Study On Actue Toxicity And Residue Determination Of Halofuginone Hydrobromide In Sturgen Muscle

Halofuginone is a halogenated analogue of the naturally occurring quinazolinone alkaloid febrifugine. In this study acute toxicity in sturgeons was developed, and an immunosorbent was prepared by covalently coupling polyclonal antibodies in the New Zealand rabbits against halofuginone to CNBr-activated Sepharose 4B. High performance Liquid chromatography was developed for determining halofuginone in sturgeon muscle.Acute toxicity in sturgeons was developed in this study. LD₅₀s were 19.41μg/L and 13.63 μg/L at 48h and 96h respectivly. Oral administration exhibited that the feedstuff additive halofuginone has intense toxicity according to the criterion of fish acute toxicity.The IgG antibodies obtained by immunizing New Zealand rabbits were purified by SAS deposition and DEME ion-exchange column. IgG concentration was measured with a 1 cm cell using a UV-Vis spectrophotometer. The purified IgG antibodies were coupled to CNBr-activated Sepharose 4B gel and the coupling percentage was 97.4%. Determined by HPLC, the dynamic column capacity was 1584 ng halofuginone per mL gel, and the specific column capacity was 222 ng halofuginone per mg immobiLized IgG. The average recovery of halofuginone standards was 99.8% at the level of 1000ng in 10mL solution.A method of immunoaffinity chromatography (IAC) technique technique combined with reversed-phase high performance liquid chromatography/ultraviolet detection was developed to determine halofuginone in sturgeon muscle. Halofuginone was extracted from trypsin-digested tissues as a free base into ethyl acetate and partitioned into ammonium acetate buffer extraction, purified by IAC column, and the elutes were determined by HPLC. pH adjusted was used to investigated the influence of the binding efficiency of IAC. The results indicate pH has a great influence on the binding efficiency of IAC. Under physiological conditions (i.e. a neutral pH application buffer with low-to-moderate ionic strength), the binding of HFG on the IAC was more stable. On account of the preparation of samples, ammonium acetate buffer (0.125 M, pH 7.4) was elected as the coupling solvent. The elution of HFG was completed after 3 mL methanol for the IAC. In the sturgeon muscle tissues were fortified at 20-200ng/g, the average recoveries were 74.6-81.0% and coefficients of variation were 0.7-8.6%. The detection limit was estimated to 10 μg/kg in a 2-g sample.A method of SPE technique combined with reversed-phase high performance liquid chromatography/ultraviolet detection was developed. Halofuginone was extracted as described above except for Oasis(?)-HLB cartridge cleanup. In the sturgeon muscle tissues were fortified at 20-200ng/g, the average recoveries were 67.5-77.5% and coefficients of variation were 0.4-15.6%. The detection limit was estimated to 15 μg/kg in a 2-g sample.The method developed in this research can be used to determine halofuginone residue in sturgeon muscle.