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Differentiation of Isomeric Ginsenosides by using Electron-Induced Dissociation Mass Spectrometry Y.-L. Elaine Wong, Xiangfeng Chen, Wan Li, Ze Wang, Y.- L. Winnie Hung, Ri Wu, and T.-W. Dominic Chan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00908 • Publication Date (Web): 14 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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Analytical Chemistry

Analytical Chemistry-Letter

Differentiation of Isomeric Ginsenosides by using Electron-Induced Dissociation Mass Spectrometry Y.-L. Elaine Wong,† Xiangfeng Chen,*,†,‡ Wan Li,† Ze Wang,† Y.-L. Winnie Hung,† Ri Wu,† T.-W. Dominic Chan†,*

†

Department of Chemistry, The Chinese University of Hong Kong, Hong Kong SAR

‡

Key Laboratory for TCM Quality Control Technology, Shandong Analysis and Test Centre, Shandong Academy of Sciences, Jinan, Shandong, P. R. China

*Address reprint requests to Professor T.-W. D. Chan, Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR. E-mail: [email protected], Dr. X. F. Chen, Shandong Academy of Sciences, Jinan, China. E-mail: [email protected].

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ABSTRACT Current phytochemical research on ginsengs focuses on the structural characterization and isomer differentiation of ginsenosides. In this letter, electron-induced dissociation (EID) was initially investigated by analyzing isomeric ginsenosides. EID provided more structural information on their differentiation than collision-induced dissociation (CID) did. Glycosyl group migration previously observed in the CID of oligosaccharide ions could also be found in the EID of protonated Rg 1 . This rearrangement reaction would show substantial ambiguities in differentiating Rg 1 from Rf. Although other charge carriers could alleviate this problem, the use of EID in dissociating deprotonated ginsenoside ions was superior to other techniques in terms of eliminating glycosyl group migration and generating diagnostic fragment ions for the differentiation of structural isomers. This study demonstrates a potential method to analyze natural products and thus help discover and evaluate novel compounds.

Keywords: ginsenoside, electron-induced dissociation, isomer, diagnostic ion, collision-induced dissociation


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Ginseng is a class of well-known traditional herbal substances. It is commonly used as a key herb in the formulation of numerous traditional Chinese medicines. Its genus name Panaxis derived from a Greek word meaning “all-healing.” Ginseng modulates multiple physiological activities, such as anti-oxidation, blood pressure regulation, metabolism, and immune functions.1,2 The therapeutic potential of ginseng is attributed primarily to the presence of various ginsenosides.3 More than 100 ginsenosides have been isolated from different Panax species.4 The structural characterization and isomer differentiation of ginsenosides have been extensively investigated in ginseng-related phytochemical research because many ginsenosides are characterized by multiple structural isomers involved in different biological activities.3,5 These methods are necessary to elucidate the relationship between the structure and bioactivity of ginsenosides and to provide relevant information for the authentication of commercial ginseng products. Wang and co-workers6 differentiated Oriental and American ginsengs by using the high ratios of a few bioactive ginsenosides, such as Rg 1 /Rf and Rc/Rb 2 . The Rg 1 /Rf and Rc/Rb 2 ratios are much higher in American ginseng than in Oriental ginseng. However, American ginsengs contain a pseudoginsenoside F 11 , which is a structural isomer of Rf and Rg 1 . The structural information of ginsenosides, including Rg 1 , Rf, Rc, Rb 2 , and pseudoginsenoside (F 11 ), is shown in Figure 1. The rather similar retention times of F 11 and Rf under many reversed-phase liquid chromatographic conditions has resulted in false identification.7–9 Tandem mass spectrometry through collision-induced dissociation (CID) as an ion activation method has been applied to structurally analyze ginsenosides. However, CID primarily involves vibrational activation and induces preferential cleavage at the weakest bond of molecular ions; as a consequence, limited structural information can 3 ACS Paragon Plus Environment


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be obtained. In the analysis of isomeric ginsenosides, such as Rg 1 /Rf and Rb 2 /Rc, the CID of protonated molecule-ions yielded identical fragment ions resulting primarily from the losses of glycan groups (Figures S-1 and S-2 in Supplementary Information). The development of a suite of electron-based dissociation methods, such as electron capture dissociation,10 electron detachment dissociation,11 and electron-induced dissociation (EID),12 has provided an alternative ion activation approach based on electron–ion interactions. Charge- and radical-directed dissociation pathways operate through these electron-based dissociation methods. Among various electron-based dissociation methods, EID can specifically activate precursor ions without altering their original charge states.12 Thus, single- and multiple-charged precursor ions can be directly analyzed through EID. A typical EID involves the interaction of target analyte ions with high-energy (> 10 eV) electrons in gas phase.13 EID activates ions by electronic excitation rather than by vibrational excitation. This technique has been applied to analyze amino acids,12 peptides,13 fatty acids,14 metabolites,15 carbohydrates,16 and other small molecules.17 This letter aimed to investigate the potential application of EID for the structural characterization of isomeric ginsenosides and 24 (R)-pseudoginsenoside. Our results demonstrated that the EID of ginsenoside ions provides more informative structural fragment ions than CID does. Glycosidic and cross-ring cleavages can be induced; as a result, diagnostic ions are formed, and these ions can be used to differentiate various pairs of isomeric compounds, such as F 11 , Rf, and Rg 1 and Rb 2 and Rc. EXPERIMENTAL SECTION Sample Preparation. Materials were obtained commercially and used without further purification. Ginsenoside standards were purchased from Sichuan WeiKeqi Biological 4 ACS Paragon Plus Environment

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Technology Co., Ltd. Analyte solutions were prepared at concentrations of 0.4–0.5 µM in 1:1 methanol (LC-MS grade)/water (LC-MS grade) (v/v). Acetic acid (0.5%) was added for positive-mode ESI. Mass spectrometry. Experiments were conducted using a 9.4 Tesla SolariX Fourier transform ion cyclotron resonance mass spectrometer (Bruker Daltonics, Germany). Samples were introduced to a homebuilt microspray source with a flow rate of 30 µL/hr. Ionization was performed in positive and negative mode with a capillary voltage of 3 kV and a transfer capillary temperature of 220°C. The singly charged precursor ions were isolated by a front-end quadrupole. The isolation width was 5 m/z. The ion accumulation time in the hexapole was 1–2 s. ESI mass spectra were acquired with Compass SolariX control (version 1.5.0, Bruker Daltonics) in broadband mode over a mass range of 50–1200m/z. For EID, the precursor ion was isolated in the quadrupole and externally accumulated in the collision cell for 1–2s. Afterward, the isolated ion was transferred to the ICR cell. The ions were then irradiated with electrons from a heated hollow cathode dispenser. The bias voltage was set between -10 and -15V, and the extraction electrode was set to 11–14V. The electron irradiation durations were 0.2s for positive ion and 5s for negative ion. Background spectra were acquired by setting the bias voltage to 0V such that no emission of electrons would occur, and all other parameters were the same. For CID, the selected precursor ions were isolated in the first quadrupole and fragmented in the collision cell with collision energies of 10–25V. The product ions were assigned through accurate mass measurement. External calibration using ESI tuning mix (Agilent Technologies Canada Inc.) produced a mass accuracy of 5 ppm. Internal calibration using confidently assigned fragments as internal calibrants of CID and EID mass spectra resulted in amass accuracy of 2 ppm or less. Data were processed by Data Analysis™ 5 ACS Paragon Plus Environment


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version 4.1 (Bruker Daltonics). RESULTS AND DISCUSSION The nomenclature for fragmentation used in this paper is based on that described by Costello and co-workers.18 The ions produced by retaining the charge at the reducing terminus are termed as Y, Z (glycoside cleavages), and X (cross-ring cleavages); by contrast, the ions retaining the charge at the non-reducing terminus are termed as B, C (glycoside cleavages), and A (cross-ring cleavages). Cross-ring cleavage ions are designated by superscript numbers indicating the two bonds cleaved. The bond cleavage at the C20 position is defined as α-chain. The C3 and C6 positions are named as β-chain. The nomenclature of the fragmentation of Rb 2 is presented in Figure S-3. Measured masses, peak assignments, and measurement errors in all of the spectral data are included in Supplementary Information. Figure 2 shows the EID mass spectra of three isomeric compounds: protonated F 11 , Rf, and Rg 1 . The predominant dissociation pathways for these sugar-containing compounds were primarily related to the cleavage of glycosidic linkages. For instance, the loss of the disaccharide units from F 11 and Rf molecular ions resulted in diagnostic fragment ions at 475.3777 and 459.3826 m/z, respectively. These fragment ions could be used to differentiate F 11 from Rf conveniently. In principle, fragment ions generated from similar glycosidic cleavage should be used to differentiate Rf from Rg 1 . This principle is commonly applied because the former contains a disaccharide unit at C6 of the protopanaxatriol ring and the latter consists of two separate glucose units at C6 and C20 of the panaxatriol ring. In Figure 2, the EID spectra and the corresponding CID spectra (Figure S-2 in Supplementary Information) of the protonated Rg 1 and Rf revealed C 2 and B 2 6 ACS Paragon Plus Environment

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fragment ions. The presence of these disaccharide ions in the spectrum of Rg 1 is tentatively attributed to the long-range glycosyl arrangement. This remote migration of the glycosyl group within molecular ions has also previously been reported in the CID of oligosaccharides.19–22 These rearranged sugar ions are also formed, as indicated by the CID and EID of [M+NH 4 ]+ ion (data not shown) because [M+H]+ ion can also be produced in the CID and EID of [M+NH 4 ]+. Migration reaction may proceed through the same pathway as that induced by the dissociation of the protonated ion. Figure 3 shows the EID spectra of the sodium ion-adducted Rf and Rg 1 . The corresponding CID spectra are shown in Figure S-4 in Supplementary Information. In both CID and EID experiments, no disaccharide ions arising from the migration of the glucose group were observed in the Rg 1 spectrum. Consistently, the EID of other metal adduct ions, such as [M+Li]+ and [M+K]+ of Rg 1 , shows no sign of the migration of the glucose group (data not shown). Therefore, the lone pair in one of the hydroxyl groups of the glucose unit attacks the nucleophilic carbon and forms a new glycosidic bond (Scheme S-1). The structure of Rg 1 obtained by molecular mechanic minimization is shown in Figure S-5. The distance between the most proximate oxygen and the carbon next to the protonated ether group is approximately 5 Å, which is a suitable distance for glycosyl transfer, as reported previously in the CID of different oligosaccharides.19–22 Protons should be considered key factors in glycosyl migration. This observation implies that transfer reaction may be mediated by protons. Scheme S-1 illustrates a possible reaction pathway. Daughter ions should be further activated (MS3) to obtain more detailed structural information because the CID of sodium ion-adducted ginsenoside species yielded a very limited number of fragment 7 ACS Paragon Plus Environment


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ions. By contrast, the EID of the same molecular ions produces more fragment ions, including some cross-ring fragments in a single-ion activation step. For example, the presence of panaxatriol ring cleavage product ions [M-C 17 H 28 O 2 ]+ at 571.2727 m/z and [M-C 16 H 26 O 2 ]+ at 585.2888 m/z in Rf provided a convenient distinction from Rg 1 . Another possible mechanism to avoid proton-mediated glycosyl migration is to work on deprotonated molecular ions. The ginsenosides used in this study exhibited a better sensitivity in a negative ion mode than in a positive ion mode. Figure 4 shows the EID spectra of the deprotonated Rf and Rg 1 . In these spectra, the disaccharide fragment ions B 2 at 323.0816 m/z and C 2 at 339.0922 m/z were only found in the EID spectrum of Rf but not in that of Rg 1 . Their absence in EID of Rg 1 supported that no migration of glucose group occurred. Therefore, these fragment ions could serve as diagnostic ions to distinguish the two isomers. Notably, the CID of these deprotonated ginsenoside ions did not generate any diagnostic fragment ions to differentiate these isomers (see Figure S-6 in the Supplementary Information). Although the CID of deprotonated ginsenoside ions also caused the cleavage on glycosidic bond, the residual charge was solely retained in the panaxatriol ring, leading to the formation of identical MS/MS spectra, in terms of the types of fragment ions. Under EID conditions, B 2 /C 2 can be generated via two possible pathways: charge-directed fragmentation of [M-H]- or radical-induced fragmentation of [M-2H]- as proposed in the previous studies.23–25 On the contrary, the EID of Rf and Rg 1 produced significantly different spectra. In addtion to the presence of B 2 at 321.0818 m/z and C 2 at 339.0924 m/z in Rf, the EID spectrum of Rg 1 displayed a number of diagnostic cross-ring cleavage product ions, such as and

1,4

0,2

X 0α/β at 679.4418 m/z

X 0α/β at 709.4523 m/z. The fragment ion at 561.2915 m/z in Rf is formed


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through the cross-ring cleavage of the panaxatriol ring and is also a diagnostic ion for their differentiation. The CID and EID MS/MS of the deprotonated Rb 2 and Rc were acquired to further explore the potential use of EID approach for structural isomer differentiation. Rb 2 and Rc are isomeric ginsenosides that differ in one of the sugar substituents. In particular, Rb 2 contains a six-membered α-L-arabinopyranose unit, but Rc consists of a five-membered α-L-arabinofuranose unit. The EID spectra are shown in Figure 5, and the corresponding CID spectra are shown in Figure S-7 in Supplementary Information. As expected, the CID of the deprotonated Rb 2 and Rc yielded identical spectra in terms of the types of fragment ions. By contrast, the EID spectra of Rb 2 and Rc displayed small but relevant differences, as marked in “red.” The more extensive ion fragmentation in EID experiments was tentatively attributed to electron-induced electronic and vibrational excitations involving charge- and radical-driven fragmentation pathways.12,26–28 The presence of [M-2H]- and other odd-electron product ions in the EID spectra supported this postulation. CONCLUSIONS Long-range glycosyl transfer occurs in the CID and EID of protonated ginsenoside species; as a result, unexpected disaccharide ions are formed. The formation of these ions complicates spectral interpretation and causes ambiguities in the differentiation of ginsenoside isomers. The use of alkali metal adduct ions or deprotonated ginsenoside ions can eliminate glycosyl rearrangement. In contrast to CID, EID generates more structural diagnostic ions for isomer differentiation. This result suggests that the proposed method can be applied to analyze natural products and to help discover novel compounds. 9 ACS Paragon Plus Environment


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ACKNOWLEDGEMENTS Financial supports from the National Natural Science Foundation of China (21205071), Research Grant Council of the Hong Kong Special Administrative Region (Research Grant Direct Allocation, Ref. 2060351), Natural Science Foundation of Shandong Province (ZR2012BQ009) and Shandong Academy of Science are gratefully acknowledged.


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REFERENCES (1) Angelova, N.; Kong, H.W.; Heijden, R.V.D.; Yang, S.Y.; Choi, Y.H.; Kim, H.K.; Wang, M.; Hankemeier, T.; Greef, J.V.D.; Xu, G.; Verpoorte,R. Phytochem Anal., 2008, 19, 2-16. (2) Fuzzatin, N. J. Chromatogr. B, 2004, 812, 119-133 (3) Leung, K.W.; Wong, A.S. Chin. Med., 2010, 5, 20-24. (4) Baek, S.H.; Bae O.N.; Park, J.H. J. Ginseng Res., 2012, 36, 119-134. (5) Dugo, P.; Favoino, O.; Tranchida, P.Q.; Dugo G.; Mondello, L. J. Chromatogr. A, 2004, 1041,135-142. (6) Wang, X.; Sakuma, T.; Asafu-Adjaye, E.; Shiu, G.K. Anal. Chem., 1999, 71, 1579-1584. (7) Chan, T.W.D.; But, P.P.H.; Cheng, S.W.; Kwok, I.M.Y.; Lau F.W.; Xu, H. X.; Anal. Chem., 2000, 72, 1281-1287. (8) Li, W.; Gu, C.; Zhang, H.; Awang, D.V.; Fitzloff, J.F.; Fong H.H.; Breemen, R.B. Anal. Chem., 2000, 72, 5417-5422. (9) Miao, X.S.; Metcalfe, C.D.; Hao C.; March, R.E. J. Mass Spectrom., 2002, 37, 495-506. (10) Zubarev, R.A.; Kelleher N.L.; McLafferty, F.W. J. Am. Chem. Soc., 1998, 120, 3265-3266. (11) Budnik, B.A.; Haselmann, K.F.; Zubarev, R.A. Chem. Phys. Lett., 2001, 342, 299-302. (12) Lioe, H.; O’Hair, R.A. Anal. Bioanal. Chem., 2007, 389,1429-1437. (13) Kalli, A.; Grigorean, G.; Hakansson, K. J. Am. Soc. Mass Spectrom., 2011, 22, 2209-2221. (14) Yoo, H.J.; Hakansson, K. Anal. Chem., 2010, 82, 6940-6946. (15) Yoo, H.J.; Liu, H.; Hakansson, Anal. Chem., 2007, 79, 7858-7866. (16) Kaczorowska, M.A.; Cooper, H.J. Chem. Commun., 2011, 47, 418-420. (17) Mosely, J.A.; Smith, M.J.P.; Prakash, A.S.; Sims, M.; Bristow, A.W.T. Anal. Chem., 2011, 83, 4068-4075. (18) Perreault, H.; Costello, C.E. Org. Mass Spectrom., 1994, 29,720-735. (19) Franz, A.H.; Lebrilla, C.B. J. Am. Soc. Mass Spectrom., 2002, 13, 325-337. (20) Brull, L.P.; Heerma, W.; Thomas-Oates J.; Haverkamp, J. J. Am. Soc. Mass Spectrom., 1997, 8, 43-49. (21) Kovac P.; Haverkamp, J. J. Mass Spectrom., 1995, 30, 949-958. 11 ACS Paragon Plus Environment


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(22) McNeil, M. Carbohydr. Res., 1983, 123, 31-40. (23) Leach, F. E.; Ly, M; Laremore, T. N.; Wolff, J. J.; Perlow, J.; Linhardt, R. J.; Amster, I. J. J. Am. Soc. Mass Spectrom., 2012, 23, 1488-1497. (24) Yu, X.; Jiang, Y.; Chen, Y.; Huang, Y.; Costello, C.E.; Lin, C. Anal Chem., 2013, 85, 10017-10021. (25) Huang, Y.Q; Xiang , Y. P.; Costello, C.E.; Lin C. J. Am. Soc. Mass Spectrom., 2016, 27, 319-328. (26) Kalli, A.; Grigorean G.; Hakansson, K. J. Am. Soc. Mass Spectrom., 2011, 22, 2209-222. (27) Kall, A.; Hess, S. J. Am. Soc. Mass Spectrom., 2011, 23, 244-263. (28) Ly, T.; Yin, S.; Loo J.A.; Julian, R.R. Rapid Commun. Mass Spectrom., 2009, 23, 2099-2101.


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Figure Captions Figure 1. Structures of (A) Rb 2 , Rc, Rf and Rg 1 and (B) Pseudoginsenoside-F 11 .

Figure 2. (A) EID mass spectrum of [M+H]+ of F 11 ; (B) EID mass spectrum of [M+H]+ of Rf; (C) EID mass spectrum of [M+H]+ of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra. o--- is the loss of H 2 O.

Figure 3. (A) EID mass spectrum of [M+Na]+ of Rf; (B) EID mass spectrum of [M+Na]+ of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra. o--- is the loss of H 2 O.

Figure 4. (A) EID mass spectrum of [M-H]- of Rf; (B) EID mass spectrum of [M-H]of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra.

Figure 5. (A) EID mass spectrum of [M-H]- of Rb 2 ; (B) EID mass spectrum of [M-H]- of Rc. An asterisk indicates electronic noise or harmonics in the spectra.



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(A)

(B)

Ginsenosides

R1

R2

R3

aglycone

Rb 2

glc[2→1]glc

H

glc[6→1]ara(p)

panaxadiol

Rc

glc[2→1]glc

H

glc[6→1]ara(f)

panaxadiol

Rf

OH

glc[2→1]glc

H

panaxatriol

Rg 1

OH

glc

glc

panaxatriol

F11

OH

glc[2→1]rha

-

-

Sugar units: glc: β-D-glucose; ara(p): α-L-arabinopyranose; ara(f): α-L-arabinofuranose; rha: α-L-rhamnose

Figure 1. Structures of (A) Rb 2 , Rc, Rf and Rg 1 and (B) Pseudoginsenoside F 11 .


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Figure 2. (A) EID mass spectrum of [M+H]+ ofF 11 ; (B) EID mass spectrum of [M+H]+ of Rf; (C) EID mass spectrum of [M+H]+ of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra. o--- is the loss of H 2 O.



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Figure 3. (A) EID mass spectrum of [M+Na]+ of Rf; (B) EID mass spectrum of [M+Na]+ of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra. o--- is the loss of H 2 O.


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Figure 4. (A) EID mass spectrum of [M-H]- of Rf; (B) EID mass spectrum of [M-H]of Rg 1 . An asterisk indicates electronic noise or harmonics in the spectra.



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Figure 5. (A) EID mass spectrum of [M-H]- of Rb 2 ; (B) EID mass spectrum of [M-H]- of Rc. An asterisk indicates electronic noise or harmonics in the spectra.


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