目的 研究牛黄上清丸全二维液相色谱指纹图谱的评价方法，并建立三维指纹图谱，为牛黄上清丸的质量控制提供新方法。方法 第一维采用Acquity UPLC HSS CYANO（100 mm×2.1 mm，1.8 μm）色谱柱，柱温40℃，流动相为0.1%甲酸水溶液-甲醇，体积流量0.1 mL/min；第二维采用Kinetex Phenyl-Hexyl（50 mm×3 mm，2.6 μm）色谱柱，流动相为0.1%甲酸水溶液-乙腈，体积流量1.5 mL/min，柱温40℃，检测波长254 nm，样品采集速率为12.5 Hz；连接第一维与第二维色谱柱的定量环体积为100 μL。建立牛黄上清丸的全二维液相色谱指纹图谱，采用欧氏距离、夹角余弦和相关系数法，分别以各共有峰峰体积的中位数、平均数的计算结果为标准对照指纹图谱，对21批市售牛黄上清丸样品进行相似度评价。结果 建立了21批市售牛黄上清丸的三维指纹图谱，标定了共有峰18个，并指认了其中的5个共有峰：栀子苷（1号峰）、胡薄荷酮（8号峰）、黄芩苷（9号峰）、欧前胡素（15号峰）、汉黄芩素（16号峰）。结论 首次建立了基于全二维液相色谱的牛黄上清丸的指纹图谱及其相似度的评价方法，说明该法可用于中药色谱指纹图谱的建立，该方法也是对传统液相色谱指纹图谱的补充和提高。

Objective To develop a new three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using Niuhuang Shangqing Pills (NSP) as an example. The developed method could offer new method for the quality control of NSP. Methods In first dimension, the separation was achieved with an Acquity UPLC HSS CYANO column (100 mm×2.1 mm, 1.8 μm), methanol-0.1% formic acid in water were used as mobile phases, flow rates were 0.1 mL/min. In second dimension, the separation was achieved with a Kinetex Phenyl-Hexyl column (50 mm×3 mm, 2.6 μm), acetonitrile-0.1% formic acid in water were used as mobile phases, flow rates were 1.5 mL/min, detection wavelength was set at 254 nm, and acquiring frequency was at 12.5 Hz. Column temperature for each dimension was 40℃ and volume of loop linking the two dimensions was 100 μL. Three similarity-calculating methods, Euclidean Distance, Cosine, and Correlation Coefficient, were employed to assess the similarities among the 21 samples on the market using medians with arithmetic means of peak volumes of the common peaks as control fingerprints. Results The three-dimensional fingerprints of 21 batches of NSP samples on the market were developed; Eighteen common peaks were assigned and five of them were identified, which were geniposide (1), pulegone (8), baicalin (9), imperatorin (15), and wogonin (16). Conclusion A three-dimensional fingerprinting method and its assessing methods based on comprehensive two-dimensional liquid chromatography using NSP as an example were successfully developed for the first time, suggesting that it is a feasible method for developing fingerprints for Chinese materia medica. This work improves and supplements the traditional liquid-chromatography fingerprints.

国家自然科学基金项目（81560641）；江西省中医药科技计划项目（2017A285）