羊红膻HPLC指纹图谱及10种成分含量测定研究

Study on HPLC fingerprint and determination of 10 components in Pimpinella thellungiana Wolff.

目的建立羊红膻药材的HPLC指纹图谱,并测定其中10种成分的含量,为羊红膻的质量控制提供依据。方法采用Agilent TC-C18(250 mm×4.6 mm,5μm)色谱柱,乙腈-混合酸(0.1%磷酸-0.1%冰醋酸)进行梯度洗脱,切换检测波长分别为265 nm和325 nm,柱温为20℃,体积流量为1.0 mL/min。应用"中药色谱指纹图谱相似度评价软件(2012版)"对10批羊红膻药材进行分析评价,建立羊红膻的HPLC指纹图谱,通过对照品比对指认共有峰,并对其中10种成分进行含量测定。结果建立了羊红膻HPLC指纹图谱,确定了20个共有峰,指认了其中13个色谱峰,分别为没食子酸、原儿茶酸、新绿原酸、绿原酸、隐绿原酸、咖啡酸、夏佛塔苷、木犀草素-7-O-β-D-葡萄糖醛酸苷、异绿原酸B、异绿原酸A、芹菜苷、芹菜素-7-O-β-D-葡萄糖醛酸苷和异绿原酸C,10批样品的相似度均大于0.936。定量测定的10种成分在各自范围内线性关系良好(r﹥0.9997),平均加样回收率98.87%~101.02%。RSD为0.47%~1.69%。结论所建立的羊红膻HPLC指纹图谱及10种成分的含量测定方法稳定、可靠,可以对羊红膻进行较全面的质量控制。

Objective To establish HPLC fingerprint and determine 10 components in Pimpinella thellungiana Wolff.,which provide evidence for quality control of Pimpinella thellungiana Wolff..Methods The chromatographic separation was performed on an Agilent TC-C18(250 mm×4.6 mm,5μm)with gradient elution using acetonitrile and mixed acid solution(0.1%phosphoric acid-0.1%glacial acetic acid)as the mobile phase.The detection wavelength was switched from 265 nm to 325 nm at 16 min.The column temperature was kept at 20℃,the flow rate was 1.0 mL/min.The HPLC fingerprint of 10 batches of Pimpinella thellungiana Wolff.was established using Similarity Evaluation Software for Chromatographic Fingerprint of Traditional Chinese Medicine(2012 edition)and the common peaks were identified by standard references.10 constituents in Pimpinella thellungiana Wolff.were quantified.Results 20 common peaks were confirmed,and 13 common peaks were identified by standard references including gallic acid,protocatechuic acid,neochlorogenic acid,chlorogenic acid,cryptochlorogenic acid;caffeic acid,schaftoside,luteolin-7-O-β-D-glucuronide,isochlorogenic acid B isochlorogenic acid A,apigetrin,apigenin-7-O-β-D-glucuronide and isochlorogenic acid C.The similarity of 10 batches samples was over 0.936.10 analytes had good separation and showed good linearity in their respective linear ranges(r>0.9997).The average recoveries ranged from 98.87%to 101.02%,and the RSDs ranged from 0.47%to 1.69%.Conclusion The established HPLC fingerprint and quantification method is stable and reliable,which could provide basis for the quality control of Pimpinella thellungiana Wolff.