| Aditoprim is a new class of animal-specific antibacterial synergist with long half-life,big apparent volume and high bioavailability.And clinical investigations showed that ADP had a better pharmacokinetic behavior than trimethoprim in many animal species.Combined with sulfonamides,it can block the folic acid synthesis and inhibit the growth and reproduction of bacteria from a dual mechanism,and has a good antibacterial effect.However,the toxicological studies of aditoprim have proved that it has a certain degree of liver toxicity,so it is particularly important to monitor the drug residues in animals.Veterinary drug residue detection is one of the important contents to evaluate the safety of animal products.The residue elimination and safety of aditoprim unilateral and compound injection in pigs have not been studied.In order to more safely and effectively apply aditoprim unilateral and compound injection to the clinic,this study carried out the residue elimination test of aditoprim unilateral and compound injection in pigs to clarify the elimination rule of ADP,SMZ and Ac SMZ and formulate its residual target tissue,residual marker and withdrawal period(WDT).At the same time,the safety test of aditoprim unilateral and compound injection in pigs were also conducted,and the safety was evaluated through clinical observation,feed conversion rate,blood physiological and biochemical,organ coefficients,and pathological changes.The research content and results were as follows:1 Establishment of ultra performance liquid chromatography tandem mass spectrometry for determination of ADP,SMZ and Ac SMZA multi-residue UPLC-MS/MS method was established for the quantitative analysis of ADP,SMZ and Ac SMZ in edible tissues of swine.Samples were extracted with 2%ammoniated acetonitrile,dissolved with 0.1%formic acid water and acetonitrile(95:5,v/v)and detected by UPLC-MS/MS.The specific liquid phase conditions were as follows: chromatography column: ACQUITY UPLC BEH C18(2.1×50 mm,1.7 μm);injection volume: 5 μL;flow rate: 0.2 m L/min;column temperature 30 ℃,with 0.1% formic acid water and acetonitrile for mobile phase gradient elution(0-3 min,5% acetonitrile;3-3.1 min,30%-5% acetonitrile;3.1-5 min,5%-30% acetonitrile).The identification of ADP,SMZ,Ac SMZ were carried out by positive electrospray ionization(ESI+)in multiple reaction monitoring(MRM)mode.The results showed aditoprim,sulfamethoxazole and acetylsulfamethoxazole had a good linearity at concentration range of 20 μg/kg-1000 μg/kg,and the correlation coefficients were over than 0.999.The detection limit and quantitation limit were 10μg/kg and 20μg/kg respectively.The average recoveries of ADP,SMZ,Ac SMZ in liver,kidney,muscle and fat were 83.7%-89.9%,84.3%-91.4% and 82.8%-90.3%respectively at three spiked concentration.The interday coefficient of variation was3.1%-6.0%,2.8%-6.8% and 3.9%-6.2% and the intraday coefficient of variation was4.8%-10.1%,4.2%-11.3% and 5.1%-9.4% respectively.The established method can meet the residue detection of ADP,SMZ and AcSMZ in swime edible tissues.2 Residue elimination of ADP and ADP-SMZ injection in swine edible tissuesForty-five healthy pigs were randomly divided into 9 groups(4 groups each for ADP and ADP-SMZ injection test groups,and 1 group for blank group).Pigs received a intramuscular injection of ADP and ADP-SMZ injection at a dose of 10 mg/kg b.w.and 25 mg/kg b.w.respectively for 7 d and twice daily.Animals were slaughtered at12 h,3 d,7 d,14 d,and 28 d after drug withdrawal collecting liver,kidney,muscle and fat for UPLC-MS/MS detection.The results were shown as following:At the withdrawal time of 12 h,ADP had the highest concentration in the kidney,with a concentration of 29283.5 μg/kg and the lowest concentration in fat was 2525.7μg/kg;At the withdrawal time of 14 d,the concentration of ADP in the kidney and liver were 90.4 μg/kg and 77.7 μg/kg respectively;At the withdrawal time of 28 d,no drug was detected.ADP had the longest residence time in the liver,with a half-life of3.01 d.It was determined that the residual target tissue and residual marker of ADP injection were liver and aitoprim respectively.According to the maximum residue limit standard of the Ministry of Agriculture,according to the above results of drug withdrawal period,the ADP injection was continuously administered to pigs for 7 d according to the recommended dose(10 mg/kg b.w.),and the recommended drug withdrawal period in pig was 23 d.At the withdrawal time 12 h,ADP,SMZ and Ac SMZ had the highest concentration in the kidney with concentrations of 27725.9 μg/kg,4440.0 μg/kg and 12806.2 μg/kg;At the withdrawal time of 7 d,the concentration of Ac SMZ in the kidney and liver were 45.4 μg/kg;At the withdrawal time 14 d,the concentrations of ADP and SMZ in liver were 85.6 μg/kg and 19.6 μg/kg respectively,the concentration of ADP and SMZ in kidney were 55.9 μg/kg and 50.7 μg/kg respectively and the concentration of SMZ in muscle was 29.3 μg/kg.At the withdrawal time of 28 d,no drug was detected.It was determined that the residual target tissue and residual marker of ADP-SMZ injection were liver and aitoprim respectively.According to the maximum residue limit standard of the Ministry of Agriculture,according to the above results of drug withdrawal period,the ADP-SMZ injection was continuously administered to pigs for 7 d according to the recommended dose(25 mg/kg b.w.),and the recommended drug withdrawal period in pig was 20 d.3 Safety of ADP and ADP-SMZ injection in swineAccording to "VICH Overview of Guiding Principles for Safety Research of Veterinary Drug Targets”,56 pigs were divided into 7 groups,including ADP injection1-fold recommended dose group(10 mg/kg b.w.),3-fold recommended dose group(30mg/kg b.w.),5-fold recommended dose group(50 mg/kg b.w.),ADP-SMZ injection1-fold recommended dose group(20 mg/kg b.w.),3-fold recommended dose group(60mg/kg b.w.),5-fold recommended dose group(100 mg/kg b.w.)and blank group(saline),with 8 pigs in each group,which were continuously administered with intramuscular injection for 9 d.During the experiment,the clinical performance and weight change of the tested pigs were observed,and the indexes of blood physiology,biochemistry and organ coefficient were determined.At the end of the experiment,the blank and high-dose groups of animals were killed,and the heart,liver,kidney and other tissues were collected to make pathological sections for pathological observation.The results showed the clinical performance of the groups of 1,3 and 5 times recommended doses of ADP injection and ADP-SMZ injection was good,the intake,drinking water,defecation and urination were normal,and there was no obvious abnormality.There was no significant differences in growth rate and feed conversion rate between experiental group of ADP injection and ADP-SMZ injection and blank group(P>0.05).Although there were some changes in blood routine and blood biochemical indexes including WBC,MCHC,MCV,CK,TP of pigs before and after administration,all the detected values were within the normal range and there was no significant differences(P>0.05).Organ coefficients revealed no significant differences(P>0.05).The tissues and cells of the were intact in morphology and normal in structure without obvious pathological changes.The study showed when ADP and ADP-SMZ injections was applied to swine at 5times of the highest recommended dose and it were safe... |
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