Method for separating sodium hyaluronate and detecting molecular weight

Abstract

The invention belongs to the technical field of biological medicine detection, and relates to a method for separating sodium hyaluronate mixtures in different molecular weight ranges and / or measuring molecular weight through gel permeation chromatography. According to the method, sodium hyaluronate mixtures with different molecular weights are subjected to separation and molecular weight determination through a gel chromatography tandem technology, key technical parameters such as selection of reference substances and types of chromatographic columns are comprehensively adjusted in the determination process, a good separation effect on the sodium hyaluronate mixtures is achieved, the accuracy of a molecular weight detection result is high, and the method is suitable for industrial production. Meanwhile, good stability and repeatability are achieved, toxic and harmful chemical reagents are not used in detection, and the safety and practicability of detection are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and in particular, relates to a method for separating and detecting the molecular weight of hyaluronic acid. Background Art

[0002] Hyaluronic acid (HA) is an acidic mucopolysaccharide, which is composed of D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) connected by β-1,3 glycosidic bonds to form a disaccharide unit structure, and then polymerized by β-1,4 glycosidic bonds to form a glycosaminoglycan. Due to the poor solubility of HA itself, it is often converted into sodium salt, namely sodium hyaluronate (SH) when used. Due to different degrees of polymerization, the average relative molecular mass of SH varies greatly, and the rheological properties of SH mainly depend on the molecular weight and determine its biological function and application scenarios. For example, hyaluronic acid with a larger molecular weight has stronger support, anti-degradation and viscoelasticity, so it is often used as a shaping filling material in medical cosmetology; sodium hyaluronate with a lower molecular weight is more easily absorbed by the body and can be used as a natural moisturizing factor in cosmetics and beauty health foods; sodium hyaluronate with a lower molecular weight has therapeutic effects such as promoting bone formation and treating bacterial corneal ulcers.

[0003] The biological activity of sodium hyaluronate is closely related to its molecular weight, so the appropriate molecular weight range should be selected according to the purpose of use. With the development of application scenarios and formulation technology, adding sodium hyaluronate of different molecular weight ranges to products to obtain multiple biological effects has become an important research direction for sodium hyaluronate products. The different molecular weights of hyaluronic acid in such products have become key detection indicators.

[0004] At present, the main methods for determining the molecular weight of sodium hyaluronate are the intrinsic viscosity method and size-exclusion chromatographic separation (SEC). The intrinsic viscosity method obtains the relative molecular mass of a single sample, which involves a mixture of sodium hyaluronate in different molecular weight ranges and is not suitable for determination by the intrinsic viscosity method. The SEC method achieves separation based on the size of the molecular volume (hydrodynamic volume) in the solution, and can simultaneously determine the average molecular weight and its distribution. Domestic and foreign pharmacopoeias generally use size-exclusion chromatography with Shodex SB-806HQ as the chromatographic column for detection, but this method is limited to the molecular weight detection of sodium hyaluronate in a narrow molecular weight range. In addition, the addition of sodium azide in the mobile phase of this method, which acts as a preservative, is a highly toxic substance and is prone to explosion, posing a major safety hazard. Summary of the invention

[0005] In order to overcome the above technical problems existing in the art, the object of the present invention is to provide a method for separating and / or measuring the molecular weight of a mixture of sodium hyaluronate in different molecular weight ranges.

[0006] The present invention adopts the following technical scheme to achieve the above-mentioned invention object: The present invention provides a method for separating and / or determining the molecular weight of a sodium hyaluronate mixture with different molecular weight ranges by gel permeation chromatography, wherein the chromatographic conditions are as follows: the chromatographic columns are Shodex OHpak SB-806HQ: 8.0mm ID*30cm, 13μm and Shodex OHpak SB-804HQ: 8.0mm ID*30cm, 10μm connected in series in sequence; the mobile phase is an ammonium acetate aqueous solution; wherein the sodium hyaluronate sample to be tested is a sodium hyaluronate mixture with a molecular weight range of 1 to 1800KDa.

[0007] Furthermore, in the method for separation and / or determination of molecular weight, the concentration of the aqueous ammonium acetate solution is 0.05 to 0.2 mol / L.

[0008] Furthermore, in the method for separation and / or determination of molecular weight, the concentration of the aqueous ammonium acetate solution is 0.08, 0.1 or 0.12 mol / L.

[0009] Furthermore, in the method for separation and / or determination of molecular weight, the temperature of the chromatographic column is 25-40°C.

[0010] Furthermore, in the method of separation and / or determination of molecular weight, the temperature of the chromatography column is 25, 30 or 35°C.

[0011] Furthermore, in the method for separation and / or determination of molecular weight, the flow rate is 0.4-0.6 ml / min; and the injection volume is 20-30 μL.

[0012] Furthermore, the method of separation and / or determination of molecular weight comprises a flow rate of 0.4, 0.5 or 0.6 ml / min; and an injection volume of 20, 25 or 30 μL.

[0013] Furthermore, in the method for separation and / or determination of molecular weight, the mobile phase is an aqueous solution of ammonium acetate with a concentration of 0.1 mol / L; the column temperature is 30° C.; the flow rate is 0.5 ml / min; and the injection volume is 25 μL.

[0014] Furthermore, in the method of separating and / or determining the molecular weight, at least 9 heparins of known molecular weight are used to prepare reference substances, which are diluted with water to prepare solutions with a concentration of 0.4 to 0.6 mg / ml.

[0015] Furthermore, in the method of separating and / or determining the molecular weight, at least 9 heparins of known molecular weight are used to prepare reference substances, which are diluted with water to prepare solutions with a concentration of 0.5 mg / ml.

[0016] Furthermore, in the method for separation and / or determination of molecular weight, the molecular weight of heparin is 600Da to 3200KDa.

[0017] Furthermore, in the method of separating and / or determining the molecular weight, the molecular weight of heparin is divided into low-middle molecular weight and high molecular weight standards, and the selection is made according to the low-middle molecular weight sodium hyaluronate and high molecular weight sodium hyaluronate in the determination sample:

[0018] For low and medium molecular weight, you can choose a full set of low molecular weight mixed standards: 600, 1200, 1800, 2400, 3000, 3600, 4200, 4800, 5400, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 32000, 36000, 40000Da.

[0019] High molecular weight options include 16K, 68K, 145K, 203K, 471K, 785K, 1207K, 2070K, and 3160KDa.

[0020] Furthermore, the method for separating and / or determining the molecular weight is to dilute the sodium hyaluronate sample to be tested with water to prepare a solution with a concentration of 0.4 mg / ml to 0.6 mg / ml.

[0021] Furthermore, in the method of separation and / or determination of molecular weight, the sodium hyaluronate sample to be tested is diluted with water to prepare a solution with a concentration of 0.5 mg / ml.

[0022] Furthermore, the method for separating and / or determining the molecular weight is to calculate the molecular weight of the sample by performing universal calibration and calculating the linear regression equation through GPC software.

[0023] Furthermore, in the method of separation and / or determination of molecular weight, the molecular weight of the sample to be measured is calculated according to the following linear regression equation:

[0024] Low and medium molecular weight linear regression equation:

[0025] F(molecular weight [n]) = -0.00275648*T 3 +0.1320164*T 2 -2.238323*T+17.17206, where T is the retention time and R 2 =0.99995.

[0026] High molecular weight linear regression equation:

[0027] F(molecular weight [n]) = 0.01252043*T 3 +0.6674033*T 2 -12.21135*T+82.13413, where T is the retention time and R 2 =0.99915.

[0028] Furthermore, in the method of separating and / or determining molecular weight, the molecular weight is a weight average molecular weight.

[0029] In the present invention, Shodex OHpak SB-806HQ: 8.0mm ID*30cm, 13μm and Shodex OHpak SB-804HQ: 8.0mm ID*30cm, 10μm are gel permeation chromatography columns produced by Showa Denko of Japan.

[0030] Due to the special viscoelasticity of sodium hyaluronate, there is no standard sample with the same characteristics in the separation and detection methods. After experiments and exploration, heparin was selected as the standard sample, which greatly improved the accuracy of the test results.

[0031]

[0032] Compared with the prior art, the present invention has the following beneficial effects: the application prospects of hyaluronic acid are very broad, and it is involved in the fields of medicine, medical devices, cosmetics, health products, etc. It is necessary to strengthen the quality control of its raw materials and preparations, especially the specificity and accuracy of the hyaluronic acid molecular weight determination method need to be improved. The present invention achieves a better separation effect on sodium hyaluronate mixtures of different molecular weight ranges through gel permeation chromatography, and the molecular weight detection results have good stability and repeatability, breaking through the existing technical limitations of only being able to detect the molecular weight of sodium hyaluronate in a single molecular weight range, and providing a method for effectively separating and detecting the molecular weight of sodium hyaluronate mixtures of different molecular weight ranges. At the same time, heparin is used as a detection standard to improve the accuracy and repeatability of the detection results. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 . Spectrum of low molecular weight heparin reference solution;

[0034] Figure 2 . Spectrum of low molecular weight sodium hyaluronate A01 solution;

[0035] Figure 3 . Spectrum of low molecular weight sodium hyaluronate B01 solution;

[0036] Figure 4 . Spectrum of low molecular weight sodium hyaluronate C01 solution;

[0037] Figure 5 . Spectrum of low molecular weight sodium hyaluronate D01 solution;

[0038] Figure 6 . Spectrum of low molecular weight sodium hyaluronate E01 solution;

[0039] Figure 7 . Spectrum of high molecular weight heparin reference solution;

[0040] Figure 8 .GPC calibration curve of high molecular weight heparin reference substance;

[0041] Fig. 9 .GPC spectrum of high molecular weight sodium hyaluronate (weight average molecular weight 1877K);

[0042] Fig.10 .GPC-Malls profile of high molecular weight sodium hyaluronate;

[0043] Fig.11 .GPC-Malls profile of high molecular weight sodium hyaluronate;

[0044] Fig.12 Spectrum of high molecular weight sodium polystyrene sulfonate reference solution;

[0045] Fig.13 GPC calibration curve of high molecular weight sodium polystyrene sulfonate reference substance;

[0046] Fig.14 GPC spectrum of high molecular weight sodium hyaluronate (weight average molecular weight 1877K). DETAILED DESCRIPTION

[0047] The following is a detailed description of the embodiments of the present invention. The embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0048] Example 1

[0049] Preparation of reference standard mixed solution: Take a low molecular weight heparin mixed standard (600, 1200, 1800, 2400, 3000, 3600, 4200, 4800, 5400, 6000, 8000, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 32000, 36000, 40000Da), dissolve and dilute with pure water to make a solution with a concentration of about 0.5mg / ml as the reference standard mixed solution;

[0050] Table 1. Low molecular weight heparin molecular weight reference substance - molecular weight distribution information

[0051]

[0052]

[0053] Preparation of test solution: Take the sample to be tested (sodium hyaluronate produced by conventional fermentation is cleaved by hyaluronidase to obtain a mixture of sodium hyaluronate with different molecular weights, with the molecular weight distribution ranging from several thousand to 10,000 Da), dissolve it in pure water and dilute it to make a solution with a concentration of about 0.5 mg / ml as the test solution; among which the weight average molecular weight of A01 solution is 1340 Da; the weight average molecular weight of B01 solution is 4441 Da; the weight average molecular weight of C01 solution is 4754 Da; the weight average molecular weight of D01 solution is 16049 Da; and the weight average molecular weight of E01 solution is 16511 Da.

[0054] The chromatographic columns were Shodex OHpak SB-806HQ (8.0mm ID*30cm, 13μm) and Shodex OHpak SB-804HQ (8.0mm ID*30cm, 10μm) connected in series; the mobile phase was ammonium acetate aqueous solution with a concentration of 0.1mol / L; the temperature of the chromatographic column (same as the detector temperature) was 30°C; the flow rate was 0.5ml / min;

[0055] Take 25 μL of the mixed standard solution of the reference substance, detect it with a differential detector (RID), record the chromatogram, and then take 25 μL of the test solution for injection (one test solution is injected each time, and 5 test solutions need to be injected 5 times), detect it with a differential detector (RID), and record the chromatogram. Use GPC software for universal correction to calculate the linear regression equation, and calculate the molecular weight of sodium hyaluronate (weight average molecular weight M) at different peaks in the test sample. w ).

[0056] The linear regression equation is as follows:

[0057] F(molecular weight [n]) = -0.00275648*T 3 +0.1320164*T 2 -2.238323*T+17.17206, where T is the retention time and R 2 =0.99995.

[0058] The experimental results are as attached Figure 1 To Attachment Figure 6 shown.

[0059] Result analysis: The reference substance series solutions showed a good universal correction linear regression relationship, R 2 is 0.99995 (with Figure 1The low molecular weight range of sodium hyaluronate 1000Da, 4000Da, 5000Da, 15000Da, and 16000Da was well separated, and their respective weight average molecular weights (M w ) value (attached Figure 2 To Attachment Figure 6 ).

[0060] Example 2

[0061] The difference from Example 1 is that the reference solution is different.

[0062] Prepare a series of reference solutions: Take high molecular weight heparin standards (16K, 68K, 145K, 203K, 471K, 785K, 1207K, 2070K, 3160KDa), dissolve and dilute with pure water to make a solution with a concentration of about 0.5 mg / ml as a series of reference solutions.

[0063] The test sample is sodium hyaluronate produced by conventional fermentation, in which the metabolic byproduct hyaluronidase degrades the target product sodium hyaluronate, and the obtained high molecular weight sodium hyaluronate is a mixture as the main component.

[0064] The chromatographic columns were Shodex OHpak SB-806HQ (8.0mm ID*30cm, 13μm) and Shodex OHpak SB-804HQ (8.0mm ID*30cm, 10μm) connected in series; the mobile phase was ammonium acetate aqueous solution with a concentration of 0.12mol / L; the chromatographic column temperature (same as the detector temperature) was 35°C; the flow rate was 0.6ml / min; and the injection volume was 20μL.

[0065] The linear regression equation is as follows:

[0066] F(molecular weight [n]) = 0.01252043*T 3 +0.6674033*T 2 -12.21135*T+82.13413, where T is the retention time and R 2 =0.99915.

[0067] The experimental results are as attached Figure 7 To Attachment Fig. 9 shown.

[0068] Result analysis: The reference substance series solutions showed a good universal correction linear regression relationship, R 2 is 0.99915 (with Figure 7 , 8 ). It can achieve better separation of sodium hyaluronate in the high molecular weight range of 1800KDa, and more accurately calculate its weight average molecular weight (Mw )Value( Fig. 9 ).

[0069] Example 3

[0070] Preparation of test solution: Take the sample to be tested in Example 2, dissolve it with the mobile phase and dilute it to a solution with a concentration of about 0.5 mg / ml as the test solution; take 100 μL of the above test solution, and calculate the molecular weight (weight average molecular weight M) of sodium hyaluronate at different peaks in the test sample by gel permeation chromatography-laser scattering (GPC-LLS) w ).

[0071] The chromatographic column was Shodex OHpak SB-806HQ (8.0 mm ID*30 cm, 13 μm); the mobile phase was sodium chloride aqueous solution-0.2‰ Proclin 200 preservative, and the concentration of sodium chloride aqueous solution was 0.2 mol / L; the chromatographic column temperature was 35°C; the flow rate was 1 ml / min; and the injection volume was 100 μL.

[0072] The experimental results are as attached Fig.10 , 11 shown.

[0073] Result analysis: The weight average molecular weight (M) of high molecular weight sodium hyaluronate was determined by the YY / T 1571-2017 sodium hyaluronate gel permeation chromatography-laser scattering method (GPC-LLS). w ) is 1953 KDa. Compared with the simple GPC method in Example 2, the relative deviation is 3.9%, which is within the error range.

[0074] This detection method achieves more efficient separation through gel chromatography tandem technology, and better separates sodium hyaluronate with different molecular weights to obtain molecular weight distribution. However, the molecular weight detection method used in the tissue engineering medical device product sodium hyaluronate YY / T 1571-2017 uses gel permeation chromatography-laser scattering spectrometry (GPC-LLS), which is expensive and costly. Therefore, we use GPC detection to determine the molecular weight and perform GPC-LLS simultaneous detection for comparison. The molecular weight ranges of the two are not much different and are both within the acceptable range of the sample nominal molecular weight.

[0075] Example 4

[0076] The difference from Example 2 is that the reference substance is different.

[0077] Prepare a series of reference solutions: Take high molecular weight sodium polystyrene sulfonate standards (150K, 620K, 980K, 2000K, 3200KDa), add mobile phase to dissolve and dilute to make a solution with a concentration of about 0.1 mg / ml as a series of reference solutions;

[0078] Prepare the test solution: Take the sample to be tested, dissolve it with the mobile phase and dilute it to a solution with a concentration of about 0.1 mg / ml as the test solution;

[0079] The chromatographic column was Shodex OHpak SB-806HQ (8.0 mm ID*30 cm, 13 μm); the mobile phase was 0.2 mol / L sodium chloride-0.1‰ sodium azide aqueous solution; the chromatographic column temperature (same as the detector temperature) was 30°C; the flow rate was 0.5 ml / min; and the injection volume was 100 μL.

[0080] The linear regression equation is as follows:

[0081] F(molecular weight [n]) = -0.0197*T 3 +1.04*T 2 -18.6*T+119, where T is the retention time and R 2 =0.997046.

[0082] The experimental results are as attached Fig.12 To Attachment Fig.14 shown.

[0083] Result analysis: The reference substance series solutions showed a good universal correction linear regression relationship, R 2 is 0.997046 (with Fig.12 , 13 The sodium hyaluronate 33960KDa in the high molecular weight range of Example 2 is very different from Examples 2 and 3, indicating that the detection method is inaccurate ( Fig.14 ).

[0084] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for separating and / or determining the molecular weight of a mixture of sodium hyaluronate in different molecular weight ranges by gel permeation chromatography, wherein: The chromatographic conditions are as follows: the chromatographic columns are Shodex OHpak SB-806HQ: 8.0mm ID*30cm, 13μm and Shodex OHpak SB-804HQ: 8.0mm ID*30cm, 10μm connected in series; the mobile phase is an aqueous solution of ammonium acetate; the sodium hyaluronate sample to be tested is a mixture of sodium hyaluronate with a molecular weight range of 1 to 1800KDa.

2. The method according to claim 1, wherein the concentration of the ammonium acetate aqueous solution is 0.05 to 0.2 mol / L.

3. method according to claim 2, wherein the concentration of ammonium acetate aqueous solution is 0.08, 0.1 or 0.12 mol / L.

4. The method according to claim 1, wherein the chromatographic column temperature is 25-40°C.

5. The method according to claim 4, wherein the temperature of the chromatography column is 25, 30 or 35°C.

6. The method according to claim 1, wherein the flow rate is 0.4-0.6 ml / min; and the injection volume is 20-30 μL.

7. The use according to claim 6, wherein the flow rate is 0.4, 0.5 or 0.6 ml / min; and the injection volume is 20, 25 or 30 μL.

8. The method according to claim 1, wherein the mobile phase is an aqueous solution of ammonium acetate with a concentration of 0.1 mol / L; the column temperature is 30°C; the flow rate is 0.5 ml / min; and the injection volume is 25 μL.

9. The method according to claim 1, wherein: At least 9 heparins of known molecular weight were used to prepare reference substances, which were diluted with water to prepare solutions with concentrations of 0.4 to 0.6 mg / ml.

10. The method according to claim 9, wherein: At least 9 heparins of known molecular weight were used to prepare control substances, which were diluted with water to prepare solutions with a concentration of 0.5 mg / ml.

11. The method according to claim 10, wherein: The molecular weight of heparin is 600Da~3200KDa.

12. The method according to claim 11, wherein: Heparin molecular weight is divided into low, medium and high molecular weight standards.

13. The method according to claim 12, wherein: The molecular weight of low and medium molecular weight heparin standards is 600Da~40KDa.

14. The method according to claim 12, wherein: The molecular weight of high molecular weight heparin standard is 16KDa~3200KDa.

15. The method according to claim 1, wherein: The sodium hyaluronate sample to be tested is diluted with water to prepare a solution with a concentration of 0.4 mg / ml to 0.6 mg / ml.

16. The method according to claim 15, wherein: The sodium hyaluronate sample to be tested was diluted with water to prepare a solution with a concentration of 0.5 mg / ml.

17. The method according to claim 1, wherein: The molecular weight of the sample was calculated by performing universal calibration and calculating the linear regression equation through the GPC software.

18. The method according to claim 17, wherein: Calculate the molecular weight of the sample according to the following linear regression equation: Low and medium molecular weight linear regression equation: F(molecular weight [n]) = -0.00275648*T 3 +0.1320164*T 2 -2.238323*T+17.17206, where T is the retention time and R 2 =0.99995. High molecular weight linear regression equation: F(molecular weight [n]) = 0.01252043*T 3 +0.6674033*T 2 -12.21135*T+82.13413, where T is the retention time and R 2 =0.99915.

19. The method according to any one of claims 1 to 18, wherein: The molecular weights stated are weight average molecular weights.

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