Combining multiple acquisition modes and computational data annotation for structural characterization in traditional Chinese medicine: Miao Nationality medicine Qijiao Shengbai Capsule as a case study

Abstract

Qijiao Shengbai Capsule (QSC) is a reputable Miao Nationality medicine used for treating leukopenia, but its chemical composition has not yet been elucidated. We herein present a strategy, by integrating multiple data acquisition, computational data annotation and processing methods to visualize and identify the complicated constituents in QSC based on ultra-high-performance liquid chromatography coupled with traveling wave ion mobility quadrupole time-of-flight mass spectrometry (UPLC-TWIMS-QTOF-MS). The multiple data acquisition modes, including data-independent mass spectrometry^Energy (MS^E), data-independent high-definition mass spectrometry^Energy (HDMS^E), and fast data-dependent acquisition (fast-DDA), in both positive and negative ion modes, were conducted on a Waters-SYNAPT G2-Si mass spectrometer with an ESI source. An in-house library built by the UNIFI platform could efficiently process the peak annotation of known compounds, whilst different structural types were clustered in the molecular networks for the analogous classification and structural annotation of the unknown ones. Neutral loss, diagnostic ions, feature fragmentation behaviors, and community curation of mass spectrometry data of known compounds helped exploit those similar neighboring nodes of unknown compounds. Moreover, by combination of the predicted CCS values from CCS platform with the experimental CCS values from HDMS^E, as well as diagnostic fragment ions, isomer compounds were annotated. By integrating reference compound comparison, a total of 202 constituents, including 94 flavonoids, 12 saponins, 30 phthalides, 38 organic acids, 3 amino acids, 7 alkaloids and 18 others, were unambiguously characterized or tentatively identified in QSC. Among them, 5 potential new compounds were detected and 12 pairs of isomers were comprehensively distinguished. Conclusively, the established multiple acquisition modes, computational data processing and analysis strategy proved to be useful for the in-depth structural identification of QSC.

Fig. 1. Chemical structures of 47 reference substance.

Fig. 2. A workflow for multiple data acquisition and computational annotation of the chemical composition of QSC.

Fig. 3. The representative base peak chromatograms (BPCs) chromatogram of QSC by UPLC-QTOF-MS^E in positive (A) and negative (B) ion modes.

Fig. 4. MS^E spectra and proposed fragmentation pathway of maackiain in QSC under positive ion mode. (A) MS/MS spectra of #123 with parent ion at m/z 285.0766 [M + H]⁺, (B) proposed fragmentation pathway of maackiain.

Fig. 5. MS^E spectra and proposed fragmentation pathway of soyasaponin I in QSC under positive ion mode. (A) MS/MS spectra of #110 with parent ion at m/z 965.5076 [M + Na]⁺, (B) proposed fragmentation pathway of soyasaponin I.

Fig. 6. The molecular network predicts the novel compounds in QSC. (A) The molecular network of isopentenyl flavonoids from SF. The blue nodes were the known compounds, while the red nodes were the unknown components. (B) MS/MS spectra and chemical structure of m/z 453 (peak 129), m/z 385 (peak 89) and m/z 369 (peak 107). (C) MS/MS spectra and chemical structure of m/z 507 (peak 171), m/z 491 (peak 184) and m/z 457 (peak 96). The groups marked with purple and red color were decreased and increased between two adjacent nodes, respectively. The green dashed line indicated that the fragment ions have the same m/z.

Fig. 7. Characterization of isomers with m/z 661.2496 and CCS prediction based on ALLCCS platform. (A) Three peaks in extracted ion chromatography (EIC) of m/z 661.2495 [M + H]⁺, (B) MS/MS spectra of three compounds (peak 122, 124 and 127), (C) fragmentation behaviors of 2′′-O-rhamnosy icarside II, 3′′-O-rhamnosy icarside II and 4′′-O-rhamnosy icarside II, (D) the RT, measured molecular weight, adducts, drift time, measured experimental CCS values and their predicted CCS values from ALLCCS platform of three compounds. Relative error represents the accuracy between measured experimental CCS values and predicted CCS values.

Fig. 8. Characterization of isomers with m/z 403.1885 and CCS prediction based on ALLCCS. (A) Twenty peaks in extracted ion chromatography (EIC) of m/z 403.1885 [M + Na]⁺, (B) MS/MS spectra of three peaks, (C) the RT, measured molecular weight, adducts, drift time, measured experimental CCS values and their predicted CCS values from ALLCCS platform of four reference compounds. Relative represents the accuracy between measured experimental CCS values and predicted CCS values, (D) fragmentation behaviors of tokinolide B, riligustilide, angelicide and levistolide A.