A large-scale qualitative method for glycosides based on liquid chromatography-mass spectrometry

A liquid chromatography and compound technology, which is applied in the field of large-scale characterization of glycoside compounds, can solve the problems of high price, limited glycoside compounds, limited scale of glycoside compound secondary mass spectrometry database, etc., to avoid redundancy and high reliability sexual effect

Active Publication Date: 2021-12-28
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

[0003] Difficulty in large-scale characterization of glycosides due to their structural diversity and complexity
Although liquid chromatography-mass spectrometry has been widely used in the analysis and identification of glycoside compounds in plants, it is mainly aimed at a small number of known glycoside compounds, and the glycoside compounds that can be identified are very limited.
In addition, commercially available glycoside standards are limited and expensive, and glycoside MS / MS databases are limited in size

Method used

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  • A large-scale qualitative method for glycosides based on liquid chromatography-mass spectrometry
  • A large-scale qualitative method for glycosides based on liquid chromatography-mass spectrometry
  • A large-scale qualitative method for glycosides based on liquid chromatography-mass spectrometry

Examples

Experimental program
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Effect test

Embodiment 1

[0034] The identification of embodiment 1 stevioside-rebaudioside N

[0035] The technical scheme that the present invention adopts is as follows:

[0036] 1. Sample configuration: the commercially available rebaudioside N standard was dissolved in pure methanol, and the sample concentration was 50ng / mL.

[0037] 2. Instrument analysis conditions:

[0038]Chromatographic conditions: ACQUITY UHPLC ultra-high performance liquid chromatography system (UPLC, Waters, Milford, MA, USA.) and Q Exactive HF high-resolution mass spectrometry (Thermo Fisher Scientific, Rockford, IL, USA.) were used as analytical instruments. The liquid chromatography conditions are: phase A and phase B are respectively 0.1% formic acid / water (volume ratio) and 0.1% formic acid / acetonitrile (volume ratio). The flow rate was 0.1 mL / min. The total analysis time is 28min. The elution gradient started with 10% B (volume ratio) and was held for 2 min. Then increase linearly to 100% B within 22min and keep...

Embodiment 2

[0056] Example 2 Large-scale characterization of glycosides in the crude stevia extract.

[0057] The technical solution is as follows:

[0058] 1. The sample extraction method is: accurately weigh 5 mg of stevia dry powder into a 1.5 mL centrifuge tube, add 1 mL of ethanol / water solution (volume ratio 50:50) to extract for 2 hours, shake on a vortex shaker for 1 minute, and place in a high-speed centrifuge Centrifuge at 4°C and 15,000rpm for 10min, take the supernatant and freeze-dry, add 1mL of methanol / water (volume ratio, 8:2) to the freeze-dried sample powder, and centrifuge at 4°C at 15,000rpm in a high-speed centrifuge. After 10 minutes, the supernatant was taken for analysis.

[0059] 2. Instrument analysis conditions: with embodiment 1.

[0060] 3. Stevioside aglycon library construction: same as Example 1.

[0061] 4. Glycosyl theoretical characteristic neutral loss library construction: Same as Example 1.

[0062] 5. Identification of glycosides in crude stevia ...

Embodiment 3

[0075] Example 3 Scale Identification of Glycoside Compounds of Corn Leaf Crude Extract

[0076] 1. Preparation method of corn leaf crude extract: Accurately weigh 50mg of corn leaf powder into a 1.5mL centrifuge tube, add 1mL of 80% methanol / water solution and vortex extract for 5min, centrifuge at 4°C and 15000rpm for 10min at high speed, and take 700μL The supernatant was lyophilized overnight, redissolved in 100 μL of 80% methanol / water, centrifuged again at 15,000 rpm for 10 min at 4°C, and the supernatant was taken for analysis.

[0077] 2. Instrument analysis conditions:

[0078] The analytical instrument used for the non-targeted LC-MS / MS metabolome data acquisition is the same as that in Example 1. In the positive ion mode, the liquid chromatography conditions are: phase A and phase B are respectively 0.1% formic acid / water (volume ratio) and 0.1% formic acid / acetonitrile (volume ratio). The flow rate was 0.35 mL / min. The total analysis time is 30 min. The elution...

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Abstract

The invention relates to a large-scale qualitative method for glycoside compounds based on ultra-high performance liquid chromatography-high resolution mass spectrum. First, obtain the aglycone or glycoside-forming substances contained in the organisms of the same biological species as the sample to be tested from the public report database or literature to establish the aglycone library, and predict the possible neutral loss of the sugar group in mass spectrometry, and construct the theoretical characteristics of the sugar group Neutral loss library; using ultra-high performance liquid chromatography-high resolution mass spectrometry to analyze the sample extract to be tested, the obtained mass spectrometry data is searched by neutral loss based on the theoretical characteristics of sugar groups, and the candidate glycoside compounds are screened out; and then through the actual measurement The neutral loss of the glycosyl characteristic was used to infer the composition of the glycosyl group, and the characteristic ions of the aglycone were obtained; then the identification of the glycoside was realized by searching the aglycone library. Then, using the secondary mass spectrometry of the candidate glycosides in the sample to be tested, a molecular network was constructed according to the similarity of their mass spectra, and the structure of the candidate glycosides that could not be identified by the search of the aglycon library was deduced. This method can realize large-scale annotation of glycoside compounds, and effectively improves the identification efficiency and reliability of glycoside compounds.

Description

technical field [0001] The invention belongs to the field of analytical chemistry, and is a large-scale qualitative method for glycoside compounds in plants based on ultra-high performance liquid chromatography-high resolution mass spectrometry. Background technique [0002] Glycosides are further metabolized by flavonoids, anthraquinones, terpenes, etc., most of which have important biological activities. Glycosylation is the most widely existing modification method in the synthesis of secondary metabolites. It has a great influence on the biological activity, solubility, stability, toxicity, transportability, subcellular localization of secondary metabolites, and mutual recognition and binding with receptors. Characteristics, etc. have an important impact, thereby affecting the growth and development of plants. The structure of glycoside compounds is mainly composed of two parts, one part is sugar residue (sugar removes hemiacetal hydroxyl group), and the other part is li...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/86
CPCG01N30/02G01N30/86
Inventor 许国旺张秀琼李在芳郑福建赵春霞赵欣捷路鑫
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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