Differentiation and identification of ginsenoside structural isomers by two-dimensional mass spectrometry combined with statistical analysis

Abstract

Background: In the current phytochemical research on ginseng, the differentiation and structural identification of ginsenosides isomers remain challenging. In this paper, a two-dimensional mass spectrometry (2D-MS) method was developed and combined with statistical analysis for the direct differentiation, identification, and relative quantification of protopanaxadiol (PPD)-type ginsenoside isomers.

Methods: Collision-induced dissociation was performed at successive collision energy values to produce distinct profiles of the intensity fraction (IF) and ratio of intensity (RI) of the fragment ions. To amplify the differences in tandem mass spectra between isomers, IF and RI were plotted against collision energy. The resulting data distributions were then used to obtain the parameters of the fitted curves, which were used to evaluate the statistical significance of the differences between these distributions via the unpaired t test.

Results: A triplet and two pairs of PPD-type ginsenoside isomers were differentiated and identified by their distinct IF and RI distributions. In addition, the fragmentation preference of PPD-type ginsenosides was determined on the basis of the activation energy. The developed 2D-MS method was also extended to quantitatively determine the molar composition of ginsenoside isomers in mixtures of biotransformation products.

Conclusion: In comparison with conventional mass spectrometry methods, 2D-MS provides more direct insights into the subtle structural differences between isomers and can be used as an alternative approach for the differentiation of isomeric ginsenosides and natural products.

Keywords: Differentiation; Ginsenoside isomers; Relative quantification; Two-dimensional mass spectrometry.

Fig. 1

The structures of isomeric ginsenosides Rb₂, Rb_3, and Rc and their possible fragmentation pathways in CID experiments. Arap, arabinopyranosyl; araf, arabinofuranosyl; CID, collision-induced dissociation; xyl, xylopyranosyl.

Fig. 2

2D-MS analyses of ginsenoside Rb₂ in the positive ion mode. The figure was obtained by overlapping the tandem MS spectra on the precursor ion at m/z 1101 using continuously varied CE in SRM mode, indicating that the intensities of the fragment ions varied with the CE. CE, collision energy; 2D-MS, two-dimensional mass spectrometry; MS, mass spectrometry; SRM, selected reaction monitor.

Fig. 3

The IF and RI distributions of the ions for ginsenosides Rb₂, Rb₃, and Rc. (A) The IF distributions of the ions at m/z 335. (B) The RI distributions of the ions at m/z 335 to m/z 789. Each of the distributions was fitted by 5^th order polynomial function with the correlation coefficients (R²) shown in the inset, the same below. IF, intensity fraction; RI, ratio of intensity

Fig. 4

The negative MS² spectra from the [M-H]^- ion at m/z 783 of ginsenosides under the CE of 12 eV. (A) Ginsenoside Rg₃. (B) Ginsenoside F₂. Both of the two isomers were fragmented by successive losses of glucose residues and alkene chain. The insets showed the possible fragmentation pathways to generate each fragment ion in the spectra. CE, collision energy.

Fig. 5

The RI distributions of the ions at m/z 459 to m/z 621 for ginsenosides Rg₃ and F₂, which can be differentiated based on the RI value at 0.9 in the CE range of 20–80 eV. CE, collision energy; RI, ratio of intensity.

Fig. 6

The IF distributions for ginsenosides Rh₂ and C-K. (A) The IF distributions of the ions at m/z 459 for ginsenoside Rh₂, which can be differentiated based on the IF values at 25% in the CE range of 12–64 eV. (B) The IF distributions of the ions at m/z 375 for ginsenoside C-K, which can be differentiated based on the IF values at 3.8% in the CE range of 32–80 eV. CE, collision energy; IF, intensity fraction.

Fig. 7

The establishment of standard curve of the first approach for the relative quantification of ginsenoside isomers. (A) The linear relationship between the IF values of the m/z 459 ion and the CE for each mixture of Rh₂ and C-K. (B) The linear relationship between the slope of each regression line in panel (A) and the molar composition of each mixture. CE, collision energy; IF, intensity fraction.

Fig. 8

The relative quantification of ginsenoside isomers in biotransformation product. (A) The mass spectrum of the biotransformation product of PPD-type ginsenosides. (B) The linear relationship between the varying CE and the IF values of the m/z 459 and m/z 621 ions for Rh₂/C-K and Rg₃/F₂, respectively. CE, collision energy; IF, intensity fraction; PPD, protopanaxadiol.