Detection method of genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate

Abstract

The present invention relates to the field of drug detection technology, specifically a method for detecting genotoxic impurities N-nitroso-vonoprazan in vonoprazan fumarate, wherein vonoprazan fumarate samples are detected by high performance liquid chromatography, mobile phase A is a formic acid aqueous solution, mobile phase B is methanol, and a specific gradient elution program is combined to successfully detect N-nitroso-vonoprazan. The present invention develops the first method for detecting genotoxic impurities N-nitroso-vonoprazan in vonoprazan fumarate, which can provide accurate guidance for the detection of nitrosamine genotoxic impurities in gastric acid suppressant vonoprazan. The method has high selectivity, good sensitivity, and a quantitative limit as low as 0.18ppm, and has a high detection efficiency in the test and separation time periods.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and in particular to a method for detecting genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate. Background Art

[0002] Helicobacter pylori (Hp) is a Gram-negative bacterium that is mainly distributed in the stomach and duodenum. Helicobacter pylori is a gastric disease that can be seen worldwide. Improper treatment can easily induce peptic ulcers, gastritis, duodenal ulcers and other diseases. It is one of the important causes of gastric cancer. The World Health Organization has classified Hp as a Class I carcinogen that induces gastric adenocarcinoma.

[0003] The treatment of Helicobacter pylori is mainly based on triple or quadruple therapy. Vonoprazan (VPZ) is the only oral potassium ion competitive acid blocker (P-CAB) approved in China, which can be used to eradicate Hp. Compared with the traditional proton pump inhibitor (PPI) triple therapy, VPZ has two main advantages. First, it has high selectivity and good affinity for receptors, and can selectively inhibit H + , K + -ATPase; secondly, the physicochemical properties of VPZ give it a higher pKa value, better acid resistance and longer half-life than PPIs.

[0004] On August 2, 2022, Phathom detected trace amounts of the nitrosamine impurity N-nitroso-vonoprazan (NVP) in vonoprazan, which attracted the attention of the FDA and manufacturers. The acceptable daily intake limit for NVP is 96 ng / day, and the maximum allowable daily dose of vonoprazan is 40 mg / day, with a limit of 1.8 ppm (based on vonoprazan fumarate).

[0005] There is currently no publicly published method for detecting NVP in vonoprazan fumarate APIs and preparations. In order to achieve better quality control and prevent the outflow of NVP-exceeding drugs, it is urgent to develop a method for detecting NVP in vonoprazan fumarate APIs and preparations to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for detecting the genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate, to detect the genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate preparations and raw materials, so as to monitor the content thereof during production and storage.

[0007] To achieve the above object, the present invention provides a method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate, comprising: detecting a vonoprazan fumarate sample by high performance liquid chromatography, wherein mobile phase A is a formic acid aqueous solution, mobile phase B is methanol, and the gradient elution procedure is as follows:

[0008] Time / min Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0.00 50±5 50±5 1.00 50±5 50±5 8.00 10±5 90±5 11.00 10±5 90±5 11.01 50±5 50±5 15.00 50±5 50±5 .

[0009] Since the nitrosamine impurity NVP was detected in the vonoprazan fumarate drug, the safety of the drug has attracted extensive attention from the FDA and manufacturers. At present, there is no publicly published method for detecting NVP in vonoprazan fumarate raw materials and preparations. In order to achieve better quality control effects and prevent the outflow of drugs with NVP exceeding the limit (1.8ppm), the present invention develops a detection method for NVP in vonoprazan fumarate raw materials and preparations to solve the above problems and provide an accurate guidance method for the detection of nitrosamine genotoxic impurities NVP.

[0010] As a further improvement of the present invention, the volume concentration of the formic acid aqueous solution is 0.03-0.10%.

[0011] As a further improvement of the present invention, the specifications of the chromatographic column of the high performance liquid chromatography are: Agilent C18, 150×3.0 mm, 2.7 μm.

[0012] As a further improvement of the present invention, the column temperature of the HPLC is 30±5°C.

[0013] As a further improvement of the present invention, the flow rate of the high performance liquid chromatography is: 0.2-0.5 mL / min; the injection volume is: 5±1 μL.

[0014] As a further improvement of the present invention, the vonoprazan fumarate sample is a vonoprazan fumarate preparation or a raw material drug; the preparation method of the vonoprazan fumarate sample comprises: adding the vonoprazan fumarate preparation or the raw material drug to a diluent, fixing the volume, filtering, and obtaining a vonoprazan fumarate sample with a concentration of 0.5-0.6 mg / mL;

[0015] The diluent is a methanol aqueous solution with a volume concentration of 45-55%.

[0016] As a further improvement of the present invention, the gradient elution procedure is as follows:

[0017] Time / min Mobile phase A volume fraction / % Mobile phase B volume fraction / % 0.00 50 50 1.00 50 50 8.00 10 90 11.00 10 90 11.01 50 50 15.00 50 50 .

[0018] As a further improvement of the present invention, high performance liquid chromatography and mass spectrometry are used for detection; the detection equipment used is a triple quadrupole liquid chromatography-mass spectrometer.

[0019] As a further improvement of the present invention, the ion source of the mass spectrometer is an electrospray ion source, the scanning mode is a positive ion mode, and the detection mode is multiple reaction monitoring MRM.

[0020] As a further improvement of the present invention, the capillary voltage of the mass spectrometer is: 3500V; the nebulizer pressure is: 40-45psi; the dryer temperature is: 250-300℃; the dryer flow rate is: 3-5L / min; the sheath gas temperature is: 250℃; and the sheath gas flow rate is: 11L / min.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. The method for detecting the genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate provided by the present invention can detect the genotoxic impurity N-nitroso-vonoprazan in vonoprazan fumarate preparations and raw materials, which is convenient for monitoring its content during production and storage, and provides an accurate guidance method for the detection of nitrosamine genotoxic impurities in the gastric acid suppressant drug vonoprazan.

[0023] 2. The present invention adopts liquid chromatography tandem mass spectrometry technology, which has high selectivity, good sensitivity and a quantitative limit as low as 0.18 ppm.

[0024] 3. The sample collection time of the present invention is 15 minutes, and the pre-treatment operation is simple, and the processing and detection of NVP in the sample to be tested can be completed within 20 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 is a blank spectrum;

[0027] Figure 2 NVP-quantitation limit spectrum;

[0028] Figure 3 This is the spectrum of NVP-test sample spiked solution;

[0029] Figure 4 This is the NVP-standard curve spectrum. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Reagents and Consumables

[0033] 1.1 Water: Ultrapure water - 18.2 megohms.

[0034] 1.2 Formic acid: mass spectrometry grade.

[0035] 1.3 Methanol: mass spectrometry grade.

[0036] 1.4 Mobile phase A (0.05% formic acid): Measure 0.5 ml of formic acid into a solvent bottle, add 1 L of ultrapure water, and shake well.

[0037] 1.5 Mobile phase B (methanol).

[0038] 1.6 Blank solution (methanol solution with a volume concentration of 50%): Measure 500 ml of methanol into a 1 L volumetric flask, make up to volume with ultrapure water, and shake well.

[0039] 1.7 Diluent (methanol solution with a volume concentration of 50%): Measure 500 ml of methanol into a 1L volumetric flask, make up to volume with ultrapure water, and shake well.

[0040] 1.8 Filter membrane: 0.22μm, PTFE.

[0041] 2. Instruments and Equipment

[0042] 2.1 Triple quadrupole liquid spectrometer: equipped with an electrospray ionization (ESI) source.

[0043] 2.2 Constant temperature water bath oscillator.

[0044] 2.3 Electronic balance.

[0045] 2.4 Medical refrigerator.

[0046] 3. Determination steps

[0047] 3.1 Preparation of test solution:

[0048] Take a sample of vonoprazan fumarate (preparation / raw material) and use a diluent to prepare a test solution containing approximately 0.536 mg of vonoprazan fumarate per 1 mL. Shake for 10 minutes, filter through a 0.22 μm PTFE filter membrane, and take the filtrate for detection.

[0049] 3.2 Preparation of blank solution

[0050] Measure 500 ml of methanol into a 1 L volumetric flask, make up to volume with ultrapure water, and shake well.

[0051] 3.3 Prepare N-nitroso-vonoprazan reference solution.

[0052] 4. Determination

[0053] 4.1 Liquid chromatography conditions

[0054] 4.1.1 Chromatographic column: Agilent C18 (150×3.0mm, 2.7μm)

[0055] 4.1.2 Column temperature: 30℃.

[0056] 4.1.3 Flow rate: 0.3 mL / min.

[0057] 4.1.4Injection volume: 5μL.

[0058] 4.1.5 Mobile phase: Mobile phase A (formic acid solution with a volume concentration of 0.05%), mobile phase B (methanol).

[0059] The mobile phase gradient program is shown in Table 1.

[0060] Table 1 Gradient elution program

[0061]

[0062]

[0063] 4.2 Mass spectrometry conditions

[0064] 4.2.1 Ion source: electrospray ionization (ESI).

[0065] 4.2.2 Scanning mode: positive ion mode.

[0066] 4.2.3 Detection method: Multiple reaction monitoring (MRM).

[0067] 4.2.4 Capillary voltage: 3500 V; Nebulizer pressure: 45 psi; Dryer temperature: 300°C; Dryer flow rate: 5 L / min; Sheath gas temperature: 250°C; Sheath gas flow rate: 11 L / min.

[0068] 4.2.5 Qualitative ion pairs, quantitative ion pairs, collision energy, and fragmentor voltage are shown in Table 2.

[0069] Table 2 Qualitative ion pairs, quantitative ion pairs, collision energy, and fragmentor voltage of N-nitrosovonoprazan and vonoprazan

[0070]

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate, characterized in that: include: The vonoprazan fumarate sample was detected by high performance liquid chromatography, wherein mobile phase A was a formic acid aqueous solution, mobile phase B was methanol, and the chromatographic column specification of the high performance liquid chromatography was: Agilent C18, 150×3.0 mm, 2.7 μm; high performance liquid chromatography and mass spectrometry were used for detection; the detection equipment used was a triple quadrupole liquid-mass spectrometer; the volume concentration of formic acid in the formic acid aqueous solution was 0.03-0.10%; and the gradient elution procedure was as follows: 。 2. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to claim 1, characterized in that: The column temperature of the HPLC is 30±5°C.

3. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to claim 1, characterized in that: The flow rate of the high performance liquid chromatography is: 0.2-0.5 mL / min; the injection volume is: 5±1 μL.

4. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to claim 1, characterized in that: The vonoprazan fumarate sample is a vonoprazan fumarate preparation or a raw material drug; The preparation method of the vonoprazan fumarate sample comprises: adding the vonoprazan fumarate preparation or the raw material drug to a diluent, fixing the volume, filtering, and obtaining a vonoprazan fumarate sample with a concentration of 0.5-0.6 mg / mL; The diluent is a methanol aqueous solution with a volume concentration of 45-55%.

5. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to any one of claims 1 to 4, characterized in that: The gradient elution program is as follows: 。 6. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to claim 1, characterized in that: The ion source of the mass spectrometer is an electrospray ion source, the scanning mode is a positive ion mode, and the detection mode is multiple reaction monitoring MRM.

7. The method for detecting N-nitroso-vonoprazan impurities in vonoprazan fumarate according to claim 6, characterized in that: The capillary voltage of the mass spectrometer is 3500 V; the nebulizer pressure is 40-45 psi; the dryer temperature is 250-300° C.; the dryer flow rate is 3-5 L / min; the sheath gas temperature is 250° C.; and the sheath gas flow rate is 11 L / min.

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