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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.