Study On The Preparation Of Reference Material And Detection Technology For Aflatoxins

Aflatoxins（AFs）are the most severely contaminated mycotoxins in grains such as maize,rice,and peanuts,as well as related foods and feed.AFs are widely recognized as strong teratogens and carcinogens,and pose significant safety risks to human health when entering the food chain.The detection,monitoring,risk assessment,and toxicological metabolism research of AFs are important measures to ensure food safety and reduce human health risks.Among them,reference materials,as the“ruler”in the analysis and measurement,are the key reagents for the management and control of food safety,and the basis for ensuring the accuracy,traceability,and reliability of detection results.Meanwhile,the accuracy of AFs detection results is seriously affected due to the low content of AFs and the complexity and diversity of food matrices.In this study,the preparation of AFs reference materials and the application of novel nanomaterials in AFs detection were taken as the entry points.Firstly,the preparation technology of reference material integrating toxin-production,purification,and characterization was established.Furthermore,MOFs nanomaterials were synthesized and used as the adsorbent during dispersed solid phase extraction for the enrichment and purification of trace AFs in cereal and dairy cow biological samples,which could provide technical support for the development of mycotoxin certified reference materials in China,especially the purity reference materials,and eventually achieve the accuracy,traceability,and comparability of measurement results.The main contents are listed as follows:（1）17 AFs-produced strains were isolated and purified from naturally polluted maize,wheat,and peanut samples using streak plate method.Morphological observation and molecular biology identified that 16 strains were Aspergillus flavus and 1 strain was Aspergillus parasiticus.The AFs production of different strains in maize,rice,wheat,and peanuts was determined using UPLC-MS/MS method.The results showed that the AFB1 yield was highest in maize by AF0109,reaching 186.34 mg kg^-1,and at the same time a small amount of AFB2 and AFM1 were produced.Using AF0109 as the AFs-producing strain,the conditions of producing AFs including water activity,culture temperature,and culture time were systematically optimized.Based on single factor experiments,a response surface methodology was used to establish a model and predict the optimal conditions for producing AFB1.The final culture conditions of culture medium,water activity,culture temperature,and culture time for producing AFs are maize medium,0.96,28°C,and 29 days,providing a raw material basis for the subsequent preparation of AFB1 reference material.（2）The high AFs yield strain（AF0109）was inoculated on the maize culture medium to obtain the high concentrated AFB1 raw material.After extraction and concentration,it was purified by Sephadex LH20 gel column chromatography,and then separated and purified in two steps by the preparation high pressure liquid chromatography（Pre-HPLC）to obtain the AFB1 candidate with purity>99%.Qualitative identification of the candidate was performed using ultraviolet spectrum（UV）,infrared spectroscopy（IR）,high resolution mass spectrum（HRMS）,and nuclear magnetic resonance（NMR）to confirm its identity as AFB1.By using two methods with different principles,namely mass balance method（MB）and quantitative nuclear magnetic resonance method（q NMR）,the purity of the reference material candidate was accurately determined,and the final purity was 99.5%.The developed AFB1 purity reference material has good homogeneity and meets stability requirements for more than 6 months.A comprehensive and systematic evaluation of various uncertainty components during the development process resulted in an extended uncertainty of 0.6%.This study combines biological fermentation with Pre-HPLC preparation to solve the technical problem of obtaining high-purity AFB1 from the origin,which could obtain a large number of high-purity and accurate AFB1 purity reference material through high-throughput preparation.（3）The maize matrix sample of high concentrated AFB1 raw material was obtained by inoculating the AFs-producing strain（AF0101）into maize medium.After crushing and sieving,it was mixed with the blank maize flour according to the principle of equal proportion dilution,and the reference material candidate for AFB1 in maize flour was obtained.Seven laboratories were organized to carry out the combined characterization by stable isotope dilution mass spectrometry.Then,the uncertainty components during the development process were comprehensively and systematically evaluated.The certified value of AFB1 reference material in maize flour is 12.7±1.2μg kg^-1,with good homogeneity and stability for more than 6 months.This reference material can be used for the qualitative and quantitative detection of AFB1 in maize flour and evaluation of AFB1detection methods,providing technical support and material guarantee for the food safety detection and establishment of monitoring traceability system.（4）MOF-808 nanomaterials were prepared by solvothermal method,and their microstructure and functional groups were characterized.The synthesized MOF-808 with a stable structure and good performance was used as adsorbent of dispersive solid-phase extraction（d SPE）for the adsorption and purification of impurities in the extraction solution of grain samples.The optimal amount of MOF-808 was 20 mg.The types and volumes of redissolved solvents are acetonitrile-water（30:70,v/v）and 1m L,respectively.The ultrasonic dispersion and vortex adsorption times are2 minutes and 5 minutes,respectively.This study developed a one-step purification method using MOF-808 as an impurity adsorbent,which could be used for accurate and quick detection of trace amounts of AFs in grain samples.（5）MIL-101（Cr）@Fe₃O₄ nanocomposite was synthesized by hydrothermal method,and their microstructure,functional groups,magnetic strength,and specific surface area were characterized.MIL-101（Cr）@Fe₃O₄,possessing the large specific surface area,strong adsorption capacity,and good magnetic properties,was used as adsorbent of magnetic solid phase extraction（M-d SPE）for the adsorption and purification of impurities in the extraction of cow biological samples.The optimal amount of MIL-101（Cr）@Fe₃O₄was 5 mg.The types and volumes of redissolved solvents are acetonitrile-water（2:98,v/v）and 1 m L,respectively.The ultrasonic dispersion and vortex adsorption times are both5 min.The types and volumes of eluting solvents are 1%formic acid acetonitrile and 1 m L,respectively.The p H of the sample solution is 6.These results indicate that MIL-101（Cr）@Fe₃O₄ could be selected as the M-d SPE adsorbent due to its food purification effect and high sensitivity,meeting the requirements for accurate detection of trace AFs in cow biological samples.