| Gamithromycin (GAM) is a new macrolide antibiotic drug. The injection made from it has been authorized to sell in foreign markets. GAM injection is uesd to treat bovine respiratory disease which is caused by Mannheimia haemolytica, Pasteurella multocida, Histophilus somni and Mycoplasma bovirhinis clinically. Rencently, GAM and its injection were copied successfully in China. The clinical research showed that cattle administered a single dose of domestic GAM injection by subcutaneous injection at 6 mg/kg b.w could be well cured if they got bovine respiratory disease caused by Pasteurella multocida. For the evaluation of residues elimination of GAM injection in cattle, the detection of GAM residues in bovine edible tissues was developed in this paper, and on this basis, the study of the residues elimination of GAM injection was carried out. The research not only priovides the technical support for domestic GAM residue’s postmarket surveilance but also gives the the basis for the formulation of withdrawal period which means a lot for the rational clinical use of drug and the ensurance for animal-derived food safety.1. Determination of GAM residues in bovine edible tissues by HPLC-MS/MSThe GAM in bovine edible tissues were extracted with acetonitrile, defatted with n-hexane and purified with Oasis(?) MCX cartridge extraction column. Sun Fire C18 column was choicen for the separation of GAM. Column temperature setted at 50℃ and the injection volume was 1 uL. The 0.01 mol/L acetate buffer (pH3.6)-methanol (30/70,v/v) was used as mobile phase.The Mass spectrometry conditions are as follows, quantitative ion pair:777.7/619.108, qualitative ion pair:777.7/619.108 and 777.7/157.768, electrospray voltage:5.5kV. ion temperature:500℃. positive electrospray ionization(ESI+), multiple reaction monitoring(MRM).The regression curves for GAM in bovine edible tissues were linear in the rangeof 6-1000 ng/mL. Mean recoveries of GAM in muscle and liver fortified at 6,200 and 800 μg/kg of GAM were from 84.17% to 94.17%; Mean recoveries of GAM in kidney fortified at 6,100 and 800 μg/kg ranged from 79.23% to 90.36%. Mean recoveries of GAM in fat fortified at 6.20 and 800 ug/kg were 84.91% to 97.73%. The inter-day and intra-day coefficients of varination were less than 10%.The limit of detection (LOD) was 2 ug/kg, and the limit of quantification (LOQ) was 6μg/kg.Stability test included the stability of the loading solutions in sample room (about 22℃), spiked tissues in blank after repeated freeze-thaw(22℃/-20℃) cycles and storage at-20℃. Dilution effect for the accuracy of determination was also studied. The result showed GAM in bovine edible tissues had a good stability, the coefficient of variation(CV) was less than 15%. Compared with the standardaddition in blank, the CV of GAM in samples diluted 100,50 and 20-folds with blank tissue extraction was under 15% which is also acceptable.2. Study on the residues elimination of GAM Injection in bovineTwenty little Holstein bulls (250-400 kg) were choicen for the study of residues elimination of GAM injection. All the cattle were administered a single dose of GAM injection by subcutaneous injection at 6 mg/kg b.w. and each four cattle were slaughtered at 10,20,30,45 and 60 d after injection. Longissimus dorsi muscle, fat, kidney, liver and muscle at injection site were collected immediately after their death. All of these samples were stored at -20℃ after labelling and package without any treatment.GAM in bovine edible tissues were determined by the established method above. The result showed, except the injection site, the concentration of GAM was highest in liver at 10 d after injection(2483.98℃96.75 μg/kg), and then in kidney(2183.0±431.25 μg/kg).The concentration in longissimus dorsi muscle and fat were lowest in all collected tissues(55.76±10.70 μg/kg and 30.66±10.31 μg/kg). Besides that, the concentration in fat from one cattle was below MRL set by EMEA. At 20 d after injection, the residues in fat from four cattle were below MRL. At 30 d after injection, except two cattle, the residues in liver were below MRL. At 45 d after injection, the average residues in liver, kidney and fat were below MRL. The results showed that liver was the target tissue of GAM in cattle. According to GAM’s MRLs set by EMEA in beef cattle and non-lactating dairy cattle, the raw data were analysed with WT1.4 program. The withdrawal period after the addnimistration of domestic GAM injection at recommended dosage was 39.54 d in liver,30 d in fat and 33.13 d in kindney. In general, we suggest that after a single dose of domestic GAM injectionby subcutaneous injection at recommended dosage for cattle, the withdrawal period of GAM was 40 day. |
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