基于HPLC指纹图谱、多指标成分含量测定及化学计量学的槐芩软膏质量评价

Quality evaluation of Huaiqin ointment based on combinative methods of HPLC fingerprint,quantitative analysis of multi-components and chemometrics analysis

目的:建立指纹图谱和多指标定量与化学计量学相结合的槐芩软膏质量评价方法。方法:采用Agilent Extend C_(18)色谱柱(4.6 mm×250 mm,5μm),柱温25℃,流动相为乙腈-0.2%磷酸水溶液,梯度洗脱,体积流量为1.0 mL·min^(-1),检测波长230 nm。利用中药色谱指纹图谱相似度评价软件(2012A版)对10批槐芩软膏建立指纹图谱,确定共有峰并进行相似度评价,并对芍药苷、芦丁、染料木苷、槐角苷、柚皮苷、新橙皮苷、黄芩苷、汉黄芩苷和丹皮酚的含量测定方法进行方法学验证。基于指纹图谱共有峰峰面积测定结果,采用聚类分析和主成分分析等化学计量学方法对不同批次的槐芩软膏进行质量评价。结果:在指纹图谱研究中,标定了21个共有峰,通过与对照品比较指认其中9个色谱峰,并对指认共有峰进行了含量测定。10批槐芩软膏与对照图谱的相似度均在0.90以上。9个成分在各自的质量浓度范围内线性关系良好,平均加样回收率为97.4%~99.2%,RSD为0.86%~1.4%;10批次样品中芍药苷、芦丁、染料木苷、槐角苷、柚皮苷、新橙皮苷、黄芩苷、汉黄芩苷和丹皮酚的含量分别为5.34~6.31、3.67~4.81、0.76~1.27、10.95~12.70、6.70~7.14、6.78~7.15、17.45~18.92、4.08~4.36、0.57~0.69 mg·g^(-1)。共聚为3类,样品1和2聚为1类,样品6聚为1类,其余样品聚为1类;主成分1~4是影响槐芩软膏质量评价的主要因子。结论:所建立的槐芩软膏质量评价方法准确、高效、重复性和稳定性好,特征性强,可用于槐芩软膏的质量控制。

Objective:To establish a quality evaluation method of Huaiqin ointment based on HPLC fingerprints,quantitative analysis of multi-components and chemometrics analysis.Methods:The chromatographic column was Agilent Extend C;column(250 mm×4.6 mm,5μm),and the column temperature was set at 25℃.The detection wavelength was set at 230 nm.The mobile phase was composed of acetonitrile-0.2%phosphate acid solution in gradient elution manner at a flow rate of 1.0 mL·min^(-1).The HPLC fingerprint of Huaiqin ointment was established,the common peaks were identified by similarity evaluation system for chromatographic fingerprint of Tradition Chinese Medicine(Version 2012),and the similarity was calculated.The content determination methods for paeoniflorin,rutin,enistin,sophoricoside,naringin,neohesperidin,baicalin,wogonoside and paeonolwere validated.The chemometrics methods such as cluster analysis and principal component analysis were used to evaluate the quality of Huaiqin ointment with different batches based on the results of fingerprint common peak area.Results:The fingerprint of Huaiqin ointment was established.Twenty-one common peaks were identified.The similarities of fingerprints of 10 batches of samples were above 0.95.Nine components had good linear relationship in the range of mass concentration,and the average recoveries were 97.4%-99.2%with the RSDs of 0.86-1.4%.The concentrations of paeoniflorin,rutin,genistin,sophoricoside,naringin,neohesperidin,baicalin,wogonoside and paeonolin 10 batches of samples were 5.34-6.31,3.67-4.81,0.76-1.27,10.95-12.70,6.70-7.14,6.78-7.15,17.45-18.92,4.08-4.36 and 0.57-0.69 mg·g^(-1),respectively.The results of cluster analysis showed that 10 batches of Huaiqin ointment were clustered into three groups,and the results of principal component analysis showed that the principal components 1-4 were the main factor affecting the quality evaluation of Huaiqin ointment.Conclusion:The method is simple,accurate and reproducible,which can be used for the quality control and evaluation of Huaiqin ointment.