基于5组分测定和6波长高效液相色谱指纹谱的双标定量指纹法建立六味地黄丸对照指纹图谱动态技术标准研究

Dual referential fingerprint quantification method for dynamic technique criteria of Liuwei Dihuang Wan standard fingerprint by determination of 5 components and 6 wavelength fingerprints by high performance liquid chromatography

目的建立双标定量指纹法精确恢复对照指纹图谱(RFP)理论和方法,实现动态指纹图谱标准的校正和复原。方法用RP-HPLC法对LWDHW中5组分进行定量分析和建立其6波长定量指纹图谱研究。结果利用HPLC-DAD在最大吸收波长处同时测定了没食子酸、5-羟甲基糠醛和丹皮酚含量,结合等吸收双波长消去法对色谱峰完全重叠的芍药苷和马钱苷进行同时定量测定,建立210、230、246、265、280和310nm LWDHW-HPLC指纹谱,用均值法整合6波长HPLC定量指纹谱。以5组分测定和6波长指纹谱鉴定结果分别聚类分析比较不同厂家产品质量差异,结果以后者更为准确可靠,在此基础上建立LWDHW双标定量指纹法技术标准。结论基于多指标定量分析和平行多波长高效液相色谱指纹谱的双标定量指纹法能够找到产生RFP的标准制剂和实现实时随行定量检测LWDHW指纹图谱。这一方法再现了中药指纹图谱标准,为中药指纹图谱工业化整体定量控制中药质量建立了可行技术方法,是中药指纹图谱评价方法发展历程中的一次技术飞跃。

Objective To establish a dual referential fingerprint quantification method （DRFQ） to correct and recover a dynamic fingerprint standard. Methods Five components of Liuwei Dihuang Wan （LWDHW） were analyzed quantita- tively by RP-HPLC and the 6-wavelength quantitative fingerprint was established. Results The contents of gallic acid, 5-hydroxymethylfurfural and paeonol were determined in the maximum absorption wavelength simultaneously with HPLC-DAD. The fully overlapped 2 components （paeoniflorin and Loganin） were detected by equal absorption with double wavelength elimination method. The LWDHW-HPLC fingerprints were determined under 210, 230, 246, 265, 280 and 310 nm, and integrated by mean method. With cluster analyzing the identification results of the 5 contents and 6-wavelength fingerprint, the differences in the product quality among different manufacturers were compared, six- wave length finger print was more accurate and reliable, on which the dynamic technique criteria of LWDHW were es- tablished by DRFQ. Conclusion Based on multi-component quantitative analyses and parallel multi-wavelength high performance liquid chromatography fingerprints, standard prescription that builds RFP will be obtained and the real- time accompanying quantitative detection of LWDHW fingerprint may be realized by DRFQ. Reproducing TCM stand- ards, the method established is effective in the quantitative quality control of TCM industrialization, which is a techni- cal breakthrough in assessing TCM fingerprints.