Method for detecting methylamine in Vonoprasan fumarate

Abstract

The invention belongs to the technical field of pharmaceutical analysis, particularly relates to a method for detecting methylamine in Vonoprasone fumarate, and provides a convenient, efficient and accurate detection method for solving the problem of detection of methylamine in Vonoprasone fumarate. Derivative products are detected by liquid chromatography, the content of methylamine in the Vonoprazan fumarate can be detected, so that medication safety is effectively guaranteed, quality control of the Vonoprazan fumarate is facilitated, the method is convenient, efficient and accurate, the system applicability, repeatability, specificity and accuracy completely conform to the guidance principle verified by methods in Chinese pharmacopoeia, and the method is suitable for popularization and application. The method can be used for quality control of the Vonoprasone fumarate.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting methylamine in vonoprazan fumarate. Background Art

[0002] Vonoprazan fumarate, chemical name: 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethylamine fumarate, structural formula: It is a new type of potassium ion competitive acid blocker used to treat erosive esophagitis, gastric ulcer, duodenal ulcer and Helicobacter pylori.

[0003] Methylamine is a material used in the synthesis process of vonoprazan fumarate, so methylamine may remain in vonoprazan fumarate. Methylamine is a low-toxic alkaline substance that is irritating and corrosive to the skin and mucous membranes, can affect the excitability of the central nervous system, and cause allergic reactions such as asthma. Therefore, the residual amount of methylamine in vonoprazan fumarate needs to be strictly controlled.

[0004] Currently, there are few literature reports on the detection method of methylamine in vonoprazan fumarate. For the safety of the drug, it is very necessary to strictly control the methylamine content in vonoprazan fumarate.

[0005] The invention discloses a method for determining the content of methylamine in vonoprazan fumarate by liquid chromatography, and provides a convenient, efficient and accurate detection method for solving the problem of detecting methylamine in vonoprazan fumarate. The method can detect the content of methylamine in vonoprazan fumarate, thereby effectively ensuring the safety of drug use and facilitating the quality control of vonoprazan fumarate. Summary of the invention

[0006] The invention provides a detection method for determining methylamine in vonoprazan fumarate by liquid chromatography, and provides a convenient, efficient and accurate detection method to solve the detection problem of methylamine in vonoprazan fumarate. The method can detect the content of methylamine in vonoprazan fumarate, thereby effectively ensuring the safety of medication and facilitating the quality control of vonoprazan fumarate. The method is convenient, efficient and accurate, and fully complies with the guiding principles of method verification of the Chinese Pharmacopoeia in terms of system applicability, repeatability, specificity and accuracy, and can be used for the quality control of vonoprazan fumarate.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for testing methylamine in vonoprazan fumarate, characterized in that the method comprises the following steps: derivatizing methylamine with a derivatizing reagent, 1-naphthyl isothiocyanate, and detecting the derivative product by liquid chromatography. The method comprises the following steps: (1) Prepare solutions including blank solution, sensitivity solution, reference solution and test solution.

[0008] (2) Determination method: The content of methylamine in vonoprazan fumarate was determined by liquid chromatography. After the system was stable, blank solution, sensitivity solution, reference solution and test solution were added respectively, and the chromatogram was recorded; The chromatographic conditions are as follows: Detector: UV detector, chromatographic column: chromatographic column with octadecylsilane bonded silica as filler, injection volume: 10-20µl, column temperature: 40±5℃, flow rate: 1.0±0.1ml / min, phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution.

[0009] Furthermore, the chromatographic column is Waters SymmetryShieldTM RP8, 4.6mm×150mm, 5µm.

[0010] Further, the elution gradient is: .

[0011] Further, the mobile phase A: 0.1% phosphoric acid solution; For the blank solution: measure an appropriate amount of diluent and boric acid buffer solution and place them in a centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix again, stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The volume ratio of the diluent: acetonitrile: water is 50:50; The boric acid buffer solution comprises the following steps: taking an appropriate amount of sodium hydroxide, placing it in a volumetric flask, adding water to dissolve it, cooling it, adding water to dilute it to the mark, and shaking it well to obtain a sodium hydroxide solution; taking an appropriate amount of boric acid, placing it in a volumetric flask, adding water to dissolve it and dilute it to the mark, and shaking it well to obtain a boric acid solution; taking an appropriate amount of potassium chloride, placing it in a volumetric flask, adding water to dissolve it and dilute it to the mark, and shaking it well to obtain a potassium chloride solution; measuring an appropriate amount of the above-mentioned boric acid solution and potassium chloride solution, placing them in the same volumetric flask, adding water to dilute it to the mark, and shaking it well; transferring all of the above-mentioned solutions to a beaker, and adjusting the pH value to 10.5 with a sodium hydroxide solution to obtain a boric acid buffer solution; The reference substance solution: measure an appropriate amount of diluent, reference substance stock solution, and boric acid buffer solution and place them in the same centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the same centrifuge tube, vortex mix again, let stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The reference substance stock solution: take an appropriate amount of methylamine hydrochloride reference substance, place it in a volumetric flask, add water to dissolve and dilute to the scale, shake well; take an appropriate amount of the above solution and place it in a volumetric flask, add diluent to dilute to the scale, shake well; The sensitivity solution: measure an appropriate amount of diluent, sensitivity reserve solution, and boric acid buffer solution, place them in a centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix again, stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; Sensitivity stock solution: Take an appropriate amount of reference stock solution, place it in a volumetric flask, add diluent to dilute to the scale, and shake well; The test solution: measure an appropriate amount of diluent, test stock solution, and boric acid buffer solution and place them in the same centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the same centrifuge tube, vortex mix again, let stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The test sample stock solution: Take an appropriate amount of the test sample, place it in a volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0012] Furthermore, the microporous membrane filtration is 0.22 μm microporous membrane filtration.

[0013] The invention discloses a method for detecting methylamine in vonoprazan fumarate, and provides a convenient, efficient and accurate detection method to solve the problem of detecting methylamine in vonoprazan fumarate. The method can detect the content of methylamine in vonoprazan fumarate, thereby effectively ensuring the safety of drug use. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the blank solution spectrum of methylamine in vonoprazan fumarate Figure 2 This is the methylamine sensitivity solution spectrum of vonoprazan fumarate Figure 3 Spectrum of methylamine reference solution in vonoprazan fumarate Figure 4 Spectrum of methylamine test solution in vonoprazan fumarate DETAILED DESCRIPTION

[0015] The present invention is further illustrated by the following examples, but are not intended to limit the present invention.

[0016] Example 1: Determination of methylamine in vonoprazan fumarate (1) Preparation of solution: Phosphoric acid: AR and above; Acetonitrile: HPLC; Water: Ultrapure water; Methylamine hydrochloride reference substance: Homemade or purchased; Sodium hydroxide: AR and above; Boric acid: AR and above; 1-naphthyl isothiocyanate: AR and above; Potassium chloride: AR and above; Diluent: acetonitrile: water = 50:50 (V / V); Boric acid buffer solution (pH 10.5): Take about 20g of sodium hydroxide, place it in a 100ml volumetric flask, add water to dissolve, let it cool, add water to dilute to the mark, shake well, and use it as sodium hydroxide solution (5mol / L). Take about 2.46g of boric acid, place it in a 100ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and use it as boric acid solution (0.4mol / L). Take about 2.98g of potassium chloride, place it in a 100ml volumetric flask, add water to dissolve and dilute to the mark, shake well, and use it as potassium chloride solution (0.4mol / L). Accurately measure 12.5ml of boric acid solution (0.4mol / L) and 12.5ml of potassium chloride solution (0.4mol / L) and place them in a 100ml volumetric flask, add water to dilute to the mark, shake well, transfer all the above solutions to a beaker, and adjust the pH value to 10.5 with sodium hydroxide solution (5mol / L) to use it as boric acid buffer solution (pH 10.5).

[0017] 1-Naphthyl isothiocyanate solution: Take 700 mg of 1-naphthyl isothiocyanate solution, weigh accurately, place in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well. (Concentration: 7 mg / ml) Methylamine stock solution: Take about 21.7 mg of methylamine hydrochloride reference substance, weigh accurately, place in a 20 ml volumetric flask, add water to dissolve and dilute to the mark, shake well; accurately measure 5 ml of the above solution, place in a 50 ml volumetric flask, add diluent to dilute to the mark, shake well. (Concentration: methylamine hydrochloride 108.5 μg / ml, equivalent to methylamine 50 μg / ml) Reference substance stock solution: Accurately measure 5 ml of methylamine stock solution, place in a 50 ml volumetric flask, add diluent to dilute to the mark, and shake well. (Concentration: methylamine hydrochloride 10.85 μg / ml, equivalent to methylamine 5 μg / ml) Sensitivity stock solution: Accurately measure 3 ml of reference stock solution, place in a 10 ml volumetric flask, add diluent to dilute to scale, and shake well. (Concentration: methylamine hydrochloride 3.255 μg / ml, equivalent to methylamine 1.5 μg / ml) Blank solution: Accurately measure 0.4 ml of the diluent and 0.6 ml of the boric acid buffer solution (pH 10.5) and place them in a 10 ml centrifuge tube. Vortex mix for 1 minute. Accurately measure 1 ml of 1-naphthyl isothiocyanate solution and place them in the centrifuge tube. Vortex mix for another 3 minutes. After standing at room temperature for 10 minutes, sonicate for 10 seconds, filter through a 0.22 μm microporous membrane, discard the initial filtrate, and take the subsequent filtrate for sampling.

[0018] Reference solution: accurately measure 0.2 ml of diluent, 0.2 ml of reference stock solution, and 0.6 ml of boric acid buffer solution (pH 10.5) and place them in a 10 ml centrifuge tube, vortex mix for 1 minute, accurately measure 1 ml of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix for another 3 minutes, let stand at room temperature for 10 minutes, ultrasonicate for 10 seconds, filter through a 0.22 μm microporous membrane, discard the initial filtrate, and take the subsequent filtrate for sampling. (Concentration: methylamine hydrochloride 1.085 μg / ml, equivalent to methylamine 0.5 μg / ml) Sensitivity solution: accurately measure 0.2ml of diluent, 0.2ml of sensitivity reserve solution, and 0.6ml of boric acid buffer solution (pH10.5) and place them in a 10ml centrifuge tube, vortex mix for 1 minute, accurately measure 1ml of 1-naphthyl isothiocyanate solution, place it in the centrifuge tube, vortex mix for another 3 minutes, let it stand at room temperature for 10 minutes, ultrasonicate for 10 seconds, filter with a 0.22μm microporous membrane, discard the initial filtrate, and take the subsequent filtrate for sampling. (Concentration: methylamine hydrochloride 0.3255μg / ml, equivalent to methylamine 0.15μg / ml) Test sample stock solution: Take about 50 mg of the test sample, weigh accurately, place in a 10 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well. (Concentration: 5 mg / ml) Test solution: accurately measure 0.2ml of diluent, 0.2ml of test stock solution, and 0.6ml of boric acid buffer solution (pH10.5) and place them in a 10ml centrifuge tube, vortex mix for 1 minute, accurately measure 1ml of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix for another 3 minutes, let stand at room temperature for 10 minutes, ultrasonicate for 10 seconds, filter through a 0.22μm microporous membrane, discard the initial filtrate, and take the subsequent filtrate for sampling. (Concentration: 0.5mg / ml) Note: The 0.22μm microporous filter membrane is a nylon filter membrane produced by Welch.

[0019] (2) Chromatographic conditions: Instruments: High performance liquid chromatograph equipped with UV detector, electronic analytical balance, precision pH meter, vortex analyzer; Chromatographic column: Use octadecylsilane bonded silica as filler (such as Waters SymmetryShield TM RP8, 4.6mm×150mm, 5µm or equivalent); Flow rate: 1ml / min; Detection wavelength: 283nm; Injection volume: 20µl; Column temperature: 40℃; Injection plate temperature: 15℃; Mobile phase A: 0.1% phosphoric acid solution (precisely measure 1000 ml of ultrapure water, add 1 ml of phosphoric acid, mix well, and sonicate to obtain); Mobile phase B: acetonitrile; Gradient table:

[0020] (3) Steps After the system is stable, inject 1 injection of blank solution (1 to 3 injections if necessary), 1 injection of sensitivity solution, 6 injections of reference solution, 1 injection of test solution, and record the chromatogram.

[0021] Require The S / N value of methylamine in the sensitivity solution should be ≥10, and the RSD of the methylamine peak area in the 6-point reference solution should be ≤10.0%.

[0022] calculate Methylamine (%) = (Ru / Rs) × (Cs / Cu) × 10 -3 ×(M1 / M2) Where: R u : The peak area of ​​methylamine in the spectrum of the test solution; R s : 6 is the average peak area of ​​methylamine in the reference solution; C s : Concentration of methylamine hydrochloride in the reference solution (μg / ml); C u : Concentration of test solution (mg / ml); M1: molecular weight of methylamine, 31.06; M2: molecular weight of methylamine hydrochloride, 67.52.

[0023] limit

[0024] Note: To confirm the stability of the system during the analysis method verification sequence, proceed as follows: After the injection of the reference solution is completed, one injection of the reference solution is performed at the end of each verification item and sequence. The RSD of the methylamine peak area in the reference solution for the next 6 injections should be ≤10.0%.

[0025] The method for detecting methylamine in vonoprazan fumarate also includes method validation before detection. The method validation is based on the chromatographic conditions of the formal detection, and the measurement results are as follows:

[0026] Example 2: System Applicability System suitability is achieved by measuring the S / N value of methylamine in the sensitivity solution and the RSD of the methylamine peak area in the reference solution. The S / N value of methylamine in the sensitivity solution should be ≥10; the RSD of the methylamine peak area in the reference solution should be ≤10.0%.

[0027]

[0028] Example 3: Specificity The specificity of the method is achieved by determining whether the blank solution interferes with the detection; the separation degree between the methylamine peak and the adjacent peaks in the selective solution. It is required that the blank solution should not interfere with the detection; the separation degree between the methylamine peak and the adjacent peaks in the selective solution should be ≥1.5.

[0029]

[0030] Example 4: Precision Precision is achieved by testing the RSD of the results of the six test solutions (spiked). The RSD of the results of the six test solutions (spiked) should be ≤10.0%.

[0031]

[0032] Example 5: Limit of Detection and Limit of Quantitation The limit of quantitation and the limit of detection are achieved by measuring the ratio of the detection response signal to the noise. The signal-to-noise ratio of the limit of quantitation should not be less than 10:1, and the signal-to-noise ratio of the limit of detection should not be less than 3:1. At the limit of quantitation concentration level, 6 limit of quantitation solutions were repeatedly examined. The LOQ should be ≤0.05%, S / N≥10; the RSD of the methylamine peak area in the 6 LOQ solutions should be ≤10.0%, and the LOD <LOQ,S / N≥3。

[0033]

[0034]

[0035] Example 6: Linearity and Range In the concentration range of LOQ to 150%, take 6 concentration points and draw a curve with concentration as the horizontal axis and peak area as the vertical axis. It is required that the curve should be linear in the concentration range of LOQ to 150%, and the square of the linear correlation coefficient R (R 2 ) should be no less than 0.99, and the absolute value of the y-intercept should be within 25% of the 100% concentration response value.

[0036]

[0037] Example 7: Accuracy Accuracy is achieved by measuring the recovery rate between the measured concentration and the theoretical concentration of the measured component and the total RSD of the recovery rate (n=9). It is required that the recovery rate of methylamine added to the accuracy solution of LOQ concentration, 100% limit concentration, and 150% limit concentration should be between 80.0% and 120.0%, and the total RSD of the recovery rate (n=9) should be ≤10.0%.

[0038]

[0039] Example 8: Durability The reference solution, test solution and selective solution were placed at low temperature (15°C) for a period of time and then injected. The change pattern of the test results over time was investigated to provide a basis for the placement time of the reference solution and test solution during the test.

[0040] Requirements: Compared with 0hr, the recovery rate of methylamine after the reference solution is placed at low temperature (15℃) for a period of time should be between 90.0% and 110.0%, so the reference solution is stable during the observation period at low temperature (15℃); If methylamine is detected in the test solution at time 0, and the test solution is placed at low temperature (15°C) for a period of time, the change value of the measurement result is within 20% of the limit, and there is no obvious change trend, then the test solution is stable during the observation period at low temperature (15°C); if methylamine is not detected in the test solution at time 0, and the test solution is placed at low temperature (15°C) for a period of time and still no methylamine is detected, then the test solution is stable during the observation period at low temperature (15°C).

[0041] When the selective solution is placed at low temperature (15°C) for a period of time, the recovery rate of methylamine should be between 80.0% and 120.0%, and the selective solution is stable during the observation period at low temperature (15°C).

[0042]

Claims

1. A method for testing methylamine in vonoprazan fumarate, characterized in that: The method comprises the following steps: derivatizing methylamine with a derivatizing agent, 1-naphthyl isothiocyanate, and detecting the derivatized product by liquid chromatography. The testing method comprises the following steps: (1) Prepare solutions, including blank solution, sensitivity solution, reference solution and test solution; (2) Determination method: The content of methylamine in vonoprazan fumarate was determined by liquid chromatography. After the system was stable, blank solution, sensitivity solution, reference solution and test solution were added respectively, and the chromatogram was recorded; The chromatographic conditions are as follows: Detector: UV detector, chromatographic column: chromatographic column with octadecylsilane bonded silica as filler, injection volume: 10-20µl, column temperature: 40±5℃, flow rate: 1.0±0.1ml / min, phosphoric acid solution as mobile phase A, acetonitrile as mobile phase B, gradient elution.

2. The inspection method according to claim 1, characterized in that: The chromatographic column is Waters Symmetry ShieldTM RP8, 4.6 mm×150 mm, 5 µm.

3. The inspection method according to claim 2, characterized in that: The elution gradient is: 。 4. The inspection method according to claim 3, characterized in that: The mobile phase A: 0.1% phosphoric acid solution; For the blank solution: measure an appropriate amount of diluent and boric acid buffer solution and place them in a centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix again, stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The diluent: The volume ratio of acetonitrile: water is 50:50; The boric acid buffer solution: measure appropriate amounts of boric acid solution and potassium chloride solution and place them in the same volumetric flask, add water to dilute to the scale, shake well, and adjust the pH value to 10.5 with sodium hydroxide solution to prepare the boric acid buffer solution; The sodium hydroxide solution: take an appropriate amount of sodium hydroxide and place it in a volumetric flask, add water to dissolve it, cool it, add water to dilute it to the scale, shake it well, and use it as the sodium hydroxide solution; Boric acid solution: Take an appropriate amount of boric acid, put it in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as the boric acid solution; Potassium chloride solution: Take an appropriate amount of potassium chloride, put it in a volumetric flask, add water to dissolve and dilute to the scale, shake well, and use it as potassium chloride solution; The reference substance solution: measure an appropriate amount of diluent, reference substance stock solution, and boric acid buffer solution and place them in the same centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the same centrifuge tube, vortex mix again, let stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The reference substance stock solution: take an appropriate amount of methylamine hydrochloride reference substance, place it in a volumetric flask, add water to dissolve and dilute to the scale, shake well; take an appropriate amount of the above solution and place it in a volumetric flask, add diluent to dilute to the scale, shake well; The sensitivity solution: measure an appropriate amount of diluent, sensitivity reserve solution, and boric acid buffer solution, place them in a centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the centrifuge tube, vortex mix again, stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; Sensitivity stock solution: Take an appropriate amount of reference stock solution, place it in a volumetric flask, add diluent to dilute to the scale, and shake well; The test solution: measure an appropriate amount of diluent, test stock solution, and boric acid buffer solution and place them in the same centrifuge tube, vortex mix, measure an appropriate amount of 1-naphthyl isothiocyanate solution, place them in the same centrifuge tube, vortex mix again, let stand at room temperature, perform ultrasound, filter with a microporous filter membrane, and take the filtrate for sampling; The test sample stock solution: Take an appropriate amount of the test sample, place it in a volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

5. The inspection method according to claim 4, characterized in that: The microporous membrane filtration is 0.22 μm microporous membrane filtration.

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