Determination Of Halofuginone Residues In Chicken Tissues By ELISA And HPLC

Halofuginone (Hal) is a widely used anti-coccidiosis agent, which is usually used as a feed additive at the medicated concentration of 3mg/kg for the prevention and treatment of the coccidiosis in poultry. In this study, halofuginone was modified to be linked with carrier protein to obtain a multiclonal antibodies. An ELISA method and a HPLC method for the determination of halofuginone in chicken tissues were developed, and the depletion of halofuginone residue from chicken tissue was investigated.An ELISA method for determination of halofuginone residue in chicken liver and muscle tissues was developed. The hydroxyl group on halofuginone was protected by N-trimethysilylimidazole, and the amino moiety readily reacted with succinic anhydride to produce the monosubstituted acid succinamide. Finally the trimethyisis moiety was removed by hydrolysis in acid condition to give a succinic acid derivative of halofuginone. This compound was coupled to BSA and OVA by NHS ester procedure,and the coupling ratio were 9.5:1 and 5:1, respectively.The linear detection of the ELISA method ranged from 0.1 ng/ml to 100 ng/ml. In the chicken liver tissue fortified at 50ng/g, 100ng/g, 500ng/g , the average recoveries ranged from 74.2% to 96.8%, with the coefficients of variation within-day were in the range of 3.8%-12.8%, and the coefficients of variation between-days were in the range of 8.8%-15.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 74.3% to 90.0%, with the coefficients of variation within-day were in the range of 2.6%-16.6%, and the coefficients of variation between-days were in the range of 5.4%-6.8%.The detection limits in chicken liver tissue and in chicken muscle tissue were 23ng/g and I5ng/g, respectively.A HPLC method for determination of halofuginone residue in chicken liver and muscle tissue was developed. The tissue samples were hydrolyzed with trypsin, extracted with ethyl acetate, and re-extracted with 0.125mol/Lacetamide buffer. The extracts were then subjected to Oasis HLB cartridge for purification, and the elutes were determined by HPLC. In the chicken liver tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 81.3% to 93.3%, with the coefficients of variation within-day were in the range of 1.5%-10.9%, and the coefficients of variation in between-days were in the range of 2.7%-6.9%. In the chicken muscle tissue fortified at 50ng/g,100ng/g, 500ng/g, the average recoveries ranged from 88.4% to 103.4%, with the coefficients of variation within-day were in the range of 1.4%-11.6%, and the coefficients of variation between-days were in the range of 9.0%-12.6%. The method detection limits in chicken liver tissue and in chicken muscle tissue were 19ng/g and 23ng/g, respectively.In this study, the ELISA method vs HPLC method for the determination of the halofuginone in chicken liver and muscle were compared. The coefficients of correlation for the detection of halofuginone in chicken liver and muscle by two methods were 0.9760 and 0.9344, respectively. Broiler chickens (AA) were fed at a medicated concentration of 3mg/kg of halofuginone-HBr for 10 days. Atwithdrawal time of 0 day, the residue levels of halofuginone in broiler chicken liver tissues detected by ELISA method and HPLC method were 105.2ng/g and 84.6ng/g, respectively. At and after 3 day, the residues were not detected by ELISA method or HPLC method. In the broiler chicken muscles, the residues were not detected by two methods at withdrawal time of 0 day. The ELISA method and the HPLC method for the determination of the halofuginone i n chicken liver and in muscle correlated well.