Research On The Metabolism Of Allocryptopine And Protopine In Laying Hens And The Elimination Of Residues In Eggs

In recent years,the abuse of antibiotics has increased the resistance of animal-derived bacteria,endangering food safety and public health safety.In order to regulate the use of antibiotics in the aquaculture industry,China is vigorously implementing the policy of banning the resistance of feed additives and reducing the resistance of veterinary drugs,and encouraging the research and development and application of natural Chinese medicine additives and veterinary drugs.Plume Poppy Total Alkaloid Powder is the second-category new veterinary drug approved by the Ministry of Agriculture.,and the main active ingredients are allocryptine（ALL）and protopine（PRO）.It has attracted great attention because of its good therapeutic effect on the diarrhea caused by salpingitis and colibacillosis of laying hens.At present,China has not yet made regulations on the residual and metabolic test for new Chinese veterinary drugs in target animals.Hence,this study used the target animal layer as the experimental object for the first time to explore the metabolism of Plume Poppy Total Alkaloid Powder in laying hens and the elimination of residues in eggs.The purpose is to make a prospective study on the safety evaluation of Plume Poppy Total Alkaloid Powder and its clinical application Provide data support and theoretical basis for promotion.The main contents of this research are as follows:（1）A liquid-mass spectrometry（UPLC-MS）analysis method of ALL and PRO in eggs was established for the first time.The chromatography conditions were separated on an Agilent ZORBAX SB-C₁₈column,with 0.1%formic acid water（A）-acetonitrile（B）.The rapid analysis method was completed within 17 min by a gradient elution program.Conventional solid liquid co-extraction was selected for sample preparation,with acetonitrile as the extraction solvent and n-hexane for degreasing.The data of method validation demonstrated that the limits of quantification of ALL and PRO were 0.1 and 0.5μg/kg,respectively,and showed good linearity（R²≥0.999）in the concentration range of0.25～500μg/kg and 1.25～500μg/kg separately.The recovery rates of all analytes were in the range of 93.17～103.68%,and the matrix effect ranged from 88.10 to 95.55%.The specificity,stability,intra-day（inter-day）accuracy and precision are all within the compliance range.（2）The validated quantitative method was successfully applied to the analysis of the residues and elimination rules of Plume Poppy Total Alkaloid Powder in eggs.A clinical dose 400 mg/kg and a clinical 5-fold high-dose 2000 mg/kg of Plume Poppy Total Alkaloid Powder was fed in the diet of Roman gray laying hens.The residues of ALL and PRO in eggs during the administration period（7 days）and the withdrawal period（14 days）were monitored.The experimental results showed that in the clinical dose group,ALL was not detected in eggs,and the residual amount of PRO reached the maximum（2.8±0.61μg/kg）on the 7th day of administration,and was below the detection limit on the 3th day after drug withdrawal.In the high-dose group,residue of ALL still existed on the 14th day of drug discontinuation,with a concentration of 1.34±0.35μg/kg.The highest residual concentration of PRO was 16.72±3.07μg/kg on the first day and it was lower than the detection limit on the seventh day after drug withdrawal.According to the analysis and comparison of data,it is found that ALL and PRO of Plume Poppy Total Alkaloid Powder deposited slowly and eliminated quickly in eggs.（3）For the first time,intravenous injection was used to study the metabolites and metabolic pathways of ALL and PRO in laying hens.Standards of ALL and PRO were administrated to Roman gray laying hens by intravenous injection at a dose of 1 mg/kg.Plasma and feces were collected at different sampling period and then were detected after pre-treatment.The structural formula of the metabolites could be inferred according to the proposed fragmentation pathways.Finally,a total of 9 metabolites of ALL and 12metabolites of PRO were identified and named as ALL-M1～M9,PRO-M1～M12,respectively.Among them,two new structural formulas of the metabolites of PRO were inferred for the first time.Through the analysis of the metabolic pathway of the metabolites,there are mainly the following reactions:methylation,demethylation,oxidation,glucuronidation and so on.