清肺汤中8种有效成分同时测定及其指纹图谱研究

Simultaneous determination of eight active components in Qingfei Decoction by HPLC and its fingerprint analysis

目的采用HPLC-VWD同时测定清肺汤市售制剂中绿原酸、苦杏仁苷、栀子苷、橙皮苷、黄芩苷、汉黄芩苷、甘草酸铵、五味子醇甲等指标成分的含量并建立清肺汤的指纹图谱,表征与区分不同来源的清肺汤市售制剂。方法采用Agilent Technologies Zorbax SB-C18(250 mm×4.6 mm,3.5μm)色谱柱,以乙腈-0.1%磷酸水溶液为流动相,梯度洗脱,柱温35℃,体积流量0.6 mL/min,进样量10μL,同时对指标成分的含量和指纹图谱进行检测。采用指纹图谱相似度评价、热图聚类分析等方法,表征与区分不同来源清肺汤市售制剂。结果8种成分专属性、线性关系(r2>0.9998)、精密度(RSD≤1.85%,n=6)、重复性(RSD≤1.82%,n=6)、稳定性(RSD≤1.49%,48h)均良好;加样回收率为(93.19±1.93)%^(102.36±4.17)%(n=6)。含量测定结果显示清肺汤市售制剂中指标成分的每日服用量在不同厂家中波动较大,如黄芩苷的波动范围59.85~224.05mg;但在相同厂家的不同剂型中波动不明显,如港香兰药厂2种制剂中黄芩苷质量为140.00~142.47 mg;同时指纹图谱相似度计算结果均大于0.945,表明不同来源的清肺汤市售制剂指纹图谱相似度较高。结论该方法准确可靠、具有良好的重复性,可用于测定清肺汤市售制剂中8种成分的含量及建立清肺汤的指纹图谱,通过成分每日服用量热图聚类分析与相似度评价,能表征与区分不同来源的清肺汤市售制剂。

Objective To simultaneously determinate the content of chlorogenic acid,amygdalin,gardenoside,hesperidin,baicalin,wogonoside,ammonium glycyrrhizate and schisandrin in Qingfei Decoction by HPLC-VWD,and establish the fingerprint of Qingfei Decoction.The results can be applied to characterize and distinguish commercially available Qingfei Decoction preparations from different manufacturers.Methods Agilent Technologies Zorbax SB-C18(250 mm×4.6 mm,3.5μm)column was used,with acetonitrile(A)-0.1%phosphoric acid aqueous solution(B)as the mobile phase for gradient elution,column temperature of 35℃,flow rate of 0.6 mL/min,injection volume of 10μL.The content of the indicator components and the fingerprints were simultaneously assayed by the above method.Fingerprint similarity evaluation,heat map cluster analysis and other methods were used to characterize and distinguish the commercially available Qingfei Decoction preparations from different sources.Results The specificity,linear relationship(r2>0.9998),precision(RSD≤1.85%,n=6),repeatability(RSD≤1.82%,n=6),stability(RSD≤1.49%,48 h)of eight components were good;The recovery rate of the sample was in the range of(93.19±1.93)%to(102.36±4.17)%(n=6).The results of content determination showed that the daily dosage of the indicator ingredients in the marketed Qingfei Decoction preparations fluctuated greatly in different manufacturers,such as baicalin,which fluctuated from 59.85 to 224.05 mg,but the fluctuation was not obvious in different dosage forms of the same manufacturer,for example,the content of baicalin in the two preparations of KPC Herbs was 140.00 to 142.47 mg.At the same time,the similarity calculation results of fingerprints were all greater than 0.945,which indicated that the fingerprints of Qingfei Decoction from different sources were highly similar.Conclusion The method is accurate and reliable with good repeatability.It can be used to determine the content of eight components in the commercial Qingfei Decoction preparations and establish the fingerprint of Qingfei Decoction.The commercial Qingfei Decoction preparations can be characterized by heat map clustering analysis of the daily dosage of the eight components and the fingerprint similarity evaluation to distinguish the commercially available preparations of Qingfei Decoction from different sources.