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Liquid chromatography-mass spectrometry analysis and detection method of polysaccharides and application thereof

A technology of liquid chromatography mass spectrometry and detection methods, which is applied in the direction of analyzing materials, measuring devices, and material separation, etc. It can solve the problems of low ion abundance of oligosaccharide fragments and limited collision efficiency, and achieve high sensitivity, interference elimination, heavy weight, etc. good effect

Inactive Publication Date: 2020-05-01
NORTHEAST NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low energy of in-source fragmentation, the collision efficiency of In-source CID is limited, and the abundance of characteristic oligosaccharide fragment ions produced is not high

Method used

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  • Liquid chromatography-mass spectrometry analysis and detection method of polysaccharides and application thereof
  • Liquid chromatography-mass spectrometry analysis and detection method of polysaccharides and application thereof
  • Liquid chromatography-mass spectrometry analysis and detection method of polysaccharides and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Weigh an appropriate amount of dextran (average molecular weight 64,650Da), dissolve and dilute to 10.0μg / mL with deionized water; take 10μL for SEC-ESI-Q-Orbitrap analysis, record chromatograms and mass spectra, and add oligosaccharide fragments Ion m / z response for qualitative and quantitative analysis of dextran.

[0028] The chromatographic conditions are:

[0029] The chromatographic column is Waters Acquity UPLC Protein BEH SEC chromatographic column (4.6×300mm, 1.7 μm, Waters Company, USA); mobile phase is isocratic elution, methanol: water=(20:80, v / v); flow rate is 0.3 mL / min; column temperature is 45°C.

[0030] The mass spectrometry conditions are:

[0031]Electrospray ion source; negative ion mode; spray voltage: 3.5kV; source temperature: 320°C; sheath gas (Sheath Gas, N2): 35a.u.; auxiliary gas (Aux Gas, N2): 15a.u.; auxiliary gas Temperature: 350°C; S-lens level: 80%; AGC: 5e6; maximum injection time: 500ms; acquisition mode: All Ion Fragmentation (AI...

Embodiment 2

[0033] Weigh 10.0 mg of dextran (average molecular weight 64,650 Da), and place it in a 5 mL volumetric flask. After adding a small amount of normal saline to dissolve, dilute to the mark with normal saline, the concentration is 2mg / mL. After administration of 5 mg / kg dextran through tail vein of rats, before administration (0h) and after administration 1, 5, 15, 30min, 1, 2, 4, 6, 8 and 12h, from rat orbital vein 0.1 mL of blood was collected from the cluster and placed in EP tubes treated with EDTA anticoagulant. Immediately centrifuge at 3,000g for 5 minutes and store at -20°C. Determination of the content of dextran in plasma after tail vein administration of dextran, the time curve of plasma drug concentration is shown in Image 6 .

[0034] The main steps are as follows:

[0035] A. Standard curve preparation

[0036] 1) Preparation of dextran stock solution: Weigh 10.0 mg of dextran standard substance, transfer 1 mL of water with a pipette, fully shake to dissolve,...

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Abstract

The invention discloses an analysis and detection method of high-molecular-weight polysaccharides, and the coupling technique of liquid chromatography and high-resolution mass spectrometry is used fordetection and analysis. The method comprises the steps of firstly separating a polysaccharide substance to be detected through size exclusion chromatography, and preliminarily fragmenting polysaccharides in an ion source of a mass spectrum in an in-source cracking mode; and transmitting all ions generated in the ion source into a collision pool by adopting an MSALL mass spectrum acquisition method, carrying out high-energy collision induced dissociation cracking to generate a series of characteristic oligosaccharide ion fragments with high abundance, acquiring the characteristic oligosaccharide fragments, and quantitatively detecting the high-molecular-weight polysaccharides according to the characteristic oligosaccharide fragments. According to the method, two ion fragmentation modes ofin-source cracking and cracking in the collision pool of the mass spectrum are combined, so that the characteristic oligosaccharide ion fragments with high abundance can be obtained. The interferenceof endogenous matrixes can be effectively eliminated by adopting high-resolution mass spectrum data. According to the method, parent ions with a specific mass-to-charge ratio do not need to be selected, and the problem that the parent ions are difficult to select due to polydispersity of the high-molecular-weight polysaccharides can be solved.

Description

technical field [0001] The invention belongs to the technical field of drug analysis and research, and relates to a biological mass spectrometry method for analyzing high molecular weight polysaccharides. Background technique [0002] With the continuous deepening of research on glycochemistry and glycobiology, the pharmaceutical research on polysaccharides has also made great progress. However, most of the current research focuses on the separation and purification, chemical composition and pharmacological activity of polysaccharides. There are still many unresolved problems in the quantitative research of polysaccharides, and there is still a lack of sensitive, accurate and reliable analysis methods. Due to the complex structure and large molecular weight of polysaccharides, similar endogenous polysaccharides in the body will also interfere with the determination. The quantitative analysis of polysaccharides has always been a difficult problem in the field of pharmaceutica...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/72G01N30/32G01N30/34G01N30/54G01N30/86
CPCG01N30/02G01N30/72G01N30/32G01N30/34G01N30/54G01N30/86G01N2030/045G01N2030/324
Inventor 王浩周义发及莉张帆宋成程
Owner NORTHEAST NORMAL UNIVERSITY
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