Residue Depletion Study Of Diaveridine In Swine, Broilers And Fish

Diaveridine(DVD) is a kind of antibacterial synergist, and with sulphonamide it can block folic acid synthesis from dual mechanism in sensitive bacteria and inhibit the growth and breeding of bacteria, so as to play a good antibacterial effect. It is used in prevention and treatment of coccidiosis of chicken and animal intestinal infection. It can also exterminate coccidiosis by itself. But toxicology studies of DVD prove that it has genotoxicity, therefore drug residue of animal tissues is closely related to human health. Drug residue determination is one of the important contents of safety evaluation of edible animal products, but the reports of residue depletion study of DVD in livestock and poultry are less. In order to improve the safety assessment of DVD and formulate its target tissue, the marker residue, maximum residue limits(MRLs) and the withdrawal time(WDT), on the basis of the radioactive tracer study, we conducted the residue depletion study of DVD and its main metabolites in pigs, broilers and fish. The achievements would provide a scientific food safety standard to monitor DVD residues.1 Preparation the reference substance of DVD1With vanillin as raw material, chloromethyl methyl ether as hydroxyl protection reagent, the mole ratio of 1:1.1, we got the buff 4-MOMO-Vanillin(yield 85%). With 4-MOMO-Vanillin and acrylonitrile as raw material, control the mole ratio of 1:1.2, after reaction we joined the guanidine nitrate and got 4-MOMO-DVD(yield 75%). With this as raw material, we slowly dropped 37% hydrochloric acid to the solution, then got 4-demethylation-diaveridine(DVD1, yield 60%). By the quality standard research, the structure was correct, and the purity was more than 97%, and the stability was good. It met the requirements of the reference substance.According to the results of early radioactive tracer study in our l2 Establishment the HPLC detection method of DVD and DVD1According to the results of early radioactive tracer study in our laboratory, we determined that the main metabolites of DVD were DVD1(4-demethylation-diaveridine) and DVD2(monoglucuronide of 4-demethylation-diaveridine). We established a high performance liquid chromatography method for simultaneous determination of DVD and DVD1 in edible tissues of pigs, chicken(liver, kidney, muscle, fat(fat+skin), heart, lung, stomach, large intestine and small intestine of 9 kinds of tissues) and carp(liver, kidney, muscle, skin and gastrointestinal tract of 5 kinds of tissues). Samples were extracted with ethyl acetate, followed purification by MCX solid phase extraction column, dissolved with 0.01% ammonia and methanol(71:29, V/V). Drugs were assayed by HPLC. When the samples had to be hydrolyzed, another 40 μL β-glucuronidase was added into the tube and the mixture was incubated at 37 o C for 12 h. When the mixture was cooled to room temperature, then the samples were extracted. HPLC conditions: HPLC column: ZORBAX Extend-C18(250 mm×4.6 mm, 5 μm); Flow rate: 1.0 m L/min; Column temperature: 30 o C; Sample volume: 30 μL; Determine wavelength: 276 nm; Mobile phase: A: 0.01% ammonia, B: methyl alcohol(71:29, V/V). The limit of quantification(LOQ) is 40 μg/kg of DVD and DVD1 in the tissues of pigs, chicken and carp in this method. The recoveries in edible tissues were greater than 70%, spiked at levels of 40-160 μg/kg, with inter-day RSD less than 14%. The residue detection method could meet the requirements of quantitative analysis and could be used for detection the residues of DVD and its main metabolites.3 Residue depletion study of DVD and its main metabolites in swine, broilers and carp24 healthy crossbred swine, 36 twenty-day-old Kebao broilers and 30 healthy carp were administrated DVD continuously at 10 mg/kg b.w. for 7 days. Animals were slaughtered at different withdrawal time, the liver, kidney, muscle, fat(fat+skin), heart, lung, stomach, large intestine and small intestine of swine and broilers, and the liver, kidney, muscle, skin and gastrointestinal tract of carp were collected. The residue content of DVD and its metabolites was quantified, and the results were shown as follows:Swine: At the withdrawal time of 6 hours, DVD and DVD1 were detected in all of the tissues, and the concentration was in the range of 63.2-519.9 μg/kg and 357.5-2297.0 μg/kg. The concentration was the highest in the kidney. The elimination of DVD and DVD1 was rapid in swine, and DVD1 was not detected in the muscle, heart, lung and small intestine, and the concentration of DVD was also greatly reduced at the withdrawal of 1 day. At the withdrawal time of 3 days, the content of DVD1 in most of the tissues was less than the LOQ except the liver, kidney and fat with the concentration of 77.6 μg/kg, 190.1 μg/kg and 51.9 μg/kg. At the withdrawal time of 5 days, DVD1 was eliminated to below the limit of quantification in all tissues. The concentration of DVD was near the LOQ in the fat, heart, stomach, large intestine and small intestine. At the withdrawal time of 7 days, in addition to the liver and the kidney, in the rest of the tissues the concentration was less than the LOQ, and the concentration of DVD was 96.5 μg/kg and 115.1 μg/kg respectively in the liver and kidney. At 14 days, all tissues had been undetectable drugs. After all tissues were treated with glucuronidase, we found that the concentration of DVD1 increased in the liver and kidney at 6 hours and 1 day. The concentration was 319.4 μg/kg and 942.8 μg/kg in the liver and kidney respectively at 6 hours. The content of DVD1 in the kidney was much more compared with not hydrolysis which showed that the concentration of DVD2 in the kidney at 6 hours was high; The concentration of DVD1 was 199.3 μg/kg and 680.5 μg/kg in the liver and kidney respectively after 1 day; The concentration of DVD1 had no obvious change after 3 days, stating DVD2 undetectable, showing that DVD2 eliminated very fast. The results showed that the content of residues was low in pigs, the elimination of the residues was rapid, and DVD prototype was the marker residue and kidney was the target tissue.Broiler: At the withdrawal time of 6 hours, DVD and DVD1 were detected in most tissues except the heart and small intestine, and the concentration was in the range of 53.6-738.5 μg/kg and 3356.4-8541.1 μg/kg. After 1 day, DVD1 could be detected in the kidney at 45.8 μg/kg. The elimination of DVD was also quick, and the concentration of DVD was dropped to 327.5-1764.6 μg/kg. DVD1 was eliminated to below the limit of quantification in every tissue on the third day. At the withdrawal time of 7 days, the concentration of DVD was 107.4 μg/kg and 118.7 μg/kg in the liver and kidney, within 100.0 μg/kg in the other tissues. At the withdrawal time of 9 days, the concentration was 68.1 μg/kg in the liver and was 77.8 μg/kg in the kidney, and the other tissues had no drugs tested. After glucuronidase processing, the content of DVD1 increased in the liver and kidney only at 6 hours, at 987.2 μg/kg and 612.3 μg/kg respectively. After 1 day, the concentration of DVD1 had no obvious change, suggesting DVD2 eliminated very fast and the residual time was quite short. Overall, the elimination of the residues was rapid. DVD had the longest residual time. Results revealed that kidney was the target tissue, and DVD prototype was the marker residue in broilers.Carp: At the withdrawal time of 6 hours, only DVD could be detected in carp tissues(liver, kidney, muscle, skin and gastrointestinal tract), DVD1 could not be detected either after enzymolysis, showing that no DVD1 and DVD2 residues in fish tissues or residual amount less than the LOQ. But the concentration of DVD in tissues was very high, the concentration ranged from 6632.9 μg/kg to 8671.0 μg/kg, with the kidney supreme. At the withdrawal time of 1 day, the concentration of DVD in tissues at 2539.6-5265.6 μg/kg. At the withdrawal time of 7 days, DVD eliminated to under 401.6 μg/kg in each tissue. At the withdrawal time of 14 days, DVD concentration was 77.6 μg/kg in the liver, and 99.7 μg/kg in the kidney, and in the rest of tissues the DVD concentration had been eliminated to less than the LOQ. Thus it could be seen that the concentration of DVD was high but the elimination of the residues was rapid in fish tissues. The marker residue was DVD and the residual target tissue was the kidney.4 Recommendation of the maximum residue limits and the withdrawal periods of DVD in edible tissues of swine and broilersIn accordance with the procedures of FDA, the withdrawal time(WDT) of DVD in pigs and broilers was 1 day. The MRL of the liver, kidney, muscle and fat in the pigs was 791, 1067, 334 and 1938 μg/kg, respectively. The MRL of the liver, kidney, muscle and fat+skin in broilers was 972, 1951, 367 and 2138 μg/kg, respectively. According to the procedures of EMEA, the WDT of DVD in pigs and broilers was 10 days and 12 days, respectively. The MRL of the liver, kidney, muscle and fat(fat+skin) in the pigs and broilers was 100 μg/kg. Comprehencive analysis of the results, it is most conservative recommended to use the results of EMEA.To sum up, the reference substance of DVD1 was prepared for the first time in this topic, a HPLC method simultaneous determination of DVD and its main metabolites in commodity and non-commodity tissues of swine, broilers and carp established, and at the same time the residue elimination rule of DVD in pigs, chicken and carp was studied systematically. The marker residue and the target tissue were further confirmed, and the MRLs and the WDT were established in swine and broilers. The study results provide a scientific basis for drug clinical use and drug safety evaluation.