Triple three-dimensional MS/MS spectrum facilitates quantitative ginsenosides-targeted sub-metabolome characterization in notoginseng

Abstract

Although serving as the workhorse, MS/MS cannot fully satisfy the analytical requirements of quantitative sub-metabolome characterization. Because more information intrinsically correlates to more structural and concentration clues, here, efforts were devoted to comprehensively tracing and deciphering MS/MS behaviors through constructing triple three-dimensional (3×3D)-MS/MS spectrum. Ginsenosides-targeted metabolomics of notoginseng, one of the most famous edible medicinal plants, was employed as a proof-of-concept. Serial authentic ginsenosides were deployed to build the correlations between 3×3D-MS/MS spectra and structure/concentration features. Through assaying ginsenosides with progressive concentrations using QTOF-MS to configure 1^st 3D spectrum, the generations of MS¹ spectral signals, particularly multi-charged multimer anions, e.g., [2M-2H]^2- and [2M+2HCOO]^2- ions, relied on both concentration and the amount of sugar chains. By programming progressive collision energies to the front collision cell of Qtrap-MS device to gain 2^nd 3D spectrum, optimal collision energy (OCE) corresponding to the glycosidic bond fission was primarily correlated with the masses of precursor and fragment ions and partially governed by the glycosidation site. The quantitative relationships between OCEs and masses of precursor and fragment ions were utilized to build large-scale quantitative program for ginsenosides. After applying progressive exciting energies to the back collision chamber to build 3^rd 3D spectrum, the fragment ion and the decomposition product anion exhibited identical dissociation trajectories when they shared the same molecular geometry. After ginsenosides-focused quantitative metabolomics, significant differences occurred for sub-metabolome amongst different parts of notoginseng. The differential ginsenosides were confirmatively identified by applying the correlations between 3×3D-MS/MS spectra and structures. Together, 3×3D-MS/MS spectrum covers all MS/MS behaviors and dramatically facilitates sub-metabolome characterization from both quantitative program development and structural identification.

Keywords: Full exciting energy ramp-MS3 spectrum; Ginsenosides; Notoginseng; Quantitative sub-metabolome characterization; Structural identification.

Graphical abstract

Figure 1

MS¹ spectra for F2 (A), Rg3 (B), and LXXV (C). Range of m/z 828−833 is zoomed to highlight the difference, and the primary signals are assigned.

Figure 2

Full concentration ramp-MS¹ spectrum (A) and the summed peak area of all primary signals or the most abundant signal (i.e., m/z 829.50 [M+HCOO]^–) against concentration profile (B) of F2.

Figure 3

FCER-MS² spectra of [M–H]^– for F2 (A), Rg3 (B), and LXXV (C), through assembling sigmoid-shaped breakdown graphs of m/z 783.5, and Gaussian-shaped breakdown graphs of m/z 621.4, 459.4, 375.3, 341.1, 323.1, 251.1, 221.1, 179.1, and 161.0. The mass fragmentation pathways being responsible for those fragment ions are proposed.

Figure 4

The correlations (R² = 0.8416) of optimal collision energy (OCE, z-coordinate) against the masses of precursor (Q1, x-coordinate) and fragment ions (Q3, y-coordinate).

Figure 5

FEER-MSⁿ spectra (n = 3 for Re and n = 2 for the other compounds) comparison between the concerned fragment ions of Re and anions of suspected decomposition products. Structure (A) and MS² spectrum (B) of Re, FEER-MSⁿ spectra of m/z 475.4 for Re and protopanoxatriol (C), FEER-MSⁿ spectra of m/z 637.4 for Re, Rh1, and F1 (D), FEER-MSⁿ spectra of m/z 783.5 for Re and Rg2 (E), and FEER-MSⁿ spectra of m/z 799.5 for Re, Rg1, and Rf (F).

Figure 6

Score scattering plot (A) and loading plot (B) after principal component analysis of the quantitative ginsenoside-focused sub-metabolome for different parts of notoginseng.

Figure 7

FEER-MSⁿ spectra (n = 3 for compounds 32, 36, and 43, and n = 2 for the other compounds) comparison among the concerned fragment ions of 32, 36, and 43 and anions of suspected decomposition products. FEER-MSⁿ spectra of m/z 459.4 (A) for 32, 36, and 43, as well as protopanoxadiol, FEER-MSⁿ spectra of m/z 621.4 (B) for 32, 36, and 43, as well as Rh2 and CK, FEER-MSⁿ spectra of m/z 783.5 (C) for 32, 36, and 43, as well as Rg3, F2, and LXXV, FEER-MSⁿ spectra of m/z 945.5 (D) for 32, 36, and 43, as well as Rd and XVII, FEER-MSⁿ spectra of m/z 1077.6 (E) for 32, 36, and 43, as well as Rc, Rb2, and Rb3, and FEER-MSⁿ spectra of m/z 1107.6 (F) for 32, 36, and 43, as well as Rb1.