| Metalaxyl has been in use for more than 40 years.It is usually used as a fungicide in farmland to treat downy mildew,black germ and late blight of cucumber,tomato,potato and other crops.In the field of aquaculture,it was approved as a new fishing drug in 2017,as an alternative to malachite green in aquaculture for the treatment of water mildews in farmed fish.At present,metalaxyl is the only antifungal agent included in Regulation No.2 of 2020 on the Clear Paper on Medicinal products for aquaculture.While playing an antibacterial role,metalaxyl also has some harm to non-target organisms.At present,the detection technology and limit standard of metalaxyl residue are only limited to crops,and there is no relevant detection technology and limit standard in aquatic products.Meanwhile,there is also a lack of relevant studies on the pharmacokinetics and identification of metabolites of metalaxyl in aquatic animals.In order to fill the gap in the research of aquatic animals,implement the supervision and control of metalaxyl residues in aquatic animals more effectively,it is necessary to establish relevant and reliable detection means to detect its residues,understand its related drug absorption,distribution,elimination and metabolism in aquatic animals,and identify and study its metabolites in aquatic animals.In this study,a method of liquid chromatography-tandem mass spectrometry was established to determine the residues of metalaxyl and its metabolites in aquatic products.The metabolites in Carassius auratus liver were detected and identified by liquid chromatography-tandem mass spectrometry.Finally,the contents of metalaxyl and its metabolites in Carassius auratus were detected by liquid chromatography-tandem mass spectrometry.The pharmacokinetic characteristics of metalaxyl and its metabolites in Carassius auratus and their migration in different tissues were studied.The main research results were as follows:(1)By optimizing extraction solvent,purification method,solid phase extraction conditions,liquid chromatography conditions and mass spectrometry parameters,the isotope dilution-liquid chromatography-tandem mass spectrometry method was established for the first time for the determination of metalaxyl residues in four aquatic products.The results showed that the linear relationship was good in the concentration range of 1-100 μg/L by internal standard method,and the correlation coefficients were more than 0.995.When different types of aquatic products were blank added at the standard levels of 1.0,2.0,10.0 μg/kg,the average recovery rates were 85.5%-110%,the RSD was less than 11.6% and the RSD was less than 12.1%.The limit of detection was 0.5 μg/kg and the limit of quantitation was 1 μg/kg.The method is simple and practical,and can be used for quantitative detection of methylaxalin residues in fish,shrimp,crab and shellfish with high fat and high protein.The addition of isotope internal criteria can also effectively correct the unstable factors in the process of experiment,improve the accuracy and stability of the method,and can meet the rapid determination of a large number of samples.(2)In order to explore the metabolites of metalaxyl in Carassius auratus,they were orally administered with 5 mg/kg of metalaxyl.After 2 hours,Q Exactive high resolution mass spectrometry was used to screen metalaxyl acid in Carassius auratus.We identified metalaxyl acid as the metabolite of metalaxyl in Carassius auratus liver for the first time.Subsequently,liquid chromatoc-tandem mass spectrometry was established to detect the above samples by metalaxyl acid standard,further verifying the above identification results,and providing technical support for the quantitative detection of subsequent pharmacokinetic experiments.(3)Through the study of the sample pretreatment method and instrument detection conditions,the liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for the detection of the residual amount of metalaxyl acid in Carassius auratus was established for the first time.In the range of concentration from 1 to 200 μg/L,the linear relationship of metalaxyl acid was good,and the correlation coefficients were all greater than 0.995.When Carassius auratus samples were added at the spiked levels of 1.0,2.0and 10.0 μg/kg,the recovery rates of methamyl were 64.5%-65.6%,the intra-batch precision was less than 2.71%,and the inter-batch precision was less than 2.38%.The limit of detection was 0.5 μg/kg and the limit of quantitation was 1 μg/kg.This method can quickly complete the quantitative detection of metalaxyl acid residue in the complex matrix of high fat and high protein of Carassius auratus,and the pretreatment time is short,so it is practical.In practice,it can satisfy the rapid determination of a large number of samples.(4)In order to study the absorption,distribution,metabolism and elimination regularity of metalaxyl and its metabolites in Carassius auratus.Carassius auratus were orally administered with 5 mg/kg of metalaxyl,and the concentrations of metalaxyl and metalaxyl acid in the plasma,muscle,liver,kidney,spleen,gonadal gland,bile,intestine and swim bladder of Carassius auratus were detected by liquid chromatography-tandem mass spectrometry.The data were analyzed by DAS 2.0software.The results showed that there were metalaxyl and metalaxyl acid in all the nine tissues of Carassius auratus.The pharmacokinetics of metalaxyl in Carassius auratus conformed to the two-compartment open model.Metalaxyl can be absorbed quickly in Carassius auratus with good permeability and is widely distributed in various tissues.In addition,there is hepatoenteral circulation in Carassius auratus.The metabolism of metalaxyl in various tissues is faster,but the residual time of metalaxyl acid is longer.The content of metalaxyl and metalaxyl acid can not be detected after 96 h and 312 h,respectively.Metalaxyl acid is mainly absorbed by the bile and stored in it.From the bile,it is gradually secreted into the intestine and excreted out of the body.Finally,according to the above pharmacokinetic data,it is concluded that the detection result of metalaxyl acid is not suitable for judging whether metalaxyl has been used in Carassius auratus. |
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