Study On Residue Elimination Of Doxycycline Hydrochloride Soluble Powder And Tilmicosin Soluble Powder In Eggs

Rrespiratory disease are common diseases in poultry production,which result in the slow growth of sick chickens and the decline of laying hens’egg production,which is harmful to the poultry industry.Doxycycline（doxycycline,DOX）and tilmicosin（TIM）are widely used in poultry breeding because of their broad-spectrum antibacterial properties and good clinical therapeutic effect on poultry respiratory tract diseases.Dox and Tim are forbidden in laying period of laying hens,but not in laying hens.If the administration time is not observed in time,cross infection of bedding,feed and water source may lead to drug residues in eggs and cause food safety risks.In view of this,it is necessary to evaluate the maximum residue limits of DOX and Tim in eggs in line with China’s national conditions,formulate an appropriate egg abandonment period,and standardize the clinical use of DOX and Tim.In this study,according to the"guidelines for veterinary drug residue test（Draft）"and clinical recommended dosage,the animal administration scheme was formulated,and the residue elimination of doxycycline and tilmicosin in egg samples was studied.A high sensitive LC-MS/MS method for the determination of doxycycline and tilmicosin in whole egg samples,egg white samples and egg yolk samples was established Objective to investigate the residual distribution characteristics of doxycycline and tilmicosin in egg white,yolk and whole egg samples,clarify the residue elimination rules of doxycycline and tilmicosin in eggs,evaluate the maximum residue limits of DOX and Tim,and formulate scientific egg abandonment period,so as to provide reference for clinical application.1 Determination of DOXand TIM residues in eggs by HPLC-MS/MSEstablishment of HPLC-MS/MS method for DOX in egg white,yolk and whole egg:DOX in egg samples（whole egg,egg white and yolk）was extracted by Na₂EDTA-Mcllvaine buffer and acetonitrile extract,and then purified by Prime HLB column.The concentration of DOX was detected by using formic acid water and acetonitrile as mobile phases.The detection limit of doxycycline was 0.5μg/kg and the limit of quantitation was 1μg/kg in whole egg samples,egg white samples and egg yolk samples.The average recoveries of DOX in egg samples were 67.6%～95.8%at the concentration levels of 1,2,4,10μg/kg,and the intra and inter assay coefficients of variation were≤12%,which met the relevant methodological requirements.Establishment of HPLC-MS/MS method for TIM in egg white,egg yolk and whole egg:after extracting Tim from egg samples with 1%formic acid acetonitrile solution and precipitating protein,the samples were purified by Prime HLB solid phase extraction column,and 0.1%formic acid water and acetonitrile were used as mobile phases.The detection limits of TIM in whole egg samples,egg white samples and egg yolk samples were 0.5μg/kg,and the limits of quantification were 1μg/kg.The average recovery rate was 67.4%～95.2%in egg samples,egg white samples and egg yolk samples with 1,2,4,160μg/kg concentrations,and the intra-batch variation coefficient and inter-batch variation coefficient were≤10%,which met the relevant methodological requirements.2 Study on residue elimination of DOX hydrochloride soluble powder in eggsThe results were as follows:the highest concentrations of doxycycline hydrochloride soluble powder were 1501.04±334.80（on the 5th day of administration）,1857.85±245.65（on the 5th day of administration）and 1137.86±324.04μg/kg（on the third day of drug withdrawal）in whole egg samples,egg white samples and egg yolk samples,respectively.The elimination half lives were 4.33,4.62and 4.95 days,respectively.Dox could not be detected in egg white samples on day35,and no DOX was detected in yolk and whole egg samples on day 42.The highest concentrations of doxycycline hydrochloride soluble powder were3747.43±690.65（on the 12th day of administration）,4945.54±1838.66（on the 12th day of administration）and 3061.65±553.87μg/kg（on the first day of drug withdrawal）in whole egg samples,egg white samples and egg yolk samples,respectively,with T_1/2of 5.78,4.62 and 6.93days,No DOX was detected in egg white,yolk and whole egg samples on day 50.3 Study on elimination of TIM soluble powder residue in eggsThe results were as follows:the highest concentrations of tilmicosin in whole egg samples,egg white samples and egg yolk samples were 76.32±12.09（on the first day of drug withdrawal）,54.32±7.34（on the fifth day of administration）and 120.25±21.09μg/kg（on the first day of drug withdrawal）,respectively.The elimination half-life period of tilmicosin was 7.70,4.95 and 7.70 days respectively,and the tim in egg white samples was 28 No Tim was detected in yolk and whole egg samples on the35th day.The highest concentrations of tilmicosin in whole egg samples,egg white samples and yolk samples were 243.77±56.7（day 12）,172.98±37.09（day 12）and372.98±53.13μg/kg（day 12）,respectively.The elimination half-life of tilmicosin was 5.78,7.70 and 6.93 days,respectively.Tim in egg white samples was 35 No Tim was detected in yolk and whole egg samples on the 40th day4 Formulation of residue control standardsAccording to the daily egg intake and ADI data of DOX and TIM in China,the maximum residue limit of DOX in eggs is calculated as 10 According to the 95%confidence limit as the standard,the analysis of doxycycline hydrochloride soluble powder in laying hens:drinking water for 5 days,doxycycline hydrochloride soluble powder for 38 days,drinking water for 12 days,doxycycline hydrochloride soluble powder for 50 days in laying hens,and the maximum residue limit of Tim in eggs was160.The results showed that tilmicosin soluble powder was administered by drinking water for 5 days in laying hens,0 days in laying hens,12 days in drinking water,and10 days in laying hens.This paper established a method for the determination of doxycycline and tilmicosin residues in eggs,revealed the residue elimination characteristics of doxycycline hydrochloride soluble powder and tilmicosin soluble powder in eggs,and proposed the egg abandonment period standards of the two products in laying hens,which provided scientific basis for the rational clinical application of the two drugs and food safety monitoring.