Detection method of atozepam

Abstract

The invention discloses a detection method of atozepam. High performance liquid chromatography is adopted, and the chromatographic conditions are as follows: a stationary phase is octadecylsilane chemically bonded silica; mobile phases: a mobile phase A is a 10-20 mmol / L phosphate aqueous solution, and a mobile phase B is acetonitrile and methanol in a ratio of 40: 60-60: 40; the elution gradient is as follows: the volume ratio of the mobile phase A to the mobile phase B is (80-85): (20-15) in 0 min; the volume ratio of the mobile phase A to the mobile phase B is (80-85): (20-15) in 3 minutes; the volume ratio of the mobile phase A to the mobile phase B is (20-15): (80-85) in 10 minutes; the volume ratio of the mobile phase A to the mobile phase B is (20-15): (80-85) in 20 minutes; the volume ratio of the mobile phase A to the mobile phase B is (80-85): (20-15) in 21min; the volume ratio of the mobile phase A to the mobile phase B in 30 minutes is (80-85): (20-15); the column temperature is 25-35 DEG C; the flow velocity is 0.8 to 1.2 mL / min; the detection wavelength is 200 to 220 nm; and the sample size is 8-10 [mu] L. According to the method disclosed by the invention, the atozepam and the related substances can be effectively separated, the purity of the atozepam is accurately determined, the determination result is reliable, the specificity is strong, the sensitivity is high, the accuracy and the linearity are good, and the product quality can be effectively controlled.

Description

Technical Field

[0001] This application relates to the technical field of pharmaceutical analysis, and particularly to a method for detecting atogepant. Background Art

[0002] Migraine is the most common recurrent neurovascular disease worldwide, affecting more than 1 billion people globally, including approximately 39 million in the United States alone. It is one of the leading causes of disability in people under 50 years old globally. The Global Burden of Disease (GBD) study group in 2016 ranked it as the second most disabling disease globally and the first among people under 50 years old. Calcitonin gene-related peptide receptor antagonist (gepant) CGRP monoclonal antibodies (mAbs) have been used for migraine prevention and provide an effective treatment for migraine prevention, but some patients have poor responses or phobias to mAbs.

[0003] Atogepant is an effective and selective oral small molecule CGRP (calcitonin gene-related peptide) receptor antagonist. In September 2021, the US Food and Drug Administration (FDA) approved the atogepant small molecule oral tablet of AbbVie Biopharmaceuticals in the United States. CGRP is a protein released around the brain nervous system, widely distributed in the central, peripheral, and other systems, and is involved in the process of pain transmission in the nervous system, causing strong inflammation of the brain meninges. During a migraine attack, the CGRP level increases, and the role of a CGRP antagonist is to prevent migraines by blocking the transmission mode of CGRP. As a small molecule CGRP receptor antagonist, atogepant can specifically inhibit the intracranial arterial vasodilation response dependent on human CGRP and does not cause human coronary vasoconstriction. Its advantages are prominent, making the development of its detection method the primary task of R & D and analysis personnel.

[0004] Currently, there is no relevant literature on the detection method of atogepant and related substances. The quality of existing products only relies on qualitative means to identify atogepant, and there is no method for quantitatively detecting atogepant. Therefore, it is urgent to develop a corresponding detection method to achieve the purpose of separation and determination of purity, so that the quality of atogepant and related substances is within a controllable range. Summary of the Invention

[0005] To address the above deficiencies in the art, this application aims to provide a method for detecting atogepant. High performance liquid chromatography (HPLC) is used to detect atogepant in order to obtain higher resolution and purity.

[0006] According to one aspect of this application, a method for detecting atogepant is provided, using high performance liquid chromatography, and the chromatographic conditions include:

[0007] Detector: Ultraviolet detector;

[0008] Stationary phase: Octadecylsilyl-bonded silica gel;

[0009] Mobile phase: Mobile phase A is an aqueous solution of phosphate at 10 - 20 mmol / L, and mobile phase B is acetonitrile:methanol = 40:60 to 60:40;

[0010] The elution gradient is:

[0011] At 0 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15;

[0012] At 3 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15;

[0013] At 10 min, the volume ratio of mobile phase A to mobile phase B is 20 - 15:80 - 85;

[0014] At 20 min, the volume ratio of mobile phase A to mobile phase B is 20 - 15:80 - 85;

[0015] At 21 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15;

[0016] At 30 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15;

[0017] Column temperature: 25 - 35 °C;

[0018] Flow rate: 0.8 - 1.2 mL / min;

[0019] Detection wavelength: 200 - 220 nm;

[0020] Sample injection volume: 8 - 10 μL.

[0021] According to some embodiments of the present application, the aqueous phosphate solution is selected from one of the following: aqueous disodium hydrogen phosphate solution, aqueous potassium dihydrogen phosphate solution, and aqueous ammonium dihydrogen phosphate solution.

[0022] According to some embodiments of the present application, mobile phase A is an aqueous solution of 20 mmol / L ammonium dihydrogen phosphate.

[0023] According to some embodiments of the present application, mobile phase B is acetonitrile:methanol = 50:50.

[0024] According to some embodiments of the present application, the elution gradient is:

[0025] At 0 min, the volume ratio of mobile phase A to mobile phase B is 80:20;

[0026] The volume ratio of mobile phase A to mobile phase B is 80:20 for 3 min;

[0027] The volume ratio of mobile phase A to mobile phase B is 20:80 for 10 min;

[0028] The volume ratio of mobile phase A to mobile phase B is 20:80 for 20 min;

[0029] The volume ratio of mobile phase A to mobile phase B is 80:20 for 21 min;

[0030] The volume ratio of mobile phase A to mobile phase B is 80:20 for 30 min.

[0031] According to some embodiments of the present application, the column temperature is 30 °C.

[0032] According to some embodiments of the present application, the flow rate is 1.0 mL / min.

[0033] According to some embodiments of the present application, the detection wavelength is 210 nm.

[0034] According to some embodiments of the present application, the injection volume is 10 μL.

[0035] According to some embodiments of the present application, the area normalization method is used to integrate the detection chromatogram to obtain the determination result of atogepant.

[0036] Compared with the prior art, the present application has at least the following beneficial effects:

[0037] The present application provides a detection method for atogepant, which can effectively separate the peaks of atogepant and related substances, accurately determine the purity of atogepant, and the determination result is stable and reliable, providing the possibility for quality control in the synthesis process of atogepant, thereby better controlling the product quality of atogepant.

[0038] The detection method of the present application can effectively separate atogepant from related substances, accurately determine the purity of atogepant, the determination result is reliable, the specificity is strong, the sensitivity is high, the accuracy is good, and the linearity is good, and can effectively control the product quality. Description of the Drawings

[0039] Figure 1 It is the detection chromatogram of the resolution of the mixed solution in Example 1 of the present application.

[0040] Figure 2 It is the detection chromatogram of the resolution of the mixed solution in Comparative Example 1 of the present application.

[0041] Figure 3 It is the detection chromatogram of the resolution of the mixed solution in Comparative Example 2 of the present application.

[0042] Figure 4 This is the detection chromatogram of the resolution of the mixed solution in Comparative Example 3 of the present application example.

[0043] Figure 5 This is the detection chromatogram of the resolution of the mixed solution in Comparative Example 4 of the present application example.

[0044] Figures 6-10 This is the detection chromatogram of the purity of atogepant in Examples 1-5 of the present application example. Detailed implementation mode

[0045] Next, the technical solutions of the present application will be clearly and completely described in conjunction with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0046] It should be particularly noted that similar replacements and modifications made to the present application are obvious to those skilled in the art, and they are all considered to be included in the present application. Relevant personnel can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present application to implement and apply the technology of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments.

[0047] If no specific conditions are indicated in the present application, they are all carried out according to conventional conditions or conditions recommended by the manufacturer. For the raw materials or excipients used, and for the reagents or instruments used without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0048] The present application will be described in detail below.

[0049] Detection raw materials: Atogepant samples and intermediates prepared in the laboratory.

[0050] Instrument: High performance liquid chromatography detector.

[0051] Structural formula of atogepant:

[0052] Structural formula of intermediate 1:

[0053] Structural formula of intermediate 2:

[0054] Structural formula of impurity:

[0055] Preparation of test solution 1: Weigh appropriate amounts of atogepant intermediate 1 and intermediate 2 precisely into the same volumetric flask, add the standard solution to dissolve and then make up to the mark to obtain the mixed solution required for the experiment.

[0056] Preparation of Test Solution 2: Accurately weigh 50.0 mg of atogepant crude product into a 100 mL volumetric flask, dissolve it with diluent (acetonitrile: water (50:50) v / v), and make up to the scale mark to obtain (the concentration of Test Solution 2 is 0.5 mg / mL).

[0057] Example 1

[0058] The test solution 1 was detected under the following detection conditions:

[0059] Mobile phase selection: Mobile phase A is 20 mmol / L ammonium dihydrogen phosphate aqueous solution, and mobile phase B is acetonitrile: methanol = 50:50;

[0060] The two solvents were used to perform gradient elution on atogepant in a certain proportion, and the elution gradient was as follows:

[0061]

[0062] The column temperature was set at 30 °C;

[0063] The flow rate was set at 1.0 mL / min;

[0064] The detection wavelength was set at 210 nm;

[0065] The injection volume was set at 10 μL;

[0066] Under the above conditions, the atogepant mixed solution was analyzed to obtain the resolution result of the mixed solution, as Figure 1 shown.

[0067] It can be seen from the chromatogram that the resolution between the related substances of atogepant is high, which are 5.65 and 13.33. The peak emergence time of 7.17 min is the peak of intermediate 2, 8.97 min is the peak of intermediate 1, and 11.08 min is the peak of the main structure. The resolution between peaks is all above 5, and the peak emergence position is moderate, and the peak shape is good, which can achieve the purpose of accurately separating and detecting atogepant and related substances.

[0068] The test solution 2 was detected under the above detection conditions, and the chromatogram was integrated by the area normalization method to obtain the purity of atogepant as 99.91%, as Figure 6 shown.

[0069] Method precision test (repeatability test): Accurately weigh 6 portions according to the preparation process of test solution 2, inject samples respectively under the above detection conditions, and integrate the chromatogram by the area normalization method to obtain the purity results of atogepant, as shown in Table 1. It can be seen from Table 1 that the RSD value of the purity of atogepant is 0.004%, which is far less than 2%, indicating that the repeatability of this method is good.

[0070] Results of precision test (repeatability test) in Table 1

[0071]

[0072]

[0073] Example 2

[0074] The test solution 2 was detected under the following detection conditions:

[0075] Detector: UV detector;

[0076] Mobile phase: Mobile phase A is an aqueous solution of 15 mmol / L ammonium dihydrogen phosphate, and mobile phase B is acetonitrile: methanol = 40:60;

[0077] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0078]

[0079] The column temperature was set at 25 °C;

[0080] The flow rate was set at 1.2 mL / min;

[0081] The detection wavelength was set at 210 nm;

[0082] The injection volume was set at 10 μL;

[0083] The chromatogram was integrated by the area normalization method, and the purity of atogepant was obtained as 99.94%, as Figure 7 shown.

[0084] Example 3

[0085] The test solution 2 was detected under the following detection conditions:

[0086] Detector: UV detector;

[0087] Mobile phase: Mobile phase A is an aqueous solution of 20 mmol / L ammonium dihydrogen phosphate, and mobile phase B is acetonitrile: methanol = 50:50;

[0088] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0089]

[0090]

[0091] The column temperature was set at 30 °C;

[0092] The flow rate was set at 1.0 mL / min;

[0093] The detection wavelength is set to 200 nm;

[0094] The injection volume is set to 8 μL;

[0095] The chromatogram was integrated by the area normalization method, and the purity of atogepant was obtained as 99.90%, as Figure 8 shown.

[0096] Example 4

[0097] The test solution 2 was detected under the following detection conditions:

[0098] Detector: UV detector;

[0099] Mobile phase: Mobile phase A is an aqueous solution of 20 mmol / L disodium hydrogen phosphate, and mobile phase B is acetonitrile: methanol = 45:55;

[0100] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0101]

[0102] The column temperature is set to 30 °C;

[0103] The flow rate is set to 1.0 mL / min;

[0104] The detection wavelength is set to 210 nm;

[0105] The injection volume is set to 10 μL;

[0106] The chromatogram was integrated by the area normalization method, and the purity of atogepant was obtained as 99.58%, as Figure 9 shown.

[0107] Example 5

[0108] The test solution 2 was detected under the following detection conditions:

[0109] Detector: UV detector;

[0110] Mobile phase: Mobile phase A is an aqueous solution of 20 mmol / L potassium dihydrogen phosphate, and mobile phase B is acetonitrile: methanol = 60:40;

[0111] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0112]

[0113] The column temperature is set to 30 °C;

[0114] The flow rate was set at 1.0 mL / min;

[0115] The detection wavelength was set at 210 nm;

[0116] The injection volume was set at 10 μL;

[0117] The area normalization method was used to integrate the chromatogram, and the purity of atogepant was obtained as 99.55%, as Figure 10 shown.

[0118] Comparative Example 1

[0119] Detector: UV detector;

[0120] Mobile phase: A: water: methanol = 40:60, B: acetonitrile: methanol = 40:60;

[0121] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0122]

[0123] Detection wavelength: 210 nm;

[0124] Flow rate: 1.0 mL / min;

[0125] Injection volume: 10 μL;

[0126] Column temperature: 30 °C;

[0127] Under the above conditions, the test solution 1 was analyzed to obtain the resolution results of the mixed solution, as Figure 2 shown.

[0128] It can be seen from the chromatogram that the peak shape of this method is good, but the resolution between the related substances of atogepant is not high, only 2.06 and 0.66. The resolution between the two peaks is relatively low. Generally, a resolution of about 1.5 is required, and the peak elution position is relatively forward, which is not suitable for the detection of atogepant.

[0129] Comparative Example 2

[0130] Detector: UV detector;

[0131] Mobile phase: A: water: methanol = 40:60, B: acetonitrile: methanol = 40:60;

[0132] The two solvents were used for gradient elution of atogepant in a certain proportion, and the elution gradient was as follows:

[0133]

[0134] Detection wavelength: 210 nm;

[0135] Flow rate: 1.0 mL / min;

[0136] Sample injection volume: 10 μL;

[0137] Column temperature: 30 °C;

[0138] Under the above conditions, the test solution 1 was analyzed to obtain the resolution results of the mixed solution, as Figure 3 shown.

[0139] It can be seen from the chromatogram that the peak shape of this method is good, but the resolution between the related substances of atogepant is not high, only 1.95 and 1.21. The resolution between the two peaks is low. Generally, a resolution of about 1.5 is required, and the peak elution position is too early, which is not suitable for the detection of atogepant.

[0140] Comparative Example 3

[0141] Detector: UV detector;

[0142] Mobile phase: A: 20 mM ammonium dihydrogen phosphate aqueous solution, B: acetonitrile: methanol = 50:50;

[0143] The two solvents were used to perform gradient elution on atogepant in a certain proportion, and the elution gradient was as follows:

[0144]

[0145] Detection wavelength: 210 nm;

[0146] Flow rate: 1.0 mL / min;

[0147] Sample injection volume: 10 μL;

[0148] Column temperature: 30 °C;

[0149] Under the above conditions, the test solution 1 was analyzed to obtain the resolution results of the mixed solution, as Figure 4 shown.

[0150] It can be seen from the chromatogram that the resolution between the related substances of atogepant is high, 18.98 and 54.05, and the resolution between the peaks is all above 5. However, the peak elution position is too late, and the baseline is not stable, and the impurities with small polarity cannot be eluted, which is not suitable for the detection of atogepant.

[0151] Comparative Example 4

[0152] Detector: differential refractometer;

[0153] Mobile phase: acetonitrile: methanol: 20 mM ammonium dihydrogen phosphate aqueous solution = 60:20:20;

[0154] Isocratic elution of the mobile phase;

[0155] Running time: 30 min;

[0156] Flow rate: 1.0 mL / min;

[0157] Sample injection volume: 10 μL;

[0158] Column temperature: 30 °C;

[0159] Under the above conditions, the test solution 1 was analyzed to obtain the resolution results of the mixed solution, as Figure 5 shown.

[0160] The peak shape of this method is poor, and the resolution between the related substances of atogepant is not high, only 1.28 and 0.73. The resolution between the two peaks is low. Generally, a resolution of about 1.5 is required, and the peak emergence position is forward, which is not suitable for detecting atogepant.

[0161] The description of the above embodiments is only used to help understand the method and its core idea of this application. It should be noted that for those of ordinary skill in the art of this technology, without departing from the principle of this application, several improvements and modifications can also be made to this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A method for detecting atogepant, characterized in that, Using high performance liquid chromatography, the chromatographic conditions include: Stationary phase: octadecylsilyl bonded silica gel; Mobile phase: mobile phase A is an aqueous solution of phosphate at 10 - 20 mmol / L, and mobile phase B is acetonitrile:methanol = 40:60 - 60:40; The elution gradient is: At 0 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15; At 3 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15; At 10 min, the volume ratio of mobile phase A to mobile phase B is 20 - 15:80 - 85; At 20 min, the volume ratio of mobile phase A to mobile phase B is 20 - 15:80 - 85; At 21 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15; At 30 min, the volume ratio of mobile phase A to mobile phase B is 80 - 85:20 - 15; Column temperature: 25 - 35 °C; Flow rate: 0.8 - 1.2 mL / min; Detection wavelength: 200 - 220 nm; Injection volume: 8 - 10 μL.

2. The detection method according to claim 1, characterized in that, The detector is an ultraviolet detector.

3. The detection method according to claim 1, characterized in that, The mobile phase A is an aqueous solution of phosphate at 20 mmol / L; Preferably, the aqueous solution of phosphate is selected from one of aqueous solution of disodium hydrogen phosphate, aqueous solution of potassium dihydrogen phosphate and aqueous solution of ammonium dihydrogen phosphate; Preferably, the aqueous solution of phosphate is an aqueous solution of ammonium dihydrogen phosphate.

4. The detection method according to claim 1, characterized in that, Mobile phase B is acetonitrile:methanol = 50:

50.

5. The detection method according to claim 1, characterized in that, The elution gradient is: At 0 min, the volume ratio of mobile phase A to mobile phase B is 80:20; At 3 min, the volume ratio of mobile phase A to mobile phase B is 80:20; At 10 min, the volume ratio of mobile phase A to mobile phase B is 20:80; At 20 min, the volume ratio of mobile phase A to mobile phase B is 20:80; At 21 min, the volume ratio of mobile phase A to mobile phase B is 80:20; At 30 min, the volume ratio of mobile phase A to mobile phase B is 80:

20.

6. The detection method according to claim 1, characterized in that, The column temperature is 30 °C.

7. The detection method according to claim 1, characterized in that, The flow rate is 1.0 mL / min.

8. The detection method according to claim 1, characterized in that, The detection wavelength is 210 nm.

9. The detection method according to claim 1, characterized in that, The injection volume is 10 μL.

10. The detection method according to claim 1, characterized in that, The area normalization method is used to integrate the detection chromatogram to obtain the purity determination result of atogepant.

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