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Authentication of Chinese medicine materials in prescriptions is extremely difficult due to the complicated chemical matrix. A strategy integrating in-depth profiling, chemical marker selection, and selected detection was established and exemplarily applied to authenticate paeony root in ShaoYao-GanCao decoction. First, an ultra-performance liquid chromatography/linear trap quadrupole-Orbitrap method was developed to probe the chemical compositions of the decoction. Second, 20 batches of decoctions prepared from white paeony root and red paeony root were compared by a metabolomics method, and multistep chemometrics analysis distinguished the chemical markers. Third, an ultraperformance liquid chromatography/QDa-selected ion monitoring method was developed to authenticate the paeony root in decoctions. As a result, 161 compounds were characterized, including 84 triterpene saponins, 42 flavonoids, and 10 monoterpenes. Four chemical markers and paeoniflorin were successfully screened out as chemical markers for white paeony root. The selected ion monitoring method easily differentiated authentic decoction (prepared from white paeony root) from fraud decoction (prepared from red paeony root) by monitoring the above five chemical markers. In conclusion, the strategy was proved effective in authentication of paeony root in ShaoYao-GanCao decoction, and it can also be applied to authenticate other Chinese medicine materials in prescriptions, which will greatly avail the quality enhancement of prescriptions.

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