| [Objectives]The safety of food is the focus issue of people's livelihood.Veterinary drugs play an important role in the disease prevention of animals,the promotion of growth,and the improvement of the quality of edible parts of animals.However,the irrational use of veterinary drugs lead to a series of safety issues,such as endanger people's health and pollute the environment.Therefore,strengthening the detection and supervision of veterinary drugs residues would be great significant for protecting the environment,promoting the orderly development of animal husbandry and safeguarding people's health.This study intended to establish a simple,sensitive,efficient and reliable multi-residue HPLC method,and detected 9 types of 4 categories veterinary drugs[Nitroimidazoles include Metronidazole(MNZ),Tinidazole(TNZ)and Ronidazole(RNZ),?-adrenergic agonists include Clenbuterol(CL),Salbutamol(STL)and Ractopamine(RAC),Benzimidazoles(BZs)include Thiabendazole(TBZ)and Albendazole(ABZ),and Diethylstilbestrol(DES)]in 10 animal foods in Jinan City.Statistical analysis of the situation exceeding the standard,in order to understand the status and level of veterinary drug residues in animal foods in Jinan City.[Methods]1 Eestablishment of the method of detecting veterinary drug residues in animal food1.1 Sample treatmentAfter homogenization,the animal sample was digested with ?-glucuronidase for 16 h under ammonium acetate,and then took out and adjusted the pH to weak alkaline.Ultrasonic extracted with acetonitrile,and removed water by anhydrous magnesium sulfate and sodium chloride.Then combined purification with PSA,C18 and GCB,the purified extract was concentrated,filtered,and then detected by high performance liquid chromatography.1.2 Chromatographic condition of Nitroimidazole and ?-adrenergic agonist combined detectionColumn:AgiLent C18 column(250mm×5mm,4.6?m);mobile phase:acetonitrile(A)-0.02M phosphate buffer(pH=6)(B),gradient elution[0?2min,20%A?25%A;2?4min,25%A;4?5min,25%?35%A;5?8min,35%A;8?8.1min,35%?50%A;8.1?15min;50%A 15?15.1min,20%A;Balanced 6min];Detection wavelength:0?4.70min:227nm,4.70?5.24min:312nm,5.24?6.40min:301nm,6.40?7.50min:311nm,7.50?9.00min 225 nm,9.00 to 10.05 min 246 nm;column temperature:30?;flow rate:1 mL/min.1.3 Chromatographic condition of Benzimidazoles and Diethylstilbestrol combined detectionColumn:AgiLent C18 column(250mm×5mm,4.6?m);Mobile phase:Acetonitrile:Phosphate buffer(pH=7)=55:45;Detection wavelength:0 to 4.40 min at 299 nm,4.40 to 5.60 min at 235 nm,5.60 to 11.00 min was 240 nm;column temperature:30?;flow rate:1 mL/min.2 Market investigation results of veterinary drug residues in animal foods of Jinan City2.1 Sampling methodIn July 2017,five administrative districts including Lixia District,Licheng District,Shizhong District,Tianqiao District,and Huaiyin District of Jinan City were selected as sampling areas.In each area,2 supermarkets and 2 large-scale farmers'markets are randomly selected as the sampling points for the two relatively-concentrated residential areas.Ten kinds of foods such as pork,pig liver,porcine kidney,chicken,chicken liver,chicken cockroach,eggs,beef,milk and lamb are collected as samples,and each product was randomly purchased two.2.2 Test itemsThis study detected 9 kinds of 4 categories veterinary drugs,they are Metronidazole(MNZ),Tinidazole(TNZ),Ronidazole(RNZ),Clenbuterol(CL),Salbutamol(STL),Ractopamine(RAC),Thiabendazole(TBZ),Albendazole(ABZ)and Diethylstilbestrol(DES),respectively.2.3 Type and quantity of animal foodsA total of 297 animal foods were tested,including 47 for pork,34 for pig liver,22 for porcine kidney,43 for chicken,9 for chicken liver,22 for chicken cockroach,40 for eggs,34 for beef,28 for milk and 18 for lamb.2.4 Data processDetected by HPLC and recorded the corresponding area value according to the retention time,calculated the content of each index in the sample through the standard curve.The results compared with the standards in the Ministry of Agriculture Notice No.235(Maximum Residue Limits of Veterinary Drugs in Animal Derived Foods),No.1624(Revision of Drug Residues in National Reference Standards for Veterinary Drug Residues),and No.1682("Clenbuterol" Special Inventory Completion of the work),etc..Calculated their detection rate and over-standard rate.2.5 Analysis and EvaluationThe software SPSS statistics version 24.0 was used for analysis.One-way analysis of variance was used to compare the mean values of groups,and the SNK test was used to compare the differences between each two groups.The ?2 test was used to compare the difference of the over-standard rates between each two groups.The difference was statistically significant at P<0.05.[Results]1 Eestablishment of the method of detecting veterinary drug residues in animal food1.1 Combined detection method of Nitroimidazole and ?-adrenergic agonist1.1.1 Detection wavelength and retention timeThe 6 kinds of veterinary drugs have maximum absorption peaks at 312nm,311nm,301nm,227nm,225nm and 246nm,respectively.The peak shapes were good and separated from each other.The retention times were as follows:STL(3.98min),MNZ(5.06min),RNZ(5.50min),TNZ(7.08min),RAC(7.85min),CL(10.16min).1.1.2 Linear relationThe regression equation of MNZ was:A=60835.89441C-269.59377,r2=0.99674;The regression equation of TNZ was:A=42508.91374C-24.42677,r2=0.99768;The regression equation of RNZ was:A=50710.97472C+163.82715,r2=0.99996;The regression equation of STL was:A=33661.19294C+309.84815,r2=0.99355;The regression equation of RAC was:A=53713.39334C-787.64839,r2=0.99614;The regression equation of CL was:A=30993.54125C+655.34738,r2=0.99742.Their line range were 0.01?1?g/ml.1.1.3 Recovery rate and RSDThe recovery rate of MNZ with three different spiked concentrations were between 69.21%and 85.06%,the intraday precision was 1.74%,and the intraday precision was 1.56%.The recovery rate of TNZ with three different spiked concentrations were between 68.57%and 89.10%,the intraday precision was 2.55%,and the intraday precision was 2.84%.The recovery rate of RNZ with three different spiked concentrations were between 76.02%and 92.36%,the intraday precision was 1.76%,and the intraday precision was 1.98%.The recovery rate of STL with three different spiked concentrations were between 80.80%and 101.56%,the intraday precision was 3.11%,and the intraday precision was 3.37%.The recovery rate of RAC with three different spiked concentrations were between 75.82%and 97.07%,the intraday precision was 4.28%,and the intraday precision was 5.04%.The recovery rate of CL with three different spiked concentrations were between 78.91%and 105.24%,the intraday precision was 3.25%,and the intraday precision was 3.48%.1.1.4 Limit of detectionThe LOD of MNZ,TNZ,RNZ,STL,RAC,CL under this experimental condition were 1.36 ?g/kg,1.95?g/kg,1.54?g/kg,2.05?g/kg,1.81?g/kg,1.59p.g/kg.1.2 Combined detection method of Benzimidazoles and Diethylstilbestrol1.2.1 Detection wavelength and retention timeThe 3 kinds of veterinary drugs have maximum absorption peaks at 299nm,235nm,240nm,respectively.The peak shapes were good and separated from each other.The retention times were as follows:TBZ(3.707min),ABZ(4.880min),DES(5.913min).1.2.2 Linear relationThe regression equation of TBZ was:A=103923.40262C+112.7958,r2=0.99726.The regression equation of ABZ was:A=71192.6097C+459.66975,r2=0.99691.The regression equation of DES was:A=82452.80985 C+156.67244,0.99803.Their line range were 2.5?100ng/mL.1.2.3 Recovery rate and RSDThe recovery rate of TBZ with three different spiked concentrations were between 81.52%and 105.31%,the intraday precision was 2.63%,and the intraday precision was 2.54%.The recovery rate of ABZ with three different spiked concentrations were between 77.07%and 104.77%,the intraday precision was 3.52%,and the intraday precision was 3.78%.The recovery rate of DES with three different spiked concentrations were between 65.73%and 96.22%,the intraday precision was 1.48%,and the intraday precision was 4.19%.1.2.4 Limit of detectionThe LOD of TBZ,ABZ,DES under this experimental condition were 0.42?g/kg,0.70?g/kg,0.72?g/kg.2 Market investigation results of veterinary drug residues in animal foods of Jinan City2.1 Situation of Nitroimidazoles residue in animal foods2.1.1 Over-standard rate and over-range of three nitroimidazole drugs in animal foodsAmong the 297 samples,the over-standard residue rate of RNZ was the highest,which was 7.41%,and the residual range was 3.34?423.80?g/kg,the over-standard residue rate of MNZ was 6.73%,and the residual range was 2.95?94.02?g/kg,the over-standard residue rate of TNZ was 2.36%,and the residual range was 1.95?18.83?g/kg.MNZ and RNZ were over-standard detected from the all 10 kinds of animal foods.TNZ was only detected excessive levels from pork and chicken,and the exceeding range was between 1.95 and 28.83 ?g/kg.The residual value of residual MNZ in swine foods was higher than chicken and cattle foods(P<0.05).The values of MNZ exceeded the standard of liver and kidney samples were significantly higher than other organ foods(P<0.05),in which the concentration of MNZ in pig liver was in the range of 8.23 to 152.93 ?g/kg,and the concentration of MNZ in pig kidney was in the range of 9.36 to 194.02 ?g/kg.The excess of RNZ in pig liver ranged from 64.08 to 423.80 ?g/kg,exceeding the standard value significantly higher than that of other organs(P<0.05).Only one sample of the lamb products was detected MNZ over-standard residue,but exceeded the standard value reached to 74.78 ?g/kg,RNZ exceeded the range of 21.63?80.98 ?g/kg in pork,RNZ exceeded the range of 41.48?145.16 ?g/kg in chicken cockroach.2.1.2 Nitroimidazole over-standard residues situation for animal foods in different sample pointsAmong the total 297 samples,158 were collected from supermarkets and 139 were collected from farmers' markets.Statistics analysis showed that there is no significant difference in the excess of the same veterinary drugs in the same food samples compared with samples collected from supermarkets and farmers' markets.In the districts,60 were collected from Shizhong District,63 from the Licheng District,53 from the Huaiyin District,and 53 from the Tianqiao District.The statistics analysis showed that the samples collected from the five administrative districts of Jinan City has no significant difference in the over-standard rate of veterinary drug residues in same kind of food.2.2 Situation of ?-adrenergic agonist residue in animal foods2.2.1 Over-standard rate and over-range of three ?-adrenergic agonist drugs in animal foodsAmong the 297 samples,the over-standard residue rate of CL was the highest,which was 4.04%,and the residual range was 3.45?337.71?g/kg,the over-standard residue rate of STL was 3.70%,and the residual range was 4.44-3058.85?g/kg,the over-standard residue rate of RAC was 1.35%,and the residual range was 33.65?442.96?g/kg.The values of CL exceeded the standard of kidney samples were significantly higher than other organ foods(P<0.05),only one sample of the porcine kidney was detected CL over-standard residue,but exceeded the standard value reached to 3058.85 ?g/kg.The residual value of residual CL in liver samples was higher than eggs(P<0.05).The over-standard rate of CL in pig liver and chicken liver were higher than 11%,respectively,and the range of CL in pig liver was 79.67-337.71 ?g/kg.Only one sample in chicken liver was detected CL exceeded the standard value,which exceeding value reached 139.42?g/kg.Two of the beef samples exceeded the standard,with a high value of 616.11 ?g/kg.Only one sample of the lamb samples exceeded the standard,and the over-standard value reached 105.94?g/kg.There was no significant difference in the rates of exceeding standard among different animals.2.2.2 ?-adrenergic agonist over-standard residues situation for animal foods in different sample pointsAmong the total 297 samples,158 were collected from supermarkets and 139 were collected from farmers' markets.Statistics analysis showed that there is no significant difference in the excess of the same veterinary drugs in the same food samples compared with samples collected from supermarkets and farmers' markets.In the districts,60 were collected from Shizhong District,63 from the Licheng District,53 from the Huaiyin District,and 53 from the Tianqiao District.The statistics analysis showed that the samples collected from the five administrative districts of Jinan City has no significant difference in the over-standard rate of veterinary drug residues in same kind of food.2.3 Situation of Benzimidazoles residue in animal foods2.3.1 Over-standard rate and over-range of three Benzimidazoles drugs in animal foodsAmong the 297 samples,ABZ was not detected exceed the standard,and the detection range was 1.52-98.65?g/kg,the over-standard residue rate of TBZ was 19.19%,and the residual range was 100.86-1493.59?g/kg.Among them,the TBZ residue was not detected over-standard rate in lamb samples,which was significantly lower than that of pigs and chickens(P<0.05).The TBZ residue detection rates of internal organs foods,such asliver,kidney and stomach were significantly higher than that of muscle,egg and dairy foods(P<0.05),and the TBZ residue detection values of liver and kidney samples were also significantly higher than other types of samples(P<0.05),and what's more,the detected value of liver was significantly higher than that of the stomach(P<0.05).The over-standard rates of TBZ residues in pig liver and pig kidneys were 44.12%and 40.91%,respectively,and the residual range were 102.97?972.09?g/kg and 100.86?384.89?g/kg,respectively.The rate of over-standard TBZ residues in chicken cockroach reached 50.00%,and the exceeded residue range was 125.93?1368.35?g/kg.The detection rate of TBZ residue in chicken liver samples even reached 100%,and the residue exceeded the range of 159.61-963.37?g/kg.ABZ was not detected exceed the standard in all samples,but the residual value of chicken samples was significantly higher than that of eggs(P<0.05).2.3.2 Benzimidazoles over-standard residues situation for animal foods in different sample pointsAmong the total 297 samples,158 were collected from supermarkets and 139 were collected from farmers' markets.Statistics analysis showed that there is no significant difference in the excess of the same veterinary drugs in the same food samples compared with samples collected from supermarkets and farmers' markets.In the districts,60 were collected from Shizhong District,63 from the Licheng District,53 from the Huaiyin District,and 53 from the Tianqiao District.The statistics analysis showed that the over-standard rate of TBZ in pig livers in Shizhong District and Huaiyin District were significantly lower than the other three administrative regions.There was no significant difference in the over-standard rate of residues of the same veterinary drug in most other types of food collected from the five administrative districts of Jinan City.2.4 Situation of Diethylstilbestrol residue in animal foods2.4.1 Over-standard rate and over-range of three Diethylstilbestrol drugs in animal foodsAmong the 297 samples,the total over-standard rate of DES was 15.15%.The highest residual rates of DES were in chicken liver and lamb,which were both 33.33%,and the range of residual exceeded were 24.31?60.73?g/kg and 3.38-24.08?g/kg,respectively.The residual range of DES in chicken cockroach was 29.38-612.74?g/kg,and the residual value was significantly higher than other organs(P<0.05).The DES exceeding standard rate of the sheep sample was 33.33%,which was significantly higher than that of the chicken and cattle samples(P<0.05).There was no significant difference in the over-standard values of different animal foods.2.4.2 Diethylstilbestrol over-standard residues situation for animal foods in different sample pointsAmong the total 297 samples,158 were collected from supermarkets and 139 were collected from farmers' markets.Statistics analysis showed that there is no significant difference in the excess of the same veterinary drugs in the same food samples compared with samples collected from supermarkets and farmers' markets.In the districts,60 were collected from Shizhong District,63 from the Licheng District,53 from the Huaiyin District,and 53 from the Tianqiao District.The statistics analysis showed that the samples collected from the five administrative districts of Jinan City has no significant difference in the over-standard rate of veterinary drug residues in same kind of food.[Conclusion]1 This study established a unified HPLC method for the simultaneous detection of nitroimidazoles and ?-adrenergic agonists,benzimidazoles and diethylstilbestrol residuesin in animal foods.The method is simple,sensitive,efficient,reproducible,with low detection-cost.This method can meet the current testing requirements of veterinary drugs residue in animal foods.2 Among the 10 kinds of animal foods from the market,the over-standard residue rates of veterinary drugs in egg and milk are relatively lower,and the over-standard values are also small;the over-standard residue rates of internal organs,such as liver,kidney and stomach is much higher,and the over-standard value is also high.3 In the commercial animal foods,albendazole(ABZ)is not detected exceeding the standard,and the other 8 drugs are detected exceeding the standard more or less.Among them,thiabendazole has the highest over-standard rate,19.91%,and ractopamine has the lowest,1.35%.4 Compared the different sampling points,most of the same food had no significant difference in the rate of exceeding with the same veterinary drug.5 The situation of the veterinary drug residues in commercial animal foods in Jinan City is not satisfactory.There are widespread veterinary drug residue violations in common species of animal foods,which require further rigorous detecting and supervision. |
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