A method for collecting enoxaparin oligosaccharides by rp-ip-hplc method

A technology of enoxaparin oligosaccharide and enoxaparin sodium, applied in the field of medicine, can solve the problems of oligosaccharide sequence destruction, extensiveness, low purity, etc.

Active Publication Date: 2016-09-14
SHENZHEN TECHDOW PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the enzymatic degradation-HPLC method mainly degrades polysaccharides into oligosaccharides and then collects them by HPLC. Generally, only short-chain oligosaccharides such as monosaccharides, disaccharides and trisaccharides can be collected, and the yield is low and the purity is not high. It can be obtained after multiple separations. At the same time, because this method cuts the oligosaccharide chain of enoxaparin, the sequence of the oligosaccharide is destroyed; the ultrafiltration method can only intercept substances in a certain molecular weight range, which is a relatively extensive method. Although the method can be combined with HPLC for purification, the collection process is relatively long, the efficiency is low and the purity is not high, and it is not suitable for general analysis experiments.

Method used

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  • A method for collecting enoxaparin oligosaccharides by rp-ip-hplc method
  • A method for collecting enoxaparin oligosaccharides by rp-ip-hplc method
  • A method for collecting enoxaparin oligosaccharides by rp-ip-hplc method

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Experimental program
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Effect test

Embodiment 1

[0096] Preparation of solutions in each step of enoxaparin sodium oligosaccharide collection

[0097] Preparation of enoxaparin sodium sample solution: Weigh 1000 mg of enoxaparin sodium sample into a clean test tube, add 15 ml of ultrapure water to dissolve, and mix well.

[0098] Preparation of 10 mg / ml total tetrasaccharide component solution: Weigh 15 mg of the collected total tetrasaccharide component sample into a clean test tube, add 1.5 ml of ultrapure water to dissolve, and mix well.

[0099] Preparation of 1 mg / ml pre-separated tetrasaccharide component solution: Weigh 3 mg of pre-separated tetrasaccharide sub-component sample into a clean test tube, dissolve in 3 ml of ultrapure water, and mix well.

[0100] Preparation of 1 mg / ml tetrasaccharide three-seed component solution: Take 1 mg of the three-step segregation sub-component samples of tetrasaccharide respectively in a clean test tube, add 1 ml, and mix well.

[0101] Solution A: Weigh 1.03g of dibutylamine an...

Embodiment 2

[0130] The collection method of the present invention is compared with the existing enzymatic degradation-HPLC collection method and ultrafiltration collection method, respectively from the aspects of collection range, time, oligosaccharide yield, purity and collection cost, etc. The comparison is shown in Table 8 below:

[0131] Table 8

[0132]

[0133]

[0134]As can be seen from the comparative results in Table 8, by comprehensively considering aspects such as collection range, time, oligosaccharide yield, purity and collection cost, the collection method of the present invention is all compared with the existing enzymatic degradation-HPLC collection method and ultrafiltration The collection method is more economical and effective.

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Abstract

The invention belongs to the field of medicine, and relates to an RP-IP-HPLC (reverse-phase ion-pair high-performance liquid chromatography) method for collecting enoxaparin oligosaccharide. The method comprises the following steps: (a) a tetrose component mixture is collected from enoxaparin, wherein an enoxaparin sodium solution with the concentration of 50-70mg / ml is separated with high-resolution gel permeation chromatography, and the tetrose component mixture is obtained; (b) single tetrose components are collected from the tetrose component mixture with RP-IP-HPLC, wherein the tetrose component mixture is taken and made into a water solution with the concentration of 10mg / ml and is pre-separated with the reverse-phase ion-pair high-performance liquid chromatography, the pre-separated tetrose components are made into water solutions of 1mg / ml respectively and then are separated and purified with the reverse-phase ion-pair high-performance liquid chromatography; (c) the collected single tetrose components are made into the water solutions of 1mg / ml and are subjected to structural analysis through ESI-MS (electrospray ionization mass spectrometry).

Description

technical field [0001] The invention belongs to the field of medicine and relates to a method for collecting enoxaparin oligosaccharides by RP-IP-HPLC. Background technique [0002] Enoxaparin is a kind of low-molecular-weight heparin, which is obtained from heparin benzyl ester through alkaline β-elimination reaction. It is a mixture of many small heparin molecules with different chain lengths, and its structure is very complex. At present, enoxaparin is mainly used clinically for the prevention of deep vein thrombosis and pulmonary embolism, the treatment of established venous thrombosis, the prevention of thrombus formation in extracorporeal circulation during hemodialysis, and the treatment of unstable angina and non-Q wave myocardial infarction. [0003] As a complex mixture of mucopolysaccharide polymers, enoxaparin contains numerous oligosaccharide chains. Most of the oligosaccharide chains contain 4,5 unsaturated uronic acid structures at their non-reducing ends, an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 李建科谭宏波邓焕跃谢贵兵
Owner SHENZHEN TECHDOW PHARM CO LTD
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