| Salbutamol and ractopamine are p-adrenergic agonist, which has widely physiological effects. It can be used as a feed additive in sheep, pigs, poultry and other livestock production, to promote growth, improve feed conversion rate and the muscle to fat ratio, to enhance carcass leanness. It will cause poisoning, when the body has excessive intake of salbutamol or ractopamine. In Chinese, European Union and other countries, the use of salbutamol and ractopamine in animal breeding have been banned. However, there are still illegal to use in the actual production, it causes a serious threat for food safety and human health. At present, the monitoring of such drugs is mostly used to detect drug residues of the animal edible tissues after slaughtering animals, resulting in unnecessary economic losses. Therefore, it becomes imperative that finding an easy and cost-saving method of monitoring. In this study, the metabolism and residue depletion in the edible tissues of salbutamol or ractopamine in pigs and goats is investigated. Based on this research, the paper studied the residues correlation between the surface tissue and edible tissues. In actual production, it can predict drug residues of edible tissues in vivo by means of detecting the body tissue, reaching "detecting in vivo" and proposing a new method of monitoring.1 Development of methods for determination and confirmation of P-agonistIt is to determine the 5 kinds of β-agonist in tissues of swine and goats by LC-MS/MS. First, Enzymatic hydrolysis of tissue samples was carried out by use of P-glucuronidase. The samples are extracted with ultrasonic using alkaline ethyl acetate, defatting with hexane. The sample was purified and concentrated by MCX column.The analytes were eluted with 5 mL of 5% ammonia in ethyl acetate. Then, the eluate was dried under a nitrogen stream and dissolved in 1 mL of 0.1% acetic acid for analysis. The limits of detection (LOD) and limit of quantitation (LOQ) of salbutamol and ractopamine in tissues of swine and goats were 0.125 μg/kg and 0.25 μg/kg, respectively. Limits of detection (LOD) and limit of quantitation (LOQ) of zilpaterol, ractopamine and phenylethanolamine A in tissues of swine and goat were 0.25 μg/kg,0.5 μg/kg, respectively. The recoveries of 5 β-agonist in hair were from 61.0% to 90.5%, the coeffieient variation were≤11.3%; The recoveries of 5 (3-agonist in tissues were from 67.7%-89.7%, the coeffieient variation were≤ 13.2%.2 Metabolism research of salbutamol and ractopamine in swine and goatsFour swine and goats were single administrated, collecting urine and feces each 24 h after administration, and collecting plasma at the first five points. The samples were processed with enzymolysis and without enzymolysis hydrolysis. The metabolites were characterized using LC/MS-IT TOF. For salbutamol, three compound (SAL、Glu-SAL and Me-SAL) were identified in in urine of swine and goat, only one compound (SAL) was found in feces of swine and goat. For ractopamine, two metabolites (RCT、Glu-RCT) were identified in urine of swine and goat, only one metabolites (RCT) was found in feces of swine and goat.3 Predicting residual of salbutamol and ractopamine in swine and goatsTwenty swine and goats were fed for 14 consecutive days with feeds containing 20 mg/kg salbutamol or ractopamine, respectively. Swine and goats were slaughtered at 6 h, 1 d,3 d,7 d,14 d of the withdrawal period. Liver, kidney, muscle, fat, lung, large intestine, small intestine, plasma, urine, feces, and hair samples were collected and stored at -20℃. The samples were processed using enzymatic and non-enzymatic hydrolysis, and measured by LC-MS/MS. According to the residues of surface and the animal edible tissues in vivo, taking hair, plasma, urine, feces as surface organizations, and researching the correlation between surface tissues and edible tissues (liver, kidney, muscle, fat and lung).In study of salbutamol in pigs and goats, the concentration of salbutamol in urine, plasma of swine were significantly different before and after the Enzymatic hydrolysis, but no significant difference in other tissues. The residues of salbutamol in swine liver were higher than those in kidney at 6 h from the last salbutamol exposure. Most tissues can’t be detected after the withdrawal of 7 days. In swine hair, salbutamol seemed to have a longer detectable periods, up to day14, is 57.26μg/kg. The eliminate half-life of swine liver, kidney, muscle, lung and hair was 0.58 d,0.63 d,0.62 d,0.45 d,5.07 d, respectively. The concentration of salbutamol in hair, feces of goat has no significant difference before and after enzymatic hydrolysis, but was significantly different in other tissues. The residues of salbutamol in goat kidney were higher than those in liver at 6h from the last salbutamol exposure. Only the hair that can be detected after the withdrawal of 14 day, is 5.43 μg/kg. The eliminate half-life of goat liver, kidney, lung and hair was 1.06 d,0.54 d, 0.58 d,7.6 d, respectively.In the study for residue correlation of salbutamol between the surface tissue and edible tissues of swine, liver, kidney, muscle, and lung vs. hair or urine, using monadic linear regression fitting, the correlation coefficients is beyond 0.9. In goats, hair vs. liver, liver (with enzymatic hydrolysis), kidney (with enzymatic hydrolysis) and lung (with enzymatic hydrolysis) vs. urine (with enzymatic hydrolysis) or feces showed a linear relationship, the correlation coefficients is beyond 0.9. Hair vs. kidney, fat and lung, using exponential function fitting, the correlation coefficients is beyond 0.94. Thus, urine and hair were selected as optimal surface tissue in swine, and urine and feces were selected as optimal surface tissue in goat.For ractopamine in pigs and goats, the concentration of ractopamine in swine urine, plasma and kidney was significantly different before and after the enzymatic hydrolysis, but no significant difference in other tissues. The residues of ractopamine in swine kidney were higher than those in liver at 6h from the last ractopamine exposure. In swine hair, ractopamine have a longer detectable periods, up to day 14, is still 131.1 μg/kg. The eliminate half-life of swine liver, kidney, lung and hair was 1.64 d,1.57 d,0.62 d,2.17 d, 6.22 d, respectively. The concentration of ractopamine in kidney, muscle, lung, urine and plasma of goat were significantly different before and after enzymatic hydrolysis, but no significant difference in other tissues. The residues of ractopamine in goat kidney were the highest at 6 h from the last salbutamol exposure, it is 125.7 μg/kg. The lowest drug concentration in goat muscle is only 3.77 μg/kg. Only the hair that can be detected after the withdrawal of 14 day, it is 8.88 μg/kg. The eliminate half-life of goat liver, kidney, muscle, fat, lung and hair was 0.7 d,0.53 d,0.91 d,0.62 d,1.16 d,7.29 d, respectively.In the study for residue correlation of ractopamine between the surface tissue and edible tissues of swine, liver, kidney and lung vs. hair, using power function fitting, the correlation coefficients is beyond 0.9. Urine vs. liver and kidney, using monadic linear regression fitting, the correlation coefficients is beyond 0.9. In goats, kidney, fat and lung vs. hair, urine or feces and liver (with enzymatic hydrolysis), kidney (with enzymatic hydrolysis), lung (with enzymatic hydrolysis) vs. plasma (with enzymatic hydrolysis), showed a linear relationship, the correlation coefficients is approximately 0.9. Thus, urine was selected as optimal surface tissue in swine, and all of hair, urine and feces were selected as optimal surface tissue in goat.In this paper, the quantitative and confirmatory method established is simple, convenient sample treatment and high sensitivity. It firstly conducted the metabolism of salbutamol in pigs, and developed the residue correlation model between surface tissue and edible tissues. The optimal surface tissue can be founded, which showed a linear relationship between it and edible tissues, the correlation coefficients is even approximately 0.9. The correlation equation can be used to detect in practice. It provides a theoretical basis for monitoring residues of salbutamol and ractopamine in food producing animals and food safety evaluation, and has important application value. |
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