Public-interest Scientific Institution Basal Research Fund, CAFS (Nos. 2019ZD0901 and 2018JBF02), the National Natural Sciences Foundation of China (No. 3150219), and China Agriculture Research System (CARS-49).

Compliance with Ethical Standards

Conflict of Interest

Yongtao Liu declares that he has no conflict of interest. Yi Song declares that he has no conflict of interest. Bo Cheng declares that he has no conflict of interest. Jing Dong declares that he has no conflict of interest. Ning Xu declares that he has no conflict of interest. Shun Zhou declares that he has no conflict of interest. Qiuhong Yang declares that he has no conflict of interest. Xiaohui Ai declares that he has no conflict of interest.

Ethical Approval

All applicable international and national/institutional guidelines for the care and use of animals were followed.

Information Consent

Not applicable.


  1. Chang SH, Lai YH, Huang CN, Peng GJ, Liao CD, Kao YM, Tseng SH, Wang DY (2019) Multi-residue analysis using liquid chromatography tandem mass spectrometry for detection of 20 coccidiostats in poultry, livestock, and aquatic tissues. J Food Drug Anal 27(3):703–716CrossRefGoogle Scholar
  2. China Institute of Veterinary Drugs Control (2010) National standard collection of veterinary medicine-local standards for veterinary medicine increased national standards (the first volume). China Agriculture Press, Beijing, p 188Google Scholar
  3. Chinese Veterinary Pharmacopoeia Committee (2015) Veterinary pharmacopoeia of the People’s Republic of China (the first part). China Agriculture Press, Beijing, p 257Google Scholar
  4. Dorne JLCM, Fernández-Cruz ML, Bertelsen U, Renshaw DW, Peltonen K, Anadon A, Feil A, Sanders P, Wester P, Fink-Germmels J (2013) Risk assessment of coccidostatics during feed cross- contamination: animal and human health aspects. Toxicol Appl Pharm 270:196–208CrossRefGoogle Scholar
  5. DrugBank (2005) p-Chlorobenzoic acid ( Accessed 08/6/2019)
  6. Dubois M, Pierret G, Delahaut P (2004) Efficient and sensitive detection of residues of nine coccidiostats in egg and muscle by liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr B 813:181–189CrossRefGoogle Scholar
  7. European Commission (2009) Commission Regulation 124/2009/EC Setting maximum levels for the presence of coccidiostats or histomonostats in food resulting from the unavoidable carry-over of these substances in non-target feed. Off J EU. L40:7–11Google Scholar
  8. European Food Safety Authority (EFSA) (2011) Scientific opinion on safety and efficacy of Cycostat® 66G (robenidine hydrochloride) for rabbits for breeding and fattening, EFSA panel on additives and products or substances used in animal feed (FEEDAP). EFSA J 9:1–32Google Scholar
  9. European Medicines Agency (EMA) (2013) European public MRL assessment report (EPMAR) Phoxim (extension to bvine species and harmonisation of MRLs) (, accessed 08/16/2019)
  10. Hansen M, Krogh KA, Brandt A, Christensen JH, Halling-Sørensen B (2009) Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products. Environ Pollut 157:474–480CrossRefGoogle Scholar
  11. Ismaiel OA, Jenkins RG, Karnes HT (2013) Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry. Drug Test Anal 5:710–715CrossRefGoogle Scholar
  12. Jia W, Chu XG, Chang J, Wang PG, Chen Y, Zhang F (2017) High-throughput untargeted screening of veterinary drug residues and metabolites in tilapia using high resolution orbitrap mass spectrometry. Anal Chimca Acta 957:29–39CrossRefGoogle Scholar
  13. Kang JW, Park SJ, Park HC, Hossain MA, Kim MA, Son SW, Lim CM, Kim TW, Cho BH (2017) Multiresidue screening of veterinary drugs in meat, milk, egg, and fish using liquid chromatography coupled with ion trap time-of-flight mass spectrometry. Appl Biochem Biotech 182:635–652CrossRefGoogle Scholar
  14. Kanrar B, Mandal S, Bhattacharyya A (2010) Validation and uncertainty analysis of a multiresidue method for 42 pesticides in made tea, tea infusion and spent leaves using ethyl acetate extraction and liquid chromatography- tandem mass spectrometry. J Chromatogr A 1217:1926–1933CrossRefGoogle Scholar
  15. Lehotay SJ, Anastassiades M, Majors RE (2010) QuEChERS, a sample preparation technique that is “catching on”: an up-to-date interview with the inventors. LCGC North AM. 28:504–516Google Scholar
  16. Liu YT, Ai XH, Wang FH, Suo WW, Yang QH, Yang H, Xu N (2015) Determination of niclosamide in aquatic animal tissue by a novel extraction procedure and high-performance liquid chromatography-heated electrospray ionization-tandem mass spectrometry. Anal Lett 48:929–943CrossRefGoogle Scholar
  17. Liu YT, Ai XH, Li L, Li JC, Yang H (2018) A fast and accurate isotope dilution GC-IT-MS/MS method for determination of eugenol in different tissues of fish: application to a depletion study in mandarin fish. Biomed Chromatogr 32:1–9Google Scholar
  18. Martinsen B, Horsberg TE, Varma KJ, Sams R (1993) Single dose pharmacokinetic study of florfenicol in Atlantic salmon (Salmo salar) in seawater at 11 °C. Aquaculture 112:1–11CrossRefGoogle Scholar
  19. Molnár K, Ostoros G (2007) Efficacy of some anticoccidial drugs for treating coccidial enteritis of the common carp caused by Goussia carpelli (Apicomplexa: eimeriiade). Acta Vet Hung 55:67–76CrossRefGoogle Scholar
  20. Mortier L, Daeseleire E, Delahaut P (2003) Simultaneous detection of five coccidiostats in eggs by liquid chromatography-tandem mass spectrometry. Anal Chim Acta 483:27–37CrossRefGoogle Scholar
  21. Peters RJB, Block YJC, Rutgers P, Stolker AAM, Nielen MWF (2009) Multi-residue screening of veterinary drugs in egg, fish and meat using high-resolution liquid chromatography accurate mass time-of-flight mass spectrometry. J Chromatogr 1216:8206–8216CrossRefGoogle Scholar
  22. Riviere JE (2011) Comparative pharmacokinetics: principles, techniques and applications, second edition. Iowa State University Press, Ames IA, pp 148–167CrossRefGoogle Scholar
  23. Sun YX, Zhao HY, Shan Q, Zhu S, Zeng DP, Liu ZC (2018) Tissue distribution and elimination of florfenicol in crucian carp (Carasslius auratus cuvieri), after a single dose intramuscular or oral administration. Aquaculture 309:82–85CrossRefGoogle Scholar
  24. Tang JF, Cai J, Huang Y, Liao JM, Qin QY, Huang YX, Jian JC (2016) Pharmacokinetics and elimination regularity of robenidine hydrochloride residues in Luthjanus sanguineus. J Guangdong Ocean U 36:33–37 (in Chinese)Google Scholar
  25. Tang JF, Huang Y, Cai J, Liao JM, Huang YX, Jian JC (2017) Analysis of pharmacokinetics and residues elimination regularity of robenidine hydrochloride in Sciaenops ocellatus. Genomics Appl Biol 36:2399–2404 (in Chinese)Google Scholar
  26. U.S. Food and Drug Administration (2018) bioanalytical method validation, guidance for industry. (, accessed 06/03/2019)
  27. U.S. National Library of Medicine (2005) 4-Chlorohippuric acid. (, accessed 06/03/2019)
  28. Wilga J, Kot-Wasik A, Namieśnik J (2007) Comparison of extraction techniques of robenidine from poultry feed samples. Talanta 73:812–819CrossRefGoogle Scholar
  29. Wilkowska A, Biziuk M (2011) Determination of pesticide residues in food matrices using the QuEChERS methodology. Food Chem 125:803–812CrossRefGoogle Scholar
  30. Wu SH, Chen KC, Dai XX, Ma LS, Zhu XP, Pan DB, Zheng GM, Yin Y, Xie WP (2011) Determination of robenidine in fishery products by high performance liquid chromatography with dispersive solid phase extraction. J Instrumental Anal 30:1356–1361Google Scholar
  31. Yamaoka K, Nakagawa T (1978) Application of Akaike’s information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J Pharmacokinet Biop 6:165–175CrossRefGoogle Scholar
  32. Yang YJ, Yin J, Yang Y, Zhou NY, Zhang J, Shao B, Wu YN (2012) Determination of bisphenol AF (BPAF) in tissues, serum, urine and feces of orally dosed rats by ultra-high-pressure liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr B 901:93–97CrossRefGoogle Scholar
  33. Yu LX, Liu YT, Su ZJ, Ding H, Ai XH (2018) Pharmacokinetics of robenidine hydrochloride in plasma of channel catfish (Ictalurus punctatus). Acta Agri Zhejiangensis 30(10):1640–1646 (in Chinese)Google Scholar
  34. Yu LX, Yongtao L, Ding H, Su ZJ, Ai XH (2019) Pharmacokinetics and elimination regularity of robenidine hydrochloride residues in Ictalurus punctatus. Acta Hydrobiol Sin 43(3):869–874 (in Chinese)Google Scholar
  35. Zhao LJ, Wang Y, Chang XQ, Fang WH, Chen JJ, Fang JL (2018) Pharmacokinetics tissue distribution and elimination of robenidine hydrochloride in Carassius auratus gibelio after oral administration via medicated feed. Mar Fish 40:227–234 (in Chinese)Google Scholar
  36. Zulalian J, Gatterdam PE (1973) Absorption, excretion, and metabolism of robenz, robenidine hydrochloride [1,3-bis (p-chlorobenzylideneamino) guanidine hydrochloride], in the rat. J Agr Chem 21:794–797CrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Yangtze River Fisheries Research InstituteChinese Academy of Fishery SciencesWuhanChina
  2. 2.Key Laboratory of Control of Quality and Safety for Aquatic ProductsMinistry of Agriculture and Rural AffairsBeijingChina
  3. 3.Hubei Province Engineering and Technology Research Center for Aquatic Product Quality and SafetyWuhanChina
  4. 4.Aquatic Products Quality and Standards Research CenterChinese Academy of Fishery SciencesBeijingChina

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