免疫亲和柱净化-在线柱后光化学衍生 HPLC-FLD 检测莲子中黄曲霉毒素B1, B2, G1, G2及其液质确证

目的:建立高效液相色谱-在线柱后光化学衍生-荧光检测器检测药食同源中药材莲子中黄曲霉毒素B1,B2,G1,G2的含量,并采用液质联用法进行确证。方法:样品以甲醇-水(80∶20)溶液提取,经免疫亲和柱净化后,利用在线柱后光化学衍生-HPLC-FLD进行分析测定。并进一步采用LC-MS/MS对阳性样品进行确证。结果:在优化条件下,黄曲霉毒素B1,G1在0.3～30μg.L-1,黄曲霉毒素B2,G2在0.09～9.0μg.L-1线性关系良好,r>0.999 9,回收率86.7%～99.1%,RSD<4.87%。黄曲霉毒素B1,B2,G1,G2的检测限(LOD)分别为0.08,0.03,0.10,0.03μg.kg-1。所测的20批莲子样品中,有14批样品的黄曲霉毒素检测结果呈阳性,其中黄曲霉毒素B1的污染水平为0.40～586μg.kg-1,黄曲霉毒素总量(B1+B2+G1+G2)的污染水平为0.40～602.5μg.kg-1。通过LC-MS/MS确证,在与对照品相同的保留时间处,样品与对照品有相同的特征离子碎片,排除了样品假阳性的可能。结论:该方法简便快速,灵敏度高,重复性好,适用于莲子中黄曲霉毒素的检测。

Determination of aflatoxins in Nelumbinis Semen by immunoaffinity column clean-up and HPLC-FLD with on-line post-column photochemical derivatization and LC-MS/MS confirmation

To determine the contents of aflatoxin B1,B2,G1 and G2 in Nelumbinis Semen using on-line post-column photochemical derivatization-HPLC-FLD method and verify the method by LC-MS method.Method: The samples were extracted with MeOH-H2O(80∶20) and purified with inmunoaffinity column,aflatoxins were analyzed by HPLC-FLD with post-column photochemical derivatizaton.The positive samples were further confirmed by LC-MS/MS.Result: On optimum conditions,aflatoxin B1,G1 ranging 0.3-30 mg·L-1 showed a good linear relationship with aflatoxin B2,G2 ranging 0.09-9.0 mg·L-1 with r>0.999 9.The recoveries ranged between 86.7% and 99.1%,with RSDs all bellow 4.87%.LOD of aflatoxin B1,B2,G1 and G2 were 0.08,0.03,0.10,0.03 μg·kg-1,respectively.Among 20 Nelumbinis Semen samples,14 were found to contain aflatoxin B1 ranging from 0.40 to 586 μg·kg-1.The total content of aflatoxin B1,B2,G1 and G2 were between 0.40 and 602.5 μg·kg-1.By LC-MS/MS method,the same fragment ions were founded in samples and the control group at the same retention times,ruling out the possibility of false positive samples.Conclusion: The method is simple,highly sensitive and reproducible for the determination of aflatoxins in Nelumbinis Semen.