Two-dimensional liquid chromatography-mass spectrometry method for mass analysis of low-molecular-weight heparin

A low molecular weight heparin, two-dimensional liquid phase technology, applied in the field of drug analysis, can solve the problems of difficult structure consistency evaluation, complex structure, lack of analysis methods, etc., to achieve excellent separation ability and meet the needs of rapid analysis.

Pending Publication Date: 2022-07-29
SUZHOU UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

Enoxaparin sodium is derived from unfractionated heparin through β-elimination of benzyl ester and chemical cleavage. It has the advantages of less bleeding side effects and

Method used

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  • Two-dimensional liquid chromatography-mass spectrometry method for mass analysis of low-molecular-weight heparin
  • Two-dimensional liquid chromatography-mass spectrometry method for mass analysis of low-molecular-weight heparin
  • Two-dimensional liquid chromatography-mass spectrometry method for mass analysis of low-molecular-weight heparin

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Experimental program
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Effect test

Embodiment 1

[0050] 1. Preparation of Enoxaparin Sodium Oligosaccharide by Column Chromatography

[0051] Using a protein purifier, the column is packed with size exclusion ( Gel) for column chromatography. Enoxaparin sodium was loaded at a concentration of 500 mg / ml, dissolved in a NaCl solution with a concentration of 0.5 mol / L in the mobile phase, and the samples were separated by isocratic elution at a flow rate of 5 ml / min. Using a UV detector, the detection wavelength was 232 nm.

[0052] see figure 2 , figure 2 It is the low molecular weight heparin column chromatography chromatogram of the present invention. like figure 2 As shown, the oligosaccharides are collected by taking the half-peak width above the online detection, and the collected oligosaccharides have a single degree of polymerization. After concentration by rotary evaporation, the salt was removed by gel column chromatography and freeze-dried. 35.6 mg of enoxaparin sodium tetrasaccharide, 45.1 mg of hexasacch...

Embodiment 2

[0076] 1. Preparation of bemiparin sodium oligosaccharide by column chromatography

[0077] Using a protein purifier, the column is packed with size exclusion ( Gel) for column chromatography. Bemiparin sodium was loaded at a concentration of 500 mg / ml, dissolved in a NaCl solution with a concentration of 0.5 mol / L in the mobile phase, and the samples were separated by isocratic elution at a flow rate of 5 ml / min. Using a UV detector, the detection wavelength was 232 nm. The oligosaccharides are collected by taking the half-peak width above the online detection, and the collected oligosaccharides have a single degree of polymerization. After concentration by rotary evaporation, the salt was removed by gel column chromatography and freeze-dried. The single polymerized bemiparin sodium tetrasaccharide 19.7 mg, the hexasaccharide 23.1 mg, the octasaccharide 28.0 mg, the deca sugar 22.4 mg, and the dodecose 18.1 mg were prepared.

[0078] 2. SAX-SEC-MS qualitative experiment ...

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Abstract

The invention discloses a two-dimensional liquid chromatography-mass spectrometry method for mass analysis of low-molecular-weight heparin, which comprises the following steps: 1) preparing and grading low-molecular-weight heparin as a raw material through medium-pressure column chromatography to obtain a plurality of low-molecular-weight heparin oligosaccharides with different polymerization degrees, and 2) qualitatively analyzing the low-molecular-weight heparin oligosaccharide by using a two-dimensional liquid phase on-line mass spectrometry, namely separating the low-molecular-weight heparin oligosaccharide with a single polymerization degree by using strong anion exchange chromatography in a first-dimensional liquid phase, cutting each SAX separation oligosaccharide peak into a second-dimensional molecular sieve chromatography, and finally, carrying out on-line mass spectrometry. And 3) comparing SAX standard maps of different samples under the same polymerization degree, and carrying out semi-quantitative analysis on oligosaccharides of each polymerization degree through peak areas. The method can be used for quickly and finely analyzing the structure and controlling the quality of the low-molecular-weight heparin, and has huge application potential.

Description

technical field [0001] The invention belongs to the field of drug analysis, and relates to a two-dimensional liquid phase-mass spectrometry combined method for quality analysis of low molecular weight heparin. Background technique [0002] Heparin is not only an anticoagulant commonly used in clinical practice, but also has the effect of preventing and treating thrombotic diseases. In the domestic market, the market size of heparin drugs has increased year by year, and by 2018, the market size has exceeded 8 billion yuan. In recent years, low molecular weight heparin has gradually replaced unfractionated heparin as the main force of heparin drugs. Among them, the market sales of enoxaparin sodium are increasing rapidly year by year. By 2018, its domestic market size has reached 2.05 billion yuan, an annual growth rate of 35% compared with 1.52 billion yuan in sales in 2017. In 2019, global oral anticoagulant drugs accounted for more than 80% of the antithrombotic market, ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/72G01N30/86
CPCG01N30/02G01N30/72G01N30/86
Inventor 张真庆陈磊欧阳艺兰易琳李笃信
Owner SUZHOU UNIV
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