A method for detecting the grafting rate of aliphatic polyester in aliphatic polyester coupled polysaccharide graft polymer

Abstract

The present application relates to a kind of aliphatic polyester coupling polysaccharide graft polymer in the detection method of grafting rate of aliphatic polyester, by chromatography respectively detects aliphatic polyester content and polysaccharide quality in graft polymer, by calculating the ratio of aliphatic polyester content and polysaccharide content obtains the grafting rate of aliphatic polyester grafting to polysaccharide.The method of the present application is strong in specificity, high accuracy, good repeatability, strong operability.According to the method of the present application, the grafting rate of aliphatic polyester in aliphatic polyester coupling polysaccharide graft polymer can be quickly measured.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical analysis, and particularly relates to a method for detecting grafting rate of aliphatic polyester in aliphatic polyester coupled polysaccharide grafting polymer. BACKGROUND

[0002] Sodium hyaluronate or hyaluronic acid (HA for short) is a glycosaminoglycan composed of disaccharide units of D-glucuronic acid through a beta-1, 4 glycosidic bond and N-acetylglucosamine through a beta-1, 3 glycosidic bond. It is widely used in the field of cosmetics or ophthalmic surgery, and can also be used as a soft tissue filler to repair wrinkles and some soft tissue defects. Sodium hyaluronate is a biomaterial in vivo, has good biocompatibility and certain biological activity, but exogenous sodium hyaluronate will be degraded by sodium hyaluronate enzyme in vivo, resulting in a shortened residence time in vivo and a shortened treatment effect, and multiple injections are required to achieve the treatment effect. In order to avoid the degradation of sodium hyaluronate by sodium hyaluronate enzyme, it is necessary to crosslink the sodium hyaluronate molecules by a chemical crosslinking agent to form a spatial network structure, which prevents the degradation of sodium hyaluronate by sodium hyaluronate enzyme and other enzymes, prolongs the residence time of exogenous sodium hyaluronate in vivo, and ensures good biocompatibility while achieving good treatment effect.

[0003] Polylactic acid (PLA) is a polyester polymerized from lactic acid. Polylactic acid has excellent biodegradability, compatibility and absorbability, and is a non-toxic and non-irritating synthetic polymer material. Poly-L-lactic acid (PLLA) is a polymer of L-lactic acid, which can be degraded into L-lactic acid in vivo, which is one of the metabolic products of human polysaccharides. Therefore, PLLA has good tissue compatibility and is widely used in bone pins, bone plates, and medical and cosmetic fields.

[0004] By using endogenous polyamine as a coupling agent, sodium hyaluronate is coupled with poly-L-lactic acid to obtain HA-b-PLLA graft copolymer. The formed HA-b-PLLA graft copolymer has amphiphilicity and can be applied in drug carriers, tissue engineering materials and soft tissue repair fields.

[0005] Considering that the performance of the polylactic acid coupled sodium hyaluronate product is closely related to the grafting rate of polylactic acid, and so far there is no exclusive sensitive, accurate and reliable detection method for characterizing the grafting rate of polylactic acid in polylactic acid coupled sodium hyaluronate.

[0006] Patent document CN114366817A discloses a method for coupling sodium hyaluronate (HA) and EPA through chemical grafting, and uses nuclear magnetic resonance (NMR) to detect the chemical grafting rate of EPA; prior art (Wang Juanqin, Modification and Functionalization of Hyaluronic Acid, *Engineering Technology I*, a full-text database of excellent master's theses in China, No. 2, 20140215) discloses the use of H... 1 -NMR characterizes the grafting rate of HA and -DOPA, and calculates the dopamine substituent in the graft by the ratio of its integrated area to the integrated area of ​​H on NHCOCH3 in the HA structural unit. Existing technology (Pitarresi G et al., Injectable insitu forming microgels of hyaluronic acid-g-polylactic acid for methylprednisolone release[J]. European Polymer Journal, 2013, 49(3): 718-725.) discloses that the grafting rate is determined by comparing the integrals of each peak measured by 1H NMR. Existing technology (Nor Azillah Fatimah Othman et al., Grafting yield determination of glycidyl methacrylate vapor on radiated kenaf fiber via FTIR spectroscopy[J]. Materials today:proceedings, 2020, 29(1): 207-211) discloses a method for calculating the grafting rate by quantitative Fourier transform infrared spectroscopy (FTIR).

[0007] However, NMR (Negative Nuclear Magnetic Resonance) analysis is affected by chemical shift, coupling constant, integral area, and splitting, with the integral area being particularly susceptible to subjective interference. Furthermore, NMR is an electromagnetic detector with relatively low sensitivity. Infrared spectroscopy is generally unsuitable for analyzing aqueous samples because the hydroxyl peaks in water interfere with the determination. Secondly, quantitative analysis using infrared spectroscopy suffers from large errors and low sensitivity. Additionally, interpretation of infrared spectra often relies on experience. This invention uses liquid chromatography (LC) to detect lactic acid content to determine polylactic acid (PLA) content, and gas chromatography (GC) to detect sodium hyaluronate content in the sample. Because gas-liquid phase detectors offer higher sensitivity and accuracy, and the GC method effectively improves the analysis of polymers in liquid-liquid phases, the determination of polylactic acid (PLA) content is significantly improved. 1 The influence of the integration factor on the results in H NMR and FTIR determinations is explained. Therefore, the detection method of the present invention has good detection accuracy, operability, and universal applicability.

[0008] In summary, existing technologies rarely provide indirect chromatographic methods for determining the grafting rate of aliphatic polyesters in aliphatic polyester-coupled polysaccharide grafted polymers. There is an urgent need to develop a specific, sensitive, accurate, and reliable method for characterizing the polylactic acid grafting rate in polylactic acid-coupled sodium hyaluronate grafted polymers. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a method for detecting the grafting rate of aliphatic polyesters in aliphatic polyester-polysaccharide-grafted polymers. The method involves detecting the aliphatic polyester and polysaccharide contents separately using chromatography, and calculating the grafting rate of the aliphatic polyester onto the polysaccharide by calculating the ratio of the aliphatic polyester content to the polysaccharide content. This method is specific, sensitive, accurate, reliable, and involves simple sample processing, and is stable and effective. The grafting rate of this invention is calculated using the following formula:

[0010]

[0011] In the formula:

[0012] L represents the grafting rate, expressed as a percentage by mass.

[0013] R represents the content of aliphatic polyester in the aliphatic polyester-coupled polysaccharide graft polymer, expressed in mg / g.

[0014] T represents the polysaccharide content in the aliphatic polyester-coupled polysaccharide graft polymer, expressed in mg / g.

[0015] Furthermore, the polysaccharide is selected from hyaluronic acid or hyaluronic acid salts; preferably, the hyaluronic acid salt is selected from potassium hyaluronic acid, sodium hyaluronic acid, and calcium hyaluronic acid, and more preferably, the polysaccharide is sodium hyaluronic acid;

[0016] Furthermore, the aliphatic polyester is polylactic acid; the polylactic acid includes polyracemic lactic acid and poly-L-lactic acid, and more preferably, the aliphatic polyester is poly-L-lactic acid.

[0017] Preferably, the aliphatic polyester-coupled polysaccharide graft polymer is a polylactic acid-coupled sodium hyaluronate graft polymer.

[0018] The grafting rate of polylactic acid in the polylactic acid-coupled sodium hyaluronate grafted polymer is preferably 0.1% to 99.9%.

[0019] The coupling agent for the polylactic acid-sodium hyaluronate grafted polymer is preferably an endogenous polyamine, including spermine, putrescine, and spermidine.

[0020] Furthermore, the method for detecting polylactic acid is as follows: the polylactic acid in the grafted polymer is degraded into lactic acid, and the lactic acid content is detected by liquid chromatography to calculate the polylactic acid content.

[0021] Furthermore, the method for detecting polylactic acid includes: taking a polylactic acid-coupled sodium hyaluronate grafted polymer sample m1, adding an enzyme, performing a single degradation, centrifuging after complete degradation, separating the solid and liquid, removing the supernatant, adding an alkaline solution to the remaining solid, performing a second degradation, adding an acidic solution to neutralize, and detecting the lactic acid concentration as C1 using liquid chromatography; blank correction is performed using purified water in the same manner.

[0022] The primary degradation conditions include: a temperature of 25–45°C and a time of 2–12 hours;

[0023] For example, the temperature can be: 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃.

[0024] For example, the time can be: 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h.

[0025] The secondary degradation conditions include a temperature of 60–100°C and a time of 0.5–4 hours.

[0026] For example, the time can be: 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h.

[0027] For example, the secondary degradation temperature can be 60℃, 70℃, 80℃, 90℃, or 100℃.

[0028] Preferably, the enzyme is hyaluronidase, and the concentration of the hyaluronidase is 100-1500 U / mL;

[0029] More preferably, the concentration of the hyaluronidase is 100–500 U / mL.

[0030] For example, the concentration of the hyaluronidase can be 100U / mL, 200U / mL, 300U / mL, 400U / mL, or 500U / mL.

[0031] Preferably, the alkaline solution is one or more of sodium hydroxide, calcium hydroxide, or potassium hydroxide solution, more preferably sodium hydroxide solution;

[0032] Preferably, the molar concentration of the alkaline solution is 3–10 mol / L;

[0033] For example, the molar concentration of the alkaline solution can be 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L, or 10 mol / L.

[0034] Preferably, the amount of alkaline solution added is 1-5 mL;

[0035] For example, the amount of alkaline solution added can be: 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL.

[0036] Preferably, the acidic solution is one or more of hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid solutions, more preferably hydrochloric acid solution;

[0037] For example, the molar concentration of the acidic solution can be 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, 7 mol / L, 8 mol / L, 9 mol / L, or 10 mol / L.

[0038] Preferably, the amount of acidic solution added is 1-5 mL;

[0039] For example, the amount of acidic solution added can be: 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL.

[0040] Preferably, the amount of acidic solution added is sufficient to neutralize the degraded alkaline solution.

[0041] Preferably, the centrifugation speed is 10000 r / min.

[0042] Furthermore, the chromatographic conditions for the liquid chromatography method are as follows: the chromatographic column is a chromatographic column packed with octadecyl bonded silica gel; a mixture of 5-100 mmol / L phosphate buffer solution and methanol is used as the mobile phase, with a volume ratio of 85:15 to 95:5, and the pH of the mobile phase is 2-4.

[0043] Preferably, the chromatographic conditions for liquid chromatography also include: flow rate: 0.5–1.0 mL / min; column temperature: 25–40 °C; detection wavelength: 210 nm; injection volume: 20 μL.

[0044] Preferably, the phosphate buffer solution includes potassium dihydrogen phosphate, sodium dihydrogen phosphate, and ammonium dihydrogen phosphate.

[0045] Furthermore, the formula for calculating the polylactic acid content R is as follows:

[0046]

[0047] In the formula:

[0048] C1 is the concentration of lactic acid in the grafted polymer sample solution calculated using the standard curve, in μg / mL;

[0049] m1 is the sample amount of the grafted polymer, in grams;

[0050] V1 is the dilution volume of the grafted polymer sample, in mL, preferably 10 mL;

[0051] f is the ratio of the polylactic acid content in the pure polylactic acid to the lactic acid content in its hydrolysate;

[0052] k is the unit conversion factor, preferably 1000;

[0053] Furthermore, the method for detecting sodium hyaluronate is as follows: sodium hyaluronate in the grafted polymer is degraded and derivatized to obtain sodium acetate, and the content of sodium acetate is detected by gas chromatography to calculate the sodium hyaluronate content.

[0054] Furthermore, the detection method for sodium hyaluronate includes: taking a polylactic acid-coupled sodium hyaluronate grafted polymer sample m2, adding an alkaline solution, degrading and derivatizing, and after the reaction is complete, adding an acidic solution to neutralize to neutral, adding sulfuric acid ethanol solution, sealing, headspace injection, and detecting by gas chromatography, the measured concentration of sodium hyaluronate is C2; blank correction is performed using purified water in the same way.

[0055] Preferably, the degradation and derivatization conditions include: a temperature of 95–100°C and a time of 0.5–4 hours;

[0056] For example, the temperature can be 95℃, 96℃, 97℃, 98℃, 99℃, or 100℃.

[0057] For example, the time can be 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, or 4h.

[0058] Preferably, the alkaline solution is one or more of sodium hydroxide, calcium hydroxide, or potassium hydroxide, more preferably a sodium hydroxide solution;

[0059] Preferably, the molar concentration of the alkaline solution is 3–10 mol / L;

[0060] Preferably, the amount of alkaline solution added is 2-20 mL;

[0061] Preferably, the acidic solution is one or more of hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid, more preferably hydrochloric acid;

[0062] Preferably, the molar concentration of the acidic solution is 3–10 mol / L;

[0063] Preferably, the amount of acidic solution added is 2-20 mL;

[0064] Preferably, the amount of acidic solution added is sufficient to neutralize the degraded alkaline solution.

[0065] Furthermore, the chromatographic conditions of the gas chromatography method are as follows: the chromatographic column is a capillary column, and the stationary phase is cyanopropylphenyl-dimethylpolysiloxane or diphenyl-dimethylpolysiloxane; when the stationary phase is cyanopropylphenyl-dimethylpolysiloxane, the content of cyanopropylphenyl in the stationary phase is 6% to 50%, and the remainder is dimethylpolysiloxane (for example, 14% cyanopropylphenyl-86% dimethylpolysiloxane, 50% cyanopropylphenyl-50% dimethylpolysiloxane, 6% cyanopropylphenyl-94% dimethylsiloxane), preferably, the medium polarity column is 6% cyanopropylphenyl-94% dimethylsiloxane.

[0066] The heating program is as follows: initial temperature 40-70℃, maintained for 5 min, then increased to 200-250℃ at a rate of 10-30℃ / min, and maintained for 5 min; the detector is a hydrogen flame ionization detector, the detection chamber temperature is 220-270℃; the vaporization chamber temperature is 180-220℃, and the split ratio is 5:1.

[0067] The chromatographic conditions for the gas chromatography method also include: headspace injection, an equilibrium temperature of 45–70°C, and an equilibrium time of 30–120 min.

[0068] Furthermore, the formula for calculating the sodium hyaluronate content T is as follows:

[0069]

[0070] In the formula:

[0071] c represents the concentration of sodium acetate in the grafted polymer sample solution, calculated using the standard curve, in μg / mL.

[0072] V represents the volume of the grafted polymer sample after dilution, in mL;

[0073] m2 is the sample mass of the grafted polymer sample, in grams;

[0074] K2 is the ratio of the molecular weight of the sodium hyaluronate disaccharide structural unit (401.3) to the molecular weight of the resulting sodium acetate (82.13), and K2 is 4.9821;

[0075] k′ is the unit conversion factor, preferably 1000.

[0076] Furthermore, the method for detecting polylactic acid also includes the preparation of standard solutions and the plotting of standard curves;

[0077] The preparation of the standard solution includes: weighing lactic acid, dissolving it in a solvent, and quantitatively diluting it to prepare a lactic acid standard solution;

[0078] Preferably, the concentration of the lactic acid standard solution is 1 mg / mL;

[0079] The standard curve is plotted as follows: Take an appropriate amount of standard solution, dilute it with water to a series of control solutions with concentrations ranging from 50 to 1000 μg / mL, with a concentration gradient of 5 or more points, add alkaline solution, seal and place under the second degradation condition for reaction. Remove and cool to room temperature, add acidic solution to neutralize to neutral, transfer the neutralized liquid to a 10 mL volumetric flask, make up to the mark, shake well, take the liquid after making up to the mark, filter it through a microporous membrane, and detect it by liquid chromatography. Perform linear regression of peak area against the concentration of the corresponding control solution to obtain the standard curve.

[0080] Preferably, among the above concentration points, the concentration difference between two adjacent concentration points is at least 50 μg / mL and at most 500 μg / mL.

[0081] Preferably, the degradation conditions are: degradation temperature 60–100°C, degradation time 0.5–4 h;

[0082] Preferably, the alkaline solution is one or more of sodium hydroxide, calcium hydroxide, or potassium hydroxide solution, more preferably sodium hydroxide solution;

[0083] Preferably, the molar concentration of the alkaline solution is 3–10 mol / L;

[0084] Preferably, the amount of alkaline solution added is 1-5 mL;

[0085] Preferably, the acidic solution is one or more of hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid solutions, more preferably hydrochloric acid solution;

[0086] Preferably, the molar concentration of the acidic solution is 3–10 mol / L;

[0087] Preferably, the amount of acidic solution added is 1-5 mL;

[0088] Preferably, the amount of acidic solution added is sufficient to neutralize the degraded alkaline solution.

[0089] Preferably, the microporous filter membrane is a 0.45μm microporous filter membrane.

[0090] Furthermore, the method for detecting sodium hyaluronate also includes the preparation of standard solutions and the plotting of standard curves;

[0091] The preparation of the standard solution includes: weighing sodium acetate reference standard, dissolving and diluting it with solvent to prepare sodium acetate standard stock solution; accurately measuring the sodium acetate standard stock solution and diluting it with water to prepare a 200 μg / mL solution as the standard working solution; accurately measuring the standard working solution and diluting it with water to prepare a series of standard solutions with concentrations of 0–200 μg / mL, with at least 5 points.

[0092] Preferably, the sodium acetate is used after being dried at 120°C for 20 hours;

[0093] Preferably, the concentration of the standard stock solution is 1000 μg / mL;

[0094] The process of constructing the standard curve involves: precisely measuring standard solutions of various concentrations, placing them in headspace vials, adding sulfuric acid ethanol solution, shaking well, sealing, injecting via headspace, and recording the chromatograms. A linear regression is then performed using peak area against the concentration of the corresponding control solution to obtain the standard curve.

[0095] Preferably, the volume of each concentration of standard solution is 1 mL;

[0096] Preferably, the volume of the sulfuric acid ethanol solution is 1 mL.

[0097] The present invention also provides an application of the aforementioned detection method in detecting the aliphatic polyester grafting rate in aliphatic polyester-coupled sodium hyaluronate grafted polymer.

[0098] The aliphatic polyester is polylactic acid.

[0099] All chemical reagents used in this invention are of analytical grade or equivalent or higher purity.

[0100] The liquid chromatography method of the present invention uses a high-performance liquid chromatograph or other similar equipment, and the gas chromatography method of the present invention uses a gas chromatograph or other similar equipment.

[0101] The beneficial effects of this invention are as follows:

[0102] This invention develops a novel method for detecting the polylactic acid (PLA) grafting rate in PLA-coupled sodium hyaluronate (NH3)-grafted polymers. Specifically, the method involves degrading NH3 in the grafted polymer to obtain sodium acetate, and then using gas chromatography to detect the sodium acetate content to calculate the NH3 content. Conversely, the method involves degrading PLA in the grafted polymer to obtain lactic acid, and then using liquid chromatography to detect the lactic acid content to calculate the PLA content. The PLA grafting rate is obtained by calculating the ratio of PLA to NH3 content. This method is highly specific, accurate, repeatable, and easy to operate. Following this method, the PLA grafting rate in PLA-coupled NH3-NH3-grafted polymers can be rapidly determined. Attached Figure Description

[0103] Figure 1 The chromatograms shown are of purified water blank solution, polylactic acid test solution, and polylactic acid reference solution.

[0104] Figure 2 The figure shown is the standard curve of lactic acid.

[0105] Figure 3 The figure shown is the standard curve of sodium acetate.

[0106] Figure 4 The chromatograms shown are of purified water blank solution, sodium hyaluronate test solution, and sodium hyaluronate reference solution.

[0107] Figure 5 As shown 1 ¹H NMR spectra of polylactic acid grafting rate in polylactic acid coupled with sodium hyaluronate grafted polymer. Detailed Implementation

[0108] The present invention will now be described in detail with reference to the embodiments and accompanying drawings. Although specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0109] In this invention, the term "standard curve" refers to a curve composed of the values ​​of a certain physicochemical property obtained by measuring a series of standard substances with known components. The standard curve is a functional relationship between the physical / chemical properties of the standard substances and the instrument response.

[0110] In this invention, the term "standard stock solution" refers to a solution of known components and concentration that can be stably stored and is used to prepare various standard solutions. In this invention, sodium acetate standard stock solution refers to a sodium acetate solution of known components and concentration that can be stably stored.

[0111] In this invention, the term "grafting rate" refers to the ratio of polylactic acid content to sodium hyaluronate content in the sample, characterized by a mass ratio.

[0112] In this invention, the term "crosslinking" refers to the process of transforming a chemically reactive linear polymer into a three-dimensional network (three-dimensional) polymer through a chemical reaction. It is commonly used in polymer modification.

[0113] In this invention, the term "derivation" refers to a process that uses chemical transformation to convert a compound into one with a similar chemical structure. Generally, when a compound with a specific function participates in a derivatization reaction, its solubility, boiling point, melting point, aggregation state, or chemical composition will deviate. The resulting new chemical properties can be used for quantification or separation. Its main function is to transform substances that are difficult to analyze into substances with similar chemical structures but are easier to analyze, facilitating quantification and separation. When a substance is difficult to detect, such as lacking ultraviolet absorption, it can be processed, for example, by adding chromophores, to generate a detectable substance.

[0114] In this invention, the term "headspace sampling" refers to the use of a headspace sampler for sample injection. A headspace sampler is a convenient and rapid sample pretreatment method in gas chromatography. Its principle is to place the sample to be tested in a sealed container, and to cause volatile components to volatilize from the sample matrix by heating. The volatile components reach equilibrium in the gas-liquid (or gas-solid) two phases, and the head gas is directly extracted for chromatographic analysis, thereby examining the composition and content of volatile components in the sample.

[0115] In this invention, the term "equilibrium time" refers to the time required for the headspace sampler to achieve gas-liquid equilibrium of the sample components.

[0116] In this invention, the term "equilibrium temperature" refers to the temperature at which the headspace sampler brings the sample components to gas-liquid equilibrium.

[0117] In this invention, the term "blank correction" refers to subtracting blank values ​​from the sample analysis results to eliminate systematic errors caused by the water used, reagents, glassware, environment, operation, etc.

[0118] In this invention, the term "precision measurement" originally refers to accurately weighing to one-thousandth of the mass taken. Precision measurement is the operation of taking a volume using a precision measuring instrument, which includes a pipette, a graduated pipette, and a pipette gun.

[0119] Example 1: Detection of polylactic acid grafting rate in samples

[0120] 1. The method for detecting polylactic acid content is as follows:

[0121] 1.1 Preparation of standard solutions and fitting of standard curve equations

[0122] Weigh 0.1250 g of L-lactic acid (80% purity, i.e., reference standard) into a 100 mL volumetric flask, dilute to the mark with purified water, shake well, and prepare a standard stock solution with a concentration of 1000 μg / mL. Accurately measure the lactic acid standard stock solution and prepare a series of reference standard solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, and 1000 μg / mL using water within the concentration range of 50–1000 μg / mL.

[0123] Take 1 mL of the series concentration reference solution and place it in a suitable-sized centrifuge tube. Add 4 mL of 4 mol / L sodium hydroxide solution, seal the tube, and heat it in a 100℃ water bath for 60 min. Remove the tube and cool it to room temperature (25℃). Add 4 mL of 4 mol / L hydrochloric acid solution for neutralization. Transfer the neutralized liquid to a 10 mL volumetric flask, dilute to the mark, and mix well. Take the diluted liquid and filter it through a 0.45 μm microporous membrane. Take the filtrate and perform liquid chromatography analysis as described in section 1.3. Record the chromatogram of the reference solution, as shown in the attached figure. Figure 1 A linear regression was performed on the peak area against the concentration of the corresponding control solution, and the standard curve is shown in the attached figure. Figure 2 .

[0124] 1.2 Determination of lactic acid content in samples

[0125] Accurately weigh 1.0 g of sample (i.e., the test sample) and place it in a centrifuge tube of appropriate size. Add 1 mL of 150 U / mL hyaluronidase, seal the tube, and place it in a 37℃ water bath for 8 hours for enzyme degradation. After complete degradation, centrifuge the tube for 15 minutes at 10000 r / min to separate the solid and liquid layers, and remove the supernatant. Add 4 mL of 4 mol / L sodium hydroxide solution to the centrifuge tube containing the remaining solids, and follow the procedure in "1.1 Construction of Standard Curve" from "Heating in a 100℃ water bath for 60 minutes after sealing" onwards. Perform blank correction with purified water using the same method. Perform liquid chromatography detection as described in section 1.3, and record the chromatograms of the test sample solution and the blank solution, as shown in the attached figure. Figure 1 .

[0126] 1.3 Liquid Chromatography Conditions

[0127] The chromatographic column was packed with octadecyl bonded silica gel (ZORBAX Eclipse plus C18 column, 250 mm × 4.6 mm, 5 μm); the mobile phase was a mixture of 10 mmol / L phosphate buffer solution (adjusted to pH = 2.2 with 50% phosphoric acid solution) and methanol at a volume ratio of 95:5; the flow rate was 0.7 mL / min; the column temperature was 25 ℃; the detection wavelength was 210 nm; and the injection volume was 20 μL.

[0128] 1.4 Calculate the polylactic acid content in the sample.

[0129] The lactic acid content in the sample was calculated based on the liquid chromatography standard curve, and the polylactic acid content R (mg / g) in the sample was calculated using the following formula:

[0130]

[0131] In Equation 1:

[0132] C1 is the concentration (μg / mL) of lactic acid in the test solution calculated using the standard curve;

[0133] m1 is the sample size (g) of the test sample.

[0134] V1 represents the dilution volume of the test sample, which is 10 mL.

[0135] f is the ratio of the polylactic acid content in the pure polylactic acid to the lactic acid content in its hydrolysate.

[0136] k is the unit conversion factor, specifically 1000;

[0137] 1.5 The ratio of polylactic acid content in pure polylactic acid to lactic acid content in its hydrolysate (confirmed by the f-value under section 1.4)

[0138] Weigh 20 mg of polylactic acid (PLA) into centrifuge tubes, add 4 mL of 4 mol / L sodium hydroxide solution, and follow the procedure in "1.1 Construction of Standard Curve" starting from "Heating in a 100℃ water bath for 60 min after sealing". Prepare 10 parallel samples and analyze them according to the chromatographic conditions in 1.3. Calculate the "ratio f of PLA content in pure PLA to lactic acid content in its hydrolysate" using the formula in 1.4, assuming a PLA content of 100% (i.e., R = 1000 mg / g). The PLA used in this batch of testing must be from the same batch as the PLA raw material used in the sample preparation. Therefore, the ratio f of PLA content in pure PLA to lactic acid content in its hydrolysate used in this product is 1.0. (The definition of f is the same as the following standard: DB22T 2105-2014 Determination of PLA Content in PLA Products by Ion Chromatography)

[0139] The results of the polylactic acid content detection in the sample, calculated by combining items 1.4 and 1.5, are shown in the table below:

[0140] Table 1. Results of polylactic acid content determination in samples.

[0141]

[0142]

[0143] 2. The method for detecting sodium hyaluronate content is as follows:

[0144] 2.1 Preparation of standard solutions and fitting of standard curve equations

[0145] Weigh an appropriate amount of sodium acetate reference standard (dried at 120℃ for 2 hours before use), accurately weigh it, place it in a 100mL volumetric flask, dissolve and dilute with water to the mark, shake well, and use as a stock solution. Store at 2–8℃. Accurately measure the stock solution and dilute with water to prepare a 200μg / mL solution as the working solution. Accurately measure the working solution and dilute with water to prepare a series of standard solutions with concentrations of 0μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 120μg / mL, 160μg / mL, and 200μg / mL. Accurately measure 1.0mL of the standard solution and place it in a 20mL headspace vial. Add 1.0mL of sulfuric acid ethanol solution, shake well, seal, and perform gas chromatography detection as described in section 2.3. Perform blank correction with 1.0mL of purified water using the same method. Record the chromatograms of the reference solution and the blank solution. Figure 4 A linear regression was performed on the peak area against the concentration of the corresponding control solution, and the standard curve is shown in the attached figure. Figure 3 .

[0146] 2.2 Determination of sodium hyaluronate content in samples

[0147] Accurately weigh 1.0 g of sample and place it in a suitable-sized non-glass test tube (such as a PTFE or polypropylene tube). Add 10 mL of 4 mol / L sodium hydroxide solution, boil in a water bath for 60 min, then remove and cool to room temperature (25°C). Add an equal volume of 4 mol / L hydrochloric acid solution to neutralize, cool to room temperature (25°C), and quantitatively transfer to a 50 mL volumetric flask. Dilute to the mark with water and mix well. Accurately measure 1.0 mL of the test solution and place it in a 20 mL headspace vial. Add 1.0 mL of sulfuric acid ethanol solution, mix well, seal, and perform gas chromatography detection as described in section 2.3. Record the chromatogram of the test solution as shown in the attached figure. Figure 4 .

[0148] 2.3 Gas Chromatography Conditions

[0149] 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 of 5:1). Headspace injection was used with an equilibration temperature of 55℃ and an equilibration time of 90 min.

[0150] 2.4 Calculate the sodium hyaluronate content in the sample.

[0151] The concentration of sodium acetate in the test solution is calculated from the standard curve based on the peak area of ​​the test solution, and the content of sodium hyaluronate in this product is calculated using the following formula:

[0152]

[0153] In Equation 2:

[0154] c represents the concentration (μg / mL) of sodium acetate in the test solution calculated using the standard curve;

[0155] V is the volume (mL) of the diluted test sample;

[0156] m2 is the sample mass (g) of the test sample;

[0157] K2 is the ratio of the molecular weight of the sodium hyaluronate disaccharide structural unit (401.3) to the molecular weight of the resulting sodium acetate (82.13), specifically 4.9821;

[0158] k′ is the unit conversion factor, specifically 1000.

[0159] Table 2. Results of sodium hyaluronate content determination in samples

[0160]

[0161] Formula for calculating the polylactic acid grafting rate of 3 samples:

[0162] The grafting rate L (in mass, %) of the sample is calculated using the following formula:

[0163]

[0164] In Equation 3:

[0165] R represents the polylactic acid content (mg / g) in the sample;

[0166] T represents the sodium hyaluronate content (mg / g) in the sample;

[0167] Six samples were tested consecutively, divided into two parts. The contents of polylactic acid (PLA) and sodium hyaluronate were measured separately, and the PLA grafting rate was calculated according to Equation 3. Table 3 shows that the measured results of PLA and sodium hyaluronate contents in the samples were relatively stable, without obvious fluctuations or decreasing trends.

[0168] Table 3 Results of polylactic acid grafting rate determination in samples

[0169]

[0170] Example 2: Investigation of Reaction Conditions

[0171] The determination was performed following the steps in Example 1, except for the amount of 4 mol / L sodium hydroxide added in the reaction conditions under section 1.1 of the polylactic acid content detection experiment in step 1. The same batch of samples was used, and the amount of sodium hydroxide added was examined at 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL, respectively, to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 4.

[0172] Table 4. Results of polylactic acid grafting rate determination in samples with different sodium hydroxide addition conditions.

[0173] Amount of sodium hydroxide added (mL) 1 2 3 4 5 Sample weight (g) 0.9985 1.0052 1.0157 1.0021 1.0088 Polylactic acid content (mg / g) 0.82 0.82 0.82 0.83 0.82 Polylactic acid grafting rate (%) 9.18 9.18 9.18 9.29 9.18

[0174] As can be seen from Table 4, the results of the polylactic acid grafting rate determination of the samples under different sodium hydroxide addition conditions are basically the same.

[0175] Example 3: Investigation of Reaction Conditions

[0176] The determination was performed following the steps in Example 1, except for the concentration of sodium hydroxide in the reaction conditions under section 1.1 of the polylactic acid content detection experiment in step 1. The same batch of samples was used, and the concentrations of sodium hydroxide were examined at 3 mol / L, 4 mol / L, 5 mol / L, 6 mol / L, and 7 mol / L, respectively, to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 5.

[0177] Table 5. Results of polylactic acid grafting rate determination in samples with different sodium hydroxide concentrations.

[0178] Concentration of sodium hydroxide (mol / L) 3 4 5 6 7 Sample weight (g) 1.0112 1.0553 1.0007 1.0051 1.0318 Polylactic acid content (mg / g) 0.81 0.82 0.82 0.83 0.82 Polylactic acid grafting rate (%) 9.07 9.18 9.18 9.29 9.18

[0179] As can be seen from Table 5, within the sodium hydroxide concentration range of 3–7 mol / L, the polylactic acid grafting rate of the samples remained stable with no significant changes.

[0180] Example 4: Investigation of Reaction Conditions

[0181] The determination was performed following the steps in Example 1, except for the alkaline hydrolysis temperature conditions under section 1.1 in the polylactic acid content detection experiment of step 1. The same batch of samples was used, and alkaline hydrolysis temperatures of 40℃, 50℃, 60℃, 80℃, and 100℃ were investigated to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 6.

[0182] Table 6. Results of polylactic acid grafting rate determination in samples under different alkaline hydrolysis temperatures.

[0183]

[0184]

[0185] Table 6 shows that the polylactic acid (PLA) content varies under different reaction temperatures. At the same alkaline hydrolysis time, temperatures below 60℃ result in incomplete PLA degradation. Between 60℃ and 100℃, the PLA content tends to stabilize. Therefore, to ensure accuracy when determining PLA content, a temperature that guarantees complete PLA degradation into lactic acid should be selected, thus accurately detecting the PLA grafting rate.

[0186] Example 5: Investigation of Reaction Conditions

[0187] The determination was performed following the steps in Example 1, except for the alkaline hydrolysis reaction time conditions under section 1.1 in the polylactic acid content detection experiment of step 1. The same batch of samples was used, and derivatization times of 0.5 h, 1 h, 2 h, 3 h, and 4 h were investigated to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 7.

[0188] Table 7. Results of polylactic acid grafting rate determination under different alkaline hydrolysis reaction times.

[0189] Alkaline hydrolysis time (h) 0.5 1 2 3 4 Sample weight (g) 1.0056 1.0089 1.0183 1.0043 1.0101 Polylactic acid content (mg / g) 0.82 0.83 0.83 0.83 0.83 Polylactic acid grafting rate (%) 9.18 9.29 9.29 9.29 9.29

[0190] As can be seen from Table 7, the results of the polylactic acid grafting rate determination of the samples were basically the same within the alkaline hydrolysis reaction time range of 0.5 to 4 hours.

[0191] Example 6: Investigation of Reaction Conditions

[0192] The determination was performed following the steps in Example 1, except for the enzyme concentration in step 1.2 of the polylactic acid content detection experiment. The same batch of samples was used, and enzyme concentrations of 100 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, and 500 U / mL were examined to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 8.

[0193] Table 8. Results of polylactic acid grafting rate determination in samples under different enzymatic hydrolysis concentrations.

[0194] Enzyme concentration (U / mL) 100 200 300 400 500 Sample weight (g) 1.0021 1.0104 1.0049 1.0118 1.0055 Polylactic acid content (mg / g) 0.82 0.83 0.83 0.83 0.83 Polylactic acid grafting rate (%) 9.18 9.29 9.29 9.29 9.29

[0195] As can be seen from Table 8, under the same enzymatic hydrolysis time, the results of the polylactic acid grafting rate determination of samples in the enzyme concentration range of 100-500U are basically the same.

[0196] Example 7: Investigation of Reaction Conditions

[0197] The determination was performed following the steps in Example 1, except for the enzymatic hydrolysis time of the samples in section 1.2 of the polylactic acid content detection experiment in step 1. The same batch of samples was used, and the enzymatic hydrolysis time was examined at 4h, 6h, 8h, 10h, and 12h, respectively, to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 9.

[0198] Table 9. Results of polylactic acid grafting rate determination under different enzymatic hydrolysis times.

[0199] Enzymatic hydrolysis time (h) 4 6 8 10 12 Sample weight (g) 1.0200 1.0133 1.0051 1.0105 1.0006 Polylactic acid content (mg / g) 0.83 0.82 0.83 0.83 0.83 Polylactic acid grafting rate (%) 9.29 9.18 9.29 9.29 9.29

[0200] As can be seen from Table 9, under the same enzyme concentration, the results of the polylactic acid grafting rate determination of the samples within the enzymatic hydrolysis time range of 4 to 12 hours are basically the same.

[0201] Example 8: Investigation of Reaction Conditions

[0202] The determination was performed following the steps in Example 1, except for the sample enzymatic hydrolysis temperature in step 1.2 of the polylactic acid content detection experiment. The same batch of samples was used, and the enzymatic hydrolysis temperature was examined at 25℃, 30℃, 35℃, 40℃, and 45℃ respectively, to calculate the polylactic acid grafting rate. The sodium hyaluronate content was calculated using the average value of 8.93 mg / g detected in Example 1. The results are shown in Table 10.

[0203] Table 10 Results of polylactic acid grafting rate determination in samples under different enzymatic hydrolysis temperatures.

[0204] Enzymatic hydrolysis temperature (°C) 25 30 35 40 45 Sample weight (g) 1.0020 1.0033 1.0122 1.0099 1.0073 Polylactic acid content (mg / g) 0.83 0.82 0.83 0.83 0.83 Polylactic acid grafting rate (%) 9.29 9.18 9.29 9.29 9.29

[0205] As can be seen from Table 10, under the same enzymatic hydrolysis time, the results of the polylactic acid grafting rate determination of the samples within the enzymatic hydrolysis temperature range of 25-45℃ are basically the same.

[0206] Example 9: Investigation of Reaction Conditions

[0207] The determination was performed following the steps in Example 1, except for the amount of 4 mol / L sodium hydroxide added in the reaction conditions under section 2.2 of the sodium hyaluronate content detection experiment in step 2. The same batch of samples was used, and the amount of sodium hydroxide added was examined at 4 mL, 6 mL, 8 mL, 12 mL, and 16 mL, respectively, to calculate the polylactic acid grafting rate. The polylactic acid content was calculated using the average value of 0.83 mg / g detected in Example 1. The results are shown in Table 11.

[0208] Table 11 Results of polylactic acid grafting rate determination in samples with different sodium hydroxide addition conditions

[0209] Amount of sodium hydroxide added (mL) 4 6 8 12 16 Sample weight (g) 1.0029 1.0134 1.0111 1.0032 1.0009 Sodium hyaluronate content (mg / g) 8.93 8.94 8.94 8.92 8.92 Polylactic acid grafting rate (%) 9.29 9.28 9.28 9.30 9.30

[0210] As can be seen from Table 11, the sodium hyaluronate content of the samples was basically consistent under different sodium hydroxide addition conditions. Therefore, the results of the polylactic acid grafting rate determination were basically the same.

[0211] Example 10 Investigation of Reaction Conditions

[0212] The determination was performed following the steps in Example 1, except for the concentration of sodium hydroxide in the reaction conditions under section 2.2 of the sodium hyaluronate content detection experiment in step 2. The same batch of samples was used, and the concentrations of sodium hydroxide were examined at 1 mol / L, 2 mol / L, 3 mol / L, 5 mol / L, and 8 mol / L, respectively, to calculate the polylactic acid grafting rate. The polylactic acid content was calculated using the average value of 0.83 mg / g detected in Example 1. The results are shown in Table 12.

[0213] Table 12 Results of polylactic acid grafting rate determination in samples with different sodium hydroxide concentrations.

[0214]

[0215] Table 12 shows that the polylactic acid (PLA) content varies under different sodium hydroxide concentrations. At the same alkaline hydrolysis time, 1 mol / L and 2 mol / L sodium hydroxide concentrations result in incomplete degradation of sodium hyaluronate in the samples. At sodium hydroxide concentrations of 3 mol / L and above, the detection results tend to be stable. Therefore, to ensure accuracy when determining sodium hyaluronate content, a concentration that guarantees complete degradation of sodium hyaluronate should be selected to accurately detect the PLA grafting rate.

[0216] Example 11 Investigation of Reaction Conditions

[0217] The determination was performed following the steps in Example 1, except for the alkaline hydrolysis reaction time conditions under section 2.2 in the sodium hyaluronate content detection experiment in step 2. The same batch of samples was used, and derivatization times of 0.5 h, 1 h, 2 h, 3 h, and 4 h were investigated to calculate the polylactic acid grafting rate. The polylactic acid content was calculated using the average value of 0.83 mg / g detected in Example 1. The results are shown in Table 13.

[0218] Table 13 Results of polylactic acid grafting rate determination under different alkaline hydrolysis reaction times.

[0219] Alkaline hydrolysis time (h) 0.5 1 2 3 4 Sample weight (g) 1.0013 1.0027 1.0109 1.0081 1.0177 Sodium hyaluronate content (mg / g) 8.92 8.93 8.94 8.93 8.92 Polylactic acid grafting rate (%) 9.30 9.29 9.28 9.29 9.30

[0220] As can be seen from Table 13, the sodium hyaluronate content of the samples was basically consistent within the alkaline hydrolysis reaction time range of 0.5 to 4 hours. Therefore, the results of the polylactic acid grafting rate determination were basically the same.

[0221] Example 12 and 1 Comparison of H NMR detection results

[0222] The same sample as in Example 1 was subjected to 400M nuclear magnetic resonance spectroscopy. 1 H-spectral scan.

[0223] Through the 1 H NMR spectrum (see details) Figure 5 The key characteristic peaks in the sample were integrated, and the actual molar ratio of polylactic acid (PLA) to sodium hyaluronate (HHA) was obtained from the integrated peak area. This was then converted to obtain the mass ratio of PLA to HHA, thus determining the grafting rate. The characteristic H atoms selected for PLA were -CH3 (3H, d = 1.404 ppm) and -CH3CHO- (1H, d = 5.022 ppm); the characteristic H atoms selected for HHA were acetaminophen-CONHCH3 (3H, 1.928 ppm) and glycosidic bonds (1H, d = 4.386 ppm, 1H, d = 4.491 ppm). Due to interference from deuteration reagents, -CH3 (3H, d = 1.404 ppm) and -CONHCH3 (3H, 1.928 ppm) were selected for calculation. The final product grafting rate was calculated using the following formula:

[0224]

[0225] Where G is the grafting rate (%), M is the number-average molecular weight of PLLA, 72 is the relative molecular mass of the PLLA structural unit -OC(CH3)-C(O)-, c is the integral area of ​​the characteristic peak -CH3CHO- of PLLA, and a is the integral area of ​​the characteristic peak acetaminophen -CONHCH3 on HA.

[0226] Calculations show that 1 The grafting rate of polylactic acid (PLA) was 9.01% as determined by ¹H NMR.

[0227] contrast 1 The results of polylactic acid grafting rate measured by H NMR and the method of the present invention (Example 1) are shown in Table 14:

[0228] Table 14 Results of polylactic acid grafting rate measured by different methods in the samples

[0229]

[0230] Table 14 shows that the polylactic acid grafting rate of the samples measured by this method is related to... 1 The results obtained by H NMR were basically the same.

[0231] 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 grafting rate of aliphatic polyesters in an aliphatic polyester-coupled polysaccharide grafted polymer, characterized in that, The aliphatic polyester content and polysaccharide content in the aliphatic polyester-polysaccharide grafted polymer were detected by chromatography, and the grafting rate of aliphatic polyester onto polysaccharide was calculated by the ratio of aliphatic polyester content to polysaccharide content. The grafting rate is expressed by the following formula: In the formula: L represents the grafting rate, expressed as a percentage by mass. R represents the content of aliphatic polyester in the grafted polymer; T represents the polysaccharide content in the grafted polymer; The polysaccharide is sodium hyaluronate, the aliphatic polyester is polylactic acid, and the aliphatic polyester-coupled polysaccharide graft polymer is a polylactic acid-coupled sodium hyaluronate graft polymer. The method for detecting polylactic acid is as follows: the polylactic acid in the grafted polymer is degraded into lactic acid, and the lactic acid content is detected by liquid chromatography to calculate the polylactic acid content. The method for detecting sodium hyaluronate is as follows: sodium hyaluronate in the grafted polymer is degraded and derivatized to obtain sodium acetate, and the content of sodium acetate is detected by gas chromatography to calculate the sodium hyaluronate content; The chromatographic conditions for the liquid chromatography method are as follows: the chromatographic column is a column packed with octadecyl bonded silica gel; the mobile phase is a mixture of 5–100 mmol / L phosphate buffer solution and methanol. The chromatographic conditions for the gas chromatography method are as follows: a capillary column is used, and the stationary phase is cyanopropylphenyl-dimethylpolysiloxane or diphenyl-dimethylpolysiloxane.

2. The detection method as described in claim 1, characterized in that, The polylactic acid includes polyracemic lactic acid or poly-L-lactic acid.

3. The detection method as described in claim 2, characterized in that, The polylactic acid is poly-L-lactic acid.

4. The method as described in claim 1, characterized in that, The method for detecting polylactic acid includes: taking a polylactic acid-coupled sodium hyaluronate grafted polymer sample m1, adding an enzyme, degrading it once, centrifuging it after complete degradation, separating the solid and liquid, removing the upper liquid, adding an alkaline solution to the remaining solid, degrading it a second time, adding an acidic solution to neutralize it, and detecting it by liquid chromatography to determine the lactic acid concentration as C1.

5. The method as described in claim 4, characterized in that, In the polylactic acid detection method, the degradation conditions for one step include: a temperature of 25~45℃ and a time of 2~12h.

6. The method as described in claim 4, characterized in that, In the method for detecting polylactic acid, the secondary degradation conditions include: a temperature of 60~100℃ and a time of 0.5~4h.

7. The method as described in claim 4, characterized in that, In the method for detecting polylactic acid, the alkaline solution is one or more of sodium hydroxide, calcium hydroxide, or potassium hydroxide solutions.

8. The method as described in claim 4, characterized in that, In the method for detecting polylactic acid, the acidic solution is one or more of hydrochloric acid, nitric acid, sulfuric acid, or phosphoric acid.

9. The method as described in claim 4, characterized in that, The formula for calculating the polylactic acid content R is as follows: In the formula: C1 is the concentration of lactic acid in the grafted polymer sample solution calculated using a standard curve; m1 is the sample volume of the grafted polymer; V1 is the dilution volume of the grafted polymer sample; f is the ratio of the polylactic acid content in the pure polylactic acid to the lactic acid content in its hydrolysate; k is the unit conversion factor.

10. The method as described in claim 1, characterized in that, The method for detecting sodium hyaluronate includes: taking a polylactic acid-coupled sodium hyaluronate graft polymer sample m2, adding an alkaline solution, degrading and derivatizing, and after the reaction is complete, adding an acidic solution to neutralize to neutral, adding sulfuric acid ethanol solution, sealing, headspace injection, and detecting by gas chromatography. The concentration of sodium hyaluronate is measured as C2.

11. The method as described in claim 10, characterized in that, In the method for detecting sodium hyaluronate, the alkaline solution is one or more of sodium hydroxide, calcium hydroxide, or potassium hydroxide.

12. The method as described in claim 10, characterized in that, In the method for detecting sodium hyaluronate, the degradation and derivatization conditions include: degradation temperature of 95~100℃ and degradation time of 0.5~4h.

13. The method as described in claim 10, characterized in that, In the method for detecting sodium hyaluronate, the acidic solution is one or more of hydrochloric acid, nitric acid, sulfuric acid, and phosphoric acid solutions.

14. The method as described in claim 10, characterized in that, The formula for calculating the sodium hyaluronate content T is as follows: In the formula: c represents the concentration of sodium acetate in the grafted polymer sample solution, calculated using the standard curve. V is the volume of the grafted polymer sample after dilution; m2 is the sampling mass of the grafted polymer sample; K2 is the ratio of the molecular weight of the sodium hyaluronate disaccharide structural unit to the molecular weight of the resulting sodium acetate. k´ is the unit conversion factor.

15. The detection method according to any one of claims 1-14, characterized in that, The detection methods for polylactic acid and sodium hyaluronate both include the preparation of standard solutions and the plotting of standard curves.

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