High performance liquid chromatography analysis method of semeglutide

Abstract

The invention discloses a high performance liquid chromatography analysis method for separating semeglutide from specific impurities B and C. The method is characterized by comprising the following steps: in a high performance liquid chromatograph, mixing a mobile phase A and a mobile phase B according to different proportions to serve as a mobile phase, and carrying out gradient elution on a semeglutide sample; wherein the mobile phase A is selected from a mixed aqueous solution of phosphate and hexafluorophosphate, and the pH value is adjusted to 2.5-3.5; the mobile phase B is acetonitrile. An impurity B and an impurity C are specific impurities of semeglutide and have similar molecular weights, and in a conventional analysis method, the two impurities have the same retention time in a liquid phase and cannot be accurately quantified. According to the analysis method disclosed by the invention, separation of specific impurities B and C can be realized under specific chromatographic conditions, and the separation degree of the impurities B and C is not lower than 1.0, so that the product quality is more favorably controlled.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides, and in particular to a high performance liquid chromatography analysis method for semaglutide and specific impurities B and C. Background Art

[0002] Semaglutide, also known as Semaglutide in English, is a new type of long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk of Denmark for the treatment of type 2 diabetes. Semaglutide has the effects of lowering blood sugar, losing weight and protecting cardiovascular system, and was recommended by FDA for approval on October 18, 2017. After the Lys side chain of semaglutide is modified with PEG, Glu and octadecanedicarboxylic acid, its hydrophilicity is greatly improved and its binding ability with albumin is enhanced; at the same time, after the Ala at the second position of the N-terminus is mutated to Aib, it effectively avoids inactivation by DPP-IV enzymatic hydrolysis, and its half-life reaches 40h. Patients only need to inject once a week. The oral dosage form of the drug is also under development. The CAS number of semaglutide is 910463-68-2, and its molecular formula is C 187 H 291 N 45 O 59 , molecular weight is 4113.64 g / mol, peptide sequence is:

[0003] H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Octadecanedioic-γ-Glu-PEG-PEG)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0004] During the synthesis and storage of semaglutide, a series of process impurities and degradation impurities are inevitably produced, such as impurity B (D-Asp 9 impurities), impurity C (Plus-Gly 4 Impurities). The presence of impurities affects the product quality of semaglutide, and the toxicological data of these impurities are still unclear, which inevitably poses potential risks to patients.

[0005] Chinese patent CN114660214 A discloses a liquid chromatography detection method for semaglutide: using phosphate buffer as mobile phase A, a mixed solution of organic solvent and water as mobile phase B, a single chromatographic column as stationary phase, and a UV detector as the detector, which can separate and detect D-His of semaglutide. 1 Racemic impurity, Des-Aib 2Defective impurities, Plus-Gly 4 Impurities, but did not mention the separation of impurities with the same retention time that may exist. Chinese patent CN112285217 A discloses an ultra-high performance liquid chromatography analysis method for semaglutide. In an ultra-high performance liquid chromatograph, mobile phase A and mobile phase B are mixed in different proportions as mobile phases, and the semaglutide sample is gradient eluted, wherein mobile phase A is selected from one or more mixed solutions of trifluoroacetate, perchlorate, and hexafluorophosphate, and the pH is adjusted to 2-4; mobile phase B is selected from methanol, acetonitrile, and isopropanol. The detection method provided can meet the method requirements in terms of separation between impurities and main peaks, tailing factor, and theoretical pedal number, but the separation between impurities has not been studied.

[0006] Impurity B is [D-Asp 9 ]Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-D-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. Under certain conditions, the Asp amino acid side chain at the S9 position of the semaglutide molecule undergoes a side reaction, forming a five-membered ring intermediate through ring closure, and then opening the ring to produce impurity B. Impurity C is [Plus-Gly 4 ]Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH. The intermediate of semaglutide contains a small amount of plus-Gly4 impurity. During the chemical synthesis of semaglutide, a small amount of plus-Gly4 semaglutide impurity, i.e., impurity C, is also produced along with the coupling process of the intermediate.

[0007] Impurities B and C are specific impurities of semaglutide with similar molecular weights. In conventional analytical methods, the two impurities have the same retention time in the liquid phase and peak at the same time, making it impossible to accurately detect the content of impurities B and C in semaglutide. Therefore, it is urgent to develop a separation and detection method specifically for impurities B and C. Summary of the invention

[0008] The present invention provides a high performance liquid chromatography analysis method for separating semaglutide from specific impurities B and C. The method adopts an ion pair reagent and a buffer salt elution technology, and can accurately and effectively separate and detect the contents of impurity B and impurity C in semaglutide under specific chromatographic conditions.

[0009] In order to achieve the purpose of the present invention, the present invention provides a high performance liquid chromatography analysis method for semaglutide, comprising the following steps:

[0010] In a high performance liquid chromatograph, mobile phase A and mobile phase B are mixed in different proportions as mobile phases to gradient elute the semaglutide test sample, wherein mobile phase A is a mixed solution of phosphate and hexafluorophosphate, and the pH is adjusted to 2.5-3.5; mobile phase B is acetonitrile.

[0011] In the present invention, the impurity B is [D-Asp 9 ]Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-D-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0012] The impurity C is [Plus-Gly 4]Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH.

[0013] In the present invention, in the high performance liquid chromatograph, the chromatographic column is an octaalkyl bonded silica gel chromatographic column.

[0014] In some embodiments of the present invention, the concentration of potassium dihydrogen phosphate in mobile phase A is 50-100 mmol / L, and the concentration of potassium hexafluorophosphate is 10-30 mmol / L.

[0015] Preferably, the concentration of potassium dihydrogen phosphate in mobile phase A is 75 mmol / L, the concentration of potassium hexafluorophosphate is 20 mmol / L, and mobile phase B is acetonitrile.

[0016] Preferably, the pH value of mobile phase A is 3.0.

[0017] In some embodiments of the present invention, the concentration of semaglutide in the test solution is 0.092 mg / ml to 1.8 mg / ml; the concentration of impurity B in the test solution is 0.4 to 1.5 μg / ml, and the concentration of impurity C is 0.4 to 1.4 μg / ml.

[0018] In some embodiments of the present invention, the column temperature of the chromatographic column is 30°C to 45°C, and preferably, the column temperature is 37°C.

[0019] In some embodiments of the present invention, the flow rate is 0.5-0.8 ml / min, and preferably, the flow rate is 0.7 mg / mL.

[0020] In some embodiments of the present invention, isocratic elution from 0 min to 65 min and gradient elution from 65 min to 95 min are used.

[0021] Among them, from 0min to 65min, the proportion of mobile phase B is 37% to 42%.

[0022] Among them, from 65min to 72min, the proportion of mobile phase B is (37-42%)→50%, from 72min to 80min, the proportion of mobile phase B is 50%; from 80min to 81min, the proportion of mobile phase B is 50%→(37-42%); from 81min to 95min, the proportion of mobile phase B is maintained at (37-42%).

[0023] In some embodiments of the present invention, the detection wavelength is 213-217 nm, and preferably, the wavelength is 215 nm.

[0024] The present invention also provides a method for use in the analysis and detection of semaglutide and its specific impurities B and C. The high performance liquid chromatography analysis method of the present invention can effectively separate semaglutide and specific impurities B and specific impurities C at the same time, and the separation degree of impurities B and impurity C can be greater than 1.0, and the tailing factor and theoretical pedal number can meet the method requirements. The method has the characteristics of strong specificity, good separation degree, fast analysis speed, high sensitivity, etc., and can be used for the detection of semaglutide raw materials and preparation-related substances, solves the technical problem that there is no method for controlling specific impurities of semaglutide in current domestic and foreign pharmacopoeias and literature, realizes the precise control of main process impurities and potential degradation impurities in the semaglutide production process, and improves the quality of semaglutide products. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the HPLC spectrum of the system suitability solution in Comparative Example 1;

[0026] Figure 2 This is the HPLC spectrum of the test solution in Comparative Example 1;

[0027] Figure 3 HPLC spectrum of the test solution in Comparative Example 2

[0028] Figure 4 HPLC spectrum of the test solution in Comparative Example 3

[0029] Figure 5 The HPLC spectrum of the system suitability solution in Example 1;

[0030] Figure 6 The HPLC spectrum of the test solution in Example 1;

[0031] Figure 7 The HPLC spectrum of the system suitability solution at pH 2.5 in Example 2;

[0032] Figure 8 This is the HPLC spectrum of the test solution at pH 2.5 in Example 2;

[0033] Fig. 9is the HPLC spectrum of the system suitability solution at pH 3.5 in Example 2;

[0034] Fig.10 The HPLC spectrum of the test solution at pH 3.5 in Example 2;

[0035] Fig.11 The HPLC spectrum of the test solution at a flow rate of 1:0.5 ml / min in Example 3;

[0036] Fig.12 The HPLC spectrum of the test solution at a flow rate of 1:0.5 ml / min in Example 3;

[0037] Fig.13 HPLC spectrum of the test solution at column temperature 1:30°C in Example 4

[0038] Fig.14 HPLC spectrum of the test solution at column temperature 1:45°C in Example 4

[0039] Fig.15 The starting ratio of mobile phase B is 1: 0min~65min, 37% system suitability HPLC spectrum

[0040] Fig.16 The starting ratio of mobile phase B is 2: 0min~65min, 42% system suitability HPLC spectrum

[0041] Fig.17 Impurity B Linear Graph

[0042] Fig.18 Impurity C Linear Graph

[0043] Fig.19 Semaglutide linearity graph DETAILED DESCRIPTION

[0044] The present invention discloses a high performance liquid chromatography analysis method for polypeptide-related substances. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention.

[0045] The system suitability solution is mainly used to examine the separation degree of impurities B and C and whether the number of theoretical plates of the main peak meets the requirements. The system suitability solution is prepared as follows:

[0046] System suitability solution: accurately weigh 5.07 mg of semaglutide reference substance, place in a 5 ml volumetric flask, accurately add 1 ml of impurity stock solution, add diluent to dissolve and dilute to scale, mix well, and prepare a mixed solution containing approximately 1 mg of semaglutide and 0.002 mg of each impurity per 1 ml;

[0047] The test solution is mainly used to determine the purity of the main peak of semaglutide, impurity B, and impurity C in the semaglutide sample solution. The test solution is prepared as follows:

[0048] Test solution: Weigh an appropriate amount of semaglutide API accurately, dissolve in water and dilute to make a solution containing approximately 1 mg per 1 ml.

[0049] Comparative Example 1

[0050] Chromatographic conditions

[0051] Instrument: Agilent 1260

[0052] Chromatographic column: YMC Triart C8 (4.6mm×250mm, 3.0μm)

[0053] Mobile phase A: 75 mmol / L potassium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid)-acetonitrile (650:273, g / g)

[0054] Mobile phase B: 75mmol / L potassium dihydrogen phosphate aqueous solution (adjust pH to 3.0 with phosphoric acid)-acetonitrile (500:391, g / g)

[0055] Flow rate: 0.7ml / min;

[0056] Column temperature: 50°C

[0057] Injection volume: 10 μL

[0058] Detection wavelength: 215nm

[0059] Elution gradient: time: 0min~50min, mobile phase B: 38→42%; 50min~60min, mobile phase B: 42%→100%; 60min~70min, mobile phase B: 100%; 70min~72min, mobile phase B: 100%→38%; 72~85min, mobile phase B: 38%.

[0060] Accurately measure 20 μl of the system suitability solution and the test solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 1 below:

[0061] Table 1

[0062] Instrument system parameters System Suitability Solutions Test solution Impurity B retention time min 34.597 32.938 Impurity C retention time min 34.597 32.938 Separation degree of impurity B and impurity C NA NA Purity of main peak of semaglutide % NA 99.85 Theoretical plate number (based on main peak) 18857 15459 Impurity B% + Impurity C% NA 0.09

[0063] Experimental results: Figure 1 , Figure 2They are the HPLC spectra of the system suitability solution and the test solution of semaglutide. Impurity B and impurity C in the system suitability solution elute simultaneously with a retention time of 34.597 min, with no separation. Only impurity B can be determined in the test solution, and the total amount of impurity C is 0.09%. The purity of semaglutide is 99.85%.

[0064] Comparative Example 2

[0065] Chromatographic conditions

[0066] Instrument: Waters e2695+2489

[0067] Chromatographic column: YMC Triart C8 (4.6mm×250mm, 3.0μm)

[0068] Mobile phase A: 75mmol / L potassium hexafluorophosphate (adjust pH to 2.5 with phosphoric acid)

[0069] Mobile phase B: acetonitrile

[0070] Column temperature: 45°C

[0071] Injection volume: 25 μL

[0072] Detection wavelength: 215nm

[0073] Elution gradient: time: 0min~60min, mobile phase B: 48→55%; 60min~62min, mobile phase B: 55%→48%; 62min~70min, mobile phase B: 48%.

[0074] Accurately measure 25 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 2 below. The HPLC chromatogram of the test solution is shown in Figure 3 .

[0075] Table 2

[0076] Instrument system parameters Test solution Impurity B retention time min 34.204 Impurity C retention time min 34.732 Separation degree of impurity B and impurity C 0.508 Purity of the main peak of semaglutide 99.42 Theoretical plate number (based on main peak) 12726 Impurity B% 0.09 Impurity C% 0.13

[0077] Experimental results: Under the chromatographic conditions of Comparative Example 2 where mobile phase A contained only potassium hexafluorophosphate, impurities B and C in the test solution eluted in sequence with retention times of 34.204 min and 34.732 min, respectively, and a separation degree of 0.508. Although this was an improvement over Comparative Example 1, the separation degree was less than 1.0, and the separation degree of impurities B and C could not meet the requirements of precise quantification.

[0078] Comparative Example 3

[0079] Chromatographic conditions:

[0080] Instrument: Waters e2695+2489

[0081] Chromatographic column: YMC triart, C8 (4.6×250 mm, 3 μm);

[0082] Mobile phase A: a mixed solution of 75 mmol / L potassium dihydrogen phosphate and 20 mmol / L potassium hexafluorophosphate (pH adjusted to 3.0 with phosphoric acid);

[0083] Mobile phase B: acetonitrile

[0084] Detection wavelength: 215nm

[0085] Flow rate: 0.6ml / min;

[0086] Column temperature: 45°C

[0087] Injection volume: 25 μl;

[0088] Elution gradient: time: 0min~70min, mobile phase B: 41~45%; 70min~90min, mobile phase B: 45%; 90min~91min, mobile phase B: 45%→41%; 91min~100min, mobile phase B: 41%.

[0089] Accurately measure 25 μl of the test solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3 below. The HPLC chromatogram of the test solution is shown in Figure 4 .

[0090] Table 3

[0091] Instrument system parameters Test solution Impurity B retention time min 38.776 Impurity C retention time min 39.615 Separation degree of impurity B and impurity C 0.642 Purity of the main peak of semaglutide 98.97 Theoretical plate number (based on main peak) 30483 Impurity B% 0.16 Impurity C% 0.19

[0092] Experimental results: Under the chromatographic conditions of comparative example 3 mobile phase A 75mmol / L potassium dihydrogen phosphate and 20mmol / L potassium hexafluorophosphate mixed solution (pH adjusted to 3.0 with phosphoric acid), impurities B and impurity C in the test solution eluted in sequence, with retention times of 38.776min and 39.615min, respectively, and a separation degree of 0.642. Although it is improved over comparative example 2, the separation degree is still less than 1.0, and the separation degree of impurities B and C cannot meet the requirements of accurate quantification.

[0093] Example 1

[0094] Chromatographic conditions

[0095] Instrument: Waters e2695+2489

[0096] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0097] Mobile phase A: a mixed aqueous solution of 75mmol / L potassium dihydrogen phosphate + 20mmol / L potassium hexafluorophosphate, with the pH adjusted to 3.0 with phosphoric acid;

[0098] Mobile phase B: acetonitrile

[0099] Flow rate: 0.7ml / min

[0100] Column temperature: 37°C

[0101] Injection volume: 10 μL

[0102] Detection wavelength: 215nm

[0103] Elution gradient: 0min~65min, the proportion of mobile phase B is 39%, 65min~72min, the proportion of mobile phase B is 39%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→39%; 81min-95min, the proportion of mobile phase B is maintained at 39%.

[0104] Accurately measure 10 μL of system suitability solution and test solution respectively, inject into the chromatograph, and record the chromatogram. Figure 5 , Figure 6 , the test results are shown in Table 4.

[0105] Table 4

[0106] Instrument system parameters System Suitability Solutions Test solution Impurity B retention time min 41.804 40.068 Impurity C retention time min 43.455 41.513 Separation degree of impurity B and impurity C 1.243 1.346 Purity of the main peak of semaglutide NA 99.74 Theoretical plate number (based on main peak) 5624 7140 Impurity B% NA 0.09 Impurity C% NA 0.12

[0107] Experimental conclusion: Under the chromatographic conditions of Example 1, in the system suitability solution, the separation degree of impurity B and impurity C is 1.243, and the separation degrees of the main peak and the front impurities and the back impurities are both greater than 1.0; the theoretical plate number meets the requirements; in the test solution, the separation degree of impurity B and impurity C is 1.346, and the contents of impurities B and C in the test solution are measured to be 0.09% and 0.12%, respectively; compared with comparative examples 1-3 and the analysis method of semaglutide in the prior art, the detection ability of impurities B and C is stronger, and the contents of impurities B and impurity C can be accurately quantified, respectively, which is more conducive to product quality control.

[0108] Example 2

[0109] Instrument: Waters e2695+2489

[0110] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0111] Mobile phase A: A mixed aqueous solution of 50mmol / L potassium dihydrogen phosphate + 10mmol / L potassium hexafluorophosphate

[0112] pH 2.5: adjust pH to 2.5 with phosphoric acid;

[0113] pH 3.5: Adjust pH to 3.5 with phosphoric acid

[0114] Mobile phase B: acetonitrile

[0115] Flow rate: 0.7ml / min

[0116] Column temperature: 37°C

[0117] Injection volume: 10 μL

[0118] Detection wavelength: 215nm

[0119] Elution gradient: 0min~65min, the proportion of mobile phase B is 39%, 65min~72min, the proportion of mobile phase B is 39%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→39%; 81min-95min, the proportion of mobile phase B is maintained at 39%.

[0120] Accurately measure 10 μL of system suitability solution and test solution respectively, inject into the chromatograph, record the chromatogram, and the test results are shown in Table 5. The HPLC spectrum of system suitability solution and test solution at pH 2.5 is shown in Figure 7 , Figure 8 ; pH 3.5 system suitability solution, test solution HPLC spectrum see Fig. 9 , Fig.10 ;

[0121] Table 5

[0122]

[0123]

[0124] Experimental conclusion: As shown in the above results, in the mobile phase A pH 2.5, the separation degree of impurity B and impurity C in the system suitability solution is 1.482, which is greater than 1.0, and the theoretical plate number meets the requirements; in the mobile phase A pH 3.5, the separation degree of impurity B and impurity C in the system suitability solution is 1.016, which is greater than 1.0, and the theoretical plate number meets the requirements. The contents of impurities B and C in the test solution measured by mobile phase A pH 2.5 and mobile phase A pH 3.5 are equivalent to those in Example 1.

[0125] Example 3

[0126] Instrument: Waters e2695+2489

[0127] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0128] Mobile phase A: 100mmol / L potassium dihydrogen phosphate + 30mmol / L potassium hexafluorophosphate mixed aqueous solution, pH adjusted to 3.0 with phosphoric acid

[0129] Mobile phase B: acetonitrile

[0130] Flow rate 1: 0.5 ml / min

[0131] Flow rate 2: 0.8 ml / min

[0132] Column temperature: 37°C

[0133] Injection volume: 10 μL

[0134] Detection wavelength: 215nm

[0135] Elution gradient: 0min~65min, the proportion of mobile phase B is 39%, 65min~72min, the proportion of mobile phase B is 39%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→39%; 81min-95min, the proportion of mobile phase B is maintained at 39%.

[0136] Accurately measure 10 μL of system suitability solution and test solution, inject into the chromatograph, record the chromatogram, and the test results are shown in Table 6. Flow rate 1: 0.5 ml / min, flow rate 2: 0.8 ml / min, the HPLC spectra of the test solution are shown in Table 6. Fig.11 , Fig.12 .

[0137] Table 6

[0138]

[0139] Experimental conclusion: As shown in the above results, the separation degree of impurity B and impurity C in the system suitability solution at flow rate 1: 0.5ml / min is 1.293, which is greater than 1.0, and the theoretical plate number meets the requirements; the separation degree of impurity B and impurity C in the system suitability solution at flow rate 2: 0.8ml / min is 1.280, which is greater than 1.0, and the theoretical plate number meets the requirements. The contents of impurities B and C in the test solution measured at flow rates 1: 0.5ml / min and 2: 0.8ml / min are equivalent to those in Example 1.

[0140] Example 4

[0141] Instrument: Waters e2695+2489

[0142] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0143] Mobile phase A: 75mmol / L potassium dihydrogen phosphate + 20mmol / L potassium hexafluorophosphate mixed aqueous solution, pH adjusted to 3.0 with phosphoric acid

[0144] Mobile phase B: acetonitrile

[0145] Flow rate: 0.7ml / min

[0146] Column temperature 1: 30°C

[0147] Column temperature 2: 45°C

[0148] Injection volume: 10 μL

[0149] Detection wavelength: 215nm

[0150] Elution gradient: 0min~65min, the proportion of mobile phase B is 39%, 65min~72min, the proportion of mobile phase B is 39%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→39%; 81min-95min, the proportion of mobile phase B is maintained at 39%.

[0151] Accurately measure 10 μL of system suitability solution and test solution, inject into the chromatograph, record the chromatogram, and the test results are shown in Table 7. Column temperature 1: 30℃, column temperature 2: 45℃ test solution HPLC spectrum is shown in Fig.13 , Fig.14 .

[0152] Table 7

[0153]

[0154] Experimental conclusion: As shown in the above results, the separation degree of impurities B and C in the system suitability solution at column temperature 1: 30°C is 1.526, which is greater than 1.0, and the number of theoretical plates meets the requirements; the separation degree of impurities B and C in the system suitability solution at column temperature 2: 45°C is 1.244, which is greater than 1.0, and the number of theoretical plates meets the requirements. The contents of impurities B and C in the test solution measured at column temperature 1: 30°C and column temperature 2: 45°C are equivalent to those in Example 1.

[0155] Example 5

[0156] Instrument: Waters e2695+2489

[0157] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0158] Mobile phase A: 75mmol / L potassium dihydrogen phosphate + 20mmol / L potassium hexafluorophosphate mixed aqueous solution, pH adjusted to 3.0 with phosphoric acid

[0159] Mobile phase B: acetonitrile

[0160] Flow rate: 0.7ml / min

[0161] Elution gradient:

[0162] The starting proportion of mobile phase B is 1: 0min~65min, 37%; 65min~72min, the proportion of mobile phase B is 37%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→37%; 81min-95min, the proportion of mobile phase B is maintained at 37%.

[0163] The starting proportion of mobile phase B is 2: 0min~65min, 42%; 65min~72min, the proportion of mobile phase B is 42%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→42%; 81min-95min, the proportion of mobile phase B is maintained at 42%.

[0164] Injection volume: 10 μL

[0165] Detection wavelength: 215nm

[0166] Accurately measure 10 μL of system suitability solution and test solution, inject into the chromatograph, record the chromatogram, and the test results are shown in Table 6. Mobile phase B starting ratio 1: 0min~65min, 37%, mobile phase B starting ratio 2: 0min~65min, 42% System suitability HPLC spectra are shown in Table 6. Fig.15 , Fig.16 .

[0167] Table 8

[0168]

[0169]

[0170] Experimental conclusion: As shown in the above results, the separation degree of impurity B and impurity C in the system suitability solution is 1.032, which is greater than 1.0, and the theoretical plate number meets the requirements; the separation degree of impurity B and impurity C in the system suitability solution is 0.961 when the mobile phase B starting ratio is 1: 0min~65min,37%, and the mobile phase B starting ratio is 2: 0min~65min,42%. The contents of impurities B and C in the test solution are equivalent to those in Example 1.

[0171] Example 6

[0172] Instrument: Waters e2695+2489

[0173] Chromatographic conditions

[0174] Chromatographic column: Waters CORTECS C8 (4.6×250mm, 2.7μm)

[0175] Mobile phase A: a mixed aqueous solution of 75mmol / L potassium dihydrogen phosphate + 20mmol / L potassium hexafluorophosphate, with the pH adjusted to 3.0 with phosphoric acid;

[0176] Mobile phase B: acetonitrile

[0177] Flow rate: 0.7ml / min

[0178] Column temperature: 37°C

[0179] Injection volume: 10 μL

[0180] Detection wavelength: 215nm

[0181] Elution gradient: 0min~65min, the proportion of mobile phase B is 39%, 65min~72min, the proportion of mobile phase B is 39%→50%, 72min~80min, the proportion of mobile phase B is 50%; 80min~81min, the proportion of mobile phase B is 50%→39%; 81min-95min, the proportion of mobile phase B is maintained at 39%.

[0182] 1. Impurity B, impurity C linear

[0183] Impurity B stock solution: Accurately weigh 10.67 mg of the impurity B reference substance, place it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, mix well, and obtain an impurity B stock solution with a concentration of 0.5 mg / ml;

[0184] Impurity C stock solution: Accurately weigh 10.31 mg of the impurity C reference substance, place it in a 20 ml volumetric flask, add diluent to dissolve and dilute to the scale, mix well, and obtain an impurity C stock solution with a concentration of 0.5 mg / ml;

[0185] Linear stock solution: Accurately pipette 1.0 ml of impurity B stock solution and impurity C stock solution into a 50 ml volumetric flask, add diluent to dilute to the mark, mix well, and obtain linear stock solutions of semaglutide, impurity B, and impurity C with a concentration of 0.01 mg / ml, respectively;

[0186] Quantitative limit solution: accurately pipette 1 ml of impurity stock solution into a 25 ml volumetric flask, dilute to the mark with diluent, and mix well;

[0187] 50% solution: accurately measure 0.5 ml of the linear stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and mix well;

[0188] 80% solution: accurately measure 0.8 ml of the linear stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and mix well;

[0189] 100% solution: accurately measure 1.0 ml of the linear stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and mix well;

[0190] 120% solution: accurately measure 1.2 ml of the linear stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and mix well;

[0191] 150% solution: accurately measure 1.5 ml of the linear stock solution, place it in a 10 ml volumetric flask, add diluent to dilute to the mark, and mix well;

[0192] Accurately measure 10 μl of each of the above solutions, inject into the chromatography system, record the chromatogram, and perform linear regression on the peak area using concentration to obtain the linear regression equation. The linear graphs of impurity B and impurity C are shown in Figure 1. Fig.17 , Fig.18 .

[0193] Impurity B has good linearity in the concentration range of 0.4μg / ml to 1.5μg / ml (quantitative limit to 150% of the limit concentration); Impurity C has good linearity in the concentration range of 0.4μg / ml to 1.4μg / ml (quantitative limit to 150% of the limit concentration);

[0194] 2. Semaglutide linearity

[0195] Semaglutide reference substance stock solution: Take an appropriate amount of semaglutide reference substance, dissolve and dilute with water to make a solution containing 10.0 mg of semaglutide per 1 ml;

[0196] 10% linear solution: Take 0.1 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0197] 50% linear solution: Take 0.5 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0198] 80% linear solution: Take 0.8 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0199] 100% linear solution: Take 1.0 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0200] 150% linear solution: Take 1.5 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0201] 200% linear solution: Take 2.0 ml of semaglutide reference stock solution in a 10 ml volumetric flask, dilute with water, and make up to volume;

[0202] Accurately measure 10 μl of the above solution, inject it into the chromatography system, record the chromatogram, and perform linear regression on the peak area using concentration to obtain the linear regression equation. The linearity of semaglutide is shown in Fig.19 .

[0203] Semaglutide has good linearity in the concentration range of 0.092 mg / ml to 1.8 mg / ml (10% to 200%).

Claims

1. A high performance liquid chromatography analysis method for separating semaglutide from specific impurities B and C, characterized in that: The following steps are involved: In a high performance liquid chromatograph, mobile phase A and mobile phase B are mixed in different proportions as mobile phases, and gradient elution is performed to detect the test solution; Wherein, the test solution is an aqueous solution containing semaglutide, the mobile phase A is a mixed aqueous solution of phosphate and hexafluorophosphate, and the pH is adjusted to 2.5-3.5; the mobile phase B is acetonitrile; Wherein, the impurity B is [D-Asp 9 ] Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-D-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; the impurity C is [Plus-Gly 4 ]Semaglutide, the structural formula is H-His-Aib-Glu-Gly-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-N6-[N-(17-carboxy-1-oxoheptadecyl)-L-γ-glutamyl[2-(2-aminoethoxy)ethoxy]acetyl[2-(2-aminoethoxy)ethoxy]acetyl]-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; In the high performance liquid chromatography, the chromatographic column is an octaalkyl bonded silica gel chromatographic column.

2. The method according to claim 1, characterized in that The mobile phase A is a mixed aqueous solution of potassium dihydrogen phosphate and potassium hexafluorophosphate, and the mobile phase B is acetonitrile.

3. The method according to claim 1 or 2, characterized in that: In the mobile phase A, the concentration of potassium dihydrogen phosphate is 50-100 mmol / L, and the concentration of potassium hexafluorophosphate is 10-30 mmol / L.

4. The method according to claim 3, characterized in that In the mobile phase A, the concentration of potassium dihydrogen phosphate is preferably 75 mmol / L, and the concentration of potassium hexafluorophosphate is preferably 20 mmol / L.

5. The method according to claim 1 or 2, characterized in that: The pH value of the mobile phase A is 3.

0.

6. The method according to claim 1, characterized in that In the test solution, the concentration of semaglutide is 0.092 mg / ml to 1.8 mg / ml mg / mL; And / or, the concentration of impurity B in the test solution is 0.4-1.5 μg / ml, and the concentration of impurity C is 0.4-1.4 μg / ml.

7. The method according to claim 1, characterized in that In the high performance liquid chromatograph, the column temperature of the chromatographic column is 30-45°C.

8. The method according to claim 7, characterized in that In the high performance liquid chromatograph, the column temperature of the chromatographic column is 37°C.

9. The method according to claim 1, characterized in that: In the high performance liquid chromatograph, the flow rate of the mobile phase is 0.5 to 0.8 ml / min.

10. The method according to claim 9, characterized in that The flow rate of the mobile phase was 0.7 ml / min.

11. The method according to claim 1, characterized in that: Isocratic elution was used from 0 min to 65 min, and gradient elution was used from 65 min to 95 min. Among them, from 0min to 65min, the proportion of mobile phase B is 37% to 42%. Among them, from 65min to 72min, the proportion of mobile phase B is (37-42%)→50%, from 72min to 80min, the proportion of mobile phase B is 50%; from 80min to 81min, the proportion of mobile phase B is 50%→(37-42%); from 81min to 95min, the proportion of mobile phase B is maintained at (37-42%).

12. The method according to claim 1, characterized in that In the high performance liquid chromatograph, the detection wavelength is 213-217 nm.

13. The method according to claim 12, characterized in that In the high performance liquid chromatograph, the detection wavelength is 215 nm.

14. Use of the method according to any one of claims 1 to 13 in the analysis and detection of semaglutide and its specific impurities B and C.

Images