坐浴二号HPLC指纹图谱及多指标成分定量研究

HPLC fingerprint and quantitative study of multi-index components of Zuoyu No.2

关键词:  坐浴二号方  指纹图谱  含量测定  

Key words:  Zuoyu  No.2 prescription, Fingerprint  spectrum, Content  determination

摘要:

摘要：目的 建立15批坐浴二号水煎液的高效液相色谱（HPLC）指纹图谱及4种成分的含量测定方法，为坐浴二号的质量控制和临床合理应用提供参考。方法 采用 Amethyst C18-H柱（4.6×250mm，5μm）进行分离，流动相A：乙腈、流动相B：0.1%磷酸水溶液，梯度洗脱；进样量为 10 μL，检测波长 214nm，柱温30℃，流速 1.0 mL/min。通过中药色谱指纹图谱相似度评价系统（2012 版）对15批次坐浴二号水煎液建立指纹图谱，基于指纹图谱相似性评价结合聚类分析、主成分分析和正交偏最小二乘-判别分析，同时测定没食子酸、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批坐浴二号水煎液有 14 个共有峰，相似度均大于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≥0.990)，没食子酸、绿原酸、羟基红花黄色素A、蒙花苷回收率的平均值分别为98.04%、99.45%、99.79%、99.52%；RSD分别为2.7%、4.8%、5.6%、5.6%（n=6），精密度、稳定性、重复性良好。没食子酸含量为1.165mg/mL-3.054mg/mL、绿原酸含量为0.426mg/mL-0.939mg/mL、羟基红花黄色素A含量为0.144mg/mL-0.360mg/mL、蒙花苷含量为0.087mg/mL-0.224mg/mL。结论 所建立的 HPLC指纹图谱和4个成分的含量测定方法具有良好的灵敏度、重复性和稳定性，可为坐浴二号水煎液的质量控制和临床安全应用提供科学依据。

Abstract:

ABSTRACT: OBJECTIVE The high performance liquid chromatography ( HPLC ) fingerprints of 15 batches of Zuo Yu No.2 decoction and the content determination methods of 4 components were established to provide reference for the quality control and clinical rational application of Zuo Yu No.2. METHODS Amethyst C18-H column ( 4.6 × 250mm, 5μm ) 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. The chromatographic fingerprint similarity evaluation system of traditional Chinese medicine (2012 edition) was used to establish the fingerprint of 15 batches of Zuo Yu No.2 decoction. Based on the fingerprint similarity evaluation, cluster analysis, principal component analysis and orthogonal partial least squares-discriminant analysis were used to simultaneously determine the contents of gallic acid, chlorogenic acid, hydroxysafflor yellow A and buddleoside. CONCLUSION The established HPLC fingerprint method met the methodological requirements. There were 14 common peaks in 15 batches of Zuo Yu No.2 decoction, and the similarity was greater than 0.900. Four components of 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 four index components had good linear relationships in their respective linear ranges ( r ≥ 0.990 ). 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 precision, stability and repeatability were good. The content of gallic acid was 1.165mg / mL-3.054mg / mL, the content of chlorogenic acid was 0.426mg / mL-0.939mg / mL, the content of hydroxysafflor yellow A was 0.144mg / mL-0.360mg / mL, and the content of linarin was 0.087mg / mL-0.224mg / mL. CONCLUSION The established HPLC fingerprint and the content determination method of the four components have good sensitivity, repeatability and stability, which can provide scientific basis for the quality control and clinical safety application of the water decoction of Zuoyu No.2.