Research On Detection Method And Degradation Products Of Aflatoxins In Food

Aflatoxin（AFT）is highly related to human health,strict limitation standards were in force in different foods to protect our human-beings.Among the types of food contaminated by AFT,most food and product matrices are complex,and it’s an urgency to develop accurate and stable methods for the determination of AFT in contaminated foods,especially a high-throughput analysis for complex matrix samples.Before entering the"table",primary agricultural products need to undergo certain processing techniques,and acid-alkaline processing is a common food processing method.Under acid or alkaline processing conditions,AFT will undergo a certain degree of degradation.However,scientific issues such as degradation products and their toxicity have not yet been clarified.In order to solve the above-mentioned issues,we use immunoaffinity column and stable isotope labelled internal standard coupled with HPLC-MS/MS to develop a detection method,which possess high sensitivity,good accuracy and precision.In addition,this study explored the degradation of aflatoxin B₁(aflatoxin B_1,AFB₁)under acid or alkaline conditions based on high-resolution mass spectrometry.In vitro experiments including simulating gastric fluid environment,liver microsome metabolism and cell viability were designed to investigate the stability and toxicity of degradation products.This study is divided into two modules,and the specific content is summarized as follows:In the first part,a high-sensitive HPLC-MS/MS method was developed for the determination of aflatoxin B₁,B₂,G₁ and G₂ in bee honey and bee pollen samples.Acetonitrile/water（70:30,v/v）was used as extraction solvent and analytes were purified by immunoaffinity chromatography in sample pretreatment.The method was systematically verified.The detection limit of this method was 0.005μg/kg⁰.01μg/kg,and the limit of quantification was 0.01μg/kg⁰.02μg/kg.At low,media and high spiked concentration level,the average recovery was between 76.6%and 114.4%,the inter-day variation coefficient in the range of 3.8%¹3.8%,and the intra-day variation coefficient was 4.2%to14.3%.In addition,this method was applied to investigate the aflatoxin contamination status of bee products in China.The results showed that 5 positive samples were detected from 25bee pollen samples,of which AFB₁ was the most widely contaminated,with the highest content of 0.32μg/kg.In the second part,we use high-resolution mass spectrometry to figure out the degradation products of AFB₁ under acid and alkaline conditions and evaluate their toxicity in the next step.The results show that AFB₁ can degrade under acidic and alkaline conditions.It is inferred that a degradation product is generated under acidic conditions,the8,9-position double bond of the AFB₁ difuran ring is destroyed and formed a structure of enol and aldehyde on the AFB₁ core by its full MS and MS/MS spectra.There are two degradation products under alkaline conditions,one is the ring opening of the lactone bond of AFB₁ coumarin,forming exposed carboxyl and hydroxyl groups.The other structure is based on the ring-opening of the lactone bond,in which the exposed carboxyl group is cleaved to further generate a decarboxylation product.In order to characterize its toxicity,in vitro experiments from different angles were carried out,including conversion tests under gastric acid conditions,liver microsomal metabolism experiments and apoptosis experiments.Results show that no cytotoxicity of two degradation products.However,basic degradation product is a masked toxin because it can’t be stable in body and will be partially returned to the form of AFB₁ under gastric acid conditions,which will play a toxic role as before.On the other hand,the toxicity site of basic degradation product still exists that could be cause potential harm to human beings.The acid degradation product,remain stable in the gastric juice environment,lose its toxic site,is no cytotoxicity,can be regarded as the detoxification product of AFB₁.