Establish And Application Of Mass Spectrometry Method For Analyzing Biotoxins In Food

Biotoxins are toxic substance secreted,metabolized or semi-biosynthesized by the organism itself.They are highly toxic and often appear in the daily diet.Exposure to these toxins can cause chronic or acute poisoning in animals and humans Therefore,controlling mycotoxins in food is of vital importance to human health.In this study,sample preparation method for different food for the detection of mycotoxins and liquid chromatography combined with electrospray ionization tandem mass spectrometry(LC-MS/MS)method for the detection of mycotoxins were established.In addition,this work we used gas chromatography tandem mass spectrometry(GC-MS)to analyze metabolites of clostridium botulinum,aims to find new detection method to identify different classifications of clostridium botulinum.Methods: Using peanut as a representative of solid or semi-solid food matrix,vegetable oil as a representative of liquid food with high fat,we established sample preparation method for mycotoxins in food.It is essential that we can extract a variety of mycotoxins from a complex food matrix,to this end,accelerated solvent extraction and solid phase extraction were used for solid,semi-solid food matrix.For the mainly impurity of liquid food with high fat is large molecule lipid,gel permeation chromatography is used for extraction and purification.Furthermore,we also established method to simultaneously identify and quantify ten mycotoxins in food using high performance liquid chromatography combined with electrospray ionization tandem mass spectrometry.In addition,we used gas chromatography-mass spectrometry-based metabonomics analysis to discover differential metabolites of clostridium botulinum.Result: 1)Accelerated solvent extraction: in the process of sample filling,sample evenly mixed with diatomite can improve the efficiency of extraction.Four factors three levels experiment table was used to obtain the optimal factors for ten mycotoxins,including acetonitrile-water(80:20,V:V)as reagent,five minutes as extraction time,100? as extraction temperature and one time as cycle.2)Solid phase extraction: most target can be successfully purified by solid phase extraction using C18 column after optimization,however,aflatoxin G2 extraction were greatly influenced by the matrix.We compared several solid phase extraction columns,and selected VRA-1 column as final condition.It can reduce the matrix effect and reach the requirement of recovery.3)Gel permeation chromatography: mobile phase: ethyl acetate and cyclohexane(50:50,V:V),flow rate: 5 ml/min,collection time: from 15 minute to 40 minute.4)liquid chromatography tandem mass spectrometry: gradient elution with 0.1% ammonia-water-acetonitrile was used as mobile phase.Separation and quantification were performed in both positive and negative modes under multiple reaction monitoring(MRM)in a single run.5)gas chromatography tandem mass spectrometry: the GC-MS data were subjected to analysis of principle component analysis(PCA),partial least squares-discriminant analysis(PLS-DA)and variance analysis(ANOVA).11 differential metabolites between Clostridium A and Clostridium B were identified;2 differential metabolites were identified between E.coli WT and E.coli with botulinum toxin.Conclusion: This work established sample preparation method for the simultaneous detection of ten mycotoxins from food and HPLC-MS/MS method.At present,the European Union,Japan and some other countries have clear requirements for the total level of aflatoxins.However,in China,the national standard of mycotoxins in oil food is only for aflatoxin B1.The sample preparation method we established is suitable for various food matrices,especially for mycotoxin extraction and purification from high fat food.This work used GC-MS-based metabonomics to identify differential metabolites of different serotypes clostridium botulinum and those of E.coli with or without botulinum toxin type A.This work had reference value for the classification of bacterial toxins in contaminated food.It is possible to turn into a new method for detection and identification of specific clostridium botulinum in contaminated foods.