| Metronidazole is a synthetic nitroimidazoles antibacterial drugs,which is used for prevention and treatment of infections caused by bacteria and protozoa of poultry blackhead and pigs treponema dysenteria.It is widely used in the veterinary clinical and the livestock industry because of the advantages of better antibacterial effect,safe,cheap,etc.But the toxicological research showed that MNZ has the carcinogenic and potential mutagenic risk to human health.It has banned for use as feed additive in food-producing animals.Moreover,the residues of MNZ is not allowed to be detected in animal foods in various jurisdictions,and only allowed to be used as therapeutic agents.However,in practice,MNZ is still abused or illegally used to prevent disease and gain high economic benefits.The residues of MNZ in animal tissues are potentially harmful to human health and of great concern for its food safety.Currently,there are still many unresolved issues about the disposition of metronidazole in pigs,chickens and other food animals,it is not very clear of metronidazole metabolism in food animals,and the residue of the target organization and residual marker on a target animal is not also yet to be determined,security application of metronidazole in food animals has large controversy.Therefore,the metabolism and tissue residue elimination of metronidazole in swine,chicken and rat were researched by using technology of LC/MS-IT-TOF combined with LC-MS/MS,LC/MS-IT-TOF was used for qualitative analysis of metabolites,LC-MS/MS comparison standard quantitative analysis of tissue metabolites,to speculate the possible metabolic pathway of metronidazole in animals,to study its residues in animal tissues,to determine the residual target tissue and residue markers,which provided clear monitoring objects and technical support for food residue monitoring of metronidazole.1 Metabolism research of metronidazole in swine,chickens and ratsFour pigs,six chickens and six rats were administered with a single oral dose of 25mg/kg of body weight.Urine and feces were collected at 6h,12h,24h and then at each day and plasma and bile(pigs,chickens)were collected at the first five points.The urine samples were processed with enzymolysis and without enzymolysis hydrolysis,using LC/MS-IT-TOF qualitative analysis of metabolites,LC-MS/MS quantitative analysis of tissue metabolites.The results show that the recoveries of drug were 75.5%-76.5%at 1d after administration.At 14d after administration,the recoveries were 85.8%,86.4%and 86.6%in the waste of swine,chicken and rat respectively,average recovery is more than 85%.Urinary excretion rates were 65.2%and 75.2%in rat and swine,respectively,feces excretion rates were 10.6%-21.4%.It can be seen,metronidazole is rapidly absorbed,and rapidly excretion,mainly excreted by urine.Six,four and four kinds of metabolites were detected in swine,chicken and rat,which MNZ,HM,MAA,MOOH,Glu-MNZ and Sul-MNZ were detected in swine,MNZ,HM,MAA and MOOH were detected in chickens,MNZ,HM,MAA and Glu-MNZ were detected in rats.According to metabolism of metronidazole in three animals,the main presumption of the metabolic characteristics of metronidazole includes:One is the side-chain hydroxylation;the second is the oxidation of hydroxylated side chain,including an alcohol-based oxidized to a carboxyl group and the 2-hydroxymethyl-oxide;Third is metronidazole combined with glucuronic acid or metronidazole combined with sulfuric acid.2 The experiment of residue depletion on swine and chickenTwenty four pigs and thirty six chikens were administered with oral dose of 25mg/kg of body weight for consecutive 7 days.Swine and chicken were slaughtered at 6h,1d,3d,5d,7d and 14d of the withdrawal period,liver,kidney,muscle,fat,heart,lung,stomach,large intestine and small intestine were collected,The samples were extracted and purified after homogenized,and the metabolites were quantitative analysised by the LC-MS/MS.In study of metronizazole in swine,the concentrations of metronidazole in swine liver and kidney were significantly different before and after the Enzymatic hydrolysis,but not significantly difference in other tissues.At the withdrawal time of 6h,MNZ,HM and MAA can be detected in all tissues,which concentrations of MNZ in the lung and stomach are highest,the concentration of HM in the kidney and muscle are highest;At the withdrawal time of 1d,MNZ and HM can be detected in fat,stomach,large intestine and small intestine,MNZ,HM and a small amount of MAA can be detected in liver,kidneys,muscles,heart and lungs;At the withdrawal time of 3d,MNZ and HM can be detected in liver,kidney,muscle,fat,lung,and small intestine,MNZ was the only detectable compound in heart,stomach,and large intestine,the concentrations of MAA were below the detection limit;All tissues are only detected MNZ at the withdrawal of 5d and 7d;At the withdrawal of 14d,only liver and kidney can detect small amounts of metronidazole,the concentration were 2.14μg/kg and 3.06μg/kg,close to the detection limit,other tissues can not detect relevant metabolites.Metronidazole eliminate slower in pig liver,kidney,elimination half-life were 3.Old,4.33d.It can be seen,metronidazole prototype is the slowest elimination compound in all swine edible tissues,kidney is the longest residual tissues of metronidazole,Thus metronidazole prototype was the marker residue in pigs,kidney was the target tissue.In study of metronizazole in chicken,at the withdrawal time of 6h,MNZ,HM and MAA can be detected in all tissues except fat which can not detecte carboxyl metronidazole,the concentrations of MNZ in the stomach are highest,the concentration of HM in muscles are highest;At the withdrawal time of 1d,MNZ,HM and MAA can be detected in kidney,muscle,heart,lung and stomach,large intestine and small intestine can only detect the MNZ and HM;At the withdrawal time of 3d,MNZ,HM and MAA could be detected in Liver,kidney,muscle,heart,lung and stomach.,the concentrations of MAA in large intestine and small intestine were below the detection limit;only MNZ can be detected in all tissues at the withdrawal of 5d and 7d;At the withdrawal of 14d,only liver kidney and muscle can detect small amounts of metronidazole,the concentrations were 5.23μg/kg,4.16μg/kg and 6.36μg/kg,it can not detect relevant metabolites in other tissues,metronidazole eliminate slower in chicken liver,kidney,and muscle,elimination half-life were 2.18μg/kg,3.37μg/kg and 1.36μg/kg.It can be seen,metronidazole prototype is the slowest elimination compound in all chicken edible tissues,kidney is the longest residual tissues of metronidazole,Thus metronidazole prototype was marker residue in the chicken,kidney was the target tissue.In summary,this paper investigated the metabolism and residual depletion of metronidazole in pigs and chickens for the first time,developed the residual analytical methods of metronidazole and its major metabolites in edible tissues of pigs and chickens.Isolated and identified the metabolites of metronidazole in urine,feces,bile and plasma of pigs,chickens and rats,and the metabolites in the edible tissue were analyzed qualitatively and quantitatively,verified residual target tissue and residues markers of metronidazole in swine and chickens.These results supplemented with relevant theoretical data for metabolism and residues research of metronidazole in food animals,and provided a theoretical basis for food safety and residue monitoring of metronidazole in food animals. |
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