| A method for high performance liquid chromatography-mass spectrometry(HPLC-MS/MS)was used to establish a 24% Salinomycin Premix in broiler tissue samples(liver,kidney,sebum,muscle).The residue elimination was studied and the chronic toxicity of the premix in SD rats was evaluated.240 healthy SPF Sprague Dawley rats,half male and half female,weighing 50～70 g,were randomly divided into blank control group(0 mg/kg·bw),low dose group(3mg/kg·bw),medium dose.The group(9 mg/kg·bw)and the high-dose group(15 mg/kg·bw)were fed for 180-day chronic toxicity test.The body weight of the rats was weighed every15 days to calculate the weight gain rate.Twenty rats(male and female)were randomly selected from each group on the 60 th,120th and 180 th day of the experiment.The blood of the rats was collected for hematological and blood biochemical indicators.The organ tissues were collected for organ coefficient and organ content.Determination of water volume and pathological examination.The results showed that the low dose addition during the whole feeding process had little effect on the physiological and biochemical functions of the animals and had a good growth-promoting effect,but long-term,high-dose addition resulted in red blood cell count,hemoglobin content and hematocrit increase in rats.High;also leads to elevated alkaline phosphatase activity in rats and affects total bilirubin,total protein content and triglyceride content.Pathological examination showed that there were more substantial lesions in the visceral tissues of the middle and high dose groups,especially in the heart,liver,lung and kidney.According to the actual feed intake of the experiment,the maximum no-effect dose of the Salinomycin Premix for each rat was 9.98 mg/(kg·d),and the maximum no-effect dose of Salinomycin was calculated according to the drug titer(NOEL)is 2.40 mg/(kg · d).Broiler tissue samples were extracted with isooctane-ethyl acetate(8:2)and assayed by HPLC-MS/MS analysis.The column was Luna C18,the mobile phase was 0.1% formic acid-methanol,gradient elution,flow rate was 0.3 m L/min;electrospray ion source,multi-stage reaction monitoring positive ion mode,external standard method single point quantification.The linear relationship of the addition of Salinomycin in the tissue samples was good,and the correlation coefficients in the range of 120～1800 μg/kg were all above0.9990.The detection limit is 60 μg/kg and the limit of quantification is 120 μg/kg.In the LOQ,1/2MRLs,MRLs(600 μg/kg for muscle and kidney,1200 μg/kg for sebum,1800μg/kg for liver)and 2 MRLs,the recovery rate of Salinomycin was 82.83%～110.68.%,the intra-assay coefficient of variation is 1.21% to 8.86%,and the inter-assay coefficient of variation is 2.33% to 6.74%.The method is accurate and sensitive,and is suitable for the detection of Salinomycin in broiler tissues.Seventy-nine yellow broilers were selected,4 to 5 weeks old,10 were randomly selected as blank control group,and the remaining 60 were 24% Salinomycin-administered group.The dose was administered by 250 g/1000 kg feed.42 d.Tissue samples(liver,kidney,muscle,sebum)were collected at 0,6,12,18,24,36,48,72,120,168 h after drug withdrawal,HPLC-MS/MS determination.The results showed that after feeding with 24%Salinomycin Premix,the residual amount in broiler tissues was much lower than the maximum residue limit after 0 h of withdrawal,and the residual elimination time was 0 h. |
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