Huang Bao-Ming, Xiao Sheng-Yuan, Chen Ting-Bo, Xie Ying, Luo Pei, Liu Liang, Zhou Hua
Faculty of Chinese Medicine, Macau University of Science and Technology, Taipa, Macau, PR China; State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology), Taipa, Macau, PR China.
School of Life Science, Beijing Institute of Technology, Beijing, PR China.
J Pharm Biomed Anal. 2017 May 30;139:193-204. doi: 10.1016/j.jpba.2017.02.055. Epub 2017 Mar 6.
Ginseng herbs comprise a group of the most popular herbs, including Panax ginseng, P. notoginseng and P. quinquefolius (Family Araliaceae), which are used as traditional Chinese medicine (TCM) and are some of the best-selling natural products in the world. The accurate quantification of ginsenoside Rg1 is one of the major aspects of its quality control. However, the purity of the commercial Rg1 chemical reference substance (CRS) is often measured with high-performance chromatography coupled with an ultraviolet detector (HPLC-UV), which is a selective detector with unequal responses to different compounds; thus, this detector introduces probable error to purity assessments. In the present study, quantitative nuclear magnetic resonance (qNMR), due to its absolute quantification ability, was applied to accurately assess the purity of Rg1 CRS. Phenylmethyl phthalate was used as the internal standard (IS) to calibrate the purity of Rg1 CRS. The proton signal of Rg1 CRS in methanol-d4 at 4.37ppm was selected to avoid interfering signals, enabling accurate quantitative analysis. The relaxation delay, number of scans, and NMR windowing were optimized for data acquisition. For post-processing, the Lorentz/Gauss deconvolution method was employed to increase the signal accuracy by separating the impurities and noise in the integrated region of the quantitative proton. The method validation showed that the developed method has acceptable sensitivity, linearity, precision, and accuracy. The purity of the commercial Rg1 CRS examined with the method developed in this research was 90.34±0.21%, which was obviously lower than that reported by the manufacturer (>98.0%, HPLC-UV). The cross-method validation shows that the commonly used HPLC-UV, HPLC-ELSD (evaporative light scattering detector) and even LC-MS (mass spectrometry) methods provide significantly higher purity values of Rg1 CRS compared with the qNMR method, and the accuracy of these LC-based methods largely depend on the amount of the sample that was loaded and the properties of the impurities.
人参类草药是最受欢迎的草药种类之一，包括人参、三七和西洋参（五加科），它们被用作传统中药，也是世界上最畅销的天然产品之一。人参皂苷Rg1的准确定量是其质量控制的主要方面之一。然而，市售Rg1化学对照品（CRS）的纯度通常采用高效液相色谱-紫外检测器（HPLC-UV）进行测定，这是一种对不同化合物响应不均等的选择性检测器；因此，该检测器会给纯度评估带来可能的误差。在本研究中，定量核磁共振（qNMR）因其绝对定量能力，被用于准确评估Rg1 CRS的纯度。邻苯二甲酸苯甲酯被用作内标（IS）来校准Rg1 CRS的纯度。选择Rg1 CRS在氘代甲醇中4.37ppm处的质子信号以避免干扰信号，从而实现准确的定量分析。对弛豫延迟、扫描次数和核磁共振窗口进行了优化以进行数据采集。在数据后处理方面，采用洛伦兹/高斯去卷积方法，通过分离定量质子积分区域中的杂质和噪声来提高信号准确性。方法验证表明，所建立的方法具有可接受的灵敏度、线性、精密度和准确度。用本研究建立的方法检测的市售Rg1 CRS的纯度为90.34±0.21%，明显低于制造商报告的纯度（>98.0%，HPLC-UV）。方法间交叉验证表明，与qNMR方法相比，常用的HPLC-UV、HPLC-蒸发光散射检测器（ELSD）甚至液相色谱-质谱（LC-MS）方法提供的Rg1 CRS纯度值明显更高，并且这些基于液相色谱的方法的准确性在很大程度上取决于进样量和杂质的性质。
