The Accumulation And Distribution Of Nitrofurazone Metabolites In Crucian Carp And Its Application

With the continuous improvement of people’s living standard,more and more people are pursuing high-quality life,and they are beginning to pay attention to food safety.Aquatic products such as fish and shrimp are rich in various nutrients（protein,amino acids,fatty acids,et al）and thus favored by the majority of consumers.In recent years,due to the occurrence of food safety problems,the quality and safety of aquatic products have also attracted widespread attentions.For example,some nitrofurans in aquatic products exceed the standard because nitrofurans are used as antibiotics in aquaculture.Nitrofurans have a potential"triple"effect that can seriously damage the growth and health of farmed animals.Therefore,there is an urgent need to monitor the metabolic residues of these drugs in aquatic products.In this study,high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）was developed for the determination of nitrofurazone metabolite semicarbazide（SEM）in Carassius auratus and various visceral tissues.Aiming to explore the law of accumulation and elimination of the original drug in the Carassius auratus tissue and the content of furazolidone in the original nitrofurazone.The main research contents are as follows:（1）A method for determining the amount of nitrofurazone metabolite residues in Carassius auratus by UPLC-MS/MS was established.The study used2-nitrobenzaldehyde for derivatization and ethyl acetate extraction of nitrofurans to optimize the pretreatment degreasing purification method.The multi-reaction monitoring mode coupled with triple quadrupole mass spectrometry was used to monitor the derivatives of nitrofuran metabolites.The results showed that the determination of nitrofurazone metabolites in the linear range from 0.5 to 1000 ng/mL（the calibration coefficients were above 0.9998）;In the three added concentration5,10,100μg/kg,mean fortification recoveries were 82.5%¹04%and their relative standard deviation（RSD）were between 2%and 8%.The experimental results showed that the established UPLC-MS/MS analysis method had the characteristics of high specificity and simple sample preparation,which can quickly and accurately determine the residual amount of nitrofurazone metabolites in Carassius auratus,and provide scientific basis for the detection of semicarbazide in aquatic products such as Carassius auratus.（2）Taking Carassius auratus as the research object,the Carassius auratus was raised by medicated bath,and the nitrofurazone was metabolized and accumulated in the body,and the content of semicarbazide in different tissues in the body were analyzed.The results showed that the residual amount of semicarbazide in the tissue samples of the squid went with a similar growth trend,and the SEM increased gradually with the accumulation time,but the growth rate also varied due to the different matrix of each tissues,including gallbladder and spleen.The SEM mass concentration was higher than that of other tissues.The SEM mass concentration in each tissue of the carp was from 144 h in the medicated bath with the order:gallbladder>spleen>liver>gills>kidney>intestines>brain>muscle.According to the analysis of the elimination test results of the squid,the drug-time curve of each visceral tissue increased to the peak and then decreased,and reached the peak drug concentration at 24 h,in which the elimination rate of gallbladder was higher than other tissues with the highest reached 7.4μg·（kg·h）^-11 The SEM residuals of each tissue were measured at 336 h:gallbladder>spleen>intestines>liver>sputum>fish head>muscle.Among them,the minimum SEM residual amount in muscle was2.07μg kg^-1,and the drug concentration was still higher than the legal detection limit.A small amount of AOZ was also detected in the viscera and muscle of the squid in the experiment,but it was much lower than the SEM content.Therefore,the actual source of AOZ was discussed.Firstly,the theoretical analysis of whether SEM will be converted into AOZ was carried out.The experimental design of nitrofurazone and its standard medicine bath carp showed that AOZ is not found in the standard medicine bath.However,a small amount of AOZ was detected in the squid of the original medicated bath,so it was confirmed that the SEM was not converted into AOZ.It was also determined that the nitrofurazone original drug contained a small amount of furazolidone（FZD）.In addition,comparisons were made between different modes of administration（injection and medicated bath）,and it was found that direct injection may have a greater impact on the survival rate of the Carassius auratus,and therefore osmotic administration was carried out by medicated bath administration.（3）Based on the previous Chapter of NFZ medicine Bath Carassius auratus experiment detected the value of AOZ,in order to explore the content of furazolidone in nitrofurazone original medicine.First of all,designed 5 groups of nitrofurazone and furazolidone standard product mixing ratio is（99.8:0.2;99.5:0.5;99:1;98:2）and compared with the same concentration of nitrofurazone raw drugs,by UPLC-UV detection comparison UV map found that a small number of FZD in the mixed label were detected,but the original drug did not detect FZD.The reason laid that FZD content is too low,and lower than UPLC-UV detection method,Therefore,on the basis of the experiment of medicine bath metabolism of Carassius auratus,the nitrofurazone original medicine of 5μg/mL and four different proportions of NFZ and FZD mixed standard were designed to test the medicine bath of Carassius auratus,and the UPLC-MS/MS and SEM in the muscle and viscera of five groups of medicine bath were tested and analyzed by AOZ method.The results showed that the concentration of AOZ measured in the muscle and viscera of Carassius auratus in nitrofurazone medicine bath was 0.16±0.1μg/kg and 6.6±0.16μg/kg respectively,and the AOZ concentration in nitrofurazone original drug was calculated by 99.938/0.062and 99.974/0.026.