A method for determining the content of polysaccharides or polysaccharide derivatives

Abstract

This invention relates to a method for detecting the content of polysaccharides or polysaccharide derivatives, wherein the structural units of the polysaccharides or polysaccharide derivatives contain acetamino groups. The method utilizes strongly alkaline conditions to degrade polysaccharide samples containing acetamino groups, producing acetate. The acetate content in the degradation solution is separated and detected using chromatographic methods. Based on the detection results, the content of the polysaccharide or polysaccharide derivative in the sample is calculated. This method offers high accuracy and precision. Compared to the traditional sulfuric acid-carbazole method, it effectively avoids interference from substances containing hexuronic acid structures, colored substances or substances that react with concentrated sulfuric acid to produce color, and reducing substances that react with concentrated sulfuric acid / carbazole. This significantly improves the specificity and accuracy of quality control testing for this type of product, and greatly reduces the consumption of concentrated sulfuric acid, effectively reducing environmental pollution.

Description

Technical Field

[0001] This invention belongs to the field of product quality inspection, and specifically relates to a method for determining the content of polysaccharides or polysaccharide derivatives. Background Technology

[0002] Most polysaccharides or polysaccharide derivatives contain acetylamino groups in their structural units, which can degrade to acetate under alkaline conditions. Hyaluronic acid (HA) is a polysaccharide containing an acetylamino group, also known as hyaluronic acid. It is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid via a β-1,4 glycosidic bond and N-acetylglucosamine via a β-1,3 glycosidic bond. It is widely used in cosmetics and ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects.

[0003] Currently, the most commonly used method for detecting hyaluronic acid content is the sulfuric acid-carbazole colorimetric method. This method is the standard method in relevant regulations for hyaluronic acid products, such as the national drug standard for sodium hyaluronate injection (WS1-(X-058)-2006Z), YY / T 0308-2015 Medical Sodium Hyaluronate Gel, YY / T 1571-2017 Tissue Engineering Medical Device Sodium Hyaluronate, and YY / T0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery.

[0004] However, when this method is used to detect hyaluronic acid content, it lacks specificity and can be significantly interfered with by other substances containing hexuronic acid structures (such as glucuronic acid, galacturonic acid, and pectin), colored substances (such as iron ions and red to purple-red pigments), substances that produce color when reacting with concentrated sulfuric acid (such as glucose, α-tocopherol, and hydroquinone), and reducing substances that react with concentrated sulfuric acid / carbazole (such as formic acid and its salts, lactic acid and its salts, oxalic acid and its salts, gallic acid and its salts, and ascorbic acid and its salts). Furthermore, this method involves harsh operating conditions and uses large amounts of concentrated sulfuric acid, resulting in high operational risks and significant environmental pollution.

[0005] In some literature, gel chromatography is commonly used to determine hyaluronic acid. However, this method has poor specificity, severe peak broadening, and stringent requirements for the sample to be tested. It is not suitable for the detection of hyaluronic acid with a wide molecular weight distribution and cross-linked hyaluronic acid.

[0006] Patent WO2005114186 A1 discloses a method for determining hyaluronic acid using hyaluronic acid-binding protein (HABP). This invention discloses that hyaluronic acid forms a hyaluronic acid / HABP complex by contacting hyaluronic acid with HABP latex particles. This complex is then reacted with a carrier carrying an anti-HABP antibody to form aggregates. The optical changes caused by the aggregates are measured, and the amount of hyaluronic acid is calculated based on the measurement data. The principle of this method is that HA binds to HABP and then specifically reacts with an anti-HABP antibody. However, the difficulty lies in the fact that anti-HABP antibodies are not readily available commercially, and the operation process is cumbersome and complex, making it difficult to perform in a routine laboratory.

[0007] Patent CN109298112 B provides a method for determining hyaluronic acid content using an enzymatic hydrolysis method combined with high-performance liquid chromatography (HPLC). However, this method requires prior enzymatic hydrolysis of the sample before detection. Incomplete hydrolysis can affect the test results, and the hydrolysis process is lengthy and complex. CN107561179A discloses a method for determining the degree of cross-linking of cross-linked hyaluronic acid or its salts, but it uses an uncommon molecular sieve column, making it unsuitable for widespread application.

[0008] Polysaccharides or polysaccharide derivatives containing acetamide groups in their structural units can degrade to form acetate under alkaline conditions. Therefore, the content of polysaccharides or polysaccharide derivatives can be indirectly calculated by detecting the amount of acetate produced during degradation. Currently, there are no reports on the application of this method to the detection of polysaccharide or polysaccharide derivative content.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] To overcome the shortcomings of existing technologies, this invention provides a novel method for detecting the content of polysaccharides or polysaccharide derivatives. The method utilizes strongly alkaline conditions to degrade polysaccharide or polysaccharide derivative samples containing acetaminophen, producing acetate. The acetate content in the degradation solution is separated and detected using chromatography. Based on the detection results, the content of polysaccharides or polysaccharide derivatives in the sample is calculated. This detection method effectively improves the anti-interference and accuracy of quality control testing for products containing polysaccharides or polysaccharide derivatives, while significantly reducing the amount of concentrated sulfuric acid used, thus lowering its harm to the environment and humans.

[0011] The first aspect of this invention provides a method for detecting the content of a polysaccharide or polysaccharide derivative, wherein the structural unit of the polysaccharide or polysaccharide derivative contains an acetylamino group, and comprises:

[0012] (1) The sample to be tested is degraded under strong alkaline conditions to generate degradation products;

[0013] (2) Determine the content of acetate in the degradation products obtained in step (1), and calculate the content of polysaccharide or polysaccharide derivative by measuring the content of acetate.

[0014] Specifically, the sample to be tested in step (1) contains polysaccharides or polysaccharide derivatives.

[0015] Specifically, the polysaccharide or polysaccharide derivative may be a cross-linked and / or uncross-linked polysaccharide or polysaccharide derivative.

[0016] Specifically, the crosslinking agent used for crosslinking can be a diepoxide (e.g., 1,3-butadiene diepoxide, 1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether (BDDE), polypropylene glycol diglycidyl ether, etc.), an unsaturated sulfone (e.g., divinyl sulfone), and active amino compounds (e.g., arginine and its derivatives, lysine and its derivatives, polyglutamic acid and its derivatives, spermine, spermidine, etc.), polyaldehyde crosslinking agents (e.g., glutaraldehyde), polyacid anhydrides or polyacrylamide halides (e.g., succinyl chloride), and complex functional group crosslinking agents (e.g., epichlorohydrin). In one embodiment of the present invention, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0017] In one embodiment of the present invention, the crosslinking agent is divinyl sulfone (DVS).

[0018] In one embodiment of the present invention, the crosslinking agent is spermidine.

[0019] Specifically, the polysaccharide or polysaccharide derivative may be selected from hyaluronic acid or hyaluronic acid salts (such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or zinc hyaluronate), preferably sodium hyaluronate.

[0020] In one embodiment of the present invention, the sample to be tested contains uncrosslinked and / or crosslinked hyaluronic acid or hyaluronic acid salts.

[0021] In one embodiment of the present invention, the sample to be tested contains both uncrosslinked and crosslinked sodium hyaluronate.

[0022] In one embodiment of the present invention, the molecular weight of the sodium hyaluronate is 200,000 to 3,000,000 Daltons (e.g., 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, 1,200,000, 1,400,000, 1,500,000, 1,600,000, 1,800,000, 2,000,000, 2,500,000, 3,000,000 Daltons). Preferably, the molecular weight of the sodium hyaluronate in the test sample containing sodium hyaluronate is 300,000 to 2,000,000 Daltons.

[0023] Specifically, the sample to be tested may also be a gel and / or a solution.

[0024] In one embodiment of the present invention, the sample to be tested is a gel.

[0025] Specifically, the strong alkaline condition in step (1) is to add a strong alkaline solution to the sample to be tested, wherein the strong alkaline solution is one or a mixture of sodium hydroxide solution, potassium hydroxide solution, and lithium hydroxide solution.

[0026] In one embodiment of the present invention, the strong alkaline solution is a sodium hydroxide solution.

[0027] Specifically, the concentration of the strong alkali solution is 0.1–10 mol / L, preferably 0.5–6 mol / L;

[0028] For example, the concentration of the strong alkali solution can be 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, 2.5 mol / L, 3.0 mol / L, 3.5 mol / L, 4.0 mol / L, 4.5 mol / L, 5 mol / L, 5.5 mol / L, or 6 mol / L.

[0029] Specifically, the degradation conditions described in step (1) are as follows: add a strong alkaline solution in an amount that is k times the number of moles of polysaccharide or polysaccharide derivative in the sample to be tested, and heat at a certain temperature T℃ for th.

[0030] In one embodiment of the present invention, the degradation conditions in step (1) are as follows: a strong alkaline solution is added to the sample to be tested in an amount k times the number of moles of sodium hyaluronate, and heated at a certain temperature T℃ for th.

[0031] To achieve complete degradation, an excess of alkali solution needs to be added, with the excess k being at least 5. More specifically, the excess k can be between 5 and 5000 (e.g., 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5). (18, 18.5, 19, 19.5, 20, 30, 40, 50, 60, 70, 80, 90, 100, 300, 500, 700, 900, 1000, 1500, 2000, 3000, 3500, 4000, 4500, 5000), preferably, the multiple k is 5 to 3000, and more preferably, the multiple k is 5 to 1500.

[0032] Preferably, the temperature T is at least 60°C, and more preferably, the temperature T is 60 to 250°C (e.g., 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 210°C, 220°C, 230°C, 240°C, 250°C).

[0033] In some embodiments of the present invention, the temperature T is 60–120°C.

[0034] Preferably, the time t is at least 0.5h, and more preferably, the time t is 0.5 to 24h (e.g., 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 8.5h, 9h, 9.5h, 10h, 15h, 20h, 22h, 24h).

[0035] In some embodiments of the present invention, the time t is 0.5 to 6 hours.

[0036] Specifically, step (1) may further include adding an acid solution to the obtained degradation product for neutralization.

[0037] Specifically, before determining the acetate content in the degradation products, step (2) may also include a step of quantitatively diluting the mixed solution obtained in step (1) with water or a buffer salt solution to prepare a test solution.

[0038] Specifically, the neutralization operation by adding acid solution involves taking out the sample after it has been completely degraded and cooling it, then adding an acid solution of equal volume to the aforementioned strong alkaline solution to neutralize the reaction system.

[0039] Preferably, the acid solution is one or a mixture of phosphoric acid, sulfuric acid, nitric acid, perchloric acid, hydroiodic acid, hydrobromic acid, or hydrochloric acid.

[0040] In one embodiment of the present invention, the acid solution is hydrochloric acid.

[0041] More preferably, the concentration of the acidic solution is 0.1 to 10 mol / L; even more preferably, the concentration of the acidic solution is 0.5 to 6 mol / L.

[0042] For example, the concentration of the acidic solution can be 0.5 mol / L, 1.0 mol / L, 1.5 mol / L, 2.0 mol / L, 2.5 mol / L, 3.0 mol / L, 3.5 mol / L, 4.0 mol / L, 4.5 mol / L, 5 mol / L, 5.5 mol / L, or 6 mol / L.

[0043] Specifically, the test solution is prepared by diluting the sample to be tested with water or buffer salt solution as a solvent after degradation and neutralization. The amount of polysaccharide or polysaccharide derivative contained therein should be within the quantitative concentration range of polysaccharide or polysaccharide derivative in this method. The polysaccharide or polysaccharide derivative is preferably hyaluronic acid or hyaluronic acid salt. Hyaluronic acid salt can be one of sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate and zinc hyaluronate, preferably sodium hyaluronate.

[0044] Preferably, the buffer salt solution is a mixed solution composed of a weak acid and its salt, and a weak base and its salt; more preferably, the buffer salt solution is one or more mixed solutions selected from phosphate buffer, formate buffer, citrate buffer, citrate buffer, carbonate buffer, borate buffer, ammonia-ammonium chloride buffer, triethylamine buffer, and tris(hydroxymethyl)aminomethane buffer; more preferably, the buffer salt solution is a phosphate buffer; more preferably, the pH of the buffer salt solution can be 1-14 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14); more preferably, the concentration of the buffer salt solution can be 0.01-1 mol / L (e.g., 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1 mol / L);

[0045] In some embodiments of the present invention, the solvent for the test solution is phosphate buffer solution with a pH of 5-9 and a concentration of 0.05-0.5 mol / L.

[0046] Preferably, the quantitative concentration range of the polysaccharide or polysaccharide derivative is 0.01 to 10 mg / g; more preferably, the quantitative concentration range is 0.01 to 5 mg / g.

[0047] Specifically, the content of acetate in step (2) is based on the acetate standard substance used to prepare the external standard method reference solution, such as sodium acetate, potassium acetate, magnesium acetate, calcium acetate, barium acetate, and zinc acetate. Preferably, the acetate in step (2) is sodium acetate.

[0048] Specifically, the detection of acetate in step (2) is performed by chromatography (such as liquid chromatography or gas chromatography), preferably by external standard method.

[0049] Specifically, the chromatographic method includes liquid chromatography and gas chromatography. Preferably, the chromatographic method is one of reversed-phase chromatography, hydrophilic interaction chromatography, ion chromatography, ion exchange chromatography, ion pair chromatography, and gas chromatography. More preferably, the chromatographic method is reversed-phase chromatography or gas chromatography.

[0050] Specifically, the detector used in the liquid chromatography method can be one of the following: ultraviolet absorption detector (UVD), photodiode array detector (PDAD), fluorescence detector (FD), refractive index detector (RID), electrochemical detector (ED), and chemiluminescence detector (CD).

[0051] Specifically, the detector used in the gas chromatography method can be one of the following: flame ionization detector (FID), nitrogen-phosphorus detector (NPD), flame photometric detector (FPD), thermal conductivity detector (TCD), photoionization detector (PID), and electron capture detector (ECD).

[0052] In some embodiments of the present invention, the chromatographic method is gas chromatography.

[0053] More preferably, the chromatographic column used in the gas chromatography is selected from common flexible quartz capillary columns; even more preferably, it is selected from flexible quartz capillary columns that retain the target component.

[0054] For example, the stationary phase can be 100% polydimethylsiloxane, 5% diphenyl (95%) dimethylpolysiloxane, 5% diphenyl 1% vinyl (94%) dimethylpolysiloxane, 50% diphenyl (50%) dimethylpolysiloxane, 14% cyanopropylphenyl (including 7% cyanopropyl 7% phenyl) (86%) dimethylpolysiloxane, 50% cyanopropylphenyl (including 25% cyanopropyl 25% phenyl) (50%) dimethylpolysiloxane, 6% cyanopropylbenzene-94% dimethylsiloxane, polyethylene glycol, and its modified forms; in some embodiments of the present invention, the chromatographic column used in the gas chromatography is an elastic quartz capillary column with 6% cyanopropylbenzene-94% dimethylsiloxane as the stationary phase.

[0055] Specifically, the chromatographic method of the present invention also includes certain pretreatment of the test solution.

[0056] Preferably, the pretreatment method is selected from acidification reaction, alkalization reaction, and derivatization reaction; in some embodiments of the present invention, the pretreatment method is a derivatization reaction;

[0057] More preferably, the reagent for the derivatization reaction is a concentrated sulfuric acid lower alcohol solution, a derivatizing reagent containing an acyl bromide group, or a silanizing reagent; for example, the reagent for the derivatization reaction can be a methanol solution of concentrated sulfuric acid, an ethanol solution of concentrated sulfuric acid, benzoyl bromide, p-nitrobenzoyl bromide, p-oxybenzoyl bromide and p-bromobenzoyl bromide, or dimethylsilane trifluoroacetamide; in some embodiments of the present invention, the reagent for the derivatization reaction is an ethanol solution of concentrated sulfuric acid.

[0058] Specifically, the external standard method described in this invention involves pretreatment and separation of a reference solution prepared using a certain amount of acetate standard material and the test solution in parallel.

[0059] Preferably, the acetate standard substance is selected from water-soluble salts formed by acetic acid and metal ions, such as sodium acetate, potassium acetate, lithium acetate, calcium acetate, magnesium acetate, barium acetate, zinc acetate, etc.

[0060] Preferably, the external standard method is selected from the external standard one-point method, the external standard two-point method, the standard curve method, and the standard addition method; more preferably, the standard curve method is selected.

[0061] Preferably, the solvent of the reference solution should be the same as the solvent of the aforementioned test solution;

[0062] Preferably, the concentration of the reference solution should be within the aforementioned quantitative concentration range.

[0063] Specifically, the content of hyaluronic acid or hyaluronic acid salt (HA) is calculated using the following formula:

[0064]

[0065] In the formula:

[0066] c represents the concentration of acetate in the test solution calculated using the external standard method;

[0067] F is the volume of the test solution;

[0068] m is the sampling mass of the sample to be tested;

[0069] α is the ratio of the molecular weight of the repeating disaccharide structural unit of hyaluronic acid or hyaluronic acid salt (HA) to the molecular weight of the standard substance acetate used in the external standard method;

[0070] β is the unit conversion factor.

[0071] In one embodiment of the present invention,

[0072] Acetate in the sodium hyaluronate test solution was separated and detected by chromatography. The acetate concentration was quantitatively calculated using sodium acetate as a standard substance according to the external standard method. The sodium hyaluronate (HA) content (mg / g) in the test sample was calculated using the following formula:

[0073]

[0074] In the formula:

[0075] c represents the concentration of acetate in the test solution (μg / ml) calculated using the external standard method;

[0076] F represents the volume (ml) of the test solution;

[0077] m is the sample mass (g) of the sample to be tested;

[0078] 4.9821 is the ratio of the molecular weight (401.3) of the repeating disaccharide structural unit of sodium hyaluronate to the molecular weight (82.13) of the standard substance (sodium acetate) used in the external standard method;

[0079] 1000 is the conversion factor.

[0080] The second aspect of the present invention provides the application of the detection method as described in the first aspect in the quality evaluation of multi-component mixtures.

[0081] Specifically, the multi-component mixture is selected from one of the following: cosmetics, health foods, pharmaceuticals, medical devices, hair care products, oral care products, and paper products.

[0082] Specifically, the multi-component mixture contains uncrosslinked and / or crosslinked polysaccharides or polysaccharide derivatives, which may be selected from hyaluronic acid or hyaluronic acid salts (such as sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or zinc hyaluronate). Preferably, the polysaccharide or polysaccharide derivative is sodium hyaluronate.

[0083] The beneficial effects of this invention are:

[0084] This invention develops a novel detection method for polysaccharides or polysaccharide derivatives. Compared to traditional detection methods (such as the sulfuric acid-carbazole colorimetric method), it effectively avoids interference from other substances containing hexuronic acid structures (such as glucuronic acid, galacturonic acid, and pectin), colored substances (such as iron ions and red to purple-red pigments), substances that produce color upon reaction with concentrated sulfuric acid (such as glucose, α-tocopherol, and hydroquinone), and reducing substances that react with concentrated sulfuric acid / carbazole (such as formic acid and its salts, lactic acid and its salts, oxalic acid and its salts, gallic acid and its salts, and ascorbic acid and its salts), thus having a wider range of applications. Furthermore, compared to other physicochemical analysis methods, such as the hyaluronic acid enzymatic hydrolysis method and the protein complexation method, the detection method of this invention is simple to operate, low in cost, and highly resistant to interference, enabling rapid and accurate determination of the sodium hyaluronate content in sodium hyaluronate products. Moreover, the consumption of organic solvents and toxic reagents during the detection process is low, especially compared to traditional detection methods (such as the sulfuric acid-carbazole colorimetric method), significantly reducing the amount of concentrated sulfuric acid used, resulting in extremely low harm to the environment and humans. Attached Figure Description

[0085] Figure 1 The chromatogram is for a blank solution;

[0086] Figure 2 The chromatogram is of the reference solution;

[0087] Figure 3 The chromatogram of the test solution;

[0088] Figure 4 This is a standard curve graph. Detailed Implementation

[0089] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0090] In this invention, the term "sample containing sodium hyaluronate" refers to a gel and / or solution sample containing uncrosslinked and / or crosslinked sodium hyaluronate; wherein, the term "crosslinking" is the process of transforming a linear polymer with chemical reactivity into a three-dimensional network (three-dimensional) polymer through a chemical reaction, and is often used in polymer modification.

[0091] In this invention, the term "digestion" is a chemical term, also known as "wet digestion," which is a method of destroying organic matter or reducing substances in a sample using acid, alkali, or oxidizing agents under heating conditions.

[0092] In this invention, the term "external standard method" is a commonly used quantitative method. It is a method that obtains the content of the analyte in the test sample solution by comparing the test sample solution with a reference solution of the analyte with a known content. In the field of chromatographic detection, the commonly used quantitative basis is peak area or peak height.

[0093] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0094] Example 1: Detection of Sodium Hyaluronate Content in Typical Samples

[0095] 1. Sample preparation

[0096] Take 10.25g of sodium hyaluronate that has been dried at 105℃ for 6h, place it in a 1000ml Schott flask, add 100ml of water for injection, seal, and stir for 6h to fully dissolve; add 12ml of 20% sodium hydroxide solution, stir to thin the gel, then add 1ml of cross-linking agent 1,4-butanediol diglycidyl ether (BDDE), stir evenly, and incubate at 25℃ for 24h in a biochemical incubator; adjust the pH to 7.0 with 2mol / L hydrochloric acid solution, seal, and stir for 6h to fully mix; add 0.1mol / L phosphate buffer solution (pH=7.0) to a final volume of 1000.52g, seal, and continue stirring for 6h to fully mix; sterilize at 121℃ for 30min, cool, and store for later use.

[0097] 2. Solution preparation

[0098] (1) Take about 0.4g of sample, accurately weigh (accurate to 0.1mg), place it in a test tube of appropriate size (such as a polytetrafluoroethylene tube or a polypropylene tube), add 10ml of 2mol / L sodium hydroxide solution, boil in a boiling water bath for 2h, take it out and let it cool, add 10ml of 2mol / L hydrochloric acid solution to neutralize, let it cool, quantitatively transfer it to a 50ml volumetric flask, add water to make up to the mark, shake well, and use it as the test solution.

[0099] (2) Select sodium acetate as the standard substance for the external standard method. Weigh approximately 109.04 mg of sodium acetate reference standard after drying at 120℃ for 2 hours. Accurately weigh the amount and place it in a 100 ml volumetric flask. Add 0.5 mol / L phosphate buffer solution (pH=7.0) to dissolve and dilute to the mark. Shake well to obtain the standard stock solution. (3) Accurately measure an appropriate amount of the standard stock solution and dilute it with 0.5 mol / L phosphate buffer solution (pH=7.0) to prepare a 200 μg / ml solution as the standard working solution. Accurately measure the standard working solution and dilute it with water according to Table 1 below to prepare the standard curve solution.

[0100] Table 1

[0101]

[0102] 3. Detection Method

[0103] (1) Chromatographic conditions

[0104] A capillary column (DB-624, 30m × 0.53mm × 3μm; or other equivalent performance) was used as the stationary phase. The temperature program was as follows: initial temperature 40℃, hold for 5 min, increase to 220℃ at 30℃ / min, hold for 5 min. The detector was a flame ionization detector (FID) with a detection chamber temperature of 250℃ and a vaporization chamber temperature of 200℃ (split ratio 1:1). Headspace injection was used with an equilibration temperature of 55℃ and an equilibration time of 90 min.

[0105] (2) Determination method

[0106] Accurately measure 1.0 ml each of the standard curve solution and the test solution, place them in 20 ml headspace vials, add 1.0 ml of 15% sulfuric acid ethanol solution to each, shake well, seal, inject via headspace, and record the chromatogram.

[0107] (3) Calculation of results

[0108] A standard curve was obtained by linearly regressing the concentration (C) of sodium acetate in the standard curve solution against the peak area (A) of the sodium acetate derivative (ethyl acetate). The concentration of sodium acetate in the test sample solution was calculated from the standard curve based on the peak area of ​​the sodium acetate derivative (ethyl acetate) in the test sample solution. The sodium hyaluronate (HA) content (mg / g) in the test sample was then calculated using the following formula:

[0109]

[0110] In the formula:

[0111] c represents the concentration (μg / ml) of sodium acetate in the test solution calculated using the external standard method.

[0112] F represents the volume of the test solution (50 ml);

[0113] m is the sample mass (g);

[0114] 4.9821 is the ratio of the molecular weight (401.3) of the repeating disaccharide structural unit of sodium hyaluronate to the molecular weight (82.13) of the standard substance sodium acetate used in the external standard method;

[0115] 1000 is the conversion factor.

[0116] 4. System suitability test

[0117] Use water instead of the sample to prepare a blank solution according to the above-described method for preparing the test solution; use the above-described standard working solution as the reference solution.

[0118] Take 1.0 ml each of the blank solution, reference solution, and the typical test solution prepared above, place them in 20 ml headspace vials, add 1.0 ml of 15% sulfuric acid ethanol solution to each, shake well, seal, inject via headspace, and record the chromatogram.

[0119] From the chromatogram of the reference solution Figure 2 Chromatograms of the test solution Figure 3 It can be seen that both methods detected acetic acid derivative peaks around 5.1 min, with no interfering peaks near this location; the theoretical plate numbers of the acetic acid derivative peaks were all no less than 5000, and the tailing factors were all between 0.8 and 1.2; while the blank solution yielded... Figure 1 No acetic acid derivative peaks were detected, indicating that this method has good system applicability for detecting sodium hyaluronate content in samples.

[0120] 5. Sample testing

[0121] Six test solutions were prepared consecutively, and the content of sodium hyaluronate (HA) in the samples was calculated. The results are shown in Table 2.

[0122] Table 2 Results of HA content detection in samples

[0123]

[0124] As can be seen from Table 2, the measured HA content in the sample is basically equivalent to the theoretical content (10.24 mg / g), and the parallelism of the results is good.

[0125] Example 2: Detection of Sodium Hyaluronate Content in Typical Samples

[0126] The content of the samples was detected according to the method of Example 1. The difference was that in step "1. Sample preparation", divinyl sulfone (DVS) was used instead of 1,4-butanediol diglycidyl ether (BDDE) to prepare sample A (the sample weight of sodium hyaluronate was 10.11 g, and the final sample weight was 1000.05 g); and 1% spermidine solution was used instead of 1,4-butanediol diglycidyl ether (BDDE) to prepare sample B (the sample weight of sodium hyaluronate was 10.02 g, and the final sample weight was 1000.08 g).

[0127] Three samples A and three samples B from the same batch were tested consecutively, and the HA content in each sample was calculated. The results are shown in Table 3.

[0128] Table 3. Detection results of HA content in samples A and B.

[0129]

[0130] As can be seen from Table 3, the measured HA content in samples treated with two different cross-linking agents is basically equivalent to the theoretical content, and the parallelism of the results is good, suggesting that the method of the present invention can be used to detect the HA content in sodium hyaluronate samples treated with different cross-linking agents.

[0131] Example 3: Detection of Hyaluronic Acid Content in Typical Samples

[0132] The content of the samples was determined according to the method described in Example 1, except for the preparation method of the test solution. Uncrosslinked sodium hyaluronate and medical zinc hyaluronate gel were used as samples, with sample amounts of 0.02 g and 5.0 g respectively. Three parallel tests were performed on each sample, and the hyaluronic acid salt (HA) content was calculated. The results are shown in Table 4 below:

[0133] Table 4. Results of HA content detection in different samples

[0134]

[0135] Note: The molecular weight of the repeating disaccharide structural unit of zinc hyaluronic acid is calculated as 410.8.

[0136] Table 4 shows that the HA content determination results in uncrosslinked sodium hyaluronate raw material (theoretical content 100% or 1000 mg / g) and zinc hyaluronate gel (theoretical content 0.103% or 1.03 mg / g) are basically equivalent to the theoretical values, and the parallelism of the results is good. This suggests that the method of the present invention can be used to detect the HA content in non-crosslinked sodium hyaluronate and other hyaluronic acid salts (such as zinc hyaluronate) samples.

[0137] Example 4: Detection of Sodium Hyaluronate Content in Typical Samples

[0138] The detection method is the same as in Example 1, except that the detection method used is reversed-phase chromatography, as detailed below:

[0139] (1) Chromatographic conditions

[0140] The chromatographic column (Xbridge C18, 5 μm, 4.6 × 250 mm; or other equivalent performance) was packed with octadecylsilane-bonded silica gel; the mobile phase was phosphoric acid solution (0.7 mL of phosphoric acid was added to 1000 mL of water, and the pH was adjusted to 2.5 with 0.1 mol / L sodium hydroxide solution) - methanol (95:5); the flow rate was 1 mL / min; the detection wavelength was 210 nm; and the injection volume was 20 μl.

[0141] (2) Determination method

[0142] Accurately measure 20 μl each of the standard curve solution and the test solution, inject them into the liquid chromatograph, and record the chromatograms.

[0143] (3) Calculation of results

[0144] A standard curve was obtained by linearly regressing the concentration (C) of sodium acetate in the standard curve solution against the peak area (A) of the sodium acetate derivative (acetic acid). The concentration of sodium acetate in the test sample solution was calculated from the standard curve based on the peak area of ​​the sodium acetate derivative (acetic acid) in the test sample solution, and the sodium hyaluronate (HA) content (mg / g) in the sample was calculated using the following formula:

[0145]

[0146] In the formula:

[0147] c represents the concentration (μg / ml) of sodium acetate in the test solution calculated using the external standard method.

[0148] F represents the volume of the test solution (50 ml);

[0149] m is the sample mass (g);

[0150] 4.9821 is the ratio of the molecular weight of the sodium hyaluronate disaccharide structural unit (401.3) to the molecular weight of the sodium acetate standard used in the external standard method (82.13);

[0151] 1000 is the conversion factor.

[0152] The test solution and standard curve were prepared according to Example 1. Six samples were tested consecutively, and the content of sodium hyaluronate (HA) in the samples was calculated. The results are shown in Table 5.

[0153] Table 5. Results of HA content detection in samples

[0154]

[0155] As can be seen from Table 5, the measured HA content in the sample is basically equivalent to the theoretical content (10.24 mg / g), and the parallelism of the results is good.

[0156] Example 5: Investigation of Digestion Conditions

[0157] The method described in Example 1 was followed, with the difference being the preparation method of the test solution. 1 mol / L sulfuric acid solution and concentrated sulfuric acid were used instead of 2 mol / L sodium hydroxide solution as digestion solutions (when using concentrated sulfuric acid, the volume added was 3.0 ml, and 6 mol / L sodium hydroxide solution was used instead of 2 mol / L hydrochloric acid solution). Three replicates of the test solution were prepared for each digestion solution, and the HA content was determined. The results are shown in Table 6.

[0158] Table 6. Results of HA content detection under strong acid digestion conditions.

[0159]

[0160] As can be seen from Table 6, when strong acid solution was used instead of strong alkali to digest the sample, the measured HA content was significantly lower than the theoretical content (10.24 mg / g). The reason for this result may be that the acetamino structure in hyaluronic acid and its salts is not completely degraded under acidic conditions, resulting in a lower amount of acetate. Therefore, acidic solution digestion is not suitable for the detection of hyaluronic acid or its salts.

[0161] Example 6: Durability Study of Digestion Conditions

[0162] The method described in Example 1 was followed, except for the preparation method of the test solution. Approximately 0.4 g (equivalent to about 0.1 mmol) of the sample prepared in Example 1 was accurately weighed, resulting in nine portions. Test solutions were prepared under different alkali addition amounts (0.5 mmol, 20 mmol, 150 mmol), digestion temperatures (60℃, 100℃, and 120℃), and digestion times (0.5 h, 2 h, and 6 h). The HA content in each sample was then calculated, and the results are shown in Table 7.

[0163] Table 7 Results of HA content detection under different sample digestion conditions

[0164]

[0165] As shown in Table 7, under the conditions of varying alkali addition (0.5–150 mmol, HA ratio 5–1500), digestion temperature (60℃–120℃), and digestion time (0.5–6 h), the HA determination results in the samples were basically equivalent to the theoretical content (10.24 mg / g). This suggests that the required alkali addition is at least 0.5 mmol (HA ratio 5), the digestion temperature is above 60℃, and the digestion time is above 0.5 h to fully digest the samples without affecting the final detection results.

[0166] Quantitative Range Examination of the Method in Example 7

[0167] Accurately measure the standard stock solution from Example 1, and dilute the series of standard solutions with 0.5 mol / L phosphate buffer (pH = 7.0) within the sodium acetate concentration range of 2–2000 μg / ml. Detect the response values ​​(based on peak area) according to the method in Example 1. The results are shown in Table 8 below:

[0168] Table 8 shows the test results of a series of concentration standard solutions.

[0169]

[0170]

[0171] Using the results from the table above, with the response value (based on peak area) as the y-axis and the corresponding sodium acetate concentration as the x-axis, perform linear regression and plot the standard curve. The results are as follows. Figure 4 As shown, the regression equation is y = 0.1236x + 0.0864, ​​R0 2 =0.9999 (n=16), indicating that sodium acetate exhibits a good linear relationship with the response value within a concentration range of 2–2000 μg / ml. Based on the compositional ratio of sodium acetate in the HA structure, the quantitative concentration range of HA obtained by the method of this invention is 0.01–10 mg / g.

[0172] Example 8: Method Recovery Rate Investigation

[0173] Take the sample from Example 1 (theoretical HA content is 10.24 mg / g), and accurately weigh approximately 0.2 g, 0.4 g, and 1.0 g at three different levels (high, medium, and low), with three replicates for each level, for a total of nine samples. Detect the HA content in each sample using the method described in Example 1 (measured amount), and compare it with the theoretical amount (theoretical amount = sample weight × theoretical HA content). Calculate the recovery rate (recovery rate = measured amount / theoretical amount × 100%). The results are shown in Table 9 below:

[0174] Table 9 Recovery Rate Results

[0175]

[0176] The results in the table show that the average recovery rate of the nine samples was 100.0%, and the relative standard deviation (RSD) was 2.2%, indicating that the method has a certain degree of accuracy and repeatability.

[0177] Comparison of results between the comparative example and the sulfuric acid-carbazole colorimetric method

[0178] Take 11.07 g of sodium hyaluronate that has been dried at 105℃ for 6 h, place it in a 1000 ml Schott flask, add 100 ml of water for injection, seal, and stir for 6 h to fully dissolve; add 12 ml of 20% sodium hydroxide solution, stir to thin the gel, then add 1 ml of cross-linking agent 1,4-butanediol diglycidyl ether (BDDE), stir evenly, and react in a biochemical incubator at 25℃ for 24 h; adjust the pH to 7.0 with 2 mol / L hydrochloric acid solution, then add 0.1 mol / L phosphate buffer solution (pH = 7.0) to a final volume of 1000.52 g, seal, and stir for 6 h to fully mix; sterilize by moist heat at 121℃ for 30 min, cool, and this is sample C.

[0179] Accurately weigh approximately 100g of sample C and place it in a 250ml Schott flask, making a total of 6 flasks. According to Table 10, accurately add approximately 10g each of the following: 0.1mol / L phosphate buffer solution (pH=7.0), 10% D-glucuronic acid solution, 10% ferrous sulfate solution, 10% glucose solution, 10% lactic acid solution, and 10% ascorbic acid solution. Seal the flasks and stir for 6 hours to ensure thorough mixing. This process yields the following samples for research: standard sample, interference sample A, interference sample B, interference sample C, interference sample D, and interference sample E.

[0180] Table 10 Composition of Samples Used in Each Study

[0181]

[0182] The above-mentioned conventional samples and each interfering sample A to E were taken and tested according to the method (sulfuric acid-carbazole colorimetric method) under Appendix C "Determination of Sodium Hyaluronate Content" of "YY / T 0962-2021 Cross-linked Sodium Hyaluronate Gel for Plastic Surgery" in Example 1. The content of sodium hyaluronate (HA) in each sample was calculated and compared. The results are shown in Table 11.

[0183] Table 11 Results of HA content measured in samples from different studies using different methods

[0184]

[0185] The above results show that when this application method and the sulfuric acid-carbazole colorimetric method are used to detect the HA content in routine samples containing hyaluronic acid prepared with phosphate buffer, the detection results of the two methods are basically equivalent. However, when the sample contains substances with hexuronic acid structure (such as interfering sample A containing glucuronic acid), colored substances (such as interfering sample B containing ferric sulfate), or substances that change color upon reaction with sulfuric acid (such as interfering sample C containing glucose), the HA content measured by the sulfuric acid-carbazole colorimetric method is significantly higher. Conversely, when the sample contains reducing substances that react with concentrated sulfuric acid / carbazole (such as interfering sample D containing lactic acid and interfering sample E containing ascorbic acid), the HA content measured by the sulfuric acid-carbazole colorimetric method is significantly lower. This result suggests that the traditional sulfuric acid-carbazole colorimetric method is easily interfered with by substances containing hexuronic acid structures, colored substances or substances that change color upon reaction with sulfuric acid, and reducing substances that can react with concentrated sulfuric acid / carbazole, and cannot accurately determine the HA content in the sample. However, the method of this application can still accurately determine the HA content in each of the interfering samples A to E containing interfering substances, indicating that the detection method of this invention can significantly avoid these interferences and has a wider range of applications.

[0186] In summary, this invention completely degrades the acetamino groups on the structural units of cross-linked or uncross-linked polysaccharides or polysaccharide derivatives (such as hyaluronic acid, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or zinc hyaluronate) under alkaline conditions to produce acetate. The content of polysaccharides or polysaccharide derivatives (such as hyaluronic acid, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, or zinc hyaluronate) is indirectly calculated by detecting the content of acetate generated during degradation. Acid degradation cannot completely degrade acetylamino groups, and it also degrades the ether bonds between D-glucuronic acid and N-acetylglucosamine during the process, affecting the detection results. In addition, the aminoglucose product after alkaline hydrolysis is highly polar and cannot be detected in conventional reversed-phase systems or gas chromatography, requiring special chromatographic columns (ion exchange columns or amino columns), which are complex, cumbersome, and costly. In contrast, this invention uses conventional reversed-phase systems or gas chromatography to detect the content of acetate generated by degradation, which has the advantages of simple detection conditions, strong operability, high accuracy, and strong anti-interference, providing a new and effective detection method for polysaccharides or polysaccharide derivatives.

[0187] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of a polysaccharide or polysaccharide derivative, wherein the structural unit of the polysaccharide or polysaccharide derivative contains an acetylamino group, comprising: (1) The sample to be tested is degraded under strong alkaline conditions to generate degradation products; (2) Determine the content of acetate in the degradation product obtained in step (1), and calculate the content of polysaccharide or polysaccharide derivative by the content of acetate; the polysaccharide or polysaccharide derivative is sodium hyaluronate. The preparation steps of the test sample are as follows: Take 10.25g of sodium hyaluronate dried at 105℃ for 6h, place it in a 1000ml Schott flask, add 100ml of water for injection, seal, and stir for 6h to fully dissolve; add 12ml of 20% sodium hydroxide solution, stir to thin the gel, then add 1ml of crosslinking agent 1,4-butanediol diglycidyl ether, stir evenly, and react in a biochemical incubator at 25℃ for 24h; adjust the pH to 7.0 with 2mol / L hydrochloric acid solution, seal, and stir for 6h to fully mix; add 0.1mol / L phosphate buffer solution at pH=7.

0. Add liquid to 1000.52g, seal, and continue stirring for 6 hours to ensure thorough mixing; sterilize at 121℃ for 30 minutes, cool, and set aside; or, in the preparation steps, use divinyl sulfone instead of 1,4-butanediol diglycidyl ether to prepare the sample to be tested, the amount of sodium hyaluronate is 10.11g, and the final mass of the sample to be tested is 1000.05g; or, in the preparation steps, use 1% spermidine solution instead of 1,4-butanediol diglycidyl ether to prepare the sample to be tested, the amount of sodium hyaluronate is 10.02g, and the final mass of the sample to be tested is 1000.08g; The strong alkaline conditions in step (1) involve adding a strong alkaline solution to the sample to be tested, and adding the strong alkaline solution in an amount that is k times the number of moles of polysaccharides or polysaccharide derivatives contained in the sample to be tested, where k is at least 5; the degradation conditions include heating at a certain temperature T after adding the strong alkaline solution, where the heating temperature T is at least 60°C; and the heating time t is at least 0.5 h. Step (1) also includes adding an acid solution to the obtained degradation product for neutralization; The detection of acetate in step (2) is performed by chromatography, specifically gas chromatography. The gas chromatography column used is an elastic quartz capillary column; the stationary phase is 6% cyanopropylbenzene-94% dimethylsiloxane; the temperature program is an initial temperature of 40℃, maintained for 5 min, then increased to 220℃ at a rate of 30℃ / min, and maintained for 5 min; the detector is a flame ionization detector.

2. The detection method according to claim 1, characterized in that, The strong alkaline solution is one or a mixture of sodium hydroxide solution, potassium hydroxide solution, and lithium hydroxide solution, and the concentration of the strong alkaline solution is 0.1~10 mol / L.

3. The detection method according to claim 2, characterized in that, The concentration of the strong alkali solution is 0.5~6 mol / L.

4. The detection method according to claim 1, characterized in that, The heating temperature T is 60~250℃.

5. The detection method according to claim 4, characterized in that, The heating temperature T is 60~120℃.

6. The detection method according to claim 1, characterized in that, The heating time t is 0.5~24h.

7. The detection method according to claim 6, characterized in that, The heating time t is 0.5~6h.

8. The detection method according to claim 1, characterized in that, The acid solution added to the obtained degradation product in step (1) is one or a mixture of phosphoric acid, sulfuric acid, nitric acid, perchloric acid, hydroiodic acid, hydrobromic acid or hydrochloric acid. Before determining the acetate content in the degradation products, step (2) further includes diluting the mixed solution obtained in step (1) with water or a buffer salt solution to prepare a test solution. The buffer salt solution is a mixed solution composed of a weak acid and its salt, and a weak base and its salt.

9. The detection method according to claim 8, characterized in that, The buffer salt solution is one or a mixture of phosphate buffer, formate buffer, citrate buffer, citrate buffer, carbonate buffer, borate buffer, ammonia-ammonium chloride buffer, triethylamine buffer, and tris(hydroxymethyl)aminomethane buffer.

10. The detection method according to claim 9, characterized in that, The buffer salt solution is a phosphate buffer solution.

11. The detection method according to claim 8, characterized in that, The detection of acetate in step (2) is performed using the external standard method.

12. The detection method according to claim 11, characterized in that, The content of sodium hyaluronate (HA) is calculated using the following formula: In the formula: c represents the concentration of acetate in the test solution calculated using the external standard method; F is the volume of the test solution; m is the sampling mass of the sample to be tested; α is the ratio of the molecular weight of the repeating disaccharide structural unit of sodium hyaluronate (HA) to the molecular weight of the standard substance acetate used in the external standard method; β is the unit conversion factor.

13. The detection method according to claim 12, characterized in that, The standard substance used in the external standard method is sodium acetate.

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