| Dinitolmide,as an effective nitroamide anticoccidial drug,has been widely applied in poultry production.However,the long-term continuous improper use of dinitolmide will make drugs in poultry tissue accumulation in the form of the original drug and metabolites,which causes a severe issue of drug residue in poultry tissues.After long-term consumption of animal-origin product containing dinitolmide and its metabolites,veterinary drugs will accumulate in the human body and bring threat to human health.At present,the analytical methods for the determination of dinitolmide and its metabolite(3-amino-2-methyl-5-nitrobenzamide,3-ANOT)in poultry tissue were mainly high performance liquid chromatography-tandem mass spectrometry.However,the residual detection of dinitolmide and its metabolite in poultry tissues analyzed by gas chromatography-tandem mass spectrometry were not been reported at home and abroad.Therefore,this study integrated new techniques or methods,for example,accelerated solvent extraction,and solid phase extraction.The present study established a GC-MS/MS detection method including pre-treatment procedures and validation process,with Jinghai Yellow chickens as test materials,to determine dinitolmide and its metabolite residue levels in chicken tissues of meat,liver,kidney,(skin and fat)and eggs,so as to provide scientific reference for the establishment of national standard on detection method of dinitolmide and its metabolite related residue as well as technological means of residue monitoring in animal-origin products.Main experimental results are shown as follows:1.Simultaneous extraction method of dinitolmide and its metabolite residue from chicken tissues of muscles,livers,kidneys,skin and fat,and eggs using technique of accelerated solvent extraction(ASE)was established and optimized for the first time,respectively.Samples of chicken tissues and eggs were mixed with diatomite and were loaded into the extract pond.The extraction process underwent at a device condition of 80 ℃ and 1500 psi with n-hexane as de-fat solvent and acetonitrile as extraction solvent of dinitolmide and its metabolite.At spiked level of limit of quantification(LOQ)、0.5MRL(Maximun redidue limits)、MRL、2MRL,the average recoveries of dinitolmide and its metabolite in chicken muscle,liver,kindey,skin and fat were 81.96%-94.31%,with the relative standard deviation(RSD)of 1.72-5.37%;the average recoveries of dinitolmide and its metabolite in albumen,yolk and whole egg were 82.74%-87.49%,with the relative standard deviation(RSD)of 1.73-4.63%.This method of extraction has advantages of less consumption of extraction solvent and matrix effect,higher extraction efficiency and recoveries,and also is convenient,fast,automatic.3.A GC-MS/MS method was established,optimized,and validated,for the first time,to determine simultaneously dinitolmide and its metabolite residue levels in tissues of muscles,livers,kidneys,skin and fat,and eggs from chickens,respectively.Electron ionization was adopted,with SCAN mode,and AutoSRM was used for qualitative or quantitative purpose.Within the dosing level of LOQ-200.0 μg/kg for dinitolmide and its metabolite in poultry muscle,kidney,skin and fat and albumen tissues,the peak area of quantificational ion showed a good linear correlation with concentration(R2>0.9993).Within the dosing level of LOQ-1600.0 μg/kg for dinitolmide and its metabolite in chicken liver,the peak area of quantificational ion showed a good linear correlation with concentration(R2>0.9993).Within the dosing level of LOQ-1800.0 μg/kg for dinitolmide and its metabolite in yolk and whole egg,the peak area of quantificational ion showed a good linear correlation with concentration(R2>0.9995).At spiked level of limit of quantification(LOQ)、0.5MRL(Maximun redidue limits)、MRL、2MRL,intra-day RSD and inter-day RSD of dinitolmide and its metabolite in chicken muscle,liver,kidney,skin and fat were within the range of 1.87%-5.74%and 3.34%-7.36%,respectively;intra-day RSD and inter-day RSD of dinitolmide and its metabolite in albumen,yolk,and whole egg were within the range of 2.96%-5.21%and 3.94%-6.34%,respectively.Under current experimental method and instrumental conditions,the limit of detection(LOD)of dinitolmide and its metabolite in chicken muscle,liver,kidney,skin and fat were within the range of 0.8-2.5 μg/kg(S/N≥3),the LOQ of dinitolmide and its metabolite in chicken muscle,liver,kidney,skin and fat were within the range of 2.7-8.0 μg/kg(S/N≥10);the LOD of dinitolmide and its metabolite in albumen,yolk and whole egg were within the range of 0.8-2.8 μg/kg(S/N≥3),the LOQ of dinitolmide and its metabolite in albumen,yolk and whole egg were within the range of 3.0-10.0 μg/kg(S/N≥10).This method is of simplicity,high precision,and high sensitivity and the validation parameters of this method meet the drug residue requirements of the Ministry of Agriculture,the European Union and the United States. |
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