黄芪药材提取纯化过程中黄芪皂苷Ⅳ含量变化及相关成分分析

Analysis of Content Variation of AstragalosideⅣand Related Chemical Components During the Extraction and Purification of Astragali Radix

该文建立了准确测定黄芪药材中游离黄芪皂苷Ⅳ含量的超高效液相色谱-串联质谱(UPLC-MS/MS)法,并研究了2015年版《中国药典》黄芪项下黄芪皂苷Ⅳ(黄芪甲苷)含量测定前处理操作对黄芪皂苷Ⅳ含量的影响。分别比较索氏提取后、正丁醇萃取后和氨试液除杂后黄芪皂苷Ⅳ的含量变化,并利用具有超高分辨率的傅里叶变换离子回旋共振质谱仪(FT-ICR-MS)分析其成分的差异性。结果表明,在优化条件下,黄芪药材中黄芪皂苷Ⅳ在0.3870~24.75 mg/L范围内呈良好的线性关系,相关系数(r)为0.9998,检出限(LOD)为1.0 mg/kg,定量下限(LOQ)为3.0 mg/kg,加标回收率为94.5%~105%,相对标准偏差(RSD,n=6)为1.4%。研究发现甲醇直接提取的黄芪皂苷Ⅰ相对含量最高,正丁醇萃取后的氨试液除杂过程会导致黄芪皂苷Ⅰ及其他黄芪皂苷Ⅳ同系物水解,黄芪皂苷Ⅰ的相对含量显著降低,而黄芪皂苷Ⅳ的含量显著升高;并发现黄芪药材中存在目前尚未报道的黄芪皂苷Ⅱ异构体,将其命名为新黄芪皂苷Ⅱ。

A validated analytical method was developed for the determination of free astragalosideⅣin Astragali Radix by ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).Effects of each step of pretreatment were studied on the determination of astragalosideⅣin Chinese pharmacopoeia(2015)(ChP).Furthermore,impacts of n-butanol extraction and ammonia impurity removal on the determination and content variation of astragalosideⅣwere investigated.The differences of components caused by pretreatment were analyzed by Fourier transform ion cyclotron resonance mass spectrometry(FT-ICR-MS).Results showed that there was a good linear relationship of the proposed method for astragalosideⅣin the range of 0.3870-24.75 mg/L,with a correlation coefficient(r)of 0.9998.The limit of detection(LOD)and limit of quantitation(LOQ)were 1.0 mg/kg and 3.0 mg/kg,respectively.The recoveries at three spiked levels ranged from 94.5%to 105%,with a relative standard deviation(RSD,n=6)of 1.4%.The relative content of astragalosideⅠwas the highest when Astragali Radix was directly extracted with methanol.The impurity removal process with ammonia after n-butanol extraction led to the homologues turned into astragalosideⅣby hydrolysis,which significantly increased the content of astragalosideⅣ.Moreover,it was found that there exists a new isomer of astragalosideⅡ,named as neoastragalosideⅡin Astragali Radix.