Method for analyzing related substances of semeglutide intermediate 29 peptide

Abstract

The invention belongs to the technical field of medicine analysis, and particularly discloses an analysis method of a semeglutide intermediate 29 peptide related substance, which comprises the following steps: by taking an octadecyl silane bonded silica gel filler chromatographic column as a stationary phase, taking a sodium perchlorate buffer solution containing acetonitrile as a mobile phase A and taking an aqueous solution of acetonitrile and methanol as a mobile phase B, carrying out gradient elution on the semeglutide intermediate 29 peptide related substance, and carrying out gradient elution on the semeglutide intermediate 29 peptide related substance, thereby obtaining the semeglutide intermediate 29 peptide related substance. Gradient elution is adopted, the system applicability, precision, limit of detection, limit of quantitation, specificity, linearity, range, accuracy, solution stability and durability of the method meet the requirements, and the reproducibility and reliability of a detection result are ensured by adopting a reference substance and a test sample with specific concentrations, a diluent with specific components and a specific pH value and a mobile phase A with a specific pH value. According to the method, the content of the semeglutide intermediate 29 peptide related substance in the test solution can be efficiently detected, the detection speed is increased, a detection result can be obtained in a relatively short time, the detection limit reaches 0.2326 mu g / mL, and the quantification limit reaches 0.4651 mu g / mL.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis and detection, and more specifically to an analysis method for semaglutide intermediate 29 peptide-related substances. Background Art

[0002] Semaglutide is a glucagon-like peptide-1 (GLP-1) receptor agonist that promotes insulin secretion and inhibits glucagon secretion in a glucose-dependent manner by mediating the GLP-1 receptor, thereby achieving the purpose of lowering blood sugar. It is commonly used to treat adult type 2 diabetes. Semaglutide can be used for diabetic patients who do not meet blood sugar standards while receiving metformin and / or sulfonylurea drugs on the basis of diet control and exercise. It is suitable for reducing the risk of major adverse cardiovascular events (cardiovascular death, non-fatal myocardial infarction or non-fatal stroke) in adult patients with type 2 diabetes and cardiovascular disease.

[0003] The long-acting mechanism of semaglutide is based on structural modification. The lysine position at the 26th position of the peptide chain is connected to an 18-carbon fatty diacid side chain. The modified carbon chain increases the molecular weight of semaglutide and enhances its affinity for albumin by 5-6 times, avoiding rapid clearance by the kidneys and metabolic degradation, thereby prolonging its half-life in vivo. Semaglutide intermediate 29 peptide [CAS number: 1169630-82-3], also known as Semaglutide 29 peptide backbone, Semaglutide Intermediate P29, GLP-1 (9-37), is a key intermediate in the synthesis of semaglutide API. At present, semaglutide intermediate 29 peptide is mainly prepared by fermentation. The obtained semaglutide intermediate 29 peptide will produce a series of impurities, which will increase the difficulty of purification of semaglutide finished products and affect the quality of the finished products.

[0004] At present, high performance liquid chromatography is often used to detect the content of related substances in the test solution. Patent application CN117805269A discloses a method for detecting related substances of semaglutide injection, which dilutes semaglutide injection with a phosphate solution to obtain a test solution; an octadecylsilane bonded silica gel column is used as the stationary phase, phosphate buffer-acetonitrile is used as the mobile phase A, and acetonitrile-isopropanol aqueous solution is used as the mobile phase B, and high performance liquid chromatography is used to detect the content of related substances of semaglutide injection in the test solution. However, this method has a slow detection speed, and the hydrophobicity difference between semaglutide and semaglutide intermediate 29 peptide is large, which is not suitable for analyzing related substances in semaglutide intermediate 29 peptide. At present, the analysis method of related substances of semaglutide intermediate 29 peptide has not been disclosed.

[0005] CN115326956A discloses a separation and detection method for homologous impurities in a semaglutide modifier, and relates to the technical field of pharmaceutical intermediate analysis. The test solution is detected by a high performance liquid chromatograph, and mobile phase A and mobile phase B are mixed in different proportions as mobile phases for gradient elution, and the impurity content is calculated by area normalization method; wherein, mobile phase A is obtained by mixing phosphate buffer and acetonitrile, and mobile phase B is obtained by mixing phosphate buffer, acetonitrile and water. By optimizing the chromatographic conditions and elution conditions, the homologous impurities in the semaglutide modifier can be effectively separated in a relatively short time, with good specificity and precision, which is beneficial to control the quality of the final product. However, in practical applications, this method still needs to further optimize the chromatographic conditions and elution conditions to improve the separation effect and detection sensitivity of homologous impurities.

[0006] Therefore, it is necessary to develop an analytical method for detecting related substances of semaglutide intermediate 29 peptide, which is simple, low-cost, good in specificity and precision, high in sensitivity and good in durability. Summary of the invention

[0007] The purpose of the present invention is to provide an analysis method for related substances of semaglutide intermediate 29 peptides. The HPLC detection method provided by the present invention has low detection limit and quantitative limit, good specificity and precision, high sensitivity, and good separation effect for impurities, which solves the problem of lack of analysis method for related substances of semaglutide intermediate 29 peptides in the prior art.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for analyzing related substances of semaglutide intermediate 29 peptide, comprising the following steps:

[0010] Step 1, taking an appropriate amount of semaglutide intermediate 29 peptide reference substance, diluting it with diluent to obtain a reference substance solution;

[0011] Step 2: Take an appropriate amount of semaglutide intermediate 29 peptide test sample, add the impurity mixed solution, and dilute with diluent to obtain a labeled test sample solution;

[0012] Step 3, using high performance liquid chromatography to detect the content of semaglutide intermediate 29 peptide-related substances in the labeled test solution;

[0013] The related substances are as follows:

[0014]

[0015]

[0016] As an embodiment of the present invention, the concentration of the reference substance is 0.5 mg / mL; the concentration of the test substance is 0.5 mg / mL.

[0017] As an embodiment of the present invention, the diluent is an aqueous solution containing tris(hydroxymethyl)aminomethane and acetonitrile; the pH value of the diluent is 8.0.

[0018] As an embodiment of the present invention, the concentration of tris(hydroxymethyl)aminomethane is 100 mmol / L; the concentration of acetonitrile is 10%.

[0019] As an embodiment of the present invention, in step three: in the high performance liquid chromatography method, mobile phase A is a sodium perchlorate buffer containing acetonitrile, and the pH value is 1.7±0.2, preferably 1.7; mobile phase B is an aqueous solution of acetonitrile and methanol.

[0020] As an embodiment of the present invention, the volume ratio of the sodium perchlorate buffer to acetonitrile is 9:1.

[0021] As an embodiment of the present invention, the concentration of the sodium perchlorate buffer is 50-100mmol / L, such as 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L; further preferably, it is 70-90mmol / L, preferably 80mmol / L.

[0022] As an embodiment of the present invention, the volume ratio of acetonitrile:methanol:water in the mobile phase B is 3:1:1.

[0023] As an embodiment of the present invention, in step three: the chromatographic column in the high performance liquid chromatography method is one of Agilent Poroshell 120EC-C18, Agilent Poroshell 120SB-C18, and Kinetex XB-C18, preferably Agilent Poroshell 120EC-C18.

[0024] As an embodiment of the present invention, in step 3: the high performance liquid chromatography method adopts a gradient program, and the gradient program is as follows:

[0025] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 70 30 5 59 41 25 50 50 30 10 90 30.5 70 30 35 70 30

[0026] Compared with the prior art, the method for analyzing the related substances of the semaglutide intermediate 29 peptide according to the present invention has the following beneficial effects:

[0027] This application uses an octadecylsilane bonded silica gel filler chromatographic column as the stationary phase, mobile phase A is a sodium perchlorate buffer containing acetonitrile, and mobile phase B is an aqueous solution of acetonitrile and methanol, and gradient elution is adopted. The system applicability, precision, detection limit, quantification limit, specificity, linearity, range, accuracy, solution stability and durability of this method meet the requirements. By using a specific concentration of reference substance and test sample, a specific component, a diluent of a specific pH value and a mobile phase A of a specific pH value, the reproducibility and reliability of the test results are ensured. The present invention can efficiently detect the content of 29 peptide-related substances of the intermediate of semaglutide in the test solution, improve the detection speed, and obtain the test results in a relatively short time, with a detection limit of 0.2326μg / mL and a quantification limit of 0.4651μg / mL. This application can accurately detect impurities in the main chain 29 peptides of semaglutide, provide direction and data for subsequent research on impurities in semaglutide finished products, reduce the difficulty of purification of semaglutide finished products, and help to ensure the safety of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the chromatogram obtained under the conditions of Example 1 of the present invention;

[0029] Figure 2 This is the chromatogram obtained in the case of screening of pH value in mobile phase A;

[0030] Figure 3 This is the chromatogram obtained in the second case of screening of pH value in mobile phase A. DETAILED DESCRIPTION

[0031] The specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, but it should be understood that the protection scope of the present invention is not limited by the embodiments.

[0032] The semaglutide intermediate 29 peptide reference substance and test substance in the examples of the present invention are from Nanjing Hanxin Pharmaceutical Technology Co., Ltd., and the related substances (impurities) are from Shanghai Newpu Biotechnology Co., Ltd. Unless otherwise specified, other experimental materials and instrument specifications used in the examples of the present invention can be purchased commercially.

[0033] Example 1

[0034] Experimental conditions:

[0035] Equipment: High performance liquid chromatography (HPLC).

[0036] Preparation of diluent: weigh 12.1 g of tris(hydroxymethyl)aminomethane, add 100 mL of acetonitrile and appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust pH to 8.0 with hydrochloric acid, mix well, and the blank solution is prepared in the same way as the diluent.

[0037] Preparation of mobile phase A: Weigh 9.8 g of sodium perchlorate, add 100 mL of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH to 1.7 with phosphoric acid, mix well, and you have it.

[0038] Preparation of mobile phase B: Take 600 mL of acetonitrile, 200 mL of methanol and 200 mL of water, mix well.

[0039] Preparation of semaglutide intermediate 29 peptide reference solution: weigh 25 mg of semaglutide intermediate 29 peptide reference into a 50 mL volumetric flask, dissolve and dilute to scale with diluent, mix well, and obtain. Prepare two portions in parallel, respectively as reference solution and inspection reference solution.

[0040] Preparation of semaglutide intermediate 29 peptide test solution: weigh 25 mg of semaglutide intermediate 29 peptide test solution into a 50 mL volumetric flask, dissolve and dilute to the scale with diluent, mix well, and obtain.

[0041] Preparation of impurity mixed solution: take appropriate amount of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, impurity 11 and impurity 12, accurately weigh them, dissolve and dilute them with diluent to make a solution containing 6.25μg of impurity 1, 2.5μg of impurity 2, 2.5μg of impurity 3, 2.5μg of impurity 4, 5μg of impurity 5, 6.25μg of impurity 6, 1.25μg of impurity 7, 2.5μg of impurity 8, 2.5μg of impurity 9, 2.5μg of impurity 10, 35μg of impurity 11 and 1.25μg of impurity 12 per 1mL.

[0042] Preparation of semaglutide intermediate 29 peptide labeled test sample: weigh 25 mg of semaglutide intermediate 29 peptide test sample into a 50 mL volumetric flask, add 10 mL of impurity mixed solution, dissolve and dilute to the scale with diluent, mix well, and obtain.

[0043] Chromatographic conditions:

[0044] Mode: LC

[0045] Wavelength: UV 214nm

[0046] Column: Agilent Poroshell 120EC-C18, 2.7 μm, 3 mm × 150 mm

[0047] Flow rate: 0.6mL / min

[0048] Injection volume: 10 μL

[0049] Column temperature: 40°C

[0050] Autosampler temperature: 5°C

[0051] Gradient Program:

[0052] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 70 30 5 59 41 25 50 50 30 10 90 30.5 70 30 35 70 30

[0053] Take 10 μL of the labeled test solution and inject it into the HPLC instrument, and record the chromatogram. Figure 1 As described, impurity 1 + impurity 6 measured in the marked test solution was 0.46%, impurity 2 was 0.09%, impurity 3 was 0.10%, impurity 4 was 0.20%, impurity 5 was 0.19%, impurity 7 + impurity 12 was 0.09%, impurity 8 was 0.10%, impurity 9 was 0.08%, impurity 10 was 0.12%, and impurity 11 was 1.36%.

[0054] Screening of pH value in mobile phase A

[0055] Test conditions:

[0056] Preparation of mobile phase A: Weigh 9.8 g of sodium perchlorate, add 100 mL of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, and adjust the pH to 8.0 with 1 mol / L sodium hydroxide.

[0057] Other parameters are consistent with those in Example 1.

[0058] Chromatographic conditions: The parameters are consistent with those in Example 1.

[0059] Take 10 μL of the labeled test solution and inject it into the HPLC instrument, and record the chromatogram. Figure 2 As described, impurity 8 was measured in the test sample at 0.09%, impurity 10 + impurity 9 at 0.21%, impurity 5 at 0.16%, impurity 3 + impurity 4 + impurity 7 at 0.34%, impurity 6 at 0.22%, impurity 11 at 1.36%, among which impurities 1, 2 and impurity 12 were not measured, and the mobile phase is not applicable when pH 8.0 is selected.

[0060] Screening of pH value in mobile phase A

[0061] Test conditions:

[0062] Preparation of mobile phase A: Weigh 9.8 g of sodium perchlorate, add 100 mL of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH to 3.5 with phosphoric acid, mix well, and you have it.

[0063] Other parameters are consistent with those in Example 1.

[0064] Chromatographic conditions: The parameters are consistent with those in Example 1.

[0065] Take 10 μL of the labeled test solution and inject it into the HPLC instrument, and record the chromatogram. Figure 3 As described, impurity 8 was measured in the test sample at 0.11%, impurity 9 + impurity 10 at 0.18%, impurity 3 + impurity 4 + impurity 5 at 0.52%, impurity 6 at 0.22%, impurity 12 at 0.06%, and impurity 11 at 1.34%. Impurity 1, impurity 2, and impurity 7 were not detected, and the mobile phase was not applicable when pH 3.5 was selected.

[0066] System suitability test

[0067] System suitability on instrument 1 (HE-41-00044):

[0068] The reference solution with 5 repeated injections and the inspection reference solution with 2 repeated injections were analyzed, and the results are shown in Tables 1 and 2. The %RSD of the peak area of ​​the 29-peptide main chain of semaglutide injected with 5 injections was 0.1%, which was less than 2.0%. The average recovery rate of the peak area of ​​the 29-peptide main chain of semaglutide in the spectrum of the inspection reference solution relative to the peak area of ​​the reference solution (the average value of the first 5 injections) was 100.4%, which was between 98.0-102.0%.

[0069] Table 1: Instrument 1 Reference Solution Results

[0070] name Peak area of ​​29 peptides in the main chain of semaglutide Reference solution-1 9048.776 Reference solution-2 9061.315 Reference solution-3 9049.097 Reference solution-4 9057.299 Reference solution-5 9056.876 Average value (N=5) 9054.673 %RSD 0.1％ Acceptable Standards Not more than 2.0% Pass / Fail qualified

[0071] Note: This summary only counts the initial 5 injections of the reference solution; subsequent injections for staged system checks are not counted in the table above. All system suitability results in the validation have been reviewed and no abnormalities were found.

[0072] Table 2: Results of Instrument 1 Checking the Reference Solution

[0073]

[0074] *The average of the peak areas of the first 5 pairs of semaglutide backbone 29 peptides in the reference solution.

[0075] System suitability on instrument 2 (HE-41-00014):

[0076] The second analyst analyzed the reference solution with 5 repeated injections and the check reference solution with 2 repeated injections on different equipment, see Table 3 and Table 4. The RSD% of the peak area of ​​the 29-peptide main chain of semaglutide in the spectrum of the reference solution was 0.04%, which was less than 2.0%. The average recovery rate of the peak area of ​​the 29-peptide main chain of semaglutide in the spectrum of the check reference solution relative to the peak area of ​​the reference solution (average of the first 5 injections) was 99.2%, between 98.0-102.0%.

[0077] Table 3: Instrument 2 reference solution results

[0078] name Peak area of ​​29 peptides in the main chain of semaglutide Reference solution-1 9382.129 Reference solution-2 9384.406 Reference solution-3 9380.515 Reference solution-4 9380.714 Reference solution-5 9378.929 Average value (N=5) 9381.339 %RSD 0.04％ Acceptable Standards Not more than 2.0% Pass / Fail qualified

[0079] Note: This summary only counts the initial 5 injections of the reference solution; subsequent injections for staged system checks are not counted in the table above. All system suitability results in the validation have been reviewed and no abnormalities were found.

[0080] Table 4: Results of Instrument 2 Checking the Reference Solution

[0081]

[0082] *The average of the peak areas of the first 5 pairs of semaglutide backbone 29 peptides in the reference solution.

[0083] Conclusion: The peak area %RSD of the semaglutide backbone 29 peptide met the acceptance criteria of no more than 2.0% for both instrument 1 and instrument 2. Therefore, the system suitability results were in compliance with the regulations.

[0084] Precision test

[0085] The precision of the analytical method of this application was demonstrated by measuring repeatability and intermediate precision.

[0086] Repeatability:

[0087] Prepare 6 labeled test solutions in parallel, and the repeatability results after testing are shown in Table 5. The %RSD of related substances in the six labeled test solutions should not be greater than 15%. The RSD% of the maximum unknown single impurity, specific single impurity (impurity 1 to impurity 12) and total impurity content in the six labeled test solutions should not be greater than 15%.

[0088] Table 5: Repeatability results

[0089]

[0090]

[0091] Intermediate precision:

[0092] Six independently prepared labeled test solutions were measured by different experimenters using different instruments. The comparison of the test results of the two experimenters is shown in Table 6. The RSD% of the maximum unknown single impurity, specific single impurity (impurity 1 to impurity 12) and total impurity content in the six labeled test solutions should not be greater than 15%. The RSD% of the maximum unknown single impurity, specific single impurity (impurity 1 to impurity 12) and total impurity content in the twelve labeled test solutions should not be greater than 20%. (The results of the first experimenter are shown in Table 5)

[0093] Table 6: Intermediate precision between two experimenters

[0094]

[0095]

[0096] Table 6: Intermediate precision between two experimenters

[0097]

[0098]

[0099] Conclusion: The RSD% of the maximum unknown single impurity, specific single impurities (impurity 1 to impurity 12) and total impurity content in the test solution determined by experimenter 1 and experimenter 2 independently in 6 samples met the acceptance standard of no more than 15%. The %RSD of the maximum unknown single impurity, specific single impurity (impurity 1 to impurity 12) and total impurity content in the test solution determined by experimenter 1 and experimenter 2 independently in 12 samples met the acceptance standard of no more than 20%. Therefore, this analytical method is precise.

[0100] Specificity test

[0101] Prepare 6 test sample solutions in parallel, and refer to Table 7 for the specificity results after detection. The blank solution should not interfere with the determination of the 29-peptide peak of the semaglutide backbone. The 29-peptide peak of the semaglutide backbone should be pure.

[0102] Table 7: Peak purity of test solution

[0103]

[0104] Conclusion: The blank solution did not interfere with the determination of the 29-peptide peak of the semaglutide backbone. The 29-peptide peak of the semaglutide backbone of the test solution was pure and met the acceptance criteria for specificity. Therefore, the method is specific.

[0105] Detection limit

[0106] Six detection limit solutions (semaglutide main chain 29 peptide concentration 0.2326 μg / mL, equivalent to 0.05% impurity level) were prepared in parallel. The test results are shown in Table 8. The semaglutide main chain 29 peptides should all be detected in the six detection limit solutions (S / N≥3).

[0107] Table 8: Detection limit results of 29 peptides in the main chain of semaglutide

[0108] repeat Main chain 29 peptide area of ​​semaglutide S / N ratio of semaglutide backbone 29 peptide 1 3.123 25.8 2 3.381 30.3 3 2.918 30.0 4 2.860 20.2 5 3.156 27.4 6 3.694 37.9 Acceptable Standards N / A Not less than 3 Pass / Fail N / A qualified

[0109] Conclusion: The 29-peptide backbone peptide of semaglutide could be detected in the 6 parallel prepared detection limit solutions, which met the requirements (S / N≥3). The concentration of the 29-peptide backbone peptide of semaglutide in the detection limit solution of the HPLC analysis method for related substances of the 29-peptide backbone peptide of semaglutide was 0.2326 μg / mL, which was equivalent to an impurity level of 0.05%.

[0110] Limit of Quantitation

[0111] Six quantitative limit solutions (semaglutide main chain 29 peptide concentration 0.4651 μg / mL, equivalent to 0.1% impurity level) were prepared in parallel, and the test results are shown in Table 9. The signal-to-noise ratio of semaglutide main chain 29 peptide in the six quantitative limit solutions should be no less than 10. The %RSD of the recovery rate of the six parallel prepared quantitative limit solutions should be no more than 15%. The average recovery rate % of the quantitative limit solution should be between 70% and 130%.

[0112] Table 9: % recovery and %RSD of semaglutide backbone 29 peptides within the limit of quantitation

[0113]

[0114] Conclusion: The average recovery of 6 parallel prepared quantitative limit solutions meets the standard of 70%-130%, the %RSD of the average recovery meets the standard of no more than 15%, and the signal-to-noise ratio of 29 peptides in the main chain of semaglutide meets the standard of no less than 10.

[0115] The concentration of semaglutide backbone 29 peptide in the quantitative limit solution in the HPLC analysis method for semaglutide backbone 29 peptide related substances was 0.4651 μg / mL, equivalent to 0.1% impurity level.

[0116] Example 2

[0117] Test conditions:

[0118] Preparation of mobile phase A: Weigh 6.1 g of sodium perchlorate, add 100 mL of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH to 1.7 with phosphoric acid, mix well, and you have it.

[0119] Other parameters are consistent with those in Example 1.

[0120] Chromatographic conditions: The parameters are consistent with those in Example 1.

[0121] 10 μL of the labeled test solution was injected into the high performance liquid chromatograph, and the impurity 1 + impurity 6 in the test sample was measured to be 0.47%, impurity 2 was 0.09%, impurity 3 was 0.11%, impurity 4 was 0.19%, impurity 5 was 0.18%, impurity 7 + impurity 12 was 0.12%, impurity 8 was 0.10%, impurity 9 was 0.09%, impurity 10 was 0.10%, and impurity 11 was 1.37%.

[0122] Example 3

[0123] Test conditions:

[0124] Preparation of mobile phase A: Weigh 12.2 g of sodium perchlorate, add 100 L of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH to 1.7 with phosphoric acid, mix well, and you have it.

[0125] Other parameters are consistent with those in Example 1.

[0126] Chromatographic conditions: The parameters are consistent with those in Example 1.

[0127] 10 μL of the labeled test solution was injected into the high performance liquid chromatograph, and the impurity 1 + impurity 6 in the test sample was measured to be 0.46%, impurity 2 was 0.10%, impurity 3 was 0.09%, impurity 4 was 0.22%, impurity 5 was 0.19%, impurity 7 + impurity 12 was 0.11%, impurity 8 was 0.08%, impurity 9 was 0.12%, impurity 10 was 0.13%, and impurity 11 was 1.39%.

[0128] Example 4

[0129] Test conditions: The parameters are the same as those in Example 1.

[0130] Chromatographic conditions: The chromatographic column was replaced with Agilent Poroshell 120SB-C18, and other parameters were consistent with those in Example 1.

[0131] 10 μL of the labeled test solution was injected into the high performance liquid chromatograph, and the impurity 1 + impurity 6 in the test sample was measured to be 0.51%, impurity 2 was 0.11%, impurity 3 was 0.12%, impurity 4 was 0.23%, impurity 5 was 0.19%, impurity 7 + impurity 12 was 0.13%, impurity 8 was 0.12%, impurity 9 was 0.10%, impurity 10 was 0.11%, and impurity 11 was 1.43%.

[0132] Example 5

[0133] Test conditions: The parameters are the same as those in Example 1.

[0134] Chromatographic conditions: The chromatographic column was replaced with Kinetex XB-C18, and other parameters were consistent with those in Example 1.

[0135] Take 10 μL of the marked test solution and inject it into the high performance liquid chromatograph, record the chromatogram, and the impurity 1 + impurity 6 in the test sample is measured to be 0.48%, impurity 2 is 0.11%, impurity 3 is 0.08%, impurity 4 is 0.21%, impurity 5 is 0.17%, impurity 7 + impurity 12 is 0.07%, impurity 8 is 0.09%, impurity 9 is 0.09%, impurity 10 is 0.14%, and impurity 11 is 1.41%.

[0136] Comparative Example 1

[0137] Test conditions:

[0138] Preparation of mobile phase A: Weigh 18.3 g of sodium perchlorate, add 100 mL of acetonitrile and an appropriate amount of water to dissolve, dilute to 1000 mL with water, adjust the pH to 1.7 with phosphoric acid, mix well, and you have it.

[0139] Other parameters are consistent with those in Example 1.

[0140] Chromatographic conditions: The parameters are consistent with those in Example 1.

[0141] 10 μL of the labeled test solution was injected into the HPLC. The impurity 1 + impurity 6 in the test sample was 0.49%, impurity 2 was 0.10%, impurity 3 was 0.09%, impurity 4 was 0.18%, impurity 5 was 0.21%, impurity 8 was 0.11%, impurity 9 was 0.08%, impurity 10 was 0.09%, and impurity 11 was 1.33%. Impurities 7 and 12 were not detected under this condition, so this condition is not applicable.

Claims

1. A method for analyzing related substances of semaglutide intermediate 29 peptide, characterized in that: The following steps are involved: Step 1, taking an appropriate amount of semaglutide intermediate 29 peptide reference substance, diluting it with diluent to obtain a reference substance solution; Step 2: Take an appropriate amount of semaglutide intermediate 29 peptide test sample, add the impurity mixed solution, and dilute with diluent to obtain a labeled test sample solution; Step 3, using high performance liquid chromatography to detect the content of semaglutide intermediate 29 peptide-related substances in the labeled test solution; The related substances are as follows:

2. The analysis method according to claim 1, characterized in that: The concentration of the reference substance is 0.5 mg / mL; the concentration of the test substance is 0.5 mg / mL.

3. The analysis method according to claim 1, characterized in that: The diluent is an aqueous solution containing tris(hydroxymethyl)aminomethane and acetonitrile; the pH value of the diluent is 8.

0.

4. The analysis method according to claim 3, characterized in that: The concentration of the tris(hydroxymethyl)aminomethane is 100 mmol / L; the concentration of the acetonitrile is 10%.

5. The analysis method according to claim 1, characterized in that In the step three: in the high performance liquid chromatography method, mobile phase A is a sodium perchlorate buffer containing acetonitrile, with a pH value of 1.7±0.2, preferably 1.7; mobile phase B is an aqueous solution of acetonitrile and methanol.

6. The analysis method according to claim 5, characterized in that: The volume ratio of the sodium perchlorate buffer to acetonitrile is 9:

1.

7. The analysis method according to claim 6, characterized in that: The concentration of the sodium perchlorate buffer is 50-100 mmol / L; preferably, the concentration of the sodium perchlorate buffer is 80 mmol / L.

8. The analysis method according to claim 5, characterized in that: The volume ratio of acetonitrile:methanol:water in the mobile phase B is 3:1:

1.

9. The analysis method according to claim 1, characterized in that In the step 3: the chromatographic column in the high performance liquid chromatography method is one of Agilent Poroshell 120EC-C18, Agilent Poroshell 120SB-C18, and Kinetex XB-C18, preferably Agilent Poroshell 120EC-C18.

10. The analysis method according to claim 1, characterized in that In the step 3: the high performance liquid chromatography method adopts a gradient program, and the gradient program is as follows: 。

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