| Mycotoxins,also known as mold(fungal)toxins,are naturally occurring toxic secondary metabolites of some fungi,which contaminate a wide range of cereals and their products during the crop planting,processing,transporting,and subsequent storage,posing a serious threat to the health of humans and animals.Mycotoxins do not usually occur singularly but as a mix since that the same fungus can produce different mycotoxins,while the same mycotoxin can also be produced by a variety of fungi.Nowadays many analytical methods used for mycotoxin detection are specific for one or a closely related group of them.Therefore,the validation and application of a method for the simultaneous determination of a large number of mycotoxins is of great significance for the related research of mycotoxin co-contamination.In this paper,10 group 25 mycotoxins,which are widely concerned,have been selected.A rapid,sensitive and reliable analytical method based on solid-phase extraction and liquid chromatography coupled to triple quadrupole mass spectrometry was successfully validated and applied for the determination of multi-mycotoxin occurrence in 104 typical cereal samples.The results are as follows:1.Development of a simultaneous determination method for the analysis of 25 mycotoxins(1)Optimization of LC-MS/MS conditionsSuitable liquid chromatography separation conditions,including mobile phase,LC column,gradient elution program,and sample solvent medium before injection,have been Selected,while MS/MS parameters,namely fragmentor,parent ions,daughter ions and their collision energy,were optimizated.Eventaully,analytes was conducted with a pentafluorophenyl column(3 ?m,150×2.1 mm i.d.),using water(0.1%formic acid)and methanol(0.1%formic acid)as mobile phase,while MS/MS detection was performed by multiple reaction monitoring(MRM)in positive ionization mode.Good linearity was achieved for 25 mycotoxins in the range of 0.2 ?g/L(AFT)?5000?g/L(DON),with R2?0.9900.The IDL was in the range of 0.075 ?g/L(AFT)?5?g/L(DON).(2)Optimization of sample preparationTaking corn as the representative matirx,with appropriate filter membrane,extraction solvent and SPE purification primarily chosen,the method of sample preparation was finally based on a double extraction approach,using a mixture of acetonitrile/water/formic acid(84:15:1,v/v/v),and Prime HLB columns for extract cleanup.When tested in 4 typical grains,ie.corn,rice,wheat and millet,the matrix effect for all mycotoxins was in the range of-30.4%-29.7%.The rcoveries calculated by 3 different quantificational methods(i.e.by external calibration,matrix matched calibration or a isotopic surrogate)were compared.Consequently,matrix matched calibration were used for quantification.Recovery experiments were carried out with blank samples.Four representative grains were chosen and spiked at 2 different levels.More than 90%of recoveries were between 70%-120%,with the relative standard deviation(n=3)less than 20%.Two mycotoxin reference samples(corn and wheat)were selected for analysis.The experimentally determined concentrations of all target analytes were in good agreement with reference values.2.Monitoring of multi-mycotoxin occurrence in cerealsThe developed method was applied to analyze 104 representative cereals samples,including 33 corn samples,32 rice samples,27 wheat samples,12 millet samples,obtained from Hainan,Fujian,Heilongjiang and other provinces of China.The results were as follows:(1)Mycotoxins occurrence in cerealsEighty-seven of the 104 samples,with a frequency of 83.65%,were positive for the following mycotoxins:fumonisins,trichothecenes,aflatoxins and enniatins etc.In the positive samples the contents of mycotoxins were in the range of MDL-20547.37 ?g/kg.(2)Mycotoxin contamination characteristics in different grain samplesTaking the positive rate and detection concentration into account,the pollution level of mycotoxins in 4 kinds of grains reversed the declining trend:corn>wheat>rice>millet.Furthermore,the main contaminants in corn were FBI,FB2,AFB1 and DON while the wheat tended to be contaminated with DON,ENB and ENA1.AFB1,FBI and DON showed the highest contamination levels in rice,and that in millet came out to be FBI,FB2 and DON.(3)Mycotoxin co-contamination in cerealsA considerable number of mycotoxin co-contamination samples(66.35%)were observed.The most co-occurrence was for 2(20.19%)and 3(19.23%)or 4(18.27%)mycotoxins,followed by combinations of 5 mycotoxins(6.54%),while combinations of 6 mycotoxins was only 1.92%.The combined contamination degree of mycotoxins in 4 typical cereals showed the following signs:corn>wheat>rice>millet.(4)Primary health risk assessmentHealth risk assessments were performed on DON and sum of FB1&FB2 by comparing DI(dietary intake)with the TDI(tolerable daily intake).The result showed that exposure estimate of DON were lower than the TDI and revealed no health risk,while the sum of FB1&FB2 with a higher DI than the guidance value indicated more attention should be paid to mycotoxin contaminations in cereals. |
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