Determination Of Deoxynivalenol And Its Biomarkers In Urine And Human Exposure Study In China

Deoxynivalenol(DON),a trichothecene mycotoxin produced by Fusarium species,mainly F.graminearum and F.culmorum,is the most frequently-detected mycotoxin contaminant in maize,wheat and barley worldwide.Deoxynivalenol(DON),deoxynivalenol-3-glucoside(DON-3-G),deoxynivalenol-15-glucoside(DON-15-G),3-acetyle-deoxynivaleol(3-A-DON),15-acetyle-deoxyni valenol(15-A-DON),deepoxy-deoxy-nivalenol(DOM-1)and nivalenol(NIV)are commonly found in food.Their toxicological effects on human health caused widespread concern on human exposure.The study was carried out to develop methods for the detection of deoxynivalenol and its biomarkers in urine,and investigated the human exposure of deoxynivalenol in Henan and Anhui province.Firstly,this study established two methods on detection of deoxynivalenol and its biomarkers in urine samples.One method was ICA coupled with UPLC-MS/MS.We detected both free DON,DOM-1(without enzyme)and total DON,DOM-1(with enzyme).Urine samples were cleared by the immune affinity column,and then applied to UPLC/MS/MS analysis.The method recoveries of DON and DOM-1 were 83%-100%,the RSDs were between 0.8% to 9.5%.The extraction recoveries were 95% and 136% respectively.The LOQ of DON and DOM-1 were 0.1 ?g/L and 0.02 ?g/L.So the method is accurate and reliable and high sensitivity was suitable for detecting low level of DON and DOM-1 in urine samples.The other method,a high-throughput sensitive method for the quantitative determination of DON,DOM-1,DON-3-G,3-A-DON and 15-A-DON in human urine using a 96-well ?Elution solid-phase extraction(SPE)followed by UPLC-MS/MS.Urinary samples were processed with and without enzyme hydrolysis to determine total and free DON and its biomarkers,respectively.The method recoveries of DON,DOM-1,3-A-DON,15-A-DON and DON-3-G were 94%-112%,80%-91%,80%-112%,88%-106% and 88%-106%.The RSD was between 2.9% to 14.1%.The extract recovery was 78.5%-112%.The matrix effect were 67% to 83%.The LOQ of all compoubd was 0.2?g/L-2?g/L.This method is accurate and reliable,and can handle a large number of samples at the same time.Secondly,the human exposure to DON was assessed in Henan and Anhui province.DON and DOM-1 could be detected both with and without enzyme hydrolysis,whereas 3-A-DON and 15-A-DON were not found in any of these samples.Among the 596 samples,all of the Henan samples were positive for total DON(100%),and 99.6% of the Anhui amples were positive for total DON.Free DON were detected in 455 samples;the positive samples were account for 95.6% and 90.7% in Anhui and Henan provinces,respectively.68.5% of Anhui samples can detect total DOM-1,while 32.2% of henan samples.53 samples detected with Free DOM-1,Aanhui and Henan samples' detection rate were 14.2% and 3.7% respectively.67 samples detected Total DON-3-G,with detection rate of 11.2%,22.4% and 0.3% of Anhui and Henan samples were positive respectively.The mean level of total DON in Anhui and Henan provinces were 117.0?g/L and 52.8 ?g/L;Free DON,mean level were 23.5?g/L and 9.6?g/L;Total DOM-1,the mean level were 2.4?g/L and 0.6?g/L.The mean level of other compound were all lower than their LOD.The difference of people were significantly associated with age(P < 0.05),region(P < 0.05),while the difference between gender were not significant(P > 0.05).Free DON and Total DON exposure levels of correlation(P < 0.05),in Henan and Anhui provinces free DON account for 21.3% and 20.6% of the total DON respectively.The level of total DON and total DOM-1 have no correlation(P > 0.05).The PDI ranged from 0.008-37.0 ?g/kg bw/d,the maximum level was 37.4 ?g/kg bw/d(a sample in anhui province).The number that exceeding the PMTDI was 403,which was about 67.6%.In Anhui,the mean and median PDI were 4.2?g/kg bw/d and 2.6 ?g/kg bw/d,while in in henan province were 1.8 ?g/kg bw/d and 1.1 ?g/kg bw/d,and all of them has exceeded the PMTDI.On the whole,the people of this two provinces are danger to DON,expecially the minority groups.To sum up,this study established the biomarker-based method in human urine sample,evaluated the human urinary exposure risk assessment of our country,and perfecting exposure assessment to deoxynivalenol in China.