Studies On Pharmacokinetics And Impact To Drug Metabolic Enzyme Of Thiamphenicol And Florfenicol In Litopenaeus Vannamei

This study establish a method to detect thiamphenicol and florfenicol in Litopenaeus vannamei plasma,muscle and hepatopancreas,using reverse phase high performance liquid chromatorgraphic method(RP-HPLC).Pharmacokinetics and residues of thiamphenicol(THA) and florfenicol(FLR) were investigated,following single oral administration at a dose of 10mg/kg in healthy Litopenaeus vannamei.The plasma,muscle and hepatopancreas were collected at different intervals after administration of thiamphenicol and florfenicol at 25.0±1.0℃.Ethyl acetate was used as extractant.The extracts were evaporated to dryness at water bath of 60℃and were dissolved in mobile phase.Then the fat of the solute was degreased by N-hexane and analysed by RP-HPLC.The mobile phase was acetonitrile and H₂O=16:84(V/V). The chromatographic column was Zorbax SB-C₁₈(5μm,150×4.6mm,I.D) column and ultraviolet detector was set at 225nm.The mean recoveries for all samples were 78.13～92.83%%,lntra-day CV and inter-day CV are under 4%and 6.5%respectively. Both LOD can meet the requirement of the residual detection.The concentration-time data of thiamphenicol and florfenicol in plasma of healthy Litopenaeus vannamei after single oral administration was fitted to two-compartment model with first order absorption.The main pharmacokinetic parameters for plasma of thiamphenicol were:distribution half-life(t_1/2α),absorption half-life(t_1/2ka) and elimination half-life(t_1/2β) were 2.448h,10.659h,0.666h respectively.The area under the curve(AUC),the time to peak concentration(T_max) and the peak concentration(C_max) were 43.963μg/(mL·h),2h and 7.956μg/mL respectively.The main pharmacokinetic parameters for plasma of florfenicol were: t_1/2α,t_1/2ka and t_1/2β were 1.364h,17.36h and 1.069h respectively.AUC,T_max and C_max were 43.963μg/(mL·h),2hå’Œ5.532μg/mL respectively.This study establish a method to detect microsomal cytochrome P450(CYP450) enzyme activity of Litopenaeus vannamei hepatopancreas,gill,stomach,muscle. Comparative study its distribution in the organization.CYP1A select 7-ethoxyyhiophene trazodone dehydrogenase(EROD) and 7-ethoxycoumarin dehydrogenase(ECOD) reaction,CYP2E select aniline 4-hydroxylase reaction (ANH),CYP3A select aminopyrine N-demethylase(AMND) and erythromycin N-demethylase(ERND) reaction,by measuring its metabolite generation to reflect enzyme activity.Activity measured in the above-mentioned tissue has different distribution.Performance of the highest in liver microsome,EROD activity was 0.771±0.026nmol/(min·mg),ECOD activity was 0.457±0.017nmol/(min·mg),AMND activity was 2.391±0.014 nmol/(min·mg),ERND activity was 1.776±0.005 nmol/(min·mg),not detected activity ANH.Followed by the gills and stomach,the muscle has the lowest activity and not detected to EROD and ECOD activity.The results showed that the major organization of Litopenaeus vannamei CYP450 isoforms with the above-mentioned activity,and their distribution and activity of body tissues and organs have a difference.The method is sensitive,reliable and reproducible,for the further study in vitro model of drug-metabolizing enzymes to provide a theoretical basis.The extraction of microsomes with thiamphenicol and florfenicol were incubated in vitro reaction,detection the enzyme activity to study the effects of drugs on the drug metabolic enzyme.The results that AMND and ERND have significant inhibition,EROD and ECOD activity did not change significantly,there is no activity detected ANH.Hepatopancreas microsomes with 100μL 200μg/mL thiamphenicol incubation reaction,EROD,ECOD,AMND and ERND activity were 0.769±0.016 nmol/(min·mg),0.476±0.025 nmol/(min·mg),1.948±0.073 nmol/(min·mg) and 1.528±0.028 nmol/(min·mg).Hepatopancreas microsomes with 100μL 200μg/mL florfenicol incubation reaction,EROD,ECOD,AMND and ERND activity were 0.765±0.015 nmol/(min·mg),0.449±0.035 nmol/(min·mg),2.062±0.017 nmol/(min·mg) and 1.506±0.023 nmol/(min·mg).