The Establishment Of Detection Methods For Methylene Blue,Brilliant Green And Its Metabolites In Aquatic Products And The Metabolism Of Brilliant Green In Crucian Carp

Methylene blue,brilliant green and their metabolites belong to dye compounds,methylene blue?MB?belongs to synthetic thiazine dyes,brilliant green?BG?belongs to triphenylmethane dyes,which is similar to the formula of malachite green molecular.They can be used to treat fish diseases in aquaculture.However,high concentrations of methylene blue and brilliant green have certain toxicity,which will cause harm to aquatic organisms and affect human health through the food chain.At present,there is a lack of methods for the simultaneous detection of these compounds,and the understanding of the metabolism in organism is poor.In this paper,a method for the determination of methylene blue,brilliant green and their metabolites in aquatic products was established by liquid chromatography-tandem mass spectrometry?LC-MS/MS?.And the metabolic elimination of brilliant green in crucian carp was studied.The main research results were as follows:?1?A liquid chromatography-tandem mass spectrometry?LC-MS/MS?method was developed for the simultaneous determination of methylene blue,brilliant green and their metabolites in aquatic products.Residues were extracted with hydroxylamine hydrochloride solution,p-toluenesulfonic acid solution,citric acid phosphate buffer solution and acetonitrile,defatted with n-hexane saturated with acetonitrile,extracted with dichloromethane,then purified by acidic alumina solid phase extraction column,Eluent was concentrated under nitrogen blowing,diluted to certain volume and determined.The targets were separated by CAPCELL PAK C₁₈ column and taking 2mmol/L aqueous ammonium acetate?containing 0.1%formic acid??acetonitrile?containing 0.1%formic acid?and methanol as mobile phase.The determination of the analytes was performed by LC-MS/MS in selected reaction monitoring?SRM?mode,and the external standard method was used for quantitation.It showed a good linearity range of from 1.0?g/kg to 100?g/kg with the correlation coefficient R²>0.991.The limit of detection?LOD,S/N?3?was 1.0?g/kg,and the limit of quantitation?LOQ,S/N?10?was 3.0?g/kg.At the spiked level of 3.0,10.0,and 25.0?g/kg in aquatic products,the average recoveries of methylene blue,brilliant green,Azure A,Azure B between 70.0%and 110%,the average recoveries of Azure C ranged from 60.0%to100%,intra-batch relative standard deviation?15%and inter-assay relative deviation?15%,respectively.The method has a high sensitivity and accuracy,and can be used for the determination of methylene blue,brilliant green and its metabolites residues in aquatic products effectively.?2?The metabolic elimination of brilliant green in crucian carp was studied.The pharmacokinetic characteristics and metabolic elimination of brilliant green were studied,After the crucian carp was exposed to brilliant green bath water of 200 ng/mL for 2 h at a water temperature varying between 18²2?.The concentrations of brilliant green in muscle,liver,kidney and blood of crucian carp were determined by liquid chromatography-tandem mass spectrometry?LC-MS/MS?,and the concentration time data was processed with pharmacokinetic software DAS 3.0.5,and the data were analyzed with non-atrioventricular model parameters.In this study,the chromatographic time of the leuco brilliant green standard solution and the leuco brilliant green in the muscles at 0 h and 4 h after the crucian carp was exposed to brilliant green bath water was consistent and the relative abundance of the characteristic ion fragments in the mass spectrometer satisfies the qualitative determination standard.It is verified that the brilliant green produces metabolites leuco brilliant green in crucian carp.In addition,the results showed that brilliant green was well absorbed,permeated and distributed in the crucian carp tissues;The concentration of brilliant green was 270.9?g/kg and 262.5?g/kg in liver and kidney,which indicated that brilliant green was easier to be accumulated in liver and kidney.The elimination half-life of brilliant green in the blood was the smallest,with a value of 23.3 h,and the total clearance rate in blood was higher than that in the other three tissues,which indicated that brilliant green was easier to be eliminated in blood.The leuco brilliant green was the highest in the liver and kidney,and the lowest in the blood,and was not detected in the blood at 48 h after the crucian carp was exposed to brilliant green bath water.The liver was not detected at 40 d,but at 40 d,the residual contents of leuco brilliant green was 6.7?g/kg?21.7?g/kg in muscle and kidney of crucian carp,which showed it was not easier to be eliminated in the kidney;The distribution data of leuco brilliant green showed a downward trend in various tissues,but there was a multi-peak phenomenon.It may be that leuco brilliant green was redistributed and accumulated in some tissues after being metabolized in crucian carp.In this study,the metabolic characteristics of brilliant green and leuco brilliant green in crucian carp were discussed,and it can also provide theoretical basis and data support for fishery culture regulatory authorities to better guide and supervise the drug use of farmers.