芍药总苷高效液相色谱指纹图谱研究

HPLC fingerprinting of total glucosides of paeony

目的 研究芍药总苷的高效液相色谱指纹图谱 ,为科学评价及有效控制其质量提供可靠方法。方法 利用HPLC DAD方法 ,梯度洗脱 ,测定了 10批芍药总苷样品。色谱条件为 :SupelcosilTM LC 18分析柱 (5 μm ,15 0mm× 4 6mm) ,柱温 2 5℃ ,流动相A为乙腈 ;流动相B为水 (磷酸调pH为 3 0 ) ,流动相A梯度洗脱 (10 %～ 4 5 %乙腈 ) ,分析时间为 5 0min ,时间为 0 ,5 ,2 5 ,2 7,38,4 0 ,5 0min ,A(% )为 10 ,15 ,18,30 ,35 ,4 0 ,4 0 %。结果 10批芍药总苷样品得到的色谱指纹图谱有 2 2个共有峰 ,可分为 3个部分 :保留时间 0～ 5min处 ,出现 3个小峰 ;保留时间 5～ 2 0min处 ,有 8个峰 ,其主要特征峰 5 ,6和 7均在此区域 ,通过与标准品的保留时间及紫外光谱比较 ,5和 6号峰分别鉴定为芍药内酯苷和芍药苷 ;保留时间 2 0～ 36min处 ,有 11个峰 ,另一个主要特征峰 12在此区域。最强峰为 6号峰 ,其次为 12 ,5和7,相对峰面积比值为 5∶6∶7∶12 =0 2 6 7～ 0 348∶1∶0 10 8～ 0 135∶0 4 0 5～ 0 5 37。相同色谱条件下测定了白芍药材的HPLC图谱 ,其结果与芍药总苷有很好相关性。结论 芍药总苷的指纹图谱特征性及专属性强 ,可结合含量测定用于全面控制芍药总苷的质量 ,确保每批产品的均一性。

Aim To establish a sensitive and specific HPLC method for controlling the quality of total glucosides from paeony (TGP). Methods HPLC method was applied for quality assessment of TGP. The preparation of sample, the HPLC column, mobile phase, elution mode (isocratic or gradient) and gradient program were optimized in order to obtain high quality HPLC profile. The HPLC system consisted of a Waters 600 pump and a 996 photodiode-array detector (DAD). Data were acquired and processed with the Millennium 2010 workstation. HPLC analysis was performed on a Supelcosil TM LC-18 column (5 μm, 150 mm×4.6 mm) with the mixture of acetonitrile (A) and H 2O (acidified to pH 3.0 with phosphoric acid) (B) as mobile phase in gradient mode. The concentrations of solvent A were 10%, 15%, 18%, 30%, 35%, 40% and 40% at 0, 5, 25, 27, 38, 40 and 50 min, respectively. The column temperature was setup at 25 ℃ and the flow-rate was 1 mL·min -1 . The reference solution of chemical standards and sample were injected into HPLC system, respectively. Results The HPLC chromatographic fingerprinting of TGP,showing 22 characteristic peaks,was established from 10 lots of TGP products. These peaks could be divided into 3 groups: From 1 to 3 min, there are 3 low peaks. From 5 to 20 min, there are 8 peaks including the main diagnostic peaks 5, 6 and 7. By comparison of the retention time and the on-line UV spectra of chemical standards, peak 5 and 6 were identified as albiforin and paeoniflorin, respectively. From 20 to 36 min, 11 peaks were observed, in which peak 12 was one of the main diagnostic peaks. The ratios of peak area between 5, 6, 7 and 12 were 0.267-0.348∶1∶0.108-0.135∶0.405-0.537. Moreover, comparison of the HPLC profiles of TGP and the roots of Paeonia lacttiflora Pall indicated that they were closely related to each other. Conclusion The chromatographicfingerprinting of TGP with high specificity can be used to control its quality and assure lot to lot consistency .