HPLC法测定中药饮片中黄曲霉毒素残留量的假阳性研究

目的:建立HPLC法检测中药饮片中黄曲霉毒素残留量的假阳性排除方法,确定易产生假阳性的中药品种,确定干扰物质的化学结构。 方法:采用免疫亲和柱净化(IAC)-HPLC-柱后光衍生荧光法测定黄曲霉毒素阳性样品在光衍生或不衍生条件下的峰面积,并计算其比值;采用不同液相色谱条件分析阳性样品的黄曲霉毒素残留量;采用HPLC-MS/MS法进行验证。 结果:71种120批中药饮片样品中有8种13批为阳性,经假阳性排除研究,阳性样品中2种3批次样品存在假阳性问题,结果和HPLC-MS/MS验证结果一致。假阳性品种占分析品种数的2.8%,假阳性样品数占分析总样品数的2.5%。补骨脂素和异补骨脂素为影响黄曲霉毒素B₁测定的干扰物。 结论:免疫亲和柱净化-HPLC分析-柱后光化学衍生方法适合大多数中药饮片中黄曲霉毒素残留量的分析测定,假阳性率较低。测定后可以采用衍生或不衍生的峰面积比值比较,结合不同色谱条件再测定的方法进行假阳性排除。

Objective: To establish an effective method of eliminating false positive results of aflatoxin residue analysis in Chinese herbal pieces by HPLC,and determine which varieties of traditional Chinese medicines (TCM) are easy to be disturbed by false positive,and define the chemical structure of disruptors. Methods: The samples were analyzed by the immunoaffinity column purification coupled with HPLC and post-column fluorescence detection under conditions of photochemical or non-photochemical derivation,and the ratios of peak areas under these two conditions were calculated and compared.Different HPLC conditions were set to analyze aflatoxin residues in the positive samples,and the analysis results were verified by HPLC-MS/MS. Results: Analysis results for samples of 71 varieties (120 batches) of Chinese herbal pieces demonstrated that there were 8 varieties (13 batches) positive,and 2 varieties (3 batches) were proved as false positive among the positive samples,which were consistent with the verification result by HPLC-MS/MS.The false positive varieties accounted for 2.8% of the total varieties and the false positive samples accounted for 2.5% of the total samples.The psoralen and isopsoralen were detected as the disruptors to aflatoxin B₁. Conclusion: The method of immunoaffinity column purification and HPLC with post column photochemical derivation and fluorescence detection is suitable for analysis of aflatoxin residues in Chinese herbal pieces,which has a lower false positive rate.The false positive result could be excluded by the methods of peak area comparison with or without photochemical derivation and re-measurement with different HPLC conditions.