Methods for isolating modified hyaluronic acid oligosaccharides

Abstract

This application provides a method for separating modified hyaluronic acid oligosaccharides. The method includes the following steps: separating the modified hyaluronic acid oligosaccharides using liquid chromatography-mass spectrometry (LC-MS), wherein the LC-MS uses a porous graphitized carbon column, with gradient elution using mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and mobile phase B is an aqueous solution of ammonium formate or ammonium acetate in acetonitrile. This separation method can effectively separate various modified hyaluronic acid oligosaccharides, providing support for further structural identification of these modified hyaluronic acid oligosaccharides.

Description

Technical Field

[0001] This application belongs to the field of detection technology, specifically, it relates to a method for separating modified hyaluronic acid oligosaccharides. Background Technology

[0002] Hyaluronic acid (HA) is a linear non-sulfated glycosaminoglycan composed of repeating disaccharide units: d-glucuronic acid (1→3)--dN-acetylglucosamine (1→4) (e.g.) Figure 1 (As shown). Hyaluronic acid is present in all vertebrates, and its primary structure is conserved, with variations only in the molecular weight and polydispersity index of the polymer. Human skin, eyes, and extracellular matrix are rich in hyaluronic acid. Due to its large negative charge, hyaluronic acid is highly hydrophilic and can therefore be used as a space filler, lubricant, and osmotic buffer. However, the poor mechanical properties of natural hyaluronic acid and its rapid degradation and elimination in vivo limit its application as a biomaterial. To improve the mechanical properties of hyaluronic acid and increase its resistance to hyaluronidase degradation, it is usually chemically modified or cross-linked to form hydrogels.

[0003] The most common method for preparing cross-linked hyaluronic acid is to react hyaluronic acid with the cross-linking agent 1,4-butanediol diglycidyl ether (BDDE) under alkaline conditions, forming a stable covalent ether bond between the hyaluronic acid and the cross-linking agent. During the cross-linking process, the epoxy groups of BDDE react with the nucleophilic groups on the hyaluronic acid molecules to form 1,4-butanediol di(propane-2,3-diol) ether (BDPE) derivatives. Some BDDE cross-linking agent molecules exist with free hydroxyl groups at both ends, some BDDE molecules are bound to hyaluronic acid at only one end, and some BDDE molecules are bound to hyaluronic acid at both ends (e.g., ...). Figure 2 (as shown in ac).

[0004] To characterize the cross-linking of cross-linked hyaluronic acid, it is necessary to separate and identify the modified oligosaccharides obtained after enzymatic hydrolysis of cross-linked hyaluronic acid. However, since there are many types of modified hyaluronic acid oligosaccharides obtained after enzymatic hydrolysis of cross-linked hyaluronic acid, there is currently no effective separation method to fully separate the modified hyaluronic acid oligosaccharides. Summary of the Invention

[0005] To address the problems existing in the prior art, this application provides a method for separating modified hyaluronic acid oligosaccharides.

[0006] Specifically, this application relates to the following aspects:

[0007] 1. A method for separating modified hyaluronic acid oligosaccharides, characterized in that the separation method comprises the following steps:

[0008] Modified hyaluronic acid oligosaccharides were separated using liquid chromatography-mass spectrometry.

[0009] The liquid chromatography-mass spectrometry (LC-MS) technique utilizes a porous graphitized carbon column with gradient elution using mobile phases A and B.

[0010] Mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and mobile phase B is an aqueous solution of ammonium formate or ammonium acetate in acetonitrile.

[0011] 2. The separation method according to item 1, wherein the concentration of ammonium formate or ammonium acetate in the aqueous solution of ammonium formate or ammonium acetate in mobile phase A is 5-10 mM.

[0012] 3. According to the separation method described in item 1, wherein the concentration of ammonium formate or ammonium acetate in the acetonitrile aqueous solution of ammonium formate or ammonium acetate in mobile phase B is 5-10 mM, and the concentration of acetonitrile is 90-95 wt%.

[0013] 4. The separation method according to item 1, wherein the column temperature of the chromatographic column in the liquid chromatography is 25-35℃.

[0014] 5. The separation method according to item 1, wherein the flow rate used in the liquid chromatography is 0.2-0.4 mL / min. -1 .

[0015] 6. The separation method according to item 1, wherein the modified hyaluronic acid oligosaccharide is a modified hyaluronic acid oligosaccharide that has been fully reduced by a reducing agent.

[0016] 7. The separation method according to item 1, wherein the modified hyaluronic acid oligosaccharide is obtained by enzymatic hydrolysis of cross-linked hyaluronic acid.

[0017] 8. The separation method according to item 7, wherein the cross-linked hyaluronic acid is hyaluronic acid cross-linked by 1,4-butanediol di(propane-2,3-diol) ether.

[0018] 9. The separation method according to item 1, wherein the mass spectrometry conditions for liquid chromatography-mass spectrometry are:

[0019] In negative ion scanning mode, the sheath gas flow rate is 10-20 arb, the auxiliary gas flow rate is 0-5 arb, the spray voltage is 3-4 kV, the capillary temperature is 250-350 ℃, and the ion lens voltage is 30-70% V.

[0020] The separation method of this application can fully separate various modified hyaluronic acid oligosaccharides, providing support for further structural identification of the modified hyaluronic acid oligosaccharides. Attached Figure Description

[0021] Figure 1The molecular structure of hyaluronic acid is shown.

[0022] Figure 2 This shows the form in which BDDE exists in cross-linked hyaluronic acid.

[0023] Figure 3 This indicates possible modification sites for unsaturated hyaluronic acid disaccharide.

[0024] Figure 4 The schematic structures of the four types of suspended (2-B, 4-B) and cross-linked modified (2-B-2, 4-B-2) fragments that account for the highest proportion in the degradation products of cross-linked hyaluronic acid are shown.

[0025] Figure 5 Showing the absolute RS configuration of the BDDE molecule.

[0026] Figure 6 This demonstrates the NaBH4 reduction of unsaturated hyaluronic acid disaccharide α and β anosomes.

[0027] Figure 7 This shows the separation of the modified hyaluronic acid oligosaccharides obtained using the separation method of Example 1 in chromatography.

[0028] Figure 8 This shows the separation of the modified hyaluronic acid oligosaccharides obtained using the separation method of Example 2 in chromatography.

[0029] Figure 9 This shows the separation of the modified hyaluronic acid oligosaccharides obtained using the separation method of Comparative Example 1 in chromatography.

[0030] Figure 10 This shows the separation of the modified hyaluronic acid oligosaccharides obtained using the separation method of Comparative Example 2 in chromatography. Detailed Implementation

[0031] The present application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present application.

[0032] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art. While similar or identical methods and materials may be applied in experimental or practical applications, materials and methods are described herein. In case of conflict, the definitions included herein shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to be limiting. The present application is further described below with reference to specific embodiments, but is not intended to limit the scope of the application.

[0033] Theoretically, each HA disaccharide unit has 7 sites that can react with BDDE, including the hydroxyl groups connected to the carbon atoms at positions 2 and 3 of uronic acid (HexA) and positions 1, 4, and 6 of N-acetylglucosamine (GlcNAc), the carboxyl group of HexA, and the amide group of GlcNAc (such as...). Figure 3 (As shown). Deprotonated hydroxyl groups are more nucleophilic than carboxyl and amide groups. Furthermore, under alkaline conditions, any ester formed with the HA carboxylic acid group may undergo hydrolysis. Therefore, the hydroxyl group is the most likely site to react with BDDE and form a stable ether bond with the crosslinking agent. The relative trends in the binding affinity of different hydroxyl groups to BDDE are not yet clear, but may be related to factors such as steric differences between primary and secondary alcohols, conformational effects of the polymer, differences in hydroxyl acidity, and differences in reaction conditions.

[0034] Because the molecular weight of HA crosslinked with BDDE is large and its solubility in water is poor, the degree of crosslinking is usually characterized by using a tool enzyme (such as hyaluronidase or chondroitin sulfate ABC) to degrade it into oligosaccharide fragments of different sizes. During enzymatic hydrolysis, a carbon-carbon double bond is formed between the C atoms at positions 4 and 5 of the oligosaccharide product hexuronic acid (e.g., ...). Figure 3 (As shown). Oligosaccharide fragments can be divided into two types: suspended modified fragments and cross-linked modified fragments. The schematic structures of the four types of suspended (2-B, 4-B) and cross-linked modified (2-B-2, 4-B-2) fragments, which account for the largest proportion of degradation products, are shown in the figure. Figure 4 .

[0035] It should be noted that, as mentioned above, there are multiple ways in which BDDE connects to the HA sugar unit. Figure 4 Only one crosslinking configuration (2-B(GlcNAc-OH6)) of a certain type of structure (e.g., 2-B) is shown. Furthermore, due to the different absolute configurations (R or S) of the C atoms at the 2' and 2' positions of the BDDE crosslinking agent (e.g., ... Figure 5 As shown, a specific oligosaccharide (such as 2-B(GlcNAc-OH6)) may also be affected by the absolute configuration of BDDE, forming different isomers.

[0036] On the other hand, the sugar units at the ends of the oligosaccharide chain undergo C1 isomerization during the formation of hemiacetals, resulting in the formation of two non-corresponding isomers (α and β). These isomers are called anomers. Taking unsaturated hyaluronic acid disaccharide as an example, the hydroxyl group attached to the C atom at the 1-position of GlcNAc may be located on the same side or opposite side of the C atom at the 6-position of the sugar ring plane, forming β and α anomers respectively (e.g., Figure 6 The presence of anosomes further increases the difficulty of isolating and structurally analyzing modified hyaluronic acid oligosaccharides.

[0037] To address the problems existing in the prior art, this application provides a method for separating modified hyaluronic acid oligosaccharides. In this application, modified hyaluronic acid oligosaccharides refer to chemically modified hyaluronic acid oligosaccharides. For example, various hyaluronic acid oligosaccharides can be obtained by enzymatic hydrolysis of cross-linked hyaluronic acid.

[0038] In one specific embodiment, the cross-linked hyaluronic acid is hyaluronic acid cross-linked with 1,4-butanediol di(propane-2,3-diol) ether. The predominant modified hyaluronic acid oligosaccharide obtained after the degradation of this cross-linked hyaluronic acid is, for example... Figure 4 As shown, it includes four types of structures: 2-B, 4-B, 2-B-2, and 4-B-2.

[0039] As mentioned above, the terminal sugar units of oligosaccharide chains undergo C1 isomerization during the formation of hemiacetals, leading to the formation of two isomers, α and β. To eliminate the influence of these two isomers on chromatographic separation, a reducing agent can be used to reduce the terminal sugar units of the oligosaccharide and form an open-ring structure (e.g., ...). Figure 6 (As shown). It should be noted that if the hydroxyl group on the anomeric C is modified with BDDE, the reduction reaction cannot occur. The reducing agent described in this application can be a commonly used reducing agent in the art, and those skilled in the art can make conventional selections, for example, sodium borohydride (NaBH4).

[0040] In one specific embodiment, the modified hyaluronic acid oligosaccharide is a modified hyaluronic acid oligosaccharide that has been fully reduced by sodium borohydride. Specifically, excess sodium borohydride can be used to react with the modified hyaluronic acid oligosaccharide, and then the remaining unreacted sodium borohydride is neutralized.

[0041] The method for isolating modified hyaluronic acid oligosaccharides according to this application includes the following steps:

[0042] Modified hyaluronic acid oligosaccharides were separated using liquid chromatography-mass spectrometry (LC-MS).

[0043] LC-MS uses liquid chromatography as the separation system and mass spectrometry as the detection system. The components in a sample are separated in the liquid chromatography section, ionized, and then the ion fragments are separated according to their mass number by the mass spectrometer, and the mass spectrum is obtained by the detector. LC-MS combines the complementary advantages of chromatography and mass spectrometry, combining the high separation capability of chromatography for complex samples with the high selectivity, high sensitivity, and ability of MS to provide relative molecular mass and structural information. It has been widely used in many fields such as pharmaceutical analysis, food analysis, and environmental analysis.

[0044] In this liquid chromatography-mass spectrometry (LC-MS) technique, a porous graphitized carbon column is used with gradient elution of mobile phases A and B. In one specific embodiment, the porous graphitized carbon column is a Hypercarb porous graphitized carbon column.

[0045] Mobile phase A is an aqueous solution of ammonium formate or ammonium acetate, and mobile phase B is an aqueous solution of ammonium formate or ammonium acetate in acetonitrile.

[0046] In one specific embodiment, the liquid chromatography-mass spectrometry technique of this application is liquid chromatography-mass spectrometry.

[0047] In one specific embodiment, the mobile phase A is an aqueous solution of ammonium formate, wherein the concentration of ammonium formate is 5-10 mM, for example, it can be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

[0048] In one specific implementation, the pH of mobile phase A can be adjusted to 2.5-3.5, for example, to 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, or 3.5. The pH of mobile phase A can be adjusted using conventional acids, bases, or buffer solutions; for example, formic acid can be used.

[0049] In one specific embodiment, the mobile phase A is an aqueous solution of ammonium acetate, wherein the concentration of ammonium acetate is 5-10 mM, for example, it can be 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM.

[0050] In one specific implementation, the pH of mobile phase A can be adjusted to 2.5-3.5, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, or 3.5. The pH of mobile phase A can be adjusted using conventional acids, bases, or buffer solutions; for example, acetic acid can be used.

[0051] In one specific embodiment, the mobile phase B is an aqueous solution of ammonium formate in acetonitrile, wherein the concentration of ammonium formate is 5-10 mM, for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. The concentration of acetonitrile is 90-95 wt%, for example, 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, or 95 wt%.

[0052] In one specific implementation, the pH of mobile phase B can be adjusted to 2.5-3.5, for example, to 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, or 3.5. The pH of mobile phase A can be adjusted using conventional acids, bases, or buffer solutions; for example, formic acid can be used.

[0053] In one specific embodiment, mobile phase B is an aqueous solution of ammonium acetate in acetonitrile, wherein the concentration of ammonium acetate is 5-10 mM, for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. The concentration of acetonitrile is 90-95 wt%, for example, 90 wt%, 91 wt%, 92 wt%, 93 wt%, 94 wt%, or 95 wt%.

[0054] In one specific implementation, the pH of mobile phase B can be adjusted to 2.5-3.5, for example, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, or 3.5. The pH of mobile phase A can be adjusted using conventional acids, bases, or buffer solutions; for example, acetic acid can be used.

[0055] In one specific embodiment, mobile phase A is a 5 mM ammonium formate aqueous solution, with the pH adjusted to 3 using formic acid; mobile phase B is a 5 mM ammonium formate aqueous solution with acetonitrile, with the pH adjusted to 3 using formic acid; and the acetonitrile concentration is 90 wt%.

[0056] In one specific embodiment, mobile phase A is a 5 mM ammonium acetate aqueous solution, with the pH adjusted to 3 using acetic acid; mobile phase B is a 5 mM ammonium acetate aqueous solution of acetonitrile, with the pH adjusted to 3 using acetic acid; and the acetonitrile concentration is 90 wt%.

[0057] In one specific implementation, the gradient elution process of this application can be divided into five stages, specifically:

[0058] In the first stage, the volume ratio of mobile phase A to mobile phase B is (90-100):(10-0);

[0059] In the second stage, the volume ratio of mobile phase A to mobile phase B is (75-85):(25-15);

[0060] In the third stage, the volume ratio of mobile phase A to mobile phase B is (40-50):(60-50);

[0061] In the fourth stage, the volume ratio of mobile phase A to mobile phase B is (0-10):(100-90);

[0062] In the fifth stage, the volume ratio of mobile phase A to mobile phase B is (90-100):(10-0).

[0063] In one specific implementation, the gradient elution process of this application can be divided into five stages, specifically:

[0064] In the first stage, the volume ratio of mobile phase A to mobile phase B is 95:5;

[0065] In the second stage, the volume ratio of mobile phase A to mobile phase B is 78:22;

[0066] In the third stage, the volume ratio of mobile phase A to mobile phase B is 45:55;

[0067] In the fourth stage, the volume ratio of mobile phase A to mobile phase B is 0:100;

[0068] In the fifth stage, the volume ratio of mobile phase A to mobile phase B is 95:5.

[0069] In one specific implementation, the gradient elution process of this application is as follows:

[0070] In the first stage, from 0 to 3 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5.

[0071] In the second stage, from 3 to 100 minutes, the volume ratio of mobile phase A to mobile phase B is 78:22.

[0072] In the third stage, from 100 to 105 minutes, the volume ratio of mobile phase A to mobile phase B is 45:55.

[0073] In the fourth stage, from 105 to 115 minutes, the volume ratio of mobile phase A to mobile phase B is 0:100.

[0074] In the fifth stage, from 115 to 140 minutes, the volume ratio of mobile phase A to mobile phase B is 95:5.

[0075] In one specific implementation, the column temperature of the chromatographic column in the liquid chromatography-mass spectrometry technique is 25-35℃, for example, it can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃.

[0076] In one specific implementation, the flow rate used in the liquid chromatography-mass spectrometry (LC-MS) technique is 0.2-0.4 mL / min. -1 For example, it can be 0.2 mL / min. -1 0.3 mL / min -1 0.4 mL / min -1 .

[0077] In one specific implementation, the mass spectrometry conditions for liquid chromatography-mass spectrometry (LC-MS) are as follows:

[0078] In negative ion scanning mode, the sheath gas flow rate is 10-20 arb, the auxiliary gas flow rate is 0-5 arb, the spray voltage is 3-4 kV, the capillary temperature is 250-350 ℃, and the ion lens voltage is 50% V.

[0079] In one specific implementation, the mass spectrometry conditions for liquid chromatography-mass spectrometry (LC-MS) are as follows:

[0080] In negative ion scanning mode, the sheath gas flow rate is 15 arb, the auxiliary gas flow rate is 3 arb, the spray voltage is 3.5 kV, the capillary temperature is 275.00 ℃, and the ion lens voltage is 50% V.

[0081] The separation method of this application can fully separate various BDDE-modified hyaluronic acid oligosaccharides, including five structures of 2-B, three structures of 4-B, and multiple structures of 2-B-2 and 4-B-2, providing support for further structural identification of the modified hyaluronic acid oligosaccharides.

[0082] Example

[0083] Example 1

[0084] (1) Enzymatic hydrolysis of cross-linked hyaluronic acid

[0085] Enzymatic hydrolysis buffer: Accurately weigh approximately 2.74 g of sodium dihydrogen phosphate dihydrate and approximately 0.35 g of anhydrous disodium hydrogen phosphate, place them in the same 100 ml volumetric flask, dissolve them in water and dilute quantitatively to the mark, shake well, accurately measure 10 ml and place it in a 100 ml volumetric flask, dilute quantitatively to the mark with water, shake well, adjust the pH to 6.6 with 1 mol / L sodium hydroxide solution, sterilize at 121℃ for 10 minutes, cool, and the solution is ready.

[0086] Hyaluronidase solution (10,000 units / ml): Take one vial of hyaluronidase (50,000 units) (Bloomage Biotechnology Co., Ltd.), transfer it to a 5ml volumetric flask, quantitatively dilute to the mark with enzyme digestion buffer, and shake well to obtain the solution.

[0087] The enzymatic hydrolysate, i.e., the test solution, is prepared as follows: Accurately weigh approximately 10 mg of cross-linked hyaluronic acid (Bloomage Biotechnology Co., Ltd.), place it in a 20 ml headspace vial, add 1 ml of enzymatic hydrolysis buffer and 0.5 ml of water, seal, cap, and sterilize in an oven at 121°C for 10 minutes. After cooling, inject 1 ml of hyaluronidase solution (10,000 units / ml) into the headspace vial using a syringe. Place the vial in a water bath at 42°C and 190 rpm for 24 hours of enzymatic hydrolysis. Transfer the solution to a 5 ml volumetric flask, wash the headspace vial once with 0.5 ml of 0.1 mol / L hydrochloric acid solution, and wash the headspace vial once with an appropriate amount of water. Combine the washings with the same volumetric flask, dilute quantitatively with water to the mark, shake well, and filter to obtain the final solution.

[0088] (2) Sample purification

[0089] The enzymatic hydrolysate obtained above was transferred to an ultrafiltration tube (500 μl, molecular weight cutoff of 10 kDa) and centrifuged at 5000 rcf for 20 min. 400 μl of H2O was added to the upper tube, and the tube was centrifuged again at 5000 rcf for 20 min. The filtrate in the lower tube was retained. These two centrifugations improved the recovery rate of the modified hyaluronic acid oligosaccharides, and the final concentration of the modified hyaluronic acid oligosaccharide sample was approximately 1 mg / ml.

[0090] (3) Sample reduction

[0091] Take 4 ml of the filtrate obtained in step (2) into the sample vial, and slowly add 0.3 ml of 0.5 M NaBH4 solution (prepared fresh for use). React at 42 °C for 1 h (shaking once every 20 minutes). After the reaction is complete, add 0.35 ml of 0.5 M acetic acid to the sample vial to neutralize the remaining unreacted NaBH4. Freeze-dry the above sample and reconstitute it in 200 μl of H2O. The final concentration of the modified hyaluronic acid oligosaccharide is approximately 20 mg / ml.

[0092] (4) LC-MS separation

[0093] The modified hyaluronic acid oligosaccharides obtained in step (3) were separated using LC-MS. The specific chromatographic conditions used in the LC-MS are as follows:

[0094] Chromatographic column: Hypercarb porous graphitized carbon column (column length 150cm, column inner diameter 4.6mm, packing particle size 5μm).

[0095] Chromatographic conditions: Mobile phase A: 5 mM ammonium formate aqueous solution, adjusted to pH 3.0 with formic acid; Mobile phase B: 5 mM ammonium formate 90% acetonitrile aqueous solution, adjusted to pH 3.0 with formic acid; Column temperature: 26℃; Flow rate: 0.2 mL / min -1 The elution gradient is shown in Table 1. The injection volume was 3 μl.

[0096] Table 1

[0097] Serial Number Time (min) A(％) B(％) curve 1 0 95 5 5 2 3 78 22 5 3 100 45 55 5 4 105 0 100 5 5 115 0 100 5 6 115.01 95 5 5 7 140 95 5 5

[0098] The eluent from 0-18 min and 115-140 min was introduced into the waste liquid, and the remainder was introduced into the mass spectrometer.

[0099] The mass spectrometry conditions (LTQ-Orbitrap, Thermo Fisher Scientific) were as follows: negative ion scanning mode; sheath gas flow rate 15 arb; auxiliary gas flow rate 3 arb; spray voltage 3.5 kV; capillary temperature 275.00 °C; ion lens voltage 50% V.

[0100] The separation of modified hyaluronic acid oligosaccharides obtained by LC-MS is as follows: Figure 7 As shown.

[0101] Depend on Figure 7 As can be seen, 2-B exhibits 5 chromatographic peaks in the chromatography, while 4-B exhibits 3 chromatographic peaks. After reduction, 2-B-2 can be further divided into two structural types. The first type has an m / z of 963.36 (z = -1), in which both GlcNAc sugar units at the disaccharide reducing end are reduced by NaBH4 to form an open-ring structure. The second type has an m / z of 961.35 (z = -1), in which only one of the two GlcNAc sugar units at the disaccharide reducing end is reduced by NaBH4 to form an open-ring structure, indicating that the hydroxyl group at position 1 of the unreduced GlcNAc sugar unit is modified by the BDDE molecule. Similar to 2-B-2, 4-B-2, after reduction, can be further divided into two types of structures. The first type has an m / z of 670.74 (z = -2), where both GlcNAc sugar units at the reducing ends of the tetrasaccharide and disaccharide are reduced by NaBH4 to form an open-ring structure. The second type has an m / z of 669.73 (z = -2), where only one of the two GlcNAc sugar units at the reducing ends of the oligosaccharide is reduced by NaBH4 to form an open-ring structure. This indicates that the hydroxyl group (-OH1) at position 1 of the unreduced GlcNAc sugar unit is modified by BDDE molecules. Based on the mechanism of hyaluronidase, the BDDE-modified GlcNAc (-OH1) structures at the reducing ends of 2-B-2 and 4-B-2 likely originate from the reducing ends of the hyaluronic acid sugar chains.

[0102] Example 2

[0103] The difference between Example 2 and Example 1 is that the sample reduction step (3) in Example 1 is not included. Instead, the modified hyaluronic acid oligosaccharide sample obtained in step (2) is directly separated by LC-MS. The other steps and detection conditions are the same as in Example 1.

[0104] The separation of modified hyaluronic acid oligosaccharides obtained by LC-MS is as follows: Figure 8 As shown. By Figure 8 It can be seen that the components cannot be fully separated. This is because the lack of sample reduction has two effects: First, due to the presence of anosomes, the structural complexity of the target analyte is doubled, and the peaks of each anosome influence each other, resulting in insufficient chromatographic separation; Second, the two different types of structures in 2-B-2 and 4-B-2 ( Figure 7 Before reduction, the mass-to-nucleus ratio is exactly the same, making it impossible to distinguish them by extracting the particle flow map (EIC), which increases the complexity of the spectrum.

[0105] Comparative Example 1

[0106] The only difference between Comparative Example 1 and Example 1 is that the specific chromatographic conditions are different; the other steps and detection conditions are the same as in Example 1.

[0107] Specifically, the only difference between Comparative Example 1 and Example 1 is the use of mobile phase A and mobile phase B. In Comparative Example 1, mobile phase A was a 0.1% aqueous formic acid solution, and mobile phase B was a 0.1% formic acid acetonitrile solution.

[0108] The separation of modified hyaluronic acid oligosaccharides obtained by LC-MS is as follows: Figure 9 As shown. By Figure 9 It can be seen that the modified hyaluronic acid oligosaccharide components were not eluted sufficiently or had poor separation, and could not be effectively separated.

[0109] Comparative Example 2

[0110] The only difference between Comparative Example 2 and Example 1 is that the chromatographic column used is different; the other steps and detection conditions are the same as in Example 1.

[0111] Specifically, the only difference between Comparative Example 2 and Example 1 is the chromatographic column used. The chromatographic column used in Comparative Example 2 is an amino column BEH Amide column (2.1 mm × 150 mm, 1.9 μm).

[0112] The separation of modified hyaluronic acid oligosaccharides obtained by LC-MS is as follows: Figure 10 As shown. By Figure 10 It can be seen that the separation of the modified hyaluronic acid oligosaccharide components is poor, and effective separation of each component cannot be achieved.

Claims

1. A method for isolating a modified hyaluronan oligosaccharide, characterized in that, The separation method comprises the following steps: The modified hyaluronic acid oligosaccharide is separated by using liquid chromatography-mass spectrometry technology, In the liquid chromatography-mass spectrometry technology, a porous graphitized carbon chromatographic column is used, and gradient elution is performed by using mobile phase A and mobile phase B, The mobile phase A is an ammonium formate or ammonium acetate aqueous solution, wherein the concentration of ammonium formate or ammonium acetate in the ammonium formate or ammonium acetate aqueous solution is 5-10mM; The mobile phase B is an ammonium formate or ammonium acetate acetonitrile solution, wherein the concentration of ammonium formate or ammonium acetate in the ammonium formate or ammonium acetate acetonitrile solution is 5-10mM, and the concentration of acetonitrile is 90-95wt%; The modified hyaluronic acid oligosaccharide is a modified hyaluronic acid oligosaccharide after sufficient reduction by a reducing agent.

2. The separation method of claim 1, wherein, The column temperature of the chromatographic column in the liquid chromatography is 25-35℃.

3. The separation method of claim 1, wherein, The flow rate used in liquid chromatography is 0.2-0.4 mL.min -1 .

4. The separation method of claim 1, wherein, The modified hyaluronic acid oligosaccharide is obtained by enzymatic hydrolysis of cross-linked hyaluronic acid.

5. The separation method of claim 4, wherein, The cross-linked hyaluronic acid is hyaluronic acid cross-linked by 1,4-butanediol di(propane-2,3-diol) ether.

6. The separation method of claim 1, wherein, The mass spectrometry conditions of the liquid chromatography-mass spectrometry technology are as follows: Negative ion scanning mode, sheath gas flow rate is 10-20arb, auxiliary gas flow rate is 0-5arb, spray voltage is 3-4KV, capillary temperature is 250-350℃, and ion lens voltage is 30-70%V.

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