Method For Identifying Chemical Active Ingredients Of Cistanche

Contact: joanna.jia@wecistanche.com

Chemical Components Identification Of Cistanche Deserticola Using The X500R QTOF System

Li Yuejie, Cheng Haiyan, Liu Ting, Li Lijun, Jin Wenhai SCIEX, Asia Pacific Application Support Center (Shanghai), China

Introduction

Cistanche deserticola was first recorded in the “Shen Nong Materia Medica”; it is also known as “Dayun,” “Rousong Rong,” and “Zong rong.” As a traditional herbal medicine, it has the properties of nourishing the kidney yang, improving blood flow, acting as a laxative, immune stimulation, and other effects[1]. In1983, the Japanese scholar H. Kobayashi and others began to study the chemical composition of Cistanche Deserticola[2], and since then it has become a popular topic in Chinese medicine research that has generated great interest both domestically and abroad over the last 30 years. Cistanche Deserticola belongs to the class of plants containing phenolic glycosides, iridoids, and their glycosides, and lignans and their glycosides.

Quadrupole time-of-flight (QTOF) mass spectrometry is a sensitive and specific tool for the identification of Chinese medicine components that have gradually become indispensable to research. This technology has overcome traditional technical challenges with retrospective analyses of single injections that permit extraction of important data and the most comprehensive acquisition of sample information. Using exact mass and high-resolution TOF-MS and TOF-MS/MS data allows for simultaneous, highly specific targeted and non-targeted qualitative analysis. However, the complexity of the instrument operation and software use has vastly limited the spread and development of this technology. Here we introduce a new QTOF system that uses a revolutionary new N-type geometry-based TOF path, intuitive software, and accurate molecular weight techniques that are easier to use in Chinese medicine component identification.

The benefits of this method are as follows:

1. The X500R uses the durable, industry-approved Turbo VTM ion source and air curtain gas interface design, which has a strong anti-contamination feature;

2. SCIEX OS Software has integrated acquisition, processing, and reporting functions on a single platform; the interface is intuitive, easy to master, and has a one-touch auto-adjust correction to ensure that analysts with any degree of expertise can obtain high quality, reliable data;

3. Using SCIEX OS Software to process data to identify Chinese medicine components are simple and permit rapid extraction of useful information, thus ;

4. It derives more accurate and reliable identification results from Chinese medicine libraries containing MS/MS spectra;

5. High-resolution MS/MS Chinese medicine databases are based on the “Chinese pharmacopeia” Part 1 TCM active ingredients; including component references in the pharmacopeia and active ingredients in the herbs, there are nearly 900 compounds

cistanche deserticola improving efficacy

Experimental Process

1. Using TOF-MS-IDA MS/MS mode, inject a sample and simultaneously obtain primary precursor ions and corresponding secondary spectra;

2. Using SCIEX OS Software targeted screening, confirmation of target compounds, and secondary spectra along with a screening of Chinese medicine standards and matching methods can increase accuracy and work productivity.

3. SCIEX OS Software’s non-targeted identification workflow uses library searches and complete unknown searches in ChemSpider to verify results, ensuring more components are identified with a simpler workflow.

Samples and Preprocessing Method

Sample source:

Purchased from Shanghai pharmacies in sliced form

Preprocessing method:

1. Slices were crushed to form powder;

2. 0.9mg was weighed and immersed in 3mL methanol for 40min;

3. The sample from step 2) was ultrasonicated 1 h;

4. Centrifugation and removal of the supernatant to use as a sample was performed.

Liquid Chromatography (LC) Conditions

Chromatographic Column: XSelect HSS T3, 2.1*150mm, 3.5µm;

Mobile phase: Gradient elution was used

Mobile phase: A is 0.1% formic acid water-2mM NH4FA

B is 95% acetonitrile-5% water-2mM NH4FA

Flow rate: 0.5mL/min

Column temperature: 40ºC

Amount inserted: 5µL

Mass Spectrometry Method：

Scanning method: TOF MS-IDA-15 MS/MS qualitative screening

ESI ion source parameters:

Air curtain gas CUR: 35psi; IS voltage: 5500V/-4500V

Source temperature: 600ºC

Atomizing gas GAS1: 55psi; Auxiliary gas GAS2: 60psi

Instrumentation mode search:

One-touch (select MS Check on the lower right), fully automated TOF-MS and TOF-MS/MS correction mode ensure that analysts of any expertise level can obtain accurate, reliable, reproducible data.

Positive and negative ion mode BPCs:

Compound Identification Procedure

1. Targeted component identification workflow

1.1: Molecular formula search

Only the chemical compound name and molecular formula are required; these can be input directly or imported using Excel’s copy and paste function to create a processing method

Once a results table is created, quantitative and qualitative results can be viewed in the same window. A red/green indicator system is used to indicate mass accuracy, retention time, isotope type, and confidence in identification by database matching.

SCIEX OS software lets users filter results and display only those compounds meeting acceptance criteria and falling within confidence intervals defined by the user. It can quickly find targeted results in large databases.

The TCM database of MS/MS spectra allows for secondary matching and yields more reliable results (grey color in the database indicates MS/MS data)

The literature contains names and molecular formulas of phenolic glycoside active ingredients. Using the above process for the identification of target components, it was determined that the sample contains 39 types of phenolic glycosides:

Besides the phenolic glycoside active components, Cistanche also contains a large number of compounds such as iridoids, glycosides, and lignans. The identification results are as follows:

The iridoids, glycosides, and lignans identification list:

1.2: Database Search:

53 compounds were obtained from matching identification in the TCM database: 43 positive ions, 22 negative ions, and 12 repeats, listed below. Besides active phenolic glycosides, iridoids, glycosides, and lignans, Cistanche also contains

2. Non-targeted component analysis

Non-targeted component identification can be performed with the built-in ChemSpider search function to determine the classification and type of unknowns. For non-targeted component identification, simply choose the “non-targeted” mode and the molecular formula search will develop a processing method. The workflow is as follows:

For complete unknowns, molecular formula search results are shown in a peak browser window in the lower part of the TOFMS mass spectrum; with ChemSpider database search, results are listed by priority, and the structural information obtained in ChemSpider is automatically compared with the MS/MS spectrum obtained, providing secondary feedback for rapid identification.

Summary

1. Rapid high-resolution data acquisition; a single injection yielded high-resolution TOF MS and MS/MS data, with 39 identifiable phenolic glycoside active components and 16 iridoids, lignans, and glycosides;

2. A TCM database of secondary spectra provides additional matching information, and the software automatically provides a database match score. Using the score, one can easily, quickly, and accurately identify Chinese medicine components;

3. The device is simple and has a one-touch auto-adjust correction to ensure that analysts with any degree of expertise can obtain high quality, reliable data;

4. The new SCIEX OS software version integrates data acquisition, processing (quantitative and qualitative), display, reporting, and database management. It solves the difficulties that many users face with an intuitive and easy-to-use interface;

5. Both the targeted and non-targeted screening workflows are simple, and the built-in method guide helps users accurately and rapidly create methods.

References

1. ISBN 978-7-5067-7337-9, PRC pharmacopeia [S]. Beijing: National pharmacopeia commission, 2015

2. Lei, Song Zhihong, Tu Pengfei; Research advances in the chemical composition of plants of the genus Cistanche, “Chinese Herbal Medicine,” Volume 34, No. 5, May 2003,473-476.