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INTRODUCTION
The efficacy of Traditional Chinese Medicine (TCM) has
been affirmed by thousands of years of history as well as by
modern clinical practice. However, understanding the material base, especially to rapidly identify all of the chemical
components from TCM and determining the mechanism of efficacy still remain to be key challenges in TCM R&D.
The example product used for this analysis was Ziziphi
Spinosae Semen (ZSS), which is used in the treatment of insomnia and palpitations in TCM. Other reported
pharmacological effects include protection of cardiovascular system, antihyperlipidemic, immunopotentiation and anxiolytic
effects. Previous studies on ZSS reported presence of flavonoids, saponins, alkaloids, terpenes and series of indole
derivatives [2,3]. However, these studies were focused on only specific group of compounds rather than overall chemical
profile of ZSS. Therefore, there is a need to develop rapid and
and effective analytical strategy to study overall chemical profile of ZSS for holistic evaluation of its quality.
Figure 1. Image of Ziziphi Spinosae Semen.
The Waters Natural Products Application Solution
(NPAS) with UNIFI provides a novel and comprehensive strategy for NP ingredient analysis. It utilizes the ACQUITY
UPLC® I-Class and Xevo G2-XS QTof MS to acquire MSE data which simultaneously provides exact mass precursor and
corresponding fragment ions for identification and structural elucidation. The obtained data is searched against the
Traditional Medicine Library (TM Library), a unique scientific library integrated within UNIFI software. The structures of the
matched components are automatically verified by
MassFragment™ with corresponding fragment ions within the MSE data set (obtained from the same injection). Additionally,
discovery tools are also available within the UNIFI informatics platform for the identification of unknown precursor ions that
did not match with TM library.
In this work, we demonstrate the use of UPLC/QTof-MS coupled to NPAS with UNIFI for the identification of total
material base including target and non-target ingredients.
A RAPID SCREENING METHOD TO IDENTIFY CHEMICAL COMPONENTS OF A TRADITIONAL HERB USING NOVEL INFORMATICS UNIFI PLATFORM
Li min1, Dai Yi1, Lirui Qiao2, Jimmy Yuk3, Mark Wrona3, Giorgis Isaac3, Kate Yu3
1Jin an University, Guangzhou, China; 2Waters Corporation, Shanghai, China; 3Corporation, 34 Maple Street, Milford, MA
METHODS
Sample Preparation
Sample was provided by Hebei Yiling Pharma Group Co., Ltd and authenticated by
Dr. Qing-Cun Tian. The powder (3.0 g) was suspended in 25 mL 60% MeOH/H2O, ultrasonically extracted for 60 min at room temperature. Then, it was filtered
through a 0.22 μm nylon filter film prior to injection. The sample was analyzed
using UPLC system coupled with a Qtof Mass Spectrometer.
LC Conditions
LC system: ACQUITY UPLC I-Class with FTN Sample Manager
Column: ACQUITY UPLC BEH 2.1 x 150 mm, 1.7 μm, 35 °C
Sample temp.: 10 °C
Mobile Phase: A: water (0.1% FA); B: acetonitrile (0.1% FA)
Flow Rate: 0.4 mL/min Gradient:
MS Conditions
MS system: SYNAPT G2 HDMS
Acquisition range: 100-1500 Da (0.1 s scan rate)
Acquisition mode: MSE, ESIˉ and ESI+ in resolution mode
Capillary voltage: 3 kV (ESI+)/2.2 kV (ESIˉ)
Cone voltage: 35 V
Collision energy (eV): Low CE: 6; High CE: 20-50
Source temp.: 100 °C
Desolvation temp.: 300 °C
Informatics Platform
The Natural Product Application Solution with UNIFI platform was used for Data
processing/Result review/Reporting. MassLynx was used for data acquisition.
Figure 2 shows the screen shot of the UNIFI Traditional Medicine (TM) Library.
1. Z.H. Yen Chinese Material Medica (II), Zu-lng, Taipei(1991), pp.671-673.
2. Niu C-Y, Wu C-S, Sheng Y-X, Zhang J-L. Identification and characterization of flavonoids from semen zizyphi spinosae by
high-performance liquid chromatography/linear ion trap FTICR hybrid mass spectrometry. Journal of Asian Natural
Products Research 2010; 12:300-312.
3. Ma J-J, Kang L-P, Zhou W-B et al. Identification and characterization of saponins in extract of Ziziphi spinosae Semen
(ZSS) by ultra-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass
spectrometry (UPLC-ESI-QTOF-MSE). Journal of Medicinal Plants Research 2011; 5:6152-6159.
4. Qiao L, Lewis R, Hooper A, Morphet J, Tan X, Yu K. Using Natural Products Application Solution with UNIFI for the
Identification of Chemical Ingredients of Green Tea Extract. Waters application note. 2013. (p/n 720004837en). 5. Qiao L and Yu K. Using the elucidation tool in UNIFI scientific information system to identify unknown compounds in
natural products. Waters application note. 2014. (p/n 720004876en).
RESULTS AND DISCUSSIONS (I)
A five-step strategy was adapted for the identification of components
from ZSS using Natural Product Application Solution with UNIFI (Figure
4).
Step 1: Integrate the NPAS with UNIFI TM library and an in-house
created library for ZSS based on the literature.
Step 2: Screen the target ingredients from the UPLC-MSE profile data,
which is automatically performed by UNIFI platform (Figure 5). Step 3: Verify the identified compounds in the initial results according
to different reference parameters and confirm by reference standards, especially for the isomers. Isomers could be distinguished by their
different MS fragmentation patterns, as well as the retention time of
reference compounds. This is followed by summarizing and proposing fragmentation pattern rules for target components.
Step 4: Screen for potential new components with similar MS fragmentation pattern as that of the indentified target ingredients using
discovery tools available in UNIFI platforms such as common fragments search and neutral loss to screen and characterize MS
fragmentation patterns of compounds of interests.
Step 5: Summarize the whole material base and holistically evaluate the
quality of ZSS TCM products
CONCLUSION
A rapid and effective strategy to holistically evaluate overall
chemical profile and quality of TCM products was
demonstrated by utilizing UPLC/Qtof MS coupled with the
UNIFI informatics platform in a workflow driven automatic streamlined process while incorporating a Traditional
Medicine Library.
The UNIFI informatics platform is easy to use and provides
an efficient analytical solution to identify chemical
ingredients from complex natural products samples.
References
RESULTS AND DISCUSSIONS (II)
A total of 113 compounds (Figure 6) including 69 flavonoids, 16 saponins,
19 alkaloids, 8 terpenes were characterized and identified with our chemical ingredients screening and identification strategy. Among those
113 compounds, 44 compounds are the new compounds identified using discovery tools such as common fragment ion search and neutral loss
search (Figure 7).
Figure 3. The base peak ion (BPI) of the extract of ZSS in the positive (a) and
negative (b) ion modes (see figure 5 for compound assignment).
Time (min.) Solvent A(%) Solvent B(%) Curves
0 98 2 6
6 76 24 6
8 68 32 6
10 49 51 6
14 0 100 6
18 0 100 6
20 98 2 1
Figure 4. Strategy for the identification of multiple components in TCM by UNIFI (illustrated for ZSS).
O
OOH
H3CO
OH
O
OHO
HO
R1O
O
OH
HOHO
R2ON
O
O
OHHO
HOHO
HO C
O
NO
O
OHHO
HOHO
HO C
O
A= B= C=N
O
O
OHHO
HOHO
C
O
16 R1=H R2=4-O-Glc-vanilloyl17 R1=H R2=4-O-Glc-benzoyl18 R1=H R2=4-O-Glc-sinapoyl19 R1=H R2=4-O-Glc-feruloyl23 R1=H R2=4-O-Rha-vanilloyl24 R1=H R2=H 27 R1=H R2=pyridyloyl 29 R1=H R2=(2 ″ ″ ,3 ″ ″ ,4 ″ ″ ,5 ″ ″ - tetramethoxyl)-(E)-cinnamoyl37 R1=H R2=vanilloyl44 R1=H R2=p-Hydroxybenzoyl49 R1=H R2=dihydrophaseoyl52 R1=H R2=sinapoyl54 R1=H R2=p-coumaroyl55 R1=H R2=feruloyl67 R1=H R2=phaseoyl73 R1=H R2=(3 ″ ″ ,4 ″ ″ ,5 ″ ″ -trimethoxyl) -(E)-cinnamoyl82 R1=H R2=cinnamoyl
87 R1=p-Hydroxybenzoyl R2=feruloyl89 R1=sinapoyl R2=feruloyl91 R1=R2=feruloyl
31 R1=H R2=A35 R1=H R2=B36 R1=A R2=H39/40 R1=H R2=C41 R1=B R2=H42/46 R1=C R2=4-O-Glc-benzoyl 56/57 R1=C R2=H 59 R1=A R2=feruloyl60 R1=B R2=feruloyl61/62 R1=C R2=vanilloyl64/66 R1=C R2=p-Hydroxybenzoyl68/69 R1=C R2=sinapoyl70/71 R1=C R2=feruloyl77/79 R1=C R2=phaseoyl81/83 R1=C R2=benzoyl
O
OOH
HO
OH
O
OHO
HO
HO
O
OHHO
HOR2O
R1
13 R1=H R2=4-O-Glc-feruloyl20 R1=R2=H 30 R1=OCH3 R2=feruloyl32/33 R1=H R2=C38 R1=H R2=sinapoyl45 R1=H R2=p-coumaroyl47 R1=H R2=feruloyl51/53 R1=C R2=H63/65 R1=C R2=feruloyl
O
OOH
H3CO
OHO
O
HO
HOR1O
OHO
HOHOR2O
21 R1=H R2=4-O-Glc-feruloyl25 R1=R2=H94 R1=sinapoyl R2=feruloyl 95 R1=R2=feruloyl
22 R=Rha43 R=H
O
OOH
HO
OH
O
ORHO
HO
OO
OHHO
HO
O
O
OOH
R1O
OH
O
OR2
HOR3O
HO
R4
11 R1=R2=R3=H R4=glu28 R1=R2=R3=R4=H34 R1=CH3 R2=R3=R4=H
Flavonoids
COOR2
HO
R1
110 R1=OH R2=H112 R1=R2=H
COOHR1
R2
HO
106 R1=COOH R2=OH R3=H108 R1=COOH R2=H R3=H109 R1=COOH R2=H R3=OH111 R1=COOH R2=H R3=H
COOH
O
113
R3
O
H3CO
COOHOHO
OHOH
OH
1
Triterpenic acids and others
O
HO
O
O
OR1
O
OR2
HOHOR3O O
O
OH
96 R1=-L-Rha R2=-D-Xyl R3=-D-Glc97 R1=-L-Rha R2=-D-Glc R3=-D-Glc98 R1=-D-Fuc R2=-D-Xyl R3=-D-Glc100 R1=-L-Rha R2=-D-Xyl R3=H (23)101 R1=-L-Rha R2=-D-Xyl R3=H102 R1=-L-Rha R2=-D-Glc R3=H103 R1=-D-Fuc R2=-D-Xyl R3=H105 R1=-L-Rha R2=-D-Xyl R3=Ac
O
HO
CH2OHO
OR1
O
OR2
HOHOR3O O
O
OH
74 R1=-L-Rha R2=-D-Xyl R3=-D-Glc78 R1=-L-Rha R2=-D-Xyl R3=H84 R1=-D-Fuc R2=-D-Xyl R3=H
O
OHHO
HOHO
O
O
HO
CH2OH
OR1
O
OR2
HOHOR3O O
O
OH
OOH
85 R1=-L-Rha R2=-D-Xyl R3=-D-Glc88 R1=-L-Rha R2=-D-Xyl R3=H
99 R1=-L-Rha R2=-D-Xyl R3=-D-Glc104 R1=-L-Rha R2=-D-Xyl R3=H
Saponins
O
HO
O
O
OR1
O
OR2
HOHOR3O O
O
OH
NO
O
OHHO
HOHO
R COOH
2 R=OH(S)3 R=OH(R)6/8 R=H
N
HO
HO
H3CO
H3CO
NH
H3CO
H3CO
HO
H3CO
159
NHO
H3CO
HO
14
NH
H3CO
R1O
R2O
4 R1=Glc R2=Glc5 R1=Glc R2=H12 R1=H R2=H
N
R1O
R2O
HO
10 R1=CH3 R2=H26 R1=H R2=CH3
NH
R1O
R2O
50 R1=CH3 R2=H 58 R1=H R2=CH3 80 R1=R2=CH3
N
O
H3CO
H3CO
92
NH
O
HNOHN
O O
N
90
Alkaloids
RESULTS AND DISCUSSIONS (III)
In this study, we proposed a multiple ingredients identification strategy
based on UPLC/Qtof-MS coupled with the UNIFI informatics platform [4].For the unknown compounds identification, the structure
elucidation tools embedded in UNIFI are used as per the previously reported method [5]. The embedded discovery tools in UNIFI also
facilitated the identification of new unknown compounds.
The compounds in a complex samples were efficiently separated by
ultra-performance liquid chromatography (UPLC). The data generated by UPLC/Qtof MS was managed by an integrated informatics platform:
UNIFI, which is a simple and efficient platform, incorporating scientific library into a streamlined workflow so that chemical ingredient
screening and identification can be easily performed for complex natural product samples.
The demonstrated strategy can be used for rapid, effective and
comprehensive screening and identification of chemical components (targeted and non-targeted) of multi-component natural products.
Figure 2. Screenshot of the UNIFI Traditional Medicine Li-
brary. Shown here is the view of compound en-try.
This library can be also searched by plant names and chemical classifica-
tion. The library is of-fered in both English and Mandarin.
HIGHLIGHTS
Rapid and effective strategy to holistically evaluate the
quality of TCM products using the UPLC/QTof MSE coupled with UNIFI informatics platform
Easy identification of known and unknown target com-
pounds belonging to various chemical classes using UNIFI informatics platform
Overview of the UNIFI informatics platform that offers
workflow driven automatic streamlined process incorporating a Traditional Medicine Library and embedded
discovery tools to discover identify of new compounds
Figure 5. Results displayed in Review tab within the UNIFI after processing. 5A) List of the avail-able workflow templates. 5B) list of the component associated with each of the workflow. Here shows the list associated with the “Good Match” filter; 5C) Extracted ion chromatograms (XIC) associated to 5B; 5D) MS spectra correlate to what’s on display in 6C, both low energy MS full
scan and high energy MSE scan.
Figure 7. The workflow of the proposed strategy for the identification of non-targeted compo-nents from ZSS using discovery tools of UNIFI
5A
5B
5C
5D
Image source:http://tsswgw.com/ProductView.asp?id=402&SortID=232
Complex extract
Acquired LC-MS data file
UNIFI data processing and identification of compounds by importing compounds from the
library
Identified compoundsTargeted screening
Unidentified compoundsNon-targeted screening
Verify and confirm identified compounds by checking
MSMS fragments from MSE
data and using reference compounds
Identification using UNIFI
discovery tools
Deduce fragmentation
pattern for identified
compounds & chemical class
Common fragments& Neutral
loss information
Creation of in-house library for chemical components of target herb (ZSS) based on
literature review and TM library
Summarize identified compounds to holistically
evaluate quality of ZSS products
Identified compounds
Identified compounds
UPLC-qTOF data acquisition
Figure 6. The structures of tentatively identified compounds in ZSS.