Study On Metabolism And Residue Of Malachite Green In Grass Carp

Malachite green (MG) is a synthetic dye industry Triphenylmethane, Because of its antimicrobial effects, and inexpensive, readily available, has been widely used in aquaculture. However, because of its potential carcinogenic effects, it has been banned in many countries as a veterinary drug. At present, there is still the phenomenon of illegal use of malachite green in aquatic products breeding and the transportation process. Therefore, it is imperative to Strength drug detection and monitoring,to optimize the detection method and to study the metabolism of main aquatic products.Based on the physical and chemical properties of malachite green (MG) and leducomalachite green (LMG) to establish a high-performance liquid chromatography visible light detection (HPLC-VIS) and high-performance liquid chromatography UV detection (HPLC-UV), The former add the oxidation column after chromatography column, Oxidate LMG to MG, Using elution time difference, thus simultaneously examines two materials ; The latter method is keen to the LMG's examination, and does not need to add oxidation column ,the flowing available methyl alcohol, the operating process is simple, and saves the cost.When detect MG and LMG of grass carp muscle samples by HPLC-VIS ,linear equations for standard curve of MG and LMG respectively are: y=150986x-389.38 and y=67995x-175.77, the correlation coefficient respectively are: 0.9995 and 0.9993, linear range in: 0.001～0.1mg/kg. When detect MG and LMG of grass carp muscle samples by HPLC- UV, linear equations for standard curve of MG and LMG respectively are: y = 57138x - 3786.4 and y = 179901x - 1199.7, the correlation coefficient respectively respectively are: 0.9927 and 0.9998, the linearity range respectively are: 0.01～0.1mg/kg and 0.001～0.1mg/kg.When detect MG and LMG of grass carp blood samples by HPLC-VIS , linear equations for standard curve of MG and LMG respectively are: y = 139259x -182.99 and y = 60093x -149.97, the correlation coefficient respectively are: 0.9991 and 0.9996, linear range in 0.005～0.5 mg/kg; When detect MG and LMG of grass carp blood samples by HPLC- UV, linear equations for standard curve of MG and LMG respectively are: y = 56401x - 3825.3 and y = 181863x -981.91, the correlation coefficient respectively respectively are: 0.9903 and 0.9985, the linearity range respectively are: 0.05～0.5 mg/kg and 0.005～0.5 rag/kg. In short, HPLC-VIS detection of the MG and LMG has strong detection sensitivity, HPLC-UV detection of the sensitivity of the LMG, the MG the detection sensitivity is not high.The optimization of the grass carp muscle and blood of the pre-treatment methods, use of acetonitrile extraction, anti-dichloromethane extraction and purification centrifugal addition to fat, decompression rotary steam enrichment method. This method is simple. In samples of muscle and blood samples after testing,When we use HPLC-VIS detection ,the relative recovery of MG in the scope of 98.341 % to 79.66 %,the relative recovery of LMG in the scope of 75.150 %to 103.6435 %;When we use HPLC-UV detection , the relative recovery of MG in the scope of 68.1355 % to 99.6754 %, the relative recovery of LMG in the scope of 76.1725 % to 97.0892 %. Meet the "technical specifications of veterinary drug residues trial (Trial)" the provisions of the method of recovery is 60% -93%, in line with residue testing requirements. When we detecte the muscle and blood samples of grass carp in the HPLC-VIS and HPLC-UV , Days precision is less than three , Intra- Days precision is lower than 8,it can meet the detection requirements.Using HPLC-UV to Study metabolism of MG and LMG in grass carp blood after intermuscular injection MG, discovered injects the malachite green 15 minutes later, MG and LMG in the blood can be detected simultaneously. The the density of MG is higher than the injection density by far in the blood, indicated that the absorption, the distribution, metabolism is very rapid in grass carp, and may store up in. The density of LMG achieves 0.54mg/kg L when 15 minutes , Its density is higher than MG after inject MG 2 hours, its erase time is longer than MG, it still achieved 51rng/kg in blood in three days inject MG. Under the 268nm examination wave length had not seen other metabolite, may determine that LMG is the main metabolite of MG in the grass carp initially.Simulation of the production of drugs commonly used in practice means: 0.3 mg/kg of the MG-bath one hour ,then detect the concentration of MG in the blood and muscle. The peak concentration of MG in muscle and blood are 1.80318 mg/kg and 1.37742 mg/kg respectively, Indicated that MG has the cumulative action in the aquatic product Muscular tissue and blood's.Malachite green's absorption half-life T1/2 in muscle and blood of grass carp respectively is 2.74999 hours and 1.93770 hours, distributed half-life T2/2 respectively is 0.20468 hours and 0.17945 hour, eliminates half-life T3/2 respectively is 100.38503 hours and 46.03478 hours, indicated that the malachite green has the strong outside blood vessel distribute ability, the malachite green distributes quickly in the grass carp Muscular tissue and blood's absorption, but eliminates slowly. The malachite green has continued for 30 days in the grass carp body muscle and the blood, the hidden color malachite green has continued separately in the grass carp blood and the muscle for 45 days and 70 days. In this research grass carp in 0.3mg/kg MG medicinal bath 1 hour later, in his/her muscle and the blood the LMG density respectively achieves: 0.006 mg/kg and 0.0053 mg/kg. Therefore LMG is entirely possible as a result of grass carp in the residual marker. HPLV-UV detection method can be used for grass carp muscle of malachite green color and implicit detection of malachite green residues.Uses the HPLC-UV examination which establisheD to grass carp of Chongqing some areas' to carry on the sampling detection, has not examined MG and LMG. Explains the Chongqing some areas aquaculture basic standard.