Synthesis And Application Of Deuterated Furazolidone Metabolite-AOZ-d₄

Furazolidone is a kind of broad-spectrum antimicrobial drug,of which metabolite is strongly carcinogenic and thus has been prohibited in the EU,the United States,Japan and many other countries.In China,the Ministry of Agriculture has also stipulated that the drug and its metabolite cannot be detected out in any agricultural products.Therefore,it is necessary to develop isotope-labeled furazolidone metabolites as an internal standard,which can be added to detect metabolites of furazolidone in agro-products in the method of LC-MS/MS.AOZ-d4 is one of labeled isotope for furazolidone metabolites.However,currently it strongly depends on imports in high cost.Therefore,studying the synthesis of isotope compounds helps to change the long-term dependence on foreign isotopes.This study contains the following four parts:(1)The synthesis of deuterated furazolidone metabolite-AOZ-d4.The synthesis route of this project stated with deuterated acetic acid and synthesize deuterated furazolidone metabolites in five steps.First,deuterated acetic acid brominate with bromine to give deuterated bromoacetic acid(intermediate 1);The intermediate 1 reacted with benzyl alcohol to furnish deuterated benzyl bromoacetate(intermediate 2)by esterification;The intermediate 2 is reduced to deuterated bromoethanol(intermediate 3)by deuterated sodium borohydride;The intermediate 3 is substituted by hydrazine hydrate to provide deuterated 2-hydroxyethylhydrazine(intermediate 4);Finally,the intermediate 4 reacts with diethyl carbonate to yield the desired final product.(2)Isolation and purification of the deuterated furazolidone metabolite-AOZ-d4.The separation and purification of each step in the reaction process were carried out using a variety of methods(e.g.,silica gel chromatography,liquid-liquid extraction,etc.).First,the intermediate 2 was separated and purified by silica gel flash column,by using n-hexane and ethyl acetate(V:V=25:1)as eluant.The intermediate product 3 was washed in sand core funnel to obtain a higher purity product by using chloroform as the filter reagent.Subsequently,the crude intermediate product 3 was separated and purified with a sephadex gel column in which a solution of methanol and methylene chloride(V:V=1:1)was used as the eluent to obtain a higher purity compound.The deuterated furazolidone metabolites were separated and purified by silica gel column chromatography which a solution of methanol and methylene chloride(V:V=1:30)were used as the elution solvent to obtain the final product in high purity.(3)Structural characterization of the deuterated furazolidone metabolite-AOZ-d4.The reaction products were identified by nuclear magnetic resonance(NMR)and mass spectrometer at each step of the test.The carbon skeleton of each step was determined by nuclear magnetic resonance spectroscopy,and the molecular weight of compounds was determined by a high resolution mass spectrometer.The purity of the final product was determined to be over 95%by quantification NMR techenics.(4)Detection in real samples.A fluorescence method was proposed to to detect the furazolidone metabolites in the chicken,since the reaction rate of 2-nitrobenzaldehyde with AOZ was efficient.The detection limit was 0.5?g/L and the limit of quantification was 1.0?g/L.In conclusion,we developed synthetic method of AOZ-d4 which was applied to the detection of drug prohibition in food sample.This study helps to improve the synthetic method of isotopes and the status of strong dependence on foreign standard isotopes.