牛黄镇惊丸中黄曲霉毒素残留检测方法研究

Methodology study on aflatoxin analysis in Niuhuang Zhenjing Pills

目的对中药复方制剂牛黄镇惊丸中黄曲霉毒素残留检测方法进行研究。方法采用免疫胶体金(GICA)方法和免疫亲和柱-高效液相色谱(IAC-HPLC)方法对牛黄镇惊丸中黄曲霉毒素残留量进行检测,并对两种方法进行方法学研究,对8批次市售牛黄镇惊丸进行检测。结果采用推荐的免疫亲和柱-高效液相色谱方法检测牛黄镇惊丸样品时,回收率仅为46.6%~67.7%,不满足试验要求。试验采用倍比稀释的方法,发现5倍稀释待测样品后,样品加样回收率为80.7%~94.0%,符合试验要求。免疫亲和柱-高效液相色谱方法线性回归方程:Y=0.656 X-0.436,R 2=0.9981;表明黄曲霉毒素B 1(AFB 1)在4~20 ng·mL-1之间线性关系良好。试验精密度RSD为0.98%~1.55%之间,重复性为0.20%。采用免疫胶体金方法进行检测,未发现高于黄曲霉毒素检测方法定量限的样品。免疫胶体金方法和免疫亲和柱-高效液相色谱方法检测结果基本一致。结论建立了牛黄镇惊丸中黄曲霉毒素残留免疫胶体金和免疫亲和柱-高效液相色谱的检测方法。牛黄镇惊丸成分复杂,对免疫亲和柱-高效液相色谱的方法检测干扰较大,可采用稀释的方法优化样品前处理过程,提高方法的回收率,保证了检测方法的准确可靠。

Objective To study the methodology on aflatoxin analysis in Niuhuang Zhenjing Pills.Methods To determine the aflatoxin residue of Niuhuang Zhenjing Pills from 8 batches of samples by immune colloidal gold technique(GICA)and immunoaffinity column-high performance liquid chromatography(HPLC),and to study both of methods.Results The recovery rates of IAC-HPLC assay,which was recommended,were 46.6%~67.7%,failed to meet the requirements.After five times dilution,the recovery rate of HPLC were 80.7%~94.0%,which was qualified for the test.The equation of linear regression of IAC-HPLC was Y=0.656 X-0.436,R 2=0.9981,indicating that AFB1 had a good linear relation between 4~20 ng.The RSD of precision ranged from 0.98%~1.55%,and the reproducibility was 0.20%.No samples above the quantification limit of aflatoxin assay were detected by GICA method.The results were basically consistent by GICA and IAC-HPLC.Conclusion GICA and IAC-HPLC were established to determine the residues in Niuhuang Zhenjing Pills.The metric complex was recognized as interference for IAC-HPLC assay,while the dilution pretreatment could effectively decrease thus effects.