Method for detecting contents of collagen and regenerated silk fibroin in composite material

Abstract

The invention provides a method for detecting the contents of collagen and regenerated silk fibroin in a composite material, which comprises the following steps: (1) hydrolyzing the composite material for a certain time, and neutralizing to obtain a solution to be detected; (2) preparing a tyrosine reference solution; and (3) detecting the peak areas of the solution to be detected and the tyrosine reference substance solution by adopting high performance liquid chromatography, and calculating to obtain the content of regenerated silk fibroin in the composite material, and (4) detecting the total nitrogen content of the composite material by using a Kjeldahl method, and calculating to obtain the collagen content in the composite material. According to the method, the high performance liquid chromatography is combined with the Kjeldahl determination method, accurate determination of two components can be realized without derivatization treatment, the operation is simple and convenient, and the detection cost is low; the limit of quantitation of the method reaches 10.35 mu g / mL (S / N is greater than or equal to 16.59), the precision RSD is less than 1%, and the method has high sensitivity and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for detecting the contents of collagen and regenerated silk fibroin in a collagen / regenerated silk fibroin composite material by combining high performance liquid chromatography with Kjeldahl nitrogen determination. Background Art

[0002] As the most abundant and important protein in the human body, collagen boasts remarkable functionality and a wide range of applications. In recent years, the market for recombinant collagen products in food, functional health supplements, skincare products, medical devices, and especially aesthetic dressings has steadily grown. The market for recombinant collagen in aesthetic dressings and skincare products far exceeds that of other application areas.

[0003] Silk fibroin, a widely available natural macromolecule, also possesses excellent biocompatibility, mechanical properties, and controlled degradability, making it a growing mainstream biomaterial. Designated a biomaterial by the U.S. Food and Drug Administration (FDA) in 1993, silk fibroin demonstrates superior biocompatibility and cell adhesion compared to other natural biopolymers, inducing cell differentiation and tissue regeneration. Its biodegradability reduces inflammation and the risk of secondary surgery, making it a crucial biomaterial in regenerative medicine research.

[0004] Collagen has strong cell interaction and behavioral regulation capabilities, but suffers from poor thermal stability, biomechanical properties, and degradation resistance. Silk fibroin, on the other hand, has high thermal stability, degradation resistance, and good flexibility and mechanical properties. The two biomaterials complement each other well in terms of performance.

[0005] Combining the functional advantages of collagen and regenerated silk fibroin, collagen / regenerated silk fibroin composite materials have become a hot topic and have been extensively researched and developed.

[0006] Collagen is a functional protein, accounting for 30% of all proteins in the body. Its molecules contain sugars and significant amounts of glycine (approximately 25% to 30%), proline (approximately 12%), and hydroxyproline (approximately 10%). Type I and type III collagen are the primary collagens in human skin. Silk fibroin contains 18 amino acids, of which glycine, alanine, and serine account for over 80% of its total composition. Regenerated silk fibroin mimics the regeneration environment of natural silk, inducing the rearrangement of its molecular chains to produce a regenerated silk fibroin with excellent mechanical properties.

[0007] The main components of collagen / regenerated silk fibroin composite materials are recombinant humanized type III collagen and regenerated silk fibroin. The protein content is usually determined by Kjeldahl method, Folin phenol method, biuret method, 2,2,-biquinoline-4,4,-dicarboxylic acid method (BCA method), Coomassie brilliant blue method and UV-visible spectrophotometry. The content of single component collagen or regenerated silk fibroin can be accurately determined by the above methods. If both exist at the same time, and both are nitrogen-containing compounds, and the two components cannot be simply separated;

[0008] Therefore, there is an urgent need to provide a method for detecting the content of collagen and regenerated silk fibroin in collagen / regenerated silk fibroin composite materials, which does not require pre-column derivatization of the sample and has simple, rapid and low-cost operation steps. Summary of the Invention

[0009] The purpose of the present invention is to overcome the problems existing in the prior art and provide a method for determining the content of collagen and regenerated silk fibroin in a collagen / regenerated silk fibroin composite material by high performance liquid chromatography combined with Kjeldahl nitrogen determination.

[0010] To achieve the above purpose, the present invention is implemented through the following technical solutions

[0011] The present invention provides a method for detecting the content of collagen and regenerated silk fibroin in a composite material, comprising the following steps:

[0012] (1) hydrolyzing the composite material with a hydrolysis reagent for a certain period of time, and then neutralizing it to obtain a test solution;

[0013] (2) Prepare tyrosine reference solution;

[0014] (3) using high performance liquid chromatography to detect the peak areas of the test solution and the tyrosine reference solution, and calculating the content of regenerated silk fibroin in the composite material;

[0015] (4) The total nitrogen content of the composite material was detected by Kjeldahl method, and the collagen content in the composite material was calculated.

[0016] In some embodiments of the present invention, the composite material is a composite material including collagen and regenerated silk fibroin.

[0017] In some embodiments of the present invention, the collagen is humanized type III collagen.

[0018] In some embodiments of the present invention, the hydrolysis reagent in step (1) is an acidic solution;

[0019] In some embodiments of the present invention, the acidic solution is one or more of a hydrochloric acid solution, a sulfuric acid solution or a nitric acid solution; in some specific embodiments of the present invention, the acidic solution is a hydrochloric acid solution; in some specific embodiments of the present invention, the acidic solution is a sulfuric acid solution; in some specific embodiments of the present invention, the acidic solution is a nitric acid solution.

[0020] In some embodiments of the present invention, the solution to be tested is obtained by hydrolyzing with sulfuric acid solution and then neutralizing with sodium hydroxide solution, followed by dilution with ultrapure water.

[0021] In some embodiments of the present invention, the hydrolysis time in step (1) is 4-24 hours, and the hydrolysis temperature is 80-130°C; in some embodiments of the present invention, the hydrolysis time in step (1) is 10-20 hours, and the hydrolysis temperature is 90°C-120°C; in some embodiments of the present invention, the hydrolysis time in step (1) is 16 hours, and the hydrolysis temperature is 120°C.

[0022] In some specific embodiments of the present invention, the hydrolysis time in step (1) is 4 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 6 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 8 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 10 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 12 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 14 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 16 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 18 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 20 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 22 h; in some specific embodiments of the present invention, the hydrolysis time in step (1) is 24 h.

[0023] In some specific embodiments of the present invention, the hydrolysis temperature in step (1) is 80°C; in some specific embodiments of the present invention, the hydrolysis temperature in step (1) is 90°C; in some specific embodiments of the present invention, the hydrolysis temperature in step (1) is 100°C; in some specific embodiments of the present invention, the hydrolysis temperature in step (1) is 110°C; in some specific embodiments of the present invention, the hydrolysis temperature in step (1) is 120°C.

[0024] In some embodiments of the present invention, the tyrosine reference solution in step (2) is prepared by adding a tyrosine reference solution to a sulfuric acid solution and then diluting the solution with ultrapure water.

[0025] In some embodiments of the present invention, in step (3), high performance liquid chromatography is used to detect the peak areas of the test solution and the tyrosine reference solution, and the content of the medical silk fibroin in the collagen / regenerated silk fibroin composite material is calculated by the external standard method.

[0026] In some embodiments of the present invention, the method of detecting the peak area of ​​the test solution by high performance liquid chromatography in step (3) comprises: injecting the test solution and the reference solution into high performance liquid chromatography for detection, and recording the peak area.

[0027] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography include: a chromatographic column is a GLSciences Inertsil ODS-3 (4.6×250 mm, 5 μm) or a chromatographic column with equivalent performance, elution is performed with a mobile phase, a detection wavelength of 200 nm, a flow rate of 1.0 mL / min, a column temperature of 35°C, and an injection volume of 10 μL.

[0028] In some embodiments of the present invention, the mobile phase consists of 20 mM sodium acetate (pH adjusted to 6.0 with dilute acetic acid) and methanol, and the volume ratio of sodium acetate to methanol is 95:5.

[0029] In some embodiments of the present invention, the total nitrogen content of the composite material is detected by Kjeldahl method, and the collagen content in the composite material is calculated.

[0030] In some embodiments of the present invention, the total nitrogen content of the collagen / regenerated silk fibroin composite material is detected by Kjeldahl method, and the nitrogen content of the regenerated silk fibroin is subtracted to calculate the collagen content in the composite material.

[0031] In some embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:20 to 20:1; in some embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:9 to 9:1; in some embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:9, 1:5, 1:3, 1:1, 3:1, 5:1 or 9:1.

[0032] In some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:20; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:17; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:13; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:9; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:5; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:3; in some specific embodiments of the present invention, the composite material The mass ratio of collagen to regenerated silk fibroin in the composite material is 1:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 3:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 5:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 9:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 13:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 17:1; in some specific embodiments of the present invention, the mass ratio of collagen to regenerated silk fibroin in the composite material is 20:1.

[0033] In some embodiments of the present invention, the concentration of the test solution in step (1) is 1 mg / mL.

[0034] In some embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 10-180 μg / mL; preferably, the concentration of the tyrosine reference solution in step (2) is 50-150 μg / mL.

[0035] In some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 10 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 10.35 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 30 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 50 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 70 μg / mL; In some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 100 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 120 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 150 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 171.93 μg / mL; in some specific embodiments of the present invention, the concentration of the tyrosine reference solution in step (2) is 180 μg / mL.

[0036] In some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 50 to 200 mg. In some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 50 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 70 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 100 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 120 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 150 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 170 mg; in some embodiments of the present invention, the sample size of the collagen / regenerated silk fibroin composite material is 200 mg.

[0037] Beneficial effects:

[0038] The present invention provides a combined detection method for the content of two components in a collagen / regenerated silk fibroin composite material. By combining high-performance liquid chromatography with the Kjeldahl method, accurate determination of the two components can be achieved without derivatization treatment. The operation is simple and the detection cost is low. The method has a quantitative limit of 10.35 μg / mL (S / N ≥ 16.59) and a precision RSD of less than 1%, demonstrating high sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 : Comparison of liquid chromatograms of blank solvent, sample, collagen and tyrosine reference (red corresponds to the liquid chromatogram of blank solvent, purple corresponds to the liquid chromatogram of sample solution, green corresponds to the liquid chromatogram of recombinant type III humanized collagen, and blue corresponds to the liquid chromatogram of tyrosine);

[0040] Figure 2 : Tyrosine standard curve;

[0041] Figure 3 : Liquid chromatogram of tyrosine in the sample of Example 1;

[0042] Figure 4 : Liquid chromatogram of tyrosine in the sample of Example 2;

[0043] Figure 5 : Liquid chromatogram of tyrosine in the sample of Example 3;

[0044] Figure 6 : Liquid chromatogram of tyrosine in the sample of Example 4;

[0045] Figure 7 : Liquid chromatogram of tyrosine in the sample of Example 5;

[0046] Figure 8 : Liquid chromatogram of tyrosine in the sample of Example 6;

[0047] Figure 9 : Tyrosine liquid phase chromatogram in embodiment 7 sample.

[0048] Specific implementation (embodiment)

[0049] In order to enable those skilled in the art to better understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of this application.

[0050] It should be noted that the terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, system, product or apparatus that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or are inherent to these processes, methods, products or apparatuses.

[0051] In the present invention, recombinant humanized type III collagen standard was provided by Jiangshan Juyuan Biotechnology Co., Ltd.; regenerated silk fibroin was provided by Nanjing Yuangu Biotechnology Co., Ltd.; tyrosine reference substance was purchased from Henan Standard Material Research and Development Center; ultrapure water was purchased from Millipore; sodium acetate was purchased from Sinopharm Chemical Reagent Co., Ltd.; acetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.; methanol was purchased from Maclean; sulfuric acid was purchased from Nanjing Chemical Reagent Co., Ltd.; potassium sulfate was purchased from Sinopharm Chemical Reagent Co., Ltd.; copper sulfate was purchased from Sinopharm Chemical Reagent Co., Ltd.; sodium hydroxide was purchased from Nanjing Chemical Reagent Co., Ltd.; zinc particles were purchased from Sinopharm Chemical Reagent Co., Ltd.; and boric acid was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. Unless otherwise specified, other raw materials were purchased commercially.

[0052] In the following examples and comparative examples, the high performance liquid chromatograph used was Agilent 1100 (DAD detector); the electronic balance was Mettler ME204E; the electric blast drying oven was Shanghai Boxun GZX-9030MBE; and the pH meter was Mettler S470-USP / EP.

[0053] In the following examples and comparative examples, the formula for calculating the regenerated silk fibroin content in the collagen / regenerated silk fibroin composite material by the external standard method is:

[0054]

[0055] Where X is the content of regenerated silk fibroin in the sample; C 对 is the tyrosine concentration in the reference solution, in milligrams per milliliter (mg / mL); A 对 A is the peak area in the chromatogram of the reference solution; 供 is the peak area in the chromatogram of the test solution; C 供 is the concentration of the solution to be tested, in milliliter (mg / mL); 0.119 is the measured proportion of tyrosine in the silk fibroin raw material.

[0056] The formula for the collagen content in the collagen / regenerated silk fibroin composite material is:

[0057]

[0058] Where Y is the collagen content in the sample; V is the volume of the titrant consumed by the test solution, in milliliters (ml); V0 is the volume of the blank titrant consumed, in milliliters (ml); 1.401 is the N that 1 ml of sulfuric acid titrant (0.05 mol / L) is equivalent to 1.401 mg; m 供is the weight of the sample to be tested, in milligrams (mg); X is the percentage of regenerated silk fibroin in the sample; 5.7644 is the conversion factor for nitrogen content to regenerated silk fibroin; and 5.4658 is the conversion factor for nitrogen content to recombinant humanized type III collagen.

[0059] Analysis of the amino acid composition of recombinant humanized type III collagen and regenerated silk fibroin revealed that tyrosine accounts for approximately 12% of regenerated silk fibroin and is stable. However, recombinant humanized type III collagen contains no tyrosine, and the tyrosine content of type I collagen is only 0.29%, far below the detection limit. Therefore, protein hydrolysis and high-performance liquid chromatography were used to determine the tyrosine content and calculate the regenerated silk fibroin content. The total nitrogen content of the composite material was determined by Kjeldahl nitrogen determination. The nitrogen content of the collagen was then calculated by subtracting the nitrogen content of the regenerated silk fibroin from the total nitrogen content, thereby calculating the collagen content.

[0060] The following are some definitions used in this invention:

[0061] In the present invention, "high performance liquid chromatography" is an analytical technique based on the principle of liquid phase distribution, in which a high-pressure pump drives the mobile phase to carry the sample through the chromatographic column, and the difference in the distribution coefficient of each component between the stationary phase and the mobile phase is used to achieve separation.

[0062] In the present invention, "peak area" is the integrated area corresponding to the target peak in the HPLC chromatogram, which is proportional to the concentration of the analyte. The present invention calculates the regenerated silk fibroin content by the ratio of the tyrosine reference peak area to the sample peak area (external standard method).

[0063] In this application, the "Kjeldahl method" is a classic method for determining total nitrogen content by converting organic nitrogen in a sample into ammonium salts through sulfuric acid digestion, followed by alkalinization and distillation, absorption with boric acid, and sulfuric acid titration. The titrant used in this application is 0.05 mol / L sulfuric acid; the conversion factor is: 1 mL of sulfuric acid is equivalent to 1.401 mg of nitrogen.

[0064] In the present invention, "RSD (Relative Standard Deviation)" refers to the degree of dispersion of multiple measurement results under repeatability conditions.

[0065] In the present invention, the "External Standard Method" refers to a quantitative method for calculating the content of the analyte by comparing the response values ​​(peak areas) of a reference substance of known concentration with those of the sample to be tested.

[0066] In the present invention, "Signal-to-Noise Ratio (S / N)" refers to the ratio of the target chromatographic peak signal intensity (peak height) to the baseline noise fluctuation amplitude.

[0067] In this application, "regenerated silk fibroin" refers to a silk fibroin material with a β-pleated crystal structure that is reconstructed by dissolving natural silk fibers (such as Bombyx mori silk) and then undergoing processes such as degumming, dialysis, and freeze-drying. Depending on the intended use, it can be classified into conventional regenerated silk fibroin and medical regenerated silk fibroin. This application is particularly suitable for the detection of medical regenerated silk fibroin.

[0068] The present invention will be further analyzed below with reference to specific examples.

[0069] Example 1

[0070] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:9): Accurately weigh 54 mg of regenerated silk fibroin and place it in a beaker, add 0.36 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 486 mg of recombinant type III humanized collagen, add 3.24 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant humanized type III collagen was 1:9.

[0071] (2) Preparation of tyrosine reference solution: Weigh 10 mg of tyrosine reference solution into a 100 ml volumetric flask, dissolve it in 1 ml of 3 mol / L sulfuric acid solution, dilute with purified water and adjust to volume, and store at room temperature.

[0072] Preparation of the test solution: Weigh 50 mg of dried collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:9) into a headspace bottle, add 5 mL of 3 mol / L sulfuric acid solution, seal with a roll cap, place in a 120°C electric blast drying oven, hydrolyze for 16 hours, take out, cool to room temperature, open the lid and transfer the entire amount of the solution in the headspace bottle to a 50 ml volumetric flask, add 5 mL of 6 mol / L sodium hydroxide solution to neutralize, add purified water to make up the volume, and store at room temperature.

[0073] (3) performing high performance liquid chromatography analysis on the tyrosine reference solution and the test solution, respectively, and recording the peak areas of the tyrosine reference solution and the test solution;

[0074] High-performance liquid chromatography (HPLC) conditions included a GL Sciences Inertsil ODS-3 (4.6 × 250 mm, 5 μm) column, a detection wavelength of 200 nm, a flow rate of 1.0 mL / min, a column temperature of 35°C, and an injection volume of 10 μL. The mobile phase consisted of 20 mM sodium acetate (pH adjusted to 6.0 with dilute acetic acid) and methanol in a 95:5 volume ratio, and the elution time was 10 min.

[0075] The content of regenerated silk fibroin in the collagen / regenerated silk fibroin composite material was calculated to be 11.7% by the peak area external standard method (recording the concentration of the tyrosine reference substance as 109.45 μg / mL, the peak area as 2650.541, the concentration of the sample as 1.158 mg / mL, the peak area as 389.776). Figure 3 .

[0076] (4) Weigh 100 mg of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:9), wrap it with filter paper, and place it in a dry 500 mL Kjeldahl flask. Then add 10 g of potassium sulfate, 0.5 g of copper sulfate, and then slowly add 20 mL of sulfuric acid along the wall of the flask. Place a small funnel at the mouth of the Kjeldahl flask and tilt the Kjeldahl flask at 45°C. Heat it slowly over a direct flame to keep the temperature of the solution below the boiling point. Wait for the boiling to stop, then heat it to boiling. After the solution turns clear green, continue heating for 30 minutes and let it cool. Slowly add 250 mL of water along the wall of the flask, shake to mix, let it cool to room temperature, add 75 mL of 40% sodium hydroxide solution, add a few zinc particles, and connect the Kjeldahl flask to the condenser with a nitrogen balloon. Take another 50 mL of 2% boric acid solution and place it in a 500 mL flask. Add 10 drops of methyl red-bromocresol green mixed indicator solution. The lower end of the condenser was inserted below the surface of the boric acid solution. The Kjeldahl flask was gently swirled to mix the solution evenly. Heat and distill until the total volume of the receiving solution was approximately 250 mL. The tip of the condenser was lifted from the liquid surface and allowed to flush with steam for approximately 1 minute. The tip was then rinsed with water and the distillation was stopped. The distillate was titrated with 0.05 mol / L sulfuric acid solution until the solution changed from bluish-green to gray-purple. The titration result was corrected using a blank test. Each 1 mL of 0.05 mol / L sulfuric acid solution was equivalent to 1.401 mg of N. The sample weight (102.2 mg), sulfuric acid solution concentration (0.04952 mol / L), volume of the consumed solution (26.90 mL), and blank volume (0.20 mL) were recorded. The collagen content of the collagen / regenerated silk fibroin composite was calculated to be 88.0%.

[0077] Example 2

[0078] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:5): Accurately weigh 90 mg of regenerated silk fibroin and place it in a beaker, add 0.6 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 450 mg of recombinant type III humanized collagen, add 3 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant humanized type III collagen was 1:5).

[0079] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated contents of regenerated silk fibroin were 18.5% and 83.4% respectively. The liquid chromatography of the sample is shown in FIG. Figure 4 .

[0080] Example 3

[0081] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:3): Accurately weigh 135 mg of regenerated silk fibroin and place it in a beaker, add 0.9 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 405 mg of recombinant type III humanized collagen, add 2.7 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it again in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant type III humanized collagen was 1:3).

[0082] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated content of regenerated silk fibroin was 26.9% and the content of collagen was 73.5%. The liquid chromatography of the sample is shown in FIG. Figure 5 .

[0083] Example 4

[0084] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 1:1): Accurately weigh 1350 mg of regenerated silk fibroin and place it in a beaker, add 9 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 1350 mg of recombinant humanized collagen, add 9 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it again in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant humanized type III collagen was 1:1.

[0085] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated contents of regenerated silk fibroin were 51.2% and 51.9% respectively. The liquid chromatography of the sample is shown in FIG. Figure 6 .

[0086] Example 5

[0087] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 3:1): Accurately weigh 405 mg of regenerated silk fibroin and place it in a beaker, add 2.7 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 135 mg of recombinant type III humanized collagen, add 0.9 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant type III humanized collagen was 3:1.

[0088] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated content of regenerated silk fibroin was 75.7% and the content of collagen was 26.2%. The liquid chromatogram of the sample is shown in FIG. Figure 7 .

[0089] Example 6

[0090] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 5:1): Accurately weigh 450 mg of regenerated silk fibroin and place it in a beaker, add 3 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 90 mg of recombinant type III humanized collagen, add 0.6 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material, where the ratio of regenerated silk fibroin to recombinant humanized type III collagen was 5:1).

[0091] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated content of regenerated silk fibroin was 84.4% and the content of collagen was 15.2%. The liquid chromatography of the sample is shown in FIG. Figure 8 .

[0092] Example 7

[0093] (1) Preparation of collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen is 9:1): Accurately weigh 486 mg of regenerated silk fibroin and place it in a beaker, add 3.24 g of water, and stir at room temperature until completely dissolved to obtain a regenerated silk fibroin solution. Accurately weigh 54 mg of recombinant type III humanized collagen, add 0.36 g of purified water, and ultrasonicate until completely dissolved to obtain a recombinant type III humanized collagen solution. Add the recombinant type III humanized collagen solution dropwise to the regenerated silk fibroin solution, stirring continuously during the addition process. After the addition of the recombinant type III humanized collagen solution is completed, continue stirring for 30 minutes to mix the two solutions evenly to obtain a mixed solution. This mixed solution is divided into vials and placed in a freeze dryer for freeze drying. After the freeze drying process is completed, take out the freeze-dried material, soak it in ethanol solution, stir and wash it twice, replace the washing solution every 1 hour, and then stir and wash it in ultrapure water 3 times, each soaking time is 1 hour. After washing, the mixed block material was placed back into a freeze dryer for freeze drying. After freeze drying, the material was mechanically crushed to obtain a collagen / regenerated silk fibroin composite material (the ratio of regenerated silk fibroin to recombinant type III humanized collagen was 9:1).

[0094] (2) The contents of collagen and regenerated silk fibroin in the collagen / regenerated silk fibroin composite material were determined according to the method in Example 1. The calculated content of regenerated silk fibroin was 91.2% and the content of collagen was 8.2%. The liquid chromatogram of the sample is shown in FIG. Figure 9 .

[0095] Example 8 Methodological Investigation

[0096] Exclusivity

[0097] Verification content: Tyrosine reference solution, sample solution, and collagen hydrolyzed solution were used as test samples for HPLC analysis to observe whether there was an absorption peak in the target tyrosine integral range of the sufficient collagen hydrolyzed solution. Acceptable standard: No absorption peak appeared in the target tyrosine integral range of the recombinant type III humanized collagen hydrolyzed solution. Verification results and conclusions: Figure 1 It can be seen that the chromatogram of recombinant type III humanized collagen has no interfering peak at the target tyrosine outlet position of the sample, and can specifically separate the regenerated silk fibroin and recombinant humanized collagen in the sample, meeting the acceptable standards.

[0098] Detection sensitivity

[0099] Verification content: The tyrosine reference solution was subjected to HPLC analysis, and the signal-to-noise ratio was calculated. Acceptable standard: The reference solution was diluted to a certain concentration for testing, with the detection limit being a signal-to-noise ratio ≥3:1, and the quantification limit being a signal-to-noise ratio ≥10:1. Verification results and conclusions: The results are shown in Table 1. When the reference solution was diluted to 10.35μg / ml, the mean signal-to-noise ratio was 16.59, which met and far exceeded the acceptable standard for the limit of quantification. When the reference solution was diluted to 4.14μg / ml, the signal-to-noise ratio was 9.13, which met and far exceeded the acceptable standard for the limit of detection. This method shows that tyrosine can still be clearly detected when the concentration is very low, and the detection sensitivity is high.

[0100] Table 1

[0101]

[0102] (3) Confirmation of the linear relationship of the curve

[0103] Verification content: Tyrosine reference substance was analyzed by HPLC at different concentrations (10.35, 40, 80, 100, 120, 150, 180 μg / mL). Linear regression was performed with sample concentration as the horizontal axis and peak area as the vertical axis to obtain the linear correlation coefficient. Acceptable standard: When the concentration was within the range of 10.35-171.93 μg / mL, the peak area was linearly correlated with its concentration, and the correlation coefficient r was not less than 0.98. Verification results and conclusions: See Figure 2 The concentration of the sample was in the range of 10.35-171.93 μg / mL, the linear regression equation was y=24.019x+257.2, the correlation coefficient was r=0.9966, which was not less than the correlation coefficient of 0.98, meeting the acceptable standard.

[0104] (4) Confirmation of injection precision

[0105] Verification: Six consecutive injections of Linear Solution 3 (103.48 μg / mL) were performed on HPLC analysis, and the retention time and peak area were recorded. Acceptance criteria: Retention time RSD <1.0% and peak area RSD <2.0% for six consecutive injections. Verification Results and Conclusions: See Table 2. Six consecutive injections of the reference solution showed retention time RSDs of 0.05% and <1.0%, respectively; and peak area RSDs of 0.98% and <2.0%, respectively, meeting the acceptance criteria.

[0106] Table 2

[0107]

[0108] (5) Accuracy confirmation

[0109] Verification Content: Sample preparation was performed. Approximately 25 mg, 40 mg, and 50 mg of sample (regenerated silk fibroin to recombinant humanized type III collagen at a 1:1 ratio) were added to approximately 11 mg, 22 mg, and 33 mg of a tyrosine reference substance, respectively. HPLC analysis was performed and the tyrosine content in the samples was calculated. The recovery rate was calculated by dividing the measured tyrosine content by the theoretical added tyrosine content. Acceptable Criteria: The test was conducted at tyrosine concentrations of (0.05, 0.1, and 0.15 mg / mL), with a recovery rate ranging from 92% to 105%. Verification Results and Conclusions: As shown in Table 3, the recovery rate was 102.9% at a tyrosine concentration of 0.05 mg / mL; 101.9% at a tyrosine concentration of 0.1 mg / mL; and 96.8% at a tyrosine concentration of 0.15 mg / mL, all meeting the acceptable criteria.

[0110] Table 3

[0111]

[0112] (6) Repeatability confirmation

[0113] Verification Details: On the same day, researcher A prepared six samples (regenerated silk fibroin to recombinant humanized type III collagen in a 1:1 ratio) for HPLC analysis. The regenerated silk fibroin content in the samples was calculated. Acceptability Criteria: The same researcher performed the test on the same day, with an RSD of ≤2%. Verification Results and Conclusions: See Table 4. The results show that the RSD of the regenerated silk fibroin content in the six sample solutions was 0.78%, meeting the acceptance criteria and demonstrating excellent reproducibility.

[0114] Table 4

[0115]

[0116]

[0117] (7) Intermediate precision confirmation

[0118] Verification content: On different days, experimenter B prepared 6 samples (the ratio of regenerated silk fibroin to recombinant type III humanized collagen was 1:1) and performed HPLC analysis. Calculate the regenerated silk fibroin content in the samples. Acceptable standard: On the same day, the same experimenter conducted the test, and the RSD of the content was ≤2%. On different days, 12 samples were tested by different experimenters, and the RSD of the content was ≤2%. Verification results and conclusions: See Table 5 and Table 6. The results in Table 5 show that the RSD of the regenerated silk fibroin content of 6 sample solutions from different experimenters on different days was 0.62%. The results in Table 6 show that the RSD of the regenerated silk fibroin content of 12 sample solutions was 0.69%, which both met the acceptable standards. The precision was very high and was not affected by factors such as personnel and time.

[0119] Table 5

[0120]

[0121] Table 6

[0122]

[0123]

Claims

1. A method for detecting the content of collagen and regenerated silk fibroin in a composite material, characterized in that: The following steps are involved: (1) hydrolyzing the composite material with a hydrolysis reagent for a certain period of time, and then neutralizing it to obtain a test solution; (2) Prepare tyrosine reference solution; (3) using high performance liquid chromatography to detect the peak areas of the test solution and the tyrosine reference solution, and calculating the content of regenerated silk fibroin in the composite material; (4) The total nitrogen content of the composite material was detected by Kjeldahl method, and the collagen content in the composite material was calculated.

2. The method according to claim 1, characterized in that The hydrolysis reagent in step (1) is an acidic solution.

3. The method according to claim 2, characterized in that The acidic solution is one or more of hydrochloric acid solution, sulfuric acid solution or nitric acid solution.

4. The method according to claim 1, wherein The hydrolysis time in step (1) is 4-24 hours, and the hydrolysis temperature is 80-130°C; preferably, the hydrolysis time in step (1) is 10-20 hours, and the hydrolysis temperature is 90°C-120°C; more preferably, the hydrolysis time in step (1) is 16 hours, and the hydrolysis temperature is 120°C.

5. The method according to claim 1, wherein The mass ratio of collagen to regenerated silk fibroin in the composite material is 1:20 to 20:1; preferably, the mass ratio of collagen to regenerated silk fibroin in the composite material is 1:9 to 9:

1.

6. The method according to claim 5, characterized in that The mass ratio of collagen to regenerated silk fibroin in the composite material is 1:9, 1:5, 1:3, 1:1, 3:1, 5:1 or 9:

1.

7. The method according to claim 1, characterized in that The concentration of the test solution in step (1) is 1 mg / mL.

8. The method according to claim 1, characterized in that The concentration of the tyrosine reference solution in step (2) is 10-180 μg / mL; preferably, the concentration of the tyrosine reference solution in step (2) is 50-150 μg / mL.

9. The method according to claim 1, characterized in that In step (4), the amount of the collagen / regenerated silk fibroin composite material to be tested is 50 to 200 mg.

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