The Synthesis Of Phenylethanolamine A And The Development Of Its Purity Reference Material

Residues and abuse of forbidden drugs is main problem in the quality and safety of animal products at home and abroad.?-agonists as one of the banned drugs in China has been listed in routine monitoring.Phenylethanolamine A is a new type of?-agonists,commonly known as"clenbuterol".The drug residue can be transferred to human by food chains,which would lead to a great threaten to human,such as nausea,dizziness,limb weakness,hand tremor and other symptoms of poisoning.Long-term consumption may lead to mutations in the body's chromosomes,induced malignant tumors,etc.Due to potential hazard to human body,the Ministry of Agriculture has forbidden the use of?-agonists in animal feed and water by the 1519 Announcement,and also issued a serial of testing standards and specifications to detect the use of phenylethanolamine A.In order to guarantee the routine monitoring of phenylethanolamine A,the certified reference material?CRM?of phenylethanolamine A is highly necessary and important.CRM can ensure the reliability,comparability and traceability of the analytical data,and realize the mutual recognition of the laboratory results.However,phenylethanolamine A CRM is not available in China.In the meantime,high purity phenylethanolamine A raw material for CRM is expensive and time-consuming to purchase from abroad.And its can not be purchased from domestic.Therefore,it has a great significance to carry out the synthesis of phenylethanolamine A and to develop the Certified reference materials.This study contains three main parts:1.The synthesis of phenylethanolamine A.The whole structure of phenylethanolamine A was supposed to be devided into two fragments:the amine fragment and the bromoacetophenone fragment.The synthesis of amine fragment was initiated with 4-nitrobenzylbromide,which underwent the dehydrogenation with ethyl acetoacetate under condition of sodium ethoxide in THF to afford the intermediate.The intermediate was subsequently hydrolyzed with hydrochloric acid and acetic acid to furnish 4-?4-nitrophenyl?butan-2-one which was applied for Leuckart reaction in two steps:heating formamide and formic acid at 120?,hydrolysis by hydrochloric acid.The bromoacetophenone fragment could be achieved from bromination of the commercially available 4-methoxyacetophenone with bromine.The S_N2 reaction was supposed to be a critical method to combine two fragments together and then reduction with KBH₄ was the final step to obtain phenylethanolamine A.In order to further promote the synthesis yield,the reaction conditions including reaction solvent,temperature and time,were optimized.On the basis of above method,an improved and convenient synthesis route was employed with Mannich reaction of amine fragment and 2-?4-methoxyphenyl?-2-oxoacetaldehyde replace the S_N2 reaction,which could be prepared from 4-methoxyacetophenone in HBr/DMSO system.2.Isolation and purification of phenylethanolamine A.A series of approaches,such as silica gel?200-300 mesh?,sephadex gel?LH-20?,reversed-phase C18?90??and anion exchange?D900?chromatography method,were applied with the isolation and purification of phenylethanolamine A.The isolation condition was established on silica gel column chromatography in which the eluent compositions were successive n-hexane eluent and ethyl acetate?V:V=3:1?,dichloromethane and acetone?V:V=5:1?,dichloromethane and acetone?V:V=3:1?and dichloromethane and methanol?V:V=3:1?,and all eluents were added 0.1%triethylamine in volume ratio to reduce adsorption effect of silica gel.After above isolation,the purity of phenylethanolamine A reached 60%,firstly.Further purification was achieved by reverse phase silica gel in condition of methanol and water?V:V=1:1,3:2and 7:3?to yield a high purity of phenylethanolamine A?99.2%?.3.Research on Phenylethanolamine A CRM.phenylethanolamine A was mixed sufficiently to ensure the homogeneity.The raw materials was characterized by high-resolution mass spectrometry and nuclear magnetic resonance instrument.In accordance with the principle of ISO Guide 34 and 35 and Chinese Technical Norm of Primary Reference Material JJF 1006-1994,approaches of the mass balance method and quantitative nuclear magnetic resonance method was established.The purity of phenylethanolamine A was determined by these two methods.The results were 98.3%and 99.0%,respectively.