颠茄草药材高效液相色谱特征图谱及多成分含量测定研究

HPLC Characteristic Chromatograms of Belladonnae Herba and Multi-Component Content Determination

目的建立颠茄草药材高效液相色谱(HPLC)特征图谱及7个主要成分的含量测定方法。方法采用HPLC法,色谱柱为Ultimate■XB-C_(18)柱(250 mm×4.6 mm,5μm),流动相为乙腈-0.05%甲酸水溶液(梯度洗脱),流速为0.8 mL/min,检测波长为290 nm,柱温为30℃,进样量为10μL。以硫酸天仙子胺峰为参照,建立16批药材样品的HPLC指纹图谱,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,并进行聚类分析、偏最小二乘-判别分析法,结合变量重要性投影值筛选主要差异成分。在上述色谱条件下测定7个主要成分含量。结果16批药材样品指纹图谱共标定了13个共有峰,相似度为0.939~0.993。经聚类分析和偏最小二乘-判别分析法,16批药材样品均可聚为3类,变量重要性投影值筛选出13个差异成分,其中7个已知。东莨菪苷、绿原酸、氢溴酸东莨菪碱、硫酸天仙子胺、东莨菪内酯、芦丁、三叶豆苷质量浓度分别在5.04~151.18μg/mL、5.11~153.41μg/mL、4.63~138.79μg/mL、5.91~177.22μg/mL、5.03~150.90μg/mL、5.00~149.98μg/mL、5.97~179.16μg/mL范围内与峰面积线性关系良好(R^(2)>0.9990,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100.82%,102.22%,101.55%,101.98%,101.50%,100.34%,100.17%,RSD分别为2.00%,1.37%,2.53%,1.11%,2.73%,2.14%,1.17%(n=9)。7个成分含量分别为0.32~0.81 mg/g、0.68~1.01 mg/g、0.33~0.46 mg/g、1.57~1.99 mg/g、0.37~0.64 mg/g、0.35~0.79 mg/g、0.74~1.90 mg/g。结论所建立的特征图谱、化学计量学方法和多成分含量测定方法,明确了颠茄草药材部分差异成分,为该药材的质量控制提供了参考。

Objective To establish the high-performance liquid chromatography(HPLC)characteristic chromatograms of Belladonnae Herba and a method for the content determination of seven main components in this medicinal herb.Methods The HPLC method was adopted,the chromatographic column was the Ultimate®XB-C_(18) column(250 mm×4.6 mm,5μm),the mobile phase was acetonitrile-0.05%formic acid aqueous solution(gradient elution),the flow rate was 0.8 mL/min,the detection wavelength was 290 nm,the column temperature was 30℃,and the injection volume was 10μL.With hyoscyamine sulfate as a reference,the HPLC fingerprints of 16 batches of medicinal herb samples were established,and the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 Version)was used for similarity evaluation.The cluster analysis(CA)and partial least square-discriminant analysis(PLS-DA)were performed,and the main differential components were identified based on variable importance in projection(VIP)values.The contents of seven main components were determined by the above chromatographic conditions.Results A total of 13 common peaks were marked in the fingerprints of 16 batches of medicinal herb samples,with the similarity in the range of 0.939 to 0.993.The CA and PLS-DA showed that 16 batches of medicinal herb samples were clustered into three categories,and 13 differential components were screened based on the VIP values,among which seven were known.The linear ranges of scopolin,chlorogenic acid,scopolamine hydrobromide,hyoscyamine sulfate,scopoletin,rutin and trifolin were 5.04-151.18μg/mL,5.11-153.41μg/mL,4.63-138.79μg/mL,5.91-177.22μg/mL,5.03-150.90μg/mL,5.00-149.98μg/mL,5.97-179.16μg/mL(R^(2)>0.9990,n=6),respectively.The RSDs of precision,stability and repeatability tests were all lower than 2.0%.The average recovery rates of the above components were 100.82%,102.22%,101.55%,101.98%,101.50%,100.34%,100.17%,with the RSDs of 2.00%,1.37%,2.53%,1.11%,2.73%,2.14%,1.17%respectively(n=9).The contents of the above seven components were in the range of 0.32 to 0.81 mg/g,0.68 to 1.01 mg/g,0.33 to 0.46 mg/g,1.57 to 1.99 mg/g,0.37 to 0.64 mg/g,0.35 to 0.79 mg/g,0.74 to 1.90 mg/g,respectively.Conclusion The established characteristic chromatograms,chemometric methods and multi-component content determination method can indentify some differential components of Belladonnae Herba,which provides a reference for the quality control of this medicinal herb.