High performance liquid detection method for blue copper peptide key intermediate and related impurities thereof

Abstract

The invention relates to the technical field of high performance liquid detection, in particular to a high performance liquid detection method for a blue copper peptide key intermediate and related impurities thereof, a sample to be detected is prepared into a sample solution, then high performance liquid chromatography is adopted for detection, and conditions of high performance liquid chromatography are as follows: a chromatographic column is a reversed-phase C18 chromatographic column; a mobile phase: a phase A is a 0.1% phosphoric acid aqueous solution; the phase B is acetonitrile; the elution mode is gradient elution. The invention provides a high-efficiency, accurate and sensitive detection method for the key intermediate of the blue copper peptide, the quality analysis of the key intermediate of the blue copper peptide is realized by optimizing the detection conditions of high performance liquid chromatography, the quality control level is improved, and the quality of a final product is ensured.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and in particular to a high-performance liquid chromatography detection method for a blue copper peptide key intermediate and related impurities thereof. BACKGROUND

[0002] Blue copper peptide (GHK-Cu) is a bioactive peptide formed by the complexation of copper ions and GHK tripeptide, which is widely used in cosmetics, medicine and other fields. The parent peptide GHK of blue copper peptide has a chemical name of glycyl-L-histidyl-L-lysine, a molecular formula of C 14 H 24 N6O4, and a structural formula as shown in formula I.

[0003] .

[0004] The synthesis of blue copper peptide (i.e., tripeptide-1 copper) mainly includes two steps: first, the parent peptide tripeptide-1 (GHK) is synthesized by a solid-phase or liquid-phase method, and then GHK is complexed with a copper salt (such as copper sulfate) to form the target product. Among them, the preparation of high-purity GHK is the core link to improve the yield and purity of blue copper peptide. However, impurities are easily introduced during the synthesis of the parent peptide due to raw material residues, degradation, etc., and the effects of impurity A (Boc-Gly-His(Trt)-Lys-OH, C 38 H 46 N6O6) and impurity B (triphenylmethanol, C 19 H 16 O) on the quality of the final product need to be focused on, wherein the structures of impurity A and impurity B are shown in formula II and formula III, respectively. 、 .

[0005] The currently disclosed liquid chromatography detection methods for blue copper peptide have significant defects and cannot meet the process requirements. For example, Chinese patent CN202411242861 (a detection method for blue copper peptide related substances) can only detect GHK, and neither impurity A nor impurity B is detected, which cannot realize the simultaneous monitoring of key impurities. Chinese patent CN202211426148 (a method for determining the content of blue copper peptide in cosmetics) has an organic phase with a maximum proportion of only 10% in gradient elution, which is insufficient in elution capacity, resulting in the inability of impurities A and B to be eluted. In addition, Chinese patent CN202211071245 (a detection method for the content of blue copper peptide) has a mobile phase of acetonitrile (0.1% trifluoroacetic acid) + water (0.1% trifluoroacetic acid) = 65:35, which improves the elution capacity, but unknown impurities in the sample easily interfere with the target peak, affecting the quantitative accuracy.

[0006] In summary, existing patented methods all have obvious defects, such as being unable to simultaneously separate GHK from process impurities (impurities A and B), having insufficient elution efficiency, or being affected by unknown impurities. They are difficult to meet the current requirements for rapid and accurate monitoring of substances related to blue copper peptide precursor peptide in the process, and there is an urgent need to develop optimized detection solutions. Summary of the Invention

[0007] The purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for the detection of key intermediates of copper peptide and related impurities, thereby solving the technical problem that existing technologies cannot simultaneously detect copper peptide precursor, impurities A and B, and thus cannot meet the needs of rapid and accurate detection in actual production.

[0008] First, this invention provides a high-performance liquid chromatography (HPLC) method for the detection of key intermediates of blue copper peptide and related impurities. The method involves preparing the sample solution and then detecting it using HPLC. The HPLC conditions are as follows: Chromatographic column: Reversed-phase C18 column (Atlantis T3 4.6*250mm, 5mm or YMC-Pack ODS-AQ 4.6*250mm, 5mm); Mobile phase: Phase A is 0.1% aqueous phosphoric acid solution; Phase B is acetonitrile; A gradient elution method was used for elution. The gradient elution program was as follows: 0–10 min, 98% A → 40% A, 2% B → 60% B; 10–20 min, 40% A → 30% A, 60% B → 70% B; 20–23 min, 30% A, 70% B; 23–23.1 min, 30% A → 0% A, 70% B → 100% B; 23.1–37 min, 0% A, 100% B; 37–37.1 min, 0% A → 98% A, 100% B → 2% B; 37.1–45 min, 98% A, 2% B.

[0009] Table 1. Gradient elution program

[0010] Furthermore, the flow rate of the mobile phase is 0.6~1.0 mL / min.

[0011] Furthermore, the column temperature is 30~40℃.

[0012] Furthermore, the sample concentration was 0.4~1.0 mg / mL.

[0013] Furthermore, by volume, the sample solvent was prepared by mixing methanol and water in a ratio of 75:25 to 85:15.

[0014] Furthermore, the injection volume is 4~10mL.

[0015] Furthermore, the detector and wavelength are UV 200~220nm.

[0016] Furthermore, it also includes standard curves for preparing the blue copper peptide precursor GHK and its related impurities.

[0017] Furthermore, the sample solution is spiked and then subjected to high-performance liquid chromatography to determine the accuracy of the method and to provide a solution for tracing process impurities.

[0018] Secondly, this method also provides the application of the high-performance liquid chromatography method for detecting the key intermediate of blue copper peptide and its related impurities provided in the first aspect above, for detecting blue copper peptide and its intermediates.

[0019] Compared with the prior art, the beneficial effects of the present invention are: First, this invention, through an innovative gradient elution program (0.1% phosphoric acid aqueous solution / acetonitrile system) adapted to a specific chromatographic column, achieves for the first time complete elution and baseline separation of blue copper peptide precursor (GHK) and key process impurities (impurity A and impurity B) in a single injection, with each peak of interest achieving a resolution of at least 1.5, thus solving the core defect of existing technologies that cannot achieve simultaneous elution and separation.

[0020] Secondly, this invention provides a highly efficient, accurate, and sensitive method for detecting key intermediates of copper peptides. By employing UV detection at 200–220 nm (preferably 205 nm), interference from various organic solvents introduced by the process is eliminated under low wavelength conditions, reducing the limit of quantitation (LOQ) to 0.04% (equivalent to the concentration of the principal component), with sensitivity far exceeding existing methods. Compared to the trifluoroacetic acid system (CN202211071245), 0.1% phosphoric acid aqueous solution significantly improves the response value, and the recovery rate of impurity A / B reaches 92.0–105.0% (RSD<2.0%), meeting the requirements for trace impurity control. Regarding applicability to production quality control, the 45-minute sample analysis time is suitable for the rapid detection needs of production lines. Key parameters (flow rate 0.6–1.0 mL / min, column temperature 30–40℃) have been validated over a wide range (RSD<2.0%), ensuring the method's transfer stability between different devices and allowing direct use in spiked sample analysis, providing a solution for tracing process impurities.

[0021] Finally, this method establishes for the first time a synchronous monitoring system for GHK and impurities A / B, accurately quantifying impurity A (Boc protecting group residue) and impurity B (triphenylmethanol byproduct), guiding the optimization of the synthesis process, providing core technical support for quality control in the production of high-purity GHK, and ultimately improving the yield and safety of blue copper peptide, ensuring its safety, effectiveness and controllable cost.

[0022] In summary, this invention, with gradient program innovation at its core, and synergistic optimization of mobile phase system and column screening, completely solves the defects of existing methods in terms of separation capability, sensitivity and anti-interference. Based on excellent separation effect and specificity, it achieves quality control of related substances, and provides the first reliable detection scheme for the blue copper peptide industry chain that can simultaneously monitor the parent peptide and key impurities, significantly improving product quality controllability and production efficiency, and improving the quality and yield of the final product. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The HPLC chromatogram of the blank solution in Example 2; Figure 2 The HPLC chromatogram of the blue copper peptide precursor peptide GHK localization solution from Example 2 is shown below. Figure 3 The HPLC chromatogram of the blue copper peptide precursor peptide impurity A localization solution in Example 2; Figure 4 The HPLC chromatogram of the blue copper peptide precursor peptide impurity B localization solution in Example 2; Figure 5 The HPLC chromatogram of the spiked test solution in Example 2; Figure 6 The graph shows the linear regression equation of GHK in Example 2; Figure 7 The graph shows the linear regression equation for impurity A in Example 2; Figure 8 The graph shows the linear regression equation for impurity B in Example 2. Detailed Implementation

[0025] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. These embodiments are merely illustrative of the technical solution of the present invention and are therefore intended to limit the scope of protection of the present invention.

[0026] Those skilled in the art will understand that the present invention can be practiced even without certain specific details. In other embodiments, methods, means, apparatus, and steps well-known to those skilled in the art have not been described in detail to highlight the spirit of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, all units used in this specification are International Standard Units (SI), and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors unavoidable in industrial production.

[0027] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents, or instruments used, unless otherwise specified by the manufacturer, are all commercially available; and the conditions not specified in the examples are all performed under conventional conditions or conditions recommended by the manufacturer. Furthermore, this invention does not limit the source of the raw materials used; unless otherwise specified, the raw materials used in this invention are all commercially available products commonly found in this technical field. Unless otherwise specified, the "ratios" referred to in the following examples are all volume ratios.

[0028] It should be specifically stated that the information on the instruments, equipment, reagents, reference solutions, test samples, and chromatographic columns used in the experiment is as follows: For instruments, the electronic balance is model XSR105A and the pH meter is model FE28, both purchased from Mettler; the high performance liquid chromatographs used are Agilent 1260 II, Waters e2695, and Shimadzu LC-2030C Plus.

[0029] All reagents and reagents were HPLC grade, including methanol (batch number 20240105001, source: Cyflure), acetonitrile (batch number: Y7WA1H, source: Honeywell), trifluoroacetic acid (batch number: 2024010301, source: Chengdu Kelon), potassium dihydrogen phosphate (batch number: 20230218, source: Comio), phosphoric acid (batch number: 2023091801, source: Chengdu Kelon), and formic acid (batch number: 2023092101, source: Chengdu Kelon).

[0030] The reference standards include blue copper peptide precursor GHK (batch number L710293, content 99.51%, sourced from Leyan), blue copper peptide precursor impurity A (batch number S-K008-E-250104, content 97.63%, sourced from self-made) and blue copper peptide precursor impurity B (batch number S-K008-F-IMP01-250301, content 99.94%, sourced from self-made).

[0031] The test sample was a self-made sample of blue copper peptide GHK, batch number S-K008-F-250303, which was self-made. There are 5 chromatographic columns in total, specifically: Waters Atlantis T3 column (4.6*250mm, 5mm) with serial number DK-RD-LC-24-21, YMC YMC-Pack ODS-AQ column (4.6*250mm, 5mm) with serial number DK-RD-LC-25-03, Agilent ZORBAX SB-C18 column (4.6*250mm, 5mm) with serial number DK-RD-LC-24-02, Agilent ZORBAX Eclipse XDB-C18 column (4.6*250mm, 5mm) with serial number DK-RD-LC-24-03, and Shimadzu Shim-pack scepter C18-120 column (4.6*150mm, 5mm) with serial number DK-RD-LC-24-13.

[0032] Example 1 The purpose of this embodiment is to provide a high-performance liquid chromatography (HPLC) method for the detection of key intermediates of copper peptide and its related impurities, solving the technical problem that existing technologies cannot simultaneously detect the copper peptide precursor (GHK) and its impurity A (Boc-Gly-His (Trt)-Lys-OH) and impurity B (triphenylmethanol), making it difficult to meet the requirements for rapid and accurate detection in actual production. The specific steps are as follows: 1.1 Sample Preparation Solvent preparation: Accurately measure methanol and ultrapure water, mix them at a volume ratio of 80:20, and use them as the sample solvent.

[0033] Preparation of the test solution: Accurately weigh about 25 mg of the test sample (GHK self-made sample), place it in a 50 mL volumetric flask, add the above solvent to dissolve and dilute to the mark, shake well, and prepare a test solution with a concentration of 0.5 mg / mL.

[0034] 1.2 Optimal chromatographic detection conditions Chromatographic conditions and column settings: reversed-phase C18 column (select Atlantis T3 4.6*250mm, 5mm or YMC-Pack ODS-AQ 4.6*250mm, 5mm).

[0035] Mobile phase: Phase A is 0.1% phosphoric acid aqueous solution (1 mL of phosphoric acid is dissolved in 1000 mL of ultrapure water and mixed well); Phase B is acetonitrile (HPLC grade).

[0036] The specific parameters of the gradient elution procedure are shown in Table 1: Table 1 Gradient elution program

[0037] Other parameters: Sample concentration: 0.5 mg / mL, flow rate: 0.8 mL / min; column temperature: 35℃; injection volume: 5 μL; detector: ultraviolet detector (UV), detection wavelength: 205 nm. Perform the injection analysis according to the above procedure. The injection volume is 5 μL. Inject the test solution into the chromatograph, record the chromatogram, and calculate the retention time, peak area and resolution of each target peak (GHK, impurity A, impurity B).

[0038] The above chromatographic conditions are the optimal chromatographic detection conditions.

[0039] 1.3 Experimental Results Using the above optimal chromatographic detection conditions, the test solution can be detected. This method can achieve a resolution of more than 1.5 between each peak of interest. Based on the excellent separation effect and specificity, it can realize the quality control of related substances, improve the quality and yield of the final product, and ensure its safety, effectiveness and controllable cost.

[0040] Example 2 The purpose of this embodiment is to validate the optimal chromatographic detection conditions provided in Example 1, including specificity, system suitability, limit of quantitation and limit of detection, linearity and range, precision (repeatability), accuracy, and solution stability, to confirm the reliability and applicability of the method, including: 1. Specificity verification (1) Experimental methods The purpose of this embodiment is to verify the specificity of the detection method provided in Embodiment 1 above. Through the detection of blank solution, positioning solution, and spiked test sample solution, it is confirmed that there are no interfering peaks and the target peak is completely separated, as detailed below: The chromatographic column used was an Atlantis T3 4.6*250mm, 5mm thick. The test solutions included: Blank solution: Diluent (methanol) (It is prepared by mixing water in a volume ratio of 8:2).

[0041] Blue copper peptide precursor GHK localization solution: Take blue copper peptide precursor GHK reference standard, add diluent to dissolve and prepare reference standard localization solution with a concentration of 0.5 mg / mL.

[0042] Blue copper peptide precursor peptide impurity A positioning solution: Take blue copper peptide precursor peptide impurity A reference standard, add diluent to dissolve and prepare a reference standard positioning solution with a concentration of 0.5 mg / mL.

[0043] Blue copper peptide precursor peptide impurity B positioning solution: Take blue copper peptide precursor peptide impurity B reference standard, add diluent to dissolve and prepare reference standard positioning solution with a concentration of 0.5 mg / mL.

[0044] Reference stock solution: Weigh appropriate amounts of each reference standard, place them in different volumetric flasks, add diluent to dissolve and quantitatively dilute to prepare a single reference stock solution of 1 mg / mL.

[0045] Mixed reference stock solution: Accurately measure 1 mL of each of the above reference stock solutions, place them in the same 20 mL volumetric flask, dilute to the mark with diluent, mix well, and the solution is ready.

[0046] Spiked test solution: Accurately weigh about 25 mg of the test sample and place it in a 50 mL volumetric flask. Accurately add 1 mL of the above mixed reference stock solution, then dilute to the mark with diluent and mix well.

[0047] According to the optimal chromatographic detection conditions determined in Example 1, 5 μL each of the blank solution, each positioning solution, and the spiked test solution were injected into the high-performance liquid chromatograph. Detection was performed under the optimal chromatographic detection conditions, and the chromatogram results were recorded. Detailed experimental results are shown in the chromatogram table. Figures 1-5 .

[0048] (2) Experimental results and analysis The chromatographic results of the blank solution are as follows: Figure 1 As shown, the experimental results indicate that there are no interfering peaks within the retention time range of the target peak.

[0049] Chromatographic detection results of each positioning solution are as follows Figures 2-4 As shown, the experimental results indicate that: GHK (see...) Figure 2 Impurity A (see Figure 3 Impurity B (see Figure 4 Peaks were observed at approximately 3.6 min, 14.1 min, and 32.5 min, respectively, with good peak shapes.

[0050] The chromatographic detection results of the spiked test solution are as follows: Figure 5 As shown, the experimental results indicate that the retention times of each target peak are consistent with those of the localization solution. The separation degree between GHK and impurity A is 1.8, and the separation degree between impurity A and impurity B is 2.1, both ≥1.5, and there is no interference from unknown peaks.

[0051] The experimental results above show that the peaks eluted by the blank solution and the test solution do not interfere with the detection of known impurities and the main peak. The resolution of each peak of interest (known impurity peak and main peak) with adjacent peaks is ≥1.5, indicating that the method has good specificity.

[0052] 2. System applicability verification The purpose of this experiment is to examine the stability and reliability of the chromatographic system (including the column, mobile phase, and instruments) under the set detection conditions, so as to ensure the accuracy and repeatability of subsequent detection results, as detailed below: (1) Experimental methods The preparation of materials is as described above; Preparation of system suitability solution: Accurately measure 1 mL of the mixed reference stock solution under the specificity section, place it in a 50 mL volumetric flask, dilute to the mark with diluent and mix well to obtain the system suitability solution.

[0053] Using the optimal chromatographic detection conditions provided in Example 1, the system suitability solution was continuously injected 5 times, and the retention time and peak area of ​​each target peak were recorded, and the RSD was calculated.

[0054] (2) Experimental results The retention times of five injections were recorded and statistically analyzed. The experimental results are shown in Table 2. Table 25 shows the statistical results of retention time in the tests.

[0055] The peak areas of five injections were recorded and statistically analyzed. The experimental results are shown in Table 3. Table 35 shows the statistical results of peak area from the tests.

[0056] The above experimental results show that in the spectra obtained by continuous injection of the system suitability solution for 5 injections, the peak area and retention time RSD of each target component are all less than 2.0%, indicating that the system suitability of this method is good.

[0057] Validation of limit of quantitation and limit of detection.

[0058] The purpose of this experiment is to determine the lowest concentration at which the method can accurately quantify the target component (limit of quantitation) and detect the target component (limit of detection), to evaluate the sensitivity of the method, and to ensure that it can detect and accurately quantify low levels of the active ingredient GHK and trace impurities A and B in the test sample, meeting the analytical needs for trace components in practical testing, as detailed below: (1) Experimental methods The preparation of materials is as described above; Preparation of limit of quantitation solution: Accurately measure 2 ml of system suitability solution (see Solution Preparation under System Suitability), place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0059] Preparation of the limit of quantitation solution: Accurately measure 3 ml of the limit of quantitation solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0060] Under the optimal chromatographic detection conditions provided in Example 1, accurately measure 5 μL each of the above-mentioned limit of quantitation solution and limit of detection solution, inject them into the liquid chromatograph, and record the chromatograms.

[0061] (2) Experimental results and analysis The results of the limit of quantitation (LOQ) and limit of detection (LOD) are shown in Table 4 and Table 5, respectively. Table 4 Results of the limit of quantitation (n=5)

[0062] Table 5 Detection limit results (n=5)

[0063] The above experimental results show that the limit of quantification (LOQ) for GHK is 214 ng / mL, for impurity A it is 201 ng / mL, and for impurity B it is 202 ng / mL. The LOQs are all equivalent to 0.04% of the concentration of the main component of the test sample, and the RSD of the peak area of ​​the target component in the chromatograms obtained by five consecutive injections is less than 5.0%, and the signal-to-noise ratio is greater than 10.

[0064] The above experimental results show that the detection limit of GHK is 64 ng / mL, the detection limit of impurity A is 60 ng / mL, and the detection limit of impurity B is 61 ng / mL. The detection limits are all equivalent to 0.01% of the concentration of the main component of the test sample, and the signal-to-noise ratio of each target component in the chromatograms obtained by three consecutive injections is greater than 3.

[0065] 4. Linearity and Range Validation The purpose of this experiment is to confirm whether the concentrations of GHK, impurities A and B, and their corresponding chromatographic peak areas exhibit a good linear relationship within a certain concentration range, to establish a reliable mathematical model for quantitative calculation, and to clarify the applicable concentration range of the method, ensuring that the concentration of the target component can be accurately calculated from the peak area within this range. Specifically: (1) Experimental methods Blank solution: diluent.

[0066] Mixed linear stock solution: Accurately measure 1 mL of each of the reference stock solutions under the specificity section and place them in the same 50 mL volumetric flask. Dilute to the mark with diluent and mix well.

[0067] Linear Solution 1: Accurately measure 2 mL of the mixed linear stock solution, place it in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0068] Linear solution 2: Accurately measure 1.5 mL of the mixed linear stock solution, place it in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0069] Linear solution 3: Accurately measure 1 mL of the mixed linear stock solution, place it in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0070] Linear solution 4: Accurately measure 5 mL of linear solution 3, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0071] Linear solution 5: Accurately measure 2 mL of linear solution 3, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0072] Under the determined chromatographic conditions, accurately measure 5 μl of each of the above linear solutions (linear solutions 1-5) and inject them into the liquid chromatograph. Record the chromatograms under the optimal chromatographic detection conditions. Plot a linear regression curve with concentration as the x-axis and peak area as the y-axis, calculate the regression equation and correlation coefficient, and see the experimental results below. Figures 6-8 .

[0073] (2) Experimental results and analysis The linear regression equation graph of GHK is shown below. Figure 6 As shown, the experimental results indicate that GHK exhibits a good linear relationship between concentration and peak area within the concentration range of 214 ng / mL to 2144 ng / mL. The linear equation is: y = 10.8115x - 1.2401, with a correlation coefficient r of 1.000 (greater than 0.990), and the ratio of the Y-axis intercept to the response value at the 100% concentration point is 0.01% (less than 10%).

[0074] The linear regression equation graph for impurity A is shown below. Figure 7 As shown, the experimental results indicate that impurity A exhibits a good linear relationship between concentration and peak area within the concentration range of 202 ng / mL to 2015 ng / mL. The linear equation is: y = 28.1098x + 376.9623, with a correlation coefficient r of 0.999 (greater than 0.990), and the ratio of the Y-axis intercept to the response value at the 100% concentration point is 1.32% (less than 10%).

[0075] The linear regression equation graph for impurity B is shown below. Figure 8 As shown, the experimental results indicate that impurity B exhibits a good linear relationship between concentration and peak area within the concentration range of 202 ng / mL to 2021 ng / mL. The linear equation is: y = 65.3372x + 2,115.6491, with a correlation coefficient r of 0.999 (greater than 0.990), and the ratio of the Y-axis intercept to the response value at the 100% concentration point is 3.14% (less than 10%).

[0076] 5. Precision (Repeatability) Validation The purpose of this experiment is to examine the consistency of results when the same test sample is repeatedly tested under the same operating conditions (same instrument, same personnel, same batch of reagents, same detection parameters), to verify the repeatability of the method, and to ensure that the test results are stable and reliable.

[0077] (1) Experimental methods Blank solution: diluent.

[0078] Reference solution: Prepare two parallel solutions using the linear solution preparation method 3.

[0079] Spiked test solution: Prepare six parallel portions according to the preparation method under the specificity section.

[0080] Under the optimal chromatographic detection conditions determined in Example 1, 5 μL of each of the above solutions were precisely measured and injected into the liquid chromatograph. The chromatograms were recorded, and the chromatographic results were statistically analyzed.

[0081] (2) Experimental results and analysis The experimental results are shown in Table 6.

[0082] Table 6 Precision test results (n=6)

[0083] The experimental results above show that the RSD of the detection amounts of each known impurity (impurity A and impurity B) in the six spiked test solutions is less than 2.0%, indicating that the method has good repeatability.

[0084] 6. Accuracy Verification The purpose of this experiment is to examine the degree of agreement between the total amount of impurities measured by the method, minus the amount of impurities introduced into the sample, and the actual amount of spiked substance, by adding a known amount of impurity reference standard to the test sample (spiking experiment). This verifies whether the method can accurately reflect the true content of impurities in the test sample and ensures the accuracy of the detection results.

[0085] (1) Experimental methods Data from six spiked test solutions at the limit concentration under the repeatability test were used, and the content of each impurity in the background sample was confirmed by separately testing the unspecified test solution. The accuracy results are shown in Table 7 below.

[0086] (2) Experimental results and analysis The experimental results are shown in Table 7: Table 7 Accuracy Verification Results

[0087] The results show that the recoveries of all known impurities are between 92.0% and 105.0%, and the RSDs of all known impurities are less than 2.0%. This indicates that the method has good accuracy in detecting impurities A and B in the sample.

[0088] 7. Solution stability verification The purpose of this experiment is to examine the changes in the peak area of ​​the target component after the reference solution and the spiked test solution are placed at room temperature for different times, to determine the stability period of the solution, and to ensure that the solution properties are stable and the test results are reliable within the actual detection cycle (such as multiple batches of samples).

[0089] (1) Experimental methods Spiked test solution: Prepared according to the preparation method under the specificity section.

[0090] Reference solution: same as the preparation method of linear solution 3.

[0091] Experimental Results and Analysis Under the determined chromatographic conditions, take the reference solution and the spiked test solution, and inject them for analysis at room temperature for 0 hours, 4 hours, 8 hours, 12 hours and 24 hours, respectively, and record the chromatograms.

[0092] Solution stability test results: After being placed at room temperature for 24 hours, the RSD of the peak areas of all target peaks in the chromatograms obtained at different time points for both the reference solution and the spiked test solution was less than 2.0%. The results indicate that both the reference solution and the spiked test solution exhibit good stability at room temperature for at least 24 hours, meeting the testing time requirements for multiple batches of test samples.

[0093] In summary, the optimal chromatographic detection conditions in the high-performance liquid chromatography (HPLC) method for the detection of key intermediates and related impurities of copper peptide provided in Example 1 have been verified for specificity, system suitability, limit of quantitation and limit of detection, linearity and range, precision, accuracy and solution stability. All indicators meet the methodological requirements and can be used for routine quality control in the production process of copper peptide.

[0094] Example 3 (Column Screening) Under the optimal chromatographic detection conditions provided in Example 1 above, the purpose of this example is to verify the influence of different chromatographic columns on the retention behavior of the target analyte, solve the defects of solvent peak interference and impurity separation, and screen out chromatographic columns that are compatible with high initial gradients of aqueous phase and free from solvent peak interference, as detailed below: (1) Experimental methods Variable settings: Five different models of C18 chromatographic columns were selected, namely: Waters Atlantis T3 (4.6*250mm, 5mm), part number DK-RD-LC-24-21; YMC-Pack ODS-AQ (4.6*250mm, 5mm), part number DK-RD-LC-25-03; Agilent ZORBAX SB-C18 (4.6*250mm, 5mm), part number DK-RD-LC-24-02; Agilent ZORBAX Eclipse XDB-C18 (4.6*250mm, 5mm), part number DK-RD-LC-24-03; Shimadzu Shim-pack scepter C18-120 (4.6*150mm, 5mm), part number DK-RD-LC-24-13.

[0095] Experimental procedure: Except for the chromatographic column, all other conditions were performed according to the optimal chromatographic detection conditions provided in Example 1. 5 μL of the mixed reference solution was precisely measured and injected into the chromatograph. The retention time of the target peak GHK and the position of the solvent peak were recorded, and the detection results were observed.

[0096] (2) Experimental results and analysis The experimental results are as follows: Waters Atlantis T3 column (DK-RD-LC-24-21): GHK retention time is 2.6 min, solvent peak does not interfere with target peak, and it is compatible with high aqueous mobile phase.

[0097] YMC-Pack ODS-AQ column (DK-RD-LC-25-03): GHK retention time is 2.7 min, solvent peak is uninterrupted, and it is compatible with high-aqueous mobile phase.

[0098] ZORBAX SB-C18 column (DK-RD-LC-24-02): GHK retention time is 2.3 min, solvent peak overlaps with GHK peak causing interference, and it is not compatible with high aqueous mobile phase.

[0099] ZORBAX Eclipse XDB-C18 column (DK-RD-LC-24-03): GHK retention time is 2.2 min, solvent peaks severely interfere with GHK peaks, incompatible with high aqueous mobile phase.

[0100] The Shim-pack scepter C18-120 column (DK-RD-LC-24-13) with GHK had a retention time of 2.4 min. The solvent peak slightly interfered with the target peak. It was compatible with high-aqueous mobile phases but had poor separation performance.

[0101] The above experimental results show that only the Waters Atlantis T3 and YMC-Pack ODS-AQ columns meet the requirements of "GHK retention time > 2.5 min, no solvent peak interference, and compatibility with high aqueous phase". The Waters Atlantis T3 will be preferred for subsequent experiments, and the applicability of YMC-Pack ODS-AQ will be verified later.

[0102] Example 4 The purpose of this embodiment is to determine the optimal mobile phase and observe and compare the effects of different acidic mobile phases A on peak shape, response value, and baseline stability, as detailed below: (1) Experimental methods Materials preparation: The instruments, reagents and reference standards are the same as in Example 1. The concentration of the mixed reference standard solution is 0.5 mg / mL (solvent is methanol-water (v / v) = 80:20); the chromatographic column used is Waters Atlantis T3 (4.6*250mm, 5mm, serial number DK-RD-LC-24-21).

[0103] Fixed experimental conditions: mobile phase B was acetonitrile; gradient elution program was the same as in Example 1; flow rate was 0.8 mL / min, column temperature was 35 °C, detection wavelength was 205 nm, and injection volume was 5 μL.

[0104] Variable settings: The mobile phase A was prepared using 0.1% formic acid aqueous solution, 0.1% phosphoric acid aqueous solution, and 0.1% trifluoroacetic acid aqueous solution, respectively.

[0105] Experimental procedure: Prepare three mobile phases A separately, and form a mobile phase system with acetonitrile. Equilibrate the chromatographic column for 30 min until the baseline is stable. Accurately measure 5 μL of the mixed reference solution and inject it into the chromatograph. Record the symmetry factor (T), peak area (relative value), and baseline fluctuation of each target peak. Repeat the injection for each condition 3 times and take the average value.

[0106] (2) Experimental results and analysis The experimental results show that: A 0.1% formic acid aqueous solution was used as phase A: GHK symmetry factor 1.20, impurity A symmetry factor 1.29, impurity B symmetry factor 1.13; the baseline showed slight fluctuations; the relative peak area was 95 (based on the phosphoric acid system as 100).

[0107] A 0.1% phosphoric acid aqueous solution was used as phase A: GHK symmetry factor 1.14, impurity A symmetry factor 1.21, and impurity B symmetry factor 1.06; the baseline showed no fluctuations and good stability; the peak area relative value was 100, and the response value was the highest.

[0108] A 0.1% trifluoroacetic acid aqueous solution was used as phase A: GHK symmetry factor 1.10, impurity A symmetry factor 1.18, impurity B symmetry factor 1.05; the baseline showed significant fluctuations; the peak area relative value was 82, and the response value was low.

[0109] Conclusion: When 0.1% phosphoric acid aqueous solution is used as mobile phase A, the target peak symmetry factor is better, the baseline is stable and the response value is the highest. It can effectively balance peak shape and detection sensitivity, so it is determined to be the optimal mobile phase A.

[0110] Example 5 The purpose of this embodiment is to adjust the elution program to observe its impact on the separation results, as detailed below: (1) Experimental methods Materials preparation: Instruments, reagents and reference standards are the same as in Example 1; the test sample is a self-made sample of blue copper peptide GHK (batch number S-K008-F-250303), with a typical sample solution concentration of 0.5 mg / mL (containing unknown impurities introduced by the process and known impurities, and the solvent is methanol-water 80:20); the chromatographic column used is Waters Atlantis T3 (4.6*250mm, 5mm).

[0111] Fixed experimental conditions: mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was acetonitrile; flow rate was 0.8 mL / min, column temperature was 35℃, detection wavelength was 205 nm, and injection volume was 5 μL.

[0112] Variable settings: A total of 6 gradient elution programs were designed (including elution program I to elution program VI), as detailed below: Washing program I: 0~5min 95% A + 5% B, 5~10min 95% A linearly decreases to 0% A, 10~20min maintains 0% A, 20~20.1min 0% A linearly recovers to 95% A, 20.1~25min maintains 95% A.

[0113] Washing program II: 0~5 min 95% A + 5% B, 5~10 min 95% A linearly decreases to 50% A, 10~15 min maintains 50% A, 15~15.1 min 50% A linearly decreases to 0% A, 15.1~25 min maintains 0% A, 25~25.1 min 0% A linearly recovers to 95% A, 25.1~30 min maintains 95% A.

[0114] Washing program III: 0~5 min 95% A + 5% B, 5~10 min 95% A linearly decreases to 40% A, 10~15 min maintains 40% A, 15~15.1 min 40% A linearly decreases to 0% A, 15.1~25 min maintains 0% A, 25~25.1 min 0% A linearly recovers to 95% A, 25.1~30 min maintains 95% A.

[0115] Washing procedure IV: 0~5 min 95% A + 5% B, 5~10 min 95% A linearly decreases to 40% A, 10~20 min 40% A linearly decreases to 30% A, 20~20.1 min 30% A linearly decreases to 0% A, 20.1~30 min maintains 0% A, 30~30.1 min 0% A linearly recovers to 95% A, 30.1~35 min maintains 95% A.

[0116] Elution program V: 0~10min 95% A linearly decreases to 40% A (phase B 5% increases to 60%), 10~20min 40% A linearly decreases to 30% A (phase B 60% increases to 70%), 20~23min maintain 30% A + 70% B, 23~23.1min 30% A linearly decreases to 0% A (phase B 70% increases to 100%), 23.1~37min maintain 0% A (phase B 100%), 37~37.1min 0% A linearly increases to 95% A (phase B 100% decreases to 5%), 37.1~45min maintain 95% A + 5% B.

[0117] Elution program VI: 0-10 min 98% A linearly decreases to 40% A (phase B 2% increases to 60%), 10-20 min 40% A linearly decreases to 30% A (phase B 60% increases to 70%), 20-23 min maintains 30% A + 70% B, 23-23.1 min 30% A linearly decreases to 0% A (phase B 70% increases to 100%), 23.1-37 min maintains 0% A (phase B 100%), 37-37.1 min 0% A linearly increases to 98% A (phase B 100% decreases to 2%), 37.1-45 min maintains 98% A + 2% B.

[0118] Experimental procedure: Equilibrate the column for 30 minutes according to the gradient program of each version. Accurately inject 5 μL of typical sample solution into the chromatograph and record the retention time, resolution (with adjacent peaks), interference of unknown peaks, and total analysis time of the target peak (GHK, impurity A, impurity B).

[0119] (2) Experimental results and analysis The experimental results are as follows: Elution program I: Impurity B was not completely eluted, and no obvious peak shape was observed at the end of the chromatogram; at the same time, the resolution between impurity A and the adjacent unknown impurity was 1.1 (<1.5), which failed to meet the separation requirements. This program has severely insufficient elution capacity and poor separation effect.

[0120] Elution Procedure II: By extending the time with a high organic phase ratio, impurity B was completely eluted. However, the separation degree between impurity A and the adjacent unknown impurity was only 1.2 (<1.5), indicating significant interference and affecting the accuracy of quantification.

[0121] Elution Procedure III: After further optimization of the gradient curve, impurity B was completely eluted. However, the separation degree between impurity A and the adjacent unknown impurity only improved to 1.3 (<1.5), and the separation effect was still unsatisfactory, indicating that the gradient change rate still did not match the requirement for this critical separation degree.

[0122] Elution Procedure IV: The gradient slope of the elution stage for impurity A was adjusted. Results showed that impurity B was completely eluted, and the resolution between impurity A and the adjacent unknown impurity reached 1.5. While this resolution just meets the basic requirements, it is at the critical point of the acceptable range. In actual quality control, even a slight decrease in column efficiency or minor fluctuations in mobile phase pH or temperature can cause the resolution to drop below 1.5, thus affecting the accuracy of quantification. This method lacks robustness and requires further optimization.

[0123] Elution Procedure V: Based on the previous procedure, the initial organic phase ratio and duration were adjusted, and the gradient steps were optimized. Although GHK, impurity A, and impurity B were all eluted, and the total analysis time was controllable, a key drawback emerged: the separation degree between GHK and the adjacent unknown impurity was 1.5, raising doubts about the reliability of the method in long-term use.

[0124] Elution Procedure VI (Final Procedure of this Invention): Addressing the shortcomings of Procedure V, the initial organic phase proportion was reduced to 2% (A phase 98%). The results were satisfactory: GHK retention time 3.6 min, impurity A retention time 14.2 min, and impurity B retention time 32.6 min; the resolutions between GHK, impurity A, and impurity B and their respective adjacent peaks were all significantly greater than 1.5; all known and unknown impurity peaks did not interfere with the detection of the peaks of interest; the total analysis time was 45 min, perfectly suited to the rapid detection requirements of the production line. This procedure achieved the best balance between separation effect, peak shape, baseline stability, and analytical efficiency.

[0125] The experimental results above show that the final gradient elution program determined in this invention can achieve complete separation and rapid elution of the target analyte, eliminate interference from unknown impurities, and is determined to be the optimal gradient program.

[0126] Example 6 The purpose of this embodiment is to optimize the detection wavelength, and to select wavelengths with high sensitivity and strong resistance to solvent interference in the range of 200~220nm, so as to ensure the stability of the target peak response.

[0127] (1) Experimental methods Materials preparation: Instruments, reagents, reference standards and test samples are the same as in Example 1; the concentration of typical sample solution is 0.5 mg / mL; the chromatographic column used is Waters Atlantis T3 (4.6*250 mm, 5 mm).

[0128] Fixed experimental conditions: mobile phase A was 0.1% phosphoric acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was the final version of Example 5; the flow rate was 0.8 mL / min, the column temperature was 35 °C, and the injection volume was 5 μL.

[0129] Variable settings: Detection wavelengths are 200nm, 205nm, 210nm, 215nm, and 220nm (intervals of 5nm).

[0130] Experimental procedure: Equilibrate the chromatographic column under fixed conditions for 30 min, inject typical sample solutions at each wavelength, record the signal-to-noise ratio (S / N) and peak area RSD (n=3) of GHK, impurity A, and impurity B, and observe the interference of solvent peaks.

[0131] (2) Experimental results and analysis The experimental results are as follows: 200nm: GHK signal-to-noise ratio 6,567,468, impurity A signal-to-noise ratio 23,595,097, impurity B signal-to-noise ratio 42,831,192; peak area RSDs are 1.2%, 1.1%, and 1.3%, respectively; solvent peak interference is slight.

[0132] 205nm: GHK signal-to-noise ratio 7217006, impurity A signal-to-noise ratio 25787723, impurity B signal-to-noise ratio 46304629; peak area RSDs are 0.8%, 1.1%, and 1.4%, respectively; no solvent peak interference, highest response.

[0133] 210nm: GHK signal-to-noise ratio 6,278,787, impurity A signal-to-noise ratio 22,176,818, impurity B signal-to-noise ratio 39,358,396; peak area RSDs are 0.9%, 1.2%, and 1.3%, respectively; no significant interference.

[0134] 215nm: GHK signal-to-noise ratio 3969353, impurity A signal-to-noise ratio 14827520, impurity B signal-to-noise ratio 28476959; peak area RSDs are 0.9%, 1.1%, and 1.2%, respectively; response value decreases by approximately 45% compared to 205nm.

[0135] 220nm: GHK signal-to-noise ratio 2958974, impurity A signal-to-noise ratio 10314797, impurity B signal-to-noise ratio 20142235; peak area RSDs are 1.0%, 1.0%, and 1.1%, respectively; response value decreases significantly.

[0136] Conclusion: The target peak signal-to-noise ratio is high at 205 nm, the peak area RSD is <2.0%, and there is no solvent interference, making it the optimal detection wavelength.

[0137] Example 7 The purpose of this embodiment is to confirm a suitable flow rate, verify the effect of flow rate on resolution and retention time within the range of 0.6~1.0 mL / min, and ensure the stability of the method, as detailed below: (1) Experimental methods Materials preparation: Instruments, reagents, reference standards and test samples are the same as in Example 1; the concentration of typical sample solution is 0.5 mg / mL; the chromatographic column used is Waters Atlantis T3 (4.6*250 mm, 5 mm).

[0138] Fixed experimental conditions: mobile phase A was 0.1% phosphoric acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was the final version of Example 5; the detection wavelength was 205 nm, the column temperature was 35 °C, and the injection volume was 5 μL.

[0139] Variable settings: flow rates of 0.6 mL / min, 0.8 mL / min, and 1.0 mL / min.

[0140] Experimental procedure: Equilibrate the column at each flow rate for 30 min, inject a typical sample solution, and record the retention time, resolution, and peak area RSD (n=3) of the target peak.

[0141] (2) Experimental results and analysis The experimental results are as follows: 0.6 mL / min: GHK retention time 4.5 min, impurity A retention time 18.3 min, impurity B retention time 42.7 min; resolution ≥1.5; peak area RSD 0.8%, 1.3%, 1.5%, respectively.

[0142] 0.8 mL / min: GHK retention time 3.6 min, impurity A retention time 14.1 min, impurity B retention time 32.5 min; resolution ≥1.5; peak area RSD 0.9%, 1.1%, 1.2%, respectively.

[0143] 1.0 mL / min: GHK retention time 2.8 min, impurity A retention time 10.6 min, impurity B retention time 27.9 min; resolution ≥1.5; peak area RSD 1.0%, 0.9%, 1.4%, respectively.

[0144] Conclusion: The separation accuracy met the standard at all three flow rates, and the peak area reproducibility was good. It was determined that the effect was comparable within the range of 0.6~1.0 mL / min.

[0145] Example 8 The purpose of this embodiment is to confirm a suitable column temperature, verify the effect of column temperature on peak shape and retention time within the range of 30~40℃, and ensure the applicability of the method.

[0146] (1) Experimental methods Materials preparation: Instruments, reagents, reference standards and test samples are the same as in Example 1; the concentration of typical sample solution is 0.5 mg / mL; the chromatographic column used is Waters Atlantis T3 (4.6*250 mm, 5 mm).

[0147] Fixed experimental conditions: mobile phase A was 0.1% phosphoric acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was the final version of Example 5; the detection wavelength was 205 nm, the flow rate was 0.8 mL / min, and the injection volume was 5 μL.

[0148] Variable settings: column temperatures are 30℃, 35℃, and 40℃.

[0149] Experimental procedure: Equilibrate each column temperature for 30 minutes, inject a typical sample solution, and record the target peak symmetry factor, retention time, and peak area RSD (n=3).

[0150] (2) Experimental results and analysis The experimental results are as follows: 30℃: GHK symmetry factor 1.15, impurity A symmetry factor 1.19, impurity B symmetry factor 1.07; peak area RSDs were 1.1%, 1.4%, and 1.2%, respectively; the separation between each peak of interest and its adjacent peaks was greater than 1.5; the retention time was extended by about 5 min compared to 35℃.

[0151] 35℃: GHK symmetry factor 1.10, impurity A symmetry factor 1.15, impurity B symmetry factor 1.01; peak area RSDs are 0.9%, 1.2%, and 1.3%, respectively; the separation degree between each peak of interest and its adjacent peak is greater than 1.5.

[0152] At 40℃: GHK symmetry factor 1.24, impurity A symmetry factor 1.21, impurity B symmetry factor 0.96; peak area RSDs were 1.0%, 1.2%, and 0.9%, respectively; the separation of impurity A from its adjacent peak was less than 1.5; the retention time was shortened by about 5 min compared to 35℃.

[0153] Conclusion: The peak shape and reproducibility were good at all three column temperatures. At 35℃, the separation between each peak of interest and its adjacent peak was greater than 1.5, and the retention time was moderate. Therefore, this was determined to be the optimal column temperature.

[0154] GHK has a symmetry factor of 1.14, impurity A has a symmetry factor of 1.21, and impurity B has a symmetry factor of 1.06. Example 9 The purpose of this embodiment is to verify the compatibility of the YMC-Pack ODS-AQ column with the optimal method and to ensure the transfer stability of the method between columns of different brands.

[0155] (1) Experimental methods Materials preparation: Instruments, reagents, reference standards and test samples are the same as in Example 1; the concentration of typical sample solution is 0.5 mg / mL; the chromatographic column used is YMC-Pack ODS-AQ (4.6*250mm, 5mm, serial number DK-RD-LC-25-03).

[0156] Fixed experimental conditions: mobile phase A was 0.1% phosphoric acid aqueous solution, and mobile phase B was acetonitrile; the gradient elution program was the final version of Example 5; the detection wavelength was 205 nm, the flow rate was 0.8 mL / min, the column temperature was 35 °C, and the injection volume was 5 μL.

[0157] Experimental procedure: Equilibrate the column for 30 min, inject a typical sample solution, record the retention time, resolution and peak area of ​​the target peak, and compare with the results of Waters Atlantis T3 in Example 8.

[0158] (2) Experimental results and analysis YMC-Pack ODS-AQ column: GHK retention time 3.8 min, impurity A retention time 14.7 min, impurity B retention time 33.2 min; the resolution of each peak of GHK, impurity A and impurity B and their adjacent peaks is greater than 1.5; the relative deviation of peak area from Waters Atlantis T3 is <2.0%; the detection capability of unknown impurities is comparable.

[0159] The experimental results show that the YMC-Pack ODS-AQ column is well compatible with the optimal method and can be used as an alternative column to ensure the stability of method transfer.

[0160] Comparative Example 1 The purpose of this comparative example is to compare the detection method of the existing patent CN202411242861 and verify whether it can simultaneously detect GHK and impurities A and B.

[0161] (1) Experimental methods Materials preparation: Same as in Example 1; the concentration of the control solution mixture was 0.5 mg / mL.

[0162] Reference chromatographic conditions: Waters HSS T3 column (4.6 mm × 250 mm, 5 μm); mobile phase: 40 mM potassium dihydrogen phosphate solution (pH 8.5, adjusted with 10 mM potassium hydroxide), isogradient elution; column temperature: 35 ℃; flow rate: 1.0 mL / min; injection volume: 10 μL; detection wavelength: 220 nm; run time: 30 min.

[0163] Experimental procedure: Equilibrate the chromatographic column according to the reference conditions, inject the mixed reference solution, and record the peaks.

[0164] (2) Experimental results and analysis The experimental results are as follows: only GHK eluted at 8.5 min, while impurities A and B did not elute (no elution).

[0165] The experimental results show that this method cannot detect impurities A and B simultaneously, which does not meet the requirements of process quality control.

[0166] Comparative Example 2: Comparison with CN202211426148 Method The purpose of this comparative example is to compare the detection method of the existing patent CN202211426148 and verify its separation effect, specifically: (1) Experimental methods Materials preparation: Same as in Example 1; the concentration of the control solution mixture was 0.5 mg / mL.

[0167] Reference chromatographic conditions: Column: C18 column (250mm × 4.6mm × 5μm); Mobile phase: A - water, B - methanol, C - 0.1% trifluoroacetic acid aqueous solution; Gradient elution program: 0~10min 5% B + 95% C, 10~15min A 0% to 90%, B 5% to 10%, C 95% to 0%, 15~20min A 90% to 95%, B 10% to 5%, C 0%, 20~25min A 95%, B 5%, C 0%, 25~30min A 95% to 0%, B 5%, C 0% to 95%, 30~35min A 0%, B 5%, C 95%; Column temperature: 25℃, flow rate: 1.0mL / min, injection volume: 5μL, detection wavelength: 220nm.

[0168] Experimental procedure: Equilibrate the chromatographic column according to the reference conditions, inject the mixed reference solution, and record the resolution and peak area.

[0169] (2) Experimental results and analysis The experimental results are as follows: Impurities A and B did not elute into sharpness under this method.

[0170] The experimental results show that this method is not suitable for the simultaneous detection of GHK, impurity A, and impurity B.

[0171] Comparative Example 3: Comparison with CN202211071245 Method The purpose of this comparative example is to compare the detection method of the existing patent CN202211071245 and verify its anti-interference ability and peak shape.

[0172] (1) Experimental methods Materials preparation: Same as in Example 1; typical sample solution concentration is 0.5 mg / mL (containing unknown impurities).

[0173] Reference chromatographic conditions: Waters XBridge BEH Amide column (4.6×75mm, 2.5μm); mobile phase: acetonitrile (0.1% trifluoroacetic acid): water (0.1% trifluoroacetic acid) = 65:35 (isogradient); column temperature 40℃, flow rate 0.4mL / min, injection volume 10μL, detection wavelength 215nm.

[0174] Experimental procedure: Equilibrate the column according to the reference conditions, inject a typical sample solution, and record the resolution and interference.

[0175] (2) Experimental results and analysis The experimental results are as follows: after 40 min, impurity B still did not elute a peak (elution was slow); the unknown impurity peak overlapped with impurity A, and the resolution was less than 1.0; the unknown impurity peak interfered with GHK, and the resolution was also less than 1.0.

[0176] The experimental results show that this method is affected by unknown impurities and has low quantitative accuracy.

[0177] The above are the embodiments listed in this example. However, this example is not limited to the optional embodiments described above. Those skilled in the art can arbitrarily combine the above methods to obtain other various embodiments. Anyone can derive other various forms of embodiments based on the inspiration of this example. The above specific embodiments should not be construed as limiting the scope of protection of this example. The scope of protection of this example should be determined by the claims, and the specification can be used to interpret the claims.

[0178] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A high-performance liquid chromatography (HPLC) method for the detection of key intermediates of blue copper peptide and related impurities, characterized in that: After preparing the sample solution, the sample was detected by high-performance liquid chromatography (HPLC). The HPLC conditions were as follows: Chromatographic column: Reversed-phase C18 column; Mobile phase: Phase A is 0.1% aqueous phosphoric acid solution; Phase B is acetonitrile; A gradient elution method is used for elution. The gradient elution procedure is as follows: 0~10min, 98%A→40%A, 2%B→60%B; 10~20min, 40%A→30%A, 60%B→70%B; 20~23min, 30%A, 70%B; 23~23.1min, 30%A→0%A, 70%B→100%B; 23.1~37min, 0%A, 100%B; 37~37.1min, 0%A→98%A, 100%B→2%B; 37.1~45min, 98%A, 2%B.

2. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The flow rate of the mobile phase is 0.6~1.0 mL / min.

3. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The column temperature is 30~40℃.

4. The high-performance liquid chromatography method for detecting key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The sample concentration ranged from 0.4 mg / mL to 1.0 mg / mL.

5. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The sample solvent, by volume ratio, is methanol:water = 75:25~85:

15.

6. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The injection volume is 4 mL to 10 mL.

7. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The detector and wavelength are UV 200~220nm.

8. The high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: It also includes standard curves for preparing blue copper peptide GHK and its related impurities.

9. The high-performance liquid chromatography method for detecting key intermediates of blue copper peptide and related impurities according to claim 1, characterized in that: The relevant impurities include at least impurity A and / or impurity B.

10. The application of the high-performance liquid chromatography method for the detection of key intermediates of blue copper peptide and related impurities according to any one of claims 1-9, characterized in that: This is used to detect key intermediates of blue copper peptide and their related impurities, wherein the related impurities include at least impurity A and / or impurity B.

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