白术UPLC指纹图谱模式识别及含量测定研究

Study on Fingerprint of Atractylodes Macrocephala Koidz and Determination of the Contents by UPLC

目的建立不同产地白术的超高效液相色谱(UPLC)指纹图谱及含量测定方法,并用于不同产地白术的质量评价。方法采用Waters ACQUITY UPLC CSH C18(2.1 mm×100 mm,1.7μm)色谱柱,流动相A为0.05%磷酸水,B为乙腈,梯度洗脱,流速为0.2 m L·min^(-1);柱温为25℃;进样量2μL。指纹图谱采用中药色谱指纹图谱相似度评价(2012版)软件,对25个不同产地的白术UPLC指纹图谱进行分析,并用SPSS版本软件进行聚类分析和主成分分析。结果 25批样品中指纹图谱标定了13个峰作为共有峰,精密度、重复性、稳定性中共有峰相对峰面积和相对保留时间的相对标准偏差(RSD)均小于2.0%。25批样品的某些样品的相似度差异较大,相似度为0.416~0.979;聚类分析将25批白术分为4类,主成分分析用2个主成分对25批样品进行综合性分析。含量测定中白术内酯Ⅰ、白术内酯Ⅱ和白术内酯Ⅲ的线性范围分别为0.086~0.60μg(r=0.999 9)、0.084~0.58μg(r=0.999)、0.090~0.62μg(r=0.999 9),加样回收率分别在95.83%~101.4%,98.61%~102.8%,97.44%~102.6%;平均加样回收率分别为98.38%,RSD 1.88%;99.54%,RSD 2.74%;98.29%,RSD 2.16%。结论建立的方法简单、可靠,精密度、稳定性、重复性良好;通过相似度和主成分对不同产地白术的差异性进行分析,可为白术的质量评价提供参考,也为白术后期深入研究奠定基础。

Objective The UPLC fingerprint and content determination methods of Atractylodes macrocephala from different producing areas were established for quality evaluation.Methods Using Waters ACQUITY UPLC CSH C18（2.1 mm×100 mm,1.7μm）chromatographic column,a gradient elution program with the mobile phase A was 0.05% phosphoric acid aqueous solution and B was acetonitrile,and a flow rate of 0.2 mL·min^-1,column temperature of 25℃,and injection volume of 2μL,the fingerprints of A.macrocephala from 25 different producing areas were analyzed by UPLC fingerprinting coupled with similarity evaluation of Chinese medicine chromatographic fingerprinting（2012 version）software;the cluster analysis and principal component analysis were performed using SPSS software.Results The fingerprints of 25 batches of samples identified a total of 13 peaks as common peaks.The RSDs of the relative peak areas and the relative retention times of the common peaks in precision,repeatability and stability were less than 2.0%.The similarity of some samples from 25 batches of samples varied largely and the similarity was 0.416-0.979.Cluster analysis classified 25 batches of A.maculata into four categories,and principal component analysis using two principal components comprehensively analyzed 25 batches samples.The linear ranges of atractyloside Ⅰ,atractyloside Ⅱ and atractylenolide Ⅲ were 0.086-0.60μg（r=0.999 9）,0.084-0.58μg（r=0.999）,and 0.090-0.62μg（r=0.999 9）.The recoveries of the samples were 95.83%-101.4%,98.61%-102.8% and 97.44%-102.6%,respectively.The average recoveries were 98.38%,RSD 1.88%;99.54%,RSD 2.74% and 98.29%,RSD 2.16%.Conclusion The established method is simple,reliable,accurate,stable and reproducible.Analysis of the differences of A.macrocephala from different habitats by similarity and principal components can provide a reference for the quality evaluation of A.macrocephalae and lay the foundation for the further study of Atractylodes.