Study On Detection Methods Of 18 ?-agonists Residues In Aquatic Products

The classes of ?-agonists possess similar chemical structure with different substituent groups on the phenyl ethanolamine,which are widely used to treat bronchial diseases.A series of animal experiments had shown that when the using dose of ?-agonists in livestock is more than 5~10 times of the recommended therapeutic dose,?-agonists have ability to enhance feed conversion and animal growth rates.Therefore,it result in the widely ?-agonists abusing in animal illegally breeding.?-agonists can be accumulated in animals and get into the body through the food chain.Humans consume animal tissues contained ?-agonists,which can cause acute poisoning and appear to be harmful to kidneys,liver and other tissues,with symptoms such as muscle tremors and palpitations.So it is necessary to develop the methods for detecting the ?-agonists in animal tissues to ensure the quality and safety of animal food.At present,the studies on detection methods of ?-agonists were mostly concentrated on livestock tissues,animal fur,urine,feeds and so on.Researches on the multi-residues of ?-agonists in aquatic products were few.These methods were not applied to all the banned ?-agonists or all the aquatic products.In order to achieve comprehensive supervision of ?-agonists in aquatic products and control the illegal abuse of ?-agonists,it is urgent to develop simultaneous detection methods for ?-agonists.In the study,salbutamol,ractopamine and tulobuterol were selected to prepare for positive samples according to eel administration experiment.The positive samples were used to study the existent morphology of 3 ?-agonists in eel muscles and extracting conditions of the ?-agonists for converting bound form to free form.The study provides a theoretical basis for the detection methods of ?-agonists residues in aquatic products.The simultaneous detection of multiple ?-agonists residues in aquatic products was carried out by the liquid chromatography-tandem mass spectrometry.Through the optimization of instrumental analysis conditions,the selection of extractants and the exploration of purificiction methods,the detection methods of 18 ?-agonists residues in aquatic products were established.The established method was simple,high sensitivity and widely application,which was as good technical support for detecting ?-agonists in aquatic products.The established methods were used to screen aquatic products in circulation field and breeding base to detect the ?-agonists residues.The main experimental results of the project are as follows:(1)The salbutamol,ractopamine and tulbuterol were prepared to positive samples by laboratory feeding.The 3 ?-agonists were presented as the bound form and free form in eel muscle.The proportion of the bound state of salbutamol,ractopamine and tulbuterol in the muscle was 79.1%,89.0% and 73.9%,respectively.(2)Hydrolysis conditions of extracting ?-agonists from positive samples were studied in order to transform ?-agonists morphology from bound form to free form.Acid hydrolysis and enzymatic hydrolysis as typical hydrolysis were selected.The acid hydrolysis optimization conditions include the temperature,time and concentration of hydrochloric acid.The enzymatic hydrolysis optimization conditions include temperature,time,p H of extracting solution and amount of the enzyme.Comparing the acid hydrolysis and enzymatic hydrolysis,the best hydrolysis conditions are follows: 50.0 ?L ?-glucuronidase/arylsulfatase enzyme were added to sample diluted by 0.2 mol/L ammonium acetate solution at 37? for 16 h.(3)The method for the detection of 18 ?-agonists residues in aquatic products was studied by using ultrahigh performance liquid chromatograph-tandem mass spectrometry.The best instrumental analysis conditions were follows: 5.0 mmol/L ammonium acetate aqueous solution(including 0.2% formic acid)– 0.1% formic acid acetonitrile was used as mobile phase gradient elution,18 ?-agonists were separated by using CAPCELL PAK MG? C18(2.0 mm I.D×100 mm,3 ?m)chromatographic column,detected by UPLC-MS/MS under selected reaction monitoring(SRM)with positive electrospray ionization(ESI+)and quantified by internal standard method.The limit of detection LOD(S/N?3)was ranged from 0.25 ?g/kg to 0.50 ?g/kg,and the limit of quantitation LOQ(S/N?10)was ranged from 0.50 ?g/kg to 1.00 ?g/kg.It showed a good linearity relationship in the ?-agonists concentration range of 0.20~200.00 ?g/L with the correlation coefficient R2>0.991.Besides,the average recoveries presented 70.0%~115.0% with the intra-batch precision less than 12.9%,and the inter-batch precision less than 13.5%.(4)47 lots of aquatic products from Shanghai,Jiangsu,Sichuan and Lasa breeding bases and circulation markets were screened ?-agonists by the established methods.The results showed that 3 aquatic products contained ?-agonists.The detection rate was 6.3%.Grass carps contained 0.38 ?g/kg of ractopamine.Large yellow croaker contained the contention of clenbuterol and clenpenterol was 1.26 ?g/kg and 0.29 ?g/kg,respectively.The eels contained the contention of ractopamine,salbutamol and tulobuterol was 2.10 ?g/kg,3.00 ?g/kg and 2.90 ?g/kg,respectively.Therefore,it was necessary to monitor ?-agonists residues in aquatic products o ensure the quality and safety.