Analysis method of nitrosamine impurities and application thereof

Abstract

The invention discloses a nitrosamine impurity analysis method and application thereof, and belongs to the technical field of pharmaceutical analysis. According to the analysis method for the nitrosamine impurities, an LC-MS / MS method is adopted for analysis, and the chromatographic conditions include that ODS C18 serves as a stationary phase, an acidic aqueous solution serves as a mobile phase A, an organic solvent serves as a mobile phase B, and gradient elution is carried out; the mass spectrum conditions comprise that a triple quadrupole mass spectrum detector is adopted, an electrospray ionization positive ion mode is adopted, and multi-reaction monitoring is carried out; the nitrosamine impurity is prepared from N-nitroso-Vonoprazan (N-nitroso-Vonoprazan). The invention also discloses an application of the analysis method, and the analysis method is used for analyzing impurities in the Vonoprazan fumarate or / and the preparation of the Vonoprazan fumarate. The method is scientific in design and ingenious in conception, and the method for analyzing the nitrosamine impurities in the Vonoprasone fumarate and the preparation Vonoprasone fumarate tablets of the Vonoprasone fumarate is provided for the first time; the analysis method has the advantages of strong specificity, high sensitivity, good accuracy and wide durability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to an analysis method for nitrosamine impurities and an application thereof. Background Art

[0002] In 1937, Freund first reported two cases of occupational exposure to N-nitrosodimethylamine (NDMA, also known as dimethylnitrosamine) poisoning, and the patients showed toxic liver damage; in 1956, two British scientists found that NDMA had strong carcinogenic activity in experimental animals. Since then, nitrosamines have begun to attract widespread attention. In 1987, NDMA and NDEA (N-nitrosodiethylamine) were listed as Class 2A carcinogens (substances with limited evidence of carcinogenicity to humans but sufficient evidence of carcinogenicity to experimental animals) by the International Agency for Research on Cancer (IARC); in 2014, ICH M7 clearly stated that nitrosamines are substances of concern and should be strictly controlled in medicines.

[0003] Since the detection of N-nitrosodimethylamine (NDMA) in valsartan API in July 2018, various nitrosamine impurities have been detected in other sartan APIs, such as NDMA, N-nitrosodiethylamine (NDEA), etc. Nitrosamine impurities belong to the "concern queue" substances mentioned in the ICH M7 (R1) (Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk) guideline, which have high carcinogenicity. Even if the intake is lower than the TTC level specified in ICH M7, there is still a potential significant carcinogenic risk in theory.

[0004] Vonoprazan fumarate, chemical name: 1-[5-(2-fluorophenyl)-1-(pyridine-3-sulfonyl)-1H-pyrrol-3-yl]-N-methylmethylamine monofumarate, molecular formula: C 17 H 16 FN3O2S·C4H4O4, molecular weight: 461.46, its structure is as follows:

[0005]

[0006] Vonoprazan fumarate tablets were first approved by Takeda Pharmaceutical Company Limited in Japan in December 2014 by PMDA for marketing in Japan under the trade name The specifications are 10mg and 20mg. In December 2019, my country's National Medical Products Administration (NMPA) approved the import and marketing of vonoprazan fumarate tablets in China under the trade name The specifications are 10mg and 20mg, and the indication is reflux esophagitis.

[0007] Vonoprazan fumarate may produce nitrosamine impurities as shown below under certain conditions:

[0008]

[0009] The nitrosamine impurity (N-nitroso-vonoprazan, NVP) in vonoprazan fumarate is not a known structure in the published nitrosamine impurity guidelines of the FDA or EMA, and its toxicological data and structural analogues have not been found. According to the relevant Q&A document of EMA (European Medicines Agency) (Questions and answers for marketing authorisation holders / applicants on the CHMP Opinion for the Article 5(3) of Regulation (EC) No 726 / 2004 referral on nitrosamine impurities in human medicinal products), the toxicological concern threshold of specific nitrosamine impurities in this product is temporarily calculated as 178ng / day. Vonoprazan fumarate tablets have been imported into China. The maximum daily dose of the original product is clearly stated as 20mg under the Usage and Dosage item in the revised instructions on October 11, 2021. The impurity limit is calculated as follows:

[0010]

[0011] Therefore, in order to ensure the safety and quality of drugs and achieve effective risk control, the level of nitrosamine impurities in vonoprazan fumarate and its similar products, vonoprazan fumarate tablets, is controlled below the safety limit of 8.9 ppm.

[0012] On January 14, 2019, the State Food and Drug Administration drafted the "Risk Warning on the Generation of Nitrosamines in the Process of APIs" and solicited public opinions, intending to provide technical references for situations where there is a risk of producing nitrosamines in the research and production of APIs. The "MAH Instructions-Nitrosamine Information" issued by EMA and the Q&A of the document emphasize that in any case, if the MAH finds nitrosamine substances in a certain drug, regardless of the amount detected, it should notify the drug regulatory authorities. This puts higher requirements on the specificity and sensitivity of the detection method of nitrosamine impurities. However, there are currently no reports on the study of nitrosamine impurities in vonoprazan fumarate and its similar varieties, vonoprazan fumarate tablets. Therefore, providing an analytical method for nitrosamine impurities for the determination of vonoprazan fumarate APIs or / and preparations, and similar products, has become a problem that needs to be solved by technicians in this field. Summary of the invention

[0013] One of the purposes of the present invention is to provide an analytical method for nitrosamine impurities, which has strong specificity, high sensitivity, good accuracy and wide durability, and can quantitatively detect nitrosamine impurities in vonoprazan fumarate and its similar varieties vonoprazan fumarate tablets.

[0014] A second object of the present invention is to provide an application of the analytical method.

[0015] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0016] The present invention discloses a method for analyzing nitrosamine impurities, which adopts LC-MS / MS method for analysis, and the chromatographic conditions include: using ODS C18 as the stationary phase, using an acidic aqueous solution as the mobile phase A, using an organic solvent as the mobile phase B, and eluting according to the following gradient:

[0017] 0min, 65~75%A, 25~35%B;

[0018] 3.5~4.5min, 65~75%A, 25~35%B;

[0019] 5.5~6.5min, 20~30%A, 70~80%B;

[0020] 7.5~8.5min, 20~30%A, 70~80%B;

[0021] 7.6~8.6min, 0~5%A, 95~100%B;

[0022] 12.5~13.5min, 0~5%A, 95~100%B;

[0023] 12.6~13.6min, 65~75%A, 25~35%B;

[0024] 14.5~17.5min, 65~75%A, 25~35%B;

[0025] The mass spectrometry conditions include: using a triple quadrupole mass spectrometer, electrospray ionization positive ion mode, and multiple reaction monitoring;

[0026] The nitrosamine impurities include N-nitroso-vonoprazan.

[0027] In some embodiments of the present invention, the elution is performed according to the following gradient:

[0028] 0min, 65~75%A, 25~35%B;

[0029] 4min, 65~75%A, 25~35%B;

[0030] 6min, 20~30%A, 70~80%B;

[0031] 8min, 20~30%A, 70~80%B;

[0032] 8.1min, 0~5%A, 95~100%B;

[0033] 13min, 0~5%A, 95~100%B;

[0034] 13.1min, 65~75%A, 25~35%B;

[0035] 15min, 65~75%A, 25~35%B.

[0036] In some embodiments of the present invention, the elution is performed according to the following gradient:

[0037] 0min, 70% A, 30% B;

[0038] 4 min, 70% A, 30% B;

[0039] 6min, 25% A, 75% B;

[0040] 8min, 25% A, 75% B;

[0041] 8.1 min, 0% A, 100% B;

[0042] 13min, 0%A, 100%B;

[0043] 13.1min, 70% A, 30% B;

[0044] 15min, 70%A, 30%B.

[0045] In some embodiments of the present invention, the acidic aqueous solution is an organic acid aqueous solution, preferably a formic acid solution, more preferably a 0.005-0.03% formic acid solution, and further preferably a 0.01% formic acid solution;

[0046] Preferably, the organic solvent is methanol and / or acetonitrile, more preferably acetonitrile.

[0047] The percentage of "% formic acid solution" in the present invention indicates that 100 ml of the solution contains a certain number of milliliters of formic acid. For example, 0.01% formic acid solution indicates that 100 ml of the solution contains 0.01 ml of formic acid.

[0048] In some embodiments of the present invention, the column temperature of the chromatographic column is 25 to 35°C, preferably 30°C;

[0049] Preferably, the flow rate is 0.3 to 1.0 ml / min; more preferably 0.6 ml / min;

[0050] Preferably, the sample is injected under low temperature conditions, more preferably, the injector temperature is 2-10°C; further preferably, it is 5°C.

[0051] In some embodiments of the present invention, the mass spectrum acquisition time is 7.3 to 9.0 minutes.

[0052] In some embodiments of the present invention, the mass-to-charge ratio of the precursor ion is 375.1, and the mass-to-charge ratios of the product ions are 315.1, 251, 173, and 78.1.

[0053] In some embodiments of the present invention, a precursor ion mass-to-charge ratio of 375.1 and a product ion mass-to-charge ratio of 315.1 are selected for quantitative analysis;

[0054] Preferably, a precursor ion mass-to-charge ratio of 375.1 and a product ion mass-to-charge ratio of at least one of 251, 173, and 78.1 are selected for qualitative analysis.

[0055] The precursor ions described in the present invention are also called parent ions, and the product ions are also called daughter ions.

[0056] The application of the above-mentioned analysis method of nitrosamine impurities disclosed in the present invention is used for the analysis of impurities in vonoprazan fumarate and / or its preparations. Preferably, the preparations include vonoprazan fumarate tablets.

[0057] In some embodiments of the present invention, the content of impurities in vonoprazan fumarate or / and its preparation is determined by an external standard method;

[0058] Preferably, when determining vonoprazan fumarate, 30-70% acetonitrile solution is used as the diluent; for example, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% acetonitrile solution;

[0059] More preferably, it is 40-60% acetonitrile solution, further preferably, it is 45-65% acetonitrile solution, and further preferably, it is 50% acetonitrile solution;

[0060] Preferably, the vonoprazan fumarate tablets are measured using a 30-70% acetonitrile solution as a diluent, and the diluent contains 0.1-10 g / L of salt, for example: 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, 5.0 g / L, 5.5 g / L, 6.0 g / L, 6.5 g / L, 7.0 g / L, 7.5 g / L, 8.0 g / L, 8.5 g / L, 9.0 g / L, 9.5 g / L, 10 g / L of salt;

[0061] More preferably, a 30-70% acetonitrile solution containing 0.5-5 g / L salt is used as the diluent; more preferably, a 50% acetonitrile solution containing 0.1-10 g / L salt is used as the diluent; further preferably, a 50% acetonitrile solution containing 0.5-5 g / L salt is used as the diluent.

[0062] Preferably, the salt is an inorganic salt;

[0063] More preferably, the salt is an inorganic potassium salt or an inorganic sodium salt; further preferably, the salt is at least one of sodium chloride, potassium chloride and potassium sulfate; and even more preferably, it is sodium chloride.

[0064] Preferably, the determination of vonoprazan fumarate tablets uses a 30-70% acetonitrile solution as a diluent, and the diluent contains 0.1-10 g / L of sodium chloride, more preferably, a 30-70% acetonitrile solution containing 0.5-5 g / L of sodium chloride is used as a diluent; more preferably, a 50% acetonitrile solution containing 0.1-10 g / L of sodium chloride is used as a diluent; further preferably, a 50% acetonitrile solution containing 0.5-5 g / L of sodium chloride is used as a diluent. In some embodiments of the present invention, when the content of impurities in vonoprazan fumarate or / and its preparations is determined by an external standard method, the concentration of the reference solution is 0.15-1 ng / ml, preferably 0.18-0.9 ng / ml; the concentration of the test solution is 0.2-3 mg / ml, preferably 0.4-2 mg / ml; the injection volume is 1-20 μl. In the technical solution of the present invention, the injection volume is 1 to 20 μl, for example, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl, 20 μl;

[0065] The preferred range is 5 to 20 μl.

[0066] As mentioned above, the percentage of "% acetonitrile solution" in the present invention indicates that 100 ml of the solution contains a certain number of milliliters of acetonitrile, for example, 50% acetonitrile solution means that 100 ml of the solution contains 50 ml of acetonitrile; and the sodium chloride concentration in the acetonitrile solution of the present invention, such as 0.1-10 g / L, means that 1 L of acetonitrile solution contains 0.1-10 g of sodium chloride. Furthermore, for example, 5 g / L sodium chloride in 50% acetonitrile solution means that 1 L of 50% acetonitrile solution contains 5 g of sodium chloride.

[0067] When 30-70% acetonitrile solution was used as a diluent to measure vonoprazan fumarate tablets, the sample recovery rate of the impurity NVP was only 66.92%, which was low and did not meet the recovery rate requirement (70%-130%). The commercially available vonoprazan fumarate tablets are film-coated tablets containing excipients such as fumaric acid and polyethylene glycol. The applicant creatively added 0.1-10 g / L salt to the 30-70% acetonitrile solution, which effectively improved the sample recovery rate, thereby making the sample recovery rate meet the requirements.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The invention is scientifically designed and ingeniously conceived, and proposes for the first time an analysis method for nitrosamine impurities in vonoprazan fumarate and its preparation vonoprazan fumarate tablets; the analysis method has strong specificity, high sensitivity, good accuracy and wide durability.

[0070] When the method of the present invention is applied to the determination of the preparation vonoprazan fumarate tablets, the determination and analysis of the impurity NVP in the vonoprazan fumarate tablets can be smoothly realized, so that the recovery rate meets the requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Attached Figure 1 The mass spectrum of the blank auxiliary material solution in Example 2, wherein the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; the lower right figure is the monitored ion ratio diagram;

[0072] Attached Figure 2 The mass spectrum of the reference solution in Example 2, wherein the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; the lower right figure is the monitored ion ratio diagram;

[0073] Attached Figure 3 The mass spectrum of the test solution in Example 2, wherein the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; the lower right figure is the monitored ion ratio diagram;

[0074] Attached Figure 4The mass spectrogram of the 50% solution recovered in Example 2, wherein the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; and the lower right figure is the monitored ion ratio diagram;

[0075] Attached Figure 5 The mass spectrogram of the 100% solution recovered in Example 2, wherein the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; and the lower right figure is the monitored ion ratio diagram;

[0076] Attached Figure 6 This is the mass spectrum of the 150% solution recovered in Example 2, where the upper figure is the total ion flow diagram, the lower left figure is the quantitative ion chromatogram; the lower middle figure is the overlapping chromatogram of all monitored ions; and the lower right figure is the monitored ion ratio diagram. DETAILED DESCRIPTION

[0077] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0078] The vonoprazan fumarate and vonoprazan fumarate tablets described in the embodiments of the present invention are commercially available products.

[0079] The LC-MS / MS involved in the embodiments of the present invention is: Agilent 1290-Agilent G6470, and the chromatographic column is: Agilent Poroshell 120EC-C18 4.6×100 mm, 2.7 μm.

[0080] Example 1

[0081] This example discloses a screening test of the method of the present invention, first determining the mass spectrometry conditions, then determining the liquid chromatography conditions, and finally optimizing them. The details are as follows:

[0082] 1. Establishment of mass spectrometry conditions

[0083] 1.1 Establishment of precursor ions

[0084] The sample was injected and analyzed under the following chromatographic and mass spectrometric conditions to obtain the mass-to-charge ratio of the NVP precursor ion.

[0085] Chromatographic conditions: chromatographic column: Agilent Poroshell EC-C18 4.6×100mm, 2.7μm; 0.01% formic acid solution-acetonitrile (30:70) as the mobile phase, isocratic elution for 4min; flow rate: 0.5ml / min; column temperature: 30℃; injection volume: 1μl; detection wavelengths: 210nm, 230nm, 254nm.

[0086] Mass spectrometry conditions: instrument: Agilent1290-Agilent G6470; ion source: electrospray ion source; monitoring mode: MS2 scan; drying gas flow rate: 5 L / min; drying gas temperature: 300°C; nebulizing gas pressure: 45 psi; sheath gas temperature: 250°C; sheath gas flow rate: 11 L / min; fragmentation voltage: 100 V; scanning range (m / z): 200-500.

[0087] Solution preparation:

[0088] Reference substance stock solution: Take an appropriate amount of NVP, accurately weigh 0.505 mg, add 10 ml of 50% acetonitrile solution to dissolve it, and obtain the reference substance stock solution.

[0089] Sampling analysis: Take the reference substance stock solution for sampling analysis.

[0090] Results: The NVP precursor ion (m / z) was 375.1.

[0091] 1.2 Establishment of the fragmentation voltage value

[0092] Monitoring mode: MS2 single ion monitoring; precursor ion: 375.1; fragmentation voltage: 10V~150V, step size: 10V; other conditions are the same as under "1.1 Establishment of precursor ion".

[0093] Sample injection analysis: The reference stock solution was taken for sample injection analysis, and the results showed that the optimal fragmentation voltage was 80V.

[0094] 1.3 Product ion determination

[0095] Monitoring mode: product ion; scanning range (m / z): 30-380; fragmentation voltage: 80 V; collision energy: 5 eV, 15 eV, 25 eV, 35 eV; other conditions are the same as those in “1.1 Establishment of precursor ions”.

[0096] Sampling analysis: The reference substance stock solution was taken for sampling analysis, and the product ions (m / z) obtained were: 78.1, 173, 315.1, 251.

[0097] 1.4 Establishment of collision energy value

[0098] Monitoring mode: multiple reaction monitoring mode; collision energy optimization range: 2eV~60eV, step size: 2eV; other conditions are the same as under "1.1 Precursor ion establishment".

[0099] Sample injection analysis: The reference stock solution was sampled and analyzed, and the optimal collision energy values ​​(product ions were 78.1, 173, 315.1, and 251, respectively) were 44, 30, 10, and 24, respectively. The mass spectrometry parameters are shown in Table 1.

[0100] Table 1 Impurity NVP mass spectrometry parameters

[0101]

[0102] 2. Establishment of liquid chromatography conditions

[0103] 2.1 Preliminary determination of liquid chromatography conditions

[0104] Chromatographic conditions: Chromatographic column: Agilent Poroshell EC-C18 4.6×100 mm, 2.7 μm; Mobile phase A: 0.01% formic acid solution; Mobile phase B: acetonitrile; Column temperature: 30°C; Injection volume: 1 μl; Flow rate: 0.6 ml / min; Gradient elution program see Table 2.

[0105] Table 2 Gradient elution program of Example 1

[0106]

[0107]

[0108] Mass spectrometry conditions: ion source: electrospray ion source; monitoring mode: multiple reaction monitoring mode; drying gas flow rate: 5 L / min; drying gas temperature: 300°C; nebulizer gas pressure: 45 psi; sheath gas temperature: 250°C; sheath gas flow rate: 11 L / min; capillary voltage: +3500 V; detector gain (+): 200 V; nozzle voltage: +500 V; dwell time: 100 ms. The parameters of precursor ion, product ion, fragmentation voltage, and collision energy are the same as those in Table 1.

[0109] Solution preparation:

[0110] Reference substance stock solution: Same as under “1. Establishment of mass spectrometry conditions”.

[0111] Test solution: Weigh an appropriate amount of vonoprazan fumarate raw material, accurately weighed to 10.67 mg, add 1 ml of 50% acetonitrile solution to dissolve it to prepare a test solution with a content of about 10 mg / ml.

[0112] Sampling analysis: Take the reference solution and the test solution for sampling analysis.

[0113] Results: Under the liquid phase conditions, the retention time of the main component of the test sample was 5.145min, and the retention time of the impurity NVP was 11.44min. NVP was well separated from the main component of the sample. However, according to the response of the reference stock solution, at the limit concentration, when the injection volume was 1μl, the response of the reference solution could not meet the quantitative requirements, and the single-needle analysis time was 20min, which was too long, and the gradient needed to be further optimized.

[0114] 2.2 Optimization of gradient elution program

[0115] Chromatographic conditions and mass spectrometry conditions: The optimized gradient elution program is shown in Table 3. The injection volume was adjusted to 5 μl. The other conditions were the same as those in method “2.1 Preliminary determination of liquid chromatography conditions”.

[0116] Table 3 Optimized gradient elution program

[0117] Time (min) 0 3.5 8 10 10.1 12 Mobile phase A (%) 70 70 10 10 70 70 Mobile phase B (%) 30 30 90 90 30 30

[0118] Solution preparation:

[0119] Reference substance stock solution: Take an appropriate amount of NVP, accurately weigh it to 2.927 mg, add 25 ml of 50% acetonitrile solution to dissolve it; obtain a solution of 0.11708 mg / ml, then accurately measure 1 ml of this solution and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve it and dilute it to the mark, to obtain a solution with a concentration of 2.3416 μg / ml. Accurately measure 1 ml of the solution with a concentration of 2.3416 μg / ml and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve it and dilute it to the mark, to obtain a reference substance stock solution with a concentration of 46.8 ng / ml.

[0120] Reference substance solution: Accurately measure 1 ml of reference substance stock solution and dilute it with 10 ml of 50% acetonitrile solution to obtain a reference substance solution with a concentration of 4.68 ng / ml.

[0121] Test solution: Preparation of test solution is the same as in “2.1 Preliminary determination of liquid chromatography conditions”.

[0122] Sampling analysis: Take the reference solution and the test solution for sampling analysis.

[0123] Results: Under the liquid phase conditions, the peak time of the main component of the test sample was 1.7-3.9 min, the retention time of the impurity NVP was 8.02 min, NVP was well separated from the main component of the sample, and the control responded well, but the control peaked at a high gradient ratio, which was easy to produce gradient peaks to interfere with impurity detection.

[0124] 3. Method Confirmation

[0125] The applicant found that vonoprazan fumarate dissolves slowly in 50% acetonitrile solution. In order to improve the detection efficiency and reduce the influence of the matrix, the sample concentration was reduced and the injection volume was increased.

[0126] Based on the chromatographic and mass spectrometric conditions optimized in "2.2 Optimized Gradient Elution Program", the concentration of the reference solution was reduced to about 0.18 ng / ml, the sample concentration was reduced to about 0.4 mg / ml, the injection volume was increased to 20 μl, the gradient elution program was slowed down, and the high-proportion organic phase flushing time was increased. The specific conditions are as follows:

[0127] Liquid chromatography conditions: chromatographic column: Agilent Poroshell EC-C18 4.6×100 mm, 2.7 μm; mobile phase A: 0.01% formic acid solution; mobile phase B: acetonitrile; column temperature: 30°C; injection volume: 20 μl; flow rate: 0.6 ml / min; gradient program see Table 4.

[0128] Table 4 Gradient program confirmed by the method of Example 1

[0129] Time (min) 0 4 6 8 8.1 13 13.1 15 Mobile phase A (%) 70 70 25 25 0 0 70 70 Mobile phase B (%) 30 30 75 75 100 100 30 30

[0130] Mass spectrometry conditions: ion source: electrospray ion source; monitoring mode: multiple reaction monitoring mode; drying gas flow rate: 5 L / min; drying gas temperature: 300°C; nebulizing gas pressure: 45 psi; sheath gas temperature: 250°C; sheath gas flow rate: 11 L / min; capillary voltage: +4000 V; detector gain (+): 500 V; nozzle voltage: +500 V; dwell time: 150 ms.

[0131] The mass spectrometry parameters are shown in Table 5.

[0132] Table 5 Mass spectrometry parameters confirmed by the method of Example 1

[0133]

[0134] Solution preparation:

[0135] Reference substance stock solution: Take an appropriate amount of NVP, accurately weigh it to 2.830 mg, add 25 ml of 50% acetonitrile solution to dissolve; obtain a solution with a concentration of 0.1132 mg / ml, then accurately measure 1 ml of the solution and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve and dilute to the mark, to obtain a solution with a concentration of 2.264 μg / ml. Accurately measure 1 ml of the solution with a concentration of 2.264 μg / ml and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve and dilute to the mark, to obtain a solution with a concentration of 45.28 ng / ml. Accurately measure 1 ml of the solution with a concentration of 45.28 ng / ml and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve and dilute to the mark, to obtain a reference substance stock solution with a concentration of 1.81 ng / ml.

[0136] Reference solution: Accurately measure 1 ml of a solution with a concentration of 1.81 ng / ml and place it in a 10 ml volumetric flask. Add 50% acetonitrile solution to dilute to the scale to obtain a reference solution with a concentration of 0.1811 ng / ml.

[0137] Test solution: Take 40 mg of vonoprazan fumarate raw material, weigh accurately, and dissolve it in 10 ml of 50% acetonitrile solution; take 1 ml of the solution, dilute it with 50% acetonitrile solution and make it up to 10 ml to obtain a test solution with a concentration of about 0.4 mg / ml.

[0138] Sample recovery solution: Weigh about 40 mg of vonoprazan fumarate raw material, weigh accurately, add 10 ml of 50% acetonitrile solution to dissolve; prepare 9 portions in parallel. Take 1 ml of the above solution and place it in a 10 ml volumetric bottle, add 0.5 ml, 1.0 ml, and 1.5 ml of the reference substance stock solution (three portions at each level), and then add 50% acetonitrile solution to dilute to the scale, shake well, filter, and take the filtrate to obtain three portions of sample recovery 50% solution, sample recovery 100% solution, and sample recovery 150% solution.

[0139] The diluent, reference solution, test solution and each sample recovery solution were sampled and analyzed. The results are shown in Table 6 below.

[0140] Table 6 Sample recovery results confirmed by the method of Example 1

[0141]

[0142] Results: The solvent blank did not interfere with the detection of impurity NVP. The average recovery rate of NVP in the recovered solution was 89.51%, between 70% and 130%, and the RSD was 5.48% (n=9), which was less than 15%, meeting the detection requirements. Therefore, it can be used as a detection method for impurity NVP in vonoprazan fumarate raw materials.

[0143] Comparative Example 1

[0144] This comparative example discloses the analysis of nitrosamine impurities in vonoprazan fumarate tablets. This comparative example uses the method confirmed in Example 1 to analyze vonoprazan fumarate tablets. The liquid chromatography conditions are the same as the liquid chromatography conditions under "3. Method Confirmation" in Example 1.

[0145] The mass spectrometry conditions were the same as those in “3. Method Confirmation” in Example 1.

[0146] Solution preparation:

[0147] Blank excipient solution: Take about 53 mg of blank excipient, add 50 ml of 50% acetonitrile solution, mix well, filter, and take the filtrate.

[0148] Reference substance stock solution: Take an appropriate amount of NVP, accurately weigh it to 2.863 mg, add 25 ml of 50% acetonitrile solution to dissolve it, and obtain a 0.11452 mg / ml solution; then accurately measure 1 ml of this solution and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve it and dilute it to the mark, and obtain a solution with a concentration of 2.2904 μg / ml. Accurately measure 1 ml of the solution with a concentration of 2.2904 μg / ml and place it in a 50 ml volumetric flask, add 50% acetonitrile solution to dissolve it and dilute it to the mark, and obtain a reference substance stock solution with a concentration of 45.8 ng / ml.

[0149] Reference substance solution: Take 53.71 mg of blank excipient + 1 ml of reference substance stock solution, place in a 50 ml volumetric flask, add 50% acetonitrile solution to dilute and make up to volume, mix well, filter, and take the filtrate.

[0150] Test solution: Take 58 mg of vonoprazan fumarate tablet powder (equivalent to 5 mg of vonoprazan), add 50 ml of 50% acetonitrile solution, mix well, filter and take the filtrate.

[0151] 50% recovery solution by adding sample: Weigh 58 mg of vonoprazan fumarate tablets fine powder (equivalent to 5 mg of vonoprazan) and 0.5 ml of reference substance stock solution, place them together in the same 50 ml volumetric flask, dilute with 50% acetonitrile solution and make up to volume, mix well, filter, and take the filtrate to obtain 50% recovery solution by adding sample. Prepare three 50% recovery solutions by the same method.

[0152] The above solutions were sampled and analyzed respectively.

[0153] The results showed that under this condition, the recovery rate of the impurity NVP was only 66.92%, which was too low and did not meet the recovery rate requirement (70% to 130%).

[0154] Example 2

[0155] This embodiment discloses the determination of nitrosamine impurities in vonoprazan fumarate tablets by the method of the present invention, using LC-MS / MS for determination, wherein the chromatographic conditions are:

[0156] Column: Agilent Poroshell 120EC-C18 4.6×100 mm, 2.7 μm;

[0157] Mobile phase A: 0.01% formic acid solution; Mobile phase B: acetonitrile;

[0158] Column temperature: 30°C; injection volume: 5 μl; flow rate: 0.6 ml / min; injector temperature: 5°C; gradient program see Table 7;

[0159] Table 7 Gradient elution program of Example 2

[0160] Time (min) 0 4 6 8 8.1 13.0 13.1 15.0 Mobile phase A (%) 70 70 25 25 0 0 70 70 Mobile phase B (%) 30 30 75 75 100 100 30 30

[0161] The mass spectrometry conditions were as follows: ion source: electrospray ion source; scanning mode: multiple reaction monitoring mode; drying gas temperature: 300°C; drying gas flow rate: 5 L / min; nebulizing gas pressure: 45 psi; sheath gas temperature: 250°C; sheath gas flow rate: 11 L / min; capillary voltage: +4000 V; nozzle voltage: +500 V; detector gain: +500 V; dwell time: 150 ms; mass spectrometry acquisition time 7.3 to 9.0 min.

[0162] The mass spectrometry parameters are shown in Table 8.

[0163] Table 8 Mass spectrometry parameters of Example 2

[0164]

[0165] Solution preparation:

[0166] Diluent: 50% acetonitrile solution (containing 5g / L sodium chloride).

[0167] Blank excipient solution: Take about 53 mg of blank excipient, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, filter, and take the filtrate.

[0168] Reference substance stock solution: Accurately weigh 2.8 mg of impurity NVP, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale to obtain a solution with a concentration of 0.112 mg / ml; accurately measure 1 ml of the solution, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and obtain a solution with a concentration of 2.24 μg / ml; accurately measure 1 ml of the solution with a concentration of 2.24 μg / ml, place it in a 50 ml volumetric flask, dilute to the scale with diluent, and obtain a reference substance stock solution with an impurity NVP content of approximately 44.8 ng / ml.

[0169] Reference substance solution: Take about 53 mg of blank auxiliary material, place in a 50 ml volumetric bottle, accurately add 1 ml of reference substance stock solution, place in a 50 ml volumetric bottle, dilute to scale with diluent, shake well, filter, and take the filtrate. (Impurity NVP is about 0.896 ng / ml)

[0170] Test solution: Take 58 mg of vonoprazan fumarate tablet powder (equivalent to 5 mg of vonoprazan), accurately weigh, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate (vonoprazan about 0.1 mg / ml).

[0171] Sample recovery solution: Weigh about 58 mg of vonoprazan fumarate tablets powder, put it in a 50 ml volumetric flask, accurately add 0.5 ml, 1.0 ml, and 1.5 ml of the reference substance stock solution, respectively, add diluent to dissolve and dilute to the scale, shake well, filter, and take the filtrate to obtain a sample recovery 50% solution, a sample recovery 100% solution, and a sample recovery 150% solution, respectively.

[0172] Take the above solutions for sampling and analysis. The results are shown in Table 9 and the attached figure. Figures 1 to 6 shown.

[0173] Table 9 Sample recovery test results of Example 2

[0174]

[0175] Note: 50%, 100%, and 150% in the above table refer to 50% recovery solution, 100% recovery solution, and 150% recovery solution, respectively.

[0176] The test results show that the diluent, main component and other impurities do not interfere with the impurity NVP detection. The average recovery rate of NVP in the recovered solution is 95.10%, between 70% and 130%, and the RSD is 2.2% (n=3), which is less than 15%, meeting the detection requirements.

[0177] Example 3

[0178] This example discloses a durability test of the method of the present invention. Based on the mass spectrometry conditions of Example 2, the chromatographic and mass spectrometry parameters were fine-tuned. The adjustment and measurement results are shown in Table 10.

[0179] Table 10 Impurity NVP method durability investigation results

[0180]

[0181] It can be seen from Table 10 above that fine-tuning the above chromatographic and mass spectrometric parameters has no interference with the detection of impurity NVP. The average recovery rate of impurity NVP is 101.90%, between 70% and 130%, and the RSD is 6.3%, which is less than 15%. The durability of the method meets the requirements.

[0182] Example 4

[0183] This example investigates the durability of a sodium chloride concentration of 0.5 g / L in a 50% acetonitrile solution as a diluent, using the conditions of Example 2. The results are shown in Table 11.

[0184] Table 11 Impurity NVP method durability investigation results

[0185] Serial number 1 2 3 4 5 6 Mean RSD Recovery rate 97.60％ 95.07％ 92.53％ 101.09％ 98.43％ 94.68％ 96.57％ 3.2％

[0186] The results showed that when the concentration of sodium chloride in the diluent was 0.5 g / L, the average recovery rate was 96.57%, between 70% and 130%, and the RSD was 3.2%, which was less than 15%, indicating that the durability of the method of the present invention met the requirements.

[0187] From the data of Examples 2 to 4 above, it can be concluded that a 50% acetonitrile solution containing 0.5-5 g / L sodium chloride can meet the detection requirements of the impurity NVP, and it can be expected that a 50% acetonitrile solution containing 0.1-10 g / L sodium chloride can meet the detection requirements of the impurity NVP.

[0188] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A method for analyzing nitrosamine impurities, characterized in that: The LC-MS / MS method was used for analysis. The chromatographic conditions included: using ODS C18 as the stationary phase, an acidic aqueous solution as the mobile phase A, and an organic solvent as the mobile phase B, and eluting according to the following gradient: 0min, 65~75%A, 25~35%B; 3.5~4.5min, 65~75%A, 25~35%B; 5.5~6.5min, 20~30%A, 70~80%B; 7.5~8.5min, 20~30%A, 70~80%B; 7.6~8.6min, 0~5%A, 95~100%B; 12.5~13.5min, 0~5%A, 95~100%B; 12.6~13.6min, 65~75%A, 25~35%B; 14.5~17.5min, 65~75%A, 25~35%B; The mass spectrometry conditions include: using a triple quadrupole mass spectrometer, electrospray ionization positive ion mode, and multiple reaction monitoring; The nitrosamine impurities include N-nitroso-vonoprazan.

2. The method for analyzing nitrosamine impurities according to claim 1, characterized in that: Elution was performed according to the following gradient: 0min, 65~75%A, 25~35%B;; 4min, 65~75%A, 25~35%B; 6min, 20~30%A, 70~80%B; 8min, 20~30%A, 70~80%B; 8.1min, 0~5%A, 95~100%B; 13min, 0~5%A, 95~100%B; 13.1min, 65~75%A, 25~35%B; 15min, 65~75%A, 25~35%B.

3. The method for analyzing nitrosamine impurities according to claim 1, characterized in that: Elution was performed according to the following gradient: 0min, 70% A, 30% B; 4 min, 70% A, 30% B; 6min, 25% A, 75% B; 8min, 25% A, 75% B; 8.1 min, 0% A, 100% B; 13min, 0%A, 100%B; 13.1min, 70% A, 30% B; 15min, 70%A, 30%B.

4. A method for analyzing nitrosamine impurities according to any one of claims 1 to 3, characterized in that: The acidic aqueous solution is an organic acid aqueous solution, preferably a formic acid solution, more preferably a 0.005-0.03% formic acid solution, and further preferably a 0.01% formic acid solution; Preferably, the organic solvent is methanol and / or acetonitrile, more preferably acetonitrile.

5. The method for analyzing nitrosamine impurities according to any one of claims 1 to 3, characterized in that: The column temperature is 25-35°C, preferably 30°C; Preferably, the flow rate is 0.3 to 1.0 ml / min; more preferably 0.6 ml / min; Preferably, the sample is injected under low temperature conditions, more preferably, the injector temperature is 2-10°C; further preferably, it is 5°C.

6. A method for analyzing nitrosamine impurities according to any one of claims 1 to 3, characterized in that: The mass spectrometry acquisition time was 7.3 to 9.0 min.

7. The method for analyzing nitrosamine impurities according to claim 6, characterized in that: The mass-to-charge ratio of the precursor ion is 375.1, and the mass-to-charge ratios of the product ions are 315.1, 251, 173, and 78.

1.

8. The method for analyzing nitrosamine impurities according to claim 7, characterized in that: The precursor ion mass-to-charge ratio was 375.1 and the product ion mass-to-charge ratio was 315.1 for quantitative analysis. Preferably, a precursor ion mass-to-charge ratio of 375.1 and a product ion mass-to-charge ratio of at least one of 251, 173, and 78.1 are selected for qualitative analysis.

9. Application of the method for analyzing nitrosamine impurities according to any one of claims 1 to 8, characterized in that: Used for the analysis of impurities in vonoprazan fumarate or / and its preparations, preferably, the preparations include vonoprazan fumarate tablets.

10. The use according to claim 9, characterized in that: The external standard method is used to determine the content of impurities in vonoprazan fumarate and / or its preparations; Preferably, when determining vonoprazan fumarate, a 30-70% acetonitrile solution is used as the diluent; more preferably, a 40-60% acetonitrile solution, further preferably, a 45-65% acetonitrile solution, and even more preferably, a 50% acetonitrile solution; Preferably, when measuring vonoprazan fumarate tablets, a 30-70% acetonitrile solution is used as a diluent, and the diluent contains 0.1-10 g / L of salt. More preferably, a 30-70% acetonitrile solution containing 0.5-5 g / L of salt is used as a diluent; more preferably, a 50% acetonitrile solution containing 0.1-10 g / L of salt is used as a diluent; further preferably, a 50% acetonitrile solution containing 0.5-5 g / L of salt is used as a diluent. Preferably, the salt is an inorganic salt; More preferably, the salt is an inorganic potassium salt or an inorganic sodium salt; further preferably, the salt is at least one of sodium chloride, potassium chloride, and potassium sulfate; more preferably, sodium chloride; Preferably, when measuring vonoprazan fumarate tablets, 30-70% acetonitrile solution is used as the diluent, and the diluent contains 0.1-10 g / L sodium chloride. More preferably, 30-70% acetonitrile solution containing 0.5-5 g / L sodium chloride is used as the diluent; more preferably, 50% acetonitrile solution containing 0.1-10 g / L sodium chloride is used as the diluent; further preferably, 50% acetonitrile solution containing 0.5-5 g / L sodium chloride is used as the diluent.

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