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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.