目的：采用免疫亲和净化手段和高效液相色谱(HPLC)检测技术,建立富含色素类中药材中黄曲霉毒素(AFT)的测定方法,为中药材中 AFT 的检测和污染调查提供技术支撑。方法中药材样品经过高速粉碎均匀后,粉末状样品经80%甲醇水高速均质提取,提取液以磷酸盐缓冲液(PBS)稀释后,用免疫亲和柱(IAC)净化,以含吐温-20的PBS淋洗柱子后,用甲醇洗脱AFT,洗脱液经三氟乙酸衍生后用HPLC分析, C18反相色谱柱分离,用荧光检测器(FLD)测定,外标法定量。结果黄曲霉毒素G1、B1、G2和B2的最低检出限(LOD, S/N＝3)分别为0.1、0.02、0.05和0.05μg/kg；以不含AFT、色素含量较高的枳实、绞股蓝、枇杷叶、鱼腥草和乌梅为基质,进行加标回收试验,加标水平为6.0～24μg/kg(以AFT总量计), AFT的平均回收率在62.3％～115%之间, RSD在0.92％～15.9%之间(n＝3)。结论 IAC净化技术和HPLC-FLD相结合,用于4种AFT的检测可取得较为满意的结果,方法的准确度高、精密度好,各项指标均满足欧盟对食品中 AFT 检测的要求,方法可满足色素含量较高的中药材中4种黄曲霉毒素(AFT)的测定。%Objective To develop a method for quantifying the levels of aflatoxins (AFTs) in the tradi-tional Chinese medicines (TCM) rich in pigments and provide the technical support for AFTs detection and contamination controlling in TCM. Method The powdered sample was extracted with 80%(v/v) methanol in water by homogeneous extraction at high speed. The diluted extract was cleaned up by the commercial immunoaffinity column (IAC) containing antibody specific to AFT. The phosphate buffer containing 0.1%Tween-20 was used to remove the interfering compounds and then methanol was used to elute AFT. The de-rivatized eluent of methanol was separated on C18 column (4.6 mm×150 mm, 3.0μm) and detected by fluo-rescence detector (FLD). Results The limits of detection (LOD, S/N=3) were in the range of 0.02~0.1μg/kg for four kinds of aflatoxins. The average recoveries varied from 62.3%to 115%when AFT was spiked to 5 kinds of TCM matrices at total levels ranged from 6.0 to 24μg/kg. And the relative standard deviation (RSD) (n=3) was in the range of 0.92% and 15.9%. Conclusion The satisfactory result was obtained by HPLC-FLD coupled with IAC column. The developed procedure can be used for the quantification of AFT in TCM rich in pigments.
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