独活的HPLC指纹图谱及4个香豆素类成分的测定

目的：建立独活HPLC指纹图谱并测定独活中二氢欧山芹醇、蛇床子素、异欧前胡素、二氢欧山芹醇当归酸酯的含量，为独活的质量控制提供参考。方法：采用SWELL Chromplus^TM C₁₈色谱柱（250 mm&#215;4.6 mm，5 μm），以乙腈（A）-0.2%醋酸水溶液（B）为流动相，梯度洗脱（0~5 min，15%→20% A；5~20 min，20%→27% A；20~45 min，27% A；45~55 min，27%→30% A；55~56 min，30%→41% A；56~60 min，41% A；60~70 min，41%→50% A；70~90 min，50%→55% A；90~95 min，55%→15% A；95~100 min，15% A），流速1.0 mL&#183;min^-1，检测波长325 nm，柱温25℃。采用"中药色谱指纹图谱相似度评价系统"建立指纹图谱并进行相似度评价，利用SPSS 20.0软件进行聚类分析。对二氢欧山芹醇、蛇床子素、异欧前胡素和二氢欧山芹醇当归酸酯的含量测定进行方法学考察。结果：建立了独活的指纹图谱，标定了21个共有峰，18批独活药材与对照图谱的相似度均≥ 0.85，聚类分析将其分为3类。二氢欧山芹醇、蛇床子素、异欧前胡素和二氢欧山芹醇当归酸酯线性范围分别为2.250~36.000、34.500~552.000、1.375~22.000和9.375~150.000 μg&#183;mL^-1，线性关系良好（r ≥ 0.999 7）；重复性试验RSD<2%，平均加样回收率在94.1%~100.6%之间。18批样品中二氢欧山芹醇、蛇床子素、异欧前胡素和二氢欧山芹醇当归酸酯的含量分别为0.009 3%~0.110 1%，0.498 9%~1.664 1%，0.007 1%~0.045 0%和0.065 4%~0.569 0%。结论：独活指纹图谱专属性强，结合4个香豆素类成分的含量测定可较全面控制独活的质量。

Objective: The aim of the study is to establish the HPLC fingerprint and determine the content of columbianetin, osthole, isoimperatorin and columbianadin in Angelica Pubescens Radix simultaneously. It provides the reference for quality control of Angelica Pubescens Radix.Methods: HPLC analysis was performed on SWELL Chromplus^TM C₁₈ column(250 mm&#215;4.6 mm, 5 μm).The mobile phase of acetonitrile(A)-0.2% acetic acid solution(B):0-5 min, 15%→20%A; 5-20 min, 20%→27%A; 20-45 min, 27%A; 45-55 min, 27%→30%A; 55-56 min, 30%→41%A; 56-60 min, 41%A;60-70 min,41%→50%A;70-90 min, 50%→55%A; 90-95 min, 55%→15%A; 95-100 min, 15%A.Detection wavelength was 325 nm;flow rate was 1.0 mL&#183;min^-1;and column temperature was 25℃.Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine was used for establishing the reference fingerprint and similarity evaluation, and cluster analysis was used in date analysis by SPSS2.0 software(SPSS 20.0).The determination method of the content of four kinds of coumarin was verified.Results: The HPLC fingerprint of Angelica Pubescens Radix from different producing areas were constructed with 21 common chromatographic peaks.The similarities of 18 batches were all over 0.85 compared with the reference fingerprint.All samples were classifiedinto 3 types by clustering analysis.The linear relationship for columbianetin, osthole, isoimperatorin and columbianadin were observed in the ranges of 2.250-36.000 μg&#183;mL^-1、34.500-552.000 μg&#183;mL^-1、1.375-22.000 μg&#183;mL^-1 and 9.375-150.000 μg&#183;mL^-1, respectively, with r ≥ 0.999 7.The RSDs of repeatability tests were<2% and the average recoveries varied from 94.1%-100.6%.The contents of columbianetin, osthole, isoimperatorin and columbianadin in 18 batches of Angelica Pubescens Radix were 0.009 3%-0.110 1%, 0.498 9%-1.664 1%, 0.007 1%-0.045 0%, 0.065 4%-0.569 0%, respectively.Coclusion: The HPLC fingerprint and the assay method of four kinds of coumarin in Angelica Pubescens Radix can be used for the quality control of Angelica Pubescens Radix.