Study On Establishment Of Simultaneous Detection Method And Combined Cytotoxic Effects Of 4 Kinds Of Mycotoxins In Grain

Part one A novel method for the simultaneous determination of four mycotoxins in grainObjective:The objective of this study was to develop a simple and reliable HPLC method for the simultaneous extraction,separation and detection of AFG1,FB1 OTA,and S T in grain crops,and to provide the basis for the quality safety control.Method:A new double pump-three access high-performance liquid chromatography method for simultaneous determination of four mycotoxins in foods with online derivation technique and coupled detector technique was established.Each analysis run contains two analytical methods.Method one:the sample was automatically injected without online derivation,analysis was made with coupled detector technique after separation by chromatographic column.The quantitative detection of ST was completed in diode-array detector(DAD).AFG1 and OTA were quantified in FLD.Method two,samples underwent derivatization procedure,and were then transfered to C18 column before finally analyzed by FLD.The simultaneous analysis of four toxins was completed by the latter method.Upon completion of the development of detection methods,four mycotoxins were simultaneously extracted in grain samples after being grinded for further studies on mycotoxin toxicity by using the QuEChERS package.Extraction solutions were analyzed using the above method after purification by PRiME HLB SPE solid phase extraction column.Results:The detection of FB1 demonstrated excellent linear correlation within a concentration range of 0.10-5.00 ug/ml(Y=1.00×106X-158792;R2=0.9981),the detection limit was 0.03 ?g/ml.The recovery of FB1 ranged from 69.10%to 81.70%with a relative standard deviation 1.03%.The key indicators of the remaining three toxins are as follows:AFG1 had a linear range of 12.50-400 ng/ml(Y=1259.40Y-860.59;R20=1.0000)and the limit of detection was 1.90 ng/ml.The average recoveries of AFG1 was 71.00%-72.90%and the RSD was 0.60%.OTA(Y=4970.60X-20651;R2=0.9989)and ST(Y=0.08X-0.11;R2=1.0000)had the same linear range 15.60-500 ng/ml.The limit of detection were 0.48 ng/ml and 1.50 ng/ml,respectively.The average recoveries of the two mycotoxins were 82.40%-84.40%and 64.00%-65.60%,and the RSD were 3.10%and 0.23%,respectively.The method was successfully applied to the detection of eleven food samples purchased in Cixian,Handan,and no toxins were detected.Conclusion:The method realized simultaneous quantification of the four mycotoxins with advantages of accurate,sensitive,rapid and simple.It was successfully applied to the determination of these mycotoxins in eleven cereal samples and the results were satisfactory.Part two Study on combined toxicity and mechanism of four kinds of mycotoxins on BRL cellsObjective:To evaluate the combined toxicity effect of the four mycotoxins,investigate whether they can affect the proliferation and apoptosis of BRL cells,utilizing the cell biology method.Method:Firstly,different concentrations of AFG1,OTA,FB1 and ST were incubated with BRL for 48 h respectively.The cytotoxicity of four mycotoxins on BRL was measured using Cell Counting Kit-8.The combined toxicity were analyzed based on dosage-effect curve establishment and Design-Expert 8.0.6 software where central composite design was run.Further Hochest 33258 and flow cytometry were employed to test apoptosis-related indexes to explore possible combined toxicity mechanism between the toxins.Results:Cytotoxic effects were found on BRL cells after incubation with each of the four toxins,and the effect of reducing cell viability displayed a concentration dependent manner.The interaction model among the four toxins was then calculated,Y48(%)=-2774.96+343.61×ST+82.31×OTA+11.82×FB1+133.07×AFG1-0.51×OTA×FB1-274.70×ST2-2.27×OTA2-0.17×FB12-2.19×AFG12.OTA and FB1 showed moderate synergistic effects on BRL,with CIs 0.74,0.71 and 0.66.These two toxins synergistically inhibit the proliferation and induce apoptosis of BRL cells.Conclusion:Co-contaminated of the four toxins in food will produce greater hepatotoxicity than the effects of these toxins alone.We need to further study the interaction of mycotoxin toxicology,and assess the health risk of crop food contamination more accurately.