Research on the Different Composition of Smilax Glabra
from Different Regions by High Resolution Mass
Spectrometry Using the SCIEX X500R QTOF System
Yu Jie, Cheng Haiyan, Liu Ting, Li Lijun, Jin Wenhai
SCIEX, Asia Pacific Application Support Center (Shanghai), China

Introduction

Experimental Process

China has a long tradition of using herbs of diverse types asmedicine. Smilax Glabra is one of the most common Chinese
herbal medicines. Smilax Glabra has many components,
including volatile ingredients, sterols, fatty acids, phenols andflavonoids. Smilax Glabra is an antitumor, anti-arteriosclerosis
drug that is used for treatment of coronary disease. It is also atreatment for angina pectoris. Smilax Glabra grows mainly in theYangtze River area and the southern provinces. Smilax Glabra
from different places varies in efficacy and quality due todifferences in environmental conditions and climate.
Differentiating between Smilax Glabra from different regions isimportant to ensure Smilax Glabra’s therapeutic efficacy.

1.

The SCIEX X500R QTOF System is a revolutionary highresolution mass spectrometry system. As the first platform
developed especially for high throughput laboratory testing, it isstable, reliable, and powerful. The fully updated hardware designoffers rapid scanning in combination with powerful informationdependent acquisition (IDA); using a single injection, TOF-MS
accurate mass and TOF-MS/MS secondary fragmentation™
spectra can be obtained. SCIEX’s unique MarkerView Software
can be used to perform statistical analysis on differences inSmilax Glabra from different regions. It can automaticallycorrelate TOF-MS and TOF-MS/MS secondary fragmentation
spectra. Chinese medicine ingredients are complex, andcurrently identification primarily relies on accurate mass (MS1),fragment ions (MS2), online and literature research. Thesemethods waste time and effort, and they produce results of poor
accuracy. SCIEX has a high-resolution MS/MS database withalmost 900 Chinese medicine ingredients. Combined with theexact mass and isotope distribution, one can quickly andaccurately make identifications.
This application note describes using the SCIEX X500R QTOF
System including updated SCIEX OS Software for mass
spectrographic control and data acquisition and analysis. With asingle sample injection, one can analyze differences with
MarkerView Software and the high-resolution MS/MS Chinese
medicine database to perform Chinese medicine component
differential analysis.

Three varieties of Smilax Glabra from four different regions Guangdong Jiangmen, Guangdong Heyuan, Guangxi
Jiazhou and Hunan - were collected, dried and crushed.

2.

Using the X500R’s high-resolution TOF-MS-IDA- mode (Top12 MS/MS per cycle), MS/MS mass spectra were obtainedin a single sample injection.

3.

MarkerView Software data analysis revealed a statisticallysignificant difference between sample groups interrogated.

4.

Using the MS, MS/MS, and the high-resolution MS/MS
Chinese medicine database for comparison, databasesearches and structural verification were performed for thedifferent samples.

X500R QTOF System High Resolution Mass
Spectrometry Screening Workflow

1.

Glabrous Greenbrier
RhizomeCollectingand Grouping

2.

TOF-MSTOF-MSTOF-MS/MS

3.

4.

DistinguishtheDifferences

Confirm
theDifferencesp1

Mass Spectrum Parameters
ESI ion source parameters:
Air curtain gas CUR: 35psi; Collision Gas CAD: 7
IS voltage: 5500V/-4500V; Source temp: 550ºC
Atomizing gas GAS1: 55psi
Auxiliary gas GAS2: 55psi
DP voltage: ± 60 VCollision energy: 40 ±20V
Dynamic Background Subtraction

Preprocessing Method

TOF-MS

Using 4 groups of Smilax Glabra from different areas, weighabout 1.0 g of Smilax Glabra powder, take 3 equal samples fromeach group, add 90%formic acid-aqueous solution 10 mL, vortex
10 min, then ultrasonicate at room temperature for 30 min,remove supernatant and centrifuge at 13000 rpm for 10 min,extract supernatant and use as sample.

IDA

TOFMS/MS

Chromatographic Conditions
Chromatographic Column: Phenomenex Kinetex C18,100 x 4.6 mm, 2.6 µm;
Mobile phase: A: 0.01% Formic acid Water/B: Acetonitrile
Flow rate: 0.4 mL/min;MarkerView’s advanced processing algorithm finds the peaks,
and data alignment compensates for small variations in theretention time and accurate mass. The differences between
samples can be normalized. Before the data is processed,
known background ion interference or uncommon ions in thesample can be removed.

Column temperature: 40ºC;
Injection volume: 10 µL

Table 1. Elution conditions

Time (min)
0.0
5.0
15.0
20.0
25.0
25.1
30.0MarkerView Data Processing

A%955520559595

B%545809595
55

Mass Spectrometry Method
Scanning method: TOF-MS –IDA (Top12 MS/MS);Ion source: ESI source double spray technology
Scanning range: m/z 50-2000

Figure 1 Advanced processing algorithm finds characteristic peaks
by MarkerView Software

CDS automatic calibration

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All samples underwent supervised principal component analysis(PCA).The scores plot highlights any differences/trends amongst
the sample groups. The loadings plot reflects the variables
responsible for the separation.

Figure 4 MarkerView software automatically associated with the raw
data

Chinese Medicine Database Searching

Figure 2 The PCA Score and loading diagram of Glabrous Greenbrier
Rhizome from different regions

The PCA Score Plot and loading chart show Smilax Glabra
samples from 4 different areas are well differentiated along thePC1 dimension, showing the largest variation between the 4groups. Taking m/z 305.0649 at retention time 5.97 min as anexample, it is clear that the the impact of this variable on theloadingss high, and the concentration of Smilax Glabra insamples from Guangdong Jiangmen is higher than those from
Guangdong Heyuan, Guangxi Jiazhou, or Hunan.

The SCIEX high resolution Chinese medicine database is basedon the “Chinese pharmacopeia” Traditional Chinese Medicine(TCM) ingredients, including almost 900 compounds such assaponins, flavonoids, flavonoid glycosides, triterpenes,
phenylethyl glycosides and organic acids. Peak extraction anddatabase matching in SCIEX OS Software’s allows a user to
identify and verify compounds in their samples. Using accuratemass, retention times, isotope ratios, database scoring, andmolecular formula scoring, an overall score is automaticallycalculated and listed. A “signal indicator” evaluates results
simply, clearly, and intuitively.

BACFig. 3 The content change of m/z 305.0654, RT5.98minin different groupsMarkerView Software intuitively shows differences between
samples in terms of accurate mass, isotope ratios, and MS/MS;
needed for confirmation.

Figure 5 The confirmed result of m/z 305.0654, RT 5.98 min

Fig. 5A is the extracted ion flow chart for a compound; its
retention time can be verified. Fig. 5B displays the measured MS
spectrum and isotope ratio. A mirror image comparison isperformed with the chemical compound’s theoretical values fromthe database. Fig. 5C shows mirror image comparisons of theMS/MS spectra, namely between acquired and database hit.
p3Summary of Identified Results
Using MarkerView data analysis and MS/MS database matching
results were verified, Smilax Glabra differences from different
areas were verified:Figure 7 The confirmed result of m/z 283.0609, RT 9.28 min

SCIEX MS/MS database helps to quickly identify and verify
compounds with confidence.
A Welch T-test has a group alignment function to enhance result
accuracy. New box plots appear automatically and identify
parallelism among samples within the group.

MarkerView Software Statistics
MarkerView Software can also use T-test statistics to finddifferent components. The volcano plot shows log fold change
versus p-value plots. The smaller the p-value, the moresignificant the difference between the log values of m/z at either
end of the X axis.

Figure 8 Box and whiskers plots figure out the parallelism between
the test samples

ChemSpider Database Search andRapid Verification
SCIEX OS Software automatically correlates with theChemSpider database, which enhances the database capability
to rapidly verify chemical compounds. The Fragment Paneanalysis function helps rapidly verify chemical compounds bytheoretically fragmenting a structure and matching the MS/MS to
the actually experimental data collected..

Figure 6 Mapping of log ratio change and p-valuep4

This study applied the SCIEX X500R high-resolution system toanalyze differences between TCM samples. The powerful SCIEXOS Software and MarkerView Software are well integrated;accurate mass, fragment ions data, and isotope ratios canquickly verify compounds. The high-resolution MS/MS databaselinks to the online ChemSpider database for automated matching
and rapid verification of differences, yielding accurate andreliable results. The efficient integration of Chinese medicine
identification solutions from SCIEX offers comprehensiveanalysis.

References
1. Chemical Constituents and Pharmacological Properties ofPoria cocos.
2. Mycology, cultivation, traditional uses, phytochemistry andpharmacology of Wolfiporia cocos (Schwein) Ryvarden et Gilb.:
A review.

Figure 9 ChemSpider database can help identifying compounds rapidly

3. Comparative study of lanostane-type triterpene acids in
different parts of Poria cocos (Schw.) Wolf by UHPLC-Fouriertransform MS and UHPLC-triple quadruple MS.

Conclusions
The SCIEX X500R high-resolution system’s rapid scan speed
and dynamic background subtraction (DBS) function can help tocomplete sample preprocessing and efficiently remove
background interference. A single injection workflow allows thecollection of high quality MS and MS/MS spectral information,
suitable for high-throughput analysis of differences in traditional
Chinese medicine. SCIEX OS Software allows for rapid dataacquisition, analysis, and database searching data acquisition,
processing and reviewing is simple, fast, and convenient.
MarkerView data analysis software applies multivariate (PCA)
and univariate (t-test) analyses to find trends/differences
between sample groups quickly and intuitively.

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© 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-02-6089-A

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