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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2014 Waters Corporation
HIGHLIGHTS
Introducing the Waters Natural Product Application
Solution with UNIFI®, which combines UPLC®/QTof MS technology with workflow-driven informatics.
Demonstrating a generic, easy-to-use, and
automated workflow that streamlines data analysis by
incorporating a Traditional Medicine (TM) Library. Facilitates the identification of chemical ingredients
and herbal composition of an unknown Traditional Chinese Medicine (TCM) commercial product.
INTRODUCTION
All Natural Products (NP) research starts from
ingredient analysis, which has always been a bottleneck. A major
challenge is to identify chemical ingredients and deduce possible
herbal composition from a completely unknown sample. It is
difficult to know where to start, especially when little background
information is provided. Even with LC/MS, researchers are still
challenged by how to narrow down the scope and to obtain
meaningful information quickly.
Classic LC/MS based workflow for profiling NP products
is: extract individual chromatographic peak; propose possible
molecular formula based on intact precursor ion; search online
libraries for potential hits. Afterwards, fragmentation pathways
are deduced based on MS/MS results so that proposed chemical
structure can be confirmed. This process is extremely time-
consuming, labor intensive, often inefficient, and demanding for
the operator’s expertise level.
The Waters Natural Products Application Solution
(NPAS) with UNIFI provides a novel and comprehensive
strategy for NP ingredient analysis (Figure 1). It utilizes the
ACQUITY UPLC® I-Class and Xevo® G2-S QTof MS to acquire MSE
data (data-independent), which are then searched against the
Traditional Medicine Library, a unique scientific library integrated
within UNIFI software. The structures of the matched components
are automatically verified by MassFragment™ with corresponding
fragment ions. Data acquisition, processing (peak picking, library
searching, structural elucidation by fragment ions), and report
generation are integrated into a simple and streamlined
workflow. This greatly improves productivity and significantly
reduces the demands for operator's technical expertise level.
Strategy on how to use the NPAS with UNIFI to identify
chemical ingredients and deduce possible herbal content for a
completely unknown Traditional Chinese Medicine (TCM)
commercial product is demonstrated in this work.
IDENTIFYING CHEMICAL AND HERBAL COMPONENTS OF AN UNKNOWN TCM PRODUCT USING LC/MS COUPLED WITH A NOVEL INFORMATICS PLATFORM
Lirui Qiao1; Dhavalkumar Narendrabhai Patel2; Jing Huang1; Diane Diehl3; Jimmy Yuk*3; Mark Wrona3; Giorgis Isaac3; Kate Yu3
1Waters China, Shanghai, China; 2Waters Singapore, Singapore; 3Waters Corporation, Milford, MA
RESULTS AND DISCUSSION (II)
Reprocessing the same data by searching against components of these four herbs from
UNIFI’s Traditional Medicine Library further indicated the presence of components from ShanZha and MuXiang. Figure 6 shows the summary plot of the confirmed components
for this unknown sample after this re-processing step.
As a result, five herbs were confirmed to present in the sample: Dan Shen (Salvia miltiorrhiza), San Qi (Panax notoginseng), Ge Gen (Lobed kudzuvine), Shan Cha
(Crataegus pinnatifida Bunge) and MuXiang (Saussurea costus). Further online search indicated that a possible TCM recipe containing these five herbs is Xinkeshu capsule.
Among the 229 compounds listed within the TM Library that are associated with the five herbs, 183 were identified and confirmed from the LC/MS data.
CONCLUSIONS
The Natural Product Application Solution with UNIFI
provides an automatic and streamlined process that is
workflow driven. It provides a realistic, easy to use,
efficient and effective pathway to solve problem that was
impossible to solve before (Figure 7).
In just two days, we were able to identify chemical ingredients and deduce
herbal composition including determination of the commercial product name
of a complete unknown TCM commercial product.
The TCM product in question is XinKeShu, which contains Dan Shen (Salvia
miltiorrhiza), San Qi (Panax notoginseng), Ge Gen (Lobed kudzuvine root),
Shan Cha (Crataegus pinnatifida Bunge), and Mu Xiang (Saussurea costus).
RESULTS AND DISCUSSION (I)
Figure 4 shows the time-aligned Base Peak Ion (BPI) chromatograms of the injected
sample at two different collision energies.
Figure 5 shows the NPAS with UNIFI results for the initial chemical profiling of the sample, which resulted in 473 components matched with UNIFI’s TM library. Among
them, 86 compounds were initially verified based on fragment analysis by MassFragment.
As shown in Figure 5D, each fragment ion that corresponds to the compound’s
precursor ion can be examined by simply clicking on the blue icon. If the fragment
structure is logical, it can be potentially concluded that the proposed compound
structure is correct, and this component can be defined as “confirmed”. If a false
positive is suspected, further identification and structural elucidation can be carried
out by using UNIFI’s Structural Elucidation Tool to search more online libraries.
From these 86 compounds, the key components identified automatically by software
included Salvianolic acid, Ginseng saponins, Notoginsenoside series, Pueraria
saponins, and large number of amino acids. This clearly indicated the presence of
three herbs: DanShen (Salvia miltiorrhiza), SanQi (Panax notoginseng), and GeGen
(Lobed kudzuvine).
Searching well-known TCM recipes from the Internet revealed that recipes containing these three herbs are typically for treating cardiovascular diseases. Other herbs that
are most commonly used along with the above three herbs are: ShanZha (Crataegus pinnatifida Bunge), MuXiang (Saussurea costus), YinXing (Ginkgo biloba), and
JiaoGuLan (Gynostemma pentaphyllum).
Figure 1. Natural Products
Application Solution with UNIFI.
Key elements of the system
solution includes: Green Tea
extract and Catachin standard
mix; ACQUITY UPLC I-Class with
Xevo G2-S QTof MS; UNIFI
Scientific Information System
with UNIFI Traditional Medicine
Library; method templates for
data acquisition, processing;
data review workflow; and
report templates.
Figure 6. S u m m a r y Plot of the i d e n t i f i e d components of unknown sample after re-processing by UNIFI.
EXPERIMENTAL CONDITIONS
SAMPLE PREPARATION
Two capsules of an unknown TCM product were grinded into power. From this
grounded powder after the removal of the coating. 600 mg powder was
dissolved in 30 mL MeOH/H2O (3/1) by ultrasonicating the solution for
10 minutes. Prior to injection, an adequate volume (ca. 2 mL) of the solution
was filtered through a 0.45 µm nylon membrane. The first 1.0 mL was dis-
carded and the remaining volume was collected and stored for injection.
INSTRUMENTAL CONDITIONS
LC CONDITIONS:
LC system: ACQUITY UPLC I-Class with FTN Sample Manager
Column: ACQUITY UPLC HSS T3 2.1 x 100 mm, 1.8 μm, 45 °C
Sample temp.: 15 °C
Mobile Phase: A: water (0.1% formic acid); B: acetonitrile
Flow Rate: 0.6 mL/min
Gradient:
MS CONDITIONS:
MS system: Xevo G2-S QTof MS
Acquisition range: 100-1500 Da (0.1 s scan rate)
Acquisition mode: MSE, ESIˉ and ESI+ in resolution mode
Capillary voltage: 3 kV (ESI+)/2.5 kV (ESIˉ)
Cone voltage: 100 V
Collision energy (eV): Low CE: 6; High CE: 20-50
Source temp.: 120 °C
Desolvation temp.: 550 °C
INFORMATICS PLATFORM:
Natural Product Application Solution with UNIFI for: System control for data
acquisition/Data processing/Result review/Reporting. Figure 2 is the screen
shot of the UNIFI Traditional Medicine (TM) Library.
Time (min.) Solvent A(%) Solvent B(%) Curves
0 95 5 Initial
1 90 10 6
7 65 35 6
9 20 80 6
10 5 95 6
13 5 95 6
15 95 5 1
Figure 2. Screen-shot of the UNIFI Traditional Medicine Library. Shown here is the view of compound entry. This library can be also be searched by plant names and chemical classifica-tion. The library is offered in both English and Mandarin.
Figure 4. UPLC/QTof MS BPI chroma-togram for the un-known TCM sample. Low CE result is used for searching compo-nent identify, high CE result is used for structural elucidation as it contains frag-ment ion information that corresponds to the low energy data. The common link of the two chroma-tograms is the reten-tion time.
4A. Low CE
4B. High CE
5A
5B
5C 5D
Figure 5. Chemical ingredient profiling results review. 5A shows the provided result review workflow templates; 5B is the component summary interface; 5C is the selected ion chromatogram of single component corresponding to 5B; and 5D is the respective mass spectrum of 5C.
Figure 7. Comparison of the traditional ap-proach vs. the NPAS with UNIFI approach.
The NPAS with UNIFI converts the cumber-some multi-step proc-ess into an automatic streamlined process with one click of a button.
METHODS
ANALYTICAL CHALLENGE
Unknown Traditional Chinese Medicine (TCM) sample in the form of capsules
without any background information provided.
Required task: 1). Deduce possible herbal plants used in these capsules;
2). Deduce capsule’s name if possible; 3). Complete the analysis with report
generated in two days.
PROBLEM-SOLVEING STRATEGY
Use the Natural Products Application Solution with UNIFI as the analytical tool.
Perform UPLC/QTof MSE data acquisition by injecting the extract of the
unknown capsule.
Adapt step-by-step approach during data mining to obtain the final answer.
Step 1: Screen major components by searching entire TM library to deduce
possible herbs in the capsule.
Step 2: Search Internet for known TCM recipes to deduce group of herbs
commonly used in synergy.
Step 3: Import target components of target herbs from TM library to verify
which herbs are actually in the capsule so that possible recipe can be deduced
based on matched herbs.
Step 4: Import components from TM library for herbs listed in the identified
recipe to confirm the finding to finalize the answer to the problem.
This approach is also shown in a workflow format below in Figure 3.
Figure 3. Workflow for identifying chemical and herbal ingredients of a completely unknown samples using the Natural Products Application Solution with UNIFI.