坐浴二号方指纹图谱的建立及多指标成分定量分析

高琼; 王鹏; 孙利萍; 曹霞; 宋耀辉; 唐佩莉

doi:10.13748/j.cnki.issn1007-7693.20261195

坐浴二号方指纹图谱的建立及多指标成分定量分析

Establishment of Fingerprints and Quantitative Analysis of Multi-Components in Zuoyu No.2 Prescription

摘要

摘要:
目的建立15批坐浴二号方水煎液的HPLC指纹图谱及4种成分的含量测定方法，为其质量控制和临床合理应用提供参考。
方法采用Amethyst C₁₈-H柱(4.6 mm×250 mm，5 μm)进行分离，流动相A为乙腈，流动相B为0.1%磷酸水溶液，梯度洗脱；进样量为10 μL，检测波长214 nm，柱温30 ℃，流速1.0 mL·min⁻¹。采用中药色谱指纹图谱相似度评价系统(2012版)建立15批坐浴二号方水煎液的指纹图谱，在相似度评价基础上进一步进行聚类分析、主成分分析和正交偏最小二乘法-判别分析，并同步测定了没食子酸、绿原酸、羟基红花黄色素A及蒙花苷4个成分的含量。
结果建立的HPLC指纹图谱方法符合方法学要求，15批坐浴二号方水煎液有14个共有峰，相似度>0.900。经对照品比对指认了没食子酸、绿原酸、羟基红花黄色素A及蒙花苷4个成分。15批样品可聚为2类，影响样品分类差异的主要成分4个，分别为没食子酸、绿原酸、羟基红花黄色素A及蒙花苷。4种指标性成分在各自线性范围内线性关系良好(r≥0.9990)，没食子酸、绿原酸、羟基红花黄色素A、蒙花苷平均回收率分别为98.04%、99.45%、99.79%、99.52%；RSD分别为2.7%、4.8%、5.6%、5.6%(n=6)，仪器精密度、样品稳定性、方法重复性均良好。没食子酸含量为1.165~3.054 mg·mL⁻¹、绿原酸含量为0.426~0.939 mg·mL⁻¹、羟基红花黄色素A含量为0.144~0.360 mg·mL⁻¹、蒙花苷含量为0.073~0.224 mg·mL⁻¹。
结论所建立的HPLC指纹图谱和4个成分的含量测定方法灵敏度高，重复性和稳定性良好，可为坐浴二号方水煎液的质量控制和临床安全应用提供科学支撑。

Abstract:
OBJECTIVE To establish HPLC fingerprints of 15 batches of Zuoyu No.2 prescription decoction and to determine the contents of 4 components, in order to provide reference for its quality control and clinical rational application.
METHODS Amethyst C₁₈-H column(4.6 mm×250 mm, 5 μm) was used for separation, mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; the injection volume was 10 μL, the detection wavelength was 214 nm, the column temperature was 30 ℃, and the flow rate was 1.0 mL·min⁻¹. The fingerprints of 15 batches of Zuoyu No. 2 prescription decoction were established using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine(Version 2012). Based on the similarity evaluation, cluster analysis, principal component analysis, and orthogonal partial least squares-discriminant analysis(OPLS-DA) were further performed, and the contents of gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin were simultaneously determined.
RESULTS The established HPLC fingerprints method met the methodological requirements. There were 14 common peaks in 15 batches of Zuoyu No.2 prescription decoction, and the similarity was greater than 0.900. Four components, namely gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin, were identified by comparison of reference substances. The 15 batches of samples could be clustered into 2 categories, and 4 main components affecting the classification of samples were gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin. The 4 main components had good linear relationships in their respective linear ranges(r≥0.9990). The average recoveries of gallic acid, chlorogenic acid, hydroxysafflor yellow A and linarin were 98.04%, 99.45%, 99.79% and 99.52%, respectively. The RSDs were 2.7%, 4.8%, 5.6% and 5.6%(n=6), respectively. The instrumental precision, sample stability, and method repeatability were all good. The content of gallic acid was 1.165−3.054 mg·mL⁻¹, the content of chlorogenic acid was 0.426−0.939 mg·mL⁻¹, the content of hydroxysafflor yellow A was 0.144−0.360 mg·mL⁻¹, and the content of linarin was 0.073−0.224 mg·mL⁻¹.
CONCLUSION The established HPLC fingerprints and the content determination method of the 4 components have good sensitivity, repeatability and stability, which can provide scientific basis for the quality control and clinical safety application of the Zuoyu No.2 prescription decoction.

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