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
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.
KeywordsRobenidine hydrochloride Metabolites Kinetic regularity Fish HPLC-HESI-MS/MS
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.
All applicable international and national/institutional guidelines for the care and use of animals were followed.
- 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
- Chinese Veterinary Pharmacopoeia Committee (2015) Veterinary pharmacopoeia of the People’s Republic of China (the first part). China Agriculture Press, Beijing, p 257Google Scholar
- DrugBank (2005) p-Chlorobenzoic acid (http://www.Drugbank.ca/drugs/DB03728. Accessed 08/6/2019)
- 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
- 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
- European Medicines Agency (EMA) (2013) European public MRL assessment report (EPMAR) Phoxim (extension to bvine species and harmonisation of MRLs) (https://www.ema.europa.eu/en/documents/mrl-report/phoxim-european-public-maximum-residue-limit-assessment-report-epmar-cvmp_en.pdf, accessed 08/16/2019)
- 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
- 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
- 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
- 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
- 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
- U.S. Food and Drug Administration (2018) bioanalytical method validation, guidance for industry. (https://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf, accessed 06/03/2019)
- U.S. National Library of Medicine (2005) 4-Chlorohippuric acid. (https://pubchem.cbi.nlm.nih.gov/ompound/4-Chlorohippuric-acid, accessed 06/03/2019)
- 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
- 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
- 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
- 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