Metabolic Transformation Of Enrofloxacin Combined With Tilmicosin And Doxycycline Combined With Florfenicol In Broilers

The combination of enrofloxacin(EF)and tilmicosin(TIM)is used to treat chronic respiratory diseases in broilers.In addition,doxycycline(DOX)is often combined with florfenicol(FF)for the treatment of colibacillosis in broilers.However,there is few study on the metabolic drug interactions and the effect of drug interactions on metabolism and residues.In this study,the effects of DDIs on metabolism and residues of broilers were evaluated on the basis of previous experiments.Firstly,two LC-MS/MS methods for the admeasurement of two groups of drugs and their metabolites in edible tissues and chicken liver microsome were established.Secondly,this study evaluated effects of drugs on cytochrome 450 enzyme(CYP450)in chicken liver microsomes.And the probe substrate method was used to evaluate the role of another drug in the combination on the drug’s key metabolic enzymes in this study.Then drugs were combined to evaluate the impact on metabolism in vitro.The effects of drugs on metabolism and residues in broilers were evaluated by LC-MS/MS.A coalescent of in vitro and in vivo trials explained the effects of DDIs on metabolism and residues.It provides a good method to evaluate the drug interaction produced.The research is as follows:1 Effect of Enrofloxacin combined with tilmicosin in vitroIn chicken liver microsomes,the relationship between EF and CYP450 enzymes was evaluated in order to find the key metabolic enzymes of EF,this study incubated EF with the inhibitor(CYP450 enzyme)by the relative activity of EF metabolism,which was the ratio of the amount of CIP and the blank group.Results showed that the key metabolic enzymes for EF were CYP3A4 and CYP2A6.To study the drug interactions produced by EF and TIM,drugs were incubated in chicken liver microsomes within 30,60,90,120 min,and the effect of TIM on EF key metabolic enzyme activity was examined,the results showed that TIM inhibited CYP3A4 activity but it did not affect the activity of CYP2A6 enzyme.To further study the metabolic effects of drug interactions produced by EF combined with TIM,EF(50 μmol/L)and TIM(50 μmol/L)were incubated into single and combined groups in chicken liver microchondria with incubation times of 0.5,1,2,4 h.Results showed that the metabolism of EF was inhibited when EF and TIM were incubated 4 h.2 Study on Residual Elimination of Enrofloxacin combined with tilmicosin in BroilersEstablishment of LC-MS/MS method for the simultaneous detection of EF,ciprofloxacin(CIP)and TIM in four edible tissues and chicken liver microsomes: the extractant of the samples was phosphate buffer,and samples were purified by HLB column.The detection limit of drugs were 5 μg/kg and the limit of quantitation were 10μg/kg in whole samples.The recoveries of EF,CIP and TIM at 3 different addition concentrations ranged from 76.45% to 100.03% respectively.The intra-batch coefficient of variation of EF,CIP and TIM was less than 10.36%,which met the relevant methodological requirements.Moreover,the inter-batch coefficient of variation of EF,CIP and TIM was less than 8.97%,which conformed to experimental conditions.Conventional recommended doses for both drugs were fed to broilers in 5 d by drinking water in vivo,and the concentrations of drugs distributed in tissues were assessed by improved LC-MS/MS.This study confirmed that EF and TIM were retained in tissues after oral administration of broilers,and the withdrawal time of both drugs were prolonged.The data obtained from the experiment,combined using WT1.4 software,performed linear regression analysis of EF residues by the 95% confidence limit in different matrix samples.The results showed that when enofloxacin soluble powder and tilmicosin soluble powder combined in broiler chickens,the WT of EF in kidney and liver were 12.05 d and 11.32 d,the WT of EF in muscle and fat were 11.32 d and 10.29 d.It is recommended that the WT of EF was 13 d in broilers after combination of 2 powder by drinking water.The WT of TIM in kidney and liver were 14.90 d and 14.09 d,the WT of TIM in muscle and fat were 10.33 d and 13.62 d.EF and TIM were extended by 8 d and10 d to 13 d and 15 d in combination.3 Effect of doxycycline combined with florfenicol in vitroIn chicken liver microsomes,the relationship between FF and CYP450 enzymes was evaluated in order to find the key metabolic enzymes of FF,this study incubated FF with the inhibitor(CYP450 enzyme)by the relative activity of FF metabolism,which was the ratio of the amount of FFA and the blank group.Results showed that the key metabolic enzymes for FF were CYP3A4 and CYP2A6.To study the drug interactions produced by FF and DOX,drugs were incubated in chicken liver microsomes within 30,60,90,120 min,and the effect of DOX on FF key metabolic enzyme activity was examined,the results showed that DOX inhibited CYP3A4 activity but it did not affect the activity of CYP2A6 enzyme.To further study the metabolic effects of drug interactions produced by DOX combined with FF,FF(50 μmol/L)and DOX(50 μmol/L)were incubated into single and combined groups in chicken liver microchondria with incubation times of 0.5,1,2,4 h.Results showed that the metabolism of FF was inhibited when FF and DOX were incubated 4 h.4 Study on Residual Eliminatioof doxycycline combined with florfenicol in broilersEstablishment of LC-MS/MS method for the simultaneous detection of DOX,florfenicolamine(FFA)and FF in four edible tissues and chicken liver microsomes: the extractant of the samples was 2% ammoniated ethylacetate,and samples were purified by MCX column.The detection limit of drugs were 5 μg/kg and the limit of quantitation were 10 μg/kg in whole samples.The recoveries of DOX,FF and FFA were between74.93% and 90.61% at three different addition concentrations.In addition,the intra-batch coefficient of variation of DOX,FF and FFA was less than 12.78%,which proved the excellent method.Morever,the inter-batch coefficient of variation of DOX,FF and FFA was less than 7.85%,which met the relevant methodological requirements.Conventional recommended doses for doxycycline hydrochloride soluble powder(10%)and florfenicol soluble powder(30%)were fed to broilers in 5 d by drinking water in vivo,and the concentrations of drugs distributed in tissues were assessed by improved LC-MS/MS.The data obtained from the experiment,combined using WT1.4 software,performed linear regression analysis of EF residues by the 95% confidence limit in different matrix samples.This study confirmed that FF and DOX were retained in tissues after oral administration of broilers,and the withdrawal time of both drugs were prolonged,the WT of FF in muscle and fat were 8.69 d and 4.54 d,and the WT of FF in kidney and liver were 13.21 d and 8.22 d.It was recommended that the WT of FF was 14 d in broilers after combination of 2 powder by drinking water.The WT of DOX in muscle and fat were 29.68 d and 25.13 d,the WT of DOX in kidney and liver were 28.94 d and5.80 d.The WT of FF and DOX were extended from 5 d and 28 d to 14 d and 30 d respectively.