巯乙胺硫解法分析缩合鞣质组成单元及硫解产物高效液相色谱行为分析

Analysis of condensed tannins' constituent units by thiolysis with cysteamine hydrochloride and analysis of thiolysis products' HPLC behavior

目的建立巯乙胺硫解法分析中成药威麦宁有效成分缩合鞣质的组成单元的方法。方法采用盐酸巯乙胺将威麦宁胶囊乙醇提取物硫解,进行高效液相色谱(HPLC)分析,并结合对照品和LC-MS法对威麦宁的8个主要硫解产物进行指认;同时考察了流动相pH值对威麦宁各硫解产物HPLC行为的影响。结果主要硫解产物分别为4β-(2-氨基乙硫基)儿茶素、4β-(2-氨基乙硫基)表儿茶素、儿茶素、4β-(2-氨基乙硫基)表儿茶素没食子酸酯、表儿茶素、4β-(2-氨基乙硫基)儿茶素没食子酸酯、表儿茶素没食子酸酯、儿茶素没食子酸酯;流动相pH值的改变对硫代物的保留时间影响较大,对儿茶素类化合物的保留时间影响较小,从而导致其出峰顺序发生改变。结论本方法是一种简便、可靠的衍生化方法,可以用来分析缩合鞣质的组成单元;在进行HPLC分析时应注意严格控制流动相pH值的稳定,以保证硫解产物保留时间的稳定。

Objective To establish a method of analyzing the structural units of condensed tannins from Chinese patent medi- cine Wei MaiNing. Methods The ethanol extract of Wei MaiNing capsule was depolymerized in the presence of cysteamine and ana- lyzed by HPLC, and eight major thiolysis products were identified by LC-MS analysis combined with reference sample. Meanwhile, the influence of mobile phases with different pH values on the HPLC behavior of thiolysis products was investgated. Results Eight major thiolysis products were 4β - （ 2 -aminoethylthio ） catechin, 4β - （ 2-aminoethylthio ） epieatechin, catechin, 4β - （ 2 -aminoethylthio ） epieate- ehin-3-O-gallate, epicateehin, 4β-（2-aminoethylthio）eateehin-3-O-gallate, epieatechin-3-O-gallate and cateehin-3-O-gallate. Varia- tion of the pH value had more impact on the retention time of cateehins＇ aminomethyhhio derivatives than that of catechins＇, which led to the changes of chromatographic peaks order. Conclusion The method is simple, reliable, and could be used to determine the structural units of condensed tannins. In order to ensure the stability of derivatives retention time, pH values of mobile phases should be controlled strictly when thiolysis products are analyzed by HPLC.