CN101825607B - Method for separating and detecting over-sulfated chondroitin sulfate in heparin sodium - Google Patents

Method for separating and detecting over-sulfated chondroitin sulfate in heparin sodium Download PDF

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CN101825607B
CN101825607B CN201010112056A CN201010112056A CN101825607B CN 101825607 B CN101825607 B CN 101825607B CN 201010112056 A CN201010112056 A CN 201010112056A CN 201010112056 A CN201010112056 A CN 201010112056A CN 101825607 B CN101825607 B CN 101825607B
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liquaemin
chondroitin polysulfate
sample
electrophoresis
separation
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CN101825607A (en
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刘榜惠
张志斌
袁红英
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Jiangsu Madsen Pharmaceutical Co ltd
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HUAIAN MDC CHEMICAL CO Ltd
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Abstract

The invention discloses a method for separating and detecting over-sulfated chondroitin sulfate in heparin sodium. The method uses a fast precipitation method to separate the over-sulfated chondroitin sulfate from the heparin sodium, and further detect an object obtained after the separation by using agarose and electrophoresis to realize the detection of the low-content over-sulfated chondroitin sulfate in the heparin sodium, wherein the sensibility reaches over 0.1 percent. The electrophoresis process and result can be directly detected and observed by using an ultraviolet lamp, and the observation is intuitive and is easy to judge. A zone after the electrophoresis is easy to be dyed, a sample can be eluted very easily to provide convenience for quantitative determination, and the sample can be preserved for a long time after being prepared into a dry film.

Description

The separation and detection method of chondroitin polysulfate in a kind of liquaemin
Technical field
The invention belongs to biological technical field, relate to the pre-service and the electrophoretic detection of chondroitin polysulfate in the liquaemin.
Background technology
China is the maximum country of liquaemin output, the liquaemin products material of international market more than 70% all from China.Along with the increase of demand, this rising all the way in several years of the price of liquaemin.In February, 2008, the U.S. is dead because of 4 examples appear in injecting heparin sodium, and the malpractice of many cases bad reaction becomes world-shaking " liquaemin incident ".Analyze through the relevant expert, above-mentioned accident possibly be because with liquaemin in to contain chondroitin polysulfate relevant.The Chinese government attaches great importance to and pays close attention to impact development, contains the chondroitin polysulfate situation in the part liquaemin to existing in the market, and national Bureau of Drugs Supervision holds ad hoc meeting, requires to strengthen management, and recalls the liquaemin that contains chondroitin polysulfate without exception.Therefore, chondroitin polysulfate in the liquaemin is detected become extremely urgent thing.
At present, nuclear magnetic resonance method, cellulose acetate electrophoresis method are generally adopted in the detection of chondroitin polysulfate in the liquaemin in the industry.The nuclear magnetic resonance method checkout equipment has high input, and chondroitin polysulfate content is less than at 1% o'clock and also is difficult for being detected.And the cellulose acetate electrophoresis detection method; Inferior separating effect is disturbed by other impurity easily, and film is opaque; Be not easy to observe; Usually the content of chondroitin polysulfate was greater than 5% o'clock in the liquaemin, and the electrophoresis detection effect is obvious, and was not easy to distinguish (causing these products to come into the market) as chondroitin polysulfate electrophoresis detection result less than 3% time.The application for a patent for invention that the applicant submits on the same day; Disclose the agarose gel electrophoresis detection method of chondroitin polysulfate in a kind of liquaemin, also can be detected even the chondroitin polysulfate content in the liquaemin is less than 1%, testing result is observed easily; Resolution is high, and detection sensitivity is high.But when the chondroitin polysulfate content in the liquaemin was less than 0.5%, this method still can not effectively detect.
Summary of the invention
The technical matters that the present invention will solve provides the separation and detection method of chondroitin polysulfate in a kind of liquaemin; Even the chondroitin polysulfate content in the liquaemin is less than at 0.5% o'clock, still can effectively detect, testing result is observed easily; Resolution is high, and detection sensitivity is high.
The present invention takes following technical scheme to be achieved:
The separation and detection method of chondroitin polysulfate comprises the following steps: in a kind of liquaemin
(1) separation of chondroitin polysulfate preparation in the liquaemin:
A, dissolution filter: will contain the liquaemin sample that weighs less than 0.5% chondroitin polysulfate, and add in the sodium chloride solution, and be warming up to 55 ℃ ± 5 ℃; Stirring is dissolved liquaemin fully; Transfer PH to 10-12 with aqueous slkali again, filter and collect filtrating, and filtrating is cooled to the freezing point to 5 ℃ of solution; Transfer PH to 1-2 with acid, filter and collect filtrating;
B, rapid precipitation: filtrating is transferred PH to 5-6 with alkali, stirs slowly to add alcohol down, and making the alcoholic weight concentration of solution is 30-38%, leaves standstill; Sound out container bottom with stirring rod, sink to the bottom, promptly can upper strata alcohol be poured out as long as have, the collecting precipitation thing, oven dry promptly gets the melange of chondroitin polysulfate, liquaemin, impurity;
(2) sample is prepared: the step (1) of identical weight is contained the liquaemin sample that weighs less than 0.5% chondroitin polysulfate, the sediment that step (1) makes, and liquaemin standard items, chondroitin polysulfate standard items are dissolved in the water respectively; The WS that makes is added acid respectively transfer PH to 1-3, carried out nitration reaction 0.5-2 hour with nitrite or nitrate then, transfer PH to 6.5-7.5 again, subsequent use;
(3) detection of chondroitin polysulfate:
C, glue plate: mix with electrophoretic buffer with agarose solution, its mixed volume ratio is 1: 0.7-1.5, and heating makes its dissolving, while hot glue fallen on the glass plate of suitable size, leaves standstill, treat that gel forms bubble-free even thin layer;
D, electrophoresis: in electrophoresis tank, add electrophoretic buffer, gel slab is placed on the electrophoresis truss, immerse damping fluid; Get the ready four kinds of sample aqueous solutions of step (1), in gel slab negative pole end difference point sample, demand working power supply, electrophoresis under the condition of the voltage gradient of setting, strength of current;
E, dyeing and decolouring: take off gel slab, with dyeing liquor dyeing, more unnecessary dyeing liquor to the background of water flush away colourless till;
F, result observe: the migration distance that observation sample and standard items showed.
The further improvement project of the present invention is, said electrophoretic buffer is acetic acid-lithium salts damping fluid, and the pH of buffer value is 2.0-4.0.
The present invention further improvement project is that the point sample amount of four kinds of sample solutions is respectively 0.5-1.5 μ l.Voltage gradient during electrophoresis is 25-35V/cm, and strength of current is 1-2mA/cm, and electrophoresis time is 15-20 minute.
Beneficial effect of the present invention:
One, the present invention separates chondroitin polysulfate with the rapid precipitation method with liquaemin, and further separating obtained thing is detected, and has realized that sensitivity reaches more than 0.1% to the detection of the lower chondroitin polysulfate of content in the liquaemin.
Two, the prepared Ago-Gel thickness of the present invention is even, water cut big (accounting for 98%-99%), and approximate free electrophoresis, sample diffusion property is good, and is atomic to sample absorption, so electrophoresis pattern is clear, and resolution is high, good reproducibility.
Three, the transparent no uv absorption of agarose, the agarose offset plate is transparence, is easy to observe, and electrophoresis process and result can directly detect, observe with ultraviolet lamp, observe intuitively, judge easily.
Four, electrophoresis back zone band easy dyeing, sample is wash-out very easily, is convenient to quantitative measurement, but and processes the dry film long preservation.
Five, the present invention is simple to operate, and electrophoretic velocity is fast.
Description of drawings
Fig. 1 is four kinds of sample electrophoresis figure of the present invention.Among the figure 1 is for containing the heparin sodium crude of weight 0.2% chondroitin polysulfate; The sediment (melange of chondroitin polysulfate, liquaemin, impurity) that among the figure 2 obtains behind precipitation separation for the heparin sodium crude that contains weight 0.2% chondroitin polysulfate; Among the figure 3 is the liquaemin standard items; Among the figure 4 is the chondroitin polysulfate standard items.Can know from diagram; Among the figure 2,4 relevant positions manifest the chondroitin polysulfate band; Among the figure 1,3 relevant positions do not manifest this band, explain when the content of chondroitin polysulfate in the liquaemin is lower than 0.5%, simply adopt the agarose gel electrophoresis detection method to detect to come out.The present invention is at first through separating the heparin sodium crude that contains weight 0.2% chondroitin polysulfate, and then the sediment that separation obtains is detected, and content is lower than 0.5% chondroitin polysulfate in the liquaemin thereby realize detecting.
Embodiment
Below only be to explanation of the present invention, but not limitation of the present invention.
(1) separation of chondroitin polysulfate preparation in the liquaemin:
A, dissolution filter: get the liquaemin sample that 10g contains weight 0.2% chondroitin polysulfate, add in the solution of 150mL water and 5g sodium chloride mixing, be warming up to 55 ℃ ± 5 ℃; Stirring is dissolved liquaemin fully; Using weight concentration again is that 20% NaOH aqueous slkali is transferred PH to 10-12, filters and collects filtrating, and filtrating is cooled to zero degree; Transfer PH to 1-2 with hydrochloric acid, filter and collect filtrating;
B, rapid precipitation: filtrating is transferred PH to 5.5 with NaOH, stirs slowly to add 95% alcohol down, and making the alcoholic weight concentration of solution is 35%, leaves standstill; Sound out container bottom with stirring rod, sink to the bottom, promptly can upper strata alcohol be poured out as long as have, collecting precipitation, oven dry promptly gets the melange of required chondroitin polysulfate, liquaemin, impurity;
(2) get the melange of the used liquaemin sample that contains weight 0.2% chondroitin polysulfate of the step (1) of identical weight, chondroitin polysulfate that step (1) makes, liquaemin, impurity; And liquaemin standard items, each 200mg of chondroitin polysulfate standard items, be dissolved in respectively in the 10ml water; The WS that makes is added hydrochloric acid respectively transfer PH to 2, carried out nitration reaction 1.5 hours with nitrite then, transfer PH to 7 again, subsequent use;
(3) detection of chondroitin polysulfate:
C, glue plate: first preparation acetic acid-lithium salts damping fluid, get glacial acetic acid 50mL, after adding water 800mL and mixing, regulate pH value to 3.0 with lithium hydroxide, it is molten surely to 1000mL to add water again; Get in the water that agarose 0.2g adds 10mL, put that heating is dissolved it fully in the water-bath, in agarose solution, add warm acetic acid-lithium salts damping fluid 10mL (its temperature and agarose solution temperature are approaching); After mixing; While hot glue is coated that (on the glass plate of 4cm * 9cm), the about 3mm of thickness leaves standstill; Treat that gel forms bubble-free even thin layer, get final product
D, point sample and electrophoresis: in electrophoresis tank, add the acetic acid-lithium salts damping fluid of this step c preparation, gel slab is placed on the electrophoresis truss, immerse damping fluid through the filter paper bridge; In the sample solution 1 μ l point sample that the gel slab negative pole end uses step (2) to obtain respectively, the demand working power supply, under the condition of the about 30V/cm of voltage gradient, strength of current 1-2mA/cm, about 20 minutes of electrophoresis, powered-down;
E, dyeing and decolour: take off gel slab, use the toluidine blue solution-dyed, more unnecessary dyeing liquor to the background of water flush away colourless till; The preparation of toluidine blue solution: general available toluidine blue 0.1g is dissolved in the 100mL water and makes;
The migration distance that f, observation caliber sample and control sample are shown, standard items are 0.8-0.9 with the ratio of the migration distance that control sample is shown.

Claims (5)

1. the separation and detection method of chondroitin polysulfate in the liquaemin is characterized in that comprising the following steps:
(1) separation of chondroitin polysulfate preparation in the liquaemin:
A, dissolution filter: will contain the liquaemin sample that weighs less than 0.5% chondroitin polysulfate, and add in the sodium chloride solution, and be warming up to 55 ℃ ± 5 ℃; Stirring is dissolved liquaemin fully; Transfer PH to 10-12 with aqueous slkali again, filter and collect filtrating, and filtrating is cooled to the freezing point to 5 ℃ of solution; Transfer PH to 1-2 with acid, filter and collect filtrating;
B, rapid precipitation: filtrating is transferred PH to 5-6 with alkali, stirs slowly to add alcohol down, and making the alcoholic weight concentration of solution is 30-38%, leaves standstill; Sound out container bottom with stirring rod, sink to the bottom, promptly can upper strata alcohol be poured out as long as have, the collecting precipitation thing, and with its oven dry;
(2) sample is prepared: the step (1) of identical weight is contained the liquaemin sample that weighs less than 0.5% chondroitin polysulfate, the sediment that step (1) makes, and liquaemin standard items, chondroitin polysulfate standard items are dissolved in the water respectively; The WS that makes is added acid respectively transfer PH to 1-3, carried out nitration reaction 0.5-2 hour with nitrite or nitrate then, transfer PH to 6.5-7.5 with alkali again, subsequent use;
(3) detection of chondroitin polysulfate:
C, glue plate: mix with electrophoretic buffer with agarose solution, its mixed volume ratio is 1: 0.7-1.5, and heating makes its dissolving, while hot glue fallen on the glass plate of suitable size, leaves standstill, treat that gel forms bubble-free even thin layer;
D, electrophoresis: in electrophoresis tank, add electrophoretic buffer, gel slab is placed on the electrophoresis truss, immerse damping fluid; Get the ready four kinds of sample aqueous solutions of step (2), in gel slab negative pole end difference point sample, demand working power supply, electrophoresis under the condition of the voltage gradient of setting, strength of current;
E, dyeing and decolouring: take off gel slab, with dyeing liquor dyeing, more unnecessary dyeing liquor to the background of water flush away colourless till;
F, result observe: the migration distance that observation sample and standard items showed.
2. the separation and detection method of chondroitin polysulfate in a kind of liquaemin as claimed in claim 1 is characterized in that: the said electrophoretic buffer of step (3) is acetic acid-lithium salts damping fluid, and the p H value of damping fluid is 2.0-4.0.
3. the separation and detection method of chondroitin polysulfate in a kind of liquaemin as claimed in claim 1 is characterized in that: the described point sample of step (3), the point sample amount of four kinds of sample solutions is respectively 0.5-1.5 μ 1.
4. the separation and detection method of chondroitin polysulfate in a kind of liquaemin as claimed in claim 1 is characterized in that: the described voltage gradient of step (3) is 25-35V/cm, and strength of current is 1-2mA/cm, and electrophoresis time is 15-20 minute.
5. the separation and detection method of chondroitin polysulfate in a kind of liquaemin as claimed in claim 1 is characterized in that: the described dyeing liquor of step (3) is a toluidine blue solution.
CN201010112056A 2010-02-12 2010-02-12 Method for separating and detecting over-sulfated chondroitin sulfate in heparin sodium Active CN101825607B (en)

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CN102830150A (en) * 2012-07-09 2012-12-19 东营天东制药有限公司 High sensitivity detection method of oversulfated chondroitin sulfate in liquaemin

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Publication number Priority date Publication date Assignee Title
CN101575385A (en) * 2008-05-09 2009-11-11 青岛九龙生物医药有限公司 Method for separating chondroitin polysulfate from heparin sodium by extraction method

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US8865412B2 (en) * 2008-05-28 2014-10-21 Baxter International Inc. Methods and assays for oversulfated glycosaminoglycans

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CN101575385A (en) * 2008-05-09 2009-11-11 青岛九龙生物医药有限公司 Method for separating chondroitin polysulfate from heparin sodium by extraction method

Non-Patent Citations (3)

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Shawn Bairstow等.Identification of a simple and sensitive microplate method for the detection of oversulfated chondroitin sulfate in heparin products.《Analytical Biochemistry》.2009,(第388期),第317-321页. *
Todd Wielgos等.Determination of impurities in heparin by capillary electrophoresis using high molarity phosphate buffers.《Journal of Pharmaceutical and Biomedical Analysis》.2009,(第49期),第319-326页. *
王皓等.污染肝素中多硫酸化硫酸软骨素的分布醋酸纤维素薄膜电泳分析.《分析化学》.2009,第37卷(第8期),第1147-1151页. *

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