A method for detecting related substances in saxagliptin

Abstract

This invention provides a method for detecting related substances in saxagliptin. The method employs high-performance liquid chromatography (HPLC) and includes the following steps: (1) preparation of the test solution and the reference solution; (2) separately measuring the test solution and the reference solution, injecting them into the HPLC instrument, recording the chromatograms, and calculating the percentage content of each related substance in the test solution using the principal component self-comparison method with correction factors. The detection method provided by this invention enables accurate qualitative and quantitative detection of related substances in saxagliptin, with a resolution of 3.75 between impurity A and impurity C, which is beneficial for effective quality control of saxagliptin and thus ensures its clinical safety. The detection method provided by this invention has the advantages of simple operation, high specificity, high sensitivity, and high accuracy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and specifically relates to a method for detecting related substances in saxagliptin. Background Technology

[0002] Saxagliptin is a highly potent dipeptidyl peptidase-4 (DPP-4) inhibitor jointly developed by Bristol-Myers Squibb and Astra Zeneca. By selectively inhibiting DPP-4, it can increase the levels of endogenous glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), thereby regulating blood glucose. In 2011, the CFDA approved its import for the treatment of type 2 diabetes. The chemical name of saxagliptin is (1S,3S,5S)-2-[(2S)-2-amino-2-(3-hydroxy-1-adamantyl)acetyl]-2-azabicyclo[3.1.0]hexane-3-nitrile monohydrate, and its structure is shown below:

[0003]

[0004] In the drug development process, the detection method for related substances is an important evaluation parameter for quality control and drug safety. Related substances refer to starting materials, intermediates, polymers, by-reaction products introduced during the production process, as well as degradation products during storage. Based on the structural characteristics of saxagliptin and its preparation process, the applicant has identified the following related substances that may be present in saxagliptin: impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4, whose structures are shown below:

[0005]

[0006] CN106568849A points out that the existing import registration standards for saxagliptin cannot effectively separate impurities IM-A and IM-B (i.e., impurities C and A in this application), and discloses a method for detecting related substances in saxagliptin using high-performance liquid chromatography. However, this method uses a ternary gradient elution procedure to separate related substances, which is relatively cumbersome, and the minimum resolution between each target peak and its nearest neighbor peak is between 1.66 and 2.05.

[0007] CN116973487A also points out that the standard testing methods for raw material pharmaceutical companies cannot accurately control saxagliptin, and discloses a method for determining the impurity content of saxagliptin, wherein the resolution of impurities BMS-537679 and BMS-554083 (i.e., impurities A and C in this application) is between 1.5 and 1.7. However, if other chromatographic conditions are kept unchanged, when the column temperature is reduced from 40℃ to 38℃, the impurity resolution drops from 1.7 to 1.5.

[0008] Therefore, in order to effectively separate the various impurity peaks, it is still necessary to develop a rapid and simple method for detecting related substances in saxagliptin. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for detecting related substances in saxagliptin.

[0010] The technical solution adopted in this invention is as follows:

[0011] A method for detecting related substances in saxagliptin, characterized in that the detection method employs high-performance liquid chromatography (HPLC), wherein the HPLC conditions include:

[0012] Chromatographic column: C18 column;

[0013] Mobile phase A: a mixed solution of potassium dihydrogen phosphate and acetonitrile, with a volume ratio of (80~99): (1~20);

[0014] Mobile phase B: a mixed solution of potassium dihydrogen phosphate and acetonitrile, with a volume ratio of (30~50): (50~70);

[0015] Linear gradient elution, wherein the gradient elution procedure is as follows:

[0016] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 <![CDATA[a1]]> <![CDATA[100-a1]]> <![CDATA[t1]]> <![CDATA[100-a1]]> <![CDATA[a1]]> <![CDATA[t1+t2]]> <![CDATA[100-a1]]> <![CDATA[a1]]> <![CDATA[t1+t2+t3]]> <![CDATA[a1]]> <![CDATA[100-a1]]> <![CDATA[t1+t2+t3+t4]]> <![CDATA[a1]]> <![CDATA[100-a1]]>

[0017] Among them, 95≤a1≤100; 30≤t1≤40, 4≤t2≤6, 1≤t3≤3, 6≤t4≤10;

[0018] Alternatively, the gradient elution procedure may be:

[0019] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 <![CDATA[b1]]> <![CDATA[100-b1]]> <![CDATA[T1]]> <![CDATA[b2]]> <![CDATA[100-b2]]> <![CDATA[T1+T2]]> <![CDATA[100-b1]]> <![CDATA[b1]]> <![CDATA[T1+T2+T3]]> <![CDATA[100-b1]]> <![CDATA[b1]]> <![CDATA[T1+T2+T3+T4]]> <![CDATA[b1]]> <![CDATA[100-b1]]> <![CDATA[T1+T2+T3+T4+T5]]> <![CDATA[b1]]> <![CDATA[100-b1]]>

[0020] Among them, 95≤b1≤100, 40≤b2≤90; 15≤T1≤25, 10≤T2≤20, 4≤T3≤12, 0.5≤T4≤1.5, 8≤T5≤10.

[0021] In one embodiment of the present invention, the relevant substances in the saxagliptin are selected from substances with the following chemical structures:

[0022] .

[0023] In one embodiment of the present invention, the C18 column is an Agilent Zorbax Eclipse AAA (150mm × 3.0mm, 3.5µm).

[0024] In one embodiment of the present invention, the concentration of potassium dihydrogen phosphate solution in mobile phase A and mobile phase B is 0.01~0.1 mol / L, preferably 0.04~0.06 mol / L, and more preferably 0.05 mol / L.

[0025] In one embodiment of the present invention, phosphoric acid is used to adjust the pH of the potassium dihydrogen phosphate solution in mobile phase A and mobile phase B to 2-4, preferably to 2.8-3.2, and more preferably to 3.

[0026] In one embodiment of the present invention, the volume ratio of the mixed solution of potassium dihydrogen phosphate and acetonitrile in the mobile phase A is (90~99):(1~10), preferably (95~99):(1~5), and more preferably 95:5; the volume ratio of the mixed solution of potassium dihydrogen phosphate and acetonitrile in the mobile phase B is (35~45):(55~65), preferably (38~42):(58~62), and more preferably 40:60.

[0027] In one specific embodiment of the present invention, the mobile phase A is a mixed solution of 0.05 mol / L potassium dihydrogen phosphate solution (with the pH adjusted to 3.0 by phosphoric acid) and acetonitrile, with a volume ratio of 95:5; the mobile phase B is a mixed solution of 0.05 mol / L potassium dihydrogen phosphate solution (with the pH adjusted to 3.0 by phosphoric acid) and acetonitrile, with a volume ratio of 40:60.

[0028] In one specific embodiment of the present invention, a1=100; t1=35, t2=5, t3=2, t4=8.

[0029] In one specific embodiment of the present invention, b1=100, b2=80; T1=20, T2=20, T3=5, T4=1, T5=9.

[0030] In one specific embodiment of the present invention, b1=100, b2=50; T1=20, T2=15, T3=5, T4=1, T5=9.

[0031] In one specific embodiment of the present invention, b1=100, b2=55; T1=20, T2=15, T3=10, T4=1, T5=9.

[0032] Preferably, in the detection method of the present invention, the chromatographic conditions optionally further include:

[0033] Detection wavelength: 210~220nm, preferably 213~217nm;

[0034] Flow rate: 0.2~1.0 ml / min, preferably 0.5~0.7 ml / min;

[0035] Column temperature: 20~50℃, preferably 38~42℃;

[0036] Injection volume: 10~60μl, preferably 15~25μl.

[0037] In one specific embodiment of the present invention, the chromatographic conditions optionally further include:

[0038] Detection wavelength: 215nm;

[0039] Flow rate: 0.6 ml / min;

[0040] Column temperature: 40℃;

[0041] Injection volume: 20 μl.

[0042] The method for detecting related substances in saxagliptin provided by this invention includes the following steps:

[0043] (1) Preparation of test solution and reference solution;

[0044] (2) Measure the test solution and the reference solution separately, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the percentage content of each relevant substance in the test sample according to the principal component self-comparison method with correction factor.

[0045] In one embodiment of the present invention, the method for preparing the test solution and the reference solution in step (1) includes:

[0046] Preparation of test solution: Take an appropriate amount of saxagliptin, dissolve it in solvent and dilute it to a concentration of 0.2~5mg saxagliptin per 1ml, and use it as the test solution;

[0047] Preparation of reference solution: Take the test solution and dilute it with solvent to 100 times the volume to obtain the reference solution.

[0048] In one embodiment of the present invention, the method for preparing the test solution in step (1) includes:

[0049] Preparation of test solution: Take an appropriate amount of saxagliptin, dissolve it in solvent and dilute it to a concentration of 0.5~2mg saxagliptin per ml, which is used as the test solution.

[0050] In one specific embodiment of the present invention, the method for preparing the test solution in step (1) includes:

[0051] Preparation of test solution: Take an appropriate amount of saxagliptin, dissolve it in solvent and dilute it to a concentration of 1 mg saxagliptin per 1 ml, and use it as the test solution.

[0052] In one exemplary embodiment of the present invention, the method for preparing the test solution and the reference solution in step (1) includes:

[0053] Preparation of test solution: Weigh approximately 20 mg of saxagliptin accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well to obtain the test solution;

[0054] Reference solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well to obtain the reference solution.

[0055] In one embodiment of the present invention, the solvent in step (1) is a hydrochloric acid solution of 0.05~0.2 mol / L, preferably a hydrochloric acid solution of 0.1 mol / L.

[0056] In one embodiment of the present invention, the method for calculating the percentage content of each relevant substance in the test sample in step (2) is as follows:

[0057]

[0058] In the formula, A i The peak area of ​​the corresponding impurity in the test solution;

[0059] A 对照 The peak area of ​​saxagliptin in the reference solution;

[0060] F i This is the correction factor for the corresponding impurities.

[0061] In one specific embodiment of the present invention, the correction factors for impurities A, B, C, intermediate 3, J, and Int3-Imp4 are 0.7, 1.0, 1.4, 1.4, 1.7, and 1.0, respectively.

[0062] Since the response values ​​of the same detector may be different for different substances, the peak areas produced when different substances of the same concentration pass through the detector are not necessarily equal. In quantitative determination by HPLC, the correction factor is usually the ratio of the absolute correction factor of a substance i to that of the selected reference substance s, i.e., the relative correction factor.

[0063] In this invention, the terms "containing" or "including (comprising)" can be open-ended, semi-closed, or closed-ended. In other words, the terms also include "consisting substantially of" or "consisting of".

[0064] In this invention, the terms "optional" or "optionally" refer to events or conditions that are possible but not necessary to occur, as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0065] In this invention, the term "about" means within ±10% of the stated value.

[0066] The European Pharmacopoeia EP11.0 specifies the maximum content of each related substance in saxagliptin, including: for impurities A, B, and C, the maximum content of each impurity is 0.15%; for unknown single impurities, the maximum content of each impurity is 0.10%; and for total impurities, the maximum content is 0.50%.

[0067] The detection method provided by this invention enables accurate qualitative and quantitative detection of related substances in saxagliptin, with a resolution of 3.75 between impurities A and C. This facilitates effective quality control of saxagliptin, thereby ensuring its safety in clinical use. The detection method provided by this invention has advantages such as simple operation, high specificity, high sensitivity, and high accuracy. Attached Figure Description

[0068] Figure 1 The high-performance liquid chromatogram of Example 7 is shown. The retention times of impurity C, impurity A, saxagliptin impurity B, impurity J, Int3-Imp4, and intermediate 3, in the order of peak elution, are 7.712 min, 8.260 min, 10.253 min, 11.550 min, 14.475 min, 21.848 min, and 24.435 min, respectively. The horizontal axis represents time (minutes / min). Detailed Implementation

[0069] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0070] Unless otherwise specified, the raw materials, reagents, and methods used in the examples are all conventional raw materials, reagents, and methods in the art, and the experimental materials and reagents used can be obtained from commercially available channels.

[0071] Example 1

[0072] High performance liquid chromatography (HPLC) was used to detect related substances (impurity A, impurity B, impurity C, and intermediate 3) in saxagliptin. The chromatographic conditions are as follows:

[0073] Column: Agilent Zorbax Eclipse AAA (150mm × 3.0mm, 3.5µm) (a C18 column)

[0074] Trapping column: Welch Ghost-Buster column (50mm×4.6mm)

[0075] Detection wavelength: 215nm

[0076] Flow rate: 0.6 ml / min

[0077] Column temperature: 40℃

[0078] Injection volume: 60 μl

[0079] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) - acetonitrile (volume ratio 95:5)

[0080] Mobile phase B: 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) - acetonitrile (volume ratio 40:60)

[0081] Linear gradient elution, the elution procedure is shown in Table 1:

[0082] Table 1

[0083] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 35 0 100 40 0 100 42 100 0 50 100 0

[0084] Solvent: 0.1 mol / L hydrochloric acid solution

[0085] Impurity A stock solution: Weigh about 5 mg of impurity A reference standard, place it in a 20 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0086] Impurity B stock solution: Weigh about 5 mg of impurity B reference standard, place it in a 20 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0087] Impurity C stock solution: Weigh about 5 mg of impurity C reference standard, place it in a 20 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0088] Intermediate 3 stock solution: Weigh about 5 mg of intermediate 3 reference standard, place it in a 20 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0089] Mixed solution: Weigh about 25 mg of saxagliptin and place it in a 50 ml volumetric flask. Add 1 ml of each of the above impurity stock solutions, dissolve in solvent, and dilute to the mark. Shake well.

[0090] Accurately measure 60 μl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram. See the attached image for the chromatogram. Figure 1 .

[0091] The results showed that all target peaks could be effectively separated. The separation degree between impurity A and impurity C was 3.43, and the separation degree between saxagliptin and impurity B was 3.58. It can be used for the detection of related substances (impurity A, impurity B, impurity C and intermediate 3) in saxagliptin.

[0092] Examples 2-4

[0093] The detection was performed using the same conditions and methods as in Example 1, only the gradient elution program was changed. The gradient elution programs for Examples 2-4 are shown in Tables 2-4, respectively.

[0094] Table 2 (Example 2)

[0095] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 80 20 40 0 100 45 0 100 46 100 0 55 100 0

[0096] Table 3 (Example 3)

[0097] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 50 50 35 0 100 40 0 100 41 100 0 50 100 0

[0098] Table 4 (Example 4)

[0099] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 20 55 45 35 0 100 45 0 100 46 100 0 55 100 0

[0100] Table 5 shows the separation degrees of impurities A and C, and saxagliptin and impurity B under different gradient elution programs.

[0101] Table 5

[0102] Example Separation degree between impurity A and impurity C Separation between saxagliptin and impurity B Example 2 3.34 4.45 Example 3 3.39 3.74 Example 4 3.42 3.86

[0103] The results show that the above method can be used for the detection of related substances (impurity A, impurity B, impurity C and intermediate 3) in saxagliptin.

[0104] Examples 5-6

[0105] The tests were performed using the same conditions and methods as in Example 1, except that the column temperature was changed. In Example 5, the column temperature was lowered to 25°C, and in Example 6, the column temperature was lowered to 30°C.

[0106] Table 7 shows the separation rates of impurities A and C, and saxagliptin and impurity B under different column temperatures.

[0107] Table 7

[0108] Example Separation degree between impurity A and impurity C Separation between saxagliptin and impurity B Example 5 2.92 2.72 Example 6 3.21 3.00

[0109] The results show that the above method can be used for the detection of related substances (impurity A, impurity B, impurity C and intermediate 3) in saxagliptin.

[0110] Example 7

[0111] The detection was performed using the same chromatographic conditions as in Example 1, with the injection volume reduced to 20 μl and the concentration of saxagliptin in the mixed solution increased to 1 mg / ml.

[0112] The results showed that all target peaks could be effectively separated and could be used for the detection of related substances (impurity A, impurity B, impurity C and intermediate 3) in saxagliptin.

[0113] Example 8

[0114] High performance liquid chromatography (HPLC) was used to detect related substances (impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4) in saxagliptin. The chromatographic conditions are as follows:

[0115] Column: Agilent Zorbax Eclipse AAA (150mm × 3.0mm, 3.5µm)

[0116] Trapping column: Welch Ghost-Buster column (50mm×4.6mm)

[0117] Detection wavelength: 215nm

[0118] Flow rate: 0.6 ml / min

[0119] Column temperature: 40℃

[0120] Injection volume: 20 μl

[0121] Mobile phase A: 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) - acetonitrile (volume ratio 95:5)

[0122] Mobile phase B: 0.05 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid) - acetonitrile (volume ratio 40:60)

[0123] Linear gradient elution, the elution procedure is the same as in Example 1:

[0124] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 35 0 100 40 0 100 42 100 0 50 100 0

[0125] Solvent: 0.1 mol / L hydrochloric acid solution

[0126] Test solution: Weigh approximately 20 mg of saxagliptin accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the test solution.

[0127] Reference solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0128] Accurately measure 20 μl each of the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatograms. Calculate the percentage content of each component using the principal component self-comparison method with correction factors. The specific calculation formula is as follows:

[0129]

[0130] In the formula, A i The peak area of ​​the corresponding impurity in the test solution;

[0131] A 对照 The peak area of ​​saxagliptin in the reference solution;

[0132] F i The correction factor is the corresponding impurity (see Table 8).

[0133] Table 8

[0134] name Correction factor Impurity A 0.7 Impurity B 1.0 Impurity C 1.4 Intermediate 3 1.4 Impurity J 1.7 Int3-Imp4 1.0

[0135] Example 9 Methodological Validation

[0136] Methodological validation was performed based on the detection method provided in Example 8.

[0137] 1. System suitability test

[0138] The location and system suitability of certain substances in saxagliptin (impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4) were investigated.

[0139] Solvent: 0.1 mol / L hydrochloric acid solution

[0140] Stock solution of impurities A, B, and C: Weigh approximately 1 mg each of impurity A, B, and C reference standards, place them in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0141] Intermediate 3 stock solution: Weigh approximately 1 mg of intermediate 3 reference standard accurately, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and the solution is ready.

[0142] Impurity J stock solution: Weigh approximately 10 mg of impurity J reference standard accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0143] Int3-Imp4 stock solution: Weigh approximately 5 mg of Int3-Imp4 reference standard accurately, place it in a 10 ml volumetric flask, add methanol-water (95:5) to dissolve and dilute to the mark, shake well, and the solution is ready.

[0144] Impurity stock solution 1: Accurately measure 3 ml each of impurity A, B, C stock solutions and intermediate 3 stock solution, place them in a 10 ml volumetric flask, dilute with solvent to the mark, and shake well to obtain the solution.

[0145] Impurity Stock Solution 2: Accurately measure 3 ml each of Impurity J Stock Solution and Int3-Imp4 Stock Solution, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0146] System suitability solution: Weigh approximately 20 mg of saxagliptin accurately and place it in a 20 ml volumetric flask. Add 2 ml each of impurity stock solution 1 and impurity stock solution 2, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0147] Precisely measure 20 μl of the system suitability solution and inject it into the liquid chromatograph. Record the chromatogram. The results are shown in Table 9.

[0148] Table 9

[0149] name Retention time / min Separation from the later peak Impurity C 7.712 3.75 Impurity A 8.260 6.35 Sagliptin 10.253 4.05 Impurity B 11.550 19.23 Impurity J 14.475 42.07 Int3-Imp4 21.848 11.99 Intermediate 3 24.435 na

[0150] The results showed that all target peaks could be effectively separated. The separation degree between impurity A and impurity C was 3.75, and the separation degree between saxagliptin and impurity B was 4.05. It can be used for the detection of related substances (impurity A, impurity B, impurity C, intermediate 3, impurity J and Int3-Imp4) in saxagliptin.

[0151] 2. Specificity

[0152] Take an appropriate amount of 0.1 mol / L hydrochloric acid solution as blank solvent, accurately measure 20 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0153] Take about 10 mg of saxagliptin and place it in a 10 ml volumetric flask. Perform forced degradation tests under acid, alkali, oxidation, high temperature and light irradiation to examine the specificity of the detection method for related substances of saxagliptin. Record the spectrum in the range of 190 nm to 400 nm and check the purity of the main peak.

[0154] The results showed that the blank solvent did not interfere with the determination, and saxagliptin was stable under light conditions. Under acidic degradation conditions, the main degradation impurities were impurity A, impurity C, and one unknown single impurity. Under alkaline degradation conditions, the main degradation impurities were also impurity A, impurity C, and one unknown single impurity. Under high-temperature conditions, impurities A, B, C, and two unknown impurities were produced. Under oxidative degradation conditions, the main impurities were impurity A, C, and two unknown single impurities. The main peak in the test solution was well separated from adjacent impurity peaks and from other impurity peaks. The purity of the main peak was 1000, indicating that they were all single peaks, with mass conservation ranging from 99.74% to 101.7%. This method has good specificity.

[0155] 3. Limit of Detection and Limit of Quantification

[0156] Accurately weigh appropriate amounts of saxagliptin, impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4 reference standard. Dissolve and dilute each with 0.1 mol / L hydrochloric acid solution to prepare mixed solutions of suitable concentrations. Accurately measure 20 μl of each solution and inject it into the liquid chromatograph. Record the chromatogram and perform serial dilutions. Use a signal-to-noise ratio of approximately 10:1 as the limit of quantitation and a signal-to-noise ratio of approximately 3:1 as the limit of detection. Inject the limit of quantitation solution six times consecutively to examine its peak area RSD. The results are shown in Tables 10 and 11.

[0157] Table 10 Results of Limit of Quantitation Test

[0158] name Concentration (μg / ml) Peak area RSD% Equivalent to the concentration (%) of the test sample solution Impurity A 0.105 0.00* 0.011 Impurity B 0.246 0.73 0.025 Impurity C 0.239 0.50 0.024 Intermediate 3 0.284 1.3 0.028 Sagliptin 0.236 0.28 0.024 Impurity J 0.253 0.72 0.025 Int3-Imp4 0.256 0.52 0.026

[0159] The applicant discovered that when the limit of quantitation solution was injected six times consecutively, the peak area of ​​impurity A was 0.122 mAU·min in all cases.

[0160] Table 11 Results of the detection limit test

[0161] name Concentration (μg / ml) Equivalent to the concentration (%) of the test sample solution Impurity A 0.0315 0.0032 Impurity B 0.0738 0.0074 Impurity C 0.0718 0.0072 Intermediate 3 0.0852 0.0085 Sagliptin 0.0708 0.0071 Impurity J 0.0759 0.0076 Int3-Imp4 0.0769 0.0077

[0162] 4. Linearity and Range

[0163] Accurately weigh appropriate amounts of saxagliptin, impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4 reference standard. Dilute with 0.1 mol / L hydrochloric acid to prepare five groups of reference standard solutions with different concentrations. The concentrations of saxagliptin and each impurity in each group of reference standard solutions are as follows: Group 1: Impurity A approximately 0.1 μg / ml, saxagliptin and other impurities approximately 0.25 μg / ml; Group 2: Saxagliptin and other impurities approximately 0.75 μg / ml; Group 3: Saxagliptin and other impurities approximately 1.5 μg / ml; Group 4: Saxagliptin and other impurities approximately 2.25 μg / ml; Group 5: Saxagliptin and other impurities approximately 3 μg / ml. Accurately inject 20 μl of each group of reference standard solution into the liquid chromatograph, record the chromatogram, and perform linear regression with the injection concentration (µg / ml) as the x-axis and the peak area (A) as the y-axis.

[0164] The results showed that impurity A exhibited good linearity in the range of 0.1009 μg / ml to 3.0259 μg / ml (y = 1.2522x + 0.0100, r = 1.0000); impurity B exhibited good linearity in the range of 0.2513 μg / ml to 3.0160 μg / ml (y = 0.7828x - 0.0040, r = 1.0000); and impurity C exhibited good linearity in the range of 0.2465 μg / ml to 2.9578 μg / ml. The linear relationship was good within the range of l, y = 0.5715x - 0.0114, r = 0.9999; Intermediate 3 showed good linearity in the range of 0.2524 μg / ml to 3.0283 μg / ml, y = 0.5631x + 0.0011, r = 0.9999; Saxagliptin showed good linearity in the range of 0.2493 μg / ml to 2.9918 μg / ml, y = 0.7831x + 0.0119, r = 1.0000; Impurity J showed good linearity in the range of 0.2530~3.0357µg / ml, y=0.4898x-0.0052, r=1.0000; Int3-Imp4 showed good linearity in the range of 0.2562~3.0743µg / ml, y=0.8112x-0.0049, r=1.0000.

[0165] 5. Sample injection precision

[0166] Inject 20 μl of the reference solution from Group 1 under “Linearity and Range” into the liquid chromatograph, repeat 6 times, and record the chromatogram.

[0167] The results showed that the peak area RSD for impurity A was 0.50%; for impurity B, it was 0.25%; for impurity C, it was 0.64%; for intermediate 3, it was 0.78%; for saxagliptin, it was 0.38%; for impurity J, it was 0.18%; and for Int3-Imp4, it was 0.21%. This method demonstrated good injection precision.

[0168] 6. Accuracy

[0169] Take appropriate amounts of saxagliptin, impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4 reference standard, and dilute with 0.1 mol / L hydrochloric acid to prepare solutions containing 1 mg of saxagliptin per ml, with three concentrations of impurity A (approximately 0.035%, 0.15%, and 0.3%), impurities B, C, and intermediate 3 (approximately 0.025%, 0.15%, and 0.2%), and impurity J and Int3-Imp4 (approximately 0.025%, 0.15%, and 0.225%). Prepare three replicates for each concentration. Determine the impurities according to the related substances analysis method, record the chromatograms, and calculate the recovery rate of each impurity using the principal component self-comparison method with correction factors, based on the original amount, the amount added, and the measured amount.

[0170] The results showed that the recovery rates of impurity A were between 103.0% and 107.6%, with an average recovery rate of 104.3% and an RSD of 1.6%; the recovery rates of impurity B were between 102.1% and 105.2%, with an average recovery rate of 104.3% and an RSD of 1.1%; the recovery rates of impurity C were between 101.8% and 112.5%, with an average recovery rate of 106.3% and an RSD of 2.6%; the recovery rates of intermediate 3 were between 101.2% and 105.4%, with an average recovery rate of 103.4% and an RSD of 1.6%; the recovery rates of impurity J were between 105.3% and 107.1%, with an average recovery rate of 106.3% and an RSD of 0.49%; and the recovery rates of Int3-Imp4 were between 99.21% and 101.1%, with an average recovery rate of 100.0% and an RSD of 0.56%. The method demonstrated good accuracy.

[0171] 7. Precision-Repeatability

[0172] Take appropriate amounts of saxagliptin, impurity A, impurity B, impurity C, intermediate 3, impurity J, and Int3-Imp4 reference standard, and dilute with 0.1 mol / L hydrochloric acid to prepare a solution containing 1 mg of saxagliptin, approximately 0.1% of each of impurities A, B, C, and intermediate 3, and approximately 0.15% of impurity J and Int3-Imp4 per ml. Prepare six parallel solutions. Determine the relevant substances according to the analytical method, record the chromatograms, and calculate using the principal component self-comparison method with correction factors.

[0173] The results showed that the repeatability RSD% for impurity A was 0.32% (n=6); for impurity B, it was 0.24% (n=6); for impurity C, it was 0.31% (n=6); for intermediate 3, it was 0.41% (n=6); for impurity J, it was 0.15% (n=6); and for Int3-Imp4, it was 0.40% (n=6). The method exhibited good repeatability.

[0174] 8. Intermediate precision

[0175] Two experimenters, A and B, conducted a set of repeatable experiments at different times and performed the measurements on different instruments (chromatograph models and manufacturers: UltiMate3000, Thermo Fisher Scientific, USA; 1260, Agilent Technologies).

[0176] The results showed that the intermediate precision RSD% for impurity A was 4.6% (n=12); for impurity B, it was 0.0% (n=12); for impurity C, it was 1.3% (n=12); for intermediate 3, it was 2.6% (n=12); for impurity J, it was 3.2% (n=12); and for Int3-Imp4, it was 6.1% (n=12). The intermediate precision of this method is good.

[0177] 9. Solution stability

[0178] Solvent: 0.1 mol / L hydrochloric acid solution

[0179] Stock solution of impurities A, B, and C: Weigh approximately 1 mg each of impurity A, B, and C reference standards, place them in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0180] Intermediate 3 stock solution: Weigh approximately 1 mg of intermediate 3 reference standard accurately, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and the solution is ready.

[0181] Impurity J stock solution: Weigh approximately 10 mg of impurity J reference standard accurately, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0182] Int3-Imp4 stock solution: Weigh approximately 5 mg of Int3-Imp4 reference standard accurately, place it in a 10 ml volumetric flask, add methanol-water (95:5) to dissolve and dilute to the mark, shake well, and the solution is ready.

[0183] Impurity stock solution 1: Accurately measure 3 ml each of impurity A, B, C stock solutions and intermediate 3 stock solution, place them in a 10 ml volumetric flask, dilute with solvent to the mark, and shake well to obtain the solution.

[0184] Impurity Stock Solution 2: Accurately measure 3 ml each of Impurity J Stock Solution and Int3-Imp4 Stock Solution, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0185] System suitability solution: Weigh approximately 20 mg of saxagliptin accurately and place it in a 20 ml volumetric flask. Add 2 ml each of impurity stock solution 1 and impurity stock solution 2, dissolve and dilute to the mark with solvent, and shake well to obtain the solution.

[0186] Test solution: Weigh approximately 20 mg of saxagliptin accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the test solution.

[0187] Reference solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0188] The system suitability solution was placed at 25°C and analyzed within 12 hours to examine its stability. The system suitability solution, test solution, and control solution were placed at 4°C and analyzed within 73 hours to examine the stability of each solution.

[0189] The results showed that at 25℃, intermediate 3 in the system suitability solution was degraded in 0.1 mol / L hydrochloric acid solution, while the peaks of other known impurities did not change. Therefore, the system suitability solution is unstable at 25℃.

[0190] At 4℃, within 73 hours, all known impurities and the main peak in the system suitability solution remained stable; all known impurities in the test solution remained stable and no new impurities appeared; the reference solution remained stable.

[0191] Therefore, the test solution, system suitability solution, and reference solution are stable at 4°C for 73 hours.

[0192] 10. Durability

[0193] Solvent: 0.1 mol / L hydrochloric acid solution

[0194] Stock solutions of impurities A, B, and C: Weigh 1 mg each of impurity A, B, and C reference standards, place them in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0195] Intermediate 3 stock solution: Weigh 1 mg of intermediate 3 reference standard, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and the solution is ready.

[0196] Impurity J stock solution: Weigh 10 mg of impurity J reference standard, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and the solution is ready.

[0197] Int3-Imp4 stock solution: Weigh 5 mg of Int3-Imp4 reference standard, place it in a 10 ml volumetric flask, add 95% methanol to dissolve and dilute to the mark, shake well, and the solution is ready.

[0198] Impurity Stock Solution 1: Accurately measure 3 ml each of impurity A, B, C stock solutions and intermediate 3 stock solution, place them in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0199] Impurity Stock Solution 2: Accurately measure 3 ml each of Impurity J Stock Solution and Int3-Imp4 Stock Solution, place them in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0200] Test solution: Weigh 10 mg of saxagliptin and place it in a 10 ml volumetric flask. Add 1 ml each of impurity stock solution 1 and impurity stock solution 2. Dissolve and dilute with solvent to prepare a solution containing approximately 1 mg of saxagliptin, 0.15 μg each of impurities A, B, C, J, intermediate 3, and Int3-Imp4 per ml.

[0201] Reference solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0202] Accurately measure 20 μl of each of the above-mentioned test solution and reference solution, inject them into the liquid chromatograph, and record the chromatograms. Investigate the effects of changes in chromatographic conditions on the determination results of related substances under different detection wavelengths (213 nm, 215 nm, 217 nm), different mobile phase flow rates (0.5 ml / min, 0.6 ml / min, 0.7 ml / min), different buffer salt pH values ​​in the mobile phase (pH 2.8, pH 3.0, pH 3.2), different column temperatures (38℃, 40℃, 42℃), and different batches of chromatographic columns.

[0203] The results showed that minor changes in detection wavelength, flow rate, pH of buffer salts in the mobile phase, column temperature, and replacement with columns from different batches had virtually no impact on the determination of related substances, and the resolution of each impurity peak met the requirements. This method exhibits good robustness.

[0204] In summary, methodological studies on specificity, limit of quantitation and limit of detection, linearity and range, injection precision, accuracy, repeatability, intermediate precision, solution stability and robustness demonstrate that this method is feasible and suitable for the detection of related substances (impurity A, impurity B, impurity C, intermediate 3, impurity J and Int3-Imp4) in saxagliptin.

[0205] Example 10 Sample Testing

[0206] According to the detection method described in Example 8, four batches of saxagliptin (S19111917, 20200301, 20200302, 20200401) samples were tested respectively.

[0207] Solvent: 0.1 mol / L hydrochloric acid solution

[0208] Test solution: Weigh approximately 20 mg of saxagliptin accurately, place it in a 20 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well to obtain the test solution.

[0209] Reference solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well to obtain the solution.

[0210] Accurately measure 20 μl each of the test solution and the reference solution, inject them into the liquid chromatograph, and record the chromatograms. Calculate the percentage content of each component using the principal component self-comparison method with correction factors.

[0211] The results showed that the contents of impurity A in multiple batches of saxagliptin samples ranged from 0.0044% to 0.016%, impurity B from undetectable to 0.0020%, impurity C from undetectable to 0.0062%, impurity J from 0.0051% to 0.073%, Int3-Imp4 from 0.0014% to 0.025%, intermediate 3 was undetectable, other single impurities ranged from 0.025% to 0.044%, and total impurities ranged from 0.034% to 0.14%. All the test results met the requirements.

[0212] Compare with Example 1

[0213] The detection was performed using the same conditions and methods as in Example 2, except that the composition of the mobile phase was changed. Specifically, the mobile phase was:

[0214] Mobile phase A: Methanol-water-trifluoroacetic acid (10:90:0.08)

[0215] Mobile phase B: Methanol-water-trifluoroacetic acid (90:10:0.08)

[0216] The results showed that, under these chromatographic conditions, impurity A and impurity C did not achieve baseline separation.

[0217] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0218] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for detecting related substances in saxagliptin, characterized in that, The detection method employs high-performance liquid chromatography (HPLC), and the HPLC conditions include: Chromatographic column: C18 column; Mobile phase A: a mixed solution of potassium dihydrogen phosphate and acetonitrile, with a volume ratio of (80~99): (1~20); Mobile phase B: A mixed solution of potassium dihydrogen phosphate and acetonitrile, with a volume ratio of (30~50): (50~70); Linear gradient elution, wherein the gradient elution procedure is as follows: Among them, 95≤a1≤100; 30≤t1≤40, 4≤t2≤6, 1≤t3≤3, 6≤t4≤10; Alternatively, the gradient elution procedure may be: Among them, 95≤b1≤100, 40≤b2≤90; 15≤T1≤25, 10≤T2≤25, 4≤T3≤12, 0.5≤T4≤1.5, 8≤T5≤10.

2. The detection method as described in claim 1, characterized in that, The concentration of potassium dihydrogen phosphate solution in mobile phase A and mobile phase B is 0.01~0.1 mol / L, preferably 0.04~0.06 mol / L, and more preferably 0.05 mol / L.

3. The detection method as described in claim 1, characterized in that, The pH of the potassium dihydrogen phosphate solution in mobile phase A and mobile phase B is adjusted to 2-4 using phosphoric acid, preferably to 2.8-3.2, and more preferably to 3.

4. The detection method as described in claim 1, characterized in that, The volume ratio of the mixed solution of potassium dihydrogen phosphate and acetonitrile in the mobile phase A is (90~99): (1~10), preferably (95~99): (1~5), and more preferably 95:5; the volume ratio of the mixed solution of potassium dihydrogen phosphate and acetonitrile in the mobile phase B is (35~45): (55~65), preferably (38~42): (58~62), and more preferably 40:

60.

5. The detection method as described in claim 1, characterized in that, a1=100; t1=35, t2=5, t3=2, t4=8; Or b1=100, b2=80; T1=20, T2=20, T3=5, T4=1, T5=9; Or b1=100, b2=50; T1=20, T2=15, T3=5, T4=1, T5=9; Or b1=100, b2=55; T1=20, T2=15, T3=10, T4=1, T5=9.

6. The detection method as described in claim 1, characterized in that, The chromatographic conditions optionally further include: Detection wavelength: 210~220nm, preferably 213~217nm; Flow rate: 0.2~1.0 ml / min, preferably 0.5~0.7 ml / min; Column temperature: 20~50℃, preferably 38~42℃; Injection volume: 10~60μl, preferably 15~25μl.

7. The detection method according to any one of claims 1-6, characterized in that, The detection method includes the following steps: (1) Preparation of test solution and reference solution; (2) Measure the test solution and the reference solution separately, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the percentage content of each relevant substance in the test sample according to the principal component self-comparison method with correction factor.

8. The detection method as described in claim 7, characterized in that, The preparation methods of the test solution and reference solution in step (1) include: Preparation of test solution: Take an appropriate amount of saxagliptin, dissolve it in solvent and dilute it to a concentration of 0.2~5mg saxagliptin per 1ml, and use it as the test solution; Preparation of reference solution: Take the test solution and dilute it with solvent to 100 times the volume to obtain the reference solution.

9. The detection method as described in claim 8, characterized in that, The method for preparing the test solution in step (1) includes: Preparation of test solution: Take an appropriate amount of saxagliptin, dissolve it in solvent and dilute it to a concentration of 0.5~2mg saxagliptin per ml, which is used as the test solution.

10. The detection method as described in claim 8, characterized in that, The solvent in step (1) is a hydrochloric acid solution of 0.05~0.2 mol / L, preferably a hydrochloric acid solution of 0.1 mol / L.

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