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目的:建立基于固相萃取和含内标HPLC特征图谱的大黄质量评价方法。方法:采用水回流提取,固相萃取净化的方法,获取大黄药材中的总蒽醌;用HPLC法建立大黄总蒽醌的特征图谱;DAD结合MSn对色谱峰进行定性,各个特征峰与内标峰的相对峰面积进行定量,从而对大黄质量进行评价。结果:大黄提取的最佳条件为料液比1∶150,回流提取60 min;提取液采用Accu BONDⅡSPE ODS-C18Cartridges固相萃取柱,上样量1 m L,3 m L 0.3%甲酸溶液分2次淋洗,然后3 m L 25%甲醇溶液(含0.3%甲酸)淋洗,3 m L甲醇洗脱,指标成分回收率为96.3%~103%;以5-羟甲基糠醛为内标物,建立了大黄总蒽醌的HPLC特征图谱,标定了12个特征峰;考察了10批大黄特征峰的相对保留时间,其RSD<3%,计算了各特征峰与内标峰的相对峰面积,通过与2015年版中国药典一部含量测定方法进行对比、主成分分析和聚类分析,表明各个特征峰与内标峰的相对峰面积可以基本反映大黄中蒽醌类成分的含量,并可以将唐古特大黄、药用大黄和掌叶大黄进行区分。结论:该方法操作简便、快捷、专属性强,通过外加内标物达到无对照品也能进行含量测定的目的,减少外界条件对特征图谱的影响,提高测定结果的准确性,可以为大黄的质量评价研究提供参考。
OBJECTIVE: To establish a method for the quality evaluation of rhubarb based on solid-phase extraction and internal standard HPLC profiles. Methods: The total anthraquinone in Rhubarb was obtained by water reflux extraction and solid-phase extraction and purification. The characteristic map of total anthraquinone in Rhubarb was established by HPLC. The peak of anthraquinone was identified by DAD and MSn. The relative peak area of the peak was quantified to evaluate the rhubarb quality. Results: The optimum conditions for the extraction of Rhubarb were as follows: the ratio of solid to liquid was 1:150, and the extraction was carried out 60 min after refluxing. The extraction solution was analyzed by Accu BONDⅡSPE ODS-C18 Cartridges. The sample volume was 1 m L, Then rinsed with 3 mL of 25% methanol solution (containing 0.3% formic acid) and eluted with 3 mL of methanol. The recoveries of the indicator components ranged from 96.3% to 103%. With 5-hydroxymethylfurfural as internal standard , The HPLC characteristic map of total anthraquinone in rhubarb was established and 12 characteristic peaks were calibrated. The relative retention time of ten rhubarb characteristic peaks was investigated, with RSD <3%. The relative peak area of each peak was calculated , Compared with a method of content determination of Chinese Pharmacopoeia 2015 edition, principal component analysis and cluster analysis showed that the relative peak area of each characteristic peak and internal standard peak can basically reflect the content of anthraquinones in rhubarb, Tangut rhubarb, rhubarb medicinal rhubarb and palm leaves to distinguish. Conclusion: The method is simple, rapid, and specific, and can achieve the purpose of determination of content without reference substance by adding internal standard substance. It can reduce the influence of external conditions on the characteristic map and improve the accuracy of determination results, Quality evaluation research for reference.