| Background:Enrofloxacin（EFX）,also known as ethyl ciprofloxacin,is a synthetic third-generation quinolone antibacterial drug.This product is designated as a special animal drug by the state.It has broad-spectrum antibacterial activity,has a strong killing effect on gram-negative bacteria,and also has a good antibacterial effect on gram-positive bacteria and mycoplasma.EFX has rapid action,high safety,strong permeability,good oral absorption,high and stable blood concentration,and its metabolite is Ciprofloxacin（CFX）,which still has a strong antibacterial effect.However,the low water solubility and drug resistance of EFX are detrimental to its application prospects,so structural modification is necessary to improve its effectiveness.Enrofloxacin-calcium complex（EFX-Ca）is a new type of EFX metal complex obtained by complexing with EFX as a ligand and chelating and coordinating with metal calcium ions.So far,although there have been some related studies on mixed metal complexes with EFX as a ligand,there are few reports on the metabolism process of related drugs in transition metal chelate with EFX as a single ligand.Therefore,it is urgent to systematically carry out the overall research in this direction.For this reason,in the process of new drug development,the study of the dynamic characteristics of the drug in the body and the law of the residue in the body has become a vital and non-negligible part of the new drug development process.In this experiment,the pharmacokinetics of EFX-Ca after oral administration in chickens was studied,and the rule of EFX-Ca residue elimination in chicken tissues was studied.Objective:1.In order to better understand the absorption,distribution,metabolism and elimination process of EFX-Ca in healthy yellow feather broilers.Establish a liquid chromatography-mass spectrometry（LC-MS）method to detect the pharmacokinetics of EFX-Ca in yellow-feather broilers.Through the study of the pharmacokinetics of EFX-Ca in chickens,it provides a reference for the clinical rational use of EFX-Ca in the prevention and treatment of chicken diseases.2.In order to better understand the changes in the concentration of EFX-Ca residues in chicken tissues after withdrawal of the drug in healthy yellow-feather broilers.To establish a LC-MS method to detect EFX-Ca residue elimination in yellow-feather broilers.By detecting the residual concentration of drug marker residues in chicken tissues,the changes of EFX and CFX residual concentrations in drug chicken tissues after drug withdrawal were obtained.It provides a theoretical basis for formulating the drug withdrawal period of the new metal complex EFX-Ca in chickens.Methods:1.Establish an LC-MS method to detect the changes in blood drug concentration of EFX-Ca in vivo,and verify the methodology.In the experiment,1%formic acid/methanol solution was used to process the plasma samples,the liquid instrument was Thermo Fisher Ulti Mate 3000 RS;the chromatographic column was Thermo Hypersil GOLD 100×2.1 mm,3μm;the flow rate:0.4m L/min.Aqueous phase:0.1%formic acid aqueous solution;organic phase:0.1%formic acid/acetonitrile;chromatographic conditions with an injection volume of 2.00μL for detection.The mass spectrometer is Thermo Fisher TSQ Quantum,electrospray ionization source（ESI）.Scan mode:positive and negative ion switching scan;detection mode:select reaction monitoring（SRM）.By oral administration,EFX and EFX-Ca were administered separately at different time points after administration to collect blood from the veins of the yellow feather broiler wings,and the plasma was separated for liquid quality testing.2.This experiment established an LC-MS detection method for the residual markers of EFX and CFX in chicken edible tissues,and carried out methodological verification.Validation was carried out in terms of method specificity,linearity,precision and recovery,detection limit and quantification limit.And prepare EFX and CFX high,medium and low three concentrations of QC samples for quality control testing.After taking the medicine with drinking water for 5 days,samples were taken at 5 time points after stopping the medicine.After the chickens were sacrificed by bloodletting from the jugular vein,tissue samples were collected from each chicken:muscle 100～200g（pectoral muscle）,whole liver,whole kidney,and 10-20g sebum.The residual concentration of tissue was measured under the validated LC/MS method.Results:1.This experiment established a liquid chromatography-tandem mass spectrometry method for the determination of EFX in broiler plasma samples,and completed the methodological verification.The method has a good linear relationship within the concentration range of 1-5000ng/m L,r2>0.99,and good specificity.At the same time,the intra-day and intra-day accuracy RE%are less than 15%,the precision RSD%is less than 5%,the recovery rate is more than90%,and the signal response is sensitive.All aspects meet the requirements of methodological investigation.The validated method was applied to the pharmacokinetic study of EFX and EFX-Ca in broiler chickens,and the pharmacokinetic parameters were obtained:the plasma Tmaxof EFX was1.50±0.57 h,and the Cmaxwas 1.55±0.73μg/m L,AUC（0-∞）is 24.58±6.00h·μg/m L,t1/2is 5.92±1.33 h,MRT（0-∞）is 15.34±4.60 h,CL is 423.25±90.72.The Tmaxof EFX-Ca is 1.83±0.40 h,Cmaxis 2.32±0.91μg/m L,AUC（0-∞）is48.05±20.81 h·μg/m L,t1/2is 10.92±0.50,MRT（0-∞）is 17.71±2.31 h,CL is251.79±128.15 m L/h/kg.2.An LC-MS method for the determination of EFX and CFX in broiler tissue samples has been established,and the methodology verification has been completed.The linear range of EFX and CFX is 1～5000 ng/m L,showing a linear correlation,the correlation coefficient is greater than 0.99,the method quantification limit is 1ng/g,and the detection limit is 0.2ng/m L and 1ng/m L,respectively.Adding EFX and CFX to chicken tissues,the recovery rate is above80%,and the precision is less than 15%.The above technical parameters can meet the detection requirements of the EFX and CFX residue test in chicken tissues.The results of the study on the rule of residue elimination show that the average amount of residues in each tissue is the highest during the zero drug withdrawal period.One day after the drug was stopped,the total residues in the chicken tissues were still higher than the national maximum residue limit（MRL）,and the total residues in skin and adipose tissue were lower than the MRL on the3rd day after the drug was stopped.After the 7th day of drug withdrawal,the total residual amount of other tissues（muscle,sebum and liver）also fell below the corresponding MRL,and no residual amount was detected after the 12th day.Conclusion:1.In this experiment,an LC-MS determination method with strong specificity,high sensitivity and good stability was established,which can be applied to the determination of EFX and EFX-Ca in chicken plasma.Through pharmacokinetic studies,it is found that oral EFX-Ca has faster absorption,wider distribution,and prolonged elimination than EFX.It further shows that EFX-Ca has good oral absorption,high blood concentration,wide distribution in the body,high bioavailability,and slow elimination in chickens.2.This experiment establishes and verifies an LC-MS method that can simultaneously determine the residual concentration of EFX and its metabolite CFX in chicken edible parts,and lays a foundation for further exploration of the EFX-Ca residue elimination rule in chicken tissues.Through the study of the rule of residue elimination,it was found that EFX-Ca was administered at the maximum recommended dose of 120 mg/L for 5 consecutive days,and the total residue of the drug in the chicken tissues was lower than the MRL on the 7th day of treatment.Using the drug withdrawal period software fitting,the drug withdrawal period is 8 days comprehensively. |
