Method for measuring content of chitosan in implant containing chitosan and collagen
Through the color development reaction of glucosamine hydrochloride standard solution and acid hydrolysis treatment, the inaccuracy problem of chitosan content determination in electrochemical implants was solved, and accurate chitosan and collagen content detection was achieved.
Patent Information
- Application Number
- CN202510909287.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-09-09
AI Technical Summary
Existing technologies have difficulty in accurately measuring the chitosan content in implants containing chitosan and collagen prepared by electrochemical deposition. Especially in the presence of collagen, the test results are unstable and black residue is easily produced, resulting in inaccurate measurements.
The method of combining the color reaction of glucosamine hydrochloride standard solution with acid hydrolysis and pH adjustment is adopted. The solution is first swelled or dissolved, then reacted with high concentration hydrochloric acid at a specific temperature, and finally fixed to volume under alkaline conditions and color reaction is carried out. The second absorbance is measured to calculate the chitosan content.
The method realizes the precise determination of chitosan content in implants containing chitosan and collagen, eliminates the interference of black residue, improves the stability and accuracy of the determination results, and takes into account the detection of chitosan and collagen.
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Figure CN120609768A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the technical field of chitosan content determination, and in particular to a method for determining the chitosan content in an implant containing chitosan and collagen. Background Art
[0002] Chitosan, also known as deacetylated chitin, is a derivative of chitin obtained by deacetylation with concentrated alkali. Through a series of experiments, the applicant deposited collagen and chitosan on electrode surfaces via electrochemical deposition, producing implantable products such as artificial corneas and drainage tubes containing chitosan and collagen.
[0003] During the process of verifying product ingredients, it is necessary to test the content of each component in the final product. However, in implants containing chitosan and collagen prepared by electrochemical deposition, the different components deposited on the electrodes vary in their proportions, making it difficult to determine the chitosan content in the final product deposited on the electrode surface. Summary of the Invention
[0004] The present application provides a method for determining the chitosan content in an implant containing chitosan and collagen, which can accurately determine the chitosan content in the implant.
[0005] This application is implemented as follows:
[0006] The present application provides a method for determining the chitosan content in an implant containing chitosan and collagen, comprising the following steps:
[0007] Providing glucosamine hydrochloride standard solutions of different concentrations, and performing a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, measuring a first absorbance at a wavelength of 535 nm, and plotting a standard curve with the concentration of the glucosamine hydrochloride solution as the abscissa and the corresponding first absorbance as the ordinate;
[0008] The implant to be tested at a constant weight is swelled or dissolved, and then subjected to an acid hydrolysis reaction. After the acid hydrolysis reaction, the pH of the solution is adjusted to alkaline to obtain a test solution, and the dilution multiple is recorded;
[0009] The test solution is subjected to a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, a second absorbance is measured at a wavelength of 535 nm, and the corresponding concentration of glucosamine hydrochloride in the test solution is calculated from the standard curve according to the second absorbance;
[0010] The chitosan content in the implant to be tested Wherein, ρ is the concentration of glucosamine hydrochloride in the test solution, ν is the volume of the test solution, m is the mass of the implant to be tested, and α is the dilution factor of the test solution.
[0011] In a possible embodiment, the step of swelling or dissolving the constant-weight implant to be tested comprises: mixing the constant-weight implant to be tested with an acid solution having a concentration of 0.001 to 0.5 mol / L.
[0012] In one possible embodiment, the acid solution in the step of swelling or dissolving the constant-weight implant to be tested comprises either hydrochloric acid or acetic acid.
[0013] In a possible embodiment, the implant to be tested at a constant weight is mixed with an acid solution and then ultrasonically treated for 10 to 90 minutes.
[0014] In a possible embodiment, the step of performing an acid hydrolysis reaction includes: mixing the swollen or dissolved sample with 3-20 mol / L hydrochloric acid and reacting the mixture at 90-120°C.
[0015] In a possible embodiment, the swollen or dissolved sample is mixed with 5-10 mol / L hydrochloric acid and then heated to 100-110° C. for reaction.
[0016] In one possible embodiment, in the acid hydrolysis step, the reaction is carried out at a temperature of 90 to 120° C. for 18 to 30 hours.
[0017] In one possible embodiment, after the acid hydrolysis reaction, the pH of the solution is adjusted to 7.5-10.
[0018] In one possible embodiment, after the acid hydrolysis reaction, the pH of the solution is adjusted to 8.2-9.2.
[0019] In one possible embodiment, the implant includes any one of a drainage tube and an artificial cornea.
[0020] This application has at least the following beneficial effects:
[0021] The present invention relates to a method for determining the chitosan content in an implant containing chitosan and collagen. The method involves subjecting a constant weight implant to be tested to swelling or dissolution treatment, allowing the implant to be tested to fully react with an acid hydrolysis reagent, and the sample to be fully hydrolyzed. After the acid hydrolysis reaction, the pH of the solution is adjusted to alkaline and the volume is fixed to the test solution. The test solution is subjected to a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound with a distinct color, which is conducive to determining a more accurate second absorbance. Moreover, experimentally verified that first performing a swelling or dissolution treatment and then an acid treatment can make the measurement result more accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 This is the standard curve in the examples of this application;
[0023] Figure 2 This is a photograph of the artificial cornea after hydrolysis of Comparative Example 1 of the present application;
[0024] Figure 3 This is a photo of the color development reaction of Comparative Example 5 of this application. DETAILED DESCRIPTION
[0025] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, the conditions are carried out according to conventional conditions or manufacturer recommendations. The reagents or instruments used are not specified by the manufacturer and are conventional products that can be purchased commercially.
[0026] The implant containing chitosan and collagen prepared by electrochemical deposition has different ratios of different components deposited on the electrode, and it is difficult to detect the chitosan content in the final product formed by deposition on the electrode surface. Moreover, the implant contains both chitosan and collagen. The inventors of this application attempted to treat the implant and determine the chitosan content using the chitosan content determination method in the national standard - high performance liquid chromatography (refer to national standard GB / T 38479-2021). However, during the test, for the same implant product, the chitosan content determination results of multiple parallel samples were very unstable. This may be because the sample needs to be acetylated during the treatment process of this method, and then subjected to gradient temperature acid hydrolysis. However, the degree of deacetylation will increase after acid hydrolysis, and the conversion ratio of glucosamine to chitosan cannot be accurately determined. The content of chitosan cannot be determined when the ratio of glucosamine to chitosan is unknown. At the same time, the chitosan content in the sample is also an unknown amount, and a single method cannot be used to determine two unknown amounts, resulting in unstable measurement results. Moreover, the inventors of the present application found in experiments that black residue is easily formed during the acid hydrolysis process, resulting in inaccurate detection of chitosan content.
[0027] The following is a detailed description of the method for determining the chitosan content in the implant containing chitosan and collagen according to the embodiment of the present application:
[0028] In a first aspect, the present invention provides a method for determining the chitosan content in an implant containing chitosan and collagen, comprising the following steps:
[0029] (1) Providing glucosamine hydrochloride standard solutions of different concentrations, and performing a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, measuring the first absorbance at a wavelength of 535 nm, and plotting a standard curve with the concentration of the glucosamine hydrochloride solution as the horizontal axis and the corresponding first absorbance as the vertical axis.
[0030] For example, the concentration of the glucosamine hydrochloride standard solution is 0.01 to 1 mg / mL, for example, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 0.8 mg / mL or 1 mg / mL. It should be noted that the concentration of the glucosamine hydrochloride standard solution can be selected according to actual conditions.
[0031] Alternatively, the acetylacetone solution can be prepared by compounding acetylacetone with a trisodium phosphate-sodium tetraborate solution. The p-dimethylaminobenzaldehyde solution can be prepared by compounding p-dimethylaminobenzaldehyde with concentrated hydrochloric acid and isopropyl alcohol.
[0032] (2) The implant to be tested is swollen or dissolved at a constant weight, and then subjected to an acid hydrolysis reaction. After the acid hydrolysis reaction, the pH of the solution is adjusted to alkaline, and the volume is constant to obtain the test solution.
[0033] Among them, the implant containing chitosan and collagen can optionally be an artificial cornea or drainage tube prepared by an electrochemical method, and the implant can also be a solution containing chitosan and collagen, which is not specifically limited in this application. Among them, the preparation method of the artificial cornea can refer to the patent with application number 2024115461659, and the preparation method of the drainage tube can refer to the patent with application number 2023116940181. The prepared implant contains chitosan and collagen. It should be noted that implants containing chitosan and collagen prepared by other processes are also suitable for the determination method of this application to detect the chitosan content therein.
[0034] By subjecting the chitosan and collagen-containing implant to swelling or dissolution, the implant to be tested can fully react with the acid hydrolysis reagent, resulting in complete hydrolysis of the sample. The inventors of this application discovered in experiments that if the constant-weight implant to be tested is not first subjected to swelling or dissolution before acid hydrolysis, the implant containing chitosan and collagen cannot be completely hydrolyzed, and some black residue is likely to remain. This results in inaccurate glucosamine content measurements, making it impossible to accurately determine the chitosan content in the chitosan and collagen-containing implant.
[0035] Illustratively, the step of subjecting the constant-weight implant to be tested to swelling or dissolution includes: mixing the constant-weight implant to be tested with an acid solution having a concentration of 0.001 to 0.5 mol / L. Optionally, the acid solution in the swelling or dissolution step includes hydrochloric acid or acetic acid. Optionally, in the step of subjecting the constant-weight implant to be tested to swelling or dissolution, the concentration of the acid solution used is any one of 0.001 mol / L, 0.005 mol / L, 0.01 mol / L, 0.05 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L, 0.4 mol / L, 0.45 mol / L, and 0.5 mol / L, or a value between any two of them.
[0036] Optionally, in order to better swell or dissolve the implant to be tested, the constant weight implant to be tested is mixed with the acid solution and then ultrasonically treated for 10 to 90 minutes. The ultrasonic treatment allows the acid solution to fully penetrate the implant containing chitosan and collagen, so that the implant containing chitosan and collagen is better swollen or dissolved. Exemplarily, the ultrasonic treatment time is any one of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 45 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, and 90 minutes, or a value between any two of them.
[0037] Furthermore, the step of subjecting the swollen or dissolved sample to an acid hydrolysis reaction comprises: mixing the swollen or dissolved sample with 3 to 20 mol / L hydrochloric acid and reacting at 90 to 120°C. Wherein, the concentration of hydrochloric acid is relatively high, and the sample can be fully hydrolyzed at a temperature of 90 to 120°C. The inventors of this application attempted to treat the implant and determine the content of chitosan using the chitosan content determination method in the national standard - high performance liquid chromatography (refer to national standard GB / T38479-2021). However, this method is not accurate enough for the detection of hydroxyproline content in implants containing collagen and chitosan. This may be because hydroxyproline can react with acetic anhydride used for acetylation treatment under acidic conditions, resulting in a decrease in the hydroxyproline detection value. The method adopted in the present application is to mix the swollen or dissolved sample with 3-20 mol / L hydrochloric acid and then directly heat it to 90-120°C for reaction, which can make the accuracy of chitosan content detection and hydroxyproline content detection (wherein, in the prior art, the collagen content in the sample is usually calculated by detecting the hydroxyproline content) higher, thereby taking into account both chitosan content detection and protein content detection, and eliminating the errors caused by separate detection of the two components in different samples.
[0038] Illustratively, the temperature in the acid hydrolysis step is any one of 90° C., 95° C., 100° C., 105° C., 110° C., 105° C., and 120° C., or any value between two thereof. In some embodiments, the temperature in the acid hydrolysis step is 100-110° C.
[0039] In the acid hydrolysis step, the reaction is carried out at a temperature of 90 to 120° C. for 18 to 30 hours. For example, the reaction time is any one of 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours and 30 hours, or a value between any two of them.
[0040] Illustratively, the concentration of hydrochloric acid used when the sample is subjected to acid hydrolysis reaction is any one of 3mol / L, 4mol / L, 5mol / L, 6mol / L, 7mol / L, 8mol / L, 9mol / L, 10mol / L, 12mol / L, 14mol / L, 16mol / L, 18mol / L and 20mol / L, or a value between any two of them.
[0041] Furthermore, the inventors of the present application discovered in their research that, after acid hydrolysis, if the solution is directly diluted to a test solution and then subsequently reacted with acetylacetone and p-dimethylaminobenzaldehyde, no red color will be displayed. However, after acid hydrolysis, if the pH of the solution is first adjusted to alkaline before being diluted to a test solution, then the solution will display a distinct red color after reacting with acetylacetone and p-dimethylaminobenzaldehyde, thereby enabling a relatively accurate measurement of the second absorbance at a wavelength of 535 nm. The dilution factor for adjusting the pH of the solution to alkaline before dilution to a test solution is α, where α is greater than or equal to 1.
[0042] Optionally, after the sample is subjected to an acid hydrolysis reaction, the pH of the solution is adjusted to 7.5-10, for example, the pH of the solution is adjusted to any one of 7.5, 7.8, 8, 8.2, 8.5, 8.8, 9, 9.2, 9.4, 9.6, 9.8 and 10, or a value between any two of them. In some embodiments, after the sample is subjected to an acid hydrolysis reaction, the pH of the solution is adjusted to 8.2-9.2.
[0043] (3) The test solution is subjected to a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, and the second absorbance is measured at a wavelength of 535 nm. The concentration of glucosamine hydrochloride in the corresponding test solution is calculated from the standard curve based on the second absorbance.
[0044] Calculation of chitosan content in the implant to be tested Where ρ is the solution to be tested
[0045] where ν is the concentration of glucosamine hydrochloride in the solution (mg / mL), m is the mass of the implant to be tested (mg), and α is the dilution factor of the solution to be tested.
[0046] The method for determining the chitosan content in the implant containing chitosan and collagen of the present application is further described in detail below with reference to the examples.
[0047] Example 1
[0048] This embodiment provides a method for determining the chitosan content in an artificial cornea produced by an electrochemical method, which comprises the following steps:
[0049] 1) Provide a glucosamine hydrochloride standard solution with a concentration of 0.1 mg / mL, take a test tube and add 0 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of the glucosamine hydrochloride standard solution in sequence, then add water to make the volume to 1 mL and mix evenly. Add 0.7 mL of acetylacetone solution to each test tube, heat at 100°C for 30 minutes, cool to room temperature and add 2 mL of p-dimethylaminobenzaldehyde solution. After 30 minutes, use the reagent blank solution as a reference and measure the first absorbance at 535 nm. Use the concentration of glucosamine hydrochloride solution as the horizontal coordinate and the corresponding first absorbance as the vertical coordinate to draw a standard curve (the standard curve is as shown in the figure). Figure 1 shown).
[0050] 2) Add 0.01 mol / L hydrochloric acid to the constant weight artificial cornea to swell it. After ultrasonic treatment for 60 minutes, add 6 mol / L hydrochloric acid to the swollen sample, mix, and react at 100°C for 24 hours. After the reaction, remove excess hydrochloric acid, adjust the pH of the solution to 8.5, and adjust the volume to the test solution. Record the dilution factor.
[0051] 3) Add 0.7 mL of acetylacetone solution to 1 mL of the test solution, heat at 100° C. for 30 min, cool to room temperature, add 2 mL of p-dimethylaminobenzaldehyde solution to carry out a color reaction, measure the second absorbance at a wavelength of 535 nm, and calculate the corresponding glucosamine hydrochloride concentration in the test solution from the standard curve based on the second absorbance;
[0052] Chitosan content in the artificial cornea to be tested Wherein, ρ is the concentration of glucosamine hydrochloride in the test solution (mg / mL), ν is the volume of the test solution (mL), m is the mass of the artificial cornea to be tested (mg), and α is the dilution factor. Furthermore, the collagen content in the artificial cornea was determined by first determining the hydroxyproline content in the artificial cornea using the hydroxyproline method, and then combining the hydroxyproline / collagen coefficient (10.8%) to determine the collagen content in the artificial cornea.
[0053] Example 2
[0054] The only difference between this embodiment and embodiment 1 is that in this embodiment, hydrochloric acid with a concentration of 6 mol / L is added to the swollen sample, mixed, and reacted at a temperature of 120° C. for 20 hours.
[0055] Example 3
[0056] The only difference between this embodiment and embodiment 1 is that in this embodiment, hydrochloric acid with a concentration of 6 mol / L is added to the swollen sample, mixed, and reacted at a temperature of 90° C. for 28 hours.
[0057] Example 4
[0058] The only difference between this embodiment and embodiment 1 is that in step (2) of this embodiment, the pH of the solution is adjusted to 9.2.
[0059] Example 5
[0060] The only difference between this embodiment and embodiment 1 is that the concentration of acid hydrolysis in step (2) of this embodiment is 12 mol / L.
[0061] Example 6
[0062] The only difference between this embodiment and embodiment 1 is that the concentration of acid hydrolysis in step (2) of this embodiment is 18 mol / L.
[0063] Example 7
[0064] The only difference between this embodiment and embodiment 1 is that the concentration of acid hydrolysis in step (2) of this embodiment is 3 mol / L.
[0065] Example 8
[0066] The only difference between this embodiment and embodiment 1 is that in step (2) of this embodiment, hydrochloric acid with a concentration of 0.1 mol / L is added to the artificial cornea to be tested at a constant weight, and then ultrasonic treatment is performed.
[0067] Example 9
[0068] The only difference between this embodiment and embodiment 1 is that in step (2) of this embodiment, acetic acid with a concentration of 0.1 mol / L is added to the artificial cornea to be tested at a constant weight, and then ultrasonic treatment is performed.
[0069] Example 10
[0070] The only difference between this embodiment and embodiment 1 is that in step (2) of this embodiment, acetic acid with a concentration of 0.3 mol / L is added to the artificial cornea to be tested at a constant weight, and then ultrasonic treatment is performed.
[0071] Example 11
[0072] The only difference between this embodiment and embodiment 1 is that in step (2) of this embodiment, hydrochloric acid with a concentration of 0.003 mol / L is added to the artificial cornea to be tested at a constant weight, and then ultrasonic treatment is performed.
[0073] Example 12
[0074] The only difference between this embodiment and embodiment 1 is that the ultrasonic treatment in step (2) is removed in this embodiment.
[0075] Example 13 to Example 24
[0076] Examples 13 to 24 all provide a method for determining the chitosan content in a glaucoma drainage tube prepared by an electrochemical method. The preparation steps of Examples 13 to 24 correspond to the preparation steps of Examples 1 to 12, respectively, except that the artificial cornea in Examples 1 to 12 is replaced by a glaucoma drainage tube.
[0077] Example 25
[0078] This embodiment provides a method for determining the chitosan content in a mixed solution containing collagen and chitosan, wherein the collagen content in the mixed solution is 12%, comprising the following steps:
[0079] 1) Provide a glucosamine hydrochloride standard solution with a concentration of 0.1 mg / mL, take a test tube and add 0 mL, 0.25 mL, 0.5 mL, 0.75 mL and 1 mL of the glucosamine hydrochloride standard solution in sequence, then add water to make the volume to 1 mL and mix evenly. Add 0.7 mL of acetylacetone solution to each test tube, heat at 100°C for 30 minutes, cool to room temperature and add 2 mL of p-dimethylaminobenzaldehyde solution. After 30 minutes, use the reagent blank solution as a reference and measure the first absorbance at 535 nm. Use the concentration of glucosamine hydrochloride solution as the horizontal coordinate and the corresponding first absorbance as the vertical coordinate to draw a standard curve (the standard curve is as shown in the figure). Figure 1 shown).
[0080] 2) Add 0.01 mol / L hydrochloric acid to the mixed solution and ultrasonicate for 60 min. Then, add 6 mol / L hydrochloric acid to the sample, mix, and react at 100°C for 24 h. After the reaction, remove excess hydrochloric acid, adjust the pH of the solution to 8.5, and dilute to the test solution. Record the dilution factor.
[0081] 3) Add 0.7 mL of acetylacetone solution to 1 mL of the test solution, heat at 100° C. for 30 min, cool to room temperature, add 2 mL of p-dimethylaminobenzaldehyde solution to carry out a color reaction, measure the second absorbance at a wavelength of 535 nm, and calculate the corresponding glucosamine hydrochloride concentration in the test solution from the standard curve based on the second absorbance;
[0082] Chitosan content in the mixed solution to be tested Wherein, ρ is the concentration of glucosamine hydrochloride in the test solution (mg / mL), ν is the volume of the test solution (mL), m is the mass of the test mixed solution (mg), and α is the dilution factor. Furthermore, the collagen content in the mixed solution was determined by first determining the hydroxyproline content in the mixed solution using the hydroxyproline method, and then combining the hydroxyproline / collagen coefficient (10.8%) to obtain the collagen content in the mixed solution.
[0083] Comparative Example 1
[0084] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 2 removes the step (2) of adding 0.01 mol / L hydrochloric acid and ultrasonically treating for 60 min in Example 1. In Comparative Example 1, after adding 6 mol / L hydrochloric acid and heating the reaction, black residue appeared at the bottom of the test tube, indicating that the artificial cornea was not completely hydrolyzed (e.g., Figure 2 In addition, referring to the steps of Comparative Example 1, the artificial cornea was replaced with a glaucoma drainage tube. Black residue also appeared after the hydrolysis was completed, indicating that the glaucoma drainage tube was not completely hydrolyzed.
[0085] It should be noted that, after the hydrolysis of the artificial corneas of Examples 1 to 12 was completed, no black residue appeared; and after the hydrolysis of the glaucoma drainage tubes of Examples 13 to 24 was completed, no black residue appeared either.
[0086] Comparative Example 2
[0087] The only difference between Comparative Example 2 and Example 1 is that, in step (2) of Comparative Example 2, a constant weight artificial cornea to be tested is added with 0.01 mol / L hydrochloric acid for swelling treatment, ultrasonicated for 60 min, and then 500 μL of methanol and 8 μL of acetic anhydride are added. After reacting for 24 h, an acetylated sample is obtained. 6 mol / L hydrochloric acid is added to the acetylated sample, and then the temperature is first raised to 55° C. for a 1.5 h incubation reaction, then raised to 75° C. for a 1.5 h incubation reaction, and then raised to 100° C. for a 4 h incubation reaction. After the reaction is completed, excess hydrochloric acid is removed, the pH of the solution is adjusted to 8.5, and the volume is constant to the test solution.
[0088] Comparative Example 3
[0089] The only difference between Comparative Example 3 and Comparative Example 2 is that the artificial cornea of Comparative Example 2 is replaced by the glaucoma drainage tube in Example 13.
[0090] Comparative Example 4
[0091] The only difference between Comparative Example 4 and Comparative Example 2 is that the artificial cornea in Comparative Example 2 is replaced by the mixed solution containing collagen and chitosan in Example 25.
[0092] Comparative Example 5
[0093] The only difference between Comparative Example 5 and Example 1 is that the step (2) of adjusting the pH of the solution to 8.5 in Example 1 is omitted in Comparative Example 5. In Comparative Example 5, the color development of the test solution with the acetylacetone solution and the p-dimethylaminobenzaldehyde solution is not obvious, which seriously affects the determination of the second absorbance (such as Figure 3 shown).
[0094] Table 1. Chitosan and collagen content determination results of Examples and Comparative Examples
[0095]
[0096]
[0097] As can be seen from the results in Table 1, the chitosan content determination method in the chitosan and collagen implants of the present invention, as measured by the embodiment, is close to 100% for the sum of the chitosan and collagen percentages. The determination method in Comparative Example 1, which omits the swelling step, easily produces black residue during hydrolysis, resulting in incomplete chitosan hydrolysis. The final sum of the chitosan and collagen percentages differs by approximately 10% from 100%, demonstrating that the determination method of the present invention, which first performs a swelling or dissolution treatment on the sample, can more accurately determine the chitosan content in the chitosan and collagen implants.
[0098] In addition, by comparing Example 1 with Comparative Example 2, Comparative Example 13 with Comparative Example 3, and Comparative Example 25 with Comparative Example 4, the samples of Comparative Examples 2 to 4 were acetylated after swelling treatment, and then the acetylated samples were treated by gradient temperature increase. The collagen content measured was much lower than that of Example 1, Example 13, and Example 25, respectively, and the sum of the collagen content and chitosan content measured in Comparative Examples 2 to 4 differed from 100% by more than 10%. This shows that the determination method of the embodiment of the present application is basically non-interfering with the detection of collagen.
[0099] Test Example 1
[0100] The artificial cornea in Example 1 was replaced with a chitosan standard solution, and the chitosan content in the chitosan standard solution was measured using the determination method of Example 1. The result was 99.24%. The artificial cornea in Comparative Example 2 was replaced with a chitosan standard solution, and the chitosan content in the chitosan standard solution was measured using the determination method of Comparative Example 2. The result was 85.14%.
[0101] It further illustrates that the detection method of the embodiment of the present application can relatively accurately determine the chitosan content in an implant containing collagen and chitosan.
[0102] The foregoing description is merely a specific embodiment of the present application and is not intended to limit the present application. Persons skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.
Claims
1. A method for determining the chitosan content in an implant containing chitosan and collagen, characterized in that: The following steps are involved: Providing glucosamine hydrochloride standard solutions of different concentrations, and performing a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, measuring a first absorbance at a wavelength of 535 nm, and plotting a standard curve with the concentration of the glucosamine hydrochloride solution as the abscissa and the corresponding first absorbance as the ordinate; The implant to be tested at a constant weight is swelled or dissolved, and then subjected to an acid hydrolysis reaction. After the acid hydrolysis reaction, the pH of the solution is adjusted to alkaline, the volume is fixed to the test solution, and the dilution multiple is recorded; The test solution is subjected to a color reaction with acetylacetone and p-dimethylaminobenzaldehyde to generate a red compound, a second absorbance is measured at a wavelength of 535 nm, and the corresponding concentration of glucosamine hydrochloride in the test solution is calculated from the standard curve according to the second absorbance; The chitosan content in the implant to be tested Wherein, ρ is the concentration of glucosamine hydrochloride in the test solution, ν is the volume of the test solution, m is the mass of the implant to be tested, and α is the dilution factor of the test solution.
2. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 1, wherein: The step of swelling or dissolving the constant-weight implant to be tested comprises: mixing the constant-weight implant to be tested with an acid solution having a concentration of 0.001-0.5 mol / L.
3. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 2, wherein: The acid solution in the step of swelling or dissolving the constant-weight implant to be tested comprises any one of hydrochloric acid and acetic acid.
4. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 2 or 3, wherein: The implant to be tested of constant weight is mixed with the acid solution and then subjected to ultrasonic treatment for 10 to 90 minutes.
5. The method for determining the chitosan content in an implant containing chitosan and collagen according to any one of claims 1 to 3, wherein: The step of performing acid hydrolysis reaction comprises: mixing the swollen or dissolved sample with 3-20 mol / L hydrochloric acid and reacting the mixture at 90-120°C.
6. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 5, wherein: The swollen or dissolved sample is mixed with 5-10 mol / L hydrochloric acid and then heated to 100-110° C. for reaction.
7. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 5, wherein: In the acid hydrolysis step, the reaction is carried out at a temperature of 90 to 120° C. for 18 to 30 hours.
8. The method for determining the chitosan content in an implant containing chitosan and collagen according to any one of claims 1 to 3, characterized in that: After the acid hydrolysis reaction, the pH of the solution is adjusted to 7.5-10.
9. The method for determining the chitosan content in an implant containing chitosan and collagen according to claim 8, wherein: After the acid hydrolysis reaction, the pH of the solution is adjusted to 8.2-9.
2.
10. The method for determining the chitosan content in an implant containing chitosan and collagen according to any one of claims 1 to 3, characterized in that: The implant includes any one of a drainage tube and an artificial cornea.