The Preparation Of Furazolidone Metabolites In Grass Carp For The Proficiency Test

Proficiency testing is the use of interlaboratory comparisons to assess participants' ability according to pre-established guidelines,also known as proficiency testing activities.Proficiency test can be used to evaluate the internal quality management of laboratory,to check the technical level of the inspectors,to detect the operating conditions of the laboratory equipment,and to increase the credibility of laboratory test results.Sample preparation is the key step in the proficiency testing activities.The successful preparation of the sample is the prerequisite for the activity proceeded smoothly.The appropriate concentration of the target in the sample also has an important impact on the implementation of the proficiency testing.The target residue concentration is too high or too low to achieve the purpose of assessment.Studies have shown that feeding medicine way,water temperature,bath time,and fish size have a great influence on drug metabolism residues in live animal experiments.However,there are few reports on how to control the above factors to achieve the required drug residual concentration in the proficiency testing sample.if the real sample is used as proficiency testing sample,there are still some issues that remain to be solved in sample preparation.First,how are the above factors related to the concentration of drug residues in animal muscles in the process of bath animals? How to control the above factors to achieve the required residual concentration of the drug for the test sample? The second is how to ensure the homogeneity and stability of the proficiency testing sample.According to the monitoring of drug residues in aquatic products by the Ministry of agriculture in recent years,we found that the metabolites of nitrofuran exceeded the limit of standard events often occurred.Nitrofuran metabolites are objects that need to be key supervision and they are also one of the test indicators of proficiency testing,which is organized by the CNCA and the Ministry of Agriculture every year.Therefore,in this paper,we studied the proficiency testing sample preparation of furazolidone metabolite AOZ with grass carp muscle as a carrier,this is of great significance to evaluate the internal quality management of laboratory,to improve the detection ability of laboratory and to improve the drug residue detection ability of aquatic products.The main results of this paper are summarized as follows:1.The Culture conditions of grass carp were studied to prepare proficiency testing samples from the muscle of grass carp furazolidone metabolite(3-amino-oxazolidinyl-one,AOZ).The effects of water temperature,fish size,time and other factors on the residual concentration of AOZ in muscle were studied after the grass carp were soaked in drug.According to the target,residual concentrations in muscle matrix were 8 ?g/kg and 25 ?g/kg,we determined the culture conditions of grass carp.The results showed that the grass carp had a slower metabolism of the drug at low temperature(20?),the fish was more likely to die at high temperature(30?),but the fish was active and rapidly accumulate metabolite at room temperature,thus the water temperature was selected at 25 ± 1 ?;the residual concentration changes relatively stable at the condition of bath time in 8 ~ 12 h,taking into account to minimize the time and the stability of the results,therefore,the bath time was determined for 10h;Body size also has great impact on the metabolism of grass carp.Ultimately the body size of 900 ~ 1100 g was confirmed by grouping test on the grass carps with different size.2.On the basis of the confirmation of culture conditions,AOZ residues of grass carp in muscle(y)is 21.57 ?g/kg,The bath concentration of x(?g/L)AOZ concentration of Y residue in muscle and grass carp(?g/kg),The linear relationship y=0.22 x was found between the initial bath concentration x(?g/L)and the AOZ residues of Grass carp in muscle(y)according to the initial concentration of medicated bath x=100 ?g/L in the pre-experiment.Take 2 groups of residual concentration of grass carp muscle low(5-10 ?g/kg),high(more than 20 ?g/kg)as the desired result,conduct 14 batches of parallel verification on relational y=0.22 x,and the results of the verification were statistically analyzed,the results showed no significant difference between the expected value and the detected value,which proves the feasibility of the relation.3.The technological process of proficiency testing samples preparation of furazolidone metabolite AOZ in grass carp was studied and established in the experiment with grass carp muscle as a carrier through low temperature lyophilization.The main steps are as follows: select grass carp at the weight of 900~1100 g,calculate the initial concentration of medicated bath according to the expected value of AOZ residues in muscle of grass carp and the relationship.Control the bath water temperature at(25 ± 1)?,bath time for 10 h,collect the muscle as the grass carp was executed,500 mg/L vitamin C solution was added to the homogenate when the muscle was put into a homogenizer to full homogenize.After freeze drying,crushing and screening,packaging,then store the homogenate at-20? with light shielded.The homogeneity and stability of the sample were detected by liquid chromatography-tandem mass spectrometry.The results showed that the sample had a good homogeneity and stability within 8 months,and the value for AOZ was based on analysis of results obtained in 10 accredited laboratories and uncertainty evaluation was conducted,and accorded with requirements for proficiency testing samples.