CN103439434A - Detection method of esterification rate of enoxaparin sodium intermediate - Google Patents
Detection method of esterification rate of enoxaparin sodium intermediate Download PDFInfo
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- CN103439434A CN103439434A CN2013104133741A CN201310413374A CN103439434A CN 103439434 A CN103439434 A CN 103439434A CN 2013104133741 A CN2013104133741 A CN 2013104133741A CN 201310413374 A CN201310413374 A CN 201310413374A CN 103439434 A CN103439434 A CN 103439434A
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Abstract
The invention discloses a detection method of the esterification rate of enoxaparin sodium intermediate. The detection method comprises the following steps: drying a mixture containing heparin benzyl ester, adding a sodium hydroxide solution to carry out hydrolysis, adding glacial acetic acid to regulate the pH value, and carrying out volume fixation to obtain a sample solution; respectively detecting a standard solution and the sample solution by utilizing a high performance liquid chromatography to obtain a spectrogram, and obtaining the esterification rate of the heparin benzyl ester according to a formula W%=(c1*b)/(a*c2)*100% and a spectrogram result. By utilizing the manner, the detection method disclosed by the invention has the advantages that the result can be accurately, rapidly, simply and conveniently obtained and is high in accuracy, the reject ratio and production cost of a product are lowered, and the quality condition of the product is improved.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to a kind of detection method of Enoxaparin Sodium intermediate ester rate.
Background technology
Heparin is a kind of Sulfated polysaccharide, belongs to glycosaminoglycan family.Heparin, by protein combination different from other, can be brought into play important biologically active, thereby be used as a kind of anticoagulant medicine, is widely used.As anticoagulation medicine, oneself has more than 70 years to heparin, for huge contribution has been made in the development of department of cardiovascular surgery and haemodialysis.Up to the present also there is no a kind of product that can replace it fully, so heparin remains one of most important anticoagulation and antithrombotic biochemical drug.
But heparin is in long-term use procedure, can produce many unpredictalbe spinoffs, as large as the dosage individual difference, be difficult to determine appropriate dose, except the LDH therapy, administration time need to carry out Laboratory Monitoring, otherwise there will be the problems such as thrombopenia that cause because of heparin.In order to solve these disadvantageous factors, people have developed novel anticoagulation medicine " low molecular weight heparin ".Low molecular weight heparin is component or the fragment that has lower molecular weight in unfraction heparin, there is the precursor structure identical with unfraction heparin, this class medicine has that chain is short, binding site is few, bioavilability is high, long half time, individual difference and uncertain factor is few, dosage is easy to determine, without carrying out the advantages such as blood concentration detection.
Enoxaparin Sodium is the best a kind of low molecular weight heparin of global marketing amount.It distributes to the most important thing is mean molecular weight and molecular weight in the Enoxaparin Sodium quality index, and the intermediate heparin esterification yield in the technological process of Enoxaparin Sodium plays conclusive effect to its molecular mass and distribution thereof.The esterification yield of the liquaemin benzyl ester intermediate in the Enoxaparin process for producing sodium refers to, in the solvent of methylene chloride, SN occurs for heparin benzethonium chloride salt and benzyl chlorine
2nucleophilic substitution, the number of the amount of the heparin benzyl ester generated.Esterification yield is higher, after the basic hydrolysis condition under, therefore the mean molecular weight after cracking is lower, only has esterification yield is controlled in certain scope and just can obtains meeting the Enoxaparin Sodium product that quality standard requires.
Summary of the invention
The technical matters that the present invention mainly solves is to provide a kind of detection method of Enoxaparin Sodium intermediate ester rate, the esterification yield that obtains liquaemin benzyl ester intermediate that this detection method can be accurate, quick, easy.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of detection method of Enoxaparin Sodium intermediate ester rate is provided, comprises that step is:
(1) heparin benzethonium chloride salt is reacted to the potpourri that contains the heparin benzyl ester obtained with benzyl chlorine dries and within 3 hours, obtains sample under 60 ℃, by described sample and 1N sodium hydroxide solution mixed hydrolysis, add again glacial acetic acid to regulate the pH value for 6-7, add the water constant volume to obtain sample solution;
(2) respectively 0.5g/L phenmethylol standard solution and the detection of 5g/L sample solution are obtained to the high-efficient liquid phase chromatogram of standard solution and the high-efficient liquid phase chromatogram of sample solution with high performance liquid chromatograph, obtain the esterification yield of heparin benzyl ester according to formula W %=(c1*b)/(a*c2) * 100%, the peak area that the high-efficient liquid phase chromatogram that wherein a is standard solution obtains, the peak area that the high-efficient liquid phase chromatogram that b is sample solution obtains, the concentration that c1 is standard solution, the concentration that c2 is sample in sample solution.
In a preferred embodiment of the present invention, described in step (1), the ratio of sample, described sodium hydroxide solution, described glacial acetic acid and described sample solution is 10mg:1ml:0.1ml:2ml, and described hydrolysis time is 2 hours.
In a preferred embodiment of the present invention, standard solution sample introduction described in step (2) repeats 5 times, and described sample solution sample introduction repeats 2 times.
In a preferred embodiment of the present invention, the condition that high performance liquid chromatograph described in step (2) detects is that mobile phase is acetonitrile, first alcohol and water, and volume ratio is 3:1:16, and flow velocity is 1.0ml/min, and column temperature is 30 ℃, and wavelength is 256nm, and sample size is 20 μ l.
In a preferred embodiment of the present invention, sample solution described in step (2) is extremely clarified with membrane filtration before sample introduction.
The invention has the beneficial effects as follows: the detection method of Enoxaparin Sodium intermediate ester rate of the present invention, the method adopts reverse-phase chromatography, the phenmethylol high performance liquid chromatograph detection level that heparin benzyl ester intermediate obtains in NaOH alkalescence Water Under solution, use external standard method accurately to calculate, obtained the reliable results of esterification yield, the method can be accurate, quick, easy obtain result, result precision is high, reduce disqualification rate and the production cost of product, improved the quality condition of product.
The accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, in below describing embodiment, the accompanying drawing of required use is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skills, under the prerequisite of not paying creative work, can also obtain other similar spectrogram according to these spectrograms, wherein:
Fig. 1 is the high-efficient liquid phase chromatogram of the detection method one preferred embodiment Plays solution of Enoxaparin Sodium intermediate ester rate of the present invention;
Fig. 2 is the high-efficient liquid phase chromatogram of sample solution in detection method one preferred embodiment of Enoxaparin Sodium intermediate ester rate of the present invention.
Embodiment
Below will the technical scheme in the embodiment of the present invention be clearly and completely described, obviously, described embodiment is only a part of embodiment of the present invention, rather than whole embodiment.Embodiment based in the present invention, those of ordinary skills, not making all other embodiment that obtain under the creative work prerequisite, belong to the scope of protection of the invention.
1) instrument and equipment: high performance liquid chromatograph, 100,000/analytical balance, turbine mixer, Ultrasound Instrument.
2) reagent: acetonitrile, methyl alcohol, pure water, NaOH, glacial acetic acid.
3) chromatographic condition: mobile phase: acetonitrile: methyl alcohol: the volume ratio of purified water is 3:1:16, thinning agent: purified water, pillar: C
8(octyl), 4.6mm * 150 mm, 5 μ m, flow velocity: 1.0 ml/min, column temperature: 30 ℃, wavelength: 256 nm, sample size: 20 μ l, time: 30 min.
4) solution preparation:
Standard solution: accurately take approximately 50 mg phenmethylols and, in 100 ml volumetric flasks, with pure water, dilute constant volume.
Sample solution: testing sample is dried 3 hours with vacuum drying chamber under 60 ℃, take out, be placed in vacuum dryer and be cooled to room temperature.Accurately take described sample approximately 50 mg in 10 ml volumetric flasks, add 5ml 1N NaOH solution, hydrolysis 2h, then add 0.5 ml glacial acetic acid, regulate pH value and be about 6-7, then use the pure water constant volume, before sample introduction with membrane filtration to clarifying.
5) system flexibility: the tailing factor at phenmethylol peak can not be greater than 1.5, and standard solution repeats sample introduction 5 times, main peak RSD%≤2.0%, and the theoretical cam curve at phenmethylol peak should be not less than 3000.
6) sample introduction: standard solution continuous sample introduction 5 times, sample solution continuous sample introduction 2 times.
7) calculate: refer to Fig. 1, the area at standard solution phenmethylol peak is a, refers to Fig. 2, and the area at sample solution phenmethylol peak is b, and standard solution phenmethylol concentration is c1 (mg/ml), and in sample solution, the concentration of sample is c2(mg/ml).
According to formula W %=(c1*b)/(a*c2) * 100%, obtain the esterification yield of heparin benzyl ester.
Disclosed by the invention is a kind of detection method of Enoxaparin Sodium intermediate ester rate, the method adopts reverse-phase chromatography, detect by the high performance liquid chromatograph with UV-detector the phenmethylol content that the heparin benzyl ester intermediate in the Enoxaparin process for producing sodium obtains in NaOH alkalescence Water Under solution, use external standard method accurately to calculate, obtained the reliable results of esterification yield, the method can be accurately, fast, the easy result that obtains, result precision is high, thereby can effectively determine the experiment condition parameter of next step technique and improve the total quality of product.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent application of utilizing description of the present invention to do, or directly or indirectly be used in other relevant technical field, all in like manner be included in scope of patent protection of the present invention.
Claims (5)
1. the detection method of an Enoxaparin Sodium intermediate ester rate, is characterized in that, comprises that step is:
(1) heparin benzethonium chloride salt is reacted to the potpourri that contains the heparin benzyl ester obtained with benzyl chlorine dries and within 3 hours, obtains sample under 60 ℃, by described sample and 1N sodium hydroxide solution mixed hydrolysis, add again glacial acetic acid to regulate the pH value for 6-7, add the water constant volume to obtain sample solution;
(2) respectively 0.5g/L phenmethylol standard solution and the detection of 5g/L sample solution are obtained to the high-efficient liquid phase chromatogram of standard solution and the high-efficient liquid phase chromatogram of sample solution with high performance liquid chromatograph, obtain the esterification yield of heparin benzyl ester according to formula W %=(c1*b)/(a*c2) * 100%, the peak area that the high-efficient liquid phase chromatogram that wherein a is standard solution obtains, the peak area that the high-efficient liquid phase chromatogram that b is sample solution obtains, the concentration that c1 is standard solution, the concentration that c2 is sample in sample solution.
2. detection method according to claim 1, is characterized in that, described in step (1), the ratio of sample, described sodium hydroxide solution, described glacial acetic acid and described sample solution is 10mg:1ml:0.1ml:2ml, and described hydrolysis time is 2 hours.
3. detection method according to claim 1, is characterized in that, standard solution sample introduction described in step (2) repeats 5 times, and described sample solution sample introduction repeats 2 times.
4. detection method according to claim 1, is characterized in that, the condition that high performance liquid chromatograph described in step (2) detects is that mobile phase is acetonitrile, first alcohol and water, volume ratio is 3:1:16, and flow velocity is 1.0ml/min, and column temperature is 30 ℃, wavelength is 256nm, and sample size is 20 μ l.
5. detection method according to claim 1, is characterized in that, sample solution described in step (2) is extremely clarified with membrane filtration before sample introduction.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175415A (en) * | 2019-12-24 | 2020-05-19 | 湖北亿诺瑞生物制药有限公司 | Method for determining benzethonium chloride residue in enoxaparin sodium |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5289618A (en) * | 1991-12-05 | 1994-03-01 | Ernst Fehrer | Apparatus for making a nonwoven web |
| CN102585037A (en) * | 2012-02-10 | 2012-07-18 | 麦科罗夫（南通）生物制药有限公司 | Enoxaparin sodium and production purification method thereof |
| CN103175925A (en) * | 2013-03-20 | 2013-06-26 | 山东辰中生物制药有限公司 | Method for detecting esterification rate of heparin benzyl ester in production process of enoxaparin sodium |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5289618A (en) * | 1991-12-05 | 1994-03-01 | Ernst Fehrer | Apparatus for making a nonwoven web |
| CN102585037A (en) * | 2012-02-10 | 2012-07-18 | 麦科罗夫（南通）生物制药有限公司 | Enoxaparin sodium and production purification method thereof |
| CN103175925A (en) * | 2013-03-20 | 2013-06-26 | 山东辰中生物制药有限公司 | Method for detecting esterification rate of heparin benzyl ester in production process of enoxaparin sodium |
Non-Patent Citations (1)
| Title |
|---|
| 金飞等: "依诺肝素钠的制备与纯化", 《中国医药工业杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111175415A (en) * | 2019-12-24 | 2020-05-19 | 湖北亿诺瑞生物制药有限公司 | Method for determining benzethonium chloride residue in enoxaparin sodium |
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Application publication date: 20131211 |