目的 建立黄芪桂枝五物汤（Huangqi Guizhi Wuwu Decoction，HGWD）基准样品UPLC指纹图谱及质量标志物（quality markers，Q-Marker）含量测定方法，对15批HGWD基准样品进行质量评价，为后续复方制剂开发提供质量控制方法和评价体系。方法 制备HGWD基准样品，采用Waters Acquity^TM T3色谱柱（100 mm×2.1 mm，1.8 μm）；以乙腈-0.15%磷酸水溶液为流动相，梯度洗脱；柱温30 ℃；检测波长：0～18.5 min，230 nm；18.5～24.5 min，260 nm；24.5～40.0 min，280 nm；体积流量0.3 mL/min；进样体积1 μL；进行指纹图谱方法学考察，采集15批HGWD基准样品的UPLC指纹图谱，将数据结果导入“中药色谱指纹图谱相似度评价系统”（2012版），对15批HGWD基准样品指纹图谱进行相似度评价，对共有峰进行归属和聚类热图分析；同时测定15批基准样品中Q-Marker（毛蕊异黄酮葡萄糖苷、芍药苷、肉桂酸、6-姜酚）的含量。结果 HGWD基准样品UPLC指纹图谱方法学验证良好，建立了15批HGWD基准样品的UPLC指纹图谱，与对照指纹图谱比较相似度均大于0.9。确定24个共有峰，经与混合对照品比对，指认出8个色谱峰，分别为8号峰氧化芍药苷、10号峰芍药苷、12号峰毛蕊异黄酮葡萄糖苷、14号峰香豆素、18号峰肉桂酸、19号峰肉桂醛、20号峰槲皮素、24号峰6-姜酚。测定15批HGWD基准样品中4个Q-Marker毛蕊异黄酮葡萄糖苷、肉桂酸、芍药苷、6-姜酚的质量分数分别为115.49～494.96、254.68～760.79、3 106.72～6 049.40、232.61～704.10 μg/g，出膏率为11.63%～14.14%，饮片-汤液中毛蕊异黄酮葡萄糖苷、肉桂酸、芍药苷及6-姜酚的转移率分别8.23%～19.66%、23.52%～41.21%、29.15%～41.23%、14.58%～30.07%；汤液-基准样品中毛蕊异黄酮葡萄糖苷、肉桂酸、芍药苷及6-姜酚的转移率分别86.35%～97.67%、90.34%～98.13%、91.51%～98.88%、93.53%～98.29%。结论 建立的HGWD基准样品UPLC指纹图谱和Q-Marker含量测定分析方法准确、可靠，可用于黄芪桂枝汤五物汤基准样品的质量评价，作为后续制剂开发和质量控制依据。

Objective To establish UPLC fingerprint and determination method for quality markers of Huangqi Guizhi Wuwu Decoction (HGWD, 黄芪桂枝五物汤) benchmark samples and evaluate the quality of 15 batches of HGWD, so as to provide quality control method and evaluation system for the subsequent development of compound preparation. Methods The benchmark samples of HGWD were prepared by Waters Acquity^TM T3 column (100 mm×2.1 mm, 1.8 μm). Acetonitrile-0.15% phosphoric acid aqueous solution was used as mobile phase with gradient elution. Column temperature: 30 ℃; Detection wavelength: 0—18 min, 230 nm; 18.5—24.5 min, 260 nm; 24.5—40 min, 280 nm; Elution rate: 0.3 mL/min; Injection volume: 1 μL; The fingerprint methodology was investigated, 15 batches of UPLC fingerprints of HGWD benchmark samples were collected, and the data results were imported into the “TCM Chromatographic Fingerprint Similarity Evaluation System” (2012 edition). The similarity evaluation was carried out on the fingerprint of 15 batches of benchmark samples of HGWD, and the common peaks were attributed and cluster analyzed. At the same time, the contents of quality markers (calycosin-7-glucoside, paeoniflorin, cinnamic acid, 6-gingerol) in 15 batches of benchmark samples were determined. Results The methodological verification of the UPLC fingerprint of the HGWD benchmark samples was good. The UPLC fingerprints of 15 batches of the benchmark samples of HGWD were well verified. The similarity between the UPLC fingerprints and the control fingerprints was greater than 0.9. A total of 24 common peaks were identified, and eight chromatographic peaks were identified by comparing with mixed controls, namely peak 8 oxypaeoniflorin, peak 10 paeoniflorin, peak 12 calycosin-7-glucoside, peak 14 coumarin, peak 18 cinnamic acid, peak 19 cinnamaldehyde, peak 20 quercetin and peak 24 6-zingerol. The mass fractions of four quality markers, including calycosin-7-glucoside, cinnamic acid, paeoniflorin and 6-gingerol, in 15 batches of HGWD were determined to be 115.49—494.96, 254.68—760.79, 3 106.72—6 049.40, 232.61—704.10 μg/g, respectively. The paste extraction rate was 11.63%—14.14%, and the transfer rates of calycosin-7-glucoside, cinnamic acid, paeoniflorin and 6-gingerol in the decoction pieces-soup liquid were 8.23%—19.66%, 23.52%—41.21%, 29.15%—41.23% and 14.58%—30.07%, respectively. The transfer rates of calycosin-7-glucoside, cinnamic acid, paeoniflorin and 6-gingerol in the benchmark samples were 86.35%—97.67%, 90.34%—98.13%, 91.51%—98.88% and 93.53%—98.29%, respectively. Conclusion The established method of UPLC fingerprint and quality marker content determination of HGWD is accurate and reliable, which can be used for the quality evaluation of HGWD benchmark samples and could be used as the basis for the development and quality control of subsequent preparations.

R283.6

黑龙江省重点研发计划指导类项目（GZ20210125）；哈尔滨中药四厂横向课题（HX2019051）