CN102323344A - Method for quantitatively detecting hyaluronic acid fragment structure change - Google Patents
Method for quantitatively detecting hyaluronic acid fragment structure change Download PDFInfo
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- CN102323344A CN102323344A CN201110202860A CN201110202860A CN102323344A CN 102323344 A CN102323344 A CN 102323344A CN 201110202860 A CN201110202860 A CN 201110202860A CN 201110202860 A CN201110202860 A CN 201110202860A CN 102323344 A CN102323344 A CN 102323344A
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 52
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 claims abstract description 36
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 10
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 11
- 102000001974 Hyaluronidases Human genes 0.000 claims description 11
- 229920002674 hyaluronan Polymers 0.000 claims description 11
- 229960003160 hyaluronic acid Drugs 0.000 claims description 11
- 229960002773 hyaluronidase Drugs 0.000 claims description 11
- 238000004811 liquid chromatography Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 238000001514 detection method Methods 0.000 claims description 7
- 239000013558 reference substance Substances 0.000 claims description 6
- 238000010812 external standard method Methods 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 239000007974 sodium acetate buffer Substances 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 10
- 230000015556 catabolic process Effects 0.000 abstract description 9
- 229920002385 Sodium hyaluronate Polymers 0.000 abstract description 8
- 229940010747 sodium hyaluronate Drugs 0.000 abstract description 8
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 abstract description 8
- 230000006378 damage Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 229920000642 polymer Polymers 0.000 abstract description 2
- 230000000593 degrading effect Effects 0.000 abstract 1
- 238000005259 measurement Methods 0.000 abstract 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 5
- 239000002253 acid Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 150000002016 disaccharides Chemical group 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000010525 oxidative degradation reaction Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 description 2
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- 238000002144 chemical decomposition reaction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- IAJILQKETJEXLJ-KLVWXMOXSA-N (2s,3r,4r,5r)-2,3,4,5-tetrahydroxy-6-oxohexanoic acid Chemical group O=C[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-KLVWXMOXSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229940097043 glucuronic acid Drugs 0.000 description 1
- -1 hyaluronic acid small molecule Chemical class 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a method for quantitatively detecting the structure change of a hyaluronic acid fragment produced by degrading a polymer sodium hyaluronate. A high performance liquid chromatography and a carbazole method are used for simultaneously measuring the content of the hyaluronic acid fragment, and a difference between the contents obtained by the two methods is used for quantifying the structure change of the hyaluronic acid fragment prepared by different methods. The method provided by the invention is suitable for the hyaluronic acid fragment prepared by different methods, can evaluate the damage degree to the hyaluronic acid structure caused by the degradation method and preliminarily judge the production method of the hyaluronic acid fragment according to the measurement result, and is an important measure for evaluating the quality of the hyaluronic acid fragment.
Description
Technical field
The present invention relates to hyaluronic assay method, the method for particularly a kind of detection by quantitative hyaluronic acid fragments structural change.
Background technology
Multiply-connected the connecing of disaccharide unit weight that hyaluronic acid is made up of glucuronic acid and acetylglucosamine forms the glutinous polysaccharide of a kind of straight chain polymer.Hyaluronic acid fragments is generally big numerator sodium hyaluronate degraded and forms; Its biodegrading process can be divided into three major types: mechanical degradation (for example heating, microwave, ultrasound wave, ray etc.), chemical degradation (for example acid, alkali, oxidation etc.) and biodegradation (for example hyaluronidase).The hyaluronidase degraded hyaluronic acid that uses animal tissue to extract has only the glycosidic bond fracture of restriction enzyme site, and its disaccharide structural unit does not change.But in mechanical degradation and chemical degradation process, hyaluronic acid is except the fracture of glycosidic bond, and the structure of glucuronic acid residue or acetylglucosamine residue also can change.For example hyaluronic acid produces two keys in heating process, in the alkaline degradation process, deacetylation reaction etc. can take place.Yet the hyaluronic acid fragments that hyaluronic acid degradation produces is the potpourri of hyaluronic acid small molecule segment different in size, uses general technology to be difficult to detect the variation and the intensity of variation of its structure.Nuclear magnetic resonance technique (NMR) can be used to measure the structure of matter, but this technology is very high to the sample purity requirement, and instrument is expensive, and testing cost is very high, can't popularize use.
Summary of the invention
The method that the purpose of this invention is to provide the structural change of a kind of detection by quantitative hyaluronic acid fragments can be carried out quantitative test to the structural change of the hyaluronic acid fragments of different biodegrading process preparation.
The present invention realizes through following measure:
The present invention relates to the method for a kind of detection by quantitative hyaluronic acid fragments structural change, it is characterized in that comprising the steps:
1) hyaluronic acid fragments is used the hyaluronidase enzymolysis, adopt the content of high effective liquid chromatography for measuring hyaluronic acid fragments then;
2) adopt the carbazole method to measure the content of hyaluronic acid fragments hyaluronic acid fragments;
3) with step 2) measured value deduct the measured value of step 1), be the amount of hyaluronic acid fragments structural change.
The detection by quantitative hyaluronic acid fragments structural change of the invention described above method, preferred, described step 1) may further comprise the steps:
A) sample pretreatment
Precision takes by weighing the hyaluronic acid reference substance and the hyaluronic acid fragments sample places the 50ml volumetric flask in right amount respectively, with sodium-acetate buffer dissolving and constant volume, adds hyaluronidase, 37 ℃ of water-bath 2h;
B) chromatographic condition
In high-performance liquid chromatogram determination of the present invention, adopt the glycan analysis post; Moving phase is the phosphate buffer of 0.5-1.0 mol/L; Flow velocity is 0.3-1.0 ml/min; Column temperature is 30 ± 5 ℃; Detect wavelength 235 ± 5nm; Sample size is 20 μ l;
C) result calculates
Adopt high performance liquid chromatography respectively reference substance enzymolysis liquid and sample enzymolysis liquid to be carried out chromatographic resolution, press external standard method with the calculated by peak area sample size.
The hyaluronidase that the present invention uses is also referred to as hyaluronidase, during its degraded hyaluronic acid, has only the glycosidic bond fracture of restriction enzyme site, and its disaccharide structural unit does not change.
The present invention uses hyaluronidase that sample is carried out pre-service; And combine the liquid chromatography isolation technics to obtain sample size; The method specificity is high; And the carbazole ratio juris is as long as exist in the sample hexuronic acid structure can with carbazole reagent generation chromogenic reaction, thereby calculate and to obtain sample size that specificity is relatively poor.These two kinds of methods result when measuring the hyaluronic acid fragments content of big numerator sodium hyaluronate and Production by Enzymes is consistent; But when the content of the hyaluronic acid fragments of measuring the preparation of physics or chemical method, the measured value of high performance liquid chromatography is starkly lower than the measured value of carbazole method.This is because of the hyaluronic acid fragments for physics or chemical method preparation, and structural change causes sample can not be detected in former retention time by the enzymolysis product of complete enzymolysis or recurring structure variation; And the carbazole method is because poor specificity is measured the result by structures of samples variation interference.Therefore the difference of two kinds of methods can be used for assessing the degree that sample structure changes.
High effective liquid chromatography for measuring hyaluronic acid fragments content specificity is high among the present invention; And carbazole method poor specificity; Utilize both mensuration difference can assess the degree of the hyaluronic acid fragments structural change of distinct methods preparation, the production and the application of hyaluronic acid fragments had certain directive function.
Embodiment
Embodiment 1:
1.1 sample: the hyaluronic acid fragments that the hyaluronidase degraded that animal tissue extracts produces, mean molecular weight is 5800.
1.2 method:
A) sample pretreatment
Precision takes by weighing the hyaluronic acid reference substance and the hyaluronic acid fragments sample places the 50ml volumetric flask in right amount respectively, with sodium-acetate buffer dissolving and constant volume, adds hyaluronidase, 37 ℃ of water-bath 2h;
B) chromatographic condition
The chromatographic condition of high performance liquid chromatography is that (4.6mm * 250mm), moving phase is the sodium phosphate buffer of 0.5 mol/L to nh 2 column; Flow velocity is 0.5ml/min; Column temperature is 30 ℃; Detect wavelength 232nm; Sample size is 20 μ L.The carbazole method is with reference to the content assaying method of Sodium Hyaluronate in the European Pharmacopoeia.
C) result calculates
Adopt high performance liquid chromatography respectively reference substance enzymolysis liquid and sample enzymolysis liquid to be carried out chromatographic resolution, press external standard method with the calculated by peak area sample size.
(above disposal route, embodiment 2-5 and last basic identical)
1.3 result: it is 92.16% that the carbazole method is measured the result; The high effective liquid chromatography for measuring result is 90.85%, and both differences are the no significant difference as a result that 1.31%, two kind of method is measured; The structural change that hyaluronic acid fragments is described is little, and promptly the method is little to hyaluronic acid structural damage effect.
Embodiment 2:
2.1 sample: the hyaluronic acid fragments that the salt acid degradation produces, mean molecular weight is 11600.
2.2 method: the chromatographic condition of high performance liquid chromatography is that (4.6mm * 250mm), moving phase is the sodium phosphate buffer of 0.5 mol/L to nh 2 column; Flow velocity is 0.5ml/min; Column temperature is 30 ℃; Detect wavelength 232nm; Sample size is 20 μ l.The carbazole method is with reference to the content assaying method of Sodium Hyaluronate in the European Pharmacopoeia.
2.3 result: it is 95.99% that the carbazole method is measured the result, and the high effective liquid chromatography for measuring result is 77.64%, and both differences are 18.35%, explain that variation has taken place the structure of hyaluronic acid fragments, i.e. salt acid degradation has destruction to the hyaluronic acid structure.
Embodiment 3:
3.1 sample: the hyaluronic acid fragments that the salt acid degradation produces, mean molecular weight is 7200.
3.2 method: the chromatographic condition of high performance liquid chromatography is that (4.6mm * 250mm), moving phase is the sodium phosphate buffer of 0.5 mol/L to nh 2 column; Flow velocity is 0.5ml/min; Column temperature is 30 ℃; Detect wavelength 232nm; Sample size is 20 μ l.The carbazole method is with reference to the content assaying method of Sodium Hyaluronate in the European Pharmacopoeia.
3.3 result: it is 95.48% that the carbazole method is measured the result, and the high effective liquid chromatography for measuring result is 62.38%, and both differences are 33.10%.In conjunction with the experimental data of embodiment 2, explain that under identical degradation condition, the mean molecular weight of hyaluronic acid fragments is low more, its structural failure degree is serious more.
Embodiment 4:
4.1 sample: the hyaluronic acid fragments that oxidative degradation produces, mean molecular weight is 9600.
4.2 method: the chromatographic condition of high performance liquid chromatography is that (4.6mm * 250mm), moving phase is the sodium phosphate buffer of 0.5 mol/L to nh 2 column; Flow velocity is 0.5ml/min; Column temperature is 30 ℃; Detect wavelength 232nm; Sample size is 20 μ l.The carbazole method is with reference to the content assaying method of Sodium Hyaluronate in the European Pharmacopoeia.
4.3 result: it is 106.4% that the carbazole method is measured the result, and the high effective liquid chromatography for measuring result is 67.96%, and both differences are 38.40%.Explain that great changes have also taken place the hyaluronic acid fragments structure that oxidative degradation produces.
Embodiment 5:
5.1 sample: the hyaluronic acid fragments that oxidative degradation produces, mean molecular weight is 2300.
5.2 method: the chromatographic condition of high performance liquid chromatography is that (4.6mm * 250mm), moving phase is the sodium phosphate buffer of 0.5 mol/L to nh 2 column; Flow velocity is 0.5ml/min; Column temperature is 30 ℃; Detect wavelength 232nm; Sample size is 20 μ l.The carbazole method is with reference to the content assaying method of Sodium Hyaluronate in the European Pharmacopoeia.
5.3 result: it is 103.2% that the carbazole method is measured the result, and the high effective liquid chromatography for measuring result is 42.14%, and both differences are 61.06%.In conjunction with the experimental data of embodiment 4, explain that under identical degradation condition, the mean molecular weight of hyaluronic acid fragments is low more, its structural failure degree is serious more.
Claims (2)
1. the method for detection by quantitative hyaluronic acid fragments structural change is characterized in that comprising the steps:
1) hyaluronic acid fragments is used the hyaluronidase enzymolysis, adopt the content of high effective liquid chromatography for measuring hyaluronic acid fragments then;
2) adopt the carbazole method to measure the content of hyaluronic acid fragments hyaluronic acid fragments;
3) with step 2) measured value deduct the measured value of step 1), be the amount of hyaluronic acid fragments structural change.
2. detection by quantitative hyaluronic acid fragments according to claim 1 structural change method, it is characterized in that described step 1) may further comprise the steps:
A) sample pretreatment
Precision takes by weighing the hyaluronic acid reference substance and the hyaluronic acid fragments sample places the 50ml volumetric flask in right amount respectively, with sodium-acetate buffer dissolving and constant volume, adds hyaluronidase, 37 ℃ of water-bath 2h;
B) chromatographic condition
In high-performance liquid chromatogram determination of the present invention, adopt the glycan analysis post; Moving phase is the phosphate buffer of 0.5-1.0 mol/L; Flow velocity is 0.3-1.0 ml/min; Column temperature is 30 ± 5 ℃; Detect wavelength 235 ± 5nm; Sample size is 20 μ L;
C) result calculates
Adopt high performance liquid chromatography respectively reference substance enzymolysis liquid and sample enzymolysis liquid to be carried out chromatographic resolution, press external standard method with the calculated by peak area sample size.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102297846A (en) * | 2011-07-28 | 2011-12-28 | 山东福瑞达生物医药有限公司 | Method for rapidly determining content of hyaluronic acid in fermentation liquor |
| CN102495154A (en) * | 2011-12-01 | 2012-06-13 | 北京爱美客生物科技有限公司 | Method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography |
| WO2013123791A1 (en) | 2012-02-21 | 2013-08-29 | 华熙福瑞达生物医药有限公司 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| CN105699578A (en) * | 2016-04-22 | 2016-06-22 | 广东东阳光药业有限公司 | Analysis method of glycoform fingerprint atlas formed by sodium hyaluronate |
| CN106053369A (en) * | 2016-07-13 | 2016-10-26 | 浙江景嘉医疗科技有限公司 | Method for detecting content of free sodium hyaluronate in medical cross-linking sodium hyaluronate gel |
| CN107561179A (en) * | 2017-08-18 | 2018-01-09 | 上海景峰制药有限公司 | A kind of assay method of the degree of cross linking of cross-linked-hyaluronic acid or its salt |
| CN108562574A (en) * | 2018-01-19 | 2018-09-21 | 常州大学 | Hyaluronic acid high throughput micro-scale rapid detection method |
| CN109298112A (en) * | 2018-12-11 | 2019-02-01 | 华熙福瑞达生物医药有限公司 | A method of measurement hyaluronic acid contents |
| CN109298113A (en) * | 2018-12-11 | 2019-02-01 | 华熙福瑞达生物医药有限公司 | A method of measurement includes the hyaluronic acid contents in the solution of citric acid |
| CN114236014A (en) * | 2021-12-27 | 2022-03-25 | 珠海溪谷医疗科技有限公司 | Method for detecting content of sodium hyaluronate in contact lens care solution |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102297846A (en) * | 2011-07-28 | 2011-12-28 | 山东福瑞达生物医药有限公司 | Method for rapidly determining content of hyaluronic acid in fermentation liquor |
| CN102495154A (en) * | 2011-12-01 | 2012-06-13 | 北京爱美客生物科技有限公司 | Method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography |
| CN102495154B (en) * | 2011-12-01 | 2014-03-26 | 北京爱美客生物科技有限公司 | Method for detecting in-vitro enzymolysis of cross-linked hyaluronic acid by utilizing water-phase gel permeation chromatography |
| WO2013123791A1 (en) | 2012-02-21 | 2013-08-29 | 华熙福瑞达生物医药有限公司 | Bacillus, hyaluronic acid enzyme, and uses thereof |
| CN105699578B (en) * | 2016-04-22 | 2017-07-07 | 广东东阳光药业有限公司 | A kind of sodium hyaluronate constitutes sugar-type fingerprint analysis method |
| CN105699578A (en) * | 2016-04-22 | 2016-06-22 | 广东东阳光药业有限公司 | Analysis method of glycoform fingerprint atlas formed by sodium hyaluronate |
| CN106053369A (en) * | 2016-07-13 | 2016-10-26 | 浙江景嘉医疗科技有限公司 | Method for detecting content of free sodium hyaluronate in medical cross-linking sodium hyaluronate gel |
| CN107561179A (en) * | 2017-08-18 | 2018-01-09 | 上海景峰制药有限公司 | A kind of assay method of the degree of cross linking of cross-linked-hyaluronic acid or its salt |
| CN107561179B (en) * | 2017-08-18 | 2020-12-04 | 上海景峰制药有限公司 | Method for measuring crosslinking degree of crosslinked hyaluronic acid or salt thereof |
| CN108562574A (en) * | 2018-01-19 | 2018-09-21 | 常州大学 | Hyaluronic acid high throughput micro-scale rapid detection method |
| CN109298112A (en) * | 2018-12-11 | 2019-02-01 | 华熙福瑞达生物医药有限公司 | A method of measurement hyaluronic acid contents |
| CN109298113A (en) * | 2018-12-11 | 2019-02-01 | 华熙福瑞达生物医药有限公司 | A method of measurement includes the hyaluronic acid contents in the solution of citric acid |
| CN109298113B (en) * | 2018-12-11 | 2021-06-18 | 华熙生物科技股份有限公司 | Method for determining content of hyaluronic acid in solution containing citric acid |
| CN109298112B (en) * | 2018-12-11 | 2021-06-18 | 华熙生物科技股份有限公司 | Method for measuring content of hyaluronic acid |
| CN114236014A (en) * | 2021-12-27 | 2022-03-25 | 珠海溪谷医疗科技有限公司 | Method for detecting content of sodium hyaluronate in contact lens care solution |
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