Simultaneous Profiling and Holistic Comparison of the Metabolomes among the Flower Buds of Panax ginseng, Panax quinquefolius, and Panax notoginseng by UHPLC/IM-QTOF-HDMSE-Based Metabolomics Analysis

Abstract

The flower buds of three Panax species (PGF: flower bud of P. ginseng; PQF: flower bud of P. quinquefolius; PNF: flower bud of P. notoginseng), widely consumed as healthcare products, are easily confused particularly in the extracts or traditional Chinese medicine (TCM) formulae. We are aimed to develop an untargeted metabolomics approach, by ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) to unveil the chemical markers diagnostic for the differentiation of PGF, PQF, and PNF. Key parameters affecting chromatographic separation and MS detection were optimized in sequence. Forty-two batches of flower bud samples were analyzed in negative high-definition MSE (HDMSE; enabling three-dimensional separations). Efficient metabolomics data processing was performed by Progenesis QI (Waters, Milford, MA, USA), while pattern-recognition chemometrics was applied for species classification and potential markers discovery. Reference compounds comparison, analysis of both HDMSE and targeted MS/MS data, and retrieval of an in-house ginsenoside library, were simultaneously utilized for the identification of discovered potential markers. Satisfactory conditions for metabolite profiling were achieved on a BEH Shield RP18 column and Vion™ IMS-QTOF instrument (Waters; by setting the capillary voltage of 1.0 kV and the cone of voltage 20 V) within 37 min. A total of 32 components were identified as the potential markers, of which Rb3, Ra1, isomer of m-Rc/m-Rb2/m-Rb3, isomer of Ra1/Ra2, Rb1, and isomer of Ra3, were the most important for differentiating among PGF, PQF, and PNF. Conclusively, UHPLC/IM-QTOF-MS-based metabolomics is a powerful tool for the authentication of TCM at the metabolome level.

Integration of Data-Dependent Acquisition (DDA) and Data-Independent High-Definition MSE (HDMSE) for the Comprehensive Profiling and Characterization of Multicomponents from Panax japonicus by UHPLC/IM-QTOF-MS

“…The effects of different stationary phases on the separation of saponins from P. japonicus were further evaluated by considering the number of resolvable peaks and peak symmetry [17,31]. Ten UHPLC columns packed with sub-2-µm particles from two major vendors (Waters Corporation and Agilent Technologies), including HSS T3, HSS C18 SB, CSH C18, BEH Shield RP18, BEH C18, CORTECS UPLC C18+ (from Waters), ZORBAX Extend C18, ZORBAX SB-C18, ZORBAX Eclipse Plus C18, and ZORBAX SB-Aq (from Agilent), which we have utilized in the analysis of the flower buds of three Panax species [17], were examined at 35 °C (Figure 2). In structure, these different stationary phases are differentiated in the silica gel core (fully porous or core-shell), the bonding technologies, and the bonding groups.…”

“…In addition, it exhibited almost the best separating capacity to Rg1/Re (comparable to the ZORBAX SB-Aq and HSS T3), a pair of ginsenosides diagnostic for discriminating different Panax species [29]. Accordingly, we selected the BEH Shield RP18 column in this experiment, consistent with the column we used in a previous chemical analysis of flower buds of P. ginseng , P. quinquefolius , and P. notoginseng [17] and the florets of Carthamus tinctorius [5]. In the next step, the effects of the column temperature varying from 25 °C to 40 °C were assessed by observing the overall resolution for ginsenosides (Figure S2).…”

“…Key parameters of the Vion IM-QTOF instrument (Waters Corporation), which may influence the ion response and the fragmentation, were further optimized. For the ion source, two parameters affecting the ion response, including spray voltage and cone voltage, had been optimized in our previous report [17], which were set at 1.0 kV and 20 V, respectively.…”

“…State-of-the-art mass spectrometers mostly enable ramp collision energy or mixed collision energies to acquire high-quality MS 2 spectra for the precursors with a wide mass span. The Vion IM-QTOF hybrid high-resolution mass spectrometer can enable ramp collision energy (RCE) in MS E /HDMS E [17,18] and mass dependent ramp collision energy (MDRCE) in the DDA mode (MS-MS/MS). No reports are currently available on how to optimize the MDRCE in DDA.…”

“…In addition, ion mobility facilitates an additional dimension of separation of ions (based on the difference of charge state, size, and shape) which is orthogonal to chromatographic retention and MS. The hybrid ion mobility/quadrupole time-of-flight mass spectrometry (IM/QTOF-MS; such as the commercial SYNAPT series and Vion IM-QTOF from Waters, 6560 QTOF from Agilent) coupled to liquid chromatography (LC) can enable three-dimensional separations providing four dimensions of information related to molecules (t R , drift time or collision cross section (CCS), MS 1 , and MS 2 ) [16,17]. IM-derived CCS can act as a stable parameter useful for more reliable structural elucidation of a metabolite [18].…”