CN114646708A - Method for measuring content of sodium hyaluronate - Google Patents
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Abstract
The invention belongs to the field of polysaccharide analysis and detection, and relates to a method for determining the content of sodium hyaluronate. And (3) hydrolyzing the sodium hyaluronate sample to be detected under an acidic condition, detecting the content of the hydrolysate, and converting and calculating to obtain the content of the sodium hyaluronate. The method adopts high performance liquid chromatography with high sensitivity, low detection limit, good accuracy and obvious separation effect to determine the monosaccharide content, and combines a universal evaporative light scattering detector, thereby overcoming the difficulties that hyaluronic acid does not have luminous groups, has weak ultraviolet absorption and is difficult to detect, has high accuracy, is simple and convenient to operate, is not influenced by the molecular weight of sodium hyaluronate, and has strong applicability. Can provide reference basis for realizing the quality control of hyaluronic acid related products, and has important theoretical and practical significance for promoting the development of hyaluronic acid industry.
Description
Technical Field
The invention belongs to the field of polysaccharide analysis and detection, and relates to a method for determining the content of sodium hyaluronate.
Background
Hyaluronic Acid (HA), also known as Hyaluronic acid and Hyaluronic acid, is one of the ingredientsDGlucuronic acid andNthe-acetylglucosamine is a linear anionic mucopolysaccharide formed by alternately connecting disaccharide units. HA was first isolated from bovine vitreous by american scientists and is widely used in the cosmetics, food and pharmaceutical industries because of its good lubricity, moisture retention and viscoelasticity, and its excellent biocompatibility. Due to the poor solubility of hyaluronic acid itself, hyaluronic acid is often converted to Sodium salt, Sodium Hyaluronate (SH) during use. SH is a white powdery solid, is also fibrous in some cases, has better moisture retention, can be dissolved in water, is not dissolved in organic solvents such as ethyl ether, acetone, ethanol and the like, has no odor, is a chain natural high molecular polysaccharide substance, is widely applied to a plurality of fields at present, and therefore, has important significance in establishing an accurate quantitative analysis method.
At present, the method for measuring the content of sodium hyaluronate comprises the following steps: sulfuric acid-carbazole method, CTAB turbidity method, gel chromatography, Elson-Morgan method, immunization method, resonance Rayleigh scattering method. The sulfuric acid-carbazole method is characterized in that sodium hyaluronate is degraded into glucuronic acid by strong acid, the glucuronic acid reacts with carbazole to form an organic complex and shows a characteristic purple color, the absorbance of the complex is in direct proportion to the concentration of the uronic acid, and the content of the sodium hyaluronate is determined by the content of the glucuronic acid. However, when the method is used for detecting a sample with a complex formula, some preservatives, emulsifiers, stabilizers and the like with complex structures in the sample may also react with a carbazole sulfate reagent, and the result deviation is large, so that the specificity is not strong. The CTAB turbidity method for measuring the content of sodium hyaluronate in fermentation liquor is characterized in that HA and cationic surfactants such as Cetyl Trimethyl Ammonium Bromide (CTAB) can perform a complex reaction to generate a turbidity phenomenon, and the method follows Lambert-beer law within a certain concentration range, but the method HAs the defect that the method is not suitable for real-time detection. The method for measuring the content of the sodium hyaluronate by utilizing the gel chromatography has the advantages of complex operation, slow analysis speed, high price and high requirement on the operation level of personnel. In addition, investigation finds that the currently commonly used SH content determination method is mainly used for determining the content of the SH according to the absorbance by means of color development after degradation and combining a visible-ultraviolet spectrophotometer to determine the content of glucuronic acid or glucosamine in the degraded product. The method has the advantages of complex operation, harsh conditions and interference of the introduction of the color developing agent on the accuracy of measurement.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for measuring the content of sodium hyaluronate.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for determining the content of sodium hyaluronate comprises the steps of hydrolyzing a sodium hyaluronate sample to be detected under an acidic condition, detecting the content of a hydrolysate, and then converting and calculating to obtain the content of sodium hyaluronate.
Sodium hyaluronate structure formula
The sodium hyaluronate is hydrolyzed under acidic condition to form salt, the hydrolyzed salt is subjected to high performance liquid chromatography to obtain the content of the salt, and the content of the sodium hyaluronate is obtained by utilizing the conversion calculation.
The high performance liquid chromatography adopts sodium hyaluronate to hydrolyze under an acidic condition to form salt as a standard substance, deionized water is added to dissolve the salt to prepare a series of standard solutions with the concentrations of 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL and 3.0 mg/mL respectively, sample injection detection is carried out under an HPLC (high performance liquid chromatography) chromatographic condition, then sodium hyaluronate is hydrolyzed under an acidic condition to form a salt concentration logarithm as a horizontal coordinate, a corresponding peak area integral logarithm is as a vertical coordinate, and a standard curve is drawn.
And (3) determination of the hydrolysate sample: taking a proper amount of prepared hydrolysate sample solution to be detected, passing the hydrolysate sample solution through an organic filter membrane of 0.45 mu m, carrying out sample injection detection under HPLC (high performance liquid chromatography) chromatographic conditions, calculating the concentration of salt formed by hydrolysis of sodium hyaluronate under acidic conditions according to a standard curve, and converting and calculating the content of the sodium hyaluronate according to a formula (1);
in the formula (1), the first and second groups,
w -sodium hyaluronate content,%;
m-sodium hyaluronate powder solid mass, g;
C -the concentration of sodium hyaluronate obtained from the standard curve hydrolyzed under acidic conditions to form salt, mg/mL;
V-acid hydrolysis of the volume of the solution to be tested, mL;
Mr 1-the molar mass of the salt formed by hydrolysis of sodium hyaluronate under acidic conditions, g/mol;
Mr 2-molar mass of sodium hyaluronate units, g/mol.
The acid is concentrated hydrochloric acid, sodium hyaluronate solid powder is taken, the concentrated hydrochloric acid and deionized water are added, the temperature is increased to 20-55 ℃ for reaction for 0.5-3.0h, the temperature is increased to 55-85 ℃ for reaction for 0.5-3.0h after the reaction, and then the temperature is increased to 85-120 ℃ for reaction for 2-16 h; removing residual hydrochloric acid after reaction, adding deionized water for dissolution, and fixing volume to obtain a sodium hyaluronate hydrolysis sample solution to be detected; wherein the ratio of the addition amount of the sodium hyaluronate solid powder to the addition amount of the concentrated hydrochloric acid and the deionized water is 10:4: 1-80: 9:2 (mg: mL: mL). The concentrated hydrochloric acid is hydrochloric acid with the concentration of 12 mol/mL.
The acid used for the hydrolysis may be sulfuric acid or phosphoric acid, in addition to hydrochloric acid.
The optimal reaction condition for obtaining the highest hydrolysis salt yield by gradient temperature rise and acid degradation is that the reaction is carried out for 1.5 h at 50 ℃, the reaction is carried out for 1.5 h when the temperature is raised to 80 ℃, and then the reaction is carried out for 10 h when the temperature is raised to 100 ℃.
The high performance liquid chromatography conditions are as follows: adopting a Syneronis Amino sugar analysis chromatographic column, wherein the detector is an evaporative light scattering detector, the mobile phase is acetonitrile-water solution (60: 40, V/V), the flow rate is 0.1-1.5mL/min, the sample injection amount is 10-80 mu L, and the column temperature is 20-60 ℃; the flow rate of carrier gas of the evaporative light scattering detector is 0.5-3.0L/min, and the temperature of the drift tube is 30-100 ℃.
Compared with the prior art, the invention has the beneficial effects that:
1. the determination method comprises the steps of performing acid hydrolysis on sodium hyaluronate to obtain a hydrolyzed solution, performing HPLC (high performance liquid chromatography) detection on glucosamine hydrochloride in the hydrolyzed solution, accurately quantifying, and then converting and calculating the content of the sodium hyaluronate according to the content of the glucosamine hydrochloride, so that the influence of different molecular weights of the sodium hyaluronate on content determination is avoided, and the method is simple, convenient and easy to implement, simple to operate and strong in applicability.
2. The invention adopts the light-emitting scattering detector, is a universal detector, avoids the defects of the common ultraviolet detector, namely the substance to be detected can be detected under the condition of no ultraviolet absorption or weak ultraviolet absorption, so the acid hydrolysis product of the sodium hyaluronate can be directly injected into a sample for HPLC (high performance liquid chromatography) to detect, and a color developing agent is not required to be introduced for pre-column modification, thereby avoiding the introduction of impurities, simplifying the operation steps, saving experimental materials and reducing the experimental cost.
3. The method obtains the content of the sodium hyaluronate through conversion calculation by measuring the content of glucosamine hydrochloride in an acid hydrolysis solution, and replaces the method for quantitatively measuring the degradation product glyoxylic acid in the market at present; and the glucosamine hydrochloride in the acid hydrolysis liquid is directly measured without any pre-biochemical column modification and introduction of interfering components, so that the content of the sodium hyaluronate obtained by measurement is closer to the actual content, the measurement result is more accurate, a new thought and method are provided for the measurement of the content of the sodium hyaluronate, and the method has better application prospect and higher practical significance.
4. According to the invention, the high performance liquid chromatography is adopted to measure the chitosan content, and an evaporative light scattering detector is adopted, so that the HPLC has the advantages of strong specificity, high sensitivity, low detection limit, good accuracy, rapidness, high efficiency, obvious separation effect and the like, and has a good application prospect; the ELSD detector belongs to a general quality detector, has strong environmental interference resistance, has no strict requirements on flow rate and temperature, can perform gradient elution, has a better analysis effect on saccharides with weak ultraviolet absorption or no ultraviolet absorption, and has wide application in high performance liquid chromatography.
Drawings
FIG. 1 is the sodium hyaluronate hydrolysis equation;
FIG. 2 is a standard curve of glucosamine hydrochloride obtained in example 1;
FIG. 3 is an HPLC chromatogram of glucosamine hydrochloride standard obtained in example 1;
fig. 4 is an HPLC chromatogram of the sodium hyaluronate acid hydrolysis sample solution obtained in example 1.
Detailed Description
The following examples are presented to further illustrate the present invention and the specific embodiments described herein are presented for the purpose of illustration and explanation and are not intended to be limiting.
According to the invention, sodium hyaluronate is hydrolyzed into monosaccharide, and the content of the sodium hyaluronate is calculated according to the content of glucosamine hydrochloride monosaccharide, so that other interference components are not introduced, and the measurement result is more accurate. In addition, the determination method adopts an evaporative light scattering detector-high performance liquid chromatography, has good accuracy, high sensitivity, low detection limit and obvious separation effect, can eliminate the interference caused by the molecular weight difference of the sodium hyaluronate, overcomes the difficulty that the sodium hyaluronate does not have a luminescent group, has weak ultraviolet absorption and is difficult to detect, is a detection method which is widely applied at present, can provide reference basis for realizing the quality control of related products of the hyaluronic acid, and has important theoretical and practical significance for promoting the development of the hyaluronic acid industry.
Example 1
A method for determining the content of sodium hyaluronate by combining acid hydrolysis with high performance liquid chromatography comprises the following steps:
HPLC chromatographic conditions: a Syneronis Amino sugar analysis chromatographic column is adopted, the mobile phase is acetonitrile-water solution (85: 15, V/V), the sample injection amount is 50 mu L, the flow rate is 1.2 mL/min, the column temperature is 35 ℃, the detector is an evaporative light scattering detector, the carrier gas flow rate is 2.0L/min, and the drift tube temperature is 70 ℃.
Drawing a standard curve: accurately weighing 0.25 g of glucosamine hydrochloride standard, adding deionized water to dissolve, transferring into a 25 mL volumetric flask, cleaning and transferring for three times, fixing the volume, shaking up, and preparing into standard stock solution with the concentration of 10 mg/mL for later use. Taking appropriate amount of stock solution, respectively, diluting to obtain a series of standard solutions with concentrations of 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL, 3.0 mg/mL, and 3.5 mg/mL. Respectively taking appropriate amount of the standard solutions with different concentrations, filtering with 0.22 μm organic filter membrane, performing sample injection detection according to set HPLC chromatographic conditions (see figure 3), drawing a standard curve with a logarithmic value X of glucosamine hydrochloride concentration as abscissa and a logarithmic value Y of corresponding peak area integral value as ordinate, and obtaining a linear equation of the standard curve with Y =1.043X +4.002 and a correlation coefficient R2Is 0.9999. The standard curve of glucosamine hydrochloride is shown in FIG. 2. As can be seen from the figure, the standard curve shows better correlation when the glucosamine hydrochloride is in the concentration range of 0.5-3.5 mg/mL.
Preparing a hydrolysate sample:
taking 55 mg of sodium hyaluronate solid powder, adding 7 mL of concentrated hydrochloric acid and 2 mL of deionized water, hydrolyzing for 2.5 h at 40 ℃, 2.5 h at 75 ℃ and 12 h at 95 ℃, after the reaction is finished, decompressing and concentrating to remove residual hydrochloric acid, adding deionized water into a 25 mL volumetric flask for preparation, and fixing the volume to obtain a sodium hyaluronate acid hydrolysis sample solution (a hydrolysis equation is shown in figure 1).
Then, taking a proper amount of sodium hyaluronate acid hydrolysis sample solution, filtering the solution through a 0.22 mu m organic filter membrane, and carrying out sample injection detection on the solution through HPLC (high performance liquid chromatography) chromatographic conditions according to the recorded conditions (see figure 4), so as to obtain the peak area of acid hydrolysis salt; then, the concentration of glucosamine hydrochloride is 0.624 mg/mL according to the linear equation Y =1.043X +4.002, and then the formula (1) is followed
Calculated, C =0.624 mg/mL, V =25 mL, Mr1=215.63 mg/mol，Mr2The content of the sodium hyaluronate can be accurately obtained by =403 mg/mol and m = 58.2 g.
Example 2
Taking 70 mg of sodium hyaluronate solid powder, adding 10 mL of concentrated hydrochloric acid and 3 mL of deionized water, hydrolyzing for 3.5 h at 35 ℃, 3.5 h at 70 ℃ and 9 h at 110 ℃, after the reaction is finished, decompressing and concentrating to remove residual hydrochloric acid, preparing with a 50 mL volumetric flask and adding deionized water, and fixing the volume to obtain the sodium hyaluronate acid hydrolysis sample solution. Then, taking a proper amount of sodium hyaluronate acid hydrolysis sample solution, filtering the solution through a 0.22 mu m organic filter membrane, and carrying out sample injection detection according to the established HPLC chromatographic conditions recorded in the embodiment 1 to obtain the peak area of acid hydrolysis salt; then the concentration of glucosamine hydrochloride is 0.397 mg/mL according to the linear equation, and then the formula (1) is followed
And calculating to accurately obtain the content of the sodium hyaluronate of 99.6 percent.
Example 3
The high performance liquid phase method of the invention is proved by methodology and stability experiment is carried out. Taking a proper amount of glucosamine hydrochloride standard sample solution (1.5 mg/ml), filtering the solution through a 0.22-micron organic filter membrane, respectively carrying out sample injection detection for 0 hour, 2 hours, 8 hours, 12 hours and 24 hours, and observing the peak area change, wherein the RSD is 1.68 percent. Stability experiments show that the glucosamine hydrochloride sample solution has good stability within 24 hours under the storage condition of 4 ℃. The results are shown in Table 1.
Table 1 stability test results
Example 4
The high performance liquid phase method of the invention is proved by methodology and is developed with reproducibility experiment. 55 mg and 5 parts of glucosamine hydrochloride are taken to prepare a glucosamine hydrochloride aqueous solution, HPLC sample injection is carried out, the peak area of the glucosamine hydrochloride is measured, the concentration is calculated by an external standard method, and the RSD is calculated to be 1.35 percent, which shows that the method has good reproducibility, and the results are shown in Table 2.
TABLE 2 results of reproducibility experiments
Example 5
The high performance liquid phase method of the invention is proved by methodology and tested by precision experiment. Taking a proper amount of glucosamine hydrochloride standard sample solution (1.5 mg/ml), passing through a 0.22 mu m organic filter membrane, carrying out HPLC sample injection, repeating for 5 times, determining the peak area integral value of the glucosamine hydrochloride, determining the concentration of the glucosamine hydrochloride by an external standard method, wherein the RSD is 0.98%, which indicates that the precision and the sample injection method of the instrument are good. The results are shown in Table 3.
TABLE 3 results of the precision test
In summary, the present invention is based on the determination of one of the hyaluronic acid sodium disaccharide unitsNThe content of the sodium hyaluronate is obtained by the content of the acetylglucosamine, specifically, the sodium hyaluronate is hydrolyzed by acid to obtain a hydrolysis salt product, then the integral peak area of the hydrolysis product is determined by combining a high performance liquid chromatography technology, and the content of the sodium hyaluronate is obtained by calculation.
Claims (6)
1. A method for measuring the content of sodium hyaluronate is characterized by comprising the following steps: and (3) hydrolyzing the sodium hyaluronate sample to be detected under an acidic condition, and converting and calculating the content of the sodium hyaluronate by using the detection content of the hydrolysate.
2. The method for determining the content of sodium hyaluronate according to claim 1, characterized in that: the sodium hyaluronate is hydrolyzed under acidic condition to form salt, the hydrolyzed salt is measured by high performance liquid chromatography to obtain the content of the salt, and the content of the sodium hyaluronate is calculated by utilizing the conversion of the salt.
3. The method for determining the content of sodium hyaluronate according to claim 2, characterized in that: the high performance liquid chromatography adopts sodium hyaluronate to hydrolyze under an acidic condition to form salt as a standard substance, deionized water is added to dissolve the salt to prepare a series of standard solutions with the concentrations of 0.5 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 2.5 mg/mL and 3.0 mg/mL respectively, sample injection detection is carried out under an HPLC (high performance liquid chromatography) chromatographic condition, then sodium hyaluronate is hydrolyzed under an acidic condition to form a salt concentration logarithm as a horizontal coordinate, a corresponding peak area integral logarithm is as a vertical coordinate, and a standard curve is drawn.
4. The method for determining the content of sodium hyaluronate according to claim 2, characterized in that: and (3) determination of the hydrolysate sample: taking a proper amount of prepared to-be-detected hydrolysate sample solution, filtering the prepared to-be-detected hydrolysate sample solution through an organic filter membrane of 0.45 mu m, carrying out sample injection detection under HPLC chromatographic conditions, calculating the concentration of salt formed by hydrolysis of sodium hyaluronate under acidic conditions according to a standard curve, and carrying out conversion calculation according to a formula (1) to obtain the content of sodium hyaluronate;
in the formula (1), the first and second groups,
w -sodium hyaluronate content,%;
m -sodium hyaluronate powder solid mass, g;
C obtained from a standard curveThe sodium hyaluronate is hydrolyzed under the acidic condition to form the concentration of salt, mg/mL;
V-acid hydrolysis of the volume of the solution to be tested, mL;
Mr 1-the molar mass of the salt formed by hydrolysis of sodium hyaluronate under acidic conditions, g/mol;
Mr 2-molar mass of sodium hyaluronate units, g/mol.
5. The method for determining the content of sodium hyaluronate according to any of claims 1 to 4, characterized in that: the acid is concentrated hydrochloric acid, sodium hyaluronate solid powder is taken, the concentrated hydrochloric acid and deionized water are added, the temperature is increased to 20-55 ℃ for reaction for 0.5-3.0h, the temperature is increased to 55-85 ℃ for reaction for 0.5-3.0h after the reaction, and then the temperature is increased to 85-120 ℃ for reaction for 2-16 h; removing residual hydrochloric acid through reaction, adding deionized water for dissolution, and fixing the volume to obtain a sodium hyaluronate hydrolysis test sample solution to be tested; wherein the ratio of the addition amount of the sodium hyaluronate solid powder to the addition amount of the concentrated hydrochloric acid and the deionized water is 10:4: 1-80: 9:2 (mg: mL: mL).
6. The method of determining the amount of sodium hyaluronate according to claim 5, wherein: the high performance liquid chromatography conditions are as follows: adopting a Syneronis Amino sugar analysis chromatographic column, wherein the detector is an evaporative light scattering detector, the mobile phase is acetonitrile-water solution (60: 40, V/V), the flow rate is 0.1-1.5mL/min, the sample injection amount is 10-80 mu L, and the column temperature is 20-60 ℃; the carrier gas flow rate of the evaporative light scattering detector is 0.5-3.0L/min, and the drift tube temperature is 30-100 ℃.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117723682A (en) * | 2023-11-20 | 2024-03-19 | 山东丰金美业科技有限公司 | Method for detecting crosslinking degree of crosslinked hyaluronic acid or salt gel thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113072651A (en) * | 2020-01-06 | 2021-07-06 | 上海利康瑞生物工程有限公司 | Preparation method of oligomeric sodium hyaluronate solution |
| CN114236014A (en) * | 2021-12-27 | 2022-03-25 | 珠海溪谷医疗科技有限公司 | Method for detecting content of sodium hyaluronate in contact lens care solution |
-
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113072651A (en) * | 2020-01-06 | 2021-07-06 | 上海利康瑞生物工程有限公司 | Preparation method of oligomeric sodium hyaluronate solution |
| CN114236014A (en) * | 2021-12-27 | 2022-03-25 | 珠海溪谷医疗科技有限公司 | Method for detecting content of sodium hyaluronate in contact lens care solution |
Non-Patent Citations (3)
| Title |
|---|
| WANG HY: "Determination of sodium hyaluronate by HPLC", 《CHIN PHARM J》 * |
| 解钰: "采用高效液相色谱法测定萘敏维滴眼液中玻璃酸钠的含量", 《山西医科大学学报》 * |
| 黄芳: "酸水解-柱前衍生化-高效液相色谱法测定面膜化妆品中透明质酸", 《理化检验-化学分册》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117723682A (en) * | 2023-11-20 | 2024-03-19 | 山东丰金美业科技有限公司 | Method for detecting crosslinking degree of crosslinked hyaluronic acid or salt gel thereof |
| CN117723682B (en) * | 2023-11-20 | 2024-06-11 | 山东丰金美业科技有限公司 | Method for detecting crosslinking degree of crosslinked hyaluronic acid or salt gel thereof |
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