High performance liquid chromatography method for determining liraglutide and specific impurities

Abstract

The invention relates to the technical field of GLP-1 detection, in particular to a high performance liquid chromatography method for determining liraglutide and specific impurities. According to the method, a C8 column serves as a chromatographic column, phosphate buffered solution-acetonitrile serves as a mobile phase, under the specific chromatographic condition, five specific impurities in liraglutide can be effectively separated at the same time, the separation degree of a main peak and the separation degree of all the impurities are all larger than 1.0, the trailing factor of the main peak is smaller than 1.0, and the method is high in sensitivity and good in repeatability. The content of liraglutide and each impurity can be quantitatively determined, and the quality of a liraglutide product can be effectively controlled.

Description

Technical Field

[0001] The present invention relates to the field of GLP-1 detection technology, and in particular to a high performance liquid chromatography method for determining liraglutide and specific impurities. Background Art

[0002] Liraglutide, also known as Liraglutide in English, is a new long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Novo Nordisk of Denmark for the treatment of type II diabetes. Liraglutide has the effects of lowering blood sugar, losing weight, and reducing the risk of major adverse cardiovascular events in diabetic patients. It was first approved for marketing in the European Union in September 2007.

[0003] Liraglutide replaces Lys34 of human GLP-1 (7-36) with Arg, and connects a 16-carbon palmitic fatty acid derivative to the side chain of Lys26. Chemically synthesized liraglutide is a 31-mer peptide with a molecular formula of C 172 H 265 N 43 O 51 , molecular weight is 3751.20g / mol. Appearance is white to off-white powder or loose block. The peptide sequence is:

[0004] H-His1-Ala2-Glu3-Gly4-Thr5-Phe6-Thr7-Ser8-Asp9-Val10-Ser11-Ser12-Tyr13-Leu14-Glu15-Gly16-Gln17-Ala18- Ala19-Lys20(N-ε-(γ-Glu(N-α-Pal))-Glu21-Phe22-Ile23-Ala24-Trp25-Leu26-Val27-Arg28-Gly29-Arg30-Gly31-OH.

[0005] Peptides are mainly composed of amino acids (including natural amino acids and unnatural amino acids), which makes peptide drugs different from general drugs in terms of quality research. Due to the uniqueness of the synthesis process and structure, the sources of process impurities in synthetic peptide APIs are different from those of general chemical drugs. The possible process impurities include: missing peptides, misconnected peptides, broken peptides, deamidated peptides, byproducts formed by incomplete deprotection of amino acid side chains, oxidized peptides, products of disulfide bond exchange, diastereoisomers, etc. At the same time, due to the structural characteristics of peptides, the stability of this type of drug is relatively poor. The main causes of instability of peptide drugs are hydrolysis, oxidation, racemization, breakage and rearrangement of disulfide bonds, β elimination, etc. Therefore, in the study of related substance inspection methods for synthetic peptide drugs, it is necessary to fully consider the detection efficiency of process impurities and degradation impurities.

[0006] Chemically synthesized liraglutide inevitably produces a series of process impurities and degradation impurities during the synthesis and storage process, such as [D-Ser12] impurity (impurity A), [D-His1] impurity (impurity D), [D-Ser8] impurity (impurity B), [Ile26] impurity (impurity F), and [des-His1-Ala2] impurity (impurity E). Among them, impurities A, D, B, and F are produced during the process. Since these impurities differ from liraglutide in structure by only one amino acid, their properties are extremely similar to those of liraglutide and are difficult to remove by purification. At the same time, since liraglutide is alkaline, it is easy to undergo racemization during storage, producing impurities A, D, B, and E. Therefore, the impurity content in the raw materials and preparations of liraglutide needs to be strictly controlled. Otherwise, once these impurities exceed the limit, the quality of the drug will be affected, bringing drug safety risks. However, these impurities are either liraglutide racemic impurities, peptide deletion impurities or misconnected peptide impurities, which are very similar to liraglutide in nature and difficult to separate from the main peak. It is difficult to determine their content. Therefore, it is necessary to develop a method that can effectively separate these impurities and determine their content. At present, no pharmacopoeia contains a detection method for liraglutide related substances. There are few reports on detection methods for liraglutide related substances in existing patents and literature. Chinese patent CN107290438A provides a detection method for liraglutide related substances, but the method does not involve the separation of any specific impurities, and the main peak tailing factor is greater than 2.0, which is serious.

[0007] Since the toxicological data of these impurities are not yet clear, there must be certain potential risks for patients to take the medicine. Therefore, in order to ensure the quality of liraglutide products and improve the safety of patients' medication, it is very important to strictly control the quality of these specific impurities in liraglutide. Summary of the invention

[0008] The object of the present invention is to provide a high performance liquid chromatography method for determining liraglutide and specific impurities, which can effectively separate liraglutide and five specific impurities at the same time under specific chromatographic conditions, and quantitatively determine the contents of liraglutide and its impurities with high sensitivity, and can effectively control the quality of liraglutide products.

[0009] In order to achieve the purpose of the present invention, the present invention provides a high performance liquid chromatography method for determining liraglutide and specific impurities, comprising the following steps:

[0010] A C8 chromatographic column was used; a mixed solution of phosphate buffer solution-acetonitrile with the pH adjusted to 4.8-5.0 was used as the mobile phase; and the elution method was gradient elution.

[0011] The specific impurities include: impurity A, impurity B, impurity D, impurity E, impurity F, and the peptide sequence is as follows:

[0012] Impurity A: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Ieu-Val-Arg-Gly-Arg-Gly-OH

[0013] Impurity B: H-His-Ala-Glu-Gly-Thr-Phe-Thr-D-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

[0014] Impurity D: HD-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

[0015] Impurity E: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH

[0016] Impurity F: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Ile-Val-Arg-Gly-Arg-Gly-OH

[0017] In the present invention, the chromatographic column is a C8 chromatographic column. Chromatographic columns with different fillers have an extremely important influence on the separation of impurities, and even affect the peak order of impurities. The preferred chromatographic column specification is 250×4.6 mm, and the filler particle size is 3 to 3.5 μm. Compared with a chromatographic column with a particle size of 5 μm, the particle size is smaller and the theoretical plate number is higher, which is more beneficial to improving the peak shape and improving the separation degree.

[0018] In the present invention, the mobile phase is a phosphate buffer solution-acetonitrile mixed solution. Different buffer salt systems have different separation effects on the main peak and impurities, and have different degrees of influence on the main peak peak shape and baseline. For example, the main peak front or tail cannot determine whether the impurity is separated from the main peak. With phosphate buffer solution-acetonitrile as the mobile phase system, the impurity separation effect is good and the main peak peak shape is good. According to some preferred embodiments of the present invention, the concentration of the phosphate buffer solution in the mobile phase is 10 to 50 mmol / L. Since the buffer salt concentration used includes a high concentration range, the present invention pre-mixes a certain proportion of acetonitrile into the mobile phase. This operation is conducive to instrument balance to make the baseline fluctuation more stable.

[0019] In some specific embodiments of the present invention, the mobile phase pH range is 4.8 to 5.0. Different pH can change the separation degree of the front and rear impurities from the main peak, and even affect the peak order of the impurities. Among them, when the pH is 4.8 to 5.0, the separation of the main peak and the front and rear impurities can achieve a good separation effect, the main peak has a good peak shape, and the impurities have a stable peak order.

[0020] In some specific embodiments of the present invention, the column temperature of the chromatographic column is 40° C. to 47° C. If the column temperature is too low, the system pressure is high; if the column temperature is too high, the separation degree of front and back impurities is small; more preferably, the column temperature is 45° C.

[0021] In some specific embodiments of the present invention, the flow rate of the mobile phase is 0.7-0.9 ml / min, more preferably 0.8 ml / min.

[0022] In some specific embodiments of the present invention, the elution gradient is mobile phase B: 26±2%→72±2% (0-60 min)→72±2% (60-90 min), more preferably 26%→72% (0-60 min)→72% (60-90 min).

[0023] The positive and progressive effects of the present invention are:

[0024] The method of the present invention can effectively separate liraglutide and its five specific impurities at the same time, and for the first time, liraglutide and five specific impurities are completed in one analytical system, and the separation between each impurity peak and between the main peak and the adjacent impurity peak is greater than 1.0. The method of the present invention adopts the impurity control method to detect the detection limit and quantitative limit of liraglutide and five specific impurities. The results show that the method can quantitatively detect liraglutide and each impurity, has the characteristics of strong specificity, good separation, fast analysis speed, high sensitivity, etc., and can be used for the detection of related substances of liraglutide raw materials and preparations, and can also be used for the detection of liraglutide content. The technical problem that there is no method for controlling specific impurities of liraglutide in current domestic and foreign pharmacopoeias and literatures is solved, and the accurate control of main process impurities and potential degradation impurities in the production process of liraglutide is achieved, and the quality of liraglutide products is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a spectrum of system suitability in Example 1;

[0026] Figure 2 is the spectrum of the quantitative limit in Example 7;

[0027] Figure 3 Spectrum showing the detection limit in Example 7;

[0028] Figure 4 is the spectrum of the test solution in Example 8;

[0029] Figure 5 This is the spectrum of Comparative Example 1 using sodium acetate / phosphoric acid aqueous solution-acetonitrile as the mobile phase;

[0030] Figure 6 This is the spectrum of Comparative Example 2 using C18 as the chromatographic column;

[0031] Figure 7 This is the spectrum of Comparative Example 3 using C4 as the chromatographic column. DETAILED DESCRIPTION

[0032] The present invention is further described below by means of specific examples, but the protection content of the present invention is not limited to the following implementation examples.

[0033] Example 1

[0034] Chromatographic conditions

[0035] Instrument: WATERS

[0036] Column: Kromasil C8 250×4.6mm, 3.5μm

[0037] Mobile phase A: 50 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.9

[0038] Mobile phase B: 50mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.9

[0039] Flow rate: 0.8ml / min

[0040] Column temperature: 45°C

[0041] Injection volume: 10 μL

[0042] Detection wavelength: 215nm

[0043] System suitability solution: Weigh appropriate amounts of liraglutide reference substance, impurity A, impurity B, impurity D, impurity E, and impurity F, and use a diluent (weigh 0.57 g of disodium hydrogen phosphate, add 500 ml of water to dissolve, and adjust the pH to 7.7 with phosphoric acid) to prepare a solution containing 1 mg / ml of liraglutide and 5 μg / ml of each impurity.

[0044] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0045] Take 10 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The HPLC spectrum is as follows: Figure 1 shown.

[0046] Table 1 System suitability separation statistics for Example 1

[0047] name Retention time Separation Tailing Factor Impurity A 43.100 -- -- Impurity F 44.408 1.796 -- Impurity B 45.669 1.465 -- Liraglutide 47.600 1.697 0.670 Impurity D 49.278 1.597 -- Impurity E 52.945 4.552 --

[0048] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0049] Example 2

[0050] Chromatographic conditions

[0051] Instrument: WATERS

[0052] Column: Kromasil C8 250×4.6mm, 3.5μm

[0053] Mobile phase A: 30mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.8

[0054] Mobile phase B: 30mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.8

[0055] Flow rate: 0.7ml / min

[0056] Column temperature: 40°C

[0057] Injection volume: 10 μL

[0058] Detection wavelength: 215nm

[0059] Elution gradient: Mobile phase B: 24% → 70% (0-60min) → 70% (60-90min)

[0060] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The data are shown in Table 2. The HPLC spectrum is similar to Figure 1 resemblance.

[0061] Table 2: System suitability separation statistics for Example 2

[0062] name Retention time Separation Tailing Factor Impurity A 47.255 -- -- Impurity F 47.682 1.584 -- Impurity B 49.211 1.665 -- Liraglutide 51.314 1.325 0.712 Impurity D 52.925 1.432 -- Impurity E 53.813 3.882 --

[0063] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0064] Example 3

[0065] Chromatographic conditions

[0066] Instrument: WATERS

[0067] Column: Besil C8-H 250×4.6 mm, 3 μm;

[0068] Mobile phase A: 10 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted pH to 5.0

[0069] Mobile phase B: 10 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 5.0

[0070] Flow rate: 0.9ml / min

[0071] Column temperature: 47°C

[0072] Injection volume: 10 μL

[0073] Detection wavelength: 215nm

[0074] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0075] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The data are shown in Table 3. The HPLC spectrum is similar to Figure 1 resemblance.

[0076] Table 3 System suitability separation statistics for Example 3

[0077] name Retention time Separation Tailing Factor Impurity A 42.112 -- -- Impurity F 43.892 1.321 -- Impurity B 44.216 1.625 -- Liraglutide 45.438 1.251 0.603 Impurity D 46.552 1.321 -- Impurity E 50.382 5.332 --

[0078] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0079] Example 4

[0080] Chromatographic conditions

[0081] Instrument: WATERS

[0082] Column: Kromasil C8 250×4.6 mm, 3.5 μm;

[0083] Mobile phase A: 30mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted pH to 5.0

[0084] Mobile phase B: 30mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 5.0

[0085] Flow rate: 0.7ml / min

[0086] Column temperature: 47°C

[0087] Injection volume: 10 μL

[0088] Detection wavelength: 215nm

[0089] Elution gradient: Mobile phase B: 24% → 70% (0-60min) → 70% (60-90min)

[0090] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The data are shown in Table 4. The HPLC spectrum is Figure 1 resemblance.

[0091] Table 4: System suitability separation statistics for Example 4

[0092]

[0093]

[0094] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0095] Example 5

[0096] Chromatographic conditions

[0097] Instrument: WATERS

[0098] Column: Besil C8-H 250×4.6 mm, 3 μm;

[0099] Mobile phase A: 50 mmol aqueous ammonium dihydrogen phosphate solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.9

[0100] Mobile phase B: 50mmol aqueous ammonium dihydrogen phosphate solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.9

[0101] Flow rate: 0.8ml / min

[0102] Column temperature: 45°C

[0103] Injection volume: 10 μL

[0104] Detection wavelength: 215nm

[0105] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0106] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The data are shown in Table 5. The HPLC spectrum is similar to Figure 1 resemblance.

[0107] Table 5: System suitability separation statistics for Example 5

[0108] name Retention time Separation Tailing Factor Impurity A 40.295 -- -- Impurity F 42.251 3.584 -- Impurity B 44.326 2.854 -- Liraglutide 45.312 1.092 0.739 Impurity D 46.226 1.003 -- Impurity E 50.821 5.892 --

[0109] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0110] Example 6

[0111] Chromatographic conditions

[0112] Instrument: WATERS

[0113] Column: Besil C8-H 250×4.6 mm, 3 μm;

[0114] Mobile phase A: 10 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.8

[0115] Mobile phase B: 10 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.8

[0116] Flow rate: 0.9ml / min

[0117] Column temperature: 40°C

[0118] Injection volume: 10 μL

[0119] Detection wavelength: 215nm

[0120] Elution gradient: Mobile phase B: 28% → 74% (0-60min) → 74% (60-90min)

[0121] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The data are shown in Table 6. The HPLC spectrum is Figure 1 resemblance.

[0122] Table 6: System suitability separation statistics for Example 6

[0123] name Retention time Separation Tailing Factor Impurity A 43.446 -- -- Impurity F 44.829 1.814 -- Impurity B 46.113 1.691 -- Liraglutide 48.125 1.984 0.670 Impurity D 49.057 1.125 -- Impurity E 53.437 2.987 --

[0124] Experimental conclusion: As shown in the above results, the separation between the main peak and impurity B and impurity D is greater than 1.0, the separation between each impurity is greater than 1.0, and the tailing factor of the main peak is less than 1.0;

[0125] Example 7

[0126] The chromatographic method of Example 1 was used to investigate the concentration of impurities that could be quantitatively determined and the minimum detection limit.

[0127] Quantitation limit solution: Accurately weigh impurity A, impurity B, impurity D, impurity E, impurity F and liraglutide reference substance, dissolve in diluent and continue to dilute until the signal-to-noise ratio is greater than 10.

[0128] Detection limit solution: Accurately weigh impurity A, impurity B, impurity D, impurity E, impurity F and liraglutide reference substance, dissolve in diluent and continue to dilute until the signal-to-noise ratio is about 2 to 4.

[0129] Accurately measure 10 μl of the quantitative limit solution and the detection limit solution and inject them into the chromatograph to record the chromatogram. The test results are shown in Table 7 and Table 8. The HPLC spectrum is shown in Figure 2 , Figure 3 shown.

[0130] Table 7 Quantitation Limit Solution

[0131]

[0132]

[0133] Table 8 Detection limit solutions

[0134] name Concentration (μg / mL) Signal-to-Noise Ratio Impurity A 0.0550 2.251 Impurity B 0.0519 3.124 Impurity D 0.0506 2.728 Impurity E 0.0559 3.868 Impurity F 0.1024 4.229 Liraglutide 0.0581 4.292

[0135] Experimental conclusion: From the above results, it can be seen that this method has high sensitivity.

[0136] Example 8

[0137] The chromatographic method of Example 1 was used to determine the contents of liraglutide and related substances.

[0138] Test solution: Weigh an appropriate amount of liraglutide raw material and use a diluent (weigh 0.57 g of disodium hydrogen phosphate, add 500 ml of water to dissolve, and adjust the pH to 7.7 with phosphoric acid) to make a 1 mg / ml solution.

[0139] Reference substance solution: Weigh an appropriate amount of liraglutide reference substance and use a diluent (weigh 0.57 g of disodium hydrogen phosphate, add 500 ml of water to dissolve, and adjust the pH to 7.7 with phosphoric acid) to prepare a 1 mg / ml solution.

[0140] Accurately measure 10 μl of the test solution and inject it into the chromatograph to record the chromatogram. The related substances of liraglutide are calculated by the area normalization method, and the content of liraglutide is calculated by the external standard method. The test results are shown in Table 9 and the HPLC spectrum is shown in Table 9. Figure 4 shown.

[0141] Table 9 Content of related substances in liraglutide test solution

[0142] name Retention time content% Impurity A 43.275 0.01 Impurity F 44.628 0.02 Impurity B 45.929 0.14 Impurity D 49.365 0.11 Impurity E 53.194 0.06 Maximum unknown impurity 43.968 0.02 Liraglutide 47.907 98.6

[0143] Comparative Example 1:

[0144] Chromatographic conditions

[0145] Instrument: WATERS

[0146] Column: Kromasil C8 250×4.6mm, 3.5μm

[0147] Mobile phase A: 10mmol sodium acetate / phosphoric acid aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted pH to 5.0

[0148] Mobile phase B: 10mmol sodium acetate / phosphoric acid aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted pH to 5.0

[0149] Flow rate: 0.8ml / min

[0150] Column temperature: 45°C

[0151] Injection volume: 10 μL

[0152] Detection wavelength: 215nm

[0153] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0154] Accurately measure 10 μl of the system suitability solution and inject it into the chromatograph. Record the chromatogram. The data are shown in Table 10. The HPLC spectrum is shown in Figure 5 shown.

[0155] Table 10 Comparative Example 1 System Suitability Separation Statistics

[0156] name Retention time Separation Tailing Factor Impurity A 53.380 -- -- Impurity F 52.868 -- -- Liraglutide 55.533 -- 0.737 Impurity D 55.533 -- -- Impurity B 56.416 -- -- Impurity E 58.199 -- --

[0157] Experimental conclusion: When using the sodium acetate system, impurity D overlaps with the main peak, each impurity has no separation from the main peak, and the baseline noise is large.

[0158] Comparative Example 2:

[0159] Chromatographic conditions

[0160] Instrument: WATERS

[0161] Column: Besil C18-H 250×4.6mm, 5μm

[0162] Mobile phase A: 50 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.9

[0163] Mobile phase B: 50mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.9

[0164] Flow rate: 1.0ml / min

[0165] Column temperature: 45°C

[0166] Injection volume: 10 μL

[0167] Detection wavelength: 215nm

[0168] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0169] Accurately measure 10 μl of the system suitability solution and inject it into the chromatograph. Record the chromatogram. The data are shown in Table 11. The HPLC spectrum is shown in Figure 6 shown.

[0170] Table 11 Comparative Example 2 System Suitability Separation Statistics

[0171] name Retention time Separation Tailing Factor Impurity A 50.616 -- -- Impurity B 52.518 1.735 -- Liraglutide 52.518 -- 0.688 Impurity D 52.518 -- -- Impurity E 54.185 0.933 -- Impurity F 58.574 2.820 --

[0172] Experimental conclusion: Using C18 chromatographic column, impurity B and impurity D coincide with the main peak.

[0173] Comparative Example 3:

[0174] Chromatographic conditions

[0175] Instrument: WATERS

[0176] Column: HALO C4 150×4.6mm, 3.4μm

[0177] Mobile phase A: 50 mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 6:4, phosphoric acid adjusted to pH 4.9

[0178] Mobile phase B: 50mmol potassium dihydrogen phosphate aqueous solution: acetonitrile = 1:1, phosphoric acid adjusted to pH 4.9

[0179] Flow rate: 0.8ml / min

[0180] Column temperature: 45°C

[0181] Injection volume: 10 μL

[0182] Detection wavelength: 215nm

[0183] Elution gradient: Mobile phase B: 26% → 72% (0-60min) → 72% (60-90min)

[0184] Accurately measure 10 μl of the system suitability solution and inject it into the chromatograph. Record the chromatogram. The data are shown in Table 12. The HPLC spectrum is shown in Figure 7 shown.

[0185] Table 12 Comparative Example 3 System Suitability Separation Statistics

[0186] name Retention time Separation Tailing Factor Impurity A 30.617 -- -- Impurity F 32.377 -- -- Impurity B 32.783 -- -- Liraglutide 34.094 -- 0.742 Impurity D 34.767 -- -- Impurity E 35.719 -- --

[0187] Experimental conclusion: When using C4 chromatographic column, there is no separation between impurities and main peak, and between impurities.

Claims

1. A high performance liquid chromatography method for determining liraglutide and specific impurities, characterized in that: A C8 chromatographic column was used; the mobile phase was a mixed solution of phosphate buffer solution and acetonitrile, and the pH was adjusted to 4.8-5.0; the elution method was gradient elution; The specific impurities include: impurity A, impurity B, impurity D, impurity E, impurity F, and the peptide sequence is as follows: Impurity A: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-D-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Ieu-Val-Arg-Gly-Arg-Gly-OH; Impurity B: H-His-Ala-Glu-Gly-Thr-Phe-Thr-D-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; Impurity D: HD-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; Impurity E: H-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH; Impurity F: H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Pal-γ-Glu)-Glu-Phe-Ile-Ala-Trp-Ile-Val-Arg-Gly-Arg-Gly-OH.

2. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The C8 chromatographic column has a specification of 250×4.6 mm.

3. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The mobile phase includes mobile phases A and B, wherein mobile phase A is a mixed solution of phosphate buffer solution-acetonitrile (3:2), and mobile phase B is a mixed solution of phosphate buffer solution-acetonitrile (1:1).

4. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1 or 3, characterized in that: The phosphate is potassium dihydrogen phosphate or ammonium dihydrogen phosphate.

5. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 3, characterized in that: The concentration of phosphate buffer solution is 10-50 mmol / L.

6. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 3 or 5, characterized in that: The concentration of phosphate buffer solution is 30mmol / L.

7. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The pH value of the mobile phase is 4.

9.

8. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The column temperature of the chromatographic column in the chromatographic conditions is 40-47°C.

9. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 8, characterized in that: The column temperature of the chromatographic column in the chromatographic conditions is 45°C.

10. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The flow rate in the chromatographic conditions is 0.7 to 0.9 ml / min.

11. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 10, characterized in that: The flow rate in the chromatographic conditions was 0.8 ml / min.

12. The high performance liquid chromatography method for specific impurities of liraglutide according to claim 1, characterized in that: The detection wavelength in the chromatographic conditions is 215 nm.

13. The high performance liquid chromatography method for liraglutide and specific impurities according to claim 1, characterized in that: The elution gradient is mobile phase B: 26±2%→72±2% (0-60 min)→72±2% (60-90 min).

14. Use of the method according to any one of claims 1 to 13 in the detection of related substances in liraglutide raw materials and preparations.

15. Use of the method according to any one of claims 1 to 13 in detecting the content of liraglutide.

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