Study On The Analytical Methods Of Azodicarbonamide And Semicarbazide In Flour And Flour Products And The Degradation Rule Of Azodicarbonamide

Azodicarbonamide（ADC or ADA）, was used as a blowing agent inrubber products previously，since it has the effect of bleaching, oxidation,and strengthen the gluten obviously, as well as the advantages of highefficiency and low price, it is substituted for potassium bromate as thedough improver and brightening agent in the United States, Canada, Chinaand other certain countries.ADC itself does not work with the flour, but when add water to theflour and form as a dough, ADC will soon release the active oxygen, thenthese sulfhydryl (-SH) containing in wheat protein is oxidized to disulfidelinkages（-S-S）, the protein chains connected to each other so thatconstituting the network structure, thereby improving the handlingproperties and the physical structure of the dough, significantly enhancedgluten strength as well. But recently, the studies found that ADC has a bigfood safety hazards as a flour additive. In the process of making flourproducts, ADC will degrade at high temperatures and high humidity treatment, generates biurea (BIU) and semicarbazide(SEM). Studies haveshown that, SEM was toxic to test animal’s uterus, ovaries, testis, thymus,spleen, thyroid, adrenal gland, pancreas, cartilage and other tissues andorgans. SEM is toxic to male and female reproductive systems of thesubject, thymus, spleen, thyroid, adrenal glands, pancreas, and cartilage andother tissues and organs. In some vitro and in vivo experiments testing alsoshowed embryo toxicity and mutagenic. This is definitely harm to peoplefor those who are long-term consumption of flour products containing SEM.Therefore, the European Union, Australia, Singapore and other regions andcountries prohibit ADC as a food additive for the processing of flour.Although China GB2760-2011"Food additives health standards"provisions that the maximum level of ADC used in wheat flour is45mg/kg,but has yet to develop an appropriate testing standards.In order to establish testing standards of ADC in flour, research thetransformation of ADC in flour and its products, to understand the currentsituation and the pollution level of SEM in flour products. This paperestablished a HPLC method for the determination of ADC in flour,developed a LC-MS/MS method for the determination of SEM in flour andits products. Using the LC-MS/MS method for the determination of ADCand the LC-MS/MS method for the determination of BIU in flour and flourproducts, which created by our research group. We studied thetransformation of ADC in flour and its products, the full text is divided into three parts.PartⅠ Determination of ADC in flour by HPLCObjective: To develop a HPLC method for the determination of ADC inflour. Methods: Samples were extracted with acetone, the extract wasconcentrated to dry, dissolved in0.5%K2HPO4(pH=8.4) solvent, anddegreased with n-hexane. using HPLC for the detection. The analyte wasseparated on a Agilent Eclipse XDB-C18column (150mm×4.6mm,5μm)using0.02%K2HPO4as mobile phase at a flow rate of0.5mL/min, thecolumn temperature was30℃, the wavelength was245nm and theinjection volumn was50μL. Result: Azodicarbonamide had a good linearrelationships in the range of1.0～90.0mg/kg, the correlation coefficientwas higher than0.99, the limit of detection was0.3mg/kg（S/N≥3）and thelimit of quantification was1.0mg/kg（S/N≥10）. Average recoveries ofADC at spiking levels of1.0,5.0,45.0mg/kg were78.0%～106.0%andrelative standard deviations were less than9.1%. Conclusion: Ananalytical method based on HPLC was developed for the determination ofADC in flour. The method is simple, rapid, accurate and reliable, it can beused for the determination of ADC in flour.Part Ⅱ Determination of SEM in flour and flour products byLC-MS/MSObjective: To develop a LC-MS/MS method for the determination of SEMin flour and flour products. Methods: Samples were hydrolyzed by0.1 mol/L hydrochloric acid and derivatized with2-nitrobenzaldehyde. Thenderivative was extracted with ethyl acetate, and the lipid was removed byliquid-liquid extraction using n-hexane. The target compound was detectedby LC-MS/MS and quantification was performed based on internalstandard. The analyte was separated on an Shimadzu VP-ODS（150mm×2.0mm，5.0μm）, eluted by gradient with mobile phase ofacetonitrile and water. The flow rate was0.3mL/min, the columntemperature was40℃and the injection volume was20μL. Conditions ofMS/MS: temperature:550.0℃； capillary voltage:5.5KV; curtain airpressure:310.3kPa； atomizing gas pressure:137.9kPa； collisionenergy(CE):68.95kPa；collision chamber entrance voltage:10V. Theanalyte detected by positive electrospray ionization tandem massspectrometry in the multiple reaction monitoring(MRM) mode. Result:Good linearity of SEM was achieved over the concentrations of0.5μg/L~100.0μg/L（R2≥0.9994）, the recoveries of SEM in flour, steamedbread and bread at the three spiking levels of0.5,1.0,5.0μg/kg were88.8%~99.4%, with relative standard deviations from4.8%to11.5%. Thelimit of quantification was0.5μg/kg. Conclusion: An analytical methodbased on LC-MS/MS was developed for SEM in flour and flour products.The method is accurate and sensitiveness, it can be used for thedetermination of SEM in flour and flour products. Part Ⅲ Study on the degradation of ADC in flour and flourproductsObjective: In order to further study the degradation of ADC in flour andflour products. Methods: Referring to the methods established by ourresearch group, HPLC-MS/MS methods were used for the analysis of ADCand BIU in samples, respectively. HPLC-MS/MS method established in thesecond part was used for the analysis of SEM. Add water to the blank andADC-spiked flour,then kneading to a dough. Determine the content of ADCat sampling points of various time(2,5,10,20,30,40,50min), analysis thedata. Make the blank and ADC-spiked flour into bread, determine thecontent of ADC, BIU and SEM at different stages and different parts of thebread.Steamed bread, noodle and friedcake were prepared by blank andADC-spiked flour, determine the content of BIU and SEM. Result: It isconclud that there was a linear correlation between the the reciprocal ofADC concentration and the reaction time, the linear equation is y=0.02975+0.0011x (y:1/c; x: time). The results showed that the sampleswere not detected in the blank during the process of making bread, but bothBIU and SEM were detected in the dough and flour products prepared byADC-spiked flour. When the ADC spiking level was20.0mg/kg, as thetreatment temperature rises, the content of SEM was increased in the rangeof160℃~210℃, which was500μg/kg at210℃. The results show that atthe same spiking level of ADC, the content of SEM in flour products indescending order is friedcake, bread, steam bread and noodle. In addition the greater of the added concentration of ADC, the higher of the residualamount of SEM. Conclusion: The reaction between ADC and flour issecondary reactions under the condition of water. It can be concluded thatSEM formation in bread making process is, ADC turning into BIU and alittle SEM in the stage of fermentation and the mixing, after baking at hightemperature, BIU turns into SEM and the concentration of SEM wasincreasd significantly. Temperature is an important factor in the ADCconversion, the higher of the treatment temperature and the greater of theresidual amount of SEM. Also, there was a direct and linear correlationbetween the level of ADC used in flour and the conctration of SEM.