Abstract

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 (t_1/2β) of ROBH and PCBA in grass carp muscle were calculated to be 17.31 h and 138.53 h, respectively.

中文翻译：

---

HPLC-HESI-MS / MS法同时测定鱼中盐酸罗布尼定及其代谢物的开发验证及在草鱼中动力学规律的探索

摘要

高效液相色谱-热电喷雾电离建立了同时测定鱼血浆和肌肉中盐酸盐酸罗苯尼定（ROBH）及其主要代谢物4-氯马尿酸（PCHA）和4-氯苯甲酸（PCBA）的原始方法使用预定义的时间段以交替的正/负模式进行串联质谱（HPLC-HESI-MS / MS）。使用改良的QuEChERS程序制备鱼肉样品，并通过液-液萃取（LLE）程序制备血浆样品。整个程序已根据美国食品和药物管理局（US Food and Drug Administration）定义的准则进行了验证。鱼血浆和肌肉的基质匹配校准曲线显示出良好的线性，且具有相关系数（R ²）≥0.9985。准确度显示出相对误差（RE）介于-14.2-8.2％之间，而被分析物的日内和日间精确度表示为相对标准偏差（RSD）在12.4％以内。检测限（检测限）和定量限（定量限）均低于2.5微克大号^-1和5μg大号^-1用于等离子体目标化合物并且不大于2.5微克千克^-1和5μg千克^-1为中的分析物肌肉。本发明的方法成功地应用于探索ROBH及其草鱼代谢物（的动力学曲线草鱼），它表明，PCBA是ROBH的草鱼血浆和肌肉中的主要代谢物。消除半衰期（t _1/2草鱼肌肉中ROBH和PCBA的_β）分别计算为17.31 h和138.53 h。