Cao Wen, Hong Liang, Yang Ming, Li Shaoping, Zhao Jing
State Key Laboratory for Quality Research of Chinese Medicine, University of Macau, Macao 999078, China.
Jiangxi Guoyao Pharmaceutical Co., Ltd., Nanchang 330096, China.
Se Pu. 2021 Sep;39(9):1006-1011. doi: 10.3724/SP.J.1123.2021.06022.
Currently, guanosine, adenosine, and uridine contents are specified as the quality criteria for related products in the quality standards for fermented powder preparations included in the 2020 edition of Chinese Pharmacopoeia. However, there are many other nucleosides in fermented powder, whose effect on the quality control has not yet been discussed. In this study, an ultra-performance liquid chromatography-ultraviolet detection (UPLC-UV) method was used for the quantitative analysis of 9 nucleosides (uracil, cytidine, guanidine, uridine, adenine, inosine, guanosine, thymidine, and adenosine) in 19 batches of fermented powder samples and products, and the corresponding fingerprints were established. In addition, a method for analyzing the index components was proposed based on statistics. By optimizing the sample extraction method, ultrasound-assisted extraction was selected to process 19 batches of samples. Chromatographic analysis was performed on an Agilent Eclipse Plus C18 column (150 mm×4.6 mm, 3.5 μm) using methanol and water as the mobile phases under gradient elution. The method was validated based on the calibration curves, accuracy, precision, repeatability, and recovery. The fingerprints of the 19 batches of samples were established, and 16 common peaks were obtained. Among them, nine nucleoside peaks were identified by standards, and their concentrations were determined by the external standard one-point method. Similarity evaluation of the fingerprints was conducted; the similarities of the 19 batches of samples were greater than 0.9. Then, chemical pattern recognition was performed. The same classification results were obtained by hierarchical clustering analysis (HCA) and principal component analysis (PCA). Thus, the samples could be segregated into five classes, and the fermented powders were classified as two types with different fermentation processes. Xinganbao capsules, Bailing capsules and Ningxinbao capsules were each separately classified into one class. This indicated that the chemical recognition pattern could effectively distinguish between the fermented powder and different products. PCA was used to calculate the weight value of each common peak for the first time, and the index components among the samples were selected according to the weight value. Finally, the selected index components were used to re-cluster the samples. The results were consistent with those obtained on the basis of the 16 common peaks, thus verifying the rationality of the index components. Therefore, uridine, guanosine, adenosine, adenine, and uracil are recommended for use as evaluation indicators for fermented powder and products, allowing for better distinction between the products on the market. In summary, the combination of liquid chromatographic fingerprints and chemical pattern recognition can provide a simple and reliable method for the analysis and quality control of fermented powder and products.
目前，《中国药典》2020年版发酵粉制剂质量标准中规定鸟苷、腺苷和尿苷含量为相关产品的质量控制指标。然而，发酵粉中还存在许多其他核苷，其对质量控制的影响尚未见讨论。本研究采用超高效液相色谱 - 紫外检测（UPLC - UV）法对19批发酵粉样品及产品中的9种核苷（尿嘧啶、胞苷、胍、尿苷、腺嘌呤、肌苷、鸟苷、胸苷和腺苷）进行定量分析，并建立了相应的指纹图谱库。此外，基于统计学提出了一种指标成分分析方法。通过优化样品提取方法，选择超声辅助提取法处理19批样品。采用甲醇和水作为流动相，在梯度洗脱条件下，于Agilent Eclipse Plus C18柱（150 mm×4.6 mm，3.5 μm）上进行色谱分析。该方法通过校准曲线、准确度、精密度、重复性和回收率进行验证。建立了19批样品的指纹图谱，共获得16个共有峰。其中，9个核苷峰通过对照品进行了指认，并采用外标一点法测定了其含量。对指纹图谱进行相似度评价，19批样品的相似度均大于0.9。然后，进行化学模式识别。层次聚类分析（HCA）和主成分分析（PCA）得到了相同的分类结果。样品可分为五类，发酵粉按发酵工艺不同分为两种类型。心肝宝胶囊、百令胶囊和宁心宝胶囊各自单独分为一类。这表明化学识别模式能够有效区分发酵粉及不同产品。首次采用PCA计算各共有峰的权重值，并根据权重值筛选样品中的指标成分。最后，用筛选出的指标成分对样品重新聚类，结果与基于16个共有峰得到的结果一致，从而验证了指标成分的合理性。因此，建议将尿苷、鸟苷、腺苷、腺嘌呤和尿嘧啶作为发酵粉及产品的评价指标，以便更好地区分市场上的产品。综上所述，液相色谱指纹图谱与化学模式识别相结合可为发酵粉及产品的分析和质量控制提供一种简单可靠的方法。
