Establishment Of HPLC-MS/MS Residue Detection Method For Three Antiviral Drugs In Animal Tissues And Study On The Elimination Of Amantadine In White Feather

Amantadine Amantadine is a kind of antiviral drug specially used for humans,and it has certain preventive and therapeutic effects on influenza A.Announcement No.560 of the Ministry of Agriculture of China in 2005 banned the use of antiviral drugs as veterinary drugs.The phenomenon of illegal addition during food animal breeding is widespread.Amantadine drugs have obvious toxic and side effects,high drug resistance rate,and are metabolized in prototype form.If amantadine drugs are abused during the cultivation of food animals,the drugs will remain in the food animal tissue in the form of parent drug and accumulate in the human body through the food chain,which will bring certain safety risks to the life and health of consumers..However,China has not yet established technical standards for the detection of amantadine,amantadine,and memantine in food animal tissues,and the monitoring of amantadine residues in food animal tissues cannot be monitored.Therefore,carrying out research on the method of amantadine residues in food animal tissues and exploring the elimination of amantadine residues in different tissues of white feather broilers can effectively supervise amantadine drugs in food animal breeding Of illegal use.The experiment improved and innovated the sample preparation method,and comprehensively optimized the chromatographic conditions and mass spectrometry conditions,and established the HPLC-MS/MS method of amantadine,amantadine,and memantine in food animal tissues.The method established in this experiment was used to study the elimination rule of amantadine in white feather broilers.The main research contents are as follows:（1）Determination of amantadine residues in food animal tissues by HPLC-MS/MSThe samples were extracted by acidifying acetonitrile and trichloroacetic acid,adding AT-d15,RT-D4 internal standard and anhydrous sodium sulfate.After vortex oscillation and ultrasonic extraction,Nitrogen was dried.After the initial mobile phase was dissolved again,the samples were purified by 30 mg C₁₈,40 mg PSA and50 mg neutral alumina dispersion solid-phase extraction agent,and then by shimpack gist C₁₈-AQ HP The column was separated and gradient elution was carried out with0.1%formic acid water and methanol as mobile phase.The results showed that the three amantadine drugs had a good linear relationship in the concentration range of2-100μg/kg,and the correlation coefficient R≥0.9992.The detection limits of amantadine,amantadine and memantine were 0.2,0.3 and 0.3μg/kg,respectively.The quantitative limits were 0.5,1.0 and 1.0μg/kg,respectively.In 9 kinds of food animal tissues,such as chicken,pork,beef and chicken liver,the correlation coefficients were2,10 and 50μg/kg The average recovery rate was between 90.6%and 100.5%,the intra day precision was between 2.45%and 6.36%,and the intra day precision was less than 8.91%.The established method is simple,fast,and highly sensitive to meet the qualitative and quantitative analysis of amantadine drug residues in food animal tissues.（2）Establishment of amantadine analysis method in white feather broiler tissuesThe detection limits of amantadine in plasma,muscle,liver and spleen were 2.0μg/L,0.2,0.3 and 0.3μg/kg,respectively.The quantitative limits were 6.0μg/L,1.0,1.0 and 1.0μg/kg,respectively.The recovery rate of amantadine was 90.0%-103.0%in all tissues of white feather broiler.The intra-day RSD and the inter-day RSD were lower than 6.12%and 9.37%,respectively.This method can be applied to the qualitative and quantitative analysis of amantadine in plasma,muscle,liver and spleen of white feather broiler.（3）Study on the elimination of amantadine in white feather broilerThe main pharmacokinetic parameters of amantadine in plasma,leg muscle,chest muscle,liver and spleen were T_max8.542,8.321,8.455,8.000 and 8.371h,T_1/2β5.35,7.32,9.98,12.5 and 10.5h,respectively,and C_max2.787μg/mL,0.221,0.196,3.937 and 2.036μg/g.The main pharmacokinetic parameters of amantadine in plasma,leg muscle,chest muscle,liver and spleen were T_max10.23,12.36,13.78,10.31 and11.25h;T_1/2β6.766,8.734,8.924,8.370 and 8.635h;C_max2.862μg/mL,0.465,0.429,4.436 and 2.484μg/g,respectively.The concentration of amantadine in plasma,leg muscle,chest muscle,liver and spleen reached the peak value in 8-9h after single dose and multi dose administration,and the residual time in vivo was longer.The results of T_1/2βand C_maxin the same tissue of single dose group and multi dose group were different,but the difference was not significant.This study established a HPLC-MS/MS method for the determination of amantadine residues in aquatic food animal tissues and studied the elimination rule of amantadine residues in white feathered broilers,which provided a theoretical basis for the supervision of the illegal use of amantadine drugs in food animal breeding.