百药煎HPLC指纹图谱的建立及其中5种成分的含量测定
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篇名: 百药煎HPLC指纹图谱的建立及其中5种成分的含量测定
TITLE: Establishment of HPLC Fingerprint of Chinese Gall Leaven and Content Determination of 5 Components
摘要: 目的:建立百药煎的高效液相色谱(HPLC)指纹图谱,同时测定其中5种成分的含量及优选百药煎的原料酒糟。方法:采用HPLC法,色谱柱为WatersSymmetryShieldTMRP18,流动相为乙腈-0.1%三氟乙酸水溶液(梯度洗脱),流速为0.8mL/min,检测波长为280nm,柱温为30℃,进样量为5μL。以没食子酸为参照,绘制14批百药煎样品的HPLC指纹图谱;采用《中药色谱指纹图谱相似度评价系统》(2012版)进行相似度评价,确定共有峰;采用SPSS20.0软件进行聚类分析。结果:14批百药煎样品共有11个共有峰,相似度为0.424~0.998,其中A1~A5相似度<0.850;B1~B9样品中为共指认出5个成分,分别为没食子酸、(-)-表没食子儿茶素、没食子酸甲酯、2,4,6-三-O-没食子酰-β-D-葡萄糖、2,4,6-三-O-没食子酰-α-D-葡萄糖。聚类分析结果显示,14批样品可聚为3类,A1~A5聚为一类,B1、B6聚为一类,B2~B5、B7~B9聚为一类。上述5种成分的质量浓度线性范围分别为29.96~599.2μg/mL(r=0.9996)、0.832~416μg/mL(r=0.9996)、0.102~51μg/mL(r=0.9998)、0.2864~143.2μg/mL(r=0.9998)、0.2864~143.2μg/mL(r=0.9998);定量限分别为0.060、0.104、0.017、0.029、0.057μg/mL,检测限分别为0.018、0.031、0.005、0.009、0.017μg/mL;精密度、稳定性、重复性、耐用性试验的RSD均小于3%;加样回收率分别为97.16%~101.88%(RSD=1.60%,n=6)、96.98%~99.24%(RSD=0.85%,n=6)、97.70%~101.64%(RSD=1.54%,n=6)、97.77%~103.08%(RSD=1.82%,n=6)、98.16%~101.88%(RSD=1.24%,n=6)。结论:本研究所建HPLC指纹图谱和聚类分析可用于评价百药煎的质量;所建含量测定方法操作简便,可用于同时测定百药煎中5种成分的含量;稻壳类酒糟不适用于发酵百药煎。
ABSTRACT: OBJECTIVE:To establis h HPLC fingerprint of Chinese gall leaven ,simultaneous determination of 5 components and optimization of distillers ’grains. METHODS :HPLC method was adopted. The determination was carried out on Waters Symmetry Shield TM RP18 column with mobile phase consisted of acetonitrile- 0.1% trifluoroacetic acid aqueous (gradient elution )at the flow rate of 0.8 mL/min. The column temperature was 30 ℃,and detection wavelength was set at 280 nm. The sample size was 5 µL. Using gallic acid as reference ,HPLC fingerprints of 14 batches of sample were drawn. The similarity evaluation was performed by using Similarity Evaluation System of Chromatogram Fingerprint of TCM (2012 edition)to determine common peaks;SPSS 20.0 software was used for cluster analysis. RESULTS :There were 11 common peaks in 14 batches of samples ,and the similarity range of 14 batches of samples was 0.424-0.998,A1-A5 was less than 0.850;5 components were identified in B 1-B9 sample,i.e. gallic acid ,(-)-epigallocatechin,methyl gallate ,2,4,6-tri-O-galloyl-α-D-glucose and 2,4,6-tri-O-galloyl- β-D-glucose. The results of cluster analysis showed that 14 batches of samples could be grouped into 3 categories,i.e. A1-A5 into one category,B1 and B 6 into one category ,B2-B5 and B 7-B9 into one category. The linear ranges of above 5 components were 29.96-599.2 μg/mL(r=0.999 6),0.832-416 μg/mL(r=0.999 6),0.102-51 μg/mL(r=0.999 8),0.286 4-143.2 μg/mL(r=0.999 8), 0.286 4- 143.2 μg/mL(r=0.999 8),respectively. The limits of quantitation were 0.060,0.104,0.017,0.029,0.057 μg/mL;the limits of detection were 0.018,0.031,0.005,0.009,0.017 μg/mL,respectively. RSDs of precision ,stability,reproducibility and durability tests were all lower than 3%. The recoveries were 97.16%-101.88%(RSD=1.60%,n=6),96.98%-99.24%(RSD= 0.85%,n=6),97.7%-101.64%(RSD=1.54%,n=6),97.77%-103.08%(RSD=1.82%,n=6),98.16%-101.88%(RSD=1.24%, n=6),respectively. CONCLUSIONS:The established HPLC fingerprint and cluster analysis can be used to evaluate the quality of Chinese gall leaven. The established method is simple to Δ 基金项目:国家重点研发计划项目(No.2018YFC1707200);公 operate and can be used to determine the contents of 5 益性行业科研专项项目(No.201507004-03);河南中医药大学研究生 components simultaneously ;rice husk distillers ’grains were 科研创新基金项目(No.YJS2018B14) *硕士研究生 。研究方向 :中药饮片及新药研究 。E-mail: not suitable for fermented Chinese gall leaven . 401327039@qq.com
期刊: 2020年第31卷第02期
作者: 陈晶晶,张振凌,曹淼淼,张颖,杨佳宁
AUTHORS: CHEN Jingjing ,ZHANG Zhenling ,CAO Miaomiao ,ZHANG Ying,YANG Jianing
关键字: 百药煎;指纹图谱;高效液相色谱法;含量测定;酒糟;聚类分析
KEYWORDS: Chinese gall leaven ; Fingerprint; HPLC;
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