Metabolism And Residues Elimination Of Robenidine Hydrochloride In Vitro And In Vivo Of Ictalurus Punctatus At Different Water Temperature

Robenidine hydrochloride,as an anti-protozoal agent,is mainly used to control coccidiosis in livestock and poultry,and it also has a good effect on the treatment of acute and chronic coccidiosis in chickens and rabbits.In aquaculture,robenidine hydrochloride is fishery drugs permitted by National Animal Husbandry and Veterinary Breau,and it is mainly used for the treatment of fish myxosporidia.But there is no research on the metabolism of robenidine hydrochloride in vitro and in vivo of Ictalurus punctatus.This article firstiy established the detection method of robenidine hydrochloride and its metabolites in various tissues of Ictalurus punctatus,and studied the metabolism and residues elimination of robenidine hydrochloride residues in vitro and in vivo ofIctalurus punctatus at different water temperature.The aimed to provide scientific basis for the rational use of robenidine hydrochloride in channel catfish farming during different seasons.According to the highest residue limit in the edible tissue,to determine the drug withdrawal period about the robenidine hydrochloride in the tissues of Ictalurus punctatus,to prevent the residues of the obenidine hydrochloride in the fish and ensure the quality and safety of the aquatic product.1.The method for determination of robenidine hydrochloride and its metabolites in Ictalurus punctatus tissues by HPLC-MSBased on the comparison of the effects different extractants,pretreatment methods and flows on the recovery and the chromatographic behavior of robenidine hydrochloride,we established a method for determination of robenidine hydrochloride and its metabolites in tissues of Ictalurus punctatus by HPLC-MS,such as plasma,muscle,skin,gill,liver,kidney,brain and intestines.The method used 1%formic acid ethyl acetate as extractant,0.1%formic acid water and methanol as mobile phase,the water in ethyl acetate and the sample were removed with anhydrous magnesium sulfate,and the n-hexane was used for grease removal.The results showed that the linear relationship between robenidine hydrochloride and its metabolites was good,R2 was greater than 0.9990.The baseline of the sample was stable,the peak shape of robenidine hydrochloride and its metabolites was good,and there was no obvious influence of impurity peaks.The average recoveries of the different tissues were 64.02%-114.17%,and the RSD was 0.05%-10.80%.Under the experimental conditions,the detection limit of robenidine hydrochloride and 4-chlorobenzldehyde was 0.5?g/kg and the limit of quantification was 1?g/kg in each tissues of Ictalurus punctatus,the detection limit of?4-Chloro-benzoylamino?-acetic acid was 2.5?g/kg and the limit of quantification was 5?g/kg.It showed that the method was simple,high recovery,good accuracy and precision to determine the robenidine hydrochloride in Ictalurus punctatus.2.The pharmacokinetics of robenidine hydrochloride of Ictalurus punctatus at different water temperatureThe pharmacokinetics of robenidine hydrochloride in the tissues of Ictalurus punctatus at different water temperatures?at 18?and 28??was investigated in this study.The concentration of robenidine hydrochloride in the tissues of Ictalurus punctatus was determined by HPLC-MS.Pharmacokinetic parameters were calculated by using the 3p97 Practical Pharmacokinetic Program software.The concentration-time data of robenidine hydrochloride in the tissues of Ictalurus punctatus were fitted to a two-compartment model at both 18?and 28?water temperature.The kinetic equation were C_18?=4.17e^–0.01t+2.48e^–0.008t–6.65e^–0.05t;C_28?=7.69e^–0.02t+0.13^e–0.01t–7.82e^–0.27t,respectively.The time point of maximum plasma concentration(T_peak)was 42.36 and10.03 h,respectively,Peak plasma concentration(C_max)was 3.51 and 5.76?g/mL,respectively.Relatively apparent distributed volume V/F_?c?was approached to 3.79 and2.78 L/kg,respectively.The elimination half-time(T_1/2???)was 85.52 and 58.63 h,respectively.The areas under the curve?AUC?was 554.18 and 326.74??g/mL?/h,respectively.The T_?peak?and C_maxax in the muscle,skin,gill,liver and kidney of Ictalurus punctatus at 18°C were 25.56,32.21,5.33,21.47,13.69 h and 1.34,1.76,1.65,0.46 and 0.25 mg/kg,respectively.The T_?peak?and C_maxax at 28°C were 15.79,11.10,2.61,12.89,7.87 h and 2.91,2.90,3.05,3.04,and 0.42 mg/kg,respectively.The elimination half-lives(T_1/2)at different water temperatures were 123.07,39.61,66.26,187.46,109.27 h,and 23.57,35.37,19.74,29.34,43.30 h,respectively.The area under the drug-time curve AUC was 275.53,176.53,167.56,135.87,42.98?mg/kg?/h,and 157.58183.72,95.09,174.82,29.85?mg/kg?/h,respectively.The results showed that the temperature could significantly affect the absorption,distribution and elimination of robenidine hydrochloride in the tissues of Ictalurus punctatus.The peak time of robenidine hydrochloride was short and the peak concentration value was high under high water temperature conditions.At low temperature,the metabolism of drugs was slowest in the liver,and the metabolism was slowest in the kidneys at high temperatures.The metabolism of drugs was slow in the tissues of Ictalurus punctatus,and the metabolism of drugs is faster than low temperatures at high temperatures.Through the determination of its main metabolites,it was found that robenidine hydrochloride existed mainly in the form of the original drug after entering the channel catfish.3.The elimination regularity of robenidine hydrochloride residues in Ictalurus punctatusThe elimination regularity of robenidine hydrochloride residues were investigated in the tissues of Ictalurus punctatus with a 30 mg/kg body weight after oral administration for continuously 5d at the water temperature of?28±1??.The result showed that the robenidine hydrochloride was rapidly distributed mainly in the intestine,gill and brain tissue,and the concentration of drug was high,which myxosporidia could infect.It provides a theoretical basis for the treatment of the disease.The elimination half-life(T_1/2)of robenidine hydrochloride in various tissues were as follow,intestines?5.33 d?>kidney?4.12 d?>liver?3.65 d?>skin?2.95 d?>muscle?2.58 d?>brain?2.54 d?>gill?2.24 d?>plasma?1.66 d?.It indicated that robenidine hydrochlori-de was slowly eliminated in tissues,and the drug was slowest eliminated in intestines and kidney.If the maximum residue limits?MRL?in edible tissue is 10?g/kg,the withdraw period should not be less than 22.75 days under this experimental condition.4.The metabolize of robenidine hydrochloride in vitro of Ictalurus punctatusThe metabolic rate of robenidine hydrochloride in vitro of Ictalurus punctatus was studied with the liver microsomes as an example.The results showed that the metabolic rate of robenidine hydrochloride in the liver microsomes increased and the liver clearance increased as the temperature increased,which was in accordance with the metabolic in the body.