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Differentiation and identification of ginsenoside isomers by electrospray ionization tandem mass spectrometry
Analytica Chimica Acta 531 (2005) 69–77
Differentiation and identification of ginsenoside isomers by electrospray ionization tandem mass spectrometry Fengrui Songa , Zhiqiang Liua , Shuying Liua , Zongwei Caib,∗ a
Laboratory of New Drug Research, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong SAR, China Received 4 June 2004; received in revised form 7 October 2004; accepted 7 October 2004 Available online 7 December 2004
Abstract Three pairs of ginsenoside isomers (Rg2 and Rg3 , Rg1 and F11 as well as Rd and Re ) were differentiated and identified through accurate mass measurement of mass spectrometry (MS) and MS–MS. [M + Li]+ and [M − H]− ions were detected in full-scan MS analyses and selected for the MS–MS experiments using positive and negative ion electrospray ionizations (ESI), respectively. The structures of aglycone and ␣- and -saccharide sugars in various ginsenosides were determined from the spectrum interpretation and accurate mass measurement. Z and C type ions were predominantly observed in the MS–MS spectra of [M + Li]+ ions, while Y type ions were the most abundant ions in the spectra obtained from the negative ion mode analysis. Furthermore, X and A ions resulted from cross-ring cleavage on the sugar directly connected to aglycone were detected in both positive and negative ion spectra, which provided the site information of the saccharide chains. The obtained MS–MS profiles were used for the structural confirmation of ginsenoside Rg2 collected from column chromatography separation of a Chinese Panax ginseng extract. The ESI–MS data with accurate mass assignment suggested that a co-eluted ginsenoside also existed in the sample fraction. The interpretation of its MS–MS spectrum and fragmentation pathways allowed the detection of the ginsenoside Rf , differentiating from its isomers Rg1 and F11 . © 2004 Elsevier B.V. All rights reserved. Keywords: Ginsenosides; Structural differentiation; MS–MS; Panax ginseng
1. Introduction Panax ginseng C.A. Meyer is the most popular herb used in traditional Chinese medicine. Many studies have shown that the ginseng posses properties against stress, diabetic, fatigue and cancer [1–3]. The pharmacological properties are mainly attributed from its major bioactive ingredients such as ginsenosides. Ginsenosides are the dammarance triterpence type saponins with (20S)-protopanaxadiol, (20S)protopanaxatriol, (24R)-pseudoginsenoside (Fig. 1) or oleanolic acid aglycone. Up to now more than 30 ginsenosides have been identified in various ginsengs.
∗
Corresponding author. Tel.: +852 34117070; fax: +852 34117348. E-mail address: [email protected] (Z. Cai).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.10.013
With its superior performances in sensitivity and selectivity, mass spectrometry (MS) has been increasingly applied for pharmaceutical research and for characterizing active components of traditional Chinese medicine [4–26]. Liquid chromatography coupled with mass spectrometry (LC–MS) with electrospray ionization (ESI) and collision induced dissociation (CID) MS–MS has been used for structure characterization of ginsenosides [8–24]. Both positive and negative ionization of ginsenosides have been studied. The glycosidic linkages, the core and the attached sugar(s) can be determined from the CID MS–MS analyses of [M + H]+ and [M − H]− ions. Moreover, some alkali and transition metals cations may form strongly bonded attachment ions with the ginsenosides, allowing the metal attachment ions being used for MS–MS studies. Metal-cationized ginsenoside molecules are found to have characteristic fragmentation. As a result, their CID
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Fragmentation pathways obtained from the interpretation of MS–MS spectrum and assignment of the accurate mass of each fragment ion can be used for differentiating ginsenoside isomers. This study identified three pairs of isomeric ginsenosides by interpreting HRMS–MS results that correspond to the elemental compositions of the saccharide sugar and aglycone. The major fragmentation pathways and mechanism were investigated with CID reactions in MS–MS mode and with the fragmentation assignment according to a traditional structure nomenclature for the ginsenosides. Structure elucidation on various ginsenosides was performed with the data from the MS–MS analyses. The developed method of interpreting MS–MS spectrum and fragmentation pathway were applied for the identification and confirmation of ginsenosides existed in a collected column chromatographic fraction of Chinese Panax ginseng.
2. Experimental 2.1. Materials Ginsenosides Rg1 , Rg2 , Rg3 , F11 , Rd , Re and Rf (Fig. 1 ) were provided by Professor X.Y. Ma of the Medicine College, Jilin University, China. HPLC-grade methanol and water were purchased from Acros (New Jersey, USA), and acetic acid glacial was purchased from Merck (Darmstadt, Germany). Other chemicals were at least of analytical grade. Fig. 1. Structures of ginsenoside analytes.
spectra show a variety of structurally characteristic fragmentation patterns. ESI–MS–MS profiles obtained from triple-quadrupole mass spectrometers have been used to differentiate several ginsenosites in methanol extracts of various ginseng roots [13,14]. Good selectivity of the LC–MS–MS method is crucial for separating and distinguishing two ginsenosides Rf and F11 that have the same molecular formula [14]. Ion trap MS and quadrupole-time of flight (Q-TOF) MS provides several advantages in structural analysis of ginsenosides and ginseng extracts. Ion trap MS with both positive and negative ionization modes have been used to rapidly analyze ginsenosides in crude plant extracts and to provide their structural information [19–22]. Although rich structural information can be obtained through CID in the course of MS–MS or MSn analysis, high-resolution mass spectrometry (HRMS) analysis provides more detailed and accurate structural elucidation, especially for unknown degradation products or metabolites of ginsenosides [23–25]. HRMS is not only useful for confirming the molecular composition, but also for studying the structures of various isomers of ginsenosides by applying CID technique [23,24]. Accurate mass measurement for both parent and fragment ions from the HRMS analysis can provide information of elemental composition of the analytes.
2.2. Preparation of Chinese Panax ginseng sample extract Ten grams of powdered root of Panax ginseng C.A. Mayer was refluxed twice with 100 ml methanol:water (95:5%) for 2 h. After the solvent was evaporated, the extract was dissolved in water and separated by D101 resin to obtain total ginsenosides. The ginsenosides were then passed through a silica gel column with various ratios of CHCl3 /MeOH as eluents. Different fractions were collected and analyzed for ginsenosides with thin-layer chromatography (TLC) by retention time comparison with the authentic standards. A fraction contained Rg2 according to the TLC analysis was selected for ESI–MS and MS–MS analyses for structural confirmation. 2.3. Mass spectrometric analyses All mass spectrometry analyses were performed on a quadrupole-time of flight (Q-TOF) tandem mass spectrometer equipped with a turbo-ion spray source (API Q-STAR Pulsar i, Applied Biosystems, Concord, Ont., Canada). Positive and negative ion modes of ESI were used for structural analyses. The ginsenoside standards and the column chromatographic fraction from the ginseng extract were dissolved in methanol:water (80:20%) containing 1 M LiCl for positive ion ESI analysis, while methanol was used as solvent for the negative ion mode analysis. The sample was infused
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into the ESI source via an infusion pump at a flow rate of 5 l min−1 . The following MS source parameters were used: ions spray voltage 4600 V; declustering potential 1 (DP1) 45 V, focusing potential (FP) 165 V and declustering potential 2 (DP2) 15 V; collision energy (CE) 12 eV. Nitrogen was used as nebulizing gas with settings of ion source gas (GS1) at 26 and (GS2) at 10. The curtain gas (CUR) was set at 12 and the collision gas (nitrogen) at 3.
3. Results and discussion 3.1. Positive and negative ion ESI–MS Fig. 1 lists structures and molecular weights of the targeted ginsenosides. Although Li et al. [14] and Miao et al. [26] have reported effects of ESI–MS parameters on ginsenoside signal intensities on a quadrupole mass spectrometer (Micromass Quattro), we examined the variable ranges of major voltage or potential values on the Q-TOF mass spectrometer. The results showed that the ideal ions spray voltage was in the range of 4500–4600 V, while the optimized values of declustering potential 1, focusing potential and declustering potential 2 were 45, 165 and 15 V, respectively. Lower voltage or potential gave low ion signal intensities and higher values resulted in some fragmentation complicating spectrum interpretation, especially when the column chromatographic fraction of the real ginseng extract was analyzed. Under the above conditions, the exact masses of [M + Li]+ and [M − H]− ions of the ginsenosides and the ginseng extract fraction were analyzed. The full-scan MS results confirmed the molecular weight of the ginsenosides with mass accuracy obtained from the comparison of the measured and theoretical values (Table 1). 3.2. ESI–MS–MS analysis Tandem mass spectrometric experiments were conducted with the Q-TOF mass analyzer for more detailed structure elucidation. Collision induced dissociation mass spectra of [M + Li]+ and [M − H]− ions were acquired by using positive and negative ion ESI, respectively. The application of [M + Li]+ ion for the CID experiments of ginsenosides was proved to be better than that of [M + H]+ and [M + Na]+ [21–23]. It was found that positive ion mode using lithium
Fig. 2. Fragmentation pathways of ginsenoside (Re as the example) according to the structural nomenclature suggested by Perreault and Costello [27].
adduct ion provided much better sensitivity than the negative ion mode for the ESI–MS and MS–MS analyses. For better understanding the structural elucidation, a nomenclature described by Perreault and Costello [27] was used as reference for the structure analysis of ginsenosides. Fig. 2 shows the fragment pathways of ginsenoside Re according to the nomenclature. The main core structure or aglycone is represented with R. The saccharide chain at C20 position (R4) was defined as ␣-chain because the bond cleavage first occurred at this position under most MS–MS conditions. The R1 at C3 and R2 at C6 position are named as the -chain sugar moieties. The Y and Z ions represent the charges retained on the main core structure, while B and C ions correspond to the complementary ions of Y and Z, respectively. Y ion is resulted from the loss of hexose or deoxyhexose ring unit and the Z ion is from the loss of complete saccharide sugar (glucose or rhamnose in the case of Re ). The most close sugar moiety to the main aglycone is named as the 0 order (e.g., Y0 and Z0 ) and the outside or terminus sugar, if any, as the
Table 1 ESI–MS data from the molecular ion detection of the ginsenosides Ginsenosideas [M + Li]+
[M - H]−
Rg2 Elemental composition Theoretical value Measured value Mass accuracy (ppm)
C42 H72 O13 Li 791.5133 791.5210 5.1
Elemental composition Theoretical value Measured value Mass accuracy (ppm)
C42 H71 O13 783.4895 783.4942 6.0
Rg3
Rg1
791.5063 −8.8
C42 H72 O14 Li 807.5082 807.5084 −0.2
783.4873 −2.8
C42 H71 O14 799.4844 799.4894 6.2
F11
Rd
Re
807.5062 −2.5
C48 H82 O18 Li 953.5661 953.5599 −6.5
953.5625 −3.7
799.4802 −5.2
C48 H81 O18 945.5423 945.5499 8.1
945.5470 5.0
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order of 1 (e.g., Y1 and Z1 ). The ion of Z1 corresponds to the loss of the outside sugar of R1 or R2 plus the loss of the ␣-chain sugar at C20 position (Fig. 2). In addition to the fragment pathways illustrated from the nomenclature, A and X type of fragment ions resulted from cross-ring cleavages on the sugar directly linked to aglycone were also discussed for the structural elucidation of the targeted ginsenosides. 3.3. CID spectra of [M + Li]+ ions Positive ion CID MS–MS spectra from [M + Li]+ ions of the six ginsenosides are presented in Fig. 3. The fragment pathways of the targeted ginsenosides were investigated and confirmed with the determination of elemental compositions
and accurate mass assignment of the fragment ions. The measured accurate masses were compared with the corresponding theoretical exact mass values of the assigned elemental compositions. Mass accuracy of less than 10 ppm was obtained not only for the lithium-adducted molecular ions but also for the major fragment ions of the ginsenosides. In the CID spectra of [M + Li]+ ions, major fragments were Z ions produced from the loss of saccharide sugars and the corresponding C ions representing the saccharide moieties (Fig. 2). The detection and interpretation of the Z and C ions allowed structure differentiation between each pair of the ginsenoside isomers, i.e., Rg2 and Rg3 , Rg1 and F11 as well as Rd and Re . For example, the observed Z0 ion at m/z 465 for Rg2 in Fig. 3a was interpreted as the fragment ion resulted from the loss of
Fig. 3. CID spectra from the [M + Li]+ ions of ginsenosides Rg2 (a), Rg3 (b), Rg1 (c), F11 (d), Rd (e) and Re (f).
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disaccharide sugar moiety (glucose–rhamnose, 326 Da) from the [M + Li]+ ion, which was confirmed with the accurate mass measurement of the corresponding elemental composition. The Z0 ion for Rg3, on the other hand, was observed at m/z 449 in Fig. 3b, showing the loss of a different disaccharide sugar of glucose–glucose (342 Da) for Rg3 . The differentiation of the disaccharide moieties in Rg2 and Rg3 was also demonstrated from the detection of the C2 ions, i.e., the lithium adducted ions of the disaccharide sugars. The [disaccharide + Li]+ ions were detected at m/z 333 and at m/z 349 for Rg2 and Rg3 , respectively, demonstrating the presence of the different disaccharide sugar moiety (glucose–rhamnose or glucose–glucose). The observation of 0,2 X0 and 0,2 A2 ions (Fig. 3a and b) resulted from cross-ring cleavage on the sugar directly linked to aglycone provided very important information for confirming the type of terminus saccharide sugar. For example, detection of 0,2 X0 ion at m/z 213 for Rg2 (Fig. 3a) and at m/z 229 for Rg3 (Fig. 3b) confirmed that terminus rhamnose and glucose present in the molecule of Rg2 and Rg3 , respectively. The 0,2 A2 ion at m/z 127 showed in Fig. 3a and b confirmed that both Rg2 and Rg3 has a glucose moiety existing on the -saccharide chain in between the aglycone and the terminus sugar. Furthermore, the mass difference of 16 Da of the observed Y1 ions at m/z 645 for Rg2 and at m/z 629 for Rg3 also suggested the difference between the -chain terminus saccharide sugar of Rg2 and Rg3 . Although the intensity of Y ion from the MS–MS analysis of [M + Li]+ ion was much less abundant than those of Z and C ions, the detected accurate masses of the Y1 ions of Rg2 and Rg3 still matched well with the corresponding theoretical values. The Y1 ion observed at m/z 645 for Rg2 showed that a deoxyhexose moiety from the rhamnose sugar was lost during the fragmentation. On the other hand, the Y1 ion at m/z 629 resulted from the loss of a hexose unit demonstrated the presence of the terminus glucose on the ginsenoside Rg3 (Fig. 3a and b). The structural difference of the ginsenoside isomers Rg1 and F11 can be illustrated from the interpretation of their MS–MS spectra shown in Fig. 3c and d. The absence of C2␣ (or C2 ) ion for Rg1 (Fig. 3c) indicated that probably no disaccharide sugar existed in the ginsenoside molecule. The observation of the Z1␣ (or Z1 ) at m/z 627 (loss of one glucose), Z0 at m/z 447 (loss of two individual glucoses) and the C1␣ (or C1 ) ion at m/z 187 ([glucose + Li]+ ) suggested that each of the ␣- and -chain has one monosaccharide sugar, which is also supported with the absence of 0,2 X0 ion (Fig. 3c). In contrast, the CID spectrum of F11 in Fig. 3d shows the Y1 ion at m/z 661, Z0 ion at m/z 481, C2 ion at m/z 333 and 0,2 X0 ion at m/z 213, confirming that the -chain of the pseudoginsenoside F11 contains one glucose sugar and one terminus rhamnose sugar. The -chain of F11 is same as that of Rg2 . The MS–MS spectra showed in Fig. 3e and f provided the evidence for differentiating the isomeric gisenosides Re and Rd . For ginsenoside Re , the observed 0,2 X0 at m/z 213 and 0,2 A2 ion at m/z 127 (Fig. 3f) are same as the 0,2 X0
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and 0,2 A2 ions in Fig. 3a, indicating that Re may have the same type of -saccharide chain as that of Rg2 . Similarly, the detection of the 0,2 X0 ion at m/z 229 for ginsenoside Rd (Fig. 3e) showed that its -saccharide chain might be same as that of Rg3 . Thus, the distinction in the -saccharide chain between Re and Rd isomers is same as the distinction between Rg2 and Rg3 . This is also supported with the observation of the C2 ions at m/z 333 for Re (and Rg2 ) and at m/z 349 for Rd (and Rg3 ). Furthermore, same Z0␣ ion for Re and Rd at m/z 773 showed that the isomers have the same ␣-chain. While other Z ions were not observed for Rd , Z0 and Z0 ions were detected for Re in Fig. 3f. The observation of Z0 ion at m/z 627 and Z0 ion at m/z 447 confirm that ginsenoside Re has one disaccharide unit (rahmnose–glucose) on the -chain and monosaccharide sugar (glucose) on the ␣-chain. The established methods of spectrum interpretation and MS–MS fragmentation pathways were applied to confirm a ginsenoside fraction collected from the column chromatographic separation of Chinese Panax ginseng extract. TLC has been commonly used for identifying and confirming ginsenosides separated and collected after column chromatography [14,28]. The TLC analysis and the comparison of the collected ginsenoside fraction with the authentic standard showed that it might be Rg2 . However, the positive ion ESI–MS analysis showed two possible ginsenoside peaks with one major [M + Li]+ ion at m/z 791.5214 and one minor at m/z 807.5108 (Fig. 4a). The accurate mass assignment of the lithium-adducted ions confirmed that two different ginsenoside isomers existed in the fraction. Structural elucidation was conducted for these two detected ginsenosides with the CID MS–MS analysis. Fragment ions from the major molecular ion peak at m/z 791 shown in Fig. 4 included m/z 645 (Y1 ), m/z 465 (Z0 ), m/z 447 (Z0 -H2 O), m/z 333 (C2 ), m/z 213 (0,2 X0 ) and m/z 127 (0,2 A2 ), which are same as those of Rg2 (Fig. 3a). Therefore, the MS–MS data together with accurate mass measurement confirmed that Rg2 existed in the collected column chromatographic fraction. The lithium-adducted molecular ion of the minor peak was observed at m/z 807 (Fig. 4a), indicating that the unknown component might have a molecular weight of 800. The exact mass assignment of the [M + Li]+ ion showed its elemental composition matched to that of ginsenoside Rg1 or F11 (C42 H72 O14 Li) with a mass difference of −3.2 ppm. The MS–MS analysis from the [M + Li]+ ion of the unknown, however, showed that the minor component was neither Rg1 nor F11 . The obtained MS–MS spectrum of the unknown showed major fragment ions at m/z 645 (Y1 ), at m/z 465 (Z0 ), at m/z 447 (Z0 -H2 O), at m/z 349 (C2 ), and at m/z 229 (0,2 X0 ) (Fig. 4b), which was quite different from those of Rg1 or F11 presented in Fig. 3c and d, respectively. In particular, the interpretation results of the MS–MS spectrum and fragmentation pathways of Rg1 or F11 according to the structural nomenclature described in Fig. 2 did not fit with the obtained results of the unknown. For example, the ions of Z0 at m/z 481 and C2 at m/z 333 were not observed in Fig. 4b, clearly indicated that the unknown was not the gin-
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Fig. 4. Positive ion ESI–MS spectrum (a) and MS–MS data obtained from the analyses of a ginsenoside fraction collected from column chromatographic separation of Chinese Panax ginseng extract. The MS–MS data of the major component M1 are listed in (a) confirming the detection of ginsenoside Rg2 from a conventional TLC test. The CID spectrum of M2 shown in (b) provided evidence of differentiating the identified ginsenoside Rf from its isomers Rg1 and F11 .
senoside F11 . Although some of the same fragment ions as those of ginsenoside Rg1 (e.g., m/z 447 and m/z 187) were seen in the MS–MS spectrum of the unknown, Fig. 4b shows the fragment ions of Z0 at m/z 465, C2 at m/z 349 and 0,2 X 0 at m/z 229 that did not exist in the CID spectrum of Rg1 . The interpretation of these fragment ions suggested that the unknown ginsenoside had a similar structure as that of Rg2 except that its -chain contained the glucose–glucose moiety rather than the glucose–rhamnose disaccharide sugar for Rg2 because of the observed 0,2 X0 ion was at m/z 229. Similar to the previous interpretation of MS–MS spectrum of Rg3 by using the established fragmentation pathways, the detection of Z0 ion at m/z 465, C2 ion at m/z 349 and 0,2 X0 ion at m/z 229 clearly indicated that the unknown ginsenoside component contained the glucose–glucose moiety. Further study of this isomer including the comparison with the authentic ginsenoside standard identified it as Rf (C42 H72 O14 , MW 800). It has been reported that the differentiation of ginsenoside isomers F11 , Rf and Rg1 plays important role in distinguishing Oriental ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) as well as variuos ginseng products [7,10,13,14]. Selected ginsenosides such as Rf was suggested to serve as specific chemical markers in
Oriental ginseng and the concentration ratio of Rg1 to Rf was used to differentiate Oriental and North American ginseng [7,10]. Thus, accurate detection on the presence of F11 and Rf in Oriental and North American ginseng appeared to be crucial for separating the ginseng roots of different species as well as various geographic origins [14]. Different results, however, have been reported [13,14]. The similar structural, chemical and chromatographic behaviors of ginsenosides F11 and Rf have posted significant analytical challenge in their separation. Due to the very close retention time under many reversed-phase liquid chromatographic conditions, problems of misidentifying the isomeric ginsneosides caused from less specific analytical methods have been reported [14]. Intensive discussions regarding the possible positive and negative detections in various ginseng products were published [7,10,14]. Recently, a LC–MS–MS method was reported for unambiguously distinguishing the Oriental and North American ginsengs [14]. In addition to the identification using tandem mass spectrometry, the method was based on the baseline chromatographic separation of Rf and F11 , along with the determination of the concentration ratio of the two-ginsenoside isomers. In this study, the interpretation of the CID MS–MS spectra from the [M + Li]+ ions and the application of the
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fragmentation pathways according to the structural nomenclature shown in Fig. 2 provided clear evidence for differentiating the ginsenoside isomers Rf , Rg1 and F11 . The method has been successfully applied for the identification and confirmation of a ginsenoside fraction collected from the column chromatographic separation of a Chinese panax ginseng extract. By directly infusing the collected ginsenoside sample into the ESI source, the Q-TOF MS–MS analysis with the accurate measurement of both molecular and fragment ions
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provided structural identification and of the ginsenoside Rf in the column chromatographic fraction from a Chinese Panax ginseng extract. 3.4. CID spectra of [M − H]− ions The MS–MS analyses of [M − H]− ions of the targeted ginsenosides produced a series of Y and Z ions resulted from glycosidic bond cleavage. Again, the accurate masses of the
Fig. 5. CID spectra from the [M − H]− ions of ginsenosides Rg2 (a), Rg3 (b), Rg1 (c), F11 (d), Rd (e) and Re (f).
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detected molecular ions and fragment ions matched the corresponding theoretical mass values within 10 ppm. The Y type ions that were found to be the dominant fragment ions in the negative ion mode CID spectra of the ginsenosides can be evidently used for elucidating the structure of saccharide chain and aglycone. Fig. 5a shows the MS–MS mass spectrum of ginsenoside Rg2 from the [M − H]− ion of m/z 783. The observation of Y1 at m/z 637 and Z1 ion at m/z 619 provided clear evidence of existing deoxyhexose (from the rhamnose) at terminus position of the -saccharide chain. The Y0 ion at m/z 475 was formed when the molecular ion lost the entire disaccharide sugar moiety on the -chain, while the [Y0 − 84]− ion resulted from the further loss of the side chain C6 H12 from the aglycone at C20 position (see Fig. 2 for the structure). The observed Y0 ion and the fragment pattern fit well with the structure of aglycone in panaxatriol ginsenosides (e.g., Rg1 , Rg2 and Re ). For panaxadiol ginsenosides (Rd and Rg3 ) and pseudoginsenoside (F11 ), Y0 (or Y0 ) ions corresponded to the [aglycone − H]− ion at m/z 459 and at m/z 491, respectively. Therefore, the panaxadiol and panaxatriol ginsenoside isomers can be immediately differentiated by the detection of Y0 and Y1 fragment ions at different masses. For example, the MS–MS data in Fig. 5a and b show Y0 ion at m/z 475 for Rg2 and at m/z 459 for Rg3 , although this pair of ginsenoside isomers have the same [M − H]− ion at m/z 783. Rg2 was also differentiated from Rg3 with the observation of the Y1 ions at m/z 637 (Fig. 5a) and at m/z 621 (Fig. 5b) for Rg2 and Rg3 , respectively. In Fig. 5e, [M − H]− ion at m/z 945 of panaxadiol ginsenoside Rd produced the same Y0␣ and Y1 ions at m/z 783, showing that the one hexose residue may exist in the ␣- or -saccharide chains. In addition, the Y0 or Y1 ion at m/z 621 resulted from the loss of 324 Da (two hexose units) and the Y0 ion at m/z 459 from the further loss of 162 Da (another hexose unit) were detected, confirming gisenoside Rd has three glucoses (two on the -chain and one on the ␣-chain). The detected Y0 ion at m/z 459 actually represented the [aglycone − H]− ion for ginsenoside Rd . The data suggested that two-hexose residues existed in the -chain and one hexose residue in ␣-chain for the ginsenoside. The CID spectrum of its isomer Re (panaxatriol ginsenoside), on the other hand, showed Y0␣ ion at m/z 783 and Y1 at m/z 799 (Fig. 5f). The detected Y0␣ and Y1 ions were resulted from the loss of one hexose and one deoxyhexose moiety from the ␣- and -saccharide chain, respectively. Additional fragment ion peaks at m/z 637 (Y0 or Y1 ) and at m/z 475 (Y0 ) suggested that the ␣-chain of ginsenoside Re contains one hexose unit and the -chain has one hexose and one deoxyohexose group. Thus, the aglycone of Re is a 20S-protopanaxatriol with its [aglycone − H]− ion at m/z 475. Similarly, different [aglycone − H]− ions were observed for isomeric ginsenosides Rg1 and F11 and their spectra interpretation can be conducted in the same way as described
above in order to differentiate various ␣- and -saccharide chains (Fig. 5c and d). Furthermore, the detection of specific 0,2 X0 and 0,2 A2 ions resulted from the cross-ring cleavage on the sugar directly linked to aglycone may also provided clear evidence for determining the number and type of the saccharide sugar. For the isomer pairs of Rg2 and Rg3 as well as Re and Rd , 0,2 X 0 ion was detected at m/z 205 for Rg2 (Fig. 5a) and Re (Fig. 5f), while at m/z 221 for Rg3 (Fig. 5b) and Rd (Fig. 5e). The data further confirmed that Rg2 and Re have the same  disaccharide sugar of glucose–rhamnose, while Rg3 and Rd has the same glucose–glucose unit on their -chain. 0,2 X0 ion was not seen in the MS–MS spectrum of Rg1 (Fig. 5c) when the mass was scanned from 50 to 1000 Da because the ginsenoside has only monosaccharide (glucose) on each of the ␣- and -chain. The -chain of the pseudoginsenoside F11 is same as that of Rg2 and Re because the peak at m/z 205 was detected for its 0,2 X0 ion (Fig. 5d). However, the pseudoginsenoside F11 has the Y0 ion at m/z 491, confirming that its aglycone contains one more oxygen or hydroxyl group than the panaxatriol aglycone of Rg2 and Re (Fig. 1).
4. Conclusions High-resolution tandem mass spectrometry analysis of ginsenosides can provide the information of molecular weight and element composition for both parent and fragment ions for the structural elucidation. The interpretation of both positive and negative ion MS–MS spectra together with the accurate mass assignment differentiated the selected ginsenoside isomers, Rg2 and Rg3 , Rg1 and F11 , as well as Rd and Re . The sensitivity of the MS–MS analysis on the [M + Li]+ ion was found much better than that on the [M − H]− ion. Positive ion mode ESI–MS–MS analysis of [M + Li]+ ions provided structure information on ␣- and -sugar chains of the ginsenosindes through the detection of predominant Z and C ions. However, the information of aglycone was not directly obtained due to the absence of Y type ions in the spectra. On the other hand, the CID experiment of [M − H]− ion produced multiple major Y type ions, providing structural information on both saccharide residues and aglycone of the isomeric ginsenosides. X and A type ions resulted from cross-ring cleavage on the sugar molecule directly connected to aglycone can be obtained from the MS–MS analyses of both [M + Li]+ and [M − H]− ions. Interpretation of the X and A ions provided strucural information of the number and type of saccharide sugar presented in the ginsenosides. The developed method has been successfully applied for analyzing a column chromatographic fraction of a Chinese Panax ginseng extract with the confirmation of ginsenoside Rg2 and identification of a co-eluted ginsenoside Rf . The method provided rapid confirmation of gisenoside detection after the traditional TLC technique.
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Acknowledgement Financial support for this work was sponsored by National Natural Science Foundation of China (Grant No. 39930210) and the Competitive Earmarked Research Grant (CERG), Research Grants Council (RGC) of Hong Kong (HKBU2017/02P).
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Differentiation and identification of ginsenoside isomers by electrospray ionization tandem mass spectrometry Fengrui Songa , Zhiqiang Liua , Shuying Liua , Zongwei Caib,∗ a
Laboratory of New Drug Research, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Kowloon, Hong Kong SAR, China Received 4 June 2004; received in revised form 7 October 2004; accepted 7 October 2004 Available online 7 December 2004
Abstract Three pairs of ginsenoside isomers (Rg2 and Rg3 , Rg1 and F11 as well as Rd and Re ) were differentiated and identified through accurate mass measurement of mass spectrometry (MS) and MS–MS. [M + Li]+ and [M − H]− ions were detected in full-scan MS analyses and selected for the MS–MS experiments using positive and negative ion electrospray ionizations (ESI), respectively. The structures of aglycone and ␣- and -saccharide sugars in various ginsenosides were determined from the spectrum interpretation and accurate mass measurement. Z and C type ions were predominantly observed in the MS–MS spectra of [M + Li]+ ions, while Y type ions were the most abundant ions in the spectra obtained from the negative ion mode analysis. Furthermore, X and A ions resulted from cross-ring cleavage on the sugar directly connected to aglycone were detected in both positive and negative ion spectra, which provided the site information of the saccharide chains. The obtained MS–MS profiles were used for the structural confirmation of ginsenoside Rg2 collected from column chromatography separation of a Chinese Panax ginseng extract. The ESI–MS data with accurate mass assignment suggested that a co-eluted ginsenoside also existed in the sample fraction. The interpretation of its MS–MS spectrum and fragmentation pathways allowed the detection of the ginsenoside Rf , differentiating from its isomers Rg1 and F11 . © 2004 Elsevier B.V. All rights reserved. Keywords: Ginsenosides; Structural differentiation; MS–MS; Panax ginseng
1. Introduction Panax ginseng C.A. Meyer is the most popular herb used in traditional Chinese medicine. Many studies have shown that the ginseng posses properties against stress, diabetic, fatigue and cancer [1–3]. The pharmacological properties are mainly attributed from its major bioactive ingredients such as ginsenosides. Ginsenosides are the dammarance triterpence type saponins with (20S)-protopanaxadiol, (20S)protopanaxatriol, (24R)-pseudoginsenoside (Fig. 1) or oleanolic acid aglycone. Up to now more than 30 ginsenosides have been identified in various ginsengs.
∗
Corresponding author. Tel.: +852 34117070; fax: +852 34117348. E-mail address: [email protected] (Z. Cai).
0003-2670/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2004.10.013
With its superior performances in sensitivity and selectivity, mass spectrometry (MS) has been increasingly applied for pharmaceutical research and for characterizing active components of traditional Chinese medicine [4–26]. Liquid chromatography coupled with mass spectrometry (LC–MS) with electrospray ionization (ESI) and collision induced dissociation (CID) MS–MS has been used for structure characterization of ginsenosides [8–24]. Both positive and negative ionization of ginsenosides have been studied. The glycosidic linkages, the core and the attached sugar(s) can be determined from the CID MS–MS analyses of [M + H]+ and [M − H]− ions. Moreover, some alkali and transition metals cations may form strongly bonded attachment ions with the ginsenosides, allowing the metal attachment ions being used for MS–MS studies. Metal-cationized ginsenoside molecules are found to have characteristic fragmentation. As a result, their CID
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Fragmentation pathways obtained from the interpretation of MS–MS spectrum and assignment of the accurate mass of each fragment ion can be used for differentiating ginsenoside isomers. This study identified three pairs of isomeric ginsenosides by interpreting HRMS–MS results that correspond to the elemental compositions of the saccharide sugar and aglycone. The major fragmentation pathways and mechanism were investigated with CID reactions in MS–MS mode and with the fragmentation assignment according to a traditional structure nomenclature for the ginsenosides. Structure elucidation on various ginsenosides was performed with the data from the MS–MS analyses. The developed method of interpreting MS–MS spectrum and fragmentation pathway were applied for the identification and confirmation of ginsenosides existed in a collected column chromatographic fraction of Chinese Panax ginseng.
2. Experimental 2.1. Materials Ginsenosides Rg1 , Rg2 , Rg3 , F11 , Rd , Re and Rf (Fig. 1 ) were provided by Professor X.Y. Ma of the Medicine College, Jilin University, China. HPLC-grade methanol and water were purchased from Acros (New Jersey, USA), and acetic acid glacial was purchased from Merck (Darmstadt, Germany). Other chemicals were at least of analytical grade. Fig. 1. Structures of ginsenoside analytes.
spectra show a variety of structurally characteristic fragmentation patterns. ESI–MS–MS profiles obtained from triple-quadrupole mass spectrometers have been used to differentiate several ginsenosites in methanol extracts of various ginseng roots [13,14]. Good selectivity of the LC–MS–MS method is crucial for separating and distinguishing two ginsenosides Rf and F11 that have the same molecular formula [14]. Ion trap MS and quadrupole-time of flight (Q-TOF) MS provides several advantages in structural analysis of ginsenosides and ginseng extracts. Ion trap MS with both positive and negative ionization modes have been used to rapidly analyze ginsenosides in crude plant extracts and to provide their structural information [19–22]. Although rich structural information can be obtained through CID in the course of MS–MS or MSn analysis, high-resolution mass spectrometry (HRMS) analysis provides more detailed and accurate structural elucidation, especially for unknown degradation products or metabolites of ginsenosides [23–25]. HRMS is not only useful for confirming the molecular composition, but also for studying the structures of various isomers of ginsenosides by applying CID technique [23,24]. Accurate mass measurement for both parent and fragment ions from the HRMS analysis can provide information of elemental composition of the analytes.
2.2. Preparation of Chinese Panax ginseng sample extract Ten grams of powdered root of Panax ginseng C.A. Mayer was refluxed twice with 100 ml methanol:water (95:5%) for 2 h. After the solvent was evaporated, the extract was dissolved in water and separated by D101 resin to obtain total ginsenosides. The ginsenosides were then passed through a silica gel column with various ratios of CHCl3 /MeOH as eluents. Different fractions were collected and analyzed for ginsenosides with thin-layer chromatography (TLC) by retention time comparison with the authentic standards. A fraction contained Rg2 according to the TLC analysis was selected for ESI–MS and MS–MS analyses for structural confirmation. 2.3. Mass spectrometric analyses All mass spectrometry analyses were performed on a quadrupole-time of flight (Q-TOF) tandem mass spectrometer equipped with a turbo-ion spray source (API Q-STAR Pulsar i, Applied Biosystems, Concord, Ont., Canada). Positive and negative ion modes of ESI were used for structural analyses. The ginsenoside standards and the column chromatographic fraction from the ginseng extract were dissolved in methanol:water (80:20%) containing 1 M LiCl for positive ion ESI analysis, while methanol was used as solvent for the negative ion mode analysis. The sample was infused
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into the ESI source via an infusion pump at a flow rate of 5 l min−1 . The following MS source parameters were used: ions spray voltage 4600 V; declustering potential 1 (DP1) 45 V, focusing potential (FP) 165 V and declustering potential 2 (DP2) 15 V; collision energy (CE) 12 eV. Nitrogen was used as nebulizing gas with settings of ion source gas (GS1) at 26 and (GS2) at 10. The curtain gas (CUR) was set at 12 and the collision gas (nitrogen) at 3.
3. Results and discussion 3.1. Positive and negative ion ESI–MS Fig. 1 lists structures and molecular weights of the targeted ginsenosides. Although Li et al. [14] and Miao et al. [26] have reported effects of ESI–MS parameters on ginsenoside signal intensities on a quadrupole mass spectrometer (Micromass Quattro), we examined the variable ranges of major voltage or potential values on the Q-TOF mass spectrometer. The results showed that the ideal ions spray voltage was in the range of 4500–4600 V, while the optimized values of declustering potential 1, focusing potential and declustering potential 2 were 45, 165 and 15 V, respectively. Lower voltage or potential gave low ion signal intensities and higher values resulted in some fragmentation complicating spectrum interpretation, especially when the column chromatographic fraction of the real ginseng extract was analyzed. Under the above conditions, the exact masses of [M + Li]+ and [M − H]− ions of the ginsenosides and the ginseng extract fraction were analyzed. The full-scan MS results confirmed the molecular weight of the ginsenosides with mass accuracy obtained from the comparison of the measured and theoretical values (Table 1). 3.2. ESI–MS–MS analysis Tandem mass spectrometric experiments were conducted with the Q-TOF mass analyzer for more detailed structure elucidation. Collision induced dissociation mass spectra of [M + Li]+ and [M − H]− ions were acquired by using positive and negative ion ESI, respectively. The application of [M + Li]+ ion for the CID experiments of ginsenosides was proved to be better than that of [M + H]+ and [M + Na]+ [21–23]. It was found that positive ion mode using lithium
Fig. 2. Fragmentation pathways of ginsenoside (Re as the example) according to the structural nomenclature suggested by Perreault and Costello [27].
adduct ion provided much better sensitivity than the negative ion mode for the ESI–MS and MS–MS analyses. For better understanding the structural elucidation, a nomenclature described by Perreault and Costello [27] was used as reference for the structure analysis of ginsenosides. Fig. 2 shows the fragment pathways of ginsenoside Re according to the nomenclature. The main core structure or aglycone is represented with R. The saccharide chain at C20 position (R4) was defined as ␣-chain because the bond cleavage first occurred at this position under most MS–MS conditions. The R1 at C3 and R2 at C6 position are named as the -chain sugar moieties. The Y and Z ions represent the charges retained on the main core structure, while B and C ions correspond to the complementary ions of Y and Z, respectively. Y ion is resulted from the loss of hexose or deoxyhexose ring unit and the Z ion is from the loss of complete saccharide sugar (glucose or rhamnose in the case of Re ). The most close sugar moiety to the main aglycone is named as the 0 order (e.g., Y0 and Z0 ) and the outside or terminus sugar, if any, as the
Table 1 ESI–MS data from the molecular ion detection of the ginsenosides Ginsenosideas [M + Li]+
[M - H]−
Rg2 Elemental composition Theoretical value Measured value Mass accuracy (ppm)
C42 H72 O13 Li 791.5133 791.5210 5.1
Elemental composition Theoretical value Measured value Mass accuracy (ppm)
C42 H71 O13 783.4895 783.4942 6.0
Rg3
Rg1
791.5063 −8.8
C42 H72 O14 Li 807.5082 807.5084 −0.2
783.4873 −2.8
C42 H71 O14 799.4844 799.4894 6.2
F11
Rd
Re
807.5062 −2.5
C48 H82 O18 Li 953.5661 953.5599 −6.5
953.5625 −3.7
799.4802 −5.2
C48 H81 O18 945.5423 945.5499 8.1
945.5470 5.0
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order of 1 (e.g., Y1 and Z1 ). The ion of Z1 corresponds to the loss of the outside sugar of R1 or R2 plus the loss of the ␣-chain sugar at C20 position (Fig. 2). In addition to the fragment pathways illustrated from the nomenclature, A and X type of fragment ions resulted from cross-ring cleavages on the sugar directly linked to aglycone were also discussed for the structural elucidation of the targeted ginsenosides. 3.3. CID spectra of [M + Li]+ ions Positive ion CID MS–MS spectra from [M + Li]+ ions of the six ginsenosides are presented in Fig. 3. The fragment pathways of the targeted ginsenosides were investigated and confirmed with the determination of elemental compositions
and accurate mass assignment of the fragment ions. The measured accurate masses were compared with the corresponding theoretical exact mass values of the assigned elemental compositions. Mass accuracy of less than 10 ppm was obtained not only for the lithium-adducted molecular ions but also for the major fragment ions of the ginsenosides. In the CID spectra of [M + Li]+ ions, major fragments were Z ions produced from the loss of saccharide sugars and the corresponding C ions representing the saccharide moieties (Fig. 2). The detection and interpretation of the Z and C ions allowed structure differentiation between each pair of the ginsenoside isomers, i.e., Rg2 and Rg3 , Rg1 and F11 as well as Rd and Re . For example, the observed Z0 ion at m/z 465 for Rg2 in Fig. 3a was interpreted as the fragment ion resulted from the loss of
Fig. 3. CID spectra from the [M + Li]+ ions of ginsenosides Rg2 (a), Rg3 (b), Rg1 (c), F11 (d), Rd (e) and Re (f).
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disaccharide sugar moiety (glucose–rhamnose, 326 Da) from the [M + Li]+ ion, which was confirmed with the accurate mass measurement of the corresponding elemental composition. The Z0 ion for Rg3, on the other hand, was observed at m/z 449 in Fig. 3b, showing the loss of a different disaccharide sugar of glucose–glucose (342 Da) for Rg3 . The differentiation of the disaccharide moieties in Rg2 and Rg3 was also demonstrated from the detection of the C2 ions, i.e., the lithium adducted ions of the disaccharide sugars. The [disaccharide + Li]+ ions were detected at m/z 333 and at m/z 349 for Rg2 and Rg3 , respectively, demonstrating the presence of the different disaccharide sugar moiety (glucose–rhamnose or glucose–glucose). The observation of 0,2 X0 and 0,2 A2 ions (Fig. 3a and b) resulted from cross-ring cleavage on the sugar directly linked to aglycone provided very important information for confirming the type of terminus saccharide sugar. For example, detection of 0,2 X0 ion at m/z 213 for Rg2 (Fig. 3a) and at m/z 229 for Rg3 (Fig. 3b) confirmed that terminus rhamnose and glucose present in the molecule of Rg2 and Rg3 , respectively. The 0,2 A2 ion at m/z 127 showed in Fig. 3a and b confirmed that both Rg2 and Rg3 has a glucose moiety existing on the -saccharide chain in between the aglycone and the terminus sugar. Furthermore, the mass difference of 16 Da of the observed Y1 ions at m/z 645 for Rg2 and at m/z 629 for Rg3 also suggested the difference between the -chain terminus saccharide sugar of Rg2 and Rg3 . Although the intensity of Y ion from the MS–MS analysis of [M + Li]+ ion was much less abundant than those of Z and C ions, the detected accurate masses of the Y1 ions of Rg2 and Rg3 still matched well with the corresponding theoretical values. The Y1 ion observed at m/z 645 for Rg2 showed that a deoxyhexose moiety from the rhamnose sugar was lost during the fragmentation. On the other hand, the Y1 ion at m/z 629 resulted from the loss of a hexose unit demonstrated the presence of the terminus glucose on the ginsenoside Rg3 (Fig. 3a and b). The structural difference of the ginsenoside isomers Rg1 and F11 can be illustrated from the interpretation of their MS–MS spectra shown in Fig. 3c and d. The absence of C2␣ (or C2 ) ion for Rg1 (Fig. 3c) indicated that probably no disaccharide sugar existed in the ginsenoside molecule. The observation of the Z1␣ (or Z1 ) at m/z 627 (loss of one glucose), Z0 at m/z 447 (loss of two individual glucoses) and the C1␣ (or C1 ) ion at m/z 187 ([glucose + Li]+ ) suggested that each of the ␣- and -chain has one monosaccharide sugar, which is also supported with the absence of 0,2 X0 ion (Fig. 3c). In contrast, the CID spectrum of F11 in Fig. 3d shows the Y1 ion at m/z 661, Z0 ion at m/z 481, C2 ion at m/z 333 and 0,2 X0 ion at m/z 213, confirming that the -chain of the pseudoginsenoside F11 contains one glucose sugar and one terminus rhamnose sugar. The -chain of F11 is same as that of Rg2 . The MS–MS spectra showed in Fig. 3e and f provided the evidence for differentiating the isomeric gisenosides Re and Rd . For ginsenoside Re , the observed 0,2 X0 at m/z 213 and 0,2 A2 ion at m/z 127 (Fig. 3f) are same as the 0,2 X0
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and 0,2 A2 ions in Fig. 3a, indicating that Re may have the same type of -saccharide chain as that of Rg2 . Similarly, the detection of the 0,2 X0 ion at m/z 229 for ginsenoside Rd (Fig. 3e) showed that its -saccharide chain might be same as that of Rg3 . Thus, the distinction in the -saccharide chain between Re and Rd isomers is same as the distinction between Rg2 and Rg3 . This is also supported with the observation of the C2 ions at m/z 333 for Re (and Rg2 ) and at m/z 349 for Rd (and Rg3 ). Furthermore, same Z0␣ ion for Re and Rd at m/z 773 showed that the isomers have the same ␣-chain. While other Z ions were not observed for Rd , Z0 and Z0 ions were detected for Re in Fig. 3f. The observation of Z0 ion at m/z 627 and Z0 ion at m/z 447 confirm that ginsenoside Re has one disaccharide unit (rahmnose–glucose) on the -chain and monosaccharide sugar (glucose) on the ␣-chain. The established methods of spectrum interpretation and MS–MS fragmentation pathways were applied to confirm a ginsenoside fraction collected from the column chromatographic separation of Chinese Panax ginseng extract. TLC has been commonly used for identifying and confirming ginsenosides separated and collected after column chromatography [14,28]. The TLC analysis and the comparison of the collected ginsenoside fraction with the authentic standard showed that it might be Rg2 . However, the positive ion ESI–MS analysis showed two possible ginsenoside peaks with one major [M + Li]+ ion at m/z 791.5214 and one minor at m/z 807.5108 (Fig. 4a). The accurate mass assignment of the lithium-adducted ions confirmed that two different ginsenoside isomers existed in the fraction. Structural elucidation was conducted for these two detected ginsenosides with the CID MS–MS analysis. Fragment ions from the major molecular ion peak at m/z 791 shown in Fig. 4 included m/z 645 (Y1 ), m/z 465 (Z0 ), m/z 447 (Z0 -H2 O), m/z 333 (C2 ), m/z 213 (0,2 X0 ) and m/z 127 (0,2 A2 ), which are same as those of Rg2 (Fig. 3a). Therefore, the MS–MS data together with accurate mass measurement confirmed that Rg2 existed in the collected column chromatographic fraction. The lithium-adducted molecular ion of the minor peak was observed at m/z 807 (Fig. 4a), indicating that the unknown component might have a molecular weight of 800. The exact mass assignment of the [M + Li]+ ion showed its elemental composition matched to that of ginsenoside Rg1 or F11 (C42 H72 O14 Li) with a mass difference of −3.2 ppm. The MS–MS analysis from the [M + Li]+ ion of the unknown, however, showed that the minor component was neither Rg1 nor F11 . The obtained MS–MS spectrum of the unknown showed major fragment ions at m/z 645 (Y1 ), at m/z 465 (Z0 ), at m/z 447 (Z0 -H2 O), at m/z 349 (C2 ), and at m/z 229 (0,2 X0 ) (Fig. 4b), which was quite different from those of Rg1 or F11 presented in Fig. 3c and d, respectively. In particular, the interpretation results of the MS–MS spectrum and fragmentation pathways of Rg1 or F11 according to the structural nomenclature described in Fig. 2 did not fit with the obtained results of the unknown. For example, the ions of Z0 at m/z 481 and C2 at m/z 333 were not observed in Fig. 4b, clearly indicated that the unknown was not the gin-
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Fig. 4. Positive ion ESI–MS spectrum (a) and MS–MS data obtained from the analyses of a ginsenoside fraction collected from column chromatographic separation of Chinese Panax ginseng extract. The MS–MS data of the major component M1 are listed in (a) confirming the detection of ginsenoside Rg2 from a conventional TLC test. The CID spectrum of M2 shown in (b) provided evidence of differentiating the identified ginsenoside Rf from its isomers Rg1 and F11 .
senoside F11 . Although some of the same fragment ions as those of ginsenoside Rg1 (e.g., m/z 447 and m/z 187) were seen in the MS–MS spectrum of the unknown, Fig. 4b shows the fragment ions of Z0 at m/z 465, C2 at m/z 349 and 0,2 X 0 at m/z 229 that did not exist in the CID spectrum of Rg1 . The interpretation of these fragment ions suggested that the unknown ginsenoside had a similar structure as that of Rg2 except that its -chain contained the glucose–glucose moiety rather than the glucose–rhamnose disaccharide sugar for Rg2 because of the observed 0,2 X0 ion was at m/z 229. Similar to the previous interpretation of MS–MS spectrum of Rg3 by using the established fragmentation pathways, the detection of Z0 ion at m/z 465, C2 ion at m/z 349 and 0,2 X0 ion at m/z 229 clearly indicated that the unknown ginsenoside component contained the glucose–glucose moiety. Further study of this isomer including the comparison with the authentic ginsenoside standard identified it as Rf (C42 H72 O14 , MW 800). It has been reported that the differentiation of ginsenoside isomers F11 , Rf and Rg1 plays important role in distinguishing Oriental ginseng (Panax ginseng C.A. Meyer) and North American ginseng (Panax quinquefolius L.) as well as variuos ginseng products [7,10,13,14]. Selected ginsenosides such as Rf was suggested to serve as specific chemical markers in
Oriental ginseng and the concentration ratio of Rg1 to Rf was used to differentiate Oriental and North American ginseng [7,10]. Thus, accurate detection on the presence of F11 and Rf in Oriental and North American ginseng appeared to be crucial for separating the ginseng roots of different species as well as various geographic origins [14]. Different results, however, have been reported [13,14]. The similar structural, chemical and chromatographic behaviors of ginsenosides F11 and Rf have posted significant analytical challenge in their separation. Due to the very close retention time under many reversed-phase liquid chromatographic conditions, problems of misidentifying the isomeric ginsneosides caused from less specific analytical methods have been reported [14]. Intensive discussions regarding the possible positive and negative detections in various ginseng products were published [7,10,14]. Recently, a LC–MS–MS method was reported for unambiguously distinguishing the Oriental and North American ginsengs [14]. In addition to the identification using tandem mass spectrometry, the method was based on the baseline chromatographic separation of Rf and F11 , along with the determination of the concentration ratio of the two-ginsenoside isomers. In this study, the interpretation of the CID MS–MS spectra from the [M + Li]+ ions and the application of the
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fragmentation pathways according to the structural nomenclature shown in Fig. 2 provided clear evidence for differentiating the ginsenoside isomers Rf , Rg1 and F11 . The method has been successfully applied for the identification and confirmation of a ginsenoside fraction collected from the column chromatographic separation of a Chinese panax ginseng extract. By directly infusing the collected ginsenoside sample into the ESI source, the Q-TOF MS–MS analysis with the accurate measurement of both molecular and fragment ions
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provided structural identification and of the ginsenoside Rf in the column chromatographic fraction from a Chinese Panax ginseng extract. 3.4. CID spectra of [M − H]− ions The MS–MS analyses of [M − H]− ions of the targeted ginsenosides produced a series of Y and Z ions resulted from glycosidic bond cleavage. Again, the accurate masses of the
Fig. 5. CID spectra from the [M − H]− ions of ginsenosides Rg2 (a), Rg3 (b), Rg1 (c), F11 (d), Rd (e) and Re (f).
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detected molecular ions and fragment ions matched the corresponding theoretical mass values within 10 ppm. The Y type ions that were found to be the dominant fragment ions in the negative ion mode CID spectra of the ginsenosides can be evidently used for elucidating the structure of saccharide chain and aglycone. Fig. 5a shows the MS–MS mass spectrum of ginsenoside Rg2 from the [M − H]− ion of m/z 783. The observation of Y1 at m/z 637 and Z1 ion at m/z 619 provided clear evidence of existing deoxyhexose (from the rhamnose) at terminus position of the -saccharide chain. The Y0 ion at m/z 475 was formed when the molecular ion lost the entire disaccharide sugar moiety on the -chain, while the [Y0 − 84]− ion resulted from the further loss of the side chain C6 H12 from the aglycone at C20 position (see Fig. 2 for the structure). The observed Y0 ion and the fragment pattern fit well with the structure of aglycone in panaxatriol ginsenosides (e.g., Rg1 , Rg2 and Re ). For panaxadiol ginsenosides (Rd and Rg3 ) and pseudoginsenoside (F11 ), Y0 (or Y0 ) ions corresponded to the [aglycone − H]− ion at m/z 459 and at m/z 491, respectively. Therefore, the panaxadiol and panaxatriol ginsenoside isomers can be immediately differentiated by the detection of Y0 and Y1 fragment ions at different masses. For example, the MS–MS data in Fig. 5a and b show Y0 ion at m/z 475 for Rg2 and at m/z 459 for Rg3 , although this pair of ginsenoside isomers have the same [M − H]− ion at m/z 783. Rg2 was also differentiated from Rg3 with the observation of the Y1 ions at m/z 637 (Fig. 5a) and at m/z 621 (Fig. 5b) for Rg2 and Rg3 , respectively. In Fig. 5e, [M − H]− ion at m/z 945 of panaxadiol ginsenoside Rd produced the same Y0␣ and Y1 ions at m/z 783, showing that the one hexose residue may exist in the ␣- or -saccharide chains. In addition, the Y0 or Y1 ion at m/z 621 resulted from the loss of 324 Da (two hexose units) and the Y0 ion at m/z 459 from the further loss of 162 Da (another hexose unit) were detected, confirming gisenoside Rd has three glucoses (two on the -chain and one on the ␣-chain). The detected Y0 ion at m/z 459 actually represented the [aglycone − H]− ion for ginsenoside Rd . The data suggested that two-hexose residues existed in the -chain and one hexose residue in ␣-chain for the ginsenoside. The CID spectrum of its isomer Re (panaxatriol ginsenoside), on the other hand, showed Y0␣ ion at m/z 783 and Y1 at m/z 799 (Fig. 5f). The detected Y0␣ and Y1 ions were resulted from the loss of one hexose and one deoxyhexose moiety from the ␣- and -saccharide chain, respectively. Additional fragment ion peaks at m/z 637 (Y0 or Y1 ) and at m/z 475 (Y0 ) suggested that the ␣-chain of ginsenoside Re contains one hexose unit and the -chain has one hexose and one deoxyohexose group. Thus, the aglycone of Re is a 20S-protopanaxatriol with its [aglycone − H]− ion at m/z 475. Similarly, different [aglycone − H]− ions were observed for isomeric ginsenosides Rg1 and F11 and their spectra interpretation can be conducted in the same way as described
above in order to differentiate various ␣- and -saccharide chains (Fig. 5c and d). Furthermore, the detection of specific 0,2 X0 and 0,2 A2 ions resulted from the cross-ring cleavage on the sugar directly linked to aglycone may also provided clear evidence for determining the number and type of the saccharide sugar. For the isomer pairs of Rg2 and Rg3 as well as Re and Rd , 0,2 X 0 ion was detected at m/z 205 for Rg2 (Fig. 5a) and Re (Fig. 5f), while at m/z 221 for Rg3 (Fig. 5b) and Rd (Fig. 5e). The data further confirmed that Rg2 and Re have the same  disaccharide sugar of glucose–rhamnose, while Rg3 and Rd has the same glucose–glucose unit on their -chain. 0,2 X0 ion was not seen in the MS–MS spectrum of Rg1 (Fig. 5c) when the mass was scanned from 50 to 1000 Da because the ginsenoside has only monosaccharide (glucose) on each of the ␣- and -chain. The -chain of the pseudoginsenoside F11 is same as that of Rg2 and Re because the peak at m/z 205 was detected for its 0,2 X0 ion (Fig. 5d). However, the pseudoginsenoside F11 has the Y0 ion at m/z 491, confirming that its aglycone contains one more oxygen or hydroxyl group than the panaxatriol aglycone of Rg2 and Re (Fig. 1).
4. Conclusions High-resolution tandem mass spectrometry analysis of ginsenosides can provide the information of molecular weight and element composition for both parent and fragment ions for the structural elucidation. The interpretation of both positive and negative ion MS–MS spectra together with the accurate mass assignment differentiated the selected ginsenoside isomers, Rg2 and Rg3 , Rg1 and F11 , as well as Rd and Re . The sensitivity of the MS–MS analysis on the [M + Li]+ ion was found much better than that on the [M − H]− ion. Positive ion mode ESI–MS–MS analysis of [M + Li]+ ions provided structure information on ␣- and -sugar chains of the ginsenosindes through the detection of predominant Z and C ions. However, the information of aglycone was not directly obtained due to the absence of Y type ions in the spectra. On the other hand, the CID experiment of [M − H]− ion produced multiple major Y type ions, providing structural information on both saccharide residues and aglycone of the isomeric ginsenosides. X and A type ions resulted from cross-ring cleavage on the sugar molecule directly connected to aglycone can be obtained from the MS–MS analyses of both [M + Li]+ and [M − H]− ions. Interpretation of the X and A ions provided strucural information of the number and type of saccharide sugar presented in the ginsenosides. The developed method has been successfully applied for analyzing a column chromatographic fraction of a Chinese Panax ginseng extract with the confirmation of ginsenoside Rg2 and identification of a co-eluted ginsenoside Rf . The method provided rapid confirmation of gisenoside detection after the traditional TLC technique.
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Acknowledgement Financial support for this work was sponsored by National Natural Science Foundation of China (Grant No. 39930210) and the Competitive Earmarked Research Grant (CERG), Research Grants Council (RGC) of Hong Kong (HKBU2017/02P).
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