Method for measuring content of sodium hyaluronate

Abstract

The invention provides a method for determining the content of sodium hyaluronate. The method comprises the following steps: (1) preparing D-glucosamine hydrochloride standard curve solutions with series concentrations; (2) preparing a sample solution; (3) respectively taking the standard curve solution and the sample solution, adding a PMP-methanol solution and a sodium hydroxide solution, reacting at a constant temperature, extracting, separating and filtering to respectively obtain a derivatization standard solution and a derivatization sample solution; (4) performing gradient elution on the derivatization solution to obtain data of a corresponding relation between the concentration and the peak area of the derivatization standard solution and data of the peak area of a target peak of the glucosamine-PMP derivative in the derivatization sample solution; and (5) calculating the content of sodium hyaluronate according to the standard curve. PMP derivatization enables glucosamine to be converted into a strong absorption derivative, and gradient elution realizes efficient separation. And the effects of small impurity interference in the complex matrix and high accuracy of low-content sample determination are realized.

Description

Technical Field

[0001] This invention relates to the field of chemical substance analysis technology, and more specifically, to a method for determining the content of sodium hyaluronate. Background Technology

[0002] Sodium hyaluronate (SH) is a natural high-molecular-weight polysaccharide composed of D-glucuronic acid and N-acetylglucosamine as disaccharide units. Due to its excellent moisturizing properties, viscoelasticity, and biocompatibility, it is widely used in cosmetics, food, and pharmaceuticals. Accurate quantification of its content is crucial for ensuring product quality and regulating market order; however, existing testing technologies still have significant limitations and cannot meet practical testing needs.

[0003] Traditional colorimetric methods (such as the sulfuric acid-carbazole method) are easily affected by impurities such as preservatives and emulsifiers in the sample, resulting in large deviations in the results. Existing methods, including HPLC-ELSD, either lack effective impurity removal steps designed for impurities and cannot accurately determine low-content samples in complex matrices.

[0004] Therefore, there is an urgent need to provide a measurement method to solve the problems in the existing technology. Summary of the Invention

[0005] The main objective of this invention is to provide a method for determining the content of sodium hyaluronate, so as to at least solve the problems of large interference from impurities and low accuracy in determining low-content samples in existing sodium hyaluronate content determination technologies.

[0006] To achieve the above objectives, the present invention provides a method for determining the sodium hyaluronate content, comprising the following steps:

[0007] Step (1): Using the prepared D-glucosamine hydrochloride standard stock solution, prepare a series of D-glucosamine hydrochloride standard curve solutions.

[0008] Step (2): Take a sodium hyaluronate sample, add hydrochloric acid solution, heat and hydrolyze, neutralize and remove the remaining hydrochloric acid, and filter to obtain the sample solution.

[0009] Step (3): Take the standard curve solution and the sample solution respectively, add PMP-methanol solution and sodium hydroxide solution and react at a constant temperature so that D-glucosamine hydrochloride in the standard curve solution and glucosamine in the sample solution are combined with PMP to generate glucosamine-PMP derivatives. Then use hydrochloric acid to stop the reaction. After extraction, separation and filtration, the derivatized standard solution and derivatized sample solution are obtained respectively.

[0010] Step (4): Perform gradient elution on the derivatized standard solution and the derivatized sample solution respectively to obtain the data on the relationship between the concentration and peak area of ​​the derivatized standard solution, and the peak area data of the glucosamine-PMP derivative target peak in the derivatized sample solution.

[0011] Step (5): Plot a standard curve based on the correlation data between concentration and peak area. Substitute the target peak area of ​​the glucosamine-PMP derivative in the derivatized sample solution into the standard curve to obtain the concentration of D-glucosamine hydrochloride in the sample. Calculate the sodium hyaluronate content accordingly.

[0012] Optionally, in step (1), the concentrations of the D-glucosamine hydrochloride standard curve solution are 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

[0013] Optionally, step (2) specifically includes:

[0014] Take the sodium hyaluronate sample extrusion, add hydrochloric acid solution, vortex mix, place in a 121 ℃ oven, heat and hydrolyze for 1.5-2 hours, remove and cool to room temperature, neutralize the remaining hydrochloric acid with sodium hydroxide solution, and make up to volume. After making up to volume, filter and take the filtrate as the sample solution.

[0015] Optionally, the filtration in step (2) includes: using a water filter with a pore size of 0.45 μm to filter the neutralized and diluted solution, discarding the initial filtrate, collecting the subsequent filtrate, and the resulting subsequent filtrate is the sample solution.

[0016] Optionally, in step (3), the reagent used for extraction is dichloromethane.

[0017] Optionally, in step (3), the extraction, separation, and filtration specifically include:

[0018] An extractant was added to the solution obtained by stopping the reaction with hydrochloric acid, followed by vortex extraction and centrifugation to obtain a layered solution. The supernatant was collected and filtered to obtain the derivatized sample solution.

[0019] Optionally, in step (3), a filter membrane with a pore size of 0.22 μm is used to filter the supernatant.

[0020] Optionally, in step (4), the gradient elution conditions are:

[0021] Mobile phase: Phase A is acetonitrile, Phase B is 0.025 mol / L ammonium acetate solution, pH of ammonium acetate solution is 4.5;

[0022] Gradient program: At 0 min, phase A accounts for 15% and phase B accounts for 85%; at 30 min, the proportion of phase A increases to 21% and the proportion of phase B decreases to 79%; at 35 min, the proportion of phase A increases to 85% and the proportion of phase B decreases to 15%; at 36 min, the proportion of phase A falls back to 15% and the proportion of phase B rises back to 85%; at 45 min, the proportion of phase A is maintained at 15% and the proportion of phase B at 85%.

[0023] Supporting conditions: flow rate 1.0 mL / min, detection wavelength 250 nm, column temperature 40 ℃, injection volume 10 μL, C18 column.

[0024] Optionally, in step (5), the formula for calculating the sodium hyaluronate content is:

[0025]

[0026] In the above formula, This refers to the sodium hyaluronate content, expressed in mg / g. The ratio of the molecular weight of the disaccharide fragment to the molecular weight of D-glucosamine hydrochloride is 1.8611. The concentration of D-glucosamine hydrochloride obtained from the regression equation is expressed in µg / mL. This is the conversion factor between micrograms and milligrams, with a value of 10. -3 V represents the volume of the sample solution in mL; S represents the mass of the sodium hyaluronate sample in g.

[0027] The present invention provides a method for determining the content of sodium hyaluronate, comprising the following steps: Step (1) using a prepared D-glucosamine hydrochloride standard stock solution, a series of D-glucosamine hydrochloride standard curve solutions are prepared; Step (2) a sodium hyaluronate sample is taken, hydrochloric acid solution is added, heated and hydrolyzed, the remaining hydrochloric acid is neutralized and removed, and the sample solution is obtained by volume adjustment and filtration; Step (3) the standard curve solution and the sample solution are taken separately, and PMP-methanol solution and sodium hydroxide solution are added and reacted at a constant temperature to make the D-glucosamine hydrochloride in the standard curve solution and the D-glucosamine hydrochloride in the sample solution equalize. All glucosamines combine with PMP to form glucosamine-PMP derivatives. The reaction is then stopped with hydrochloric acid. After extraction, separation, and filtration, derivatized standard solutions and derivatized sample solutions are obtained, respectively. Step (4): Gradient elution is performed on the derivatized standard solutions and derivatized sample solutions to obtain the correlation data between the concentration and peak area of ​​the derivatized standard solutions and the peak area data of the glucosamine-PMP derivative target peak in the derivatized sample solutions. Step (5): A standard curve is plotted based on the concentration-peak area data. The glucosamine-PMP derivative target peak area of ​​the derivatized sample solutions is substituted into the standard curve to obtain the concentration of D-glucosamine hydrochloride in the sample. The sodium hyaluronate content is then calculated. Acid hydrolysis ensures complete hydrolysis of macromolecules, PMP derivatization converts glucosamine, which has no UV absorption, into a strongly absorbing derivative, extraction removes excess PMP and lipid-soluble impurities, and gradient elution achieves efficient separation. It effectively solves the problems of large interference from impurities and inaccurate measurement of low-content samples in traditional methods. The recovery rate is stable at 94.8%-105.1% across the entire concentration range, with RSD≤2.8%, making it suitable for complex matrix samples. Attached Figure Description

[0028] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0029] Figure 1 This is a flowchart of a method for determining sodium hyaluronate content according to the present invention. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0031] The measurement principle of this invention is as follows:

[0032] First, the macromolecular polysaccharide structure of sodium hyaluronate is destroyed by acid hydrolysis, which hydrolyzes it to generate glucosamine (a small molecule monomer). Then, 1-phenyl-3-methyl-5-pyrazolone (PMP) is used as a derivatizing reagent to react with glucosamine to generate glucosamine-PMP derivative with strong ultraviolet absorption. Finally, the derivative is separated by high performance liquid chromatography. By detecting the peak area of ​​the derivative and combining it with a standard curve, the content of sodium hyaluronate is calculated.

[0033] Example

[0034] I. Instruments and Equipment:

[0035] Analytical balance (accuracy 0.1mg): with an accuracy of one ten-thousandth, it can accurately weigh trace amounts of substances;

[0036] High performance liquid chromatograph (equipped with a UV detector or equivalent): includes a chromatographic separation system and a UV detection module. Equivalent performance equipment must have the same separation efficiency and a detection sensitivity of 250nm wavelength.

[0037] Vortex mixer (or equivalent): capable of generating high-speed vortex motion; equivalent performance equipment must have the same mixing intensity and operational stability.

[0038] II. Reagents and Materials

[0039] Concentrated hydrochloric acid, analytical grade;

[0040] Sodium hydroxide, analytical grade;

[0041] D-glucosamine hydrochloride reference standard (CAS: 66-84-2).

[0042] 1-Phenylacetyl-3-methyl-5-pyrazolone (CAS: 89-25-8), analytical grade;

[0043] Ammonium acetate, chromatographic grade;

[0044] Acetonitrile, chromatographic grade.

[0045] III. Solution Preparation

[0046] 3.1 0.5 mol / L PMP-methanol solution: Weigh 8.71 g of 1-phenyl-3-methyl-5-pyrazolone, add 100 mL of methanol to dissolve, and the solution is obtained.

[0047] 3.2 5 mol / L hydrochloric acid solution: Take 40 mL of concentrated hydrochloric acid, add 60 mL of water, and mix well.

[0048] 3.3 0.3 mol / L hydrochloric acid solution: Take 1 mL of concentrated hydrochloric acid, add 39 mL of water, and mix well.

[0049] 3.4. 5 mol / L sodium hydroxide solution: Weigh 20g of sodium hydroxide and dissolve it in 100mL of water to obtain the solution.

[0050] 3.5. 0.3 mol / L sodium hydroxide solution: Weigh 1.2 g of sodium hydroxide and dissolve it in 100 mL of water to obtain the solution.

[0051] 3.6 0.025 mol / L ammonium acetate solution (pH=4.5): Weigh 1.93 g of ammonium acetate, dissolve it in a small amount of water, and bring the volume to 1000 mL. Adjust the pH to 4.5 with glacial acetic acid.

[0052] 3.7 D-glucosamine hydrochloride standard stock solution (100 μg / mL): Accurately weigh 10 mg of D-glucosamine hydrochloride reference standard (calculated as pure product), place it in a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and use as the stock solution.

[0053] 3.8. D-Glucosamine Hydrochloride Standard Curve Series: Accurately measure 0 mL, 1 mL, 2 mL, 3 mL, 4 mL, and 5 mL of D-glucosamine hydrochloride standard stock solution into 10 mL volumetric flasks, dilute to the mark with water, and mix well to prepare a series of D-glucosamine hydrochloride standard curve solutions with concentrations of 0, 10, 20, 30, 40, and 50 μg / mL. This concentration series covers the common detection range of glucosamine in actual samples, and can establish standard curves with good linearity, providing accurate quantitative basis for sodium hyaluronate samples as low as 0.5 mg / g.

[0054] IV. Chromatographic Conditions

[0055] Chromatographic column: C18 column, 4.6 mm x 250 mm, 5 mm, or equivalent column.

[0056] Mobile phase: Phase A was acetonitrile, and Phase B was 0.025 mol / L ammonium acetate solution (pH=4.5). The gradient elution program is shown in Table 1.

[0057] Table 1 Gradient elution conditions

[0058]

[0059] V. Sample Preparation

[0060] 5.1. Weigh approximately 1.0 g of the sample to be tested precisely using an analytical balance with an accuracy of 0.1 mg, and then place it in a 20 mL headspace vial. The headspace vial is chosen to accommodate the sealing required for subsequent heating and hydrolysis.

[0061] 5.2 Add 5 mL of 5 mol / L hydrochloric acid solution to the headspace vial. Sodium hyaluronate is a large polysaccharide formed by alternating links of D-glucuronic acid and N-acetylglucosamine, and cannot be directly detected by liquid chromatography. The role of hydrochloric acid is to provide a strongly acidic environment, creating conditions for breaking the glycosidic bonds (the key chemical bonds connecting monosaccharide units) of the polysaccharide macromolecule. Immediately after adding the reagent, seal the vial to prevent the hydrochloric acid from evaporating during subsequent heating, thus preventing a decrease in concentration, and also to avoid contamination of the sample by external impurities.

[0062] 5.3 Place the sealed headspace vial on a vortex mixer to mix thoroughly, ensuring complete contact between the sodium hyaluronate in the sample and the hydrochloric acid solution, guaranteeing uniform hydrolysis. Then, place the headspace vial in a 121°C oven for 1.5 hours to hydrolyze. The high temperature significantly accelerates the breaking of glycosidic bonds, allowing the large molecular chains of sodium hyaluronate to completely decompose and generate glucosamine, one of its constituent units.

[0063] 5.4 After hydrolysis, remove the headspace flask and allow it to cool to room temperature before proceeding with subsequent operations. This is because directly transferring the high-temperature solution will cause volume errors in the volumetric flask due to the sudden temperature change. Additionally, the reaction between the overheated solution and the subsequent sodium hydroxide will be highly exothermic, posing a risk of splashing. After cooling, transfer all the hydrolysate from the flask to a 25mL volumetric flask using pure water. Then add 5mL of 5mol / L sodium hydroxide solution. The sodium hydroxide will neutralize the excess hydrochloric acid from the hydrolysis reaction, adjusting the pH of the system to neutral. An acidic environment will inhibit the subsequent derivatization reaction, while a neutral system ensures derivatization efficiency.

[0064] 5.5. Continue adding pure water to the volumetric flask until the solution level reaches the mark. Tighten the stopper and shake thoroughly. This volume adjustment process dilutes the sample solution to a fixed volume, facilitating subsequent calculation of the glucosamine content per unit volume. Shaking ensures a uniform glucosamine concentration in the solution, avoiding detection errors caused by localized concentration differences.

[0065] 5.6. Filter the well-mixed solution through a 0.45μm aqueous filter. Discard the initial filtrate and collect the subsequent filtrate as the final sample solution. A small amount of insoluble impurities may be generated during hydrolysis, and trace amounts of solid particles may remain in the sample extrusion. If these substances enter the high-performance liquid chromatograph (HPLC), they may clog the column or interfere with the detector signal. The 0.45μm aqueous filter effectively traps these impurities, ensuring smooth subsequent chromatographic separation and detection.

[0066] VI. Measurement

[0067] 6.1. Accurately measure 1 mL each of the D-glucosamine hydrochloride standard curve solution and the sample solution prepared above, and place them in separate 15 mL centrifuge tubes. The standard curve solution is used to establish the concentration-peak area correspondence, therefore its sampling volume is consistent with that of the sample solution. Add 1.0 mL of 0.5 mol / L PMP-methanol solution and 1.0 mL of 0.3 mol / L sodium hydroxide solution to each centrifuge tube sequentially, and then mix thoroughly. PMP can undergo nucleophilic substitution with the amino group of glucosamine; the sodium hydroxide solution provides an alkaline reaction environment, which activates the reactivity of PMP and promotes derivatization; meanwhile, methanol, as a solvent for PMP, ensures its uniform dispersion in the system and guarantees sufficient contact with glucosamine.

[0068] 6.2. Place the mixed centrifuge tubes in a 70℃ constant temperature water bath for 100 min. Too low a temperature will result in a slow reaction rate, preventing the reaction from completing within 100 min; too high a temperature may trigger side reactions such as PMP self-polymerization, producing impurities that interfere with detection. The 100 min holding time ensures complete reaction between glucosamine and PMP, generating a stable glucosamine-PMP derivative. This derivative exhibits significant UV absorption characteristics (maximum absorption wavelength 250 nm), solving the problem of glucosamine's lack of UV absorption and difficulty in direct detection.

[0069] 6.3 Remove the centrifuge tubes from the constant temperature water bath and cool to room temperature. After cooling, add 1.0 mL of 0.3 mol / L hydrochloric acid solution and vortex to terminate the reaction. The derivatization reaction is carried out under alkaline conditions. Adding an equal volume and concentration of hydrochloric acid can completely neutralize the excess sodium hydroxide, restoring the pH of the system to neutral, fundamentally terminating the reaction process and preventing the derivative concentration from becoming unstable due to the continued reaction in subsequent operations. At the same time, the neutral system can also prevent the alkaline environment from adversely affecting the subsequent extraction solvent and chromatographic column.

[0070] 6.4. Add 5 mL of extractant, i.e., dichloromethane, to the solution after the reaction has been terminated. Vortex extract for 1 min, followed by centrifugation at 3500 r / min for 5 min to remove excess PMP reagent. PMP is a lipid-soluble substance, readily soluble in dichloromethane, while glucosamine-PMP derivatives are water-soluble and mainly present in the aqueous phase. Vortex extraction enhances the contact between the aqueous and organic phases, allowing excess PMP to fully transfer to the dichloromethane layer; centrifugation accelerates the separation of the two phases; a speed of 3500 r / min can achieve clear separation within 5 min, avoiding emulsification. After separation, discard the lower layer of dichloromethane (containing excess PMP) and collect the upper aqueous supernatant to obtain the purified derivatized sample solution. Dichloromethane, as an extractant, has higher extraction efficiency, faster separation speed, and lower toxicity compared to reagents such as trichloromethane.

[0071] 6.5. Filter the collected supernatant through a 0.22 μm filter membrane. After filtration, transfer the filtrate to the injection vial of the high-performance liquid chromatograph (HPLC) for instrument analysis. The 0.22 μm filter membrane can retain any small particles that may remain in the solution (such as trace amounts of organic phase impurities introduced during extraction), further purifying the sample, preventing clogging of the chromatographic column sieve, and ensuring the stability of chromatographic separation and the accuracy of detection results.

[0072] 6.6 Using the autosampler or manual injection method of the high-performance liquid chromatograph (HPLC), inject 10 μL each of the derivatized standard solution (D-glucosamine hydrochloride series) and the derivatized sample solution sequentially into the chromatographic system and perform gradient elution. Record the complete chromatograms for each solution simultaneously. Using 10 μL as the injection volume ensures that the detector captures a clear glucosamine-PMP derivative peak signal, avoiding excessively small peak areas and increased detection errors due to insufficient injection volume; it also prevents column overload caused by excessive injection volume, which can lead to peak broadening and decreased separation efficiency. The gradient elution program accurately separates the target derivative from impurities, providing clear chromatographic peak signals for subsequent data extraction.

[0073] 6.8. Extract core data from the recorded chromatograms. For derivatized standard solutions, interference peaks such as solvent peaks and impurity peaks need to be excluded. Characteristic peaks are matched based on the retention time of the target derivative, and the peak areas of the glucosamine-PMP derivative corresponding to each concentration of standard are extracted to form a one-to-one correspondence data set of standard concentration (unit: µg / mL) and characteristic peak area. For derivatized sample solutions, the target peak is located based on the same retention time, and its characteristic peak area data is extracted. Subsequently, a peak area-concentration standard curve is plotted using linear regression with standard concentration as the x-axis and the corresponding characteristic peak area as the y-axis, and a regression equation is established.

[0074] 6.9. Substituting the characteristic peak area of ​​the glucosamine-PMP derivative in the derivatized sample solution into the regression equation of the standard curve above, the concentration of D-glucosamine hydrochloride in the sample solution can be calculated. This process reverses the conversion of the sample's detection signal (peak area) into the substance concentration, providing data support for the subsequent calculation of sodium hyaluronate content. After obtaining the concentration of D-glucosamine hydrochloride in the sample solution, the formula for calculating the sodium hyaluronate content is as follows:

[0075]

[0076] In the above formula, This refers to the sodium hyaluronate content, expressed in mg / g. The ratio of the molecular weight of the disaccharide fragment to the molecular weight of D-glucosamine hydrochloride is 1.8611. The concentration of D-glucosamine hydrochloride in the sample solution, obtained from the regression equation, is expressed in µg / mL. This is the conversion factor between micrograms and milligrams, with a value of 10. -3 V represents the volume of the sample solution in mL; S represents the mass of the sodium hyaluronate sample in g.

[0077] VII. Experimental Instructions

[0078] Common interfering substances found in cosmetics and food (preservatives: phenoxyethanol; emulsifiers: glyceryl stearate; moisturizers: glycerin) were selected. Simulated samples containing interfering substances and standard samples with known content were prepared, and the results were determined using this method and the control method, respectively.

[0079] 7.1 Experimental Materials and Grouping

[0080] Standard samples: Weigh out sodium hyaluronate standard with a purity of 99.5% and prepare pure standard samples with low concentration (0.5 mg / g), medium concentration (5 mg / g), and high concentration (50 mg / g) respectively (free from interfering substances).

[0081] Simulated interference samples: Phenoxyethanol (0.5%), glyceryl stearate (1.0%), and glycerin (5.0%) were added to the standard samples of the above concentrations by mass percentage to simulate the complex matrix environment of cosmetics.

[0082] 7.2 Experimental Grouping: Each sample was measured in parallel 6 times, for a total of 3 groups.

[0083] Experimental group: The method in this invention (acid hydrolysis + PMP derivatization + HPLC-UV);

[0084] Control group 1: Traditional sulfuric acid-carbazole method (no special impurity removal step);

[0085] Control group 2: conventional HPLC-UV method (simple filtration for impurity removal only, without PMP derivatization).

[0086] 7.3 Key Validation Indicators and Measurement Methods

[0087] 7.3.1 Comparative Experiment 1: Comparative Experiment on Impurity Interference

[0088] Experimental materials: Simulated cosmetic samples containing sodium hyaluronate (1.0 mg / g) + common interfering substances (phenoxyethanol 0.5% + glyceryl stearate 1.0%) were prepared and divided into 3 parallel groups.

[0089] Experimental group: The content was determined by performing the complete procedure according to this protocol (including dichloromethane extraction for impurity removal and PMP derivatization), in 6 parallel runs.

[0090] Control group 1: The same batch of samples was determined using the traditional sulfuric acid-carbazole method (without special impurity removal), in 6 parallel trials.

[0091] Control group 2: The same batch of samples was measured using conventional HPLC-UV method (without derivatization, only simple filtration to remove impurities), in 6 parallel runs.

[0092] Results Comparison: The relative standard deviation (RSD) of the three groups was calculated, with a focus on comparing the deviation rate of the interference on the measurement results.

[0093] Interference impact rate: (interference sample measurement value - pure sample measurement value) ÷ pure sample measurement value × 100%. The smaller the absolute value, the stronger the anti-interference ability.

[0094] Relative standard deviation (RSD): The standard deviation of 6 parallel measurements ÷ the average value × 100%, which reflects the precision of the method. RSD ≤ 3.0% is excellent.

[0095] 7.3.2 Comparative Experiment Two: Verification of Accuracy Across the Entire Concentration Range (Covering Low, Medium, and High Concentrations)

[0096] Experimental materials: Prepare standard samples of sodium hyaluronate at low concentration (0.5 mg / g), medium concentration (5 mg / g), and high concentration (50 mg / g) (with known accurate content), and divide each sample into 3 parallel groups.

[0097] Results Comparison: Using known concentrations as a baseline, the determination error, recovery rate, and RSD of the three groups were calculated.

[0098] 7.4 Data and Conclusions

[0099] The samples with low (0.5 mg / g), medium (5 mg / g), and high (50 mg / g) concentrations were measured, and the data are shown in the table below:

[0100] Table 2 Comparison of the performance of sodium hyaluronate samples with different concentrations

[0101]

[0102] As shown in Table 2, the recoveries of pure samples and simulated samples containing interfering substances such as phenoxyethanol and glyceryl stearate were consistently between 94.8% and 105.1%, with a relative standard deviation (RSD) of ≤2.8% and an interference rate of only 1.2% to 3.2%. In contrast, the traditional sulfuric acid-carbazole method, due to its lack of targeted impurity removal and the susceptibility of the colorimetric reaction to concentration, resulted in a recovery rate of 79.6% to 124.5% for high-concentration interfering samples, with an RSD of 7.1%. The conventional HPLC-UV method, due to the lack of specific derivatization leading to overlapping impurity peaks, had an interference rate of 6.3% to 15.9%, all of which have significant performance limitations. These advantages stem from the synergistic effect of the method design: high-temperature acid hydrolysis at 121℃ ensures the complete hydrolysis of sodium hyaluronate macromolecules into glucosamine; PMP derivatization reacts specifically with the target monomer to generate a strong UV-absorbing derivative; combined with the targeted removal of lipid-soluble impurities by dichloromethane and efficient separation by gradient elution, it achieves excellent results in accurate detection of low-concentration samples, stable response of high-concentration samples, and anti-interference determination of complex matrix samples. In the actual sample testing of commercially available moisturizing creams, the measured values ​​deviate from the measured data of third-party testing institutions with CNAS accreditation using the national standard method by ≤2%, fully demonstrating its reliable applicability in the detection of complex matrices such as cosmetics and food.

[0103] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for determining the content of sodium hyaluronate, characterized in that, Includes the following steps: Step (1): Using the prepared D-glucosamine hydrochloride standard stock solution, prepare a series of D-glucosamine hydrochloride standard curve solutions. Step (2): Take a sodium hyaluronate sample, add hydrochloric acid solution, heat and hydrolyze, neutralize and remove the remaining hydrochloric acid, and filter to obtain the sample solution. Step (3): Take the standard curve solution and the sample solution respectively, add PMP-methanol solution and sodium hydroxide solution and react at a constant temperature so that D-glucosamine hydrochloride in the standard curve solution and glucosamine in the sample solution are combined with PMP to generate glucosamine-PMP derivatives. Then use hydrochloric acid to stop the reaction. After extraction, separation and filtration, the derivatized standard solution and derivatized sample solution are obtained respectively. Step (4): Perform gradient elution on the derivatized standard solution and the derivatized sample solution respectively to obtain the data on the relationship between the concentration and peak area of ​​the derivatized standard solution, and the peak area data of the glucosamine-PMP derivative target peak in the derivatized sample solution. Step (5): Plot a standard curve based on the correlation data between concentration and peak area. Substitute the target peak area of ​​the glucosamine-PMP derivative in the derivatized sample solution into the standard curve to obtain the concentration of D-glucosamine hydrochloride in the sample. Calculate the sodium hyaluronate content accordingly.

2. The method for determining the sodium hyaluronate content according to claim 1, characterized in that, In step (1), the concentrations of the D-glucosamine hydrochloride standard curve solution are 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL, respectively.

3. The method for determining the sodium hyaluronate content according to claim 1, characterized in that, Step (2) specifically includes: Take the sodium hyaluronate sample extrusion, add hydrochloric acid solution, vortex mix, place in a 121 ℃ oven, heat and hydrolyze for 1.5-2 hours, remove and cool to room temperature, neutralize the remaining hydrochloric acid with sodium hydroxide solution, and make up to volume. After making up to volume, filter and take the filtrate as the sample solution.

4. The method for determining the sodium hyaluronate content according to claim 3, characterized in that, The filtration in step (2) includes: using a water filter with a pore size of 0.45 μm to filter the neutralized and diluted solution, discarding the initial filtrate, collecting the subsequent filtrate, and the resulting subsequent filtrate is the sample solution.

5. The method for determining the sodium hyaluronate content according to claim 1, characterized in that, In step (3), the reagent used for extraction is dichloromethane.

6. The method for determining the sodium hyaluronate content according to claim 5, characterized in that, In step (3), the extraction, separation, and filtration specifically include: An extractant was added to the solution obtained by stopping the reaction with hydrochloric acid, followed by vortex extraction and centrifugation to obtain a layered solution. The supernatant was collected and filtered to obtain the derivatized sample solution.

7. The method for determining the sodium hyaluronate content according to claim 6, characterized in that, In step (3), the supernatant is filtered using a filter membrane with a pore size of 0.22 μm.

8. The method for determining the sodium hyaluronate content according to claim 1, characterized in that, In step (4), the gradient elution conditions are as follows: Mobile phase: Phase A is acetonitrile, Phase B is 0.025 mol / L ammonium acetate solution, pH of ammonium acetate solution is 4.5; Gradient program: At 0 min, phase A accounts for 15% and phase B accounts for 85%; at 30 min, the proportion of phase A increases to 21% and the proportion of phase B decreases to 79%; at 35 min, the proportion of phase A increases to 85% and the proportion of phase B decreases to 15%; at 36 min, the proportion of phase A falls back to 15% and the proportion of phase B rises back to 85%; at 45 min, the proportion of phase A is maintained at 15% and the proportion of phase B at 85%. Supporting conditions: flow rate 1.0 mL / min, detection wavelength 250 nm, column temperature 40 ℃, injection volume 10 μL, C18 column.

9. The method for determining the sodium hyaluronate content according to claim 1, characterized in that, In step (5), the formula for calculating the sodium hyaluronate content is as follows: In the above formula, This refers to the sodium hyaluronate content, expressed in mg / g. The ratio of the molecular weight of the disaccharide fragment to the molecular weight of D-glucosamine hydrochloride is 1.8611. The concentration of D-glucosamine hydrochloride obtained from the regression equation is expressed in µg / mL. This is the conversion factor between micrograms and milligrams, with a value of 10. -3 V represents the volume of the sample solution in mL; S represents the mass of the sodium hyaluronate sample in g.

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