Method for determining content of human epidermal growth factors in sodium hyaluronate composite gel

Abstract

The invention relates to the technical field of polypeptide detection, and particularly discloses a method for determining the content of human epidermal growth factors in sodium hyaluronate composite gel. The method comprises the following steps: S1, carrying out enzymolysis on sodium hyaluronate in the sodium hyaluronate composite gel by using an enzyme to obtain a test solution; s2, measuring a blank matrix solution containing no human epidermal growth factors, and adding enzyme for enzymolysis to obtain a blank matrix solution; s3, measuring the human epidermal growth factor, and diluting the human epidermal growth factor with the blank matrix solution to obtain a reference substance solution; and S4, detecting the test solution and the reference solution by using a high performance liquid chromatography and an ultraviolet detector, and determining the content of the human epidermal growth factor by using an external standard method. According to the method, hyaluronidase is used for pre-treating the test sample, macromolecular and high-viscosity sodium hyaluronate is subjected to enzymolysis to form micromolecular sugar, the method is compatible with the same liquid chromatography separation system, the content of the human epidermal growth factors in the test sample is measured by adopting an HPLC (High Performance Liquid Chromatography) combined ultraviolet detector, and the method is high in specificity, high in accuracy, low in detection limit and good in repeatability.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide detection, and in particular to a method for determining the content of human epidermal growth factor in a sodium hyaluronate composite gel.

[0002] The method of the invention is a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel by combining an enzymatic pretreatment method with a high performance liquid chromatography method. Background Art

[0003] Hyaluronic acid (HA) is a high-molecular-weight, linear mucopolysaccharide composed of disaccharide units consisting of D-glucuronic acid and N-acetylglucosamine. It is an important component of human and animal skin, vitreous body, joint lubricating fluid and cartilage tissue. The hydroxyl groups on the uronic acid groups in the hyaluronic acid molecules are free. When combined with sodium ions, the polysaccharide takes the form of a sodium salt, called sodium hyaluronate. The molecular weight of its polymer in the body generally ranges from tens of thousands to millions of Daltons. Hyaluronic acid is soluble in water but insoluble in ethanol, ether and acetone. It is widely used in industries such as pharmaceuticals, medical devices and cosmetics.

[0004] Sodium hyaluronate is a polysaccharide with functions such as space filling, structural stabilization, cell coating, and cell protection. It possesses excellent lubricity, moisture retention, viscoelasticity, and biocompatibility. It is currently widely used in various ophthalmic surgeries and in the treatment of traumatic or degenerative bone and joint problems, as a cosmetic product for wrinkle filling and wrinkle reduction. Sodium hyaluronate gel is degraded by hyaluronidase in the body, requiring repeated injections for long-term efficacy. Sodium hyaluronate composite gel, primarily composed of sodium hyaluronate, also contains human epidermal growth factor, which promotes cell proliferation and growth, further increasing the secretion of hyaluronic acid and glycoproteins, providing filling, repairing, and anti-inflammatory properties, potentially achieving long-lasting wrinkle-filling and wrinkle-removing effects.

[0005] Human epidermal growth factor (hEGF), also known as human oligopeptide-1, is an active substance in the human body. It is an active polypeptide composed of 53 amino acids. Human epidermal growth factor (hEGF) can be used to repair and heal wounds such as corneal injuries, burns and surgery, and can achieve good therapeutic effects. At present, commonly used methods for peptide drug detection include reversed-phase HPLC, ion-exchange HPLC, and size-exclusion HPLC. High-performance liquid chromatography detection methods are very common and highly accurate analytical methods for peptide drugs. However, when sodium hyaluronate is present, due to the high viscosity and large molecular weight of sodium hyaluronate, high-molecular-weight polymers and small-molecule peptides are incompatible in the same separation chromatography system, so conventional HPLC methods cannot be used to directly detect hEGF. Summary of the Invention

[0006] Due to the high viscosity, large molecular weight, and incompatibility of high-molecular-weight polymers and small-molecular-weight peptides in the same chromatographic separation system of sodium hyaluronate, existing detection methods are unable to solve the problems of sodium hyaluronate with high viscosity entering the chromatographic system, which easily causes chromatographic column blockage, reduced column efficiency, inability to separate impurities, and short chromatographic column service life.

[0007] The present invention aims to address the problem that existing detection methods are not suitable for detecting human epidermal growth factor in highly viscous sodium hyaluronate. The present invention provides a high-performance liquid chromatography detection method for human epidermal growth factor. This method does not affect the stability of hEGF, so that HPLC can be used to better detect the purity of hEGF, thereby solving the above-mentioned problem.

[0008] The present invention is achieved through the following technical solutions:

[0009] Provided is a method for determining the content of human epidermal growth factor in a sodium hyaluronate composite gel, the method comprising the following specific steps:

[0010] S1. Preparation of test solution:

[0011] Enzymatically hydrolyzing the sodium hyaluronate in the sodium hyaluronate composite gel to obtain a test solution;

[0012] S2. Preparation of blank matrix solution:

[0013] A blank matrix solution without human epidermal growth factor was measured, and then enzyme was added for enzymatic hydrolysis to obtain a blank matrix solution;

[0014] S3. Preparation of reference solution:

[0015] measuring human epidermal growth factor, and diluting it with the blank matrix solution to obtain a reference solution;

[0016] S4. Detection:

[0017] The test solution and the reference solution were detected by high performance liquid chromatography and an ultraviolet detector, and the content of human epidermal growth factor was determined by an external standard method.

[0018] Specifically, the method of the present invention also provides a new approach for liquid chromatography analysis of samples containing both high molecular weight polymers and small molecular weight polypeptide components.

[0019] The method of the present invention uses hyaluronidase to pre-treat a sample and combines it with liquid chromatography separation technology to measure the human epidermal growth factor content in the sample. The method has high accuracy, low detection limit, and good reproducibility. The method of the present invention can avoid the interference of sodium hyaluronate in the determination of human epidermal growth factor content, improve the method's specificity, sensitivity, and accuracy, and reduce the detection limit. At the same time, it can protect the chromatographic system from damage and extend the service life of the chromatographic column.

[0020] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: Step S1, preparation of test solution: wherein the enzyme used is hyaluronidase.

[0021] Specifically, the hyaluronidase is an endoglycosidase extracted from mammalian testicles.

[0022] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: Step S1, preparation of test solution: wherein the temperature during enzymatic hydrolysis is 30-40°C, the oscillation speed during enzymatic hydrolysis is 100-300 rpm, and the enzymatic hydrolysis time is 2-4 hours.

[0023] Preferably, the temperature is 37° C., the shaking speed is 200 rpm, and the enzymatic hydrolysis time is 3 hours.

[0024] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: in step S1, the mass volume ratio of sodium hyaluronate composite gel to hyaluronidase solution is 1.0 g: (25.0-40.0) μL.

[0025] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: Step S2, preparation of blank matrix solution: Take 1.0 g of blank matrix solution without human epidermal growth factor into a centrifuge tube, then add 25.0-50.0 μL of hyaluronidase solution, and enzymatically hydrolyze at a speed of 150-250 rpm in a constant temperature shaking incubator for 2-4 hours to obtain a blank matrix solution.

[0026] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: the concentration of the hyaluronidase solution is 4000-6000 IU / ml.

[0027] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: Step S3, preparation of reference solution: measuring human epidermal growth factor, diluting it with the blank matrix solution to contain 5.0 to 15.0 μg of human epidermal growth factor per milliliter, thereby obtaining a reference solution.

[0028] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: Step S4, detection: the detection conditions are as follows:

[0029] The chromatographic column used is an octadecylsilane bonded silica gel column, and the column temperature is 20-30°C (preferably 25°C);

[0030] Based on 100 volumes of the mobile phase, a 0.1% trifluoroacetic acid-water solution and a 0.1% trifluoroacetic acid-acetonitrile solution are used in a volume ratio of (90-98): (2-10);

[0031] Chromatographic conditions used: flow rate 1.0-1.5 ml / min (preferably 1.5 ml / min);

[0032] The detection wavelength is 220-280 nm (preferably 278 nm); the injection volume is 50.0-100.0 μL (preferably 100.0 μL).

[0033] Preferably, the chromatographic column is a Poroshell 120-EC18 chromatographic column or a ZORBAX300SB-C18 chromatographic column. Preferably, the volume ratio of the mobile phase is 95:5, comprising 0.1% trifluoroacetic acid-water solution and 0.1% trifluoroacetic acid-acetonitrile solution.

[0034] Furthermore, a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel: the detection limit of human epidermal growth factor in the sodium hyaluronate composite gel is greater than or equal to 0.2 ppm.

[0035] The method of the invention is a method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel by combining an enzymatic pretreatment method with a high performance liquid chromatography method.

[0036] The applicant has found through research that when sodium hyaluronate is degraded using hyaluronidase, the stability of hEGF will not be affected, so the purity of hEGF can be better detected using HPLC.

[0037] Beneficial effects of the present invention:

[0038] The method of the present invention uses hyaluronidase to pre-treat the test sample, enzymatically hydrolyzes the large molecular weight, high-viscosity sodium hyaluronate into small molecular sugars, is compatible with the same liquid chromatography separation system, and uses HPLC combined with an ultraviolet detector to measure the human epidermal growth factor content in the test sample. The method of the present application has strong specificity, high accuracy, low detection limit, and good repeatability.

[0039] The method provided in this application can avoid the interference of high-viscosity sodium hyaluronate on the determination of human epidermal growth factor content, improve the sensitivity and accuracy of the method, and reduce the detection limit; at the same time, it can protect the chromatography system and extend the service life of the chromatographic column. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 is the chromatogram of the reference substance solution in Example 1;

[0042] Figure 2 is the chromatogram of the blank matrix solution in Example 1;

[0043] Figure 3 The chromatogram of the test solution in Example 1. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is in no way intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0045] In the description of the present invention, it should be understood that the terms "upper", "lower", "left", "right", "top", "bottom", etc., indicating directions or positional relationships, are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific direction, be constructed and operate in a specific direction, and therefore should not be understood as limiting the present invention. In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Moreover, the terms "first", "second", etc. are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchangeable where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein.

[0046] Example 1

[0047] Provided is a method for determining the content of human epidermal growth factor in a sodium hyaluronate composite gel, the method comprising the following specific steps:

[0048] S1. Preparation of test solution (this step is also the sample pretreatment step):

[0049] Using hyaluronidase, enzymatically hydrolyze the sodium hyaluronate in the sodium hyaluronate composite gel to obtain a test solution;

[0050] Wherein: the hyaluronidase is an endoglycosidase extracted from mammalian testicles, the mass volume ratio of sodium hyaluronate composite gel to hyaluronidase solution is 1.0 g:30.0 μL, and the concentration of the hyaluronidase solution is 5000 IU / ml;

[0051] The enzymatic hydrolysis in step S1 is performed in a shaking incubator, and the temperature during the enzymatic hydrolysis is 37° C., the shaking speed during the enzymatic hydrolysis is 200 rpm, and the enzymatic hydrolysis time is 3 hours;

[0052] S2. Preparation of blank matrix solution:

[0053] Accurately measure 1.0 g of a blank matrix solution without human epidermal growth factor into a centrifuge tube, then add 30.0 μL of a hyaluronidase solution (the concentration of the hyaluronidase solution is 5000 IU / ml), and then perform enzymatic hydrolysis at 200 rpm in a constant temperature shaking incubator at 37°C for 3 hours to obtain an enzymatically hydrolyzed blank matrix solution;

[0054] S3. Preparation of reference solution:

[0055] Accurately measure human epidermal growth factor, and dilute it with the blank matrix solution to contain 10.0 μg of human epidermal growth factor per milliliter, thereby obtaining a reference solution;

[0056] S4. Detection:

[0057] The test solution and the reference solution were detected by high performance liquid chromatography and ultraviolet detection, and the content of human epidermal growth factor was determined by external standard method;

[0058] Among them: The specific testing conditions are as follows:

[0059] The chromatographic column used is an octadecylsilane bonded silica gel column (specifically a ZORBAX300SB-C18 chromatographic column with a size of 4.6×250 mm and a column length of 5 μm, high sensitivity, high resolution, and a wide acid-base tolerance range), and the column temperature is 25°C; based on the mobile phase volume of 100, it uses 0.1% trifluoroacetic acid-water solution (mobile phase A) and 0.1% trifluoroacetic acid-acetonitrile solution (mobile phase B) with a volume ratio of 95:5; the chromatographic conditions used are: a flow rate of 1.5 ml / min; a detection wavelength of 278 nm; and an injection volume of 100.0 μL.

[0060] The detection run time of Example 1 is 7 minutes, and the gradient elution program is as follows:

[0061] Time (min) Mobile phase A Mobile phase B 0 5 95 5 26 74 25 31 69 30 70 30

[0062] Result calculation: The formula for calculating the content of human epidermal growth factor in the test sample is as follows:

[0063]

[0064] Where: Ci is the concentration of human epidermal growth factor in the reference solution (μg / mL); A sample is the peak area of ​​the test solution; A control is the peak area of ​​the reference solution; C control is the concentration of human epidermal growth factor in the reference solution (μg / mL); m is the sample weight (g); 0.03 is the amount of enzyme added (g);

[0065] Test results: The content of human epidermal growth factor in the hyaluronic acid composite solution in Example 1 is as follows:

[0066]

[0067] This Example 1 utilizes the above-described chromatographic conditions to more accurately determine the content of human epidermal growth factor with good repeatability and high accuracy. Furthermore, the detection method provided by the present invention utilizes hyaluronidase to specifically enzymatically hydrolyze the sodium hyaluronate in the sodium hyaluronate composite gel, thereby reducing the interference of sodium hyaluronate with the detection of human epidermal growth factor, achieving a detection limit of 0.2 ppm and high detection accuracy.

[0068] Example 2

[0069] Provided is a method for determining the content of human epidermal growth factor in a sodium hyaluronate composite gel, the method comprising the following specific steps:

[0070] S1. Preparation of test solution (this step is also the sample pretreatment step):

[0071] Using hyaluronidase, enzymatically hydrolyze the sodium hyaluronate in the sodium hyaluronate composite gel to obtain a test solution;

[0072] Wherein: the hyaluronidase is an endoglycosidase extracted from mammalian testicles, the mass volume ratio of sodium hyaluronate composite gel to hyaluronidase solution is 1.0 g:40.0 μL, and the concentration of the hyaluronidase solution is 4000 IU / ml;

[0073] The enzymatic hydrolysis in step S1 is performed in a shaking incubator, and the temperature during the enzymatic hydrolysis is 30° C., the shaking speed during the enzymatic hydrolysis is 300 rpm, and the enzymatic hydrolysis time is 4 hours;

[0074] S2. Preparation of blank matrix solution:

[0075] Accurately measure 1.0 g of a blank matrix solution without human epidermal growth factor into a centrifuge tube, then add 50.0 μL of a hyaluronidase solution (the concentration of the hyaluronidase solution is 6000 IU / ml), and then perform enzymatic hydrolysis in a constant temperature shaking incubator at 37°C and 150 rpm for 4 hours to obtain an enzymatically hydrolyzed blank matrix solution;

[0076] S3. Preparation of reference solution:

[0077] Accurately measure human epidermal growth factor, and dilute it with the blank matrix solution to contain 15.0 μg of human epidermal growth factor per milliliter, thereby obtaining a reference solution;

[0078] S4. Detection:

[0079] The test solution and the reference solution were detected by high performance liquid chromatography and ultraviolet detection, and the content of human epidermal growth factor was determined by external standard method;

[0080] Among them: The specific testing conditions are as follows:

[0081] The chromatographic column used was an octadecylsilane bonded silica gel column (specifically a Poroshell 120-EC18 column with dimensions of 4.6×150 mm and an inner diameter of 2.7 μm. Generally, the smaller the inner diameter of the filler particles of the chromatographic column, the better the column efficiency of the chromatographic column, but the service life of the column will be shortened as the column pressure increases). The column temperature was 30°C. Based on the mobile phase volume of 100, a 0.1% trifluoroacetic acid-water solution (mobile phase A) and a 0.1% trifluoroacetic acid-acetonitrile solution (mobile phase B) with a volume ratio of 90:10 were used. The chromatographic conditions used were: a flow rate of 1.0 ml / min; a detection wavelength of 230 nm; and an injection volume of 50.0 μL.

[0082] Example 3

[0083] Repeatability determination: Prepare six test sample solutions and control solutions according to the test sample treatment method of Example 1, and the test conditions are the same as Example 1;

[0084] The results of human epidermal growth factor detection in Example 3 are shown in the following table:

[0085]

[0086] Example 4

[0087] Linearity determination: Prepare linear solutions of varying concentrations according to the procedures and test conditions of Example 1; Linearity stock solution: Accurately measure 1 ml of hEGF stock solution and dilute to 10 ml with diluent; Linearity solutions are prepared in two steps: first, dilute an appropriate amount of hEGF stock solution to obtain linearity stock solution 1, and then dilute sequentially to prepare linearity stock solutions at each linear point. Accurately measure 0.2 ml of each linearity stock solution and add 0.8 ml of substrate solution to obtain each linearity solution;

[0088] The linearity test results of Example 4 are shown in the following table:

[0089]

[0090] Example 5

[0091] Accuracy determination: Sodium hyaluronate composite gels containing 50%, 100%, and 150% human epidermal growth factor were prepared according to the test sample treatment method in Example 1. Two replicates were prepared for each concentration, and the testing conditions were the same as in Example 1.

[0092] The accuracy test results of Example 5 are shown in the following table:

[0093]

[0094]

[0095] In this invention, "enzymatic hydrolysis" refers to the hydrolysis of specific substances using active enzymes. Enzymes are proteins or RNA produced by living cells that are highly specific and catalytic for their substrates, making them an important and highly effective class of biocatalysts. The addition of human epidermal growth factor to the sodium hyaluronate composite gel promotes cell proliferation and growth, increasing the secretion of hyaluronic acid and glycoproteins, and possessing repair, conditioning, and acne-removing properties.

[0096] The instruments and equipment used in the present invention include: a chromatograph (Agilent 1260 high performance liquid chromatograph), a constant temperature shaking incubator, a vortex mixer and an analytical balance.

[0097] The above preferred embodiments of the present invention are only used to explain the present invention and are not used to limit the present invention. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. A method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel, characterized in that: The method comprises the following steps: S1. Preparation of test solution: Enzymatically hydrolyzing the sodium hyaluronate in the sodium hyaluronate composite gel to obtain a test solution; S2. Preparation of blank matrix solution: A blank matrix solution without human epidermal growth factor was measured, and then enzyme was added for enzymatic hydrolysis to obtain a blank matrix solution; S3. Preparation of reference solution: measuring human epidermal growth factor, and diluting it with the blank matrix solution to obtain a reference solution; S4. Detection: The test solution and the reference solution were detected by high performance liquid chromatography and an ultraviolet detector, and the content of human epidermal growth factor was determined by an external standard method.

2. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: Step S1, preparation of test solution: the enzyme used is hyaluronidase.

3. A method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1 or 2, characterized in that: Step S1, preparation of the test solution: wherein the temperature during enzymatic hydrolysis is 30-40° C., the shaking speed during enzymatic hydrolysis is 100-300 rpm, and the enzymatic hydrolysis time is 2-4 hours.

4. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: The mass-to-volume ratio of the sodium hyaluronate composite gel to the hyaluronidase solution in step S1 is 1.0 g: (25.0-40.0) μL.

5. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: Step S2, preparation of blank matrix solution: 1.0 g of blank matrix solution without human epidermal growth factor was taken into a centrifuge tube, and then 25.0 to 50.0 μL of hyaluronidase solution was added, and enzymatic hydrolysis was performed at a speed of 150 to 250 rpm in a constant temperature shaking incubator for 2 to 4 hours to obtain a blank matrix solution.

6. A method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 4 or 5, characterized in that: The concentration of the hyaluronidase solution is 4000-6000 IU / ml.

7. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: Step S3, preparation of reference solution: human epidermal growth factor is measured, and it is diluted with the blank matrix solution to contain 5.0-15.0 μg of human epidermal growth factor per milliliter, thereby obtaining a reference solution.

8. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: Step S4, detection: the detection conditions are as follows: The chromatographic column used was an octadecylsilane bonded silica gel column, and the column temperature was 20-30°C; Based on 100 volumes of the mobile phase, a 0.1% trifluoroacetic acid-water solution and a 0.1% trifluoroacetic acid-acetonitrile solution are used in a volume ratio of (90-98): (2-10); The chromatographic conditions used were: flow rate 1.0–1.5 ml / min; The detection wavelength is 220-280 nm; the injection volume is 50.0-100.0 μL.

9. The method for determining the content of human epidermal growth factor in sodium hyaluronate composite gel according to claim 1, characterized in that: The detection limit of human epidermal growth factor in the sodium hyaluronate composite gel is greater than or equal to 0.2 ppm.

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