Discovery of minor quality evaluation marker ....

Chinese patent medicines (CPMs) are popularly used in clinical practice. Though the composition is complex, the quality of CPM is usually evaluated by the contents of a few main compounds. In this study,a two-leveled metabolomics strategy was proposed to discover minor marker compounds for different CPM products. Zhenqi Fuzheng (ZQFZ) granule was studied an example, where 15 batches from 3 producers were analyzed. The samples were separated using UHPLC on an Acquity UPLC® HSS T3 column,and then detected using Q-Orbitrap-MS. In the first level, 1475 common peaks were extracted and 95 compounds were identified using diagnostic ions and a homemade database. In the second level, the data were subjected to a two-way hierarchical clustering analysis and screened by variable importance value. In total 14 marker compounds were discovered which were responsible for the grouping of different ZQFZ products. Echinacoside (22), oleoside (13), loganic acid (5), salidroside (7), ligustrosidic acid (42), 6α-hydroxygeniposide (28), and oleoside 11-methyl ester (15) could be used to reflect the quality difference for ZQFZ granule products. The proposed strategy could also contribute to the discovery of quality control markers for other CPMs.

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2. Experimental 

2.1. Chemicals and reagents

The reference compounds salidroside (R1), echinacoside (R2), oleuropein (R3), specnuezhenide (R4), nuezhenoside G13 (R5), astragaloside I (R6), astragaloside III (R7), calycosin (R8) and calycosin-7-O-β-d-glucoside (R9) were purchased from Chengdu DeSiTe Biological Technology Co., Ltd. (Chengdu, China);

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