Development And Application Of Stable Isotope Internal Standard Reagent For Food Safety Testing

In recent years,the food safety incident occurs frequently,which not only causes great harms to the general people’s health and life safety,but also damages our international image and export trade.Therefore,it is important to quickly and accurately establish food safety testing methods and prepare reagents for testing.Isotope dilution mass spectrometry is not susceptible to interference from the complex matrix of the sample to be measured,and can accurately determine,eliminate detection instrument response errors,and improve the quantitative accuracy of tests and is now commonly used in food safety field.The internal standard reagents used in the field of food safety testing in China are basically monopolized by foreign companies such as Sigama,Dr.E,LGC,etc.Foreign countries rarely disclose their preparation methods for reasons such as technical confidentiality and product monopoly.In the above context,this paper aims to develop stable isotope labeling reagents for food safety testing urgently needed in China.In this paper,the preparation method of deuterated reagents such as plasticizer residues,clenbuterol residues,pyrethroid pesticide residues and dairy allergens is explored.The two typical internal standard reagents obtained were studied in practical application in food.The research includes:（1）Preparation of deuterium labeled phthalate plasticizer internal standard reagent.The key intermediate phthalate-D₄ was obtained by selective oxidation of the basic reagent o-xylene-D₁₀,and the different oxidants,oxidation time,co-solvent types and other conditions were discussed to study the effects on the yield and abundance of phthalate-D₄.Relatively optimal process conditions are determined.Subsequently,with phthalate-D₄as raw material,and corresponding alcohol under the catalytic action of condensation agents,sixteen deuterated plasticizer internal standard compounds were prepared.Choosing typical dimethyl phthalate-D₄ as an example,the conditions such as the type of condensate,the molar ratio of the reactants,and the reaction time were explored,and the effects on the yield and abundance of phthalates were studied,and the relatively optimal process conditions were determined.The structure and purity of the compounds of the 16 stable isotope labeling reagents synthesized were characterized by Fourier transform infrared spectroscopy,nuclear magnetism,liquid phase,mass spectrometry.The chemical purity of the compounds is more than 99.0%and isotope abundance is more than 99.5atom%D.（2）Preparation of deuterium labeled pyrethroid internal standard reagents.Taking phenol-D₆as the labeling precursor,the dehydration reaction was given to sodium phenol-D₅,and then the important intermediate m-phenoxybenzaldehyde-D₅was obtained by the reaction of sodium phenol-D₅ with m-bromobenzaldehyde under the catalysis of cuprous chloride.The reaction time,temperature,catalyst,etc.were investigated to obtain the optimal synthesis process of m-phenoxybenzaldehyde-D₅.The home-made synthesis of m-phenoxybenzaldehyde-D₅ was used as an intermediate,and the next step of the reaction with bromocyanate chloride and sodium cyanide was rapidly prepared under the action of the phase transfer catalyst to obtain cypermethrin-D₅.The interrelationship of cypermethrin-D₅ yields and the catalyst addition amount,reaction time,sodium cyanide concentration and so on was discussed.The optimal process of cypermethrin-D₅ was finally determined.Using the same process,cypermethrin-D₅,cypermethrin-D₅ and kung fumethrin-D₅ of deuterium marking products were prepared.The synthetic five deuterium-labeled pyrethroids were tested for product purity and abundance by Fourier transform infrared spectroscopy,nuclear magnetism,liquid phase,mass spectrometry,etc.,and the compound was determined to be the target product,with chemical purity of more than 98%and isotope abundance of more than 98atom%D.（3）Preparation of internal standard reagents for deuterium label allergen detection.Alkylation reaction with diphenylmethylene aminoacetonitrile and sodium hydroxide and bromoisopropane-D₆ under the action of a phase transfer catalyst was used to obtain valine intermediate-D₆.The relatively optimal synthesis conditions were explored that affected the yield of the compound.Valine-D₆ was obtained after hydrolysis of valine intermediate-D₆.The optimized synthesis process of valine intermediates-D₆ and valine-D₆ was applied to alanine-D₃ and phenylalanine-D₅ and leucine-D₆.For the four kinds of deuterium-labeled amino acid reagents synthesized,Fourier transform infrared spectroscopy,nuclear magnetism,liquid phase,mass spectrometry,etc.were tested for product purity and abundance.and the compounds were determined to be the target products with chemical purity of more than 98%and isotope abundance of more than 99.0atom%D.Then,using the obtained leucine-D₆ as raw material,the solid phase synthesis method was used,and the dairy detection characteristic peptide IDAL*NENK-D₆ was synthesized.The optimal synthesis conditions were explored.The structure and purity of the compound were characterized by liquid phase,mass spectrometry and other means of the synthesized stable isotope labeled peptide,and the compound was determined to be the target product,with chemical purity of more than 98%and isotope abundance of more than 99.0atom%D.（4）Preparation of serial deuterium labelingβ-receptor agonist internal standard reagents.3,5-N,N-dimethylcarbamoyloxyacetophenone was used as the starting material,and after bromination reaction,the Bambuterol-D₉ brominated intermediate was obtained.The correlation of yields of bambuterol-D₉bromide intermediates and the types of brominating agents,reaction solvents,material ratios were discussed,and the optimal synthesis process of Bambuterol-D₉ brominated intermediates was finally determined.Bambuterol-D₉ bromide intermediate was reduced to obtain bambuterol-D₉ reducing intermediate,and then an amination reaction with the labeled precursor tert-butylamine-D₉ was used to obtain the target product Bambuterol-D₉.The correlation between the reaction mode,the type of binder,the molar ratio of the reactants and the yield of bambuterol-D₉ was discussed,and the optimal preparation process of bambuterol-D₉ was finally determined.The optimal synthesis process of Bambuterol-D₉ intermediates and Bambuterol-D₉ was applied to synthesize Terbutaline-D₉,Oscinalin-D₆,and cloprenaline-D₆ ofβreceptor agonists with similar structure.The structure and purity of the compound were characterized by nuclear magnetism,liquid phase,mass spectrometry and other means of the synthesized four stable isotope label internal standard reagents.The chemical purity of compounds is more than 98%and the isotope abundance of compounds is more than 99.0atom%D.（5）Established an accurate quantitative analysis method for six kinds of plasticizers such as DMP,DEP and DBP in bottled drinking water.The addition of the homemade isotope internal standard reagent before the sample is processed fully guarantees the specificity and accuracy of the detection method.Through experimental data such as precision and scaling recovery rate,it is found that isotope dilution mass spectrometry is significantly better than external standard method in terms of scaling recovery,linearity of regression curve and accuracy.The standard-up recovery rate of the external standard method is only between 10.8-82.7%,but the standard-up recovery rate of the isotope internal standard method is between 85.22-103.92%,the standard-up recovery rate is good,the detection limit is between 0.06-0.33μg/L,the quantitative limit is between 0.20-1.11μg/L.The isotope internal standard method is the preferred method for rapid and accurate detection of plasticizers in commercially available bottled drinking water.（6）The accurate quantitative detection method of the milkβ-lactoglobulin was established.The addition of the homemade isotope internal standard reagent before sample processing,fully guarantees the specificity and accuracy of the test method.The univariate and orthogonal tests were used to optimize and establish the optimal digestion conditions.Comparing the precision and standard recovery results of the internal standard method and the external standard method,it is easy to see that the isotope dilution mass spectrometry method is obviously superior to the external standard method in terms of the standard recovery rate,linearity or accuracy of the regression curve,and for dairy products with complex matrix,the isotope dilution mass spectrometry method has an incomparable advantage in reducing the matrix effect.The use of the isotopic internal standard method instead of the external standard method has significantly improved the intraday precision and the daytime precision（RSD<8%）,the scaling recovery rate of the method is 90%-105%,the detection limit is 0.3 mg/100 m L,and quantitative limit is 0.8 mg/100 m L.The created isotopic internal standard method is applied to five commercially available dairy products quantitative detection ofβ-lactoglobulin.Liquid phase-Isotope dilution mass spectrometry method is with high accuracy,convenient and operability.