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.