生地黄HPLC指纹图谱的建立及其HPLC-ESI-MS分析

Establishment of HPLC fingerprint of Rehmanniae Radix and its HPLC-ESI-MS analysis

目的 建立基于HPLC指纹图谱的生地黄的质量评价方法，并通过HPLC-ESI-MS技术对生地黄药材中的化学成分进行定性分析。方法 生地黄样品采用甲醇超声提取，采用Agilent Extend C18（250 mm×4.6 mm，5 μm），以乙腈-0.1%磷酸水溶液为流动相梯度洗脱，检测波长为215 nm，对15批次不同来源生地黄药材进行相似度评价分析，并采用UPLC-LTQ-Orbitrap MS技术对生地黄的化学成分进行全面定性分析。结果 建立15批生地黄药材HPLC指纹图谱，获得10个共有色谱峰且相似度在0.910~0.982。通过对照品指认、文献比对以及高分辨质谱数据解析，初步鉴定了其中67个化学成分。结论 所建立的生地黄的HPLC指纹图谱专属性强，并结合液质联用技术的定性分析，为生地黄药材的质量控制提供了可行的方法。

Objective To establish a quality assessment method for Rehmanniae Radix based on HPLC fingerprint and qualitatively analyze the chemical constituents by HPLC-ESI-MS.Methods The methanol extract of Rehmanniae Radix by ultrasonication was separated on an Agilent Extend C18 column （250 mm×4.6 mm,5 μm） with gradient elution.The mobile phase consisted of acetonitrile-0.1% phosphoric acid.The detection wavelength was 215 nm.The similarity evaluation of 15 batches of Rehmanniae Radix from different sources was carried out by the ＂Similarity Evaluation System for Chromatographic Fingerprints of TCM＂（Chinese Pharmacopoeia Commission,version 2004A）.Ultra high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer （UPLC-LTQ-Orbitrap MS） was used to characterize the chemical constituents of the methanol extract of Rehmanniae Radix.Results The chromatographic fingerprints of 15 batches of Rehmanniae Radix were generated with 10 common peaks.The similarity scores between each material batch and the reference fingerprint ranged from 0.910-0.982.A total of 67 components were putatively identified along with six unknown components via referring to reference components and literatures and analyzing MS/MS data.Conclusion The established method of fingerprint is specific and can be a reliable method used for quality assessment of Rehmanniae Radix.