Disposition Of Tritium Labeled Sulfamethoxazole In Swine,Broiles And Rats

Sulfamethoxazole（SMZ）is a medium effective sulfonamide antibacterial drug with a broad antibacterial spectrum and good antibacterial effect and it is widely used in veterinary clinics.With the widespread use of sulfamethoxazole,its residues in the tissues are becoming more and more serious,causing great safety risks to food safety.At present,there are still many unresolved problems about the metabolism and residual elimination of sulfamethoxazole.The mass balance is not clear,the metabolic pathway is not explicit,and the residual target tissue is not determined in swine and broilers.Therefore,in order to fully understand the metabolic transformation process and distribution and elimination characteristics of sulfamethoxazole in animals,and to control its drug residues,this study used radiolabeling technology to synthesize tritium-labeled sulfamethoxazole,and its metabolism,excretion,distribution and elimination in swine,broilers and rats were studied.The metabolites of sulfamethoxazole in the three animals were qualitatively and quantified,the main metabolic pathways were inferred,the residue elimination rules in different tissues were analyzed,and the species differences between different species were compared.Further confirmed the residue markers and target tissues of sulfamethoxazole in animals,providing a basis for the food safety assessment of sulfamethoxazole.1 Synthesis of tritium-labeled sulfamethoxazole and its quality standardFirst,using meta-bromoaniline as raw material,the amino group is protected by trifluoroacetic anhydride and then reacted with chlorosulfonic acid to obtain intermediate metabolite.Then reacted with 3-amino-5-methylisoxazole condensation,prepared 2-bromo-sulfamethoxazole after alkaline hydrolysis and deprotection.And then tritium-halogen exchange occurs with tritium gas under the action of palladium carbon catalyst and alkali acceptor to prepare 2-[³H]-sulfamethoxazole.The synthesized product was purified by preparing liquid phase to obtain tritium labeled sulfamethoxazole with high specific activity（52.9 Ci/g）,high radiochemical purity（≥98%）and high chemical purity（≥99%）.2 Study on the mass balance and metabolism of sulfamethoxazole in swine,broilers and rats4 swine,6 rats and 6 broilers each at 25 mg/kg b.w.single intramuscular injection（broilers were given by gavage）³H-SMZ（specific activity 0.1 Ci/g）and collected urine and feces at different time.A part of the collected samples were measured by liquid scintillation counter（LSC）,and another sample of a certain quality was extracted and purified and then the radioactive substances in the sample are qualitatively and quantitatively analyzed byν.ARC and LC/MS-IT-TOF.The results showed that the total radioactive recovery rates of sulfamethoxazole in swine,broilers and rats were 95.31%,93.39%and 97.48%during the 14 d recovery period respectively.The peak excretion appeared on the 0-0.5 d after drug withdrawal and the cumulative recovery rate of the three animals all exceeded 59%.After 1 d post dose,the cumulative recovery rate exceeded 75%,and the elimination rate gradually slowed down thereafter.At 3 d,the cumulative recovery rate in the three animals exceeded 88%,indicating that the excretion of sulfamethoxazole in the animals was mainly concentrated in the first 3 d.In swine and rats,more than 75%of the dose is recovered in urine,indicating that sulfamethoxazole is mainly excreted in urine.Three,six and three radioactive compounds including prototype were detected in swine,broilers and rats.The metabolites in swine and rats were the same,both parent S0,acetylation metabolite S1 and trace amounts of glucuronide metabolite S5.In addition,5-methylhydroxylated metabolite S2,N₄-hydroxyamine metabolite S3 and sulfate metabolite S4 were also detected in broilers.The metabolic pathways of SMZ in swine,broilers and rats mainly include acetylation,hydroxylation,sulfate and glucuronide of N₄-amino,and hydroxylation of the oxazole ring methyl group.Acetylation reaction was the most important metabolic mode in the three animals.There are species differences in the metabolism of sulfamethoxazole in different animals.3 Study on the distribution and elimination of sulfamethoxazole in swine,broilers and ratsSwine（24 divided into 6 groups）,broilers（36 divided into 6 groups）and rats（42divided into 7 groups）at 25 mg/kg b.w.³H-SMZ was injected intramuscularly（broiler by gavage）for 7 consecutive days（single administration for rats）,and the specific activity was 0.1 Ci/g.At different time points after administration（12 h,3 d,7 d,14 d and 28 d for swine,6 h,3 d,7 d,14 d and 28 d for broilers,1 h,2 h,6 h,1 d,3 d and 7d for rats）collected various organs,tissues,bile and plasma.Randomly slaughter a group of animals to collect various tissues,bile and plasma.Part of the collected tissue samples were digested and faded,and then LSC was used to determine the total radioactivity.Another tissue sample of a certain quality was extracted with methanol water and then LC-ν.ARC and LC/MS-IT-TOF were used to determine the content of each radioactive material in the tissue.The structure of metabolites was identified,and the distribution and elimination of sulfamethoxazole in different tissues of three animals were obtained.Sulfamethoxazole was widely distributed in swine,broilers and rats.The drug concentration in blood,bile,kidney and liver were higher at 6 h after administration（swine 12 h）.The radioactivity in each tissue decreased rapidly at 3 d,and it was about 10%of the concentration for 6 h（12 h for swine）.At 14 d,radioactivity could still be detected in the tissues of swine and broilers.At 28 d,radioactivity was not detected in most of the tissues,only the liver,muscles at the injection site,bile and blood,and broiler kidneys and muscles could be detected.Only two radioactive compounds were detected in the liver,kidney,muscle and fat of swines and rats,the prototype drug S0 and the acetylated metabolite S1 respectively.The five radioactive substances detected in broilers kidneys were S0,S1,S2,S3 and S4,and only S0 and S1 were detected in liver,muscle and fat of broilers.Species differences exists in the elimination of sulfamethoxazole in the three animals.Sulfamethoxazole has the slowest elimination in swine liver,the elimination half-life was 5.76 d,and the fastest elimination speed in muscle,the elimination half-life was 3.84 d.Sulfamethoxazole was eliminated slowly in broiler muscle and liver,and the elimination half-life of the two tissues was 5.16 d.It had the slowest elimination rate and the longest elimination half-life in rat kidney.The above results indicated that there are species differences in the elimination of sulfamethoxazole in animals.Among the four tissues,the radioactive substance with the longest elimination half-life was S0.S0 persisted in each tissue（14-28 d）.We recommended that S0 was used as residual marker of sulfamethoxazole in animals.In summary,tritium labeled sulfamethoxazole was synthesized in this study and its quality standard was investigated.The metabolism,excretion,distribution and residue elimination of sulfamethoxazole in swine,broilers and rats were studied by using radioactive tracer and liquid chromatography-mass spectrometry technology.Four metabolic pathways and five metabolites of sulfamethoxazole in broilers were discovered for the first time,the toxic metabolites and toxic target organs were pointed out,the species differences of sulfamethoxazole metabolism in swine,broilers and rats were revealed,and the residual marker of sulfamethoxazole was confirmed as the prototype.The results of this paper provide a strong reference for the detection of sulfamethoxazole residue in food,and provide theoretical reference for revealing the pharmacological and toxicological mechanism of sulfamethoxazole.