五淋丸高效液相色谱指纹图谱研究及7种成分含量测定

HPLC Fingerprint of Wulin Pills and Determination of Seven Components

目的为五淋丸的质量控制提供依据。方法采用高效液相色谱(HPLC)法建立6批五淋丸样品的指纹图谱,色谱柱为Anpel Athena C_(18)柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.1%磷酸水溶液(梯度洗脱),流速为1.0 mL/min,检测波长为240 nm(指纹图谱建立及栀子苷、芍药苷、甘草酸铵含量测定)及327 nm(绿原酸、盐酸巴马汀、盐酸小檗碱、藁本内酯含量测定),柱温为25℃,进样量为10μL。采用中药色谱指纹图谱相似度评价系统(2012版)进行相似度评价,确定共有峰及归属,指认成分并测定其含量。结果6批样品指纹图谱与对照指纹图谱的相似度均在0.992~1.000之间。标定共有峰12个,均确定归属药材,并指认出其中7个共有峰成分。栀子苷、芍药苷、甘草酸铵、绿原酸、盐酸巴马汀、盐酸小檗碱、藁本内酯质量浓度分别在2.77~27.74μg/mL、16.10~161.04μg/mL、8.54~85.42μg/mL、0.71~7.05μg/mL、2.75~27.54μg/mL、3.14~31.35μg/mL、5.43~54.27μg/mL范围内与峰面积线性关系良好(R^(2)≥0.9995,n=6);精密度、稳定性、重复性试验结果的RSD均小于2.0%;平均加样回收率分别为93.48%,103.15%,104.68%,93.53%,95.76%,104.85%,97.25%,RSD分别为2.50%,1.90%,1.67%,2.05%,1.99%,1.48%,2.33%(n=6)。结论本研究中所建方法可用于五淋丸HPLC指纹图谱的建立及其中7个成分的含量测定,可更全面地为五淋丸的质量控制提供依据。

Objective To provide a basis for the quality control of Wulin Pills.Methods High-performance liquid chromatography(HPLC)method was used to establish the fingerprint of six batches of Wulin Pills.The chromatographic column was the Anpel Athena C_(18)column(250 mm×4.6 mm,5μm),the mobile phase was acetonitrile-0.1%phosphoric acid aqueous solution(gradient elution),the flow rate was 1.0 mL/min,the detection wavelengths were 240 nm(fingerprint establishment and determination of geniposide,paeoniflorin,ammonium glycyrrhizinate)and 327 nm(determination of chlorogenic acid,palmatine hydrochloride,berberine hydrochloride,ligustilide),the column temperature was 25℃,and the injection volume was 10μL.The similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine(version 2012)was used for similarity evaluation,the common peaks were marked and attributed,the components were identified and determined.Results The similarity between the fingerprint of the six batches of samples and the reference fingerprint was in the range of 0.992-1.000.Twelve common peaks were marked and attributed to medicinal herbs,and seven components were identified.The linear ranges of geniposide,paeoniflorin,ammonium glycyrrhizinate,chlorogenic acid,palmatine hydrochloride,berberine hydrochloride and ligustilide were 2.77-27.74μg/mL,16.10-161.04μg/mL,8.54-85.42μg/mL,0.71-7.05μg/mL,2.75-27.54μg/mL,3.14-31.35μg/mL,5.43-54.27μg/mL(R^(2)≥0.9995,n=6)respectively.The RSDs of precision,stability and repeatability tests were all lower than 2.0%.The average recovery rates of the above seven components were 93.48%,103.15%,104.68%,93.53%,95.76%,104.85%,97.25%,with RSDs of 2.50%,1.90%,1.67%,2.05%,1.99%,1.48%,2.33%(n=6)respectively.Conclusion The established method in this study can be used to establish the HPLC fingerprint of Wulin Pills and determine the contents of seven components,which provides a basis for more comprehensive quality control of Wulin Pills.