液相色谱-串联质谱法测定人工模拟猪消化液中3种霉菌毒素

Rapid and Sensitive LC-MS / MS Determination of Aflatoxin B1,Deoxynivalenol,Zearalenone in Artificial Porcine Gastrointestinal Digested Juices

建立了液相色谱-串联质谱法(LC-MS/MS)同时测定人工模拟猪胃和小肠消化液中黄曲霉毒素B1(AFB1)、脱氧雪腐镰刀菌烯醇(DON)和玉米赤霉烯酮(ZEA)的快速、灵敏方法,并将此方法应用于霉菌毒素吸附剂吸附率体外法评价。通过模拟猪的消化道对饲料基质进行体外消化获得猪胃和小肠消化液,分别向其中按一定比例添加霉菌毒素吸附剂和3种霉菌毒素,孵育、离心后,经进样液10倍稀释后测定。采用反相C18色谱柱分离,以0.2 mmol/L乙酸铵溶液和0.1%甲酸-甲醇溶液作为流动相,梯度洗脱,多反应监测离子模式(MRM)检测,同位素内标法定量。在优化条件下,对AFB1,DON和ZEA在人工猪胃和小肠消化液中的定量限分别是1,50,40μg/L和0.3,50,20μg/L,相对偏差(RSD)<5.0%。并且,在39℃±0.5℃,10 h内测定结果稳定,能够满足霉菌毒素吸附剂吸附率的测定。采用本方法对市售8种蒙脱石类吸附剂和5种酵母细胞壁类吸附剂进行吸附率评价。

A rapid liquid chromatography-tandem mass spectrometric（ LC-MS / MS） method was developed for the determination of aflatoxin B1（ AFB1）,Deoxynivalenol（ DON）,Zearalenone（ ZEA） in artificial porcine gastrointestinal digested juices,as pigs reacted most sensitively to these mycotoxins. The formula feed was digested by artificial gastric and intestinal juices respectively,then the mycotoxins and adsorbent were added in ratio. After incubation and centrifugation,the supernatant was diluted 10-folds by injection solution and analyzed by LC-MS / MS. The 3 analytes were separated on a reversed phase C18 column using a gradient elution program of aqueous solution containing 0. 2 mmol / L ammonium acetate and 0. 1% formic acid methanol. Qualitative analysis was performed using multiple-reaction monitoring（ MRM）,and quantitative analysis was by internal standard method. Under optimum conditions,the limit of quantitation（ LOQ） of AFB1,DON,ZEA was 1,50,40 and 0. 3,50,20 μg /L in artificial gastric and intestinal digested juices respectively,and the relative standard deviations（ RSDs） were below 5. 0%. Then,the thermal stability was studied by incubating the analytes at 39 ℃ ± 0. 5 ℃ for 1,2,5 and 10 h,and the results showed 3 analytes were stable under the conditions. Furthermore,the method was applied to evaluate the binding efficacy of 8mineral benders and 5 organic adsorbents. The adsorbents demonstrated binding efficacy of 85. 1%- 96. 5%,8. 1%- 14. 7%,13. 7%- 30. 0% and 7. 4%- 16. 6%,6. 7%- 16. 2%,18. 6%- 39. 0% in gastric digested juice,and 76. 2%- 93. 0%,12. 3%- 31. 3%,0%- 23. 2% and 8. 6%- 13. 4%,3. 8%- 23.5%,24. 9%- 34. 8% in intestinal digested juice for these 3 mycotoxins,respectively,with 2 kinds of adsorbents.