Methods and applications for detecting nitrogen oxide impurities in vonoprazan fumarate tablets

Abstract

This invention relates to the field of drug detection technology, specifically to a method for detecting nitrogen oxide impurities in vonoprazan fumarate tablets and its application. The invention employs liquid chromatography-mass spectrometry (LC-MS) to detect the nitrogen oxide impurities in the sample. The detection method provided by this invention can achieve qualitative and quantitative analysis of nitrogen oxide impurities in vonoprazan fumarate tablets, and has advantages such as high impurity separation, good specificity, high precision, good repeatability, and high sensitivity. The method is accurate and reliable and can be widely applied to the quality testing of vonoprazan fumarate tablets.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and in particular to a method for detecting nitrogen oxide impurities in vonoprazan fumarate tablets and its application. Background Technology

[0002] Vonoprazan fumarate tablets are a potassium-competitive acid blocker (P-CAB), whose important clinical applications include use in combination with antibiotics to eradicate Helicobacter pylori and to treat gastroesophageal reflux disease. The active compound of vonoprazan fumarate tablets is 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate (this compound can also be called "vonoprazan fumarate"), with the following chemical structural formula: .

[0003] Vonoprazan fumarate tablets or their raw materials (such as the aforementioned compounds before being processed into finished drugs, or semi-finished products during processing) are easily oxidized during storage to generate nitrogen oxides with genotoxicity warning structures. Generally, the pathways for nitrogen oxide impurities in vonoprazan fumarate tablets include: under aerobic conditions, vonoprazan undergoes the following oxidation reactions, generating two different types of nitrogen oxide impurities:

[0004]

[0005] Unlike other impurities (such as hydrolyzed impurities), the aforementioned nitrogen oxide impurities have lower control limits (for example, the acceptable limit for genotoxic impurities is usually at the ppm or even ppb level). They are often difficult to detect using conventional detection methods when their content is below the control limit, posing a significant challenge to the quality control and testing of products.

[0006] However, there are currently no reports on the qualitative and quantitative detection of nitrogen oxide impurities in the two types of fumarate vonoprazan tablets mentioned above in the existing technology. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies in the lack of targeted and accurate detection methods for genotoxic nitrogen oxide impurities in vonoprazan fumarate tablets, which are present in extremely low amounts, and to provide a method for detecting nitrogen oxide impurities in vonoprazan fumarate tablets and its application.

[0008] Typically, the limits for genotoxic impurities are established based on ICH M7 assessment and control of mutagenic impurities, with an intake dose of 1.5 μg / day. The finished product formulation of vonoprazan fumarate tablets is 20 mg (equivalent to 26.72 mg of vonoprazan fumarate), with a maximum daily dose of 2 tablets. Calculations show that the limit for genotoxic impurities is 0.0028% by weight (28 ppm), meaning the limit for genotoxic impurities in vonoprazan fumarate tablets must not exceed 28 ppm. Therefore, it is evident that the detection of genotoxic impurities in vonoprazan fumarate tablets during drug quality control presents significant challenges due to the low limit.

[0009] The inventors of this invention have conducted extensive research to address the aforementioned problems and have provided a detection method using liquid chromatography-mass spectrometry (LC-MS), in which liquid chromatography achieves good separation, and mass spectrometry qualitatively identifies nitrogen oxide impurities, thereby enabling qualitative and quantitative analysis of nitrogen oxide impurities in fumarate flavoxate tablets. The method of this invention has advantages such as high impurity separation, good specificity, high precision, good repeatability, and high sensitivity. The method is accurate and reliable and can be applied to the quality detection of fumarate flavoxate tablets or their active ingredient raw materials.

[0010] To achieve the above objectives, the present invention provides the following technical solution: Through extensive research, the inventors of this invention discovered that a specific liquid chromatography-mass spectrometry (LC-MS) method can achieve better separation when detecting vonoprazan fumarate tablets, thereby enabling the detection and analysis of extremely low levels of nitrogen oxide impurities in further mass spectrometry analysis.

[0011] Based on this, the first aspect of the present invention provides a method for detecting nitrogen oxide impurities in a 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate sample, the method comprising: detecting the sample using liquid chromatography-mass spectrometry to obtain the content of nitrogen oxide impurities in the sample, wherein the mobile phase used in the liquid chromatography includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate with a concentration of 0.5-1.5 g / L, and mobile phase B is acetonitrile; The nitrogen oxide impurities include any one or a combination of methylamine nitrogen oxides and pyrrole ring nitrogen oxides.

[0012] In the method provided by this invention, the 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate sample (which may also be referred to as "sample" in this invention) may be vonoprazan fumarate tablets (finished drug), its raw materials (such as compounds, active pharmaceutical ingredients, etc.) or semi-finished products.

[0013] According to some preferred embodiments of the present invention, the sample comprises vonoprazan fumarate tablets (finished drug) and / or its active pharmaceutical ingredient.

[0014] In this invention, "active pharmaceutical ingredient" refers to any substance or mixture of substances used in the manufacture of a pharmaceutical product, which, when used in pharmaceutical manufacturing, becomes an active ingredient of the drug. This substance has pharmacological activity or other direct effects in the diagnosis, treatment, symptom relief, management, or prevention of a disease, or can affect the function and structure of the body. For example, it can be a chemically synthesized active pharmaceutical ingredient (compound), or a pharmaceutical composition containing the active ingredient and (at least partially) excipients, but does not form a finished drug product.

[0015] In this invention, the concentration of the ammonium acetate aqueous solution, which is the fluid A, can be 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, or 1.5 g / L, or it can be any range consisting of any two of the above values, or any intermediate value within that range.

[0016] In this invention, the acetonitrile used as flowable component B preferably has a purity of not less than 95%, for example, it can be 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 100%, or any range consisting of any two of the above values, or any intermediate value within that range. Purity refers to the mass fraction of acetonitrile in flowable component B.

[0017] According to a preferred embodiment of the present invention, the nitrogen oxide impurities include any one or a combination of at least two of methylamine nitrogen oxides and pyrrole ring nitrogen oxide impurities having genotoxic structures.

[0018] In some preferred embodiments, the nitrogen oxide impurities comprise the structures shown in formula (1) and / or formula (2): Equation (1); Equation (2).

[0019] For ease of description, impurities having the structure shown in formula (1) can also be referred to as "impurity 1" and impurities having the structure shown in formula (2) can be referred to as "impurity 2".

[0020] According to a preferred embodiment of the present invention, the pH value of the mobile phase A is 4.5-5. For example, the pH value of the mobile phase A can be 4.5, 4.6, 4.7, 4.8, 4.9, or 5, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0021] Preferably, the pH value of fluid A can be adjusted using acetic acid.

[0022] According to a preferred embodiment of the present invention, the chromatographic column used in the liquid chromatography is a hybrid matrix phenyl column.

[0023] Preferably, the chromatographic column used in the liquid chromatography has a particle size of 1.8-5 μm, a column length of 100-250 mm, and an inner diameter of 4-6 mm.

[0024] More preferably, the chromatographic column used in the liquid chromatography has a particle size of 2.5-3 μm and a column length of 100-150 mm.

[0025] For example, the particle size of the chromatographic column used in the liquid chromatography can be 2.5μm, 2.55μm, 2.6μm, 2.65μm, 2.7μm, 2.75μm, 2.8μm, 2.85μm, 2.9μm, 2.95μm, or 3μm, or it can be any range consisting of any two of the above values, or any intermediate value within that range.

[0026] For example, the column length of the chromatographic column used in the liquid chromatography can be 100mm, 105mm, 110mm, 115mm, 120mm, 125mm, 130mm, 135mm, 140mm, 145mm, or 150mm, or it can be any range of any two of the above values, or any intermediate value in that range.

[0027] For example, the inner diameter of the chromatographic column used in the liquid chromatography can be 4 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, or 6 mm, or it can be any range of any two of the above values, or any intermediate value within that range.

[0028] In a particularly preferred embodiment, the liquid chromatography uses a column with a particle size of 2.7 μm, a column length of 100 mm, and an inner diameter of 4.6 mm.

[0029] According to a preferred embodiment of the present invention, the liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows: During the first 0-5 minutes, the volume of fluidity B remained at 4%. Over 5-20 minutes, the volume of mobile phase B changed from 4% to 13%. Over 20-25 minutes, the volume of mobile phase B changed from 13% to 15%. Between 25 and 29 minutes, the volume of mobile phase B changed from 15% to 4%. During 29-35 minutes, the volume of mobile phase B was maintained at 4%.

[0030] According to a preferred embodiment of the present invention, the column temperature of the liquid chromatogram is 30-40°C. For example, it can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0031] According to a preferred embodiment of the present invention, the injection flow rate of the liquid chromatograph is 0.5-0.7 mL / min. For example, it can be 0.5 mL / min, 0.52 mL / min, 0.54 mL / min, 0.55 mL / min, 0.56 mL / min, 0.58 mL / min, 0.6 mL / min, 0.62 mL / min, 0.64 mL / min, 0.65 mL / min, 0.66 mL / min, 0.68 mL / min, or 0.7 mL / min, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0032] According to a preferred embodiment of the present invention, the ultraviolet detection wavelength of the liquid chromatography is 255-265 nm. For example, it can be 255 nm, 256 nm, 257 nm, 258 nm, 259 nm, 260 nm, 261 nm, 262 nm, 263 nm, 264 nm, or 265 nm, or it can be any range consisting of any two of the above values, or any intermediate value in the range.

[0033] According to a preferred embodiment of the present invention, the injection volume of the liquid chromatograph is 1-5 μL. For example, it can be 1 μL, 1.5 μL, 2 μL, 2.5 μL, 3 μL, 3.5 μL, 4 μL, 4.5 μL, 5 μL, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0034] According to a preferred embodiment of the present invention, the concentration of vonoprazan fumarate in the sample is 0.5-1.5 mg / mL. The concentration of "vonoprazan fumarate in the sample" refers to the concentration of vonoprazan fumarate in the sample solution prepared for loading during the detection. Based on the content of nitrogen oxide impurities detected in the prepared sample solution, those skilled in the art can calculate the content of nitrogen oxide impurities in the sampled sample (such as vonoprazan fumarate tablets or its raw material), and the specific calculation process will not be elaborated here. For example, the concentration of vonoprazan fumarate in the sample (solution) can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, or a range consisting of any two of the above values, or any intermediate value within that range.

[0035] According to a preferred embodiment of the present invention, the content of nitrogen oxide impurities in the sample is 0.1 ppm or more, preferably 1 ppm or more, and more preferably 1-10 ppm. "The content of nitrogen oxide impurities in the sample" refers to the content of nitrogen oxide impurities (preferably impurities having the structure of the aforementioned formula (1) and / or formula (2)) in the sampled sample (such as vonoprazan fumarate tablets or its raw material). As mentioned above, the content of nitrogen oxide impurities in the sample is calculated based on the detection results and the concentration of the prepared sample solution. In the present invention, unless otherwise specified, "ppm" refers to parts per million by mass, i.e., 1 ppm = 0.0001 by weight.

[0036] In the method of the present invention, any mass spectrometry detection method suitable for coupling with the aforementioned liquid chromatography can be used to detect nitrogen oxide impurities in the sample.

[0037] According to some preferred embodiments of the present invention, the mass spectrometry is performed using a triple quadrupole mass spectrometer.

[0038] Preferably, the mass spectrometry is performed using an ESI mass spectrometer.

[0039] More preferably, the ion source of the mass spectrometer is set to ESI positive ion mode.

[0040] According to a preferred embodiment of the present invention, the sheath gas pressure of the mass spectrometer is 60-65 arb. For example, it can be 60 arb, 60.5 arb, 61 arb, 61.5 arb, 62 arb, 62.5 arb, 63 arb, 63.5 arb, 64 arb, 64.5 arb, or 65 arb, or it can be a range consisting of any two of the above values, or any intermediate value within that range.

[0041] The second aspect of the present invention provides the application of the method described in the first aspect in the quality testing of pharmaceutical products or raw materials containing 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate or the same.

[0042] In this invention, the pharmaceutical product containing 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate or its raw material refers to a vonoprazan fumarate product or a pharmaceutical raw material containing vonoprazan fumarate.

[0043] According to a preferred embodiment of the present invention, the pharmaceutical product containing 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate or its raw materials include vonoprazan fumarate (such as vonoprazan fumarate tablets, etc.) and its raw material.

[0044] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The detection method of liquid chromatography-mass spectrometry provided by the present invention can simultaneously determine at least two nitrogen oxide impurities in fumarate tablets (such as impurity 1 and impurity 2 in the present invention). The method is interference-free and has the advantages of good specificity, high impurity separation, high precision, good durability, and suitability for quality control. The method is accurate and reliable.

[0045] (2) The detection method provided by the present invention has high sensitivity. For example, based on fumarate formora, the detection sensitivity limits of nitrogen oxide impurities (impurity 1 and impurity 2) in the present invention can reach 0.002 μg / mL and 0.003 μg / mL, respectively, and the detection limits can be as low as 0.0006 μg / mL and 0.001 μg / mL, respectively. Attached Figure Description

[0046] Figure 1 This is the liquid chromatography spectrum of the Poroshell 120 Phenyl-Hexyl column (hybrid matrix phenyl column) used in Example 1.

[0047] Figure 2 This is the liquid chromatography spectrum obtained using the Agilent ZORBAX Silica column (porous silica column) in Example 1.

[0048] Figure 3 This is a liquid phase spectrum of the mobile phase ratio used in Example 3 with Group 1.

[0049] Figure 4This is the liquid phase spectrum of mobile phase A with a pH of 4.0 in Example 4.

[0050] Figure 5 This is the liquid phase spectrum of Example 5 with a column temperature of 40°C.

[0051] Figure 6 This is the mass spectrometry qualitative spectrum of impurity 1 in the specificity test of Example 8.

[0052] Figure 7 This is the mass spectrometry qualitative spectrum of impurity 2 in the specificity test of Example 8. Detailed Implementation

[0053] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following embodiments are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0054] Example 1: Determination of Chromatographic Column In this embodiment, ultrapure water was used to avoid impurities introduced by the water from affecting the test results.

[0055] This embodiment compares the test results using different types of chromatographic columns in liquid chromatography, specifically including: (1) Solution preparation Test solution: Take vonoprazan fumarate raw material (i.e., crystalline powder of vonoprazan fumarate with a purity of about 98% synthesized, purchased from Chongqing Boteng Pharmaceutical Technology Co., Ltd.) that has been stored at 40℃ for 30 days, accurately weigh it, dissolve it in water and dilute it quantitatively to prepare a solution with a concentration of about 1.0 mg / mL, as the test solution.

[0056] (2) Chromatographic conditions Mobile phase A: 1 g / L ammonium acetate aqueous solution (pH adjusted to 4.5 with acetic acid); Mobile phase B: Anhydrous acetonitrile (purity approximately 99.8%, i.e., acetonitrile mass fraction approximately 99.8%). Flow rate: 0.6 mL / min; Detection wavelength: 260 nm; Column temperature: 35℃; The elution gradient of the mobile phase is shown in Table 1, where the proportions of mobile phases A and B change at a constant rate from 5 to 20 min and from 25 to 29 min. Table 1 Elution gradient conditions

[0057] (3) Experimental results The chromatographic column type used and the detection results are shown in Table 2: Table 2 Detection results of different chromatographic columns

[0058] Conclusion: Using column 1, the peaks of impurity 1 (structure as shown in formula (1)) and impurity 2 (structure as shown in formula (2)) can be effectively detected. Figure 1 Impurities 1 and 2 were well separated from their adjacent peaks; however, when using column 2, impurities 1 and 2 did not elute, indicating that the hybrid matrix phenyl column can detect nitrogen oxide impurities (such as impurities 1 and 2 in this invention) in fumarate tablets.

[0059] Example 2: Determination of the chromatographic mobile phase This embodiment compares different mobile phases in gradient elution in high performance liquid chromatography, specifically including: (1) Based on the detection conditions in Example 1, using chromatographic column 1, the type of mobile phase was changed according to the conditions described below, while other conditions remained unchanged: Group 1: Same mobile phase as in Example 1; Group 2: The only difference from the mobile phase in Example 1 is that mobile phase B is replaced with anhydrous methanol (purity of about 99.9%, i.e., methanol mass fraction of about 99.9%).

[0060] (2) Experimental results The corresponding detection results using the mobile phases of Group 1 and Group 2 are shown in Table 3: Table 3 Detection results for different mobile phases

[0061] Conclusion: When using ammonium acetate buffer-acetonitrile as the mobile phase, the peaks of the target impurities (impurity 1 and impurity 2) are well separated from the adjacent peaks. However, when using ammonium acetate buffer-methanol as the mobile phase, the peak shapes of the target impurities are poor.

[0062] Example 3: Determination of the ratio of chromatographic mobile phase This embodiment compares the proportions of mobile phases in gradient elution in high-performance liquid chromatography, specifically including: (1) Based on the detection conditions of Example 1, using chromatographic column 1, the proportion of the mobile phase was changed according to the conditions described below, while other conditions remained unchanged: Group 1: The only difference from the mobile phase elution gradient in Example 1 is that at the 20th minute, the volume percentage of mobile phase A is 83% and the volume percentage of mobile phase B is 17%. Group 2: The only difference from the mobile phase elution gradient in Example 1 is that at the 29th minute, the volume percentage of mobile phase A is 94% and the volume percentage of mobile phase B is 6%. Group 3: Same elution gradient as in Example 1; Group 4: The only difference from the mobile phase elution gradient in Example 1 is that at the 20th minute, the volume percentage of mobile phase A is 87% and the volume percentage of mobile phase B is 13%. Group 5: The only difference from the mobile phase elution gradient in Example 1 is that at the 29th minute, the volume percentage of mobile phase A is 98% and the volume percentage of mobile phase B is 2%.

[0063] (2) Experimental results The detection results under different gradient elution procedures for different groups are shown in Table 4: Table 4. Detection results for different mobile phase elution programs

[0064] Conclusion: At the 20th minute, the proportion of mobile phase A was 85-87%, indicating good separation; at the 29th minute, the proportion of mobile phase A was 96%, indicating good separation.

[0065] Example 4: Determination of pH value of chromatographic mobile phase A This embodiment compares the pH value of mobile phase A in liquid chromatography, specifically including: (1) Based on Example 1, using chromatographic column 1, the pH value of mobile phase A was changed to 4.0, 4.5, 5.0 and 5.5 respectively, while other conditions remained unchanged; (2) Experimental results The test results for different pH values ​​are shown in Table 5: Table 5. Detection results of mobile phase A at different pH values

[0066] Conclusion: When the pH of mobile phase A is between 4.5 and 5.0, the peak shape of the target impurity is good.

[0067] Example 5: Determination of the column temperature range for chromatography This embodiment compares column temperatures in high-performance liquid chromatography, specifically including: (1) Based on Example 1, using chromatographic column 1, the column temperature of the chromatographic column was changed to 30℃, 35℃ and 40℃ respectively, while other conditions remained unchanged; (2) Experimental results The test results at different column temperatures are shown in Table 6: Table 6. Detection results at different column temperatures

[0068] Conclusion: When the column temperature is between 30-35℃, the separation between the target impurity peak and the adjacent peak is good.

[0069] Example 6: Determination of chromatographic conditions and flow rate This embodiment compares the injection flow rate in high-performance liquid chromatography (HPLC) detection, specifically including: (1) Based on Example 1, using column 1, the injection flow rate was changed to 0.4 mL / min, 0.5 mL / min, 0.7 mL / min and 0.8 mL / min, while keeping other conditions unchanged; (2) Experimental results The detection results for different injection flow rates are shown in Table 7: Table 7 Detection results at different injection flow rates

[0070] Conclusion: When the flow rate is between 0.5 and 0.7 mL / min, the chromatographic method has good system suitability (peak shape, resolution, and sensitivity, etc.).

[0071] Example 7: Determination of sheath gas pressure in mass spectrometry detection This embodiment compares the corresponding detection sensitivities for different sheath gas pressures in mass spectrometry detection, specifically including: (1) Based on Example 1, using column 1, detection was performed according to the chromatographic conditions provided in Example 1. The mass spectrometry conditions are as follows: Ion source: ESI+; Spray voltage: 3500V(+); Sheath gas pressure: Select 55 arb, 60 arb, and 65 arb respectively; Auxiliary gas pressure: 10 arb; Evaporation temperature: 350℃; Ion transfer tube temperature: 350℃; Scan mode: FulMS-ddMS2; Ful MS: Scan range is 200-3000 m / z, resolution is 70000; MS2: Resolution is 17500; Step NCE: 20, 40, 60; TopN: 3.

[0072] (2) Experimental results The detection sensitivity results under different sheath gas pressures are shown in Table 8: Table 8. Detection results under different sheath gas pressures

[0073] Conclusion: The mass spectrometry response sensitivity is good when the sheath gas pressure is between 60 and 65 arb.

[0074] Example 8 Specificity Test (1) Chromatographic conditions Same as Example 7.

[0075] (2) Mass spectrometry conditions Sheath gas pressure: 60 arb Other conditions are the same as in Example 7.

[0076] Ion source: ESI+; Spray voltage: 3500V(+); Sheath gas pressure: 60 arb; Auxiliary gas pressure: 10 arb; Evaporation temperature: 350℃; Ion transfer tube temperature: 350℃; Scan mode: FulMS-ddMS2; Ful MS: Scan range is 200-3000 m / z, resolution is 70000; MS2: Resolution is 17500; Step NCE: 20, 40, 60; TopN: 3.

[0077] (3) Test method Blank solvent: Mobile phase A: Mobile phase B = (80:20, V / V); System suitability solution: Accurately weigh vonoprazan fumarate (high temperature 40℃, 30 days), dissolve in water and quantitatively dilute to prepare a solution with a concentration of about 1.0 mg / mL, which is used as the system suitability solution.

[0078] The test results are shown in Table 9: Table 9 Specificity Detection Results

[0079] Conclusion: The blank solvent did not interfere with the sample determination; the analyte peaks were of a single composition; and the separation between the impurity peaks was good, indicating that the detection method of this invention has good specificity. The mass spectrometry qualitative spectra corresponding to impurity 1 and impurity 2 are as follows: Figure 6 and Figure 7 .

[0080] Example 9 Sensitivity Test The testing method is as follows: Blank solvent: Mobile phase A: Mobile phase B = (96:4, V / V); Sensitivity test solution: Take impurity 1 and impurity 2 reference standards, accurately weigh them, dissolve them in water and dilute them quantitatively. When the signal-to-noise ratio (S / N) is 10:1 and the signal-to-noise ratio (S / N) is 3:1, the determination is carried out. These are the limits of quantitation and the limits of detection of the method of the present invention. The chromatographic and mass spectrometry conditions are as described in Example 8.

[0081] The test results are shown in Table 10: Table 10 Sensitivity Detection Results

[0082] Conclusion: The limits of quantitation (LOQ) for nitrogen oxide impurities 1 and 2 are 2 ppm and 3 ppm, respectively. However, using the method of this invention, the detection limits for impurities 1 and 2 reach 0.6 ppm and 0.1 ppm, respectively. This demonstrates that the method of this invention possesses extremely high sensitivity, accurately detecting the content of impurities 1 and 2 in samples within the LQ limits, thus providing a foundation for high-level quality control testing of vonoprazan fumarate tablets and their active pharmaceutical ingredients.

[0083] Example 10 Repeatability Test The testing method is as follows: Blank solvent: Mobile phase A: Mobile phase B = (96:4, V / V); Repeatability test solution: A mixed standard solution (containing vonoprazan fumarate, impurity 1, and impurity 2, with vonoprazan fumarate concentration of 1 mg / mL and impurity 1 and impurity 2 concentrations of 3 μg / mL) was prepared for repeatability testing. The concentration of the test sample solution was 1.0 mg / mL. The preparation method of the test sample solution was the same as in Example 1.

[0084] The chromatographic and mass spectrometry conditions are as described in Example 8.

[0085] The test was repeated 6 times, and the results are shown in Table 11: Table 11 Repeatability Test Results

[0086] Conclusion: The detection data of different groups of nitrogen oxide impurities 1 and 2 are stable, indicating that the method of the present invention has good repeatability.

[0087] Example 11 Stability Test The testing method is as follows: Blank solvent: Mobile phase A: Mobile phase B = (96:4, V / V); Repeatability test solution: A mixed standard solution (containing vonoprazan fumarate, impurity 1, and impurity 2, with vonoprazan fumarate concentration of 1 mg / mL and impurity 1 and impurity 2 concentrations of 3 μg / mL) was prepared for repeatability testing at the limit concentration. The test sample concentration was 1.0 mg / mL. The preparation method of the test sample solution was the same as in Example 1.

[0088] The chromatographic and mass spectrometry conditions are as described in Example 8.

[0089] The test results are shown in Table 12: Table 12 Stability Test Results

[0090] Conclusion: The results showed that the peak areas of impurity 1 and impurity 2 did not change significantly when the test sample was tested at different times after preparation, indicating that the test sample solution was stable within 16 hours.

[0091] The above embodiments are merely illustrative of specific implementations of the present invention, but the scope of protection of the present invention is not limited thereto. Those skilled in the art should understand that any variations or substitutions made by those skilled in the art based on the technical solutions disclosed in this invention fall within the scope of protection and disclosure of this invention.

Claims

1. A method for detecting nitrogen oxide impurities in a sample of 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate, the method comprising: The sample was analyzed by liquid chromatography-mass spectrometry to obtain the content of nitrogen oxide impurities in the sample. The liquid chromatography method is characterized in that the mobile phase includes mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate with a concentration of 0.5-1.5 g / L, and mobile phase B is acetonitrile. The nitrogen oxide impurities include any one or a combination of methylamine nitrogen oxides and pyrrole ring nitrogen oxides.

2. The method according to claim 1, wherein, The nitrogen oxide impurities include structures shown in formula (1) and / or formula (2): Equation (1); Equation (2).

3. The method according to claim 1 or 2, wherein, The pH value of the mobile phase A is 4.5-5.

4. The detection method according to any one of claims 1-3, wherein, The liquid chromatography column used is a hybrid matrix phenyl column; Preferably, the chromatographic column used in the liquid chromatography has a particle size of 1.8-5 μm, more preferably 2.5-3 μm; a column length of 100-250 mm, more preferably 100-150 mm; and an inner diameter of 4-6 mm.

5. The detection method according to any one of claims 1-4, wherein, The liquid chromatography employs a gradient elution method, and the gradient elution procedure is as follows: During the first 0-5 minutes, the volume of fluidity B remained at 4%. Over 5-20 minutes, the volume of mobile phase B changed from 4% to 13%. Over 20-25 minutes, the volume of mobile phase B changed from 13% to 15%. Between 25 and 29 minutes, the volume of mobile phase B changed from 15% to 4%. During 29-35 minutes, the volume of mobile phase B was maintained at 4%.

6. The detection method according to any one of claims 1-5, wherein, The column temperature of the liquid chromatography column is 30-40℃; And / or, the injection flow rate of the liquid chromatography is 0.5-0.7 mL / min; And / or, the ultraviolet detection wavelength of the liquid chromatography is 255-265 nm; And / or, the injection volume of the liquid chromatograph is 1-5 μL.

7. The method according to any one of claims 1-6, wherein, The concentration of 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate in the sample was 0.5-1.5 mg / mL; And / or, in the sample, the content of the nitrogen oxide impurities is above 0.1 ppm, preferably above 1 ppm, and more preferably 1-10 ppm.

8. The detection method according to any one of claims 1-7, wherein, The mass spectrometry is performed using a triple quadrupole mass spectrometer; preferably, the mass spectrometry is performed using an ESI mass spectrometer; more preferably, the ion source of the mass spectrometer is set to ESI positive ion mode.

9. The detection method according to any one of claims 1-8, wherein, The sheath gas pressure of the mass spectrometer is 60-65 alb.

10. The method of any one of claims 1-9 is used in the quality control of pharmaceutical products or raw materials containing 1-[5-(2-fluorophenyl)-1-(pyridin-3-sulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate.

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