高效液相色谱法与荧光光度法检测中药材中黄曲霉毒素的比较

Comparison between the post-column derivatization with bromine by HPLC and the fluorometric analysis for determination of aflatoxins in medicinal herbs and plant extracts

目的 比较研究了免疫亲合柱(IAC)净化 HPLC柱后溴化衍生荧光法检测黄曲霉毒素B1,B2,G1和G2的 含量与溴化荧光光度法(SFB法)检测黄曲霉毒素总量的方法。方法 在IAC HPLC柱后溴衍生荧光法中,药材经甲 醇 水(70∶30)溶剂系统提取后,采用免疫亲合柱净化、富集黄曲霉毒素,净化后的样品溶液通过高效液相色谱柱分离 后,进入柱后衍生管中与过溴化溴化吡啶溶液发生反应,最后进入荧光检测器分别检测黄曲霉毒素B1,B2,G1和G2 含量。在SFB法中,样品经甲醇 水(70∶30)提取后,再经免疫亲合柱净化、富集,在处理后的样品溶液中添加一定量 一定浓度的溴水溶液后,迅速置于荧光光度计中读数。结果 IAC HPLC柱后溴衍生荧光法中,考察了3种不同药 材中添加2个浓度水平的混合对照品的回收率实验,回收率平均值在93%-97%之间。黄曲霉毒素B2和G2的最低 检出限为0.06μg·kg-1,黄曲霉毒素B1和G1的最低检出限为0.20μg·kg-1。精密度实验的RSD值在0.8%- 1.4%之间。运用此高效液相色谱法检测了39种中药中黄曲霉毒素的含量。同时运用SFB法检测了上述39种中药 中黄曲霉毒素的总量。结论 SFB法不适合用来检测中药中黄曲霉毒素的总量。

Aim To compare the post-column derivatization technique (IAC-PCD-HPLC) for the determination of aflatoxins B 1, B 2,G 1 and G 2 and the rapid procedure with fluorometric analysis (SFB) for the determination of total aflatoxins. Methods The method of post-column derivatization with bromine by HPLC consisted of extraction of the sample with MeOH-H 2O ( 70∶30) followed by clean-up of the extracts with immunoaffinity columns and finally,HPLC determination with fluorescence detection. Aflatoxins B 1 and G 1 were determined as their bromine derivatives,produced in an on-line post-column derivatization system. In SFB method,samples were ground and extracted with methanol-water ( 70∶30). A portion of the extract was cleaned up by passage through a immunoaffinity column,One mL of purified extract was derivatized with a bromine reagent,and fluorescence of the solution was immediately quantified with a calibrated fluorometer containing a broad wavelength pulsed xenon light source. Results In IAC-HPLC method,the overall average recoveries for three different medicinal herbs spiked at levels of 1.3 and 2.6 ng·g -1 of total aflatoxins ranged from 93% to 97%. The detection limit was 0.06 μg·kg -1 for both G 2 and B 2 and 0.20 μg·kg -1 for both G 1 and B 1,based on a signal/noise 3∶1 and the precision (within-laboratory relative standard deviation) ranged from 0.8% to 1.4%. Each of aflatoxins B 1,B 2,G 1 and G 2 in 39 kind medicinal materials were determined by IAC-PCD-HPLC,and the total aflatoxins were determined by SFB. Conclusion The SFB method is not the suitable method for the determination of total aflatoxins in medicinal herbs and plant extracts.