The Residue Depletion Of Olaquindox In Swine,Broilers And Carp

Olaquindox(OLA),an quinoxaline antibacterial and growth promoting feed additive,first synthesized by Bayer A.G in 1967,has been widely used in livestock breeding.However,there is a severe abuse of olaquindox,which seriously affecting food safety monitoring and human health for its excessive residues in body.Olaquindox is disabled in US and EU,it was stipulated only for pigs less than 35kg with 35 days of the withdrawal period in China,and banned from fish and poultry,furthermore,MQCA(3-methyl-quinoxaline-2-carboxylic acid)is residual marker,which maximum residue limits(MRL)for muscle and liver are 4μg/kg and 50μg/kg respectively.Nevertheless,all previous studies did not provide systematic residue depletion data of olaquindox in food animals and formulation basis of residual standard,it is necessary to comprehensively study its residue depletion.According to the results of radiotracer analysis of olaquindox in pigs,chickens,fish and rats,it was determined that its main metabolites in these animals,which contained 01(N1 deoxy olaquindox),02(N],N4-Deoxy olaquindox),04(3-methyl-quinoxaline-2-carboxylic acid),05(De-dioxo-3-methyl-quinoxaline-2-carboxamide acetate),07(N4 deoxy-olaquindox),and olaquindox(O0)along with original residual marker 06(3-methyl-quinoxaline-2-carboxylic acid,MQCA)were detection objects according to guideline of VICH,the reference substance of the main metabolites were first synthesized,then laws of residue elimination were studied to confirm the residual marker and its target tissues.Presently,liver,kidney,muscle and fat were internationally recognized as commodity tisues,however,the non-commodity tissues which contained heart,lung,stomach,large intestine and small intestine were brought into study considering the life style and customs of Chinese residents.In this paper,six main metabolites of olaquindox were first synthesized,then HPLC detection methods of which and the prototype of olaquindox in various commodity and non-commodity tissues were established with swine,broiler and carp as subject investigated,based on which laws of residue depletion of olaquindox and its metabolites were researched in the three objective animals,and further food safety evaluation was conducted.The results identified that kidney was residual target tissue of olaquindox in swine,however,it was liver for broiler and carp with Deoxy olaquindox acted as residual marker.It was respectively drew up of residue limits and withdrawal standards according to JECFA,FDA and the EMEA program,which provided scientific basis for drug residue monitoring of olaquindox,and theoretical and technical support for revision of its residue standards.1 Preparation of olaquindox and reference substances of the main metabolitesReactions were carried out with olaquindox as raw material,N2S2O4 as the reducing agent,and mol ratio between them was 1:1,sodium hydrosulfite solution was added slowly,the reaction temperature was controlled at about 55℃,mixture of two deoxy olaquindox were obtained after a time,the crude product was purified by silica gel column,followed by recrystallization from ethyl acetate for two times,the purity of the final product was not less than 97%.Reactions were conducted with olaquindox as raw material,N2S2O4 as the reducing agent,the mol ratio between them was determined as 1:4,sodium hydrosulfite solution was added slowly,the temperature was controlled at about 60℃,Deoxy olaquindox was acquired after five hours,which purity was not less than 99%by means of recrystallization from ethyl acetate for three times.Intermediate product 3-methyl quinoxaline-2-carboxylate was obtained with BFO and ethyl acetoacetate as raw materials and triethylamine as the catalyst at 40℃reacted for 24 hours,the mol ratio between raw material and catalyst was controlled at 1:1.5.And then N1 Deoxy-3-methyl-quinoxaline-2-ethyl formate was generated via deoxygenation reaction with the intermediate product as raw material and N2S2O4 as the reducing agent,which mol ratio was 1:1,and the product is Deoxy-3-methyl-quinoxaline-2-ethyl formate when adding an excess of N2S2O4,afterwards the ester bond of which was hydrolyzed with NaOH,MQCA and N1 Deoxy MQCA were obtained,purity of MQCA was not less than 99%after recrystallization from ethyl acetate two times.Where after condensation reaction was conducted between MQCA and glycine ethyl ester,then further treated with alkaline hydrolysis,3-methyl-quinoxaline-2-carboxylic acid and De-dioxo-3-methyl-quinoxaline-2-carboxamide acetate were obtained,which purity were not less than 97%after recrystallization from ethyl acetate for two times.2 Establishment the HPLC residue detection method of olaquindox and its main metabolitesIt was determined that 01,02,04,05 and 07 were main metabolites of olaquindox in terms of our previous radiotracer analysis in pig,chicken,fish and rat.The HPLC detection methods of olaquindox and its five main metabolites as well as original marker residue MQCA in the nine commodity and non-commodity tissues of swine and broiler were established,meanwhile,methods of six tissues that is liver,kidney,muscle,fat,skin and gastrointestinal tract towards carp were studied.Specifically,chromatographic column:Aglient ZORBAX SB-C18(4.6×250mm,5μm);mobile phase:A phase:0.6%formic acid water,B phase:acetonitrile.the elution condition:0-5min,acetonitrile 10%,formic acid water 90%;5-25min,acetonitrile 30%,formic acid water 70%;25.01 min,acetonitrile 10%,formic acid water 90%:32min,acetonitrile 10%,formic acid water 90%,detected by HPLC at 320 nm,sample injection volume was 40μL,column temperature was 30 C.The tissues were extracted by a complex reagent comprised of O.lmol/L hydrochloric acid.50%methanol and 40%ethyl acetate,defatted with hexane,and purified by hydrophilic-lipophilic balance copolymer(HLB)SPE column,followed by redissolving with acetonitrile:0.6%formic acid water(v/v 10/90),finally,the residues were detected by HPLC at 320 nm.In this method,the limit of quantification(LOQ)of 01 and 02 was 20μg/kg in liver,kidney,muscle,fat,heart,lung,stomach,large intestine and small intestine of swine and broiler,and in muscle,skin and gastrointestinal tract of carp.The LOQ of 04,05,06.and 07 in tissues of swine,broiler and carp was 40:g/kg,and LOQ of O0 was 50μg/kg.The recovery range was 63.1%-86.5%when administrated with different drugs that more that LOQ.and inter-day CV was inferior to 12.6%.The residue detection method established in this study meet the requirements of quantitative analysis,can be used for residual elimination and detection analysis of olaquindox and the metabolites.3 The depletion study of olaquindox in Swine,Broilers and CarpPrimarily.25 healthy crossbred swines,30 twenty-day-old Kebao broilers and 30 healthy carps were randomly divided into five groups,swines were administrated continuously with 100 mg/kg olaquindox for 30 days,broilers and carps were treated with 50 mg/kg olaquindox for 30 days.Animals were slaughtered at different withdrawal time,the liver.kidney,muscle,fat,heart,lung,stomach,large intestine and small intestine were collected.The content of olaquindox and its metabolites were quantified according to the described methods,and the results are shown as follows:Swine:Six hours after withdrawal time,prototype O0,O1,O2and MQCA were detected in all tissues,concentration ranges of which were 108.4-212.4 μg/kg,36.6-219.1μg/kg,73.7-934.0μg/kg and 47.6-402.8.μg/kg,02 is highest in kidney.And prototype and O1 were eliminated more rapidly in swine.Three days after withdrawal time,prototype and O1 were eliminated to LOQ in all tissues,MQCA was detected in liver and kindey with 35.8μg/kg and 101.0μg/kg,02 was detected in liver,kidney and lung with 86.5μg/kg,293.6μg/kgand 61.1μg/kg respectively,and all detection objects were eliminated to LOQ in other tissues.Seven days after withdrawal time,only 02 was detected in kidney,eoncentration was 111.0 μg/kg.Fourteen days after withdrawal time,prototype and all metabolites were eliminated to LOQ in all tissues.Thereby,it was found that residence time of olaquindox and its metabolites were all above three time points in liver,kidney,muscle and lung,02 and 06 had longest stay.Broiler:Six hours after withdrawal time,in no tissues of broiler was 07 detected compared with swine,O0,01 and 02 were detected in all tissues,concentration of which were 51.8-298.9μg/kg,38.2-178.1μg/kg,23.4-164.6μg/kg respectively,and concentration in liver was the highest.Three days after withdrawal time,02 was detected in liver with 57.8μg/kg and kidney with 57.7μg/kg,01 and 02 were detected with 22.0 and 38.5μg/kg in lung respectively.A small amount of 02 was detected in kidney five days after withdrawal time and concentration was 40.5μg/kg,concentration of which in liver was 43.6μg/kg.No product of metabolism was detected in all the tissues ten days after withdrawal time.It showed that residence time of olaquindox and its metabolites were all above three time points in liver,kidney,muscle and lung,01,02 and 06 had longest stay.Carp:Less metabolism product were detected six hours after withdrawal time in carp compared with swine and broiler,only prototype O0,O2 and O6 were detected in liver,kidney,muscle,skin,and gastrointestinal tract,and only O0 and 02 were detected in fat.One day after withdrawal time,02 and 06 were detected only in liver and kidney,concentration ranges of which were 39.8-62.4μg/kg,38.4-39.9μg/kg respectively,majority of 02 and 06 were found existed in liver,and 02 was also detected in gastrointestinal tract with 46.2μg/kg.Prototype O0 and 06 were eliminated to LOQ in all tissues three days after withdrawal time,only a small amount of 02 was detected in liver and eoncentration was 36.3μg/kg.Seven days after withdrawal time,in no tissues of carp were metabolism products detected.It was found that residence time of olaquindox and its metabolites were above three time points only in liver,among which 02 had longest stay.4 Recommendation of maximum residue limits and withdrawal times of olaquindox in edible tissues of swines,broilers and carpThe maximum residue limits(MRLs)of olaquindox in edible tissues of swine,broiler and carp were calculated according to procedures and methods which JECFA recommended.Also,three maximum residue limits standards of olaquindox were proposed in reference with procedures and methods of US,FDA and EU.Based on which,the MRLs of olaquindox in edible tissues of the three objective animals were raised that compliance with China s national conditions.In accordance with the procedures of JECFA,withdrawal time of olaquindox in swine,broiler and carp were respectively 7 d,10 d and 4 d,and the withdrawal time of olaquindox were 14 d,15 d,and 8 d respectively according to EMEA.Comprehensive analysis of the results of the procedures,the MRLs of olaquindox in edible tissues of swine,broiler and carp that conservatively recommended was 10 μg/kg,and the withdrawal time were respectively 14 d,15 d and 8d.In summary,reference substances of olaquindox main metabolites were prepared for the first time,a HPLC detection method of olaquindox and the six main metabolites in various commodity and non-commodity tissues of swine,broiler and carp was established.Meanwhile,laws of residue elimination of olaquindox were firstly systematically studied,residual marker and target tissues were further corroborated,and MRLs together with withdrawal standards were further put forward.It supplied base values for pharmacological,toxicological effects,drug safety evaluation and risk assessment,and provided scientific basis for food security monitoring and comparison of residue depletion of olaquindox in different animals,as well as theoretical support for clinical safety rational use of drugs and establishment of healthy culture environment.