Determination Of Residues Of Flunixin In Swine Edible Tissues

Flunixin,which is usuallyusedwith its meglumine salt(FM)in veterinary practice,belongs to the animal-special non-steroidal anti-inflammatory drug(NSAID).And like most NSAIDs,its mechanism is related to its ability of inhibiting cyclooxygenase activity leading to decrease of prostaglandin and thromboxane A2,which could alleviate the organism of pyrexia,inflammation and pain.The maximum residue limits(MRL)of flunixin in the edible swine tissue have been established by the European Unionand USA,however,the MRLof flunixin in swine have not been set by China.Meanwhile,there has very few published report about the determination of flunixin residue in edible swine tissues,besides,the methods were low reproducibility,low sensitivity and labor-intensive.In order to assure the safety of animal food products for human consumption and avoid potential hazards to health,it is highly necessary to establish a reliablemethod for determination of flunixinresidues in edible swine tissues.The purposes of the study were to establish areliable,rapid and sensitive UPLC-MS/MS method for determination of flunixin in edible swine tissues(muscle,skin+fat,liver and kidney),plasma,urine and feces,and to study the related residue depletion profile in edible swine tissues,as well asits’ metabolism in swine plasma,urine and feces.The results are as following:1.Development of an UPLC-MS/MS method for the determination of flunixin in edible swine tissuesA reliable,rapid and sensitive UPLC-MS/MS method was developed and optimized for determination of flunixin in edible swine tissue.The sample was extracted with acetonitrile/ethyl acetate(1:1,v:v),and then purified via a MCXcartridge.Chromatographic separation was performed on an Acquity UPLC BEH C18 column.Analysis was carried out on UPLC-ESI-MS/MS working with multiple reaction monitoring mode.Stable isotope internal standard was used for quantitation of target drug.The analyte has a good linearity at the range of 1～200μg·kg-1,with related coefficient of R2>0.999.Recoveries of spiked samples ranged from73.7%to 124.3%with relative standard deviation(RSD)less than 17.8%.The limits of detection(LODs)and quantification(LOQs)were 0.5 and 1 μg·kg-1 respectively.2.Development of an UPLC-MS/MS method for the determination of flunixin in swine plasma,urine and fecesA reliable,rapid and sensitive UPLC-MS/MS method was developed and optimized for determination of flunixin in swine plasma,urine and feces.The plasma sample is extracted with acetonitrile;the urine sample is extracted with acetonitrile/2%phosphoric acid aqueous solution(1:4,v:v),and then purified via an Oasis MCX solid-phase extraction cartridge;the feces sample is extracted with acetonitrile/ethyl acetate(1:1,v:v),and further purified via a MCX cartridge.Chromatographic separation was performed on an Acquity UPLC BEH C18 column.Analysis was carried out on UPLC-ESI-MS/MS working with multiple reaction monitoring mode.Stable isotope internal standard was used for quantitation of target drug.The analyte has a good linearity at the range of 0.5～500μg·kg-1 or μg L-1,with related coefficient of R2>0.999.Recoveries of spiked samples ranged from 68.9%to 118.6%with relative standard deviation(RSD)less than 18.3%.The LOD and LOQ in plasma were 0.25 and 0.5 μg·L-1,in urine were 0.25 and 0.5 μg·L-1,and in feces were 0.5 and 1 μg·kg-1,respectively.3.Residue depletion profile of flunixin in edible swine tissues after intramuscular administrationTwenty-seven healthy pigs were divided randomLy into five treatment groups(5 pigs/group)and one control group(1 male:1 female).Flunixin-treated pigs were given doses of 2.2 mg·kg-1 once a day for 3 consecutive days,control animals did not receive any treatment.Five medicated pigs were sacrificed at 0.5,6,11,16,and 23 days after the last dose,and another two control pigs were sacrificed at 7 days.Tissue samples were obtained,homogenized and stored at-20℃ prior to the UPLC-MS/MS analysis.The results showed thatthe developed UPLC-MS/MS method in this study was successfully applied to monitor flunixin residue depletion in edible swine tissues.After intramuscular administration of flunixin meglumine,flunixin residues in all tissues were below the MRL at 16-days post last dose,and were around or lower than the LOQ at 23-days post last dose.Considering Flunixin residue concentrations in muscle,skin+fat,liver,kidney,injection site muscle and injection site skin+fat,corresponding withdrawal periods of 5.15,23.37,9.78,19,70,18.72,and 26.76 days were determined based on MRL set by EMEA by the software WT1.4.4.Metabolism profile of flunixin in swine plasma,urine and feces after intramuscular administrationNine healthy pigswere divided randomLy into one treatment groups(4 castrated males:3 females)and one control group(1 male:1 female).Flunixin-treated pigs were given doses of 2.2 mg·-kg-1 once a day for 3 consecutive days,control animals did not receive any treatment.Plasma,urine and feces samples were obtained,vortexed and stored at-20℃prior to the UPLC-MS/MS analysis.The results showed that the developed UPLC-MS/MS method in this study was successfully applied to monitor flunixin metabolism in swine plasma,urine and feces.After intramuscular administration of flunixin meglumine,The half-life(ti/2λz)of flunixin after the last dose in pigs was 7.37±1.60 h,mean peak plasma concentration(Cmax)was 2.79±1.29N.g·mL-1,mean time to peak(tmax)was 0.57±0.19 h,and approximately 6.9±1.6%and 1.9±0.4%of the administered dose was excreted as parent compound in urine and feces respectively.