Detection method of vonoprazan fumarate related substances

Abstract

The invention relates to a method for detecting related substances of Vonoprazan fumarate. The method comprises the following steps: (1) pretreating a sample to be detected by using a formic acid methanol solution; (2) determining related substances in the pretreated sample by liquid chromatography-mass spectrometry; the high performance liquid chromatography detection conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, a 0.2% formic acid aqueous solution is used as a mobile phase A, a 0.2% formic acid methanol solution is used as a mobile phase B, and gradient elution is performed; the mass spectrum conditions are as follows: an ion source is an electrospray ion source, an acquisition mode is a positive ion mode, a monitoring mode is multi-reaction monitoring, the parent ion is 375.3, the daughter ion is 315.4, and the collision voltage is 15V. The method for detecting the impurities of the Vonoprazan fumarate NVP, provided by the invention, has the advantages of high sensitivity and high recovery rate.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for detecting vonoprazan-related substances in fumarate. Background Technology

[0002] Vonoprazan fumarate (1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrolo-3-yl]-N-methylmethylamine monofumarate) is a novel proton pump inhibitor primarily used to treat acid-related gastrointestinal disorders such as gastric ulcers and gastroesophageal reflux disease. Developed jointly by Takeda Pharmaceutical and Otsuka Pharmaceutical, it was approved for marketing in Japan in 2014 and received marketing authorization in China in December 2019 (trade name: Walker). In 2021, global sales of this drug reached US$820 million.

[0003] On January 11, 2023, the FDA temporarily suspended approval for vonoprazan fumarate, developed by Phathom, a company founded by Takeda and Frazier, for the treatment of erosive esophagitis. This was due to a discovery in August 2022 of trace amounts of the nitrosamine impurity N-nitroso-vonoprazan (NVP) in Phathom's commercial batches of vonoprazan fumarate. N-nitroso-vonoprazan is a genotoxic impurity, and the FDA has set the acceptable daily intake limit for NVP at 96 ng / day. Therefore, strict control of NVP levels in vonoprazan fumarate is crucial, as sensitive and accurate detection of NVP is vital for patient health. Summary of the Invention

[0004] This invention provides a highly sensitive and high-recovery method for detecting vonoprazan fumarate NVP impurities.

[0005] The present invention solves the above-mentioned technical problems through the following technical solution:

[0006] This invention provides a method for detecting vonoprazan fumarate-related substances, characterized in that the method includes the following steps: 1) pretreatment of the sample to be tested;

[0007] 2) The relevant substances in the pretreated sample were determined by liquid chromatography-mass spectrometry; the solvent for pretreatment was formic acid methanol solution.

[0008] The concentration of formic acid in the formic acid methanol solution used as a pretreatment solvent in this invention is 0.1%-0.2%.

[0009] The present invention employs high performance liquid chromatography-tandem mass spectrometry for detection. The high performance liquid chromatography detection conditions are as follows: using octadecylsilane-bonded silica gel as the packing material, 0.2% formic acid aqueous solution as mobile phase A, and 0.2% formic acid methanol solution as mobile phase B, and performing gradient elution.

[0010] The gradient elution conditions described in this invention are as follows:

[0011] time min Mobile phase A% Mobile phase B% 0 35-40 60-65 3 30 70 5 30 70 5.01 35-40 60-65 7 30-40 60-70

[0012] Preferred options are:

[0013] time min Mobile phase A% Mobile phase B% 0 40 60 3 30 70 5 30 70 5.01 40 60 7 40 60

[0014] or

[0015] time min Mobile phase A% Mobile phase B% 0 40 60 3 30 70 5 30 70 5.01 40 60 7 30 70

[0016] or

[0017] time min Mobile phase A% Mobile phase B% 0 35 65 3 30 70 5 30 70 5.01 35 65 7 35 65

[0018] More preferably:

[0019] time min Mobile phase A% Mobile phase B% 0 40 60 3 30 70 5 30 70 5.01 40 60 7 40 60 .

[0020] In the high-performance liquid chromatography detection conditions described in this invention, the column temperature is 30°C.

[0021] The flow rate in the high-performance liquid chromatography detection conditions described in this invention is 0.25 ml / min.

[0022] This invention employs high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) for detection. The mass spectrometry conditions are as follows: the ion source is an electrospray ion source, the acquisition mode is positive ion mode, the monitoring mode is multiple reaction monitoring (MRM), the precursor ion is 375.3, the daughter ion is 315.4, and the collision voltage is 15V.

[0023] The method for preparing the test solution described in this invention is as follows: accurately weigh vonoprazan fumarate tablets and grind them into a fine powder; accurately weigh an appropriate amount of the fine powder, containing approximately 100 mg of vonoprazan, add 10 ml of 0.1%-0.2% formic acid-methanol solution, shake well, centrifuge, filter the supernatant, and collect the filtrate.

[0024] The detection method of the present invention includes the following steps:

[0025] 1) Preparation of test solution: Accurately weigh vonoprazan fumarate tablets and grind them into a fine powder; accurately weigh an appropriate amount of the fine powder, containing about 100 mg of vonoprazan, add 10 ml of 0.1%-0.2% formic acid-methanol solution, shake well, centrifuge, filter the supernatant, and collect the filtrate.

[0026] 2) Content determination: Inject the test solution into a liquid chromatography column for detection. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material (WatersACQUITY). CSH TM A C18 column, 2.1 mm × 150 mm, 1.7 μm or equivalent in performance; mobile phase A: 0.2% formic acid aqueous solution; mobile phase B: 0.2% formic acid methanol solution; flow rate: 0.25 mL / min; column temperature: 30 °C; injection volume: 2 μL; gradient elution conditions:

[0027]

[0028] The mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the acquisition mode was positive ion mode, the monitoring mode was multiple reaction monitoring, the precursor ion was 375.3, the daughter ion was 315.4, and the collision voltage was 15V.

[0029] The vonoprazan fumarate tablets of this invention contain: vonoprazan fumarate, mannitol, microcrystalline cellulose, croscarmellose sodium, fumaric acid, and magnesium stearate; preferably: vonoprazan fumarate, mannitol, microcrystalline cellulose, croscarmellose sodium, hydroxypropyl cellulose, fumaric acid, and magnesium stearate.

[0030] The inventors of this invention detected NVP impurities in Vocinti and were able to achieve extraction recovery rates of over 90% with an RSD of less than 5%; signal-to-noise ratio of over 40 with an RSD of less than 5%, demonstrating the advantages of high sensitivity and high recovery rate.

[0031] The method for detecting VNP impurities in fumarate provided by this invention has the advantages of high sensitivity and high recovery rate. Attached Figure Description

[0032] Figure 1 Example 1: Ion spectrum of water as pretreatment solvent

[0033] Figure 2 Example 1: Ion spectrum of methanol as pretreatment solvent

[0034] Figure 3 Example 1: Ion spectrum of acetonitrile as pretreatment solvent

[0035] Figure 4 Example 1: Ion spectrum of 0.1% trifluoroacetic acid methanol solution as pretreatment solvent

[0036] Figure 5 Example 1: Ion spectrum of 0.1% formic acid methanol solution as pretreatment solvent

[0037] Figure 6Example 1: Ion spectrum of 0.2% formic acid methanol solution as pretreatment solvent

[0038] Figure 7 Example 2 Gradient elution conditions 1 Ion spectrum

[0039] Figure 8 Example 2 Gradient elution conditions 2 Ion spectrum

[0040] Figure 9 Example 2 Gradient elution conditions 3 Ion spectrum

[0041] Figure 10 Example 2 Gradient elution conditions 4 Ion spectrum

[0042] Figure 11 Example 2 Gradient elution conditions 5 Ion spectrum

[0043] The peaks in the ion diagram are numbered as follows: Peak 1 - Unknown impurity; Peak 2 - NVP. Specific Implementation

[0044] The following embodiments further illustrate the content of the present invention in detail, but should not be used to limit the scope of protection of the present invention.

[0045] The following examples of vonoprazan fumarate tablets contain: vonoprazan fumarate, mannitol, microcrystalline cellulose, croscarmellose sodium cellulose, hydroxypropyl cellulose, fumaric acid, and magnesium stearate. Preparation method: The above raw materials and excipients are mixed and then compressed into tablets.

[0046] Example 1

[0047] 1. Testing conditions

[0048] The determination was performed by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition) and mass spectrometry (General Chapter 0431, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0049] Pretreatment solvent:

[0050] Serial Number solvent 1 water 2 methanol 3 Acetonitrile 4 0.1% TFA (trifluoroacetic acid) methanol solution 5 0.1% formic acid in methanol solution 6 0.2% formic acid in methanol solution

[0051] Test solution: Accurately weigh 20 vonoprazan fumarate tablets and grind them into a fine powder. Accurately weigh an appropriate amount of the fine powder (containing approximately 100 mg of vonoprazan), accurately add 10 ml of pretreatment solvent, shake well, centrifuge, filter the supernatant, and collect the filtrate.

[0052] Reference solutions: Accurately weigh an appropriate amount of N-nitrosovonorazine reference standard, dissolve it in 0.2% formic acid methanol solution and quantitatively dilute it to prepare solutions containing approximately 12 ng, 24 ng, 48 ng, 72 ng, and 96 ng per ml.

[0053] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Waters ACQUITY). CSH TM C18 column (2.1 mm × 150 mm, 1.7 μm or equivalent performance); use 0.2% formic acid aqueous solution as mobile phase A and 0.2% formic acid methanol solution as mobile phase B, and perform gradient elution according to the table below; flow rate is 0.25 ml per minute; column temperature is 30 °C; injection volume is 2 μl.

[0054]

[0055]

[0056] Mass spectrometry conditions: Detection was performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization (ESI) source, positive ion mode, multiple reaction monitoring (MRM) mode, precursor ion of 375.3 g / L, daughter ion of 315.4 g / L, and collision voltage (CE) of 15 V.

[0057] 2. Test Results

[0058]

[0059]

[0060] Example 2

[0061] The determination was performed by high performance liquid chromatography (General Chapter 0512, Part IV, Chinese Pharmacopoeia 2020 Edition) and mass spectrometry (General Chapter 0431, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0062] Pretreatment solvent: 0.2% formic acid in methanol solution.

[0063] Test solution: Accurately weigh 20 vonoprazan fumarate tablets and grind them into a fine powder. Accurately weigh an appropriate amount of the fine powder (containing approximately 100 mg of vonoprazan), accurately add 10 ml of pretreatment solvent, shake well, centrifuge, filter the supernatant, and collect the filtrate.

[0064] Reference solutions: Accurately weigh an appropriate amount of N-nitrosovonorazine reference standard, dissolve it in 0.2% formic acid methanol solution and quantitatively dilute it to prepare solutions containing approximately 12 ng, 24 ng, 48 ng, 72 ng, and 96 ng per ml.

[0065] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Waters ACQUITY). CSH TMC18 column (2.1 mm × 150 mm, 1.7 μm or equivalent performance); use 0.2% formic acid aqueous solution as mobile phase A and 0.2% formic acid methanol solution as mobile phase B, and perform gradient elution according to the elution gradient of conditions 1-5 in the table below; flow rate is 0.25 ml per minute; column temperature is 30 °C; injection volume is 2 μl.

[0066] Mass spectrometry conditions: Detection was performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization (ESI) source, positive ion mode, multiple reaction monitoring (MRM) mode, precursor ion of 375.3 g / L, daughter ion of 315.4 g / L, and collision voltage (CE) of 15 V.

[0067] Table 1 Screening of elution gradients

[0068]

[0069]

[0070] Example 3

[0071] 1. Solution preparation

[0072] Same as Example 2.

[0073] 2. Exclusivity

[0074] Take solvent, blank excipient solution, reference solution and test solution respectively, and detect them under the chromatographic and mass spectrometric conditions described in Example 1, and record the chromatograms.

[0075] Experimental results show that the solvent and blank excipient do not interfere with the detection of NVP. The retention time of the NVP peak in the test solution (4.38 min) is consistent with that in the reference solution (4.38 min).

[0076] name solvent Blank excipient solution Reference solution Test solution Retention time N / A N / A 4.38min 4.38min

[0077] 3. Accuracy

[0078] Accurately weigh approximately 1000 mg of blank excipient, add 10 ml of solvent, and add an appropriate amount of NVP reference stock solution (a solution containing approximately 1 μg of NVP per 1 ml) to prepare solutions containing approximately 50%, 100%, and 150% of the limit concentration of NVP per 1 ml. Prepare three parallel samples for each concentration, and simultaneously prepare a blank excipient solution without impurities.

[0079] Accurately measure the test solution and standard curve solution, perform the determination under the planned chromatographic conditions, record the chromatograms, and calculate the recovery rate and RSD value of NVP by peak area using the standard curve method.

[0080]

[0081]

[0082] 4. Precision

[0083] Take an appropriate amount of the fine powder of this product and prepare 6 parallel test solutions. Perform the determination under the planned chromatographic conditions, record the chromatograms, and calculate the content and its RSD value based on the peak area using the standard curve method.

[0084]

Claims

1. A method for detecting vonoprazan fumarate-related substances, characterized in that, The method includes the following steps: 1) Pretreatment of the sample to be tested; 2) The relevant substances in the pretreated sample were determined by liquid chromatography-mass spectrometry; the solvent for pretreatment was formic acid methanol solution.

2. The detection method according to claim 1, characterized in that, The concentration of formic acid in the formic acid methanol solution used as a pretreatment solvent is 0.1%-0.2%.

3. The detection method according to claim 1, characterized in that, The detection was performed using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The HPLC detection conditions were as follows: octadecylsilane-bonded silica gel was used as the stationary phase, 0.2% formic acid aqueous solution was used as mobile phase A, and 0.2% formic acid methanol solution was used as mobile phase B, with gradient elution.

4. The detection method according to claim 3, characterized in that, The gradient elution conditions are as follows: Preferred options are: or or ； More preferably 。 5. The detection method according to claim 3, characterized in that, The column temperature in the high-performance liquid chromatography detection conditions is 30℃.

6. The detection method according to claim 3, characterized in that, The flow rate in the high-performance liquid chromatography detection conditions is 0.25 ml / min.

7. The detection method according to claim 1, characterized in that, The detection was performed using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). The mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the acquisition mode was positive ion mode, the monitoring mode was multiple reaction monitoring (MRM), the precursor ion was 375.3, the daughter ion was 315.4, and the collision voltage was 15V.

8. The detection method according to claim 1, characterized in that, The preparation method of the test solution is as follows: accurately weigh vonoprazan fumarate tablets and grind them into a fine powder; accurately weigh an appropriate amount of the fine powder, containing about 100 mg of vonoprazan, add 10 ml of 0.1%-0.2% formic acid-methanol solution, shake well, centrifuge, filter the supernatant, and collect the filtrate.

9. The detection method according to any one of claims 1-8, characterized in that, Includes the following steps: 1) Preparation of test solution: Accurately weigh vonoprazan fumarate tablets and grind them into a fine powder; accurately weigh an appropriate amount of the fine powder, containing about 100 mg of vonoprazan, add 10 ml of 0.1%-0.2% formic acid-methanol solution, shake well, centrifuge, filter the supernatant, and collect the filtrate. 2) Content determination: Inject the test solution into a liquid chromatography column for detection. The chromatographic conditions are as follows: octadecylsilane-bonded silica gel as the packing material (Waters ACQUITY). CSH TM A C18 column, 2.1 mm × 150 mm, 1.7 μm or equivalent in performance; mobile phase A: 0.2% formic acid aqueous solution; mobile phase B: 0.2% formic acid methanol solution; flow rate: 0.25 mL / min; column temperature: 30 °C; injection volume: 2 μL; gradient elution conditions: ； The mass spectrometry conditions were as follows: the ion source was an electrospray ion source, the acquisition mode was positive ion mode, the monitoring mode was multiple reaction monitoring, the precursor ion was 375.3, the daughter ion was 315.4, and the collision voltage was 15V.

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