CN101825608A - Agarose gel electrophoresis detection method for oversulfated chondroitin sulfate in heparin sodium - Google Patents
Agarose gel electrophoresis detection method for oversulfated chondroitin sulfate in heparin sodium Download PDFInfo
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- CN101825608A CN101825608A CN201010112072A CN201010112072A CN101825608A CN 101825608 A CN101825608 A CN 101825608A CN 201010112072 A CN201010112072 A CN 201010112072A CN 201010112072 A CN201010112072 A CN 201010112072A CN 101825608 A CN101825608 A CN 101825608A
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- liquaemin
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Abstract
The invention discloses an agarose gel electrophoresis detection method for oversulfated chondroitin sulfate in heparin sodium. The heparin sodium is degraded through the nitration reaction according to the characteristic that the molecular structures of the oversulfated chondroitin sulfate and the heparin sodium are different, and the oversulfated chondroitin sulfate is not easy to degrade and is detected in electrophoresis detection. By adopting agarose gel electrophoresis, the invention realizes the detection on the oversulfated chondroitin sulfate with lower content in the heparin sodium and has the sensitivity of more than 0.5%, clear electrophoretogram, high resolution, intuitionistic observation, easy determination and good repeatability. The zone subjected to electrophoresis is easy to dye, and samples are easy to elute, are convenient for quantitative determination, and can be stored for a long time when made into dry films. The invention has simple operation and high electrophoresis speed.
Description
Technical field
The invention belongs to biological technical field, relate to the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin.
Background technology
China is the country of liquaemin output maximum, the liquaemin products material of international market more than 70% all from China.Along with the increase of demand, the price rising all the way of this several years liquaemins.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 may 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 at 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, the detection of chondroitin polysulfate in the liquaemin is generally adopted nuclear magnetic resonance method, the cellulose acetate electrophoresis method in the industry.Nuclear magnetic resonance method checkout equipment input is bigger, 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, disturbed by other impurity, 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 as chondroitin polysulfate electrophoresis detection result less than 3% time.
Summary of the invention
The technical problem to be solved in the present invention provides the agarose gel electrophoresis detection method of chondroitin polysulfate in a kind of liquaemin, overcome the defective that prior art exists, even being less than 1%, the chondroitin polysulfate content in the liquaemin also can be detected, testing result of the present invention is observed easily, the resolution height, the detection sensitivity height.
The present invention takes following technical scheme to be achieved:
The agarose gel electrophoresis detection method of chondroitin polysulfate comprises the following steps: in the liquaemin
(1) sample dissolution: get identical weight the heparin sodium crude that does not contain chondroitin polysulfate, contain the heparin sodium crude, liquaemin standard items, the chondroitin polysulfate standard items that weigh less than 1% chondroitin polysulfate, be dissolved in the water respectively, contain sample 5-20mg in every ml soln;
(2) nitrated: to four kinds of aqueous solution that step (1) obtains, add acid respectively and 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, standby;
(3) glue plate: mixes with electrophoretic buffer with agarose solution, its mixed volume ratio is 1: 0.7-1.5, and heating dissolves it, while hot with glue on the glass plate of suitable size, leave standstill, treat that gel forms bubble-free even thin layer;
(4) 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;
(5) dyeing and decolouring: take off gel slab, with dyeing liquor dyeing, the more unnecessary dyeing liquor of water flush away to background colourless till;
(6) result observes: the migration distance that observation sample and standard items are shown.
The further improvement project of the present invention is, step (3), the described electrophoretic buffer of step (4) are acetic acid-lithium salts damping fluid, and the pH value of damping fluid is 2.0-4.0.
The present invention further improvement project is, the described point sample of step (4), and the point sample amount of four kinds of sample solutions is respectively 0.5-1.5 μ l.Described voltage gradient is 25-35V/cm, and strength of current is 1-2mA/cm, and electrophoresis time is 15-20 minute.
Chondroitin polysulfate is a kind of material that contains glucuronic acid derivant and acetylamino galactosamine derivant; its molecular structure is made up of 2-O-sulfate-glucuronic acid-(1 → 3)-2-N-acetyl group-2-deoxidation-4 or 6-O-sulfate galactose, and liquaemin does not have such special molecular structure.The amino mucopolysaccharide that nitrite or nitrous acid category material can only be degraded and be connected with sulfate, and the acetylamino galactosamine that can not degrade and be connected with acetyl group.Therefore nitration reaction makes the liquaemin degraded, and chondroitin polysulfate is not degraded or be not easy degraded, is detected in electrophoresis detection.By nitrite or nitrous acid and so on material the liquaemin sample that contains chondroitin polysulfate is handled; Again by the chondroitin polysulfate in the agarose gel electrophoresis detection liquaemin.
Beneficial effect of the present invention:
One, the present invention has realized that sensitivity reaches more than 0.5% 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 is good, and is atomic to sample absorption, so electrophoresis pattern is clear, resolution height, 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 makes 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 among the present invention.Among the figure 1 be not for containing the heparin sodium crude of chondroitin polysulfate; Among the figure 2 is the heparin sodium crude that contains the weight 0.6% of chondroitin polysulfate; Among the figure 3 is the liquaemin standard items, and 4 among the figure is the chondroitin polysulfate standard items.From diagram as can be known, 2 among the figure, 4 relevant positions manifest the chondroitin polysulfate band, and 1 among the figure, 3 relevant positions do not manifest this band.Illustrating that the present invention can detect to contain in the liquaemin is lower than 1% chondroitin polysulfate (being higher than this number percent more can detect).
Specific embodiment
Below only be to explanation of the present invention, but not limitation of the present invention.
(1) sample dissolution: get the heparin sodium crude that does not contain chondroitin polysulfate, heparin sodium crude (0.6% chondroitin polysulfate that adds weight in the heparin sodium crude that does not contain chondroitin polysulfate obtains), liquaemin standard items, each 200mg of chondroitin polysulfate standard items that contains weight 0.6% chondroitin polysulfate, be dissolved in respectively in the 10ml water.
(2) nitrated: to four kinds of sample solutions that step (1) obtains, add hydrochloric acid respectively and transfer PH to 2, carried out nitration reaction 1.5 hours with sodium nitrite then, transfer PH to 7 with NaOH again, standby;
(3) 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, add water again and decide molten to 1000mL; Get in the water of agarose 0.2g adding 10mL, putting to heat in the water-bath dissolves it fully, 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;
(4) point sample and electrophoresis: in electrophoresis tank, add the acetic acid-lithium salts damping fluid of step (3) 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 (1) 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;
(5) dyeing and decolouring: take off gel slab, use the toluidine blue solution-dyed, the more unnecessary dyeing liquor of water flush away to background colourless till; The preparation of toluidine blue solution: general available toluidine blue 0.1g is dissolved in the 100mL water and makes;
(6) the shown migration distance of observation caliber sample and control sample, the ratio of the migration distance that standard items and control sample are shown is 0.8-0.9.
Claims (5)
1. the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin is characterized in that comprising the following steps:
(1) sample dissolution: get identical weight the heparin sodium crude that does not contain chondroitin polysulfate, contain the heparin sodium crude, liquaemin standard items, the chondroitin polysulfate standard items that weigh less than 1% chondroitin polysulfate, be dissolved in the water respectively;
(2) nitrated: to four kinds of aqueous solution that step (1) obtains, add acid respectively and 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, standby;
(3) glue plate: mixes with electrophoretic buffer with agarose solution, its mixed volume ratio is 1: 0.7-1.5, and heating dissolves it, while hot with glue on the glass plate of suitable size, leave standstill, treat that gel forms bubble-free even thin layer;
(4) 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;
(5) dyeing and decolouring: take off gel slab, with dyeing liquor dyeing, the more unnecessary dyeing liquor of water flush away to background colourless till;
(6) result observes: the migration distance that observation sample and standard items are shown.
2. the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin as claimed in claim 1 is characterized in that: step (3), the described electrophoretic buffer of step (4) are acetic acid-lithium salts damping fluid, and the pH value of damping fluid is 2.0-4.0.
3. the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin as claimed in claim 1 is characterized in that: the described point sample of step (4), the point sample amount of four kinds of sample solutions is respectively 0.5-1.5 μ 1.
4. the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin as claimed in claim 1 is characterized in that: the described voltage gradient of step (4) is 25-35V/cm, and strength of current is 1-2mA/cm, and electrophoresis time is 15-20 minute.
5. the agarose gel electrophoresis detection method of chondroitin polysulfate in the liquaemin as claimed in claim 1 is characterized in that: the described dyeing liquor of step (5) is a toluidine blue solution.
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| CN201010112072A CN101825608A (en) | 2010-02-12 | 2010-02-12 | Agarose gel electrophoresis detection method for oversulfated chondroitin sulfate in heparin sodium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
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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 |
| US20090298705A1 (en) * | 2008-05-28 | 2009-12-03 | Baxter International Inc. | Methods and assays for oversulfated glycosaminoglycans |
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2010
- 2010-02-12 CN CN201010112072A patent/CN101825608A/en active Pending
Patent Citations (2)
| 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 |
| US20090298705A1 (en) * | 2008-05-28 | 2009-12-03 | Baxter International Inc. | Methods and assays for oversulfated glycosaminoglycans |
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| 《Journal of Chromatography B》 20060310 Nicola Volpi等 Electrophoretic approaches to the analysis of complex polysaccharides 第1-13页 1-5 , 第834期 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698426A (en) * | 2013-12-12 | 2014-04-02 | 中国海洋大学 | Method for degrading chondroitin sulfate and hyaluronic acid to obtain chondroitin sulfate disaccharide and hyaluronic acid disaccharide and detecting chondroitin sulfate disaccharide and hyaluronic acid disaccharide |
| CN103698426B (en) * | 2013-12-12 | 2015-08-05 | 中国海洋大学 | A kind of method obtaining and detect chondroitin sulfate and hyaluronic acid disaccharides of degrading |
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Application publication date: 20100908 |