The Disposition Of Olaquindox In Swine,Broilers,Carps And Rats

Olaquindox is a kind of feed additives first synthesized by Germany's Bayer in 1967 for growth promotion.Olaquindox is widely used in the livestock industry,but at the same time,due to the extensive use of olaquindox,it results in excessive residue in tissues which has been seriously alfected at food security monitoring and human health.According to the Joint of FAO/WHO Expert Committee on Food Additives(JECFA)report,an acceptable daily intake(ADI)was not set for olaquindox because of the absence of the information about the mutagenic potential of the drug and special toxic effects of its metabolites.There are still some new and old issues with the absorption,distribution,excretion and metabolism of olaquindox unresolved.Therefore,the excretion,metabolism,distribution and residue elimination of olaquindox in swine,broilers,carps and rats were researched by the use of 3H labeling and high performance liquid chromatography combined with ion trap time-of-flight mass spectrometry(LC/MS-IT-TOF)in this study,by which residues were determined in animal tissues,metabolites were identified and the possible pathway was speculated.Based on this study,it can provide a strong basis for the development of animal food safety(residue limits,withdrawal),pharmacology and toxicology.1 The synthesis of 3H-olaquindoxThe synthesis of tritium labeled olaquindox was based on the tritium labeled o-nitroaniline under alkaline conditions,in which sodium hypochlorite solution(containing 5%available chlorine)was added for 4h and tritium labeled benzofurazan-1-oxide was obtained;secondly,acetyl acetateester and ethanolamine reacted and N-hydroxyethyl-acetoacetamide was obtained;lastly,tritium labeled olaquindox was produced by the reaction of tritium labeled benzofurazan-1-oxide and N-hydroxyethyl-acetoacetamide at 40? for 48h.In this study,the chemical purity,radiochemical purity and specific activity of the target compound were determined by using high-performance liquid chromatography combined with liquid scintillation counter.The results showed that the yield of synthesis of tritium labeled olaquindox was 60.5%,chemical purity and radiochemical purity were higher than 98%,and specific activity was 2.65Ci/g,which meet the requirements for ani-mal experiment.2 Mass balance and metabolism research of olaquindox in swine,broilers,carps and ratsFour pigs and six chickens were administered with a single oral dose of 3H-olaquindox in accordance with the feed added olaquindox 200mg/kg and 50mg/kg dose,respectively;six carps and six rats were treated with a single oral dose of 3H-olaquindox at 10mg/kg of body weight.The specific activity was 0.1Ci/g.Urine and feces were collected at 6h,12h,24h and then at each day after dosing until none of radioactivity was detected,by which the excretion of olaquindox in animals was calculated.Urine,feces,blood and bile were extracted and purified,and then the metabolites of the drug were identified and quantified by a combination of LC/MS-IT-TOF and liquid scintillation coumting(LSC).The results showed that the recoveries of total radioactivity were 95.07%,94.92%,and 96.95%in the excreta of pigs,chickens,and carps,and the recoveries of total radioactivity in the excreta of male and female rats were 94.89%and 91.79%,respectively.More than 85%of total radioactivity excreted in 24h after administration;the recovery of radioactivity in pigs urine was 93.08%and the recovery of radioactivity was also more than 85%in rats urine.The results concluded that the absorption and excretion of olaquindox were rapid in pigs,chickens,carps and rats,and it was mainly excreted in the urine.Nine,nine,five and ten kinds of radioactive compounds were detected in pigs,chickens,carps and rats;the metabolites detected in pigs and chickens were the same,09 was only detected in rats,and 00,01,02,04 and 07 were detected in carps.According to the concentrations of metabolites in pigs,chickens,carps and rats during different time periods,it can be deduced that N1-deoxy olaquindox and N4-deoxy olaquindox were generated by deoxygenation of olaquindox firstly,and then bis-N-oxide of olaquindox was generated by the deoxygenation of mono-N-oxide of olaquindox;at the same time,mono-N-oxide of olaquindox were oxidized to the carboxylic acid derivatives,and these compounds were deoxidized to 2-carboxymethylaminocarbonyl-3-methylquinoxaline.3 Distribution and elimination research of olaquindox in swine,broilers,carps and ratsPigs(twenty-four divided into six groups)and chickens(thirty divided into five groups)were administered with a single daily oral dose of 3H-olaquindox corresponded to a feed inclusion of about 100mg/kg and 50mg/kg dose for growth promotion for seven consecutive days,respectively;fish(thirty-six divided into six groups)and rats(thirty divided into five groups)were administered with a single daily oral administration of lOmg/kg body weight 3H-olaquindox for seven consecutive days.The specific activity was 0.1Ci/g.Pigs and carps were sacrificed at 6h,1d,3d,7d,14d and 28d(chickens at 6h.Id,3d,7d and 14d,rats at 6h,1d,3d,7d and 14d)after dosing.Heart,liver,spleen,lung,kidney,blood,bile et.al were collected and digested for the determination of total radioactivity by LSC,by which the distribution of olaquindox the in vivo were studied.Excreta and edible tissues were extracted and purified,and then the metabolites of the drug were identified and quantified by a combination of LC/MS-IT-TOF and liquid scintillation counter.Olaquindox and its metabolites were distributed widely in pigs,chickens,carps and rats.At 6h after administration,higher radioactivity were detected in swine bile,kidney,stomach,bladder and liver;the radioactivity concentrations in bile,kidney and liver were higher in chickens;higher radioactivity were found in the blood,liver,kidney and intestines in carps;the radioactivity concentrations in blood,kidney,liver,bladder,large intestine and small intestine were higher in rats.At 7d(6d for chickens)after dosing,the radioactivity concentrations were higher in liver and kidney than that in other organizations;at 14d after administration,trace amounts of radioactivity were detected in the liver and kidney in pigs and carps.The metabolites in tissues were widely distributed.In pigs,three metabolites(02,05 and 08)were detected in the liver;eight kinds of metabolites(01,02,03,04,05,06,07 and 08)were found in the kidney;six radioactive compounds(O0,01,02,03,06 and 07)were determined in the muscle;eight kinds of radioactive compounds(O0,01,02,03,04,05,07 and 08)were observerd in the fat.In chickens,the metabolites detected in liver and kidney were the same that were 01,02,03,04,05,08 and 09;five kinds of radioactivity compounds(O0,O1,02,03 and 08)were identified in muscle;seven kinds of radioactivity compounds(O0,01,02,03,05,07 and 08)were detected in fat.In carps,the radioactivity compounds in liver and skin were the same that were O0,O1,02,04 and 07;five kinds of metabolites(O1,02,04,05 and 07)were found in kidney;five kinds of radioactive compounds(O0,01,02,06 and 07)were determined in muscle.In rats,the metabolites in liver,kidney and muscle were the same that were 01,02,03,04,05,07 and 08;four kinds of metabolites(01,02,05 and 08)were detected in fat.The results showed that olaquindox was metabolized quickly and widely in pigs,chickens,carps and rats.Although there are some differences in the types of metabolites in different tissues,the major metabolite was the same that was 02 in pigs,chickens and rats,and the main metabolites were 01 and 02 in carps based on AUC calculated.The elimination of the drug in liver and kidney were slower than that in other organizations in pigs,chickens,carps and rats.In pigs,the half-time of total residues were 2.87d and 4.94d in liver and kidney,respectively;in chickens,the half-time of total residues were 3.13d and 2.57d in liver and kidney,respectively;in carps,the half-time of total residues were 3.28d and 3.22d in liver and kidney,respectively;in rats,the half-time of total residues were 1.36d and 3.28d in liver and kidney,respectively.Thus,kidney was concluded as the residue tissue in pigs,chickens and carps for the residues of olaquindox.And metabolite 02 was identified as the residue marker in pigs,chickens,carps because of the similar concentration-time curve for metabolite 02 to that of total residues.In sum,the synthesis of 3H-olaquindox and research of its quality criteria were studied for the first time.And the excretion,metabolism,distribution and elimination of olaquindox in pigs,chickens,carps and rats were illustrated in this study.The metabolites in urine,feces,blood and bile were separated and identified,and the metabolites in the edible tissues were identified and quantified in pigs,chickens,carps and rats for illustrating the residue tissues and residue marker.The results would provide more comprehensive information for olaquindox and provide a theoretical basis for the clinical use of drugs.