The Residue Depletion Of Cyadox In Swine, Chicken And Fish

Cyadox (Cyx) is a new kind of antimicrobial growth-promoter of the quinoxalines. Compared with other members of the quinoxalines, cyadox could improve the growth and feed conversion efficiency with lower toxicity and more safety, has an extensive application prospects. As the drug for food animals, food safety is one of the important study contents. The residue depletion study is the core content of the safety evaluation for drugs used in food-producing animals, is also the direct scientific basis to determine the residual marker, the target tissue and to formulate the withdrawl period. This study based on the radioactive tracer studies, feed cyadox with the recommended dose and production conditions in long-term, further confirmed the residual marker and target tissue in food animals, which provided clear monitoring of objects and technical standards for food residue monitoring of cyadox. Besides, the degradation of cyadox and its metabolites in the environment was carried out to provide the scientific basis for the rational evaluation of environmental safety of cyadox.1. Methods of quantitative analysis of cyadox and its metabolites in the edible tissues of swine, chicken and fishMethods of quantitative analysis of cyadox and its metabolites:A method of high performance liquid chromatography with UV have been established for the simultaneous quantitative determination of cyadox and its five major metabolites (Cy1, Cy2, Cy4, Cy6and Cy12) in edible tissues of swine, chicken and fish. For tissues (liver, kidney, muscle and fat), samples were extracted with1%metaphosphoric acid in methanol/acetonitrile/water(50:10:40, v/v/v), followed by HLB solid-phase extraction clean-up and liquid-liquid extraction. Analysis was performed on a ZORBAX SB C18liquid chromatography column by UV detection with a gradient program of wavelength of320nm. In the edible tissues of swine, chicken and fish, the LOQ for Cyx, Cy1, Cy2, Cy6, Cy12were20μg/mL, Cy4was20μg/mL. The recoveries in edible tissues, spiked at levels of20μg/mL～40μg/mL, were68.0%～79.5%, with inter-day RSD less than9.6%.Methods of quantitative analysis of Cy1:A method of high performance liquid chromatography with UV have been established for the simultaneous quantitative determination of Cy1in edible tissues of swine, chicken and fish. For tissues (liver, kidney, muscle and fat),2g samples were extracted with ethyl acetate, then evaporated to dryness and performed on a ZORBAX SB C18liquid chromatography column by UV detection with a gradient program of wavelength of280nm. In the edible tissues of swine, chicken and fish, the LOQ for Cyl was20p.g/mL.The recoveries in edible tissues, spiked at levels of20μg/mL～40μg/mL, were71.4%～78.4%%, with inter-day RSD less than10.0%.2. The experiment of residue depletion on swine, chicken and fish25healthy hybridized Sanyuan swine,30one-day-old Kebao broiler and50healthy carp were fed with cyadox at the level of150mg/kg (cyadox at the level of100mg/kg) for60days,40days,30days. animals were killed at different withdrawal time, liver, kidney, muscle, fat (sebum or skin) and intestinal of fish were taken, the residual quantity of cyadox and its metabolites were determined by above-mentioned methods. The results are as follows:Swine:At the withdrawal time of6h, Cyx, Cy1and Cy6were detected in liver and kidney, the residual concentration were21.8μg/kg to55.0μg/kg. Cy1can be detected in kidney for3days. Cyx and Cy1can be detected for6h or1day in kidney and liver. In muscle, Cyx and Cy6were detected at the withdrawal time of6h, the residual concentration were27.5μg/kg to37.6μg/kg; only Cy6could be detected at the withdrawal time of1day, related residues were below LOQ after a withdrawal time of1d. Only few Cyx and Cy4could be detected in fat at the withdrawal time of6h, the residual concentration were22.2μg/kg to34.6μg/kg, related residues were below LOQ after a withdrawal time of6h. The results showed the residual quantity of cyadox was low in swine, residue depletion was quickly, Cyl was its marker residue, and kidney was the target tissue. Based on the toxicology and metabolism results, the MRL of cyadox was calculated to be32.4mg/kg in kidney of swine, the withdrawal time of cyadox in swine was0day.Chicken:At the withdrawal time of6h, Cyx, Cy1, Cy6and Cy12were detected in liver, the residual concentration were24.7-49.7μg/kg. Cy1and Cy6were below LOQ after the withdrawal time of1d while Cyx and Cy12could be detected for6 hours. Cyx, Cy1, Cy4, Cy6and Cy12were detected in kidney, the residual concentration were20.5-53.6μg/kg. Cy1and Cy6could be detected for3and1day, but other metabolites were below LOQ after the withdrawal time of6h. In muscle and fat, only Cy12was detected at the withdrawal time of6h, the residual concentrations were24.7-53.6μg/kg. At the withdrawal time of12h, few Cy4could be detected in fat, but no metabolites could be detected in muscle. Based on the toxicology and metabolism results, the MRL of cyadox was calculated to be32.4mg/kg in kidney of chicken, the withdrawal time of cyadox in chicken was0day.Fish:At the withdrawal time of6h, Cyx, Cy1, Cy4and Cy6were detected in5tissues, the residual concentration were22.5-42.8μg/kg. In liver and kidney, Cy4was below LOQ after the withdrawal time of6h, while Cyx and Cy6were below LOQ after a withdrawal time of1d. Cy1could be detected in liver for3days, the residual concentration were21.0±4.3μg/kg, but disappeared in kidney at3days; metabolites were below LOQ after the withdrawal time of6h in muscle. In skin, Cy1and Cy6could be detected at the withdrawal time of Id, Cyx and Cy4could be only detected after the withdrawal time of6h. The results showed the residual quantity of cyadox was low in swine, residue depletion was quickly, Cy1was its marker residue, intestinal was the target tissue.3. The degradation of cyadox and its metabolites in the environmentTritium labeled cyadox was added in swine manure to study the degradation and transformation of cyadox in swine manure, soil and cabbage. Tritium labeled cyadox was added in200g swine manure, to make drug specific activity reach22.5mCi/g, covered the manure with membrane, another same sample was mixed with soil in proportion of1:1, and cabbage was planted, swine manure, soil and cabbage were collected at different time every day, detected after they were mixed. The results indicated that Cyx could be transformed into Cy2and Cyl in the environment. They would be degraded quickly in the manure with the extension of time and were blew the limit of detection after6days; Cy1had the longest residual time and it could last for6days, the t1/2of which is1.49d. The residual time of Cyx, Cy2and Cy1in soil were short, they were blew the limit of detection only for3days after exposure to the environment. From the results, we concluded that Cyx and its metabolites degraded quickly in the environment, and they had excellent environment security. Cyadox was administrated to3healthy broilers in medicated feed at the level of400mg/kg, the intaken quantity of every broiler every day was converted into quantity of one time gavage, based on drug radioactive specific activity26.25mCi/g, the manure of the first3days was collected. Divided into two parts after they were mixed, covered the manure with membrane, another same sample was mixed with soil in proportion of1:4, and cabbage was planted, swine manure, soil and cabbage were collected at different time every day, detected after they were mixed. The results showed only Cyx, Cy2and Cy6could be detected in the beginning of manure, and they eliminated quickly, there was new generating metabolite Cyl, the reason might be generated by Cyx or Cy2, but it also eliminated quickly, it eliminated completely at5th day. Cy6had the highest content in the manure, and had the highest residual time, the eliminate rate constant was0.224d-, the eliminate half-life was2.84d, it degradated quickly and was below LOQ at the7th day. Because concentration of compounds in manure was low, so no compounds could be detected in soil after they were mixed with soil, no compounds could be detected in cabbage, too. So the generated excreta which chicken metabolized cyadox would not harm soil and plants.In conclusion, this paper developed the residual analytical methods of cyadox and its metabolites in tissues of swine, chicken and fish, and studied the residue depletion experiments on swine, chicken and fish. This study also clarified the residue depletion characteristics of cyadox in swine, chicken and fish, verified residual target tissue and marker residue, established the withdrawal time, clarify degradation pattern of cyadox in the environment. These results provided reliable technical information for food safety evaluation of cyadox, provided scientific technology standard and methods for food residual monitor of cyadox, and provided scientific basis to evaluate comprehensively food safety and environment safety of cyadox, had important academic value and application prospects.