Method for detecting content of hexadecapeptide in self-assembled peptide hydrogel

Abstract

The present application relates to a kind of detection method of self-assembled peptide hydrogel hexadecapeptide content, belong to biological material detection technical field.The method includes the following steps: first instrument preparation, using high performance liquid chromatography (HPLC) as detection equipment;Secondly, preparation of test solution, about 0.2 g self-assembled peptide hydrogel test sample is placed in centrifugal tube, after being dissolved with 2 mL primary water, drying is carried out in vacuum freeze-drying box;The dry powder obtained is added to 0.2 mL formic acid, extracts 30 min under the condition of 37.0 ℃, then add primary water to constant volume to 10 mL, and vortex mixing, obtain test solution;Finally, the test solution is detected by HPLC and the content of hexadecapeptide is calculated.The method is simple in operation, good in repeatability, accurate in detection result, can effectively realize the quantitative detection of self-assembled peptide hydrogel hexadecapeptide content, and has good application value.

Description

Technical Field

[0001] This invention relates to the field of self-assembled peptide hydrogel detection, specifically a method for detecting the content of hexadecapeptide in self-assembled peptide hydrogels. Background Technology

[0002] Self-assembled peptide hydrogels are a class of functional materials in which short peptide molecules spontaneously form a three-dimensional network structure through non-covalent forces such as hydrogen bonds, hydrophobic interactions, and π-π stacking. Due to their excellent biocompatibility, biodegradability, and structural designability, they have broad application prospects in tissue engineering, drug delivery, and biomedical materials. As the application of these materials deepens, higher requirements are placed on the determination of the content of their key components and quality control.

[0003] Hexapeptides, as an important component in self-assembled peptide hydrogels, significantly influence the gelation behavior, mechanical properties, and biological activity of the hydrogels. However, the stable network structure formed within self-assembled peptide hydrogels through various non-covalent interactions results in the high aggregation or embedding of hexapeptide molecules within the gel system. Under conventional solvent conditions, it is difficult to effectively disrupt this structure and release the target peptide, thus increasing the difficulty of accurately detecting the hexapeptide content.

[0004] In existing technologies, high-performance liquid chromatography (HPLC) and other analytical methods are often used for the detection of peptide content. However, during sample pretreatment, only buffer solutions or water systems are typically used for dissolution or dilution, which is insufficient to fully disassemble the three-dimensional network structure of the self-assembled peptide hydrogel. This results in incomplete extraction of hexapeptides, lower detection results, and poor repeatability and accuracy. In addition, some methods employ strong physical destruction or complex chemical treatment conditions, which, while improving extraction efficiency to some extent, can easily cause peptide chain degradation or structural changes, hindering stable and accurate quantitative analysis of hexapeptides.

[0005] Therefore, there is an urgent need to provide a method for detecting the content of hexapeptides in self-assembled peptide hydrogel systems. By introducing appropriate extraction techniques, the non-covalent forces inside the hydrogel can be effectively disrupted while ensuring the stability of the peptide structure, so as to achieve full release and efficient extraction of hexapeptides, thereby improving the accuracy and repeatability of the detection results and meeting the actual needs of product development and quality control of self-assembled peptide hydrogels. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies, adapt to practical application needs, and provide a method for detecting the content of hexapeptides in self-assembled peptide hydrogels. This addresses the technical problems in existing technologies for detecting hexapeptide content in self-assembled peptide hydrogel systems, such as the complex structure of the hydrogel matrix and the tendency of hexapeptide molecules to self-assemble and aggregate, leading to difficulties in fully extracting the target peptide during pretreatment. These problems result in significant matrix interference, poor repeatability of detection results, and insufficient accuracy and stability of quantitative analysis. Therefore, this invention aims to achieve accurate and reliable detection of the hexapeptide content in self-assembled peptide hydrogels. To achieve the purpose of this invention, the technical solution adopted is as follows: A method for detecting the content of hexapeptides in self-assembled peptide hydrogels is designed, comprising the following steps:

[0007] S1. Preparation of the test solution:

[0008] Weigh approximately 0.2 g of the self-assembled peptide hydrogel sample into a centrifuge tube, add 2 mL of primary water to dissolve it completely, and then place it in a vacuum freeze dryer for drying; add 0.2 mL of formic acid to the dried powder and extract at 37.0 °C for 30 min, then add primary water to make up to 10 mL, vortex to mix, and obtain an intermediate solution; dilute the intermediate solution 5 times to obtain the test solution;

[0009] S2. Preparation of the method validation solution:

[0010] S21, Hexapeptide Standard Stock Solution: Accurately weigh the hexapeptide standard, dissolve it in primary water and dilute to volume to prepare a standard stock solution with a mass concentration of 114.8 μg / mL.

[0011] S22, Linear solutions: Take the above standard stock solution and dilute it stepwise with primary water to prepare a series of linear solutions with concentrations of 11.48 μg / mL, 22.96 μg / mL, 45.92 μg / mL, 68.88 μg / mL and 114.8 μg / mL, respectively.

[0012] S23. Accuracy solution (spiked recovery solution): Take the test solution of the known low concentration of hexapeptide, accurately add an appropriate amount of hexapeptide standard stock solution, vortex mix and make up to volume to prepare solutions of three spiking concentration levels (11.48 μg / mL, 22.96 μg / mL and 34.44 μg / mL, respectively), and prepare three copies of each concentration level in parallel;

[0013] S24. Repeatability solution: Prepare 7 sample solutions in parallel according to the preparation method of the test solution described in step S1;

[0014] S25. Limit of detection and limit of quantitation solutions: Take the hexadecapeptide standard stock solution and dilute it stepwise with primary water to prepare a limit of detection solution with a concentration of approximately 2.296 μg / mL and a limit of quantitation solution with a concentration of approximately 5.740 μg / mL, respectively.

[0015] S3. High-performance liquid chromatography analysis:

[0016] High performance liquid chromatography (HPLC) was used with a C4 column and gradient elution with 0.1% trifluoroacetic acid aqueous solution and 0.1% trifluoroacetic acid in acetonitrile solution as mobile phases. The column temperature was 30℃, the detection wavelength was 220nm, the flow rate was 1.0mL / min, and the injection volume was 20μL. Various solutions prepared in step S2 and test solution prepared in step S1 were detected.

[0017] S4. Methodological Validation and Content Calculation:

[0018] Using the detection results of the series of linear solutions in step S22, a standard curve was plotted with hexapeptide concentration as the abscissa and peak area as the ordinate to investigate the linear relationship. The accuracy, repeatability, limit of detection, and limit of quantitation of the method were investigated using the solutions in steps S23-S25. After the method validation met the requirements, the detection peak area of ​​the test solution was substituted into the standard curve to calculate its hexapeptide concentration. Based on the sampling amount, volume, and dilution factor, the mass percentage content of hexapeptide in the self-assembled peptide hydrogel was calculated.

[0019] Preferably, in step S3, the chromatographic column is a Kromasil 100-3.5-C4 column with dimensions of 4.6 mm × 150 mm.

[0020] Preferably, in step S3, the instrument preparation involves a mobile phase gradient of:

[0021]

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. This invention introduces formic acid as an extraction agent during sample pretreatment, which effectively disrupts the non-covalent interactions within the self-assembled peptide hydrogel under mild conditions, promoting the full release of hexapeptide from the hydrogel network structure and significantly improving the extraction efficiency of hexapeptide.

[0024] 2. The detection method described in this invention can effectively reduce the interference of the complex matrix of self-assembled peptide hydrogel on the detection results, improve the stability and repeatability of the detection process, and thus ensure the accuracy and reliability of the hexadecapeptide content determination results.

[0025] 3. The method of the present invention has simple sample pretreatment steps and mild operating conditions, which are not likely to cause degradation of the hexapeptide structure or change of properties, and is suitable for quantitative analysis of hexapeptide content in self-assembled peptide hydrogels.

[0026] 4. This invention combines high-performance liquid chromatography for detection, which has the advantages of high sensitivity, good separation effect and accurate quantification. It can meet the detection needs of self-assembled peptide hydrogels in the research, development, production and quality control process, and has good application prospects. Attached Figure Description

[0027] Figure 1 This is the standard curve spectrum of the hexapeptide of the present invention.

[0028] Figure 2 This is a typical spectrum of the hexadecapeptide in the test sample of this invention.

[0029] Figure 3 This is a typical spectrum of the hexadecapeptide in the blank of this invention.

[0030] Figure 4 This is a typical spectrum of the hexadecapeptide standard solution of the present invention. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0032] A method for detecting the content of hexadecapeptide in a self-assembled peptide hydrogel includes the following steps:

[0033] S1. Instrument and reagent preparation: High performance liquid chromatograph: Agilent 1260 Infinity II;

[0034] Column: Kromasil 100-3.5-C4, 4.6mm × 150mm;

[0035] Electronic balance: METTLER XSR105DU / A, accuracy 0.00001g;

[0036] Vacuum freeze dryer: CTFD-12S-U;

[0037] Hexapeptide reference standard;

[0038] Trifluoroacetic acid (TFA, chromatographic grade);

[0039] Acetonitrile (chromatographic grade);

[0040] Formic acid (chromatographic grade);

[0041] The water used in the experiment was Grade I water;

[0042] S2. Preparation of the test solution

[0043] Weigh approximately 0.2 g of the test sample into a centrifuge tube, add 2 mL of primary water to dissolve it, and then dry it in a vacuum freeze dryer. Add 0.2 mL of formic acid to the dried powder, extract it at 37.0℃ for 30 min, then add primary water to make up to 10 mL, vortex to mix, and dilute 5 times to obtain the test sample solution.

[0044] S3. Preparation of the method validation solution

[0045] Hexapeptide Standard Stock Solution: Accurately weigh a certain amount of hexapeptide standard, dissolve it in primary water, and prepare a standard stock solution of a certain concentration.

[0046] Linear solutions: Prepare multiple linear solutions with different concentration gradients using hexadecapeptide stock solution and primary water;

[0047] Accuracy solution: Add an appropriate amount of hexadecapeptide standard stock solution to the test sample solution, vortex mix and dilute to volume to obtain the accuracy solution;

[0048] Repeatability solution: Prepare the solution according to the preparation method of the test sample solution, and prepare multiple parallel samples;

[0049] Detection limit solution: Prepare a detection limit solution of a certain concentration by stepwise dilution of the hexadecapeptide standard solution;

[0050] Limit of quantitation solution: Prepare a limit of quantitation solution of a certain concentration by stepwise dilution of the hexadecapeptide standard solution;

[0051] S4. HPLC was used to validate the detection method.

[0052] S5. The content of hexapeptide in the self-assembled peptide hydrogel was determined by HPLC.

[0053] Specifically, in S1, the instrument preparation involves adjusting all parameters to the working state, and the chromatographic column is a Kromasil 100-3.5-C4 4.6 × 150 mm.

[0054] More specifically, the mobile phase in S1 is 0.1% trifluoroacetic acid solution - 0.1% trifluoroacetic acid acetonitrile solution.

[0055] Furthermore, in S1, the instrument preparation is based on the following mobile phase gradient:

[0056]

[0057] Furthermore, in S2, approximately 0.2 g of the test sample was weighed into a centrifuge tube, dissolved in 2 mL of primary water, and dried in a vacuum freeze dryer. The dried powder was then extracted with 0.2 mL of formic acid at 37.0 °C for 30 min, and then diluted to 10 mL with primary water. The mixture was vortexed and diluted 5 times.

[0058] It is worth noting that the mass concentration of the hexapeptide standard stock solution in S3 is 114.8 μg / mL, and its linear solutions are prepared using the hexapeptide standard stock solution with concentration gradients of 11.48 μg / mL, 22.96 μg / mL, 45.92 μg / mL, 68.88 μg / mL and 114.8 μg / mL, which are designated as STD-1, STD-2, STD-3, STD-4 and STD-5, respectively.

[0059] It is worth noting that the accuracy solutions in S3 were prepared at concentration levels of 11.48 μg / mL, 22.96 μg / mL, and 34.44 μg / mL, with three parallel samples prepared for each concentration level. The repeatability solution was prepared according to the method for preparing the test sample solution, with seven parallel samples prepared. The hexapeptide concentration corresponding to the limit of detection solution was 2.296 μg / mL, and the hexapeptide concentration corresponding to the limit of quantitation solution was 5.740 μg / mL.

[0060] It is worth emphasizing that the method validation items in S4 include limit of detection, limit of quantitation, linearity and range, repeatability, and accuracy. HPLC is used to detect hexapeptides in the standard linear solution and the test solution, the peak area of ​​the hexapeptides is recorded, and the content of hexapeptides in the test solution is calculated based on the hexapeptide standard curve.

[0061] Example 1

[0062] Instrument conditions

[0063] The HPLC instrument parameter settings are shown in Table 1.

[0064] Table 1. HPLC Instrument Conditions

[0065]

[0066] Solution preparation

[0067] Preparation of test solution

[0068] Weigh approximately 0.2 g of the test sample into a centrifuge tube, add 2 mL of primary water to dissolve it, and then dry it in a vacuum freeze dryer. Add 0.2 mL of formic acid to the dried powder, extract it at 37.0℃ for 30 min, then add primary water to make up to 10 mL, vortex to mix, and dilute 5 times to obtain the test sample solution.

[0069] Method validation solution preparation

[0070] Hexapeptide Standard Stock Solution: Accurately weigh a certain amount of hexapeptide standard, dissolve it in primary water, and prepare a standard stock solution of a certain concentration.

[0071] Linear solutions: Prepare multiple linear solutions with different concentration gradients using hexadecapeptide stock solution and primary water;

[0072] Accuracy solution: Add an appropriate amount of hexadecapeptide standard stock solution to the test sample solution, vortex mix and dilute to volume to obtain the accuracy solution;

[0073] Repeatability solution: Prepare the solution according to the preparation method of the test sample solution, and prepare multiple parallel samples;

[0074] Detection limit solution: Prepare a detection limit solution of a certain concentration by stepwise dilution of the hexadecapeptide standard solution;

[0075] Limit of quantitation solution: Prepare a limit of quantitation solution of a certain concentration by stepwise dilution of the hexadecapeptide standard solution;

[0076] The detection method was validated using HPLC.

[0077] The content of hexapeptides in self-assembled peptide hydrogels was determined by HPLC. The method validation results for one of the methods for detecting the content of hexapeptides in self-assembled peptide hydrogels are as follows:

[0078] linear

[0079] The linear solution was tested according to the conditions in Table 1. A standard curve was plotted with concentration on the x-axis and response value on the y-axis. The linear correlation coefficient r should be ≥0.99. The test results are shown in Table 2. As can be seen from the results in Table 2, this method has good linearity.

[0080] Table 2 Linear Results

[0081]

[0082] Limit of detection (LOD) and limit of quantitation (LOQ)

[0083] One portion of LOD solution and one portion of LOQ solution were each injected three times for testing. The specific test results for the limits of detection and quantitation are shown in Table 3.

[0084] Table 3 LOD and LOQ Results

[0085]

[0086] Repeatable results

[0087] Take a repeatable solution and test it according to the instrument conditions shown in Table 1. The results are shown in Table 4. According to GB / T27417-2017, the RSD should be ≤5.3%. As can be seen from the results in Table 2, this method has good repeatability.

[0088] Table 4 Repeatability Results

[0089]

[0090] Accuracy

[0091] Nine accuracy solutions (low, medium, and high concentrations) were taken and tested according to the conditions in Table 1. According to GB / T27417-2017, the acceptable recovery rate of the target analyte in the solution for low, medium, and high concentrations is 90%–110%, as shown in Table 5. The results in the table indicate that this method has good accuracy.

[0092] Table 5 Accuracy Results

[0093]

[0094] Summary of method validation results

[0095] The validation results of the hexadecapeptide method are summarized in Table 6. As can be seen from the results in Table 6, the validation results of each indicator meet the requirements of the evaluation criteria.

[0096] Table 6 Summary of Method Validation Results

[0097]

[0098] Hexapeptide content test

[0099] Using the instrument conditions shown in Table 1, the hexapeptide content in the test solution was determined by HPLC. The measured hexapeptide content was then converted to the hexapeptide concentration using the following formula. The detection results are shown in Table 7.

[0100] W=C×V / m / 10 -6 ×f*100%

[0101] In formula (1):

[0102] W – The content of hexapeptide in the sample, in %

[0103] C—Concentration of hexadecapeptide in the test solution, in μg / mL;

[0104] V – constant volume, in mL;

[0105] f—Dilution factor;

[0106] m—Sample weight, in grams;

[0107] 10 -6 —Unit conversion factor.

[0108] Table 7. Content of hexapeptide (%)

[0109]

[0110] In addition, all components designed in this invention are general standard parts or components known to those skilled in the art. Their structure and principle can be known to those skilled in the art through technical manuals or conventional experimental methods. Those skilled in the art can fully implement them, so there is no need to elaborate. The content protected by this invention does not involve improvements to the internal structure and method.

Claims

1. A method for detecting the content of hexapeptide in a self-assembled peptide hydrogel, characterized in that, Includes the following steps: S1. Preparation of the test solution: Weigh the self-assembled peptide hydrogel test sample, dissolve it in primary water, and freeze-dry it to obtain a dry powder; add formic acid to the dry powder and extract it at 37°C; after extraction, add water to make up the volume and dilute to obtain the test solution; S2. Preparation of standard curve solutions: Accurately weigh the hexapeptide standard, dissolve and dilute it with primary water to prepare a series of standard solutions with at least five different concentrations. S3. High-performance liquid chromatography (HPLC) analysis: The test solution and a series of standard solutions were injected into the HPLC instrument for analysis. The chromatographic conditions included: using a C4 column, gradient elution with an aqueous solution containing 0.1% trifluoroacetic acid and an acetonitrile solution containing 0.1% trifluoroacetic acid as the mobile phase, and a detection wavelength of 220 nm. S4. Methodological Validation and Content Calculation: A standard curve was plotted with the concentration of hexapeptide in the series of standard solutions as the x-axis and the corresponding chromatographic peak area as the y-axis, and the linearity was verified. The chromatographic peak area of ​​the test solution was substituted into the standard curve to calculate the concentration of hexapeptide in the test solution, and the content of hexapeptide in the self-assembled peptide hydrogel was further calculated.

2. The detection method according to claim 1, characterized in that, In step (1), the amount of sample weighed is 0.2g, the amount of formic acid added is 0.2mL, and the extraction time is 30min.

3. The detection method according to claim 1, characterized in that, In step (1), the volume of the final volume is 10 mL and the dilution factor is 5 times.

4. The detection method according to claim 1, characterized in that, In step (2), the concentration range of the series of standard solutions includes 11.48 μg / mL, 22.96 μg / mL, 45.92 μg / mL, 68.88 μg / mL and 114.8 μg / mL.

5. The detection method according to claim 1, characterized in that, In step (3), the chromatographic column is a Kromasil 100-3.5-C4 column with a specification of 4.6×150 mm.

6. The detection method according to claim 1, characterized in that, In step (3), the gradient elution program is as follows: 0-20 min, the volume percentage of the acetonitrile solution containing 0.1% trifluoroacetic acid increases linearly from 5% to 40%, and the volume percentage of the aqueous solution containing 0.1% trifluoroacetic acid decreases linearly from 95% to 60%; 20.1-25 min, the initial ratio is restored and equilibrium is maintained.

7. The detection method according to claim 1, characterized in that, In step (3), the column temperature of the high performance liquid chromatography analysis is 30℃, the flow rate is 1.0 mL / min, the injection volume is 20 μL, and the detection wavelength is 220 nm.

8. The detection method according to claim 1, characterized in that, In step (4), the methodology validation also includes accuracy validation, repeatability validation, limit of detection and limit of quantitation validation.

9. The detection method according to claim 8, characterized in that, The accuracy verification was performed by adding different concentrations of hexapeptide standards to a test sample solution with known background, and conducting a spike recovery experiment with a recovery rate required to be between 90% and 110%.

10. The detection method according to claim 8, characterized in that, The repeatability verification was performed by preparing and measuring at least six test solutions in parallel, with a relative standard deviation of no more than 5.3% for the determination of hexadecapeptide content.

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