Panax quinquefolius (American ginseng) is globally popular serving as herbal medicine or tonifying agent. Compared with the root, studies on the flower of Panax quinquefolius (PQF) in respect of the multicomponent characterization and quality evaluation is scarce. This work was designed, by feat of two liquid chromatography–mass spectrometry platforms, to characterize and quantitatively assay the saponins of PQF. Ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) combined with data-independent high-definition MSE (HDMSE) was developed to acquire the structure information of PQF saponins. Consequently, efficient and more accurate identification (higher identification accuracy and much lower false positives rate) of the involved saponins were accomplished by the intelligent peak annotation workflows of the UNIFI bioinformatics platform incorporated with a “Multi-dimensional Information Library of Ginsenosides” (GinMIL). Totally 128 ginsenosides were identified or tentatively characterized, and 45 thereof were identified by comparing with the reference standards. Additionally, an efficient UHPLC/QTrap-MS (triple quadrupole-ion trap mass spectrometry) method enabling quantitative assay of 21 ginsenosides in PQF was established and validated by scheduled multiple reaction monitoring (sMRM). It gave low limit of detection (0.011–0.65 pg) and limit of quantification (0.053–4.1 pg), good linearity (R2 > 0.99), intra-/inter-day precision (RSD < 13.6 %), stability (RSD < 3.5 %), repeatability (RSD < 7.5 %), and recovery (RSD < 13.5 %). It application to quantify 21 ginsenosides in 14 batches of PQF samples revealed differences in their saponin content. Conclusively, systematic multi-component characterization and multi-indicator quantitation of PQF were achieved, thus providing the methodological reference for the quality investigation of functional herbs.
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