CN108333273A - A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution - Google Patents
A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution Download PDFInfo
- Publication number
- CN108333273A CN108333273A CN201810258732.9A CN201810258732A CN108333273A CN 108333273 A CN108333273 A CN 108333273A CN 201810258732 A CN201810258732 A CN 201810258732A CN 108333273 A CN108333273 A CN 108333273A
- Authority
- CN
- China
- Prior art keywords
- sodium hyaluronate
- molecular weight
- solution
- weight distribution
- detection method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a kind of detection methods of sodium hyaluronate molecular weight and molecualr weight distribution, include the following steps：(1) sodium hyaluronate test sample is weighed, the solution of a concentration of 1g/L of sodium hyaluronate is diluted to mobile phase, as test solution；(2) kayexalate control series product PSS 140K, PSS 280K, PSS 1M and PSS 2M are taken, the solution of a concentration of 1g/L is diluted to mobile phase, as a contrast product solution；Mobile phase is the ammonium acetate aqueous solution of 100mol/L；(3) liquid chromatographic system is used, 100 μ L sample introductions of reference substance solution is taken, is detected with electron spray detector, record chromatogram I；100 μ L sample introductions of the test solution are taken again, are detected with method, record chromatogram II, with GPC Software on Drawing calibration curves, result of calculation obtains molecular weight and molecualr weight distribution coefficient；There is higher detection stability and repeatability compared to Composition distribution.
Description
Technical field
The present invention relates to chemical analysis technology fields, and in particular to a kind of inspection of sodium hyaluronate molecular weight and molecualr weight distribution
Survey method.
Background technology
Sodium hyaluronate (Sodiumhyaluronate, SH) is by (1y4)-O-B-D- glucuronic acids-(1y3) -2- acetyl ammonia
The sodium salt for the glycosaminoglycan that -2 deoxidation-B-D- glucose disaccharide unit weights of base connect and compose again is acid mucopolysaccharide.SH can be used as
There is higher clinical value, SH to be additionally operable to soft tissue repair and as drug for the protective agent of ophthalmology and intraarticular injection agent
Carrier etc. especially achieves greater advance in preventing and reducing surgical postoperative adhesion.
Molecular mass size is closely related corresponding thereto for above-mentioned each function of SH.The clinical efficacy of SH and its molecular mass are tight
Close correlation, different purposes have different molecular weight ranges, and the weight for eye drops, orthopaedics, ophthalmologic operation and pharmaceutic adjuvant is equal
Molecular weight and molecular weight distribution have different regulations respectively.So molecular weight control just becomes the important indicator of SH Quality Controls, and
Difficult point and emphasis.
Method for sodium hyaluronate molecular weight determination mainly has intrinsic viscosity method, size exclusion chromatography (SEC methods) to swash
Light scattering-gel permeation chromatography combination method (SEC-MALLS methods).SEC methods are the molecular mass controls that domestic Extra Pharmacopoeia Martindale generally uses
Method processed is currently limited to use Composition distribution (RID), is mobile phase, Composition distribution with 0.2mol/L sodium chloride solutions
(RID) affected by environment larger, data acquisition is harsher, and there are result stability difference and poor repeatability to lack for detection molecules amount
It falls into.It is more expensive using multi-angle laser light scattering instrument, it is difficult to universal to use.
CN105548420A discloses a kind of MOPS (3- (N- morpholines) propane sulfonic acid, 3- (N-Morpholino) propane-
Sulfonic acid) detection method, this method is detected with high performance liquid chromatography, using liner sum up method pair
MOPS is quantified, and chromatographic column is Acclaim Trinity P1, although this high performance liquid chromatography also uses electron spray to detect
Device, but it is a kind of method of the micro MOPS of detection, it is therefore an objective to overcoming the prior art that cannot detect, micro MOPS is remaining to be lacked
It falls into, is capable of detecting when MOPS residual quantities in HPV vaccine finished products, improve detection precision, detection sensitivity and accuracy.
CN106290614A discloses Sodium Hyaluronate weight average molecular weight and molecule in a kind of Medical sodium hyaluronate gel
Measure the detection method of breadth coefficient.First, the accurate pharmaceutical preparation solution that measures is appropriate, and mobile phase is added to be diluted to the confession of required concentration
Test solution shakes up, sample introduction, records chromatogram.Kayexalate reference substance separately is taken, is measured in the same method, is calibrated with GPC Software on Drawing
Curve, using universal calibration method to obtain the final product.It is medical transparent using laser light scattering-gel permeation chromatography combination method measurement in professional standard
Sodium Hyaluronate weight average molecular weight and molecular weight distribution index in matter acid sodium gel, because multi-angle laser light scattering instrument is relatively more high
It is expensive, it is difficult to universal to use.It is detected using the method, the instrument generality used is strong, and cost of determination is low, as a result accurately, reliably, surely
It is qualitative good.But what it was used is still that the acquisition of Composition distribution (RID) data is harsher, and that there are results is steady for detection molecules amount
The defect of qualitative difference and poor repeatability.
Invention content
For problems of the prior art, an object of the present invention is to provide one kind can be to sodium hyaluronate molecule
The method that amount and molecular weight distribution are effectively analyzed has good result stability and detection repeatability.It can overcome and show
Difference detector (RID) larger disadvantage affected by environment, for sodium hyaluronate molecular weight and molecular weight distribution detection method provide according to
According to.
For this purpose, the present invention adopts the following technical scheme that：
The present invention provides a kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution, includes the following steps：
(1) test solution is prepared：Sodium hyaluronate test sample is weighed, it is a concentration of to be diluted to sodium hyaluronate with mobile phase
The solution of 0.9-1.1g/L, as test solution；
(2) reference substance solution is prepared：Take kayexalate control series product PSS 140K, PSS 280K, PSS 1M
With PSS 2M, the solution of a concentration of 0.9-1.1g/L is diluted to mobile phase, as a contrast product solution；
(3) use liquid chromatographic system, take the reference substance solution 90-110 μ L sample introductions, for example, 90 μ L, 92 μ L, 95 μ L,
98 μ L, 100 μ L, 102 μ L, 105 μ L, 108 μ L or 110 μ L etc., are detected with electron spray detector, record chromatogram I；It takes again
The test solution 90-110 μ L sample introductions, for example, 90 μ L, 92 μ L, 95 μ L, 98 μ L, 100 μ L, 102 μ L, 105 μ L, 108 μ L or
110 μ L etc., are detected with electron spray detector, and record chromatogram II uses pervasive school with GPC Software on Drawing calibration curves
It executes, it is 0.65 that the K values of reference substance, which are 0.0018, α values,；The K values of sodium hyaluronate are .00057, and α values are 0.75；Result of calculation,
Obtain the molecular weight and molecualr weight distribution coefficient of sodium hyaluronate；
The mobile phase is the ammonium acetate aqueous solution of 90-110mol/L, such as mol/L, 9mol/L, 95mol/L, 98mol/
L, 100mol/L, 102mol/L, 105mol/L, 108mol/L or 110mol/L etc..
It is right present invention employs the method that EFI fog detector (CAD) measures sodium hyaluronate molecular weight and molecular weight distribution
Sodium hyaluronate molecular weight and molecular weight distribution are effectively analyzed, and the concentration of test solution and reference substance solution is 1g/L,
With good detection stability and repeatability.Solve that Composition distribution (RID) is affected by environment larger, and equilibration time is long,
The low problem of checkability.Effectively detection and analysis skill is provided to obtain sodium hyaluronate molecular weight and the accurate result of molecular weight distribution
Art means.
It is not disclosed in the prior art and replaces Composition distribution to carry out sodium hyaluronate molecular weight with electron spray detector
And the technical solution of molecular weight distribution, such as CN105548420A discloses a kind of MOPS (3- (N- morpholines) propane sulfonic acid, 3- (N-
Morpholino) propane-sulfonic acid) detection method, this method is detected with high performance liquid chromatography,
MOPS is quantified using liner sum up method, chromatographic column is Acclaim Trinity P1, although this high performance liquid chromatography
Also EFI fog detector is used, but it is a kind of method of the micro MOPS of detection, it is therefore an objective to overcome the prior art that cannot detect
The micro remaining defects of MOPS are capable of detecting when that MOPS residual quantities in HPV vaccine finished products, raising detection precision, detection are sensitive
Degree and accuracy.The application to the measurement of content, is not directed to the detection side of sodium hyaluronate molecular weight and molecualr weight distribution
Method, for determinand be sodium hyaluronate, belong to entirely different technical problem with the content detection of above-mentioned MOPS.Due to glass
Sour sodium belongs to carbohydrate, not only without UV absorption but also has not generated fluorescence, therefore existing detection sodium hyaluronate molecular weight and molecular weight distribution
Technology is currently limited to use Composition distribution, is not disclosed and replaces Composition distribution to carry out Hyaluronic Acid with electron spray detector
The technical solution of sodium molecule amount and molecular weight distribution.
Preferably, the chromatographic column in the liquid chromatographic system is Shodex 8.0mm × 300mm gel chromatographic columns.
To further increase accuracy, it is preferable that a concentration of 0.98-1.02g/ of sodium hyaluronate in the test solution
L, such as 0.98g/L, 0.99g/L, 1.00g/L, 1.01g/L or 1.02g/L etc..Preferably, the concentration of the reference substance solution
For 0.98-1.02g/L, such as 0.98g/L, 0.99g/L, 1.00g/L, 1.01g/L or 1.02g/L etc..Preferably, the control
The sample size of product solution and test solution is each independently 95-105 μ L.
To further increase the separating effect of sample, it is preferable that the mobile phase is that the ammonium acetate of 98-102mol/L is water-soluble
Liquid.Preferably, the evaporation tube temperature in the liquid chromatographic system is 50 DEG C.Preferably, the liquid phase of the liquid chromatographic system
Frequency acquisition is in 4-5Hz, such as 4Hz, 4.2Hz, 4.5Hz, 4.8Hz or 5Hz etc..Preferably, in the liquid chromatographic system
Liquid phase flow rate is 0.5mL/min.Preferably, the column temperature in the liquid chromatographic system is 35 DEG C.
Preferably, step (1) described test solution shakes 8-12h at 1-5 DEG C, fully dissolves.
As currently preferred technical solution, the detection method packet of the sodium hyaluronate molecular weight and molecualr weight distribution
Include following steps：
(1) test solution is prepared：It weighs sodium hyaluronate test sample and is diluted to a concentration of 1g/L of sodium hyaluronate with mobile phase
Solution, 8-12h is shaken at 1-5 DEG C, as test solution；
(2) reference substance solution is prepared：Take kayexalate control series product PSS 140K, PSS 280K, PSS 1M
With PSS 2M, the solution of a concentration of 1g/L is diluted to mobile phase, as a contrast product solution；
(3) liquid chromatographic system is used, 100 μ L sample introductions of the reference substance solution is taken, is detected with electron spray detector,
Record chromatogram I；100 μ L sample introductions of the test solution are taken again, are detected with electron spray detector, and chromatogram II is recorded,
With GPC Software on Drawing calibration curves, using universal calibration method, it is 0.65 that the K values of reference substance, which are 0.0018, α values,；Sodium hyaluronate
K values are .00057, and α values are 0.75；Result of calculation obtains the molecular weight and molecualr weight distribution coefficient of sodium hyaluronate；
Chromatographic column in the liquid chromatographic system is Shodex 8.0mm × 300mm gel chromatographic columns；Column temperature is 35 DEG C；
Evaporation tube temperature is 50 DEG C；Liquid phase frequency acquisition is in 4-5Hz；Time constant is 3.6s；Liquid phase flow rate is 0.5mL/min；Flowing
It is mutually the ammonium acetate aqueous solution of 100mol/L.
Compared with prior art, the present invention at least has the advantages that：
1. present invention employs EFI fog detectors (CAD), and Composition distribution in the prior art to be replaced to measure sodium hyaluronate
Molecular weight and molecular weight distribution have good detection stability and repeatability, solve Composition distribution (RID) by environment shadow
Larger disadvantage is rung, effective detection and analysis technology hand is provided to obtain sodium hyaluronate molecular weight and the accurate result of molecular weight distribution
Section；
2. by selecting EFI fog detector to coordinate suitable chromatographic condition, detection stability and repetition are further increased
Property.
Description of the drawings
Fig. 1 is that 1 different molecular weight PSS reference substances CAD of embodiment analyzes collection of illustrative plates；
Fig. 2 is the PSS reference substance molecular weight M and retention time t obtained by CAD detectors of embodiment 1RStandard curve；
Fig. 3 is that the CAD detectors of embodiment 1 acquire molecular weight 152000PSS reference substances repetition linearity curve；
Fig. 4 is that embodiment 1 acquires sodium hyaluronate S1-S3 analysis of spectra with CAD detectors.
Specific implementation mode
Technical solution to further illustrate the present invention below with reference to the accompanying drawings and specific embodiments.But following reality
The simple example that example is only the present invention is applied, the scope of the present invention, protection model of the invention are not represented or limit
It encloses and is subject to claims.
Embodiment 1
A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution, includes the following steps：
(1) test solution is prepared：It weighs sodium hyaluronate test sample and is diluted to a concentration of 1g/L of sodium hyaluronate with mobile phase
Solution, 12h is shaken at 4 DEG C, as test solution；
(2) reference substance solution is prepared：Take kayexalate control series product PSS 140K, PSS 280K, PSS 1M
With PSS 2M (molecular weight is respectively 15.2 ten thousand, 25.8 ten thousand, 113.7 ten thousand, 226.7 ten thousand), a concentration of 1g/L is diluted to mobile phase
Solution, product solution as a contrast；
(3) chromatographic condition is established：
Chromatographic column in the liquid chromatographic system is Shodex 8.0mm × 300mm gel chromatographic columns；Column temperature is 35 DEG C；
Evaporation tube temperature is 50 DEG C；Liquid phase frequency acquisition is in 4Hz；Time constant is 3.6s；Liquid phase flow rate is 0.5mL/min；Mobile phase
For the ammonium acetate aqueous solution of 100mol/L.
It measures：
100 μ L sample introductions of the reference substance solution are taken, are detected with electron spray detector, chromatogram is recorded；It takes again described
100 μ L sample introductions of test solution, are detected with electron spray detector, record chromatogram, with GPC Software on Drawing calibration curves,
Using universal calibration method, it is 0.65 that the K values of reference substance, which are 0.0018, α values,；The K values of sodium hyaluronate are .00057, and α values are 0.75；
Result of calculation obtains the molecular weight and molecualr weight distribution coefficient of sodium hyaluronate.For analysis repeatability, detected with same procedure as above
Tri- samples of S1, S2, S3.
Table 1 is with CAD detector different molecular weight PSS reference substance retention times.
Table 1
| Molecular weight M | Retention time tR, min |
| 2267000 | 15.48 |
| 1137000 | 16.24 |
| 258000 | 17.82 |
| 152000 | 18.55 |
The RID detector different molecular weight PSS reference substance retention times of table 2.
Table 2
| Molecular weight M | Retention time tR, min |
| 2267000 | 15.44 |
| 1137000 | 16.26 |
| 258000 | 17.72 |
| 152000 | 18.58 |
Table 3 is sodium hyaluronate S1-S3 molecular weight analysis results.
Table 3
| Sample | S1 | S2 | S3 |
| Retention time (t at summitR,min) | 14.76 | 14.68 | 14.84 |
| Number-average molecular weight (Number Average Mn) | 1965726 | 2018511 | 1982064 |
| Weight average molecular weight (Weight Average Mw) | 9436850 | 9794656 | 9327296 |
| Z-average molecular weight Z Average Mz | 29529845 | 30591933 | 28587028 |
| (Z+1)-average molecular weight ((Z+1) Average Mz+1) | 52907218 | 54659342 | 49992392 |
| Molecular weight at summit | 4925379 | 5360220 | 4515248 |
| Distribution Breadth Index (Polydispersity PD (Mw/Mn)) | 4.8 | 4.85 | 4.71 |
Interpretation of result：
A) EFI fog detector (CAD) can be to different molecular weight PSS (as shown in Figure 1) and sodium hyaluronate sample (such as Fig. 4
It is shown) it is analyzed, molecular weight is carried out by GPC softwares and molecular weight distribution calculates, obtains retention time and molecular weight logarithm
Correspondence thus draw standard curve as shown in table 1, as shown in Fig. 2, the equation of curve be logM=-0.3877 × tR
+ 12.35, related coefficient R2=0.998；And control Tables 1 and 2 is it is found that the retention time that EFI fog detector (CAD) obtains
It is consistent with Composition distribution (RID)；
B) as shown in Table 3, the weight average molecular weight M of three sodium hyaluronate sample S1-S3wSubstantially all 940,000 or so；
C) as shown in figure 3, by taking 152000 molecular weight PSS reference substances as an example, retention time repeatability RSD=0.02%；Such as
Shown in Fig. 4, three sodium hyaluronate sample S1-S3 retention time repeatability RSD=0.42% illustrate EFI fog detector of the present invention
(CAD) sodium hyaluronate molecular weight and molecular weight distribution can effectively be analyzed, there is good detection repeatability.
Embodiment 2
With differing only in for embodiment 1：Chromatographic condition is different：
A concentration of 0.98g/L of test solution, a concentration of 1.02g/L of reference substance solution, sample size are 105 μ L,
Mobile phase is the ammonium acetate aqueous solution of 98mol/L.Chromatographic column in liquid chromatographic system is Shodex8.0mm × 300mm gels
Chromatographic column；Column temperature is 30 DEG C；Evaporation tube temperature is 45 DEG C；Liquid phase frequency acquisition is in 4.5Hz；Time constant is 3.6s；Liquid phase stream
Speed is 0.7mL/min.
Embodiment 3
With differing only in for embodiment 1：Chromatographic condition is different：
A concentration of 1.02g/L of test solution, a concentration of 0.98g/L of reference substance solution, sample size are 95 μ L, stream
Dynamic is mutually the ammonium acetate aqueous solution of 102mol/L.Chromatographic column in liquid chromatographic system is Shodex8.0mm × 300mm gel colors
Compose column；Column temperature is 35 DEG C；Evaporation tube temperature is 55 DEG C；Liquid phase frequency acquisition is in 4.8Hz；Time constant is 3.6s；Liquid phase flow rate
For 0.5mL/min.
Embodiment 2-3 is similar with the result of embodiment 1, and details are not described herein.
Embodiment 4
With differing only in for embodiment 1：Chromatographic condition is different：
A concentration of 0.9g/L of test solution, a concentration of 1.1g/L of reference substance solution, sample size are 110 μ L, stream
Dynamic is mutually the ammonium acetate aqueous solution of 90mol/L.Chromatographic column in liquid chromatographic system is Shodex8.0mm × 300mm gel colors
Compose column；Column temperature is 40 DEG C；Evaporation tube temperature is 40 DEG C；Liquid phase frequency acquisition is in 5Hz；Time constant is 3.6s；Liquid phase flow rate is
0.4mL/min。
By taking 152000 molecular weight PSS reference substances as an example, retention time repeatability RSD=0.05%, three sodium hyaluronate samples
Product S1-S3 retention time repeatabilities RSD=0.45%.
Embodiment 5
With differing only in for embodiment 1：Chromatographic condition is different：
A concentration of 1.1g/L of test solution, a concentration of 0.9g/L of reference substance solution, sample size are 90 μ L, influent stream
Dynamic is mutually the ammonium acetate aqueous solution of 110mol/L.Chromatographic column in liquid chromatographic system is Shodex8.0mm × 300mm gel colors
Compose column；Column temperature is 25 DEG C；Evaporation tube temperature is 60 DEG C；Liquid phase frequency acquisition is in 4Hz；Time constant is 3.6s；Liquid phase flow rate is
0.8mL/min。
By taking 152000 molecular weight PSS reference substances as an example, retention time repeatability RSD=0.04%, three sodium hyaluronate samples
Product S1-S3 retention time repeatabilities RSD=0.52%.
Comparative example 1
With differing only in for embodiment 1：Detector is Composition distribution.
As a result repeatability is deteriorated compared to embodiment 1.By taking 152000 molecular weight PSS reference substances as an example, retention time repeats
Property RSD=1.56%, three sodium hyaluronate sample S1-S3 retention time repeatabilities RSD=1.23%.
Embodiment 6
With differing only in for embodiment 1：A concentration of 1.3g/L of test solution.
Embodiment 7
With differing only in for embodiment 1：A concentration of 0.7g/L of test solution.
Embodiment 8
With differing only in for embodiment 1：A concentration of 1.3g/L of reference substance solution.
Embodiment 9
With differing only in for embodiment 1：A concentration of 1.3g/L of reference substance solution.
Embodiment 10
With differing only in for embodiment 1：The sample size of reference substance solution is 80 μ L.
Embodiment 11
With differing only in for embodiment 1：The sample size of reference substance solution is 120 μ L.
Embodiment 12
With differing only in for embodiment 1：The sample size of test solution is 80 μ L.
Embodiment 13
With differing only in for embodiment 1：The sample size of test solution is 120 μ L.
In embodiment 6, by taking 152000 molecular weight PSS reference substances as an example, retention time repeatability RSD=0.08%, three
The result of sodium hyaluronate sample S1-S3 retention time repeatability RSD=0.89%, embodiment 7-13 are similar to embodiment 6, herein
It repeats no more.
Comparative examples 1 are with embodiment 6-13, comparative example 1 it is found that present invention employs EFI fog detector (CAD) replacements
Composition distribution in the prior art measures sodium hyaluronate molecular weight and molecular weight distribution, with good detection stability and again
Renaturation solves Composition distribution (RID) larger disadvantage affected by environment, sodium hyaluronate molecular weight and molecular weight distribution result
Accuracy is high, reproducible.And worked in coordination by the concentration and sample size of reference substance and sample, control glass in test solution
A concentration of 0.98-1.02g/L of glass acid sodium, a concentration of 0.98-1.02g/L reference substance solutions and test sample of reference substance solution are molten
The sample size of liquid is 95-105 μ L, can advanced optimize accuracy and repeatability.
Applicant states that the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment,
But the invention is not limited in above-mentioned detailed process equipment and technological processes, that is, it is above-mentioned detailed not mean that the present invention has to rely on
Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention,
The addition of equivalence replacement and auxiliary element to each raw material of product of the present invention, the selection etc. of concrete mode all fall within the present invention's
Within protection domain and the open scope.
Claims (10)
1. a kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution, which is characterized in that include the following steps：
(1) test solution is prepared：Sodium hyaluronate test sample is weighed, a concentration of 0.9- of sodium hyaluronate is diluted to mobile phase
The solution of 1.1g/L, as test solution；
(2) reference substance solution is prepared：Take kayexalate control series product PSS 140K, PSS 280K, PSS 1M and PSS
2M is diluted to the solution of a concentration of 0.9-1.1g/L with mobile phase, as a contrast product solution；
(3) liquid chromatographic system is used, the reference substance solution 90-110 μ L sample introductions is taken, is detected with electron spray detector,
Record chromatogram I；The test solution 90-110 μ L sample introductions are taken again, are detected with electron spray detector, and chromatogram is recorded
II, with GPC Software on Drawing calibration curves, using universal calibration method, it is 0.65 that the K values of reference substance, which are 0.0018, α values,；Hyaluronic Acid
The K values of sodium are .00057, and α values are 0.75；Result of calculation obtains the molecular weight and molecualr weight distribution coefficient of sodium hyaluronate；
The mobile phase is the ammonium acetate aqueous solution of 90-110mol/L.
2. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as described in claim 1, which is characterized in that the liquid
Chromatographic column in phase chromatographic system is Shodex 8.0mm × 300mm gel chromatographic columns.
3. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
State a concentration of 0.98-1.02g/L of sodium hyaluronate in test solution.
4. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
State a concentration of 0.98-1.02g/L of reference substance solution.
5. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
The sample size for stating reference substance solution and test solution is each independently 95-105 μ L.
6. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
State the ammonium acetate aqueous solution that mobile phase is 98-102mol/L.
7. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
It is 40-60 DEG C to state the evaporation tube temperature in liquid chromatographic system.
8. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
The liquid phase frequency acquisition of liquid chromatographic system is stated in 4-5Hz；
Liquid phase flow rate in the liquid chromatographic system is 0.4-0.8mL/min.
9. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that institute
It is 25-40 DEG C to state the column temperature in liquid chromatographic system.
10. the detection method of sodium hyaluronate molecular weight and molecualr weight distribution as claimed in claim 1 or 2, which is characterized in that step
Suddenly (1) described test solution shakes 8-12h at 1-5 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810258732.9A CN108333273A (en) | 2018-03-27 | 2018-03-27 | A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810258732.9A CN108333273A (en) | 2018-03-27 | 2018-03-27 | A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108333273A true CN108333273A (en) | 2018-07-27 |
Family
ID=62931726
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810258732.9A Pending CN108333273A (en) | 2018-03-27 | 2018-03-27 | A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108333273A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113341028A (en) * | 2021-07-08 | 2021-09-03 | 山东诺明康药物研究院有限公司 | Method for measuring sodium hyaluronate molecular weight and molecular weight distribution by size exclusion chromatography |
| CN115932083A (en) * | 2022-11-24 | 2023-04-07 | 华熙生物科技股份有限公司 | Method for detecting glycerin and 1, 4-butanediol in cross-linked sodium hyaluronate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102980951A (en) * | 2012-11-15 | 2013-03-20 | 上海景峰制药股份有限公司 | Detection method of sodium hyaluronate and molecular weight in preparation of sodium hyaluronate |
| CN106290614A (en) * | 2016-07-27 | 2017-01-04 | 浙江景嘉医疗科技有限公司 | A kind of detection method of Medical sodium hyaluronate gel molecular weight and molecualr weight distribution coefficient |
-
2018
- 2018-03-27 CN CN201810258732.9A patent/CN108333273A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102980951A (en) * | 2012-11-15 | 2013-03-20 | 上海景峰制药股份有限公司 | Detection method of sodium hyaluronate and molecular weight in preparation of sodium hyaluronate |
| CN106290614A (en) * | 2016-07-27 | 2017-01-04 | 浙江景嘉医疗科技有限公司 | A kind of detection method of Medical sodium hyaluronate gel molecular weight and molecualr weight distribution coefficient |
Non-Patent Citations (7)
| Title |
|---|
| DAWEN KOU 等: "Size exclusion chromatography with Corona charged aerosol detector for the analysis of polyethylene glycol polymer", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| KA-MAN YIP 等: "Ultrasound-Assisted Extraction May Not Be a Better Alternative Approach than Conventional Boiling for Extracting Polysaccharides from Herbal Medicines", 《MOLECULES》 * |
| 刘立洋 等: "一种新型的通用型检测器——电喷雾检测器", 《现代科学仪器》 * |
| 刘莉莉 等: "医用透明质酸钠凝胶的绝对分子量及其分布的测定方法研究", 《药物分析杂志》 * |
| 宋玉娟 等: "玻璃酸钠及制剂分子质量与分子质量分布测定方法研究", 《药物分析杂志》 * |
| 李心怡 等: "电喷雾检测器在药物HPLC分析中的优势及应用进展", 《中国药房》 * |
| 王海英 等: "分子排阻色谱法测定玻璃酸钠相对分子质量及其分布", 《食品与药品》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113341028A (en) * | 2021-07-08 | 2021-09-03 | 山东诺明康药物研究院有限公司 | Method for measuring sodium hyaluronate molecular weight and molecular weight distribution by size exclusion chromatography |
| CN115932083A (en) * | 2022-11-24 | 2023-04-07 | 华熙生物科技股份有限公司 | Method for detecting glycerin and 1, 4-butanediol in cross-linked sodium hyaluronate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dasgupta et al. | Microrheology of cross-linked polyacrylamide networks | |
| CN108333273A (en) | A kind of detection method of sodium hyaluronate molecular weight and molecualr weight distribution | |
| CN102507381B (en) | Colloid bismuth pectin compound and quality control method of pharmaceutical compositions thereof | |
| CN112730720B (en) | Liquid phase detection method for vitronectin in cosmetics | |
| CN106290614A (en) | A kind of detection method of Medical sodium hyaluronate gel molecular weight and molecualr weight distribution coefficient | |
| CN105092513B (en) | A kind of method of the content of drug ingedient in measurement hybrid medicine | |
| CN109580428A (en) | A kind of method of hyaluronan molecule amount in simplicity Accurate Determining solution | |
| CN106841413A (en) | A kind of ticagrelor enantiomter, the method for separating and detecting of diastereoisomer | |
| Thevarajah et al. | Towards a less biased dissolution of chitosan | |
| CN105699375B (en) | The method for measuring Glucosamine using spectrophotometry | |
| Burç et al. | Voltammetric determination of curcumin in spices using platinum electrode electrochemically modified with poly (vanillin-co-caffeic acid) | |
| Cave et al. | A flow system for the on-line quantitative measurement of the retention of dosage forms on biological surfaces using spectroscopy and image analysis | |
| Chen et al. | Absolute molecular weight determination of hypromellose acetate succinate by size exclusion chromatography: Use of a multi angle laser light scattering detector and a mixed solvent | |
| CN110736796B (en) | Method for detecting molecular weight of pneumococcal capsular polysaccharide | |
| CN113341028A (en) | Method for measuring sodium hyaluronate molecular weight and molecular weight distribution by size exclusion chromatography | |
| CN105372204A (en) | Near infrared spectrum online detection method of etimicin sulfate column separation process | |
| CN106680062B (en) | Utilize the method for resonance rayleigh light scattering method measurement anionic surfactant concentration | |
| CN105067719A (en) | HPLC measuring method for three ingredients of trinary mycin for injection | |
| CN108872179A (en) | The measuring method of liquid refractivity under a kind of pressurized conditions | |
| CN110274972A (en) | A method of series connection gel chromatography Dendrobium nobile polysaccharide molecular weight distribution | |
| CN106018401A (en) | Method for determining content of chlorine ions in water | |
| CN107421975B (en) | The detection method of glycerol content in a kind of injection | |
| CN104931617A (en) | Method for measuring polyester molecular weight and molecular weight distribution thereof | |
| CN107084929B (en) | The quantitative detecting method of pneumococal polysaccharide | |
| WO2020048269A1 (en) | Method for measuring content of chitosan oligosaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180727 |
|
| RJ01 | Rejection of invention patent application after publication |