Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization

A low-molecular-weight heparin, completely degraded technology, used in measurement devices, material separation, analysis of materials, etc., can solve problems such as qualitative analysis and no reducing hemiacetal hydroxyl groups

Active Publication Date: 2014-03-12
HEBEI CHANGSHAN BIOCHEM PHARMA
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Problems solved by technology

The non-reducing end of dalteparin sodium is a saturated uronic acid, which does not have characteristic UV absorption and cannot be detected by traditional analytical methods, but it can be combined with a derivatizing reagent by pre-column derivatization to carry a fluorescent group or Has characteristic UV absorption; and the reducing end structures of dalteparin sodium and enoxaparin sodium do not have reducing hemiacetal hydroxyl groups, so they can

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  • Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization
  • Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization
  • Reverse-phase chromatography and mass-spectrometry combined detection method for complete low-molecular-heparin degradation product through precolumn derivatization

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[0006] A reversed-phase chromatography-mass spectrometry detection method for complete degradation products of low-molecular-weight heparin combined with pre-column derivatization, the steps are as follows:

[0007] (1) Dissolve the ammonium acetate reagent in deionized water, and adjust the pH to 3-6 to prepare mobile phase A with an ammonium acetate concentration of 20-80 mM;

[0008] (2) methanol as mobile phase B;

[0009] (3) Add 5-10 μL of 0.1M 2-aminoacridone (AMAC) solution to 10-50 μg of low-molecular-weight heparin complete enzymatic hydrolysis sample containing internal standard, let it stand at room temperature for 10-20 minutes, and then add 5-10 10 μL of 1 M sodium cyanoborohydride (NaBH 3 CN) solution, incubate at 45°C for 2-4 hours to obtain the derivatized sample;

[0010] (4) Dilute the derivatized sample prepared in step (3) with a 50% dimethyl sulfoxide (DMSO) solution to obtain a test solution with a derivatized sample concentration of 1-5 μg / μL. After ...

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Abstract

The invention relates to a reverse-phase chromatography and mass-spectrometry combined detection method for a complete low-molecular-heparin degradation product through precolumn derivatization. All components of the complete low-molecular-heparin degradation product are detected by combining reversed-phase chromatography and a high-resolution mass spectrum; all the components in a sample are separated through the reversed-phase chromatography, and a precision molecular weight can be obtained through the high-resolution mass spectrum; besides detection of eight common heparin disaccharides, tail end modification structures and special structures such as 3-O-tetrose-hydrosulfates relevant to anticoagulation and trisaccharides obtained through peeling reaction can be also detected; therefore, the structure of each complete low-molecular-heparin degradation product can be precisely represented. According to the reverse-phase chromatography and mass-spectrometry combined detection method, the problem that only the eight common heparin disaccharides can be detected in the prior art is solved; the reverse-phase chromatography and mass-spectrometry combined detection method has an extremely large practical value in research, development and production control of low-molecular-heparin imitating drugs and guarantee of the safety of the drugs.

Description

technical field [0001] The invention relates to a combined detection method of low-molecular-weight heparin complete degradation products combined with pre-column derivatization by reversed-phase chromatography and high-resolution mass spectrometry, and belongs to the technical field of medicine, raw material medicine and raw material detection. Background technique [0002] Heparin is a heterogeneous glycosaminoglycan with anticoagulant effect and can be used as a clinical anticoagulant drug for the prevention and treatment of thrombotic diseases. However, as an anticoagulant drug, heparin can easily cause side effects such as bleeding, osteoporosis and thrombocytopenia, which limits its clinical application. Low-molecular-weight heparin is a new type of anticoagulant drug prepared by controlled enzymatic and chemical degradation of heparin. Due to different production processes, each low-molecular-weight heparin has a different terminal modification structure, making its s...

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Application Information

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IPC IPC(8): G01N30/89
Inventor 迟连利王章杰
Owner HEBEI CHANGSHAN BIOCHEM PHARMA
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