A qualitative and quantitative detection method for the amino acid composition of GLP-1 polypeptide drugs

Abstract

The application discloses a qualitative and quantitative detection method for GLP-1 polypeptide drug amino acid composition, and relates to the technical field of drug analysis chemistry, and the method comprises the following steps: a first derivative detection step, the contents of natural amino acids and linker AEEA in the GLP-1 polypeptide drug are detected simultaneously through high performance liquid chromatography; and a second derivative detection step, the content of unnatural amino acid Aib in the GLP-1 polypeptide drug is detected through high performance liquid chromatography. According to the different structural characteristics of natural amino acids, linker AEEA and unnatural amino acid Aib in tirzepatide, independent detection is carried out by using a phenyl isothiocyanate derivative system and a combined chloroformic acid fluorenyl methyl ester derivative system of o-phthaldehyde, so that the problem that Aib cannot be accurately determined with amino acids and AEEA in the same system due to the difference between the reactivity and stability of Aib derivatization is effectively solved.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analytical chemistry, specifically to a qualitative and quantitative method for detecting the amino acid composition of GLP-1 polypeptide drugs. Background Technology

[0002] Thilborpeptide is a GLP-1 / GIP dual receptor agonist composed of 39 amino acid residues. Its sequence contains two non-natural amino acids, Aib, and a hydrophilic linker, AEEA (2-[2-(2-aminoethoxy)ethoxy]acetic acid), is introduced through side chain modification. The proportions of natural amino acids, the accurate content of Aib, and the residual level of AEEA collectively determine the structural correctness, process purity, and safety of thiolborpeptide. Therefore, establishing reliable analytical methods for accurate quantification of these multiple components is crucial for the quality control of thiolborpeptide.

[0003] Currently, the analysis of amino acid composition in peptide drugs commonly employs pre-column derivatization-high performance liquid chromatography (HPLC). In existing technologies, natural amino acids and AEEA can be determined by HPLC using the same pre-column derivatization conditions, enabling the quantitative analysis of multiple targets in a single derivatization and injection. However, due to its unique structure, with two methyl groups substituting at the α-position, and steric hindrance, Aib exhibits significantly different derivatization reactivity and stability compared to other common amino acids and AEEA. In particular, when Aib is placed in the same derivatization system with natural amino acids and AEEA, the derivatization efficiency of Aib is low, and the derivative stability is poor, making it difficult to meet the requirements for accurate quantification in terms of detection sensitivity and reproducibility. Therefore, there is currently no mature method in the technology to simultaneously determine natural amino acids, AEEA, and Aib in a single derivatization system.

[0004] For the detection of Aib, existing methods usually employ specialized derivatization conditions. However, there is no systematic integration of derivatization systems applicable to natural amino acids and AEEA with derivatization systems specifically for Aib, and no optimization of their derivatization and chromatographic conditions to achieve a comprehensive, accurate, and highly sensitive quantification of all amino acid compositions in telpolide. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a qualitative and quantitative detection method for the amino acid composition of GLP-1 peptide drugs, so as to solve the technical problem in the prior art that the activity and stability of Aib derivatization reaction are significantly different from other amino acids and AEEA, which makes it impossible to accurately, sensitively and reproducibly determine the amino acid composition of telpoide simultaneously in the same system.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a qualitative and quantitative detection method for the amino acid composition of a GLP-1 peptide drug, the method comprising: a first derivatization detection step, wherein phenyl isothiocyanate (PITC) is used as a pre-column derivatizing agent, and the contents of natural amino acids and linker AEEA in the GLP-1 peptide drug are simultaneously detected by high performance liquid chromatography; and a second derivatization detection step, wherein o-phthalaldehyde (OPA) and fluorene methyl chloroformate (FMOC) are used as pre-column derivatizing agents, and the contents of non-natural amino acid Aib in the GLP-1 peptide drug are detected by high performance liquid chromatography; wherein the GLP-1 peptide drug is telpolide.

[0007] The present invention is further configured such that the first derivatization detection step includes: S1: Hydrolysis of the test sample: hydrolyzing the telpoeptide sample with hydrochloric acid solution to obtain a hydrolysate, and then drying it; S2: Derivatization treatment: redissolving the dried hydrolysate with a solvent, adding triethylamine solution and phenyl isothiocyanate solution sequentially to carry out a derivatization reaction, extracting with n-hexane after the reaction, and taking the lower layer solution to obtain the first test solution; S3: High performance liquid chromatography detection: performing high performance liquid chromatography analysis on the first test solution, using an ultraviolet detector at a wavelength of 254 nm, and calculating the content of natural amino acids and AEEA using the external standard method.

[0008] The present invention is further configured such that, in step S1, the hydrolysis conditions are: the hydrolysate is a 6M hydrochloric acid solution, the hydrolysis temperature is 110℃, the hydrolysis time is 24 hours, and the hydrolysate is freeze-dried; in step S2, the derivatization reaction conditions are: the triethylamine concentration is 1 mol / L, the phenyl isothiocyanate concentration is 0.1 mol / L, and the reaction is allowed to stand at room temperature for 1 hour.

[0009] The present invention further specifies that, in step S3, the high-performance liquid chromatography (HPLC) conditions are as follows: the chromatographic column is an octadecylsilane-bonded silica column, and the column temperature is 25°C; mobile phase A is a 50 mmol / L sodium acetate aqueous solution with a pH of 7.5 ± 0.05; mobile phase B is a mixed solution of methanol, acetonitrile, and water in a volume ratio of 200:600:200; gradient elution is employed, and the elution program is as follows: from 0 to 39 minutes, mobile phase A linearly decreases from 95% to 52%, and mobile phase B linearly increases from 5% to 48%; from 39 to 40 minutes, mobile phase A decreases from 52% to 0%, and mobile phase B increases from 48% to 100%; from 40 to 45 minutes, mobile phase A is maintained at 0%, and mobile phase B at 100%; from 45 to 46 minutes, mobile phase A increases from 0% to 95%, and mobile phase B decreases from 100% to 5%; from 46 to 60 minutes, mobile phase A is maintained at 95%, and mobile phase B at 5%; the flow rate is 1... The injection rate was mL / min, and the injection volume was 10 μL.

[0010] The present invention is further configured such that the second derivatization detection step includes: S11: Hydrolysis of the test sample: hydrolyzing the telpoide sample with hydrochloric acid solution to obtain a hydrolysate, and then drying it; S21: Derivatization treatment: redissolving the dried hydrolysate with a solvent, and sequentially adding borate buffer solution, o-phthalaldehyde solution and fluorene methyl chloroformate solution to carry out a derivatization reaction to obtain a second test solution; S31: High performance liquid chromatography detection: performing high performance liquid chromatography analysis on the second test solution, using a UV detector at dual wavelengths of 338 nm and 262 nm, and calculating the content of Aib using the external standard method.

[0011] The present invention is further configured such that, in S11, the hydrolysis conditions are: the hydrolysate is a 6M hydrochloric acid solution, the hydrolysis temperature is 110℃, the hydrolysis time is 24 hours, and the hydrolysate is freeze-dried; in S21, the derivatization reaction conditions are: the borate buffer solution has a pH of 10.2, first add o-phthalaldehyde solution to derive primary amino acids, let stand for 30 seconds, then add fluorene methyl chloroformate solution to derive secondary amino acids, and let stand for 30 seconds.

[0012] The present invention further specifies that, in step S31, the high-performance liquid chromatography (HPLC) conditions are as follows: the chromatographic column is an octadecylsilane-bonded silica column, and the column temperature is 40°C; mobile phase A is 0.05 mol / L sodium acetate solution-triethylamine-tetrahydrofuran, with a volume ratio of 950:0.14:5, and the pH is adjusted to 6.8 with 2% acetic acid; mobile phase B is 0.16 mol / L sodium acetate solution, methanol, and acetonitrile, with a volume ratio of 200:400:400, wherein 0.16 mol / L sodium acetate solution... The pH of the mol / L sodium acetate solution was adjusted to 6.8 with 2% acetic acid. Gradient elution was used, with the following elution program: 0 to 27 minutes, mobile phase A decreased linearly from 92% to 90%, and mobile phase B increased linearly from 8% to 10%; 27 to 31 minutes, mobile phase A decreased from 90% to 0%, and mobile phase B increased from 10% to 100%; 31 to 38 minutes, mobile phase A was maintained at 0%, and mobile phase B at 100%; 38 to 41 minutes, mobile phase A increased from 0% to 92%, and mobile phase B decreased from 100% to 8%; 41 to 45 minutes, mobile phase A was maintained at 92%, and mobile phase B at 8%; the flow rate was 1 mL / min, and the injection volume was 20 μL.

[0013] The present invention is further configured to include a data analysis step: comparing the measured molar number of natural amino acids, AEEA, and Aib with the theoretical number of sequences, and calculating the error percentage to verify the structural correctness of telpoeptide.

[0014] The present invention is further configured such that, for the data obtained in the first derivatization detection step, the total number of measured moles of aspartic acid, glutamic acid, alanine, leucine, isoleucine and lysine is divided by the total number of their theoretical amino acids as the denominator to calculate the measured number of other amino acids and AEEA and the error percentage.

[0015] The present invention is further configured such that, for the data obtained in the second derivatization detection step, the measured number of Aib and the error percentage are calculated by dividing the sum of the measured molar numbers of aspartic acid, glutamic acid, alanine, leucine, and isoleucine by the sum of their theoretical amino acid numbers. In summary, the present invention has the following main beneficial effects: This invention addresses the different structural characteristics of natural amino acids, the linker AEEA, and the non-natural amino acid Aib in telpolide. It employs separate derivatization systems using phenyl isothiocyanate and phthalaldehyde combined with fluorene chloroformate, effectively solving the problem of inaccurate determination of Aib in the same system as amino acids and AEEA due to differences in the reactivity and stability of Aib derivatization. This achieves comprehensive, accurate, and highly sensitive quantitative analysis of the entire amino acid composition of telpolide. The method is simple and rapid, with mild derivatization conditions, good derivative stability, and minimal interference during detection. It exhibits good reproducibility and specificity, providing a systematic and reliable analytical tool for the structural confirmation and quality control of telpolide and its analogues. Furthermore, this method has wide applicability and can be extended to the amino acid composition analysis of other peptide drugs containing Aib or AEEA structural units. Attached Figure Description

[0016] Figure 1 This is a high-performance liquid chromatogram of the amino acid standard after derivatization in Example 1 of the present invention; Figure 2 This is a high-performance liquid chromatogram of the hydrolyzed sample of telpoide in Example 1 of the present invention after derivatization; Figure 3 This is a high-performance liquid chromatogram of the amino acid standard after derivatization in Example 2 of the present invention; Figure 4 This is a high-performance liquid chromatogram of the hydrolyzed sample of telpoeptide in Example 2 of the present invention. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0018] The embodiments of the present invention will now be described.

[0019] This invention provides a qualitative and quantitative method for the amino acid composition of GLP-1 peptide drugs. This method comprises two independent pre-column derivatization-high performance liquid chromatography (HPLC) detection systems: the first derivatization system uses phenyl isothiocyanate (PITC) as a pre-column derivatizing agent to simultaneously detect the content of natural amino acids and the linker AEEA in telpolide; the second derivatization system uses o-phthalaldehyde (OPA) and fluorene methyl chloroformate (FMOC) as pre-column derivatizing agents to specifically detect the content of the non-natural amino acid Aib in telpolide. The two detection systems are described in detail below through specific examples. Example 1:

[0020] This embodiment uses PITC as a pre-column derivatizing agent, combined with a UV detector for quantitative detection of 17 natural amino acids and AEEA. The working principle is as follows: PITC (phenyl isothiocyanate) reacts with the amino group of amino acids under alkaline conditions to generate phenylaminothiocarbamoyl amino acid derivatives (PTC-amino acids). These derivatives exhibit strong UV absorption at 254 nm. After separation by high-performance liquid chromatography, qualitative analysis is based on retention time, and quantitative analysis is based on peak area using the external standard method.

[0021] 1. Solution preparation First, prepare various solutions. Mobile phase A is a 50 mmol / L sodium acetate aqueous solution: Weigh 8.2 g of anhydrous sodium acetate, dissolve it in 1800 mL of water, adjust the pH to 7.5 ± 0.05 with glacial acetic acid, then add water to a final volume of 2000 mL, filter through a 0.22 μm filter membrane, and sonicate for 30 min. Mobile phase B is a mixed solution of methanol, acetonitrile, and water in a volume ratio of 200:600:200. Preparation of 6 MHCl solution: Measure 50 mL of concentrated hydrochloric acid into a 100 mL volumetric flask, add water to a final volume, and mix well. 0.1 mol / L PITC solution: Pipette 148.2 μL of acetonitrile into a 1.5 mL EP tube, add 1.8 μL of PITC, vortex, and use immediately. 1 mol / L Triethylamine Solution: Pipette 129 μL of acetonitrile into a 1.5 mL EP tube, then add 21 μL of triethylamine, vortex, and use immediately. AEEA Standard Solution: Prepare a 1 mmol / L AEEA standard solution. A mixed standard solution of 17 amino acids was used, including aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, and proline, with known concentrations of each amino acid.

[0022] 2. Blank Derivative Processing Take 25 μL of mobile phase A, add 12.5 μL of 1 mol / L triethylamine and vortex mix. Then add 12.5 μL of 0.1 mol / L PITC and vortex mix. Let stand at room temperature for 1 hour. Add 100 μL of n-hexane and mix vigorously for 1 min. Let stand for 10 min. Take 20 μL of the lower layer solution and add 180 μL of mobile phase A. Mix and filter through a 0.22 μm filter tube to obtain a blank derivatization solution for testing.

[0023] 3. Derivatization of Standard Products Take 25 μL of the amino acid mixed solution standard, add 12.5 μL of 1 mol / L triethylamine and vortex to mix. Then add 12.5 μL of 0.1 mol / L PITC and vortex to mix. Let stand at room temperature for 1 hour. Add 100 μL of n-hexane and mix vigorously for 1 min. Let stand for 10 min. Take 20 μL of the lower layer solution and add 180 μL of mobile phase A. Mix and filter through a 0.22 μm filter tube to obtain the amino acid standard derivatization solution. Similarly, take 25 μL of AEEA standard solution and perform derivatization treatment according to the same steps to obtain the AEEA standard derivatization solution.

[0024] 4. Hydrolysis of the test sample Add 1 mg of telpolide sample to a hydrolysis tube, add 6 M HCl solution, and purge the top of the hydrolysis tube with high-purity nitrogen for 1 min. Immediately tighten the cap and transfer the tube to an oil bath at 110°C for 24 hours for hydrolysis. After hydrolysis, cool the hydrolysis tube to room temperature and transfer the hydrolysate to a 10 mL volumetric flask. Rinse the hydrolysis tube several times with pure water, transferring all the rinsing solution sequentially to the volumetric flask. Then, add an appropriate amount of pure water to bring the volume to 10 mL. After mixing thoroughly, transfer 1 mL of the final volumetric hydrolysate to a 1.5 mL EP tube, freeze at -80°C until solid, and then freeze-dry overnight in a pre-cooled freeze dryer.

[0025] 5. Derivatization treatment of the test sample Take 25 μL of the completely dried sample after redissolving in mobile phase A and place it in a 1.5 mL EP tube. Add 12.5 μL of 1 mol / L triethylamine solution and vortex to mix. Then add 12.5 μL of 0.1 mol / L PITC solution and vortex to mix. Let it stand at room temperature for 1 hour. Add 100 μL of n-hexane and mix vigorously for 1 min. Let it stand for 10 min. Take 20 μL of the lower layer solution into a 1.5 mL EP tube and add 180 μL of mobile phase A solution. Mix and filter through a 0.22 μm filter tube to obtain the derivatized solution of the test sample, which is ready for testing.

[0026] 6. Liquid chromatography for the detection of chromatographic parameters This embodiment uses an Agilent high-performance liquid chromatograph for detection. The chromatographic column is a Diamonsil AAA 5μm column, 250×4.6mm. The column temperature is set to 35℃. Mobile phase A is 50 mmol / L sodium acetate aqueous solution, and mobile phase B is a mixture of methanol, acetonitrile, and water (200:600:200). The flow rate is 1 mL / min. The UV detection wavelength is 254 nm. The injection volume is 10 μL. Gradient elution is used, and the elution program is as follows:

[0027] 7. Data Processing Using OpenLab software, the molar number of amino acid residues in the sample was automatically calculated using the external standard method. Since telpoeptide has a theoretical sequence, the measured amino acid composition data was compared with the theoretical sequence for calculation. Specifically, the calculation method was as follows: the sum of the measured molar numbers of the six amino acids (Aspartic acid (Asp), Glutamic acid (Glu), Alanine (Ala), Leucine (Leu), Isoleucine (Ile), and Lysine (Lys)) was divided by the sum of their theoretical amino acid counts as the denominator. It should be noted that during hydrolysis, asparagine (Asn) and glutamine (Gln) are converted to aspartic acid (Asp) and glutamic acid (Glu), respectively. Therefore, when calculating the theoretical amino acid count, the counts of Asparagine and glutamine were included in the calculations for Aspartic acid and glutamic acid (Glu), respectively. The measured molar numbers of the other amino acids were divided by this denominator to obtain the measured number of amino acids in the sample. The difference between the theoretical and measured amino acid counts was then compared with the theoretical amino acid count to obtain the percentage error. An error within 20% is generally considered acceptable.

[0028] 8. Data Analysis Results The two batches of telpoeptide samples (Test1 and Test2) were tested according to the above method. The amino acid composition data obtained were compared with the theoretical data in the table below.

[0029] Comparison of amino acid composition data and theoretical data of telpopeptide:

[0030] Figure 1 This is the chromatogram of amino acid standards after derivatization. Figure 2 This is the chromatogram of the derivatized sample after hydrolysis of telpoeptide. As can be seen from the figure, the chromatographic peaks of each amino acid and AEEA are well separated, with no significant interference.

[0031] 9. Analysis of Experimental Results The data above show that the measured number of each amino acid in the test sample is in high agreement with the theoretical calculation, and the errors for all amino acids and AEEA are within 20%. This verifies the correctness of the telpolide sequence structure. It also demonstrates that the pre-column derivatization-high performance liquid chromatography method established in this embodiment is suitable for the amino acid composition analysis and structural confirmation of telpolide. Example 2:

[0032] This embodiment uses OPA and FMOC as pre-column derivatizing agents to derivatize primary and secondary amino acids, respectively, and specifically detects the content of the non-natural amino acid Aib in telpolide using high-performance liquid chromatography (HPLC). The working principle is as follows: OPA (o-phthalaldehyde) reacts rapidly with primary amino acids under alkaline conditions to form isoindole derivatives with strong UV absorption at 338 nm; FMOC (fluorene methyl chloroformate) reacts with secondary amino acids to form FMOC-amino acid derivatives with characteristic absorption at 262 nm. Aib is a secondary amino acid (α-dimethyl substitution) and cannot be derivatized by OPA, but it can react with FMOC to form a stable derivative, thus achieving specific detection.

[0033] 1. Solution preparation Mobile phase A: 0.05 mol / L sodium acetate solution - triethylamine - tetrahydrofuran, volume ratio 950:0.14:5, pH adjusted to 6.8 with 2% acetic acid. Mobile phase B: 0.16 mol / L sodium acetate solution (pH adjusted to 6.8 with 2% acetic acid), a mixture of methanol and acetonitrile, volume ratio 200:400:400. The 6 M HCl solution was prepared as in Example 1. OPA solution: Weigh 40 mg of o-phthalaldehyde, add 3.5 mL of borate buffer, 0.5 mL of acetonitrile, and 64 μL of 3-mercaptopropionic acid, dissolve and mix thoroughly. FMOC solution: Weigh 40 mg of FMOC, add 8 mL of acetonitrile, dissolve and mix thoroughly. Aib standard solution: Prepare a 1 mmol / L Aib standard solution. The mixed amino acid standard solution was prepared as in Example 1.

[0034] 2. Blank Derivative Processing Take 10 μL of ultrapure water, add 40 μL of pH 10.2 boric acid buffer solution and vortex mix. Then add 20 μL of OPA solution (for derivatizing primary amino acids), mix well and let stand for 30 seconds. Then add 10 μL of FMOC solution (for derivatizing secondary amino acids), mix well and let stand for 30 seconds. Add 110 μL of water, mix and filter through a 0.22 μm filter tube to obtain a blank derivatization solution for testing.

[0035] 3. Derivatization of Standard Products Take 10 μL of amino acid mixed solution standard, add 40 μL of pH 10.2 boric acid buffer solution and vortex mix. Then add 20 μL of OPA solution, mix well, and let stand for 30 seconds. Next, add 10 μL of FMOC solution, mix well, and let stand for 30 seconds. Add 110 μL of water, mix, and filter through a 0.22 μm filter tube to obtain the amino acid standard derivatization solution. Take 10 μL of Aib standard solution and perform derivatization treatment according to the same procedure to obtain the Aib standard derivatization solution.

[0036] 4. Hydrolysis of the test sample The hydrolysis procedure for the test sample was exactly the same as in Example 1: 1 mg of telpolide sample was added to a hydrolysis tube, followed by the addition of 6M HCl solution. After filling with high-purity nitrogen for 1 min, the cap was tightened, and the sample was hydrolyzed in an oil bath at 110°C for 24 hours. After cooling, the sample was transferred to a 10 mL volumetric flask, brought to a final volume, and 1 mL was lyophilized overnight.

[0037] 5. Derivatization treatment of the test sample Take 10 μL of ultrapure water to reconstitute the completely dried sample, add 40 μL of pH 10.2 boric acid buffer solution and vortex mix. Then add 20 μL of OPA solution, mix well and let stand for 30 seconds. Next, add 10 μL of FMOC solution, mix well and let stand for 30 seconds. Then add 110 μL of water, mix and filter through a 0.22 μm filter tube to obtain the derivatized solution of the test sample, which is ready for testing.

[0038] 6. Liquid chromatography for the detection of chromatographic parameters This embodiment also uses an Agilent high-performance liquid chromatograph. The chromatographic column is a Welch Ultimate-C18 (4.6 mm × 250 mm, 5 μm) or a column with equivalent performance. The column temperature is set to 40 °C. Mobile phase A is 0.05 mol / L sodium acetate solution-triethylamine-tetrahydrofuran (950:0.14:5, pH 6.8), and mobile phase B is 0.16 mol / L sodium acetate solution-methanol-acetonitrile (200:400:400, pH 6.8). The flow rate is 1 mL / min. The UV detection wavelengths are: 338 nm for primary amino acids and 262 nm for secondary amino acids. The injection volume is 20 μL. Gradient elution is used, and the elution program is as follows:

[0039] 7. Data Processing Using OpenLab software, the molar number of amino acid residues in the sample was automatically calculated using the external standard method. The amino acid composition data obtained from the test was compared with the theoretical sequence for calculation. Specifically, the calculation method was as follows: the sum of the measured molar numbers of the five amino acids (aspartic acid (Asp), glutamic acid (Glu), alanine (Ala), leucine (Leu), and isoleucine (Ile)) was divided by the sum of their theoretical amino acid numbers as the denominator. Similarly, the hydrolysis products of asparagine (Asn) and glutamine (Gln) were included in the calculation for aspartic acid and glutamic acid, respectively. The measured molar numbers of other amino acids (especially Aib) were divided by the denominator to obtain the measured number of amino acids in the test sample. The percentage error was then calculated, and an error within 20% was considered acceptable.

[0040] 8. Data Analysis Results The two batches of telpolide samples were tested using the method described above, and the results of the comparison between the obtained data and the theoretical data are shown in the table below.

[0041] Comparison of amino acid composition data and theoretical data of telpopeptide:

[0042] Figure 3 This is the chromatogram of amino acid standards after derivatization. Figure 4 This is the chromatogram of the derivatized sample after hydrolysis of telpoide. As can be seen from the figure, the chromatographic peaks of Aib are well separated and free from interference.

[0043] 9. Analysis of Experimental Results The above data show that the measured number of Aib in the test sample is in high agreement with the theoretical calculation value, and the error between the two tests is within 20%, verifying the accurate content and structural correctness of Aib in the telpolide sequence. This indicates that the OPA / FMOC pre-column derivatization-high performance liquid chromatography method established in this embodiment is suitable for the specific determination of Aib in telpolide.

[0044] In summary, this invention addresses the different structural characteristics of natural amino acids, the linker AEEA, and the non-natural amino acid Aib in telpolide by employing both PITC and OPA combined with FMOC derivatization systems for independent detection. This effectively solves the problem of inaccurate determination of Aib in the same system as amino acids and AEEA due to differences in the reactivity and stability of Aib derivatization. This achieves comprehensive, accurate, and highly sensitive quantitative analysis of all amino acid components (including natural amino acids, non-natural amino acids, and linkers) in telpolide. The method is simple and rapid, with mild derivatization conditions, good derivative stability, and minimal interference during detection. It exhibits good reproducibility and specificity, providing a systematic and reliable analytical tool for the structural confirmation and quality control of telpolide and its analogues. Furthermore, this method has wide applicability and can be extended to the amino acid composition analysis of other peptide drugs containing Aib or AEEA structural units.

[0045] Although embodiments of the present invention have been shown and described, these specific embodiments are merely explanations of the invention and are not intended to limit it. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. After reading this specification, those skilled in the art may make modifications, substitutions, and variations to the embodiments as needed without departing from the principles and spirit of the invention, but such modifications, substitutions, and variations are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A qualitative and quantitative method for detecting the amino acid composition of a GLP-1 polypeptide drug, characterized in that, The method includes: The first derivatization step uses phenyl isothiocyanate (PITC) as a pre-column derivatizing agent to simultaneously detect the content of natural amino acids and the linker AEEA in the GLP-1 peptide drug by high-performance liquid chromatography; and, The second derivatization detection step uses o-phthalaldehyde (OPA) and fluorene methyl chloroformate (FMOC) as pre-column derivatizing agents to detect the content of non-natural amino acid Aib in the GLP-1 peptide drug by high performance liquid chromatography. The GLP-1 polypeptide drug is selected from at least one of telpolide, smegglutide, and retaloglutide.

2. The detection method according to claim 1, characterized in that, The first derivative detection step includes: S1: Hydrolysis of the test sample: Hydrolyze the telpoide sample with hydrochloric acid solution to obtain a hydrolysate, and then dry it; S2: Derivatization treatment: The dried hydrolysis product is reconstituted with solvent, and triethylamine solution and phenyl isothiocyanate solution are added sequentially to carry out the derivatization reaction. After the reaction, hexane is added for extraction, and the lower layer solution is taken to obtain the first test solution. S3: High performance liquid chromatography detection: The first test solution was analyzed by high performance liquid chromatography, and the detection was performed at a wavelength of 254 nm using an ultraviolet detector. The content of natural amino acids and AEEA was calculated using the external standard method.

3. The detection method according to claim 2, characterized in that, In S1, the hydrolysis conditions are: the hydrolysate is a 6M hydrochloric acid solution, the hydrolysis temperature is 110℃, the hydrolysis time is 24 hours, and the hydrolysate is freeze-dried after hydrolysis; in S2, the derivatization reaction conditions are: the triethylamine concentration is 2 mol / L, the phenyl isothiocyanate concentration is 0.5 mol / L, and the reaction is allowed to stand for 1 hour.

4. The detection method according to claim 2, characterized in that, In S3, the high-performance liquid chromatography (HPLC) conditions are as follows: the column is an octadecylsilane-bonded silica column, and the column temperature is 25℃; mobile phase A is a 50 mmol / L sodium acetate aqueous solution with a pH of 7.5±0.05; mobile phase B is a mixture of methanol, acetonitrile, and water in a volume ratio of 200:600:200; gradient elution is used, and the elution program is as follows: from 0 to 39 minutes, mobile phase A linearly decreases from 95% to 52%, and mobile phase B linearly increases from 5% to 48%; from 39 to 40 minutes, mobile phase A decreases from 52% to 0%, and mobile phase B increases from 48% to 100%; from 40 to 45 minutes, mobile phase A is maintained at 0%, and mobile phase B at 100%; from 45 to 46 minutes, mobile phase A increases from 0% to 95%, and mobile phase B decreases from 100% to 5%; from 46 to 60 minutes, mobile phase A is maintained at 95%, and mobile phase B at 5%; the flow rate is 1 mL / min, and the injection volume is 10 mL / min. μL.

5. The detection method according to claim 1, characterized in that, The second derivative detection step includes: S11: Hydrolysis of the test sample: Hydrolyze the telpoide sample with hydrochloric acid solution to obtain a hydrolysate, and then dry it; S21: Derivatization treatment: The dried hydrolysis product is reconstituted with a solvent, and boric acid buffer solution, o-phthalaldehyde solution and fluorene methyl chloroformate solution are added in sequence to carry out the derivatization reaction to obtain the second test solution; S31: High performance liquid chromatography detection: The second test solution was analyzed by high performance liquid chromatography using a UV detector at dual wavelengths of 338 nm and 262 nm. The content of Aib was calculated using the external standard method.

6. The detection method according to claim 5, characterized in that, In S11, the hydrolysis conditions are as follows: the hydrolysate is a 6M hydrochloric acid solution, the hydrolysis temperature is 110℃, the hydrolysis time is 24 hours, and the hydrolysate is freeze-dried after hydrolysis. In S21, the derivatization reaction conditions are as follows: the borate buffer solution has a pH of 10.2, first add o-phthalaldehyde solution to derive primary amino acids, let stand for 30 seconds, then add fluorene methyl chloroformate solution to derive secondary amino acids, and let stand for 30 seconds.

7. The detection method according to claim 5, characterized in that, In S31, the high-performance liquid chromatography (HPLC) conditions are as follows: the chromatographic column is an octadecylsilane-bonded silica column, and the column temperature is 40℃; mobile phase A is 0.05 mol / L sodium acetate solution-triethylamine-tetrahydrofuran, with a volume ratio of 950:0.14:5, and the pH is adjusted to 6.8 with 2% acetic acid; mobile phase B is 0.16 mol / L sodium acetate solution, methanol, and acetonitrile, with a volume ratio of 200:400:400, wherein 0.16 mol / L sodium acetate solution... The pH of the mol / L sodium acetate solution was adjusted to 6.8 with 2% acetic acid. Gradient elution was used, with the following elution program: 0 to 27 minutes, mobile phase A decreased linearly from 92% to 90%, and mobile phase B increased linearly from 8% to 10%; 27 to 31 minutes, mobile phase A decreased from 90% to 0%, and mobile phase B increased from 10% to 100%; 31 to 38 minutes, mobile phase A was maintained at 0%, and mobile phase B at 100%; 38 to 41 minutes, mobile phase A increased from 0% to 92%, and mobile phase B decreased from 100% to 8%; 41 to 45 minutes, mobile phase A was maintained at 92%, and mobile phase B at 8%; the flow rate was 1 mL / min, and the injection volume was 20 μL.

8. The detection method according to any one of claims 1-7, characterized in that, It also includes a data analysis step: comparing the measured molar number of natural amino acids, AEEA, and Aib with the theoretical number of sequences, calculating the percentage of error, to verify the structural correctness of telpoeptide.

9. The detection method according to claim 8, characterized in that, For the data obtained in the first derivatization detection step, the total number of measured moles of aspartic acid, glutamic acid, alanine, leucine, isoleucine, and lysine is divided by the total number of their theoretical amino acids as the denominator to calculate the measured number of other amino acids and AEEA and the error percentage.

10. The detection method according to claim 8, characterized in that, For the data obtained in the second derivatization detection step, the measured number of Aib and the error percentage are calculated by dividing the sum of the measured moles of aspartic acid, glutamic acid, alanine, leucine, and isoleucine by the sum of their theoretical amino acid numbers.

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