Studies On Method Of HPLC For Determination Of Avermectins Residues And Their Residues Depletion In Muscle Of Carassius Auratus

Avermectin B_1a(AVM) and ivermectin H₂B_1a(IVM) are the macrocyclic lactones anti-parasitic agents most widely used in the treatment of food producing animals, poultry and aquaculture. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. In order to determine AVM and IVM residues in muscle adhering skin of Carassius auratus and offer scientific gist for constituting withdrawal time (WDT). A multi-residue method was developed and validated for the quantitation and confirmation simultaneously of marker residues AVM and IVM in Carassius auratus muscle adhering skin by use of high-performance liquid chromatography with fluorescent detection (HPLC-FLD). Study the residues depletion of AVM and IVM in muscle adhering skin of Carassius auratus, respectively, and determine residues of AVM and IVM in it from some part of Chongqing..The multi-residue determination method of AVM and IVM in Carassius auratus muscle tissue: Samples were prepared by adding AVM and IVM into extracts of Carassius auratus muscle tissues, which were prepared by acetonitrile extraction of the Carassius auratus muscle homogenate followed degreasing by n-hexane, derivated by 1-methylimidazole and trifluoroacetic anhydride in acetonitrile and hydrolyzed in methanol in the condition of protect from light and room temperature, subsequently. The doramectin were used as internal standard. HPLC-FLD condition: The mobile phase used was methanol /water (94/6, v: v) at a flow rate of 1mL/min. The fluorescence detector setting were excitation wavelength of 365nm, emission wavelength of 475 nm. The column temperature was 35℃.The injection volume was 20μL. HPLC-FLD results showed that the calibration curves were linear over the working range from 5 to 100 ng/g for the two drugs, which regression coefficients were 0.9998. For the quantitative analysis, the limit of detection of the method for AVM and IVM was 0.8 ng/g and 1.0 ng/g, while the limit of quantitation for AVM and IVM was 2.7 ng/g and 3.4ng/g, respectively. The overall extraction recoveries of AVM and IVM in the Carassius auratus tissue ranged from 92.15% to 104.53% and 91.32% to 103.03%, and method recoveries ranged from 90.50% to 104.54% and 94.08% to 106.38%, respectively. The intra-day coefficient variations (CV) were less than 9.29% and 7.74, and inter-day CV less than 5.84% and 6.71%, respectively. These results suggest that the method reported herebe was effective, sensitive, simple, convenient and precise for the multi-residue determination of AVM and IVM in Carassius auratus muscle tissues.Study on residues depletion of AVM and IVM in Carassius auratus muscle tissue: Actual aquacultural conditions were imitated in this study. Under 27-31℃, two experiments were performed using Carassius auratus. There were 100 fish (mean body weight of 45 g) in test group of each experiment and 50 fish in the control group. In the first experiment, sprinkling AVM in methanol into water once made its concentration 1.32 ng/mL. In the second experiment, concentration of IVM was 1.32 ng/mL in water. Untreated Carassius auratus in control group served as negative control. Fish were sampled on days 0, 1, 3, 5, 8, 10, 11, 14, 16, 21, 25, 28, 32 and 35 after administration. The residues of AVM and IVM which were detected in muscle adhering skin on post-dose day 0(at post-dose 4 and 5h as zero WDT) were 13.68±4.68 ng/g and 8.1±1.67 ng/g, respectively. Maximum residues of both AVM (36.38±6.20 ng/g) and IVM(25.47±3.16 ng/g) were detected in muscle adhering skin on post-dose day 3, and residues of AVM and IVM reduced by half about on post-dose day 10 and 11, respectively. The residue of AVM was below the detection limit on post-dose day 32, and IVM on post-dose day 28. The main pharmacokinetic parameters of AVM in muscle adhering skin were: Maximum residue (C_max) of 31.42 ng/g, the time to C_max of 3.53 d., the area under the curve (AUC) of 469.88 d.ng/g, the depletion half-life (t_2/2) of 7.48 d. The depletion equations of AVM was LnC = -0.0922t + 3.8985, and the depletion half-life (t_1/2e) was 7.52 d. The main pharmacokinetic parameters of IVM in muscle adhering skin were: C_max of 24.51 ng/g, T_max of 3.91 d, AUC of 293.13 d ng/g, t_1/2k of 4.42 d. The depletion equations of IVM was LnC = -0.1382t + 3.9202, and t_1/2e was 5.01 d. The results showed that : relatively rapid uptake and slow depletion of the two drugs were observed ,and the two drugs do not strongly bioconcentrate in muscle adhering skin of Carassius auratus. At present, there are no regulation about maximum residue limit (MRL) and WDT in Carassius auratus tissues at home and abroad. Consulting MRL of 20μg/kg and 25μg/kg of AVM in ovine muscle for official pronunciamento of European Community (EC) and China, WDT of AVM which calculated from the formula of WDT was 9.79 d and 7.37 d, respectively, or from the program WT1.4, WDT was 14.97 d and 12.64 d, respectively. Considering the difference between formula calculation and fact, it was suggested WDT added to 19 d. Consulting MRL of 20μg/kg of IVM in ovine muscle for both of official pronunciamento of EC and China, WDT of IVM which calculated from the formula of WDT was 6.69 d, or from the program WT1.4, WDT was 11.04 d, it was suggested WDT added to 16d.Multi-residue determination of AVM and IVM in Carassius auratus muscle tissue from some part of Chongqing: Determination rates of both of the two drugs were less than 5%, and residues were less than 10 ng/g. The results indicated that: Both of the two drugs were used normatively in aquaculture of these areas, and residues of their were lower MRL. Citizens could set one's heart at rest at purchase and eating it.