Identification of Ginsenosides Using the SCIEX X500R
QTOF System
Wang Sha, Cheng Haiyan, Liu Ting, Li Lijun, Jin Wenhai[Author]
SCIEX, Pacific Applications Support Center (Beijing). China

Background
Ginseng is one of the most valued herbs. It has properties ofnourishing vital strength, tranquilizing the mind, promoting
secretions, and supplementing deficiencies. Modern medical
research shows that ginseng is effective in preventing cancer,
countering aging, acting as an antiarrhythmic agent, and has
hypoglycemic, hypolipidemic, and immune-stimulating
properties. Its main active components are ginsenosides.
Ginsenosides are triterpenoid chemical compounds; based on
the glycosyl structure, they can be divided into tetracyclic
triterpenes of the dammarane type and pentacyclic triterpenesof the oleanane type. The dammarane type can be divided into
ginseng diols and ginseng triols. Because ginsenosides have
many components, different species and sources yield
differences in composition [1], so a full identification of theginsenoside composition and accurate analysis of its structure
currently requires extensive literature and document research.
At the same time, the analytical result obtained by different
technologies can be quite difficult to verify with data, which cancomplicate quality evaluation and material basis.

The software has a simple user interface, and the workflow is
intuiative.

Experimental Process
1.

2.
3.
4.
5.

Using TOF-IDA- mode (Top 10 MS/MS per cycle), inject asample and simultaneously obtain primary precursor ionsand secondary daughter ion information. This allows
confidence in the compound identification and allows alldata to be acquired ina single shot alleviating time
involved in a two-injection workflow.
Search known ginsenoside components; according to theaccurate mass, isotope distribution, and Chinese medicine
matching data, identify the compounds.
Use Ginsenoside Rg2 as an internal standard to verify
accuracy of match results.
Use the accurate mass, characteristic fragment ions, andrelative retention times to enhance identification ofginsenoside isoforms.
A total of 51 commonly observed ginsenoside components
have already been identified.

The SCIEX X500R QTOF high resolution mass spectrometerrequires a single injection to collect high quality MS andMS/MS data. Combined with an expansive high resolution
MS/MS database of Traditional Chinese Medicine (TCM) active
ingredients, SCIEX OS software automatically determines thetheoretical molecular weight and isotope pattern distribution
and simultaneously matches it with the MS/MS database.
Comprehensive scoring allows intuitive, rapid, and accurate
ginsenoside component identification.
The SCIEX high resolution database of Chinese medicine isbased on “Chinese pharmacopeia” Part 1, TCM active
ingredients. It includes almost a thousand compounds such assaponins, flavonoids, flavonoid glycosides, triterpenes,phenylethyl glycosides, and organic acids.
This document describes the workflow for analysis ofginsenosides in Chinese medicine using the SCIEX OS ultraefficient data processing software and the high resolution
Chinese medicine MS/MS database on the SCIEX X500R
QTOF high resolution mass spectrometer system.

Figure 1 Workflow for using the SCIEX OS X500R QTOF highresolution mass spectrometer and the Chinese medicine MS/MS
database to identify ginsenoside components

Sample Preparation
1.

Accurately measure 5.0g ginseng powder into a 50mL
centrifugation tube.

2.

Add 25mL 90% methanol water, agitate 5 min.

3.

Immerse in an ice bath overnight.

4.

Ultrasonicate 30 min, at 4 deg. C, then centrifuge at10000r/min for 12 min.
p1

5.

Remove the supernatant and pass through a 0.22µm filter.

Liquid Chromatography (LC) Conditions
Chromatographic Column: Phenomenex Kinetex C18,2.1*100mm, 2.6µm;
Mobile phase: Gradient elution is used
Negative ions: A is H2O (containing 0.05% formic acid); B is
acetonitrile;
Flow rate: 0.25mL/mLColumn temperature: 40℃

Data acquisition is performed on extracted samples according
to conditions described. The Explorer data processing options
can be used to open the acquired high-resolution data, andperform any data QC as in Fig. 2.

Injection volume: 3µL
Table 1. Elution conditions

Time (min)

A%

B%

00.5
5.0
35.0
40.0
40.1
45.0

9090501009090101050901001010Mass Spectrometry Conditions

Figure 2. Acquired high-resolution TOF MS-IDA-TOF MS/MS data.
Fig. 2A shows a full TOF MS scan, and Fig. 2B is an IDA TOF MS/MSspectrogram of a sample.

2. Editing of data processing methods
Using targeted component analysis, one can enter or copy
known ginsenosides to the component options, including theirname and molecular composition, as shown in Fig. 3.

Scanning method: TOF-IDA-10 MS/MS qualitative;
ESI ion source parameters:
Air curtain gas CUR: 35psi;
IS voltage: -4500V: Source temperature: 550℃
Cone voltage: -80V;
Atomizing gas GAS1: 55psi; Auxiliary gas GAS2: 55psi

Figure 3. Input chemical compound list

At the same time, select the database to search (this study
uses the TCM MS/MS Library) and configure the confidence
levels, as shown in Fig. 4.

Application of SCIEX OS Software for
Ginsenoside Analysis
SCIEX OS Software platform provides
simultaneous mass spectrometer control,
method editing, data analysis, and result
reporting.

Figure 4. Setting SCIEX OS Software confidence levels

1. Data acquisition2

Confidence intervals are primarily used in compound
identification and verification, including theoretical mass
numbers, isotope distribution, retention times (can be omitted if
unavailable), and MS/MS spectrum matching in the database.
Each score is calculated based on the configuration, and anoverall score is determined based on the weight of the fourparameters.

theoretical MS pattern, and the two match well. Fig. D is themirror image display for the MS/MS database match results:
the upper, blue portion is the MS/MS spectrum acquired forginsenoside, and the lower, gray portion shows the MS/MS
spectrum from the database. Results are compared clearly.
The lower right corner shows database search results, and withan overall score above 90, matching results are excellent.

3. Data processing and Results Viewing

Verification of identification resultsUse built-in processing methods to processing data. Select“process,” and the software will list results based on 4
established confidence intervals. Use the “signal indicator” toeasily obtain the results, including MS/MS matchedresults.Identification of Ginsenoside Rg2 is given as anexample; results are shown in Fig.5.

For ginsenoside in the negative ion mode, the excimer ion
peaks are mainly present at [M-H] ￣and [M+HCOOH-H]; thestructure of the saponin component is described by its
secondary signature fragment loss of HCOOH and daughterions of sugars, e.g.: -46 -162 (Loss HCOOH& Glu); -46-146(Loss HCOOH& Rhamnose) or -46-132 (Loss HCOOH&arabinose), and fragment 161 forms readily. For ginsenoside
Rg2 secondary fragment structure analysis, see Fig. 6.

Figure 6. Secondary structural analysis spectrogram forginsenoside Rg2

Using MS/MS information, a neutral loss (NL) loss of 162 and146, and the signature fragment for dammarane triol at m/z475, this structure can be described as dammarane triol+glucose+rhamnose, which is similar to ginsenoside Rg2 andmatches search results.

Figure 5. Screening results display

Fig. A is the display of the data processing results; B is thedisplay of an extracted ion chromatogram. Retention time isuseful for compound verification. Fig. C shows the mass
spectrum and isotope distributions; the upper portion is theactual measured value, the lower, gray part shows theGinsenosides are made of saponins and sugars such as
glucose, rhamnose, and arabinose that may be linked atvarious positions on the saponin. At the same time, sincesaponins and sugars have different structures, they may form
stereoisomers. In database searches, isomers link tosecondary signature fragment ion information, retention time,and relevant literature［2］. Verification of known ginsenosides
is necessary, and secondary fragment information is critical for
structural identification of Chinese medicine components

3

Table 2. List of identified ginsenosides
Index

Compound name

Molecular
formula[M+HCOOH-H]-

ss Error(ppm)Retention
time (min)

MS/MS

12
3
4
5
6
7
8
910
11
12
13
14
15
16
1718
1920
21
22
23
24
25
26
27
28
2930
31323334353637383940414243
4445
46
47
48

20R-ginsenoside Rg2
20S-ginsenoside Rg2
20S-ginsenoside F2
20R-ginsenoside F2
20S-ginsenoside Rg3
20R-ginsenoside Rg3
Ginsenoside Rg4
Ginsenoside F4
Ginsenoside Ic
Ginsenoside Rk1
Ginsenoside Rk1 (isomer)
Pseudoginsenoside F11
Ginsenoside Rf
Ginsenoside Rf (isomer)
Ginsenoside Rg7
Ginsenoside Rg1(Ginsenoside A2)
Ginsenoside F1
20(S)-Ginsenoside Rh1
20(R)-Ginsenoside Rh1
Ginsenoside Re
Ginsenoside Rd
Ginsenoside Rd (isomer)
pseudo-Ginsenoside RT2
Ginsenoside Rb220(S)-Ginsenoside Rc
20(R)-Ginsenoside Rc
Ginsenoside Rb1
20(S)-Ginsenoside-Rh220(R)-Ginsenoside-Rh2
Ginsenoside Rd+Acetylation
Ginsenoside Re+Acetylation
Pseudoginsenoside RT5
Ginsenoside Ra1
Ginsenoside Ra2
Chikusetsusaponin III
Ginsenoside Rs2
Ginsenoside Rs2 (isomer)
Ginsenoside Rs1
Ginsenoside Rs1 (isomer)
Ginsenoside R1
Ginsenoside F3
Ginsenoside F3 (isomer)
Pseudo-ginsenoside RT1
Ginsenoside Rs3
Ginsenoside R2
Ginsenoside Ra3
Ginsenoside Ra3 (isomer)
Ginsenoside Rb3

C42H72O13C42H72O13C42H72O13C42H72O13C42H72O13C42H72O13C42H70O12C42H70O12C42H70O12C42H70O12C42H70O12C42H72O14C42H72O14C42H72O14C42H72O14C42H72O14C36H62O9C36H62O9C36H62O9C48H82O18C48H82O18C48H82O18C41H70O14C53H90O22C53H90O22C53H90O22C54H92O23C36H62O8C36H62O8C50H84O19C50H84O19C36H62O10C58H98O26C58H98O26C47H80O17C55H92O23C55H92O23C55H92O23C55H92O23C47H80O18C41H70O13C41H70O13C47H74O18C44H74O14C41H70O13C59H100O27C59H100O27C53H90O22

829.4944
829.4944
829.4944
829.4944
829.4944
829.4944
811.4838
811.4838
811.4838
811.4838
811.4838
845.4893
845.4893
845.4893
845.4893
845.4893
683.4365
683.4365
683.4365
991.5472
991.5472
991.5472
831.47371123.5895
1123.5895
1123.5895
1153.6001
667.4416
667.4416
1033.5578
1033.5578
699.4314
1255.6317
1255.6317
961.5367
1165.6001
1165.6001
1165.6001
1165.6001
977.5316
815.4788
815.4788
971.4846
871.5050
815.4788
1285.6423
1285.6423
1123.5895

2.30
1.78
1.50
1.10
-0.80
-0.80
-1.20
-1.70
-0.90
-1.20
1.40
-1.60
-1.90
-1.60
-2.00
-1.60
0.90
0.70
0.90
-0.84
-0.70
-0.70
-1.50
-1.90
-2.10
-1.90
-1.00
-0.50
-0.50
-1.40
-1.52
-1.10
-0.63
-0.70
-1.00
1.60
1.84
1.00
1.00
-1.20
-1.40
-1.44
0.40
0.40
-1.40
-2.09
-2.10
-1.00

13.93
14.01
21.05
20.10
26.09
26.54
7.79
12.22
28.58
29.04
41.33
12.52
8.63
8.35
7.64
7.40
15.85
14.23
14.10
8.47
21.22
22.83
7.41
18.45
19.44
19.76
17.99
29.52
30.50
20.26
20.74
7.75
17.23
18.40
23.65
20.17
19.57
18.67
17.73
7.87
13.04
11.22
9.64
25.25
10.52
15.97
17.52
18.45

m/z783, 637, 475,391,161
m/z783, 637, 475,391,161
m/z783, 621, 459,161
m/z783, 621, 459,161
m/z783, 621,459,375,161
m/z783, 621,459,375,161
m/z765,619,457,161
m/z765,619,457,161
m/z765,619,457,161
m/z765,603,441,161
m/z765,603,441,161
m/z799,637,475,161
m/z799,637,475,161
m/z799,637,475,161
m/z799,637,475,161
m/z799,637,475,161
m/z637,475,391,161
m/z637,475,391,161
m/z637,475,391,161
m/z945, 799,637,475
m/z945,783,621,459,375,161
m/z945,783,621,459,161
m/z785,653,491,391
m/z1077,945,783,621,459
m/z1077,945,783,621,459
m/z1077,945,783,621,459
m/z1107, 945,783,621,459
m/z621,459,375
m/z621,459,375
m/z987, 945, 928, 783, 621,459
m/z987, 945, 928, 783, 621,459
m/z699,653,491,329,161m/z1209,1077,945,783,621,459
m/z1209,1077,945,783,621,459
m/z915,783,621,459,375
m/z1119,1077,1059,945,783,621,459
m/z1119,1077,1059,945,783,621,459
m/z1119,1077,1059,945,783,621,459
m/z1119,1077,1059,945,783,621,459
m/z 931, 799, 637, 475, 161
m/z 161, 391, 475, 637, 769
m/z 161, 391, 475, 637, 769
m/z 161, 763
m/z 161, 459, 621,783
m/z 161, 391, 475, 637, 769
m/z 1239, 1077, 945, 783, 621
m/z 1239, 1077, 945, 783, 621
m/z 1077, m/z 1123

4950
51
5253

Ginsenoside Rb3 (isomer）
Ginsenoside Rk3
Protopanaxatriol
Protopanaxatriol(isomer)
Ginsenoside Ro

C53H90O22
C36H60O8
C30H52O4
C30H52O4
C48H76O19

1123.5895-1.13

665.4259
521.3837
521.3837
1001.4952

-1.30
-1.00
-1.03
2.40

19.44
20.68
21.26
22.05
25.07

m/z 1077, m/z 1123
m/z 161, 619
m/z521,475,391
m/z521,475,391
m/z 955,793,631,455

p4

Conclusions
This study used the high-resolution SCIEX X500R QTOF
System for identification of ginsenoside components. It uses
SCIEX OS software along with the TCM MS/MS database forrapid, accurate identification of 53 ginsenoside components,
showing strong resolving power and illustrating the benefits of
the high-resolution SCIEX database in traditional Chinese
medicine analysis. The high-resolution MS/MS TCM database
contains almost a thousand TCM active ingredient MS/MSspectra; automatic data extraction can be used for matching
and greatly decreases the identification time for Chinese
medicines. It also allows for simple and accurate component
identification.
The SCIEX X500R QTOF high-resolution system is the righttool when identifying Chinese medicine components. The IDA
workflow can be used to ensure the integrity of the acquisition,and TOF MS and MS/MS data can be obtained for allcomponents. The X500R’s front end has all the advantages ofa triple quadrupole mass spectrometer, greatly improving its
quantification capabilities, sensitivity, stability, and linear range.
SCIEX OS system software integrates instrument control,
method editing, data acquisition, and reporting. It can perform
simultaneous qualitative and quantitative analysis, wirelesslyconnect to other software, and simplify analytic workflow.

References
1.

Shen Dan, Tang Shihuan, Lu Peng, et al. Ginsengcontaining Chinese medicine pattern analysis [J]. Chinese
Journal of Traditional Chinese Medicine, 2013, 38(12),2027-2032.

2.

Zhang Xiaoxu, Wang Hongping, Yang Yang, et al. Rapid
identification of saponins in sunflower ginseng by ultra
performance liquid chromatography tandem quadrupole
time-of-flight mass spectrometry [J]. China MedicalJournal, 2015, 12(9), 130-136.

AB Sciex is doing business as SCIEX.
© 2017 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
AB SCIEX™ is being used under license.
Document number: RUO-MKT-02-6090-A

p5