Glycoside Fragmentation in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Natural Products of Ginsenosides

Abstract

I investigated the tandem mass spectrometry (MS/MS) fragmentation of ginsenoside glycosides using matrix-assisted laser desorption/ionization MS for ginsenosides Rg1, Rh1, Rb1, and Rb3, focusing on their sodium adduct molecules [M+Na]⁺. The glycosidic linkage at the C-20 position cleaved more readily than those at C-3 and C-6. These glycosides fragmented on their glucosyl acceptor sides, exhibiting C- and Z-type fragmentation, although generally B/Y-type fragment ions are dominant in MS/MS spectra of neutral oligosaccharides. These results suggest that, due to the hydrophobic triterpene skeleton of the aglycone, sodium cations cannot effectively coordinate with the aglycone moiety.

INTRODUCTION

Ginsenosides are natural steroidal glycosides belonging to the class of triterpenoid saponins and are the major bioactive components found in the roots of Panax ginseng. The roots of P. ginseng have long been used as a traditional herbal medicine,¹⁾ and reliable quality control is essential for their commercialization. Structural analyses of ginsenosides have been conducted using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) and tandem MS/MS techniques.^2–7) Matrix-assisted laser desorption/ionization-MS (MALDI-MS) has also been applied for the rapid and simple differentiation of Panax species based on their fingerprinting patterns,⁸⁾ and subsequently for the localization of ginsenosides within P. ginseng roots.^9–11) Ginsenosides possess three glycosidic sites at C-3 (R1), C-6 (R2), and C-20 (R3), as shown in Fig. 1. In this study, we investigated the MS/MS fragmentation behavior at these three glycosidic sites in MALDI-MS using sodium adduct molecules [M+Na]⁺ as precursor ions.

Fig. 1. Structure of ginsenosides.

MATERIALS AND METHODS

Materials

High-performance liquid chromatography-grade methanol was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). 2,5-Dihydroxybenzoic acid (DHB) for MALDI-time of flight/MS (MALDI-TOF/MS) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Vancomycin hydrochloride was purchased from Nacalai Tesque Inc. (Kyoto, Japan). Ginsenosides Rg1, Rh1, Rb1, and Rb3 were obtained from LKT Laboratories Inc. (St. Paul, MN, USA). Analytes were prepared at 1 mg/mL in 60% methanol, and DHB was prepared at 10 mg/mL in 60% methanol. A 1 μL aliquot of each analyte and matrix solution was spotted onto a MALDI-MS sample plate.

MALDI-MS

All mass spectra were acquired using a RapifleX MALDI-TOF/TOF MS instrument (Bruker Corp., Billerica, MA, USA) operated in reflectron and positive-ion modes within the 0–2 kDa range. A Smartbeam 3D laser (355 nm wavelength, Bruker Corp., Billerica, MA, USA) was used for desorption/ionization. MS/MS fragmentation spectra (post-source decay) were obtained from the sodium adduct ions of each analyte. Fragmentation nomenclature followed the systematic scheme for carbohydrate fragmentation proposed by Domon and Costello (Fig. 2).¹²⁾

Fig. 2. Systematic nomenclature of carbohydrate fragmentation.¹²⁾.

RESULTS AND DISCUSSIONS

Ginsenoside Rg1

Ginsenoside Rg1 contains two glucose (Glc) units attached at the R2 (C-6) and R3 (C-20) glycosylation sites (Fig. 1). The MS/MS product ion spectrum of ginsenoside Rg1 was obtained from the sodium adduct ion [M+Na]⁺ at m/z 823 (Fig. 3). Prominent fragment ions appeared at m/z 643 and 203, corresponding to the loss of a glucose unit and the formation of [Glc+Na]⁺ (180 + 23 = 203), indicating cleavage of a glycosidic bond. Based solely on this spectrum, it was not possible to determine which of the two glycosidic bonds (R2 or R3) underwent cleavage. To clarify this, we examined ginsenoside Rh1, which contains a single glucose at the R2 site (Fig. 4). Its MS/MS spectrum exhibited relatively weak fragment ions at m/z 481 and 203, derived from the loss of Glc and Glc+Na (C- and Z-type fragmentation). The markedly lower intensity compared to Rg1 suggests that the glucose at the R3 position (C-20) cleaves more readily than that at R2, indicating preferential fragmentation at the R3 site.

Fig. 3. The MS/MS product ion spectrum of ginsenoside Rg1 was obtained from [M+Na]⁺. MS/MS, tandem mass spectrometry.

Fig. 4. The MS/MS product ion spectrum of ginsenoside Rh1 was obtained from [M+Na]⁺. MS/MS, tandem mass spectrometry. *The peak labeled with an asterisk was not generated by only the cleavage of glycosidic linkages. Possible ion species are listed in Supporting Information 1.

Ginsenoside Rb1 and Rb3

Ginsenoside Rb1 possesses two glucose disaccharide (Glc–Glc) moieties at R1 (C-3) and R3 (C-20), while Rb3 contains one glucose disaccharide (Glc–Glc) and one xylosyl–glucose (Xyl–Glc) moiety (Fig. 1). In the MS/MS spectrum of Rb3, fragment ions at m/z 789 and 335 were observed, corresponding to the loss of Xyl–Glc and the intact Xy–Glc sodium adduct, respectively (Fig. 5). These ions resulted from C/Z-type cleavage of the glycosidic bond at the R3 site rather than at R1. Wang et al. reported a similar fragmentation tendency between the R3 and R1 glycosidic bonds in ginsenosides Rb1, Rb2, and Rc in their electrospray triple-quadrupole collision-induced dissociation MS/MS spectra.²⁾

Fig. 5. The MS/MS product ion spectrum of ginsenoside Rb3 obtained from [M+Na]⁺. MS/MS, tandem mass spectrometry.

Several mechanistic pathways can explain the formation of the observed fragment ions. Because ginsenoside Rb3 possesses two glycosidic linkages at the C-3 (R1) and C-20 (R3) positions, the [M+Na]⁺ precursor ion is expected to exist as at least two coordination isomers, in which Na⁺ is attached either to the R1 or the R3 sugar chain. The C-type fragment ion [R3+Na]⁺ is most reasonably generated from the precursor ion in which Na⁺ is coordinated to the C-20 sugar chain (Xyl–Glc–; R3). In this isomer, Na⁺ is stabilized by coordination with the hydroxyl groups of the sugar moiety at C-20 (R3), and cleavage of the aglycone side of the glycosidic bond—likely facilitated by hydrogen transfer and/or electron migration—produces the C-type fragment ion [R3+Na]⁺.

The Z-type fragment ion [Aglycone-R1+Na]⁺ can arise from at least two mechanistic possibilities. First, when Na⁺ is initially localized on the R1 sugar chain at C-3, cleavage of the C-20 glycosidic bond (R3), initiated by hydrogen transfer or electron migration, results in retention of Na⁺ on the aglycone–R1 portion, yielding a Z-type product ion. Second, even when Na⁺ is initially localized on the R3 sugar chain at C-20, cleavage of the glycosidic bond may produce a transiently destabilized Na⁺ environment. Under such conditions, there is a possibility that Na⁺ migration to the R1 sugar chain on the aglycone side can occur, and subsequent coordination to this more favorable site can stabilize the resulting Z-type fragment ion.

A similar fragmentation pattern was also observed for Rb1 in MALDI-MS (Fig. 6).

Fig. 6. The MS/MS product ion spectrum of ginsenoside Rb1 obtained from [M+Na]⁺. MS/MS, tandem mass spectrometry.

Generally, B/Y-type fragment ions are dominant in MS/MS spectra of neutral oligosaccharides derived from sodium adducts [M+Na]^+13–16) because sodium cations can coordinate with multiple oxygen atoms (hydroxyl and ring oxygens) near the glycosidic bonds.¹⁶⁾ In such cases, sodium coordinates with both donor and acceptor residues, allowing cleavage via B/Y-type fragmentation. Certain natural product glycosides, such as vancomycin, display B/Y-type fragmentation (see Supporting Information 2) because their aglycones contain multiple hydroxyl groups capable of sodium coordination. In contrast, the aglycone of ginsenosides is a triterpene skeleton possessing only one hydroxyl group at C-12 near the R3 site. Consequently, in ginsenosides, the glycosidic bonds cleave at the acceptor side, leading predominantly to C/Z-type fragmentation, which is believed to be induced by hydrogen and/or electron transfer after sodium cation coordination.^17–19) Fragmentation from [M+Na]⁺ ions occurs mainly at the R3-glycoside rather than at the R1 and R2 glycosides. The sodium-adduct spectra provide valuable information on site-specific cleavage, complementing data from protonated species. Together, MS/MS data from both ion types offer a more complete basis for structural elucidation of ginsenosides.

Mass Spectrom (Tokyo) 2025; 14(1): A0183

SUPPORTING INFORMATION

Supporting Information 1 Possible ion species of the ion at m/z 333 in the MS/MS spectrum of ginsenoside Rh1.

Supporting Information 2 The MS/MS product ion spectrum of vancomycin from [M+Na]⁺ and the structure of vancomycin.