Safety And Depletion Of Doxycycline Hydrochloride-Flufenicol Compound Injection In Pigs

Pneumonia diseases have always been a serious hazard to the pig industry.doxycycline hydrochloride and fluorophenicol have been widely used in the treatment of these diseases due to their wide antibacterial spectrum,strong antibacterial activity and excellent pharmacokinetic characteristics in pigs.As the Pneumonia diseases are often manifested as mixed infection of multiple pathogenic bacteria,it is difficult to effectively control with single drug in clinical,so combination of drugs is needed to improve the efficacy and avoid the production of drug-resistant bacteria.Combined application of doxycycline hydrochloride and fluorobenicol can increase the antibacterial spectrum and enhance the antibacterial effect.Therefore,the development of the doxycycline hydrochloride-fluorobenicol compound injection has important clinical application value.As a new compound injection,it must undergo rigorous phase?,?,and?clinical trials before being put on the market.The safety and depletion of target animal are the core research content.The purposes are to observe the tolerance and adverse reactions of target animal to the new drug,reveal the elimination law in the target animal,and propose the standard of withdrawal time.According to the"VICH Veterinary Drug Target Animal Safety Guidelines"and"Veterinary Drug Residue Test Guidelines?Draft for Comment?",the study on the safety and depletion of doxycycline hydrochloride-flufenicol compound injection in pigs were carried out.1. The safety of doxycycline hydrochloride-florfenicol compound injection in pigs32 healthy ternary hybrid pig were selected,weighing 19±0.47 kg.After adapting to feeding for one week,they were randomly divided into four groups,namely 1?8mg/kg?,3?24 mg/kg?,5?40 mg/kg?times the recommended dose group and blank control group,each group had 8 animals.Administered by intramuscular injection once a day for 6 consecutive days.During the experiment,the clinical manifestations of pigs in each group were observed,and blood routine and blood biochemical indexes were detected before drug administration,0d after withdrawal and 3d drug withdrawal.At the end of the experiment,autopsy and histopathological examination were performed on the animals.Clinical observation found that the injection site of the 5-fold recommended dose group?40 mg/kg?appeared red and swollen,and occurred diarrhea.None of other groups saw any abnormal symptoms.Blood routine detection result showed that the WBC levels and HGB concentration of the 5-fold recommended dose group?40 mg/kg?were significantly reduced at the withdrawal time of 0 d,and the difference was significant compared with blank control group?P<0.05?.When time was extended to the withdrawal time of 3 d,the WBC and HGB values slightly increased.at this time,the significant difference from the blank control group disappeared.Except for WBC and HGB,the other blood routine indicators were not significantly different between groups?P>0.05?.Through comparative analysed of blood biochemical indicators in each group,ALP,AST,TG,CREA,UREA,TP,ALT,GLB,LDH,P,MG,TC,CA values of each dose group were not significantly different from blank control group?P>0.05?;but with the increase of the administered dose,the changes of CK,GGT,and GLU levels became more and more obvious,and the difference between the 5-fold recommended dose group?40 mg/kg?and blank control group were significant?P<0.05?.The results of autopsy and histopathological examination showed that the heart,liver,kidney,lung,spleen and other organs in the 5-fold recommended dose group?40 mg/kg?did not show any pathological changes.In conclusion the doxycycline hydrochloride-florfenicol compound injection had no significant effect on the indicators of blood routine and blood biochemical in pigs,and had no obvious pathological effects on pigs'heart,liver,kidney,lung,spleen.2. Establishment of UPLC-MS/MS method for simultaneous detection of doxycycline hydrochloride,fluorophenicol and fluorophenicomide in pig edible tissues0.1%ammoniated acetonitrile+Na₂EDTA-Mac Ilvaine buffer was used as the extractant,the extraction was repeated twice.The extractant was blown dry at 60?with nitrogen.Reconstituted with the initial mobile phase.UPLC-MS/MS and external standard methods were used for detection and quantification,respectively.The method showed good linearity?r>0.99?in the range of 10-1000?g/kg,and the LOD and LOQ was 5?g/kg and 10?g/kg,respectively.The average recovery was between 68.85%?113.54%.The overall intra-batch variation coefficient was less than11.7%,and the inter-batch variation coefficient was less than 9.5%,The stock solution and working solution could be stored in the dark at-20?for three months,and the spiked samples could be kept in the dark at-20?for 30 days.3. Depletion of doxycycline hydrochloride-florfenicol compound injection in pig edible tissues25 healthy ternary hybrid pig were selected,weighing 29.0±0.55 kg.After a week of routine feeding and adaptation,they were randomly divided into 2 groups,namely blank control group?5 heads?and residual group?20 heads?.The residual group was injected with doxycycline hydrochloride and florfenicol compound injection in the neck muscle at a dose of 20 mg/kg,once a day,for consecutive 7 d,A group of pigs were slaughtered at the withdrawal times of 0.5,3,7,14,28 d and 42 d,respectively,of which including 1 blank pig and 4 residual pigs.The liver,kidney,muscle,sebum of each pig were collected,and the samples were detected by UPLC-MS/MS after pretreatment.At the withdrawal time of 0.5 d,except for the injection site,the highest concentrations of doxycycline hydrochloride and florfenicol were observed in kidney,with concentrations of 10853?g/kg and 4591?g/kg,respectively,While in liver the concentration of florfenicol amine was the highest,with concentration of 1178.51?g/kg;the t_1/2of doxycycline hydrochloride in pig's kidney,liver,muscle,and sebum was 4.90 d,5.72 d,6.79 d,and 5.81 d,respectively,and florfenicol was 2.49 d,3.40 d,2.74 d,and 1.72 d,respectively.Florfenicol amine could only be detected in sebum at two time points,so couldn't be plotted as a elimination curve and the t_1/2in the kidney,liver,and muscle was 3.89d,4.41d,and3.62 d,respectively.The withdrawal time of florfenicol in kidney,liver,muscle,sebum and the injection site muscle was calculated by WT1.4 software to be 14 d,7 d,11 d,5 d and 17 d,respectively,and those of doxycycline hydrochloride were 30 d,34d,44 d,21 d and 38 d,respectively.It was suggested that doxycycline-fluorophenicol compound injection be given to pigs once a day at 20 mg/kg b.w for 7 consecutive days,the withdrawal time should be at least 44 days.In This paper,the safety of doxycycline hydrochloride-flufenicol compound injection in pigs was evaluated and the rule of depletion of this compound injection in edible tissues of pigs was clarified,and a withdrawal time was formulated.These results provide reliable data support for the approval of doxycycline hydrochloride-flufenicol compound injection as new veterinary drugs,and have important guiding significance for its future application in veterinary clinical.