Study On Development Methods Of Determination Multi-residue Of Avermectins

Avermectins belong to the family of macrocyclic lactone, which have been found wide usage in livestock against a board spectrum of nematodes and arthropods. However, AVMs are taken as poison substances. So, the AVMs residue in animal tissue should be considered. In this study, based the antibody of AVMs, an ELISA method and immunoaffinity chromatography-high performance liquid chromatography-fluorescence detector (IAC-HPLC-FLD) method were developed to detect the AVMs residue in animal tissue.Avermectin (AVM), Ivermectin (IVM) and Doramectin (DOR) were modified to make the haptens: 4"-o-succinoylAVM, 4"-o-succinoyIVM, and 4"-o-succinoylDOR. The molecular weights were 995.79 (M+Na+), 997.8 (M+Na+), and 998.6 (M-H⁺) through mass spectrum detection. The haptens were coupled to BSA as immunogen, and to OVA as coated antigen. The conjugates were identified by UV-VIS spectrophotometer, and the ratio of hapten and carrier protein was calculated. The ratio ranged from 3.4 to31.6.Among the polyclonal antibodies, one anti-AVM polyclonal antibody had the high cross-reaction with Eprinomectin (EP, 145.4%), AVM (100%), IVM (25.0%), and DOR (12.3%). Based on the antibody, an ELISA method was developed to detect AVM, IVM, and EP residue in animal tissue. The optional working dilution of antibody and coated antigen were all 1:5000, and the dilution of enzyme-conjugate was 1:2000. The formula was y = -45.613x + 81.191, R2 = 0.9951, and the 50% inhibition concentration (IC₅0) was 4.8 ng/mL. The linear detecting range of calibration curve was from 1.1 ng/mL to 21.9 ng/mL. When the spiked concentrations of AVMs were 20 ng/g, 50 ng/g, and 100 ng/g in bovine liver, the recovery of AVMs was 53.8-80.4% with coefficients of variation (CV) of 3.4-17.9%. When the spiked concentrations of AVMs were 5 ng/g,10 ng/g, and 20 ng/g in bovine muscle, the recovery of AVMs was 70.9-108.6% with CV of 3.7-17.1 %.The enzyme immunoassay kit was developed through choosing the appropriate solutions and the production techniques. The technique parameters as follows: IC50 ranged from 3.0 ng/mL to 7.0 ng/mL; the limits of detection of blank bovine liver ranged from 8.4 ng/g to 9.6 ng/g, and the limits of detection ranged from 0.5 ng/g to 0.8 ng/g in blank bovine muscle; the cross reaction with AVM was 100%, IVM was 25.0%, and EP was 145.4%; the accuracy and precision accorded with the kit standards. The kit could be reserved for 6 months at 2-8 ℃.Immunoaffinity chromatography columns were prepared by coupling purified the antibody to CNBr activated Sepharose 4B. The dynamic column capacity of AVM, IVM, DOR, and EP were 3531, 3542, 3543, and 3284 ng/mL gel, and the specific column capacity were 495, 499, 493, and 460 ng/mg IgG respectively. After 15 cycles in a month, the capacity of IAC also was above 1300 ng/mL gel for every drug.The IAC-HPLC-FLD method was developed to detect the residue of AVM, IVM, EP, and DORin animal tissue. Recoveries ranged from 79.4 to 113.8% with CV of 1.1-19.4% when AVM, IVM, DOR, and EP were spiked in bovine liver at levels of 5-100 ng/g. Recoveries ranged from 77.3 to 119.5% with CV of 1.5-18.9% when AVM, IVM, DOR, and EP were spiked in bovine muscle at levels of 5-100 ng/g. The limit of detection of the method was 0.8 ng/g in bovine tissue.