阳春砂中黄曲霉毒素的免疫亲和层析净化高效液相色谱法测定

建立了高效液相色谱法测定中药材阳春砂中黄曲霉素(AFT)B_1、B_2、G_1、G_2的方法.样品经提取、免疫亲和层析净化,采用荧光检测器,以柱后衍生系统结合HPLC检测.结果表明,阳春砂中4种黄曲霉素在1.00 μg/kg,10.00 μg/kg和32.00 μg/kg 3个水平的加标回收率均在84.0%～101.0%之间,相对标准偏差均小于1.7%;方法最低检出限AFB1与AFG1为0.35 μg/kg,AFB2与AFG2为0.06 μg/kg;标准曲线良好,相关系数大于0.998 7.本方法可用于阳春砂中黄曲霉素的检验.

Method for determining the aflatoxin B_1, B_2, G_1, G_2 Content in the Amomum villosum-clean-up by immuno-affinity chromatography and HPLC

The paper is to present a quick and effective detection method for determining the aflatoxins B_1, B_2, G_1 and G_2 in Chinese herbal medicine Amomum villosum. The samples of the herbal medicine we take as the case study were first extracted by using acetonitrile:water (9:1) and then cleaned-up by using an immuno-affinity column with the analysis done by high performance liquid chromatography (HPLC), including post-column derivatization for fluorescence detection. While doing the experiments, we have also studied the optimal conditions for the extraction and chromatographic separation, which was made possible to be finished in 10 min by adopting an isocratic chromatographic system with a mobile phase comprising acetonitrile:methanol:water (7:28:65, V/V/V). In addition, we have made it possible for all the samples to recover each aflatoxin B_1, B_2, G_1 and G_2 spiked to Amomum villosum at three levels of 1.00 μg/kg, 10.00 μg/kg and 32.00 μg/kg between 84.0%-101.0%. With all the relative standard deviations made lower than 1.7%, the lowest detection limits of aflatoxins in Chinese herbal medicine were 0.35 μg/kg for AFB1 and AFG1, and 0.06 μg/kg for AFB2 and AFG2. The calibration curves of all the above tested samples demonstrate perfect linearity with the correlation coefficient being ≥0.998 7. Thus, the method displays nice stability, high sensitivity and high selectivity, which suggests a great potential for the method to get extensively adopted for determining the aflatoxins in Amomum villosum. The results show that all the seven samples we have used were contaminated with aflatoxins, which ranges from 0.21 μg/kg to 4.92 μg/kg whereas the total aflatoxin levels in one sample proves to be beyond the standard limit of the European Union of 4 μg/kg.