Establishment And Application Of Furosine Rapid Detection Method Based On UPLC

In order to ensure the safety of food,milk before entering the market must be subject to heat treatments.The heat treatment of milk has great influences on the flavor formation,p Hysical and chemical properties and nutritional value of milk.In addition,transportion and storage of milk will also affect the biological active substances in milk and other nutrients.In addition,storage and heat treatment process will lead to a large number of biologically active substances losing their activity,and some nutrients losing their nutritional value.The furosine content are affected by many factors.The three main factors are the heating temperature and heating time,the storage temperature and time,and the p Hysical and chemical properties of milk.Therefore,development of a more efficient,simple and accurate method for the detection of furosine,study of the influencial factors of the content of furosine has essential significances.Based on HPLC,UPLC and UPLC-MS/MS equipments,this experiment developed a high efficient method for detection of furosine.In addition,storage stability and effects of concentration of hydrochloric acid on the furosine standards were investigated,to find out the potential influential factors that affect the final results of furosine standards.What's more,different heat treatments,storage and transportation conditions were set to study the study the heat stability,storage stability and transportation stability of furosine in milk samples.Experiment 1: In this paper,the method based on HPLC,UPLC and UPLCMS/MS was developed.The main procedures of the UPLC method were as follows: 2 m L milk and 1 m L solution of 6 mol/L HCl were added to the hydrolysis tube.The hydrolysis tube is kept isothermally at 110? for at least 12 hours.After hydrolysis,hydrolysed milk solution was filtered using ordinary filter paper.The filtrate was then diluted 6 times using 0.6% sodium hydroxide solution.Finally,ultra high performance liquid chromatograp Hy(UPLC)was used to detect furosine at the wavelength of 280 nm.The mobile p Hase was 0.6% ammonium acetate and 0.1% trifluoroacetic acid isocratic elution.Flow rate was 0.4 m L/min and the column temperature is at 40?.Results showed that method developed in this study can not only shorten the testing time,simplify the pretreatment process,improve the detection efficiency,reduce the corrosion damage to the experimental apparatus,but also led to good repeatability.The intra-day precision and inter-day precision were 2% and 1.14%.The recovery rate of the method ranging from 91.4% to 102.3%.Experiment 2: This study investigated whether furosine standard can maintain its structure and characteristics at different temperatures and different storage time conditions,by comparing the peak area of furosine standard under the different temperature and different storage time.Results showed that the furosine standards solution was stable under the acid solution.Experiment 3: Effects of the concentration of hydrochloric acid on the quantitative detection of furosine was studied in this part.Results showed that the molar concentration of hydrochloric acid has great influence on the quantitative determination of furosine.In the range of 1~9 mol/L of hydrochloric acid,with the increase of hydrochloric acid concentration,furosine content increased gradually,and showed a linear relationship.Under the condition of 9~11mol/L hydrochloric acid concentration,the content of furosine tends to be stable,which may be due to the fact that the concentration of hydrochloric acid at 9 mol/L and above can make the furosine completely free.Finally,in this paper,the same concentration of hydrochloric acid was used to hydrolyze milk,the results of the quantitative detection of furosine are reproducible.When the concentration of hydrochloric acid in hydrolyzed milk was greater than or equal to 5,there was no statistical difference between the results of the three test.Experiment 4: Effects of storage and transportation conditions,and heat treatment temperature on the content of furosine in milk were studied.And the correlation between the content of furosine and lactulose,vitamin A and vitamin C was also analysed.Firstly,it was found that with the increasing of the processing temperature,the growth rate of the furosine increased gradually.When the processing temperature is lower than 100?,the growth rate is relatively slow.When the processing temperature is higher than 100?,the content of furosine increases dramatically.Secondly,furosine in both home and abroad brand milk was determined.Results showed that furosine in domestic and abroad brand UHT milk and pasteurized milk in average is relatively consistent.Then,we studied the statistical relationship between the content of furosine and the content of lactulose.The results showed that there was a statistical correlation between the content of furosine and lactulose,the correlation coefficient was 0.85.Finally,the statistical relationship between furosine content and vitamin A,vitamin E content was evaluated.The study found that furosine content was not correlated with vitamin A and vitamin E,which may be due to that vitamin A and vitamin E are with good thermal stability.