Method for detecting N, N-dimethyl nitrosamine in neostigmine methylsulfate and injection

Abstract

The invention discloses a method for detecting N, N-dimethyl nitrosamine in neostigmine methylsulfate and injection, and belongs to the technical field of medicine quality detection.The method comprises the following steps that 1, a test solution is prepared, specifically, the test solution is dissolved with methyl alcohol and diluted into a solution with the concentration of 0.3 mg / ml to 0.8 mg / ml; (2) preparation of a reference substance solution: taking a proper amount of an N, N-dimethyl nitrosamine reference substance (standard substance), dissolving with methanol, and diluting into a 5-15ng / ml solution; (3) detecting by adopting an ultra-high performance liquid chromatography-mass spectrometry method; and (4) inspection and measurement: measuring 10-50 [mu] l of the applicable solution, 10-50 [mu] l of the reference solution and 10-50 [mu] l of the test solution, respectively injecting the solutions into an ultra-high performance liquid chromatography-mass spectrometer, recording chromatograms, and calculating according to an external standard method. The detection method disclosed by the invention is high in specificity, high in sensitivity, good in precision, stable in solution, rapid and sensitive in detection and accurate and reliable in result, and the controllability of the quality of the medicine is improved.

Description

Technical Field

[0001] The present invention relates to a method for detecting N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, belonging to the technical field of drug quality detection. Technical Background

[0002] In 1931, the stimulatory effect of neostigmine on the intestine was first applied clinically; in 1935, it was confirmed to be effective for myasthenia gravis. The methylsulfate of neostigmine can form the most stable aqueous solution. Neostigmine methylsulfate injection was first approved for marketing in Japan in October 1936, with a specification of 1 ml: 0.5 mg, and the license holder was Shionogi & Co., Ltd. In March 2001, approval was obtained to add the muscle relaxation effect of non-depolarizing muscle relaxants. In March 2003, approval was obtained to add a large specification of 4 ml: 2 mg. In December 2016, the license holder was transferred from Shionogi & Co., Ltd. to Kyowa Pharmaceutical Industry Co., Ltd.; in October 2019, Shionogi & Co., Ltd. transferred both the production and sales of neostigmine methylsulfate injection to Kyowa Pharmaceutical Industry Co., Ltd.

[0003] On May 31, 2013, neostigmine methylsulfate injection was launched in the United States under the trade name: BLOXIVERZ, with specifications of 5MG / 10ML and 10MG / 10ML, and the license-holding manufacturer: EXELA PHARMA.

[0004] Neostigmine methylsulfate injection, as an anticholinesterase drug, is used to antagonize the residual muscle relaxation effect of non-depolarizing muscle relaxants at the end of surgery, for myasthenia gravis, postoperative functional flatulence and urinary retention, etc.

[0005] The specifications included in the national essential medicine list in China are 1 ml: 0.5 mg and 2 ml: 1 mg; the specifications of domestic marketed generic preparations are 1 ml: 0.5 mg and 2 ml: 1 mg. There are no original research imported and original research domesticated products of neostigmine methylsulfate injection on the domestic market. Neostigmine methylsulfate injection is included in the "National Basic Medical Insurance, Work-related Injury Insurance and Maternity Insurance Drug List" in 2024, belonging to Class A medical insurance, and is widely used, so it is of great significance to effectively control the product quality.

[0006] Refer to the notice (No. 1, 2020) of the Center for Drug Evaluation of the National Medical Products Administration, "Technical Guidelines for the Study of Nitrosamine Impurities in Chemical Drugs (Trial)". During the production process of neostigmine methylsulfate raw materials, during the storage process of neostigmine methylsulfate, and during the storage process of neostigmine methylsulfate injection, N,N-dimethylnitrosamine (NDMA) may be degraded. According to the carcinogen list of the International Agency for Research on Cancer of the World Health Organization, NDMA belongs to Group 2A carcinogens, that is, substances with limited evidence of carcinogenicity to humans but sufficient evidence of carcinogenicity to experimental animals. At the same time, nitrosamine compounds belong to substances with high carcinogenicity ("concerned cohort") clearly mentioned in the ICH M7 guideline. Therefore, such compounds should be strictly controlled in drugs.

[0007] At present, there are no reports at home and abroad on the detection methods for N,N-dimethylnitrosamine impurities in neostigmine methylsulfate and neostigmine methylsulfate injection. Therefore, developing an economical, practical, and efficient detection method for N,N-dimethylnitrosamine in neostigmine methylsulfate and neostigmine methylsulfate injection, which can accurately detect N,N-dimethylnitrosamine in neostigmine methylsulfate and neostigmine methylsulfate injection, in order to achieve good economic and social benefits and provide convenience for the quality research and impurity detection of neostigmine methylsulfate and neostigmine methylsulfate injection, has become an urgent technical problem to be solved in this technical field. Summary of the Invention

[0008] The purpose of the present invention is to provide a detection method for N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection. By using ultra-high performance liquid chromatography-mass spectrometry, suitable chromatographic conditions, sample preparation solvents, and appropriate mobile phases are creatively developed, and the developed analytical method is subjected to methodological verification, proving that it can be used for the detection of N,N-dimethylnitrosamine in neostigmine methylsulfate and neostigmine methylsulfate injection, and the limit of quantification can reach 2 ppm. The method of the present invention has strong specificity, high sensitivity, good precision, stable solution, rapid and sensitive detection, and accurate and reliable results for the detection of N,N-dimethylnitrosamine.

[0009] The above object of the present invention is achieved by the following technical solutions:

[0010] A detection method for N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, the steps are as follows:

[0011] (1) Chromatographic conditions of the ultra-high performance liquid chromatograph:

[0012] Chromatographic column: Kinetex F5 (4.6 mm × 150 mm, 2.6 μm);

[0013] Flow rate: 0.1 ml / min to 0.8 ml / min;

[0014] Column temperature: 10°C to 40°C;

[0015] Injector temperature: 2°C to 6°C;

[0016] Mobile phase:

[0017] Mobile phase A: 2 mmol / L to 10 mmol / L ammonium formate solution;

[0018] Mobile phase B: Methanol solution of formic acid, mass percentage of formic acid is 0.05% to 0.15%;

[0019] Mobile phase ratio: Mobile phase A: Mobile phase B = 30:70 to 95:5;

[0020] (2) Mass spectrometry conditions:

[0021] Corona needle current: 3 μA to 8 μA;

[0022] Desolvation gas temperature: 400°C to 600°C;

[0023] Desolvation gas: 600 L / Hr to 900 L / Hr;

[0024] Cone gas: 40 L / Hr to 60 L / Hr;

[0025] Ionization mode: APCI+;

[0026] The collected ion pairs are: 75>43;

[0027] (3) Preparation of test solution: Take neostigmine methylsulfate or neostigmine methylsulfate injection, dissolve it with methanol and dilute it to a solution of 0.3 mg / ml to 0.8 mg / ml;

[0028] (4) Preparation of reference solution: Take an appropriate amount of N,N-dimethylnitrosamine reference substance (standard substance), weigh it, dissolve it with methanol and dilute it to a solution of 5 ng / ml to 15 ng / ml;

[0029] (5) Preparation of suitability solution: Measure appropriate amounts of neostigmine methylsulfate or neostigmine methylsulfate injection and N,N-dimethylnitrosamine reference substance (standard substance), dissolve and dilute with methanol, mix and make a solution containing neostigmine methylsulfate: 0.45 mg / ml, N,N-dimethylnitrosamine: 10 ng / ml;

[0030] (6) Test measurement: Measure 10 μl to 50 μl of the suitability solution, reference solution and test solution respectively, inject them into an ultra-high performance liquid chromatography-mass spectrometer, record the chromatogram, and calculate by the external standard method.

[0031] Preferably, in step (1), the packing material in the chromatographic column is a pentafluorophenyl material.

[0032] Preferably, in step (1), the chromatographic column is a pentafluorophenyl column Kinetex F5 (4.6 mm × 150 mm, 2.6 μm).

[0033] Preferably, in step (1), the ultra-high performance liquid chromatography-mass spectrometer is an ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometer of brands such as Waters, SCIEX, Shimadzu, and Agilent.

[0034] Preferably, in step (1), mobile phase A: 5 mmol / L ammonium formate solution; mobile phase B: methanol solution of 0.1% formic acid.

[0035] Preferably, in step (1), mobile phase ratio: mobile phase A: mobile phase B = 40:60.

[0036] Preferably, in step (1), the column temperature is 35°C ± 5°C.

[0037] Preferably, in step (1), the flow rate is 0.4 ml / min.

[0038] Preferably, in step (2), the corona needle current: 5 μA.

[0039] Preferably, in step (2), the desolvation gas temperature: 500°C.

[0040] Preferably, in step (2), the desolvation gas: 800 L / Hr.

[0041] Preferably, in step (2), the cone gas: 50 L / Hr.

[0042] Preferably, in step (3), the concentration of the test solution is 0.5 mg / ml.

[0043] Preferably, in step (4), the concentration of the reference solution is 10 ng / ml.

[0044] Preferably, in steps (3) and (4), the sample preparation solvents for the test solution and the reference solution are methanol.

[0045] Preferably, in step (5), the suitability solution contains neostigmine methylsulfate at 0.45 mg / ml and N,N-dimethylnitrosamine (NDMA) at 10 ng / ml.

[0046] Preferably, in step (6), the injection volume is 50 μl for each of the suitability solution, the reference solution, and the test solution.

[0047] Beneficial effects:

[0048] The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection of the present invention creatively adopts the ultra-high performance liquid chromatography-mass spectrometry method with a pentafluorophenyl chromatographic column. Through research, key factors such as the type of mobile phase, the proportion of mobile phase, column temperature, flow rate, injection volume, liquid preparation solvent, mass spectrometry corona needle current, desolvation gas temperature, desolvation gas, and cone orifice gas are confirmed, and an effective determination method is obtained. This method for determining N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection has the advantages of strong specificity, high sensitivity, good precision, stable solution, rapid and sensitive detection, and accurate and reliable results, increasing the controllability of the drug quality and providing an effective and stable analysis method for the detection of N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection.

[0049] The detection method of N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection of the present invention can accurately detect N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, enabling the detection ability of N,N-dimethylnitrosamine to reach 2 ppm, having good economic and social benefits, and providing convenience for the quality research and impurity detection of neostigmine methylsulfate and its injection.

[0050] The present invention will be further described below through the drawings and specific embodiments, but it does not mean limiting the protection scope of the present invention. Description of the Drawings

[0051] Figure 1 It is the detection chromatogram of the N,N-dimethylnitrosamine reference substance solution in Example 1 of the present invention;

[0052] Figure 2 It is the detection chromatogram of the applicability solution in Example 1 of the present invention. Detailed Embodiments

[0053] Unless otherwise specified, the raw materials, auxiliaries or equipment used in the embodiments of the present invention are all products that can be purchased on the market, and their models are the conventional models in the art; the testing methods used are all the conventional methods in the art.

[0054] Example 1

[0055] A detection method for N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection is as follows:

[0056] Ultra-high performance liquid chromatography conditions:

[0057] Chromatographic column: Kinetex F5 (4.6 mm × 150 mm, 2.6 μm);

[0058] Flow rate: 0.4 ml per minute;

[0059] Column temperature: 35 °C;

[0060] Sample injection volume: 50 μl;

[0061] Sample injector temperature: 4 °C;

[0062] Mobile phase:

[0063] Mobile phase A: 5 mmol / L ammonium formate solution;

[0064] Mobile phase B: Methanol solution of 0.1% formic acid;

[0065] Mobile phase A: Mobile phase B = 40:60;

[0066] Mass spectrometry conditions:

[0067] Corona needle current: 5 μA;

[0068] Desolvation gas temperature: 500 °C;

[0069] Desolvation gas: 800 L / Hr;

[0070] Cone gas: 50 L / Hr;

[0071] Ionization mode: APCI+;

[0072] Acquired ion pair is: 75>43;

[0073] Preparation of solutions:

[0074] Solvent: Methanol;

[0075] Test solution: Take neostigmine methylsulfate, dissolve and dilute with methanol to prepare a solution containing 0.5 mg / ml of neostigmine methylsulfate;

[0076] Reference solution: Weigh the reference substance of N-nitrosodimethylamine (standard substance), dissolve and dilute with methanol to prepare a reference solution containing 10 ng / ml of N-nitrosodimethylamine;

[0077] Suitability solution: Measure neostigmine methylsulfate and the reference substance of N-nitrosodimethylamine (standard substance), dissolve and dilute with methanol to prepare a solution containing 0.45 mg / ml of neostigmine methylsulfate and 10 ng / ml of N-nitrosodimethylamine;

[0078] Inspection and measurement: Take the suitability solution, reference solution, and test solution, inject them for analysis respectively, and record the chromatograms.

[0079] Example 2

[0080] A method for detecting N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, the steps are as follows:

[0081] Ultra-high performance liquid chromatography conditions:

[0082] Column: Kinetex F5 (4.6 mm × 150 mm, 2.6 μm)

[0083] Flow rate: 0.1 ml per minute;

[0084] Column temperature: 10 °C;

[0085] Injection volume: 10 μl;

[0086] Injector temperature: 2 °C;

[0087] Mobile phase:

[0088] Mobile phase A: 2 mmol / L ammonium formate solution;

[0089] Mobile phase B: Methanol solution of 0.1% formic acid;

[0090] Mobile phase A: Mobile phase B = 30:70;

[0091] Mass spectrometry conditions:

[0092] Corona needle current: 3 μA;

[0093] Desolvation gas temperature: 400 °C;

[0094] Desolvation gas: 600 L / Hr;

[0095] Cone gas: 40 L / Hr;

[0096] Ionization mode: APCI+;

[0097] Acquired ion pair is: 75>43;

[0098] Preparation of solutions:

[0099] Solvent: Methanol;

[0100] Test solution: Take neostigmine methylsulfate, dissolve and dilute with methanol to prepare a test solution containing 0.3 mg / ml of neostigmine methylsulfate;

[0101] Reference solution: Weigh the reference substance of N-nitrosodimethylamine (standard), dissolve and dilute with methanol to prepare a reference solution containing 5 ng / ml of N-nitrosodimethylamine (abbreviation: NDMA);

[0102] Suitability solution: Measure neostigmine methylsulfate injection and the reference substance of N-nitrosodimethylamine (standard), dissolve and dilute with methanol to prepare a solution containing 0.45 mg / ml of neostigmine methylsulfate and 10 ng / ml of N-nitrosodimethylamine;

[0103] Test measurement: Take the applicability solution, reference solution, and test solution, inject them for analysis respectively, and record the chromatogram.

[0104] Example 3

[0105] A method for detecting neostigmine methylsulfate and N,N-dimethylnitrosamine in injection, the steps are as follows:

[0106] Ultra-high performance liquid chromatography conditions:

[0107] Chromatographic column: Kinetex F5 (4.6mm×150mm, 2.6μm);

[0108] Flow rate: 0.8 ml per minute;

[0109] Column temperature: 40°C;

[0110] Injection volume: 50 μl;

[0111] Injector temperature: 6°C;

[0112] Mobile phase:

[0113] Mobile phase A: 10 mmol / L ammonium formate solution;

[0114] Mobile phase B: Methanol solution of 0.5% formic acid;

[0115] Mobile phase A: Mobile phase B = 95:5;

[0116] Mass spectrometry conditions:

[0117] Corona needle current: 8 μA;

[0118] Desolvation gas temperature: 600°C;

[0119] Desolvation gas: 900 L / Hr;

[0120] Cone gas: 60 L / Hr;

[0121] Ionization mode: APCI+;

[0122] The collected ion pair is: 75>43;

[0123] Preparation of solution:

[0124] Solvent: Methanol;

[0125] Test solution: Take neostigmine methylsulfate, dissolve it with methanol and dilute it to a test solution of 0.8 mg / ml;

[0126] Reference solution: Weigh the reference standard of N-nitrosodimethylamine, dissolve and dilute it with methanol to prepare a reference solution containing 15 ng / ml of the reference standard of N-nitrosodimethylamine;

[0127] System suitability solution: Measure neostigmine methylsulfate and the reference standard of N-nitrosodimethylamine, dissolve and dilute them with methanol to prepare a system suitability solution containing neostigmine methylsulfate: 0.45 mg / ml and N-nitrosodimethylamine (NDMA): 10 ng / ml;

[0128] Inspection and measurement: Inject the system suitability solution, reference solution, and test solution for analysis respectively, and record the chromatogram.

[0129] Methodology verification:

[0130] Taking Example 1 as an example, conduct methodology verification from aspects of specificity, detection limit and quantitation limit, linearity and range, accuracy, repeatability, intermediate precision, solution stability and robustness. The results are as follows:

[0131] (1) Specificity

[0132] Precisely measure 50 μl of blank solvent (methanol), mixed solution (test solution: reference solution = 1:1), test solution, and reference solution respectively, and inject them into an ultra-high performance liquid chromatography-mass spectrometer to record the chromatogram; the blank solvent, test solution, and mixed solution do not interfere with the detection of NDMA, meeting the verification requirements, and this method has good specificity;

[0133] (2) Quantitation limit and detection limit

[0134] Take the reference solution of N,N-nitrosodimethylamine, dissolve and dilute it with methanol for injection, and prepare a solution with a concentration 50% lower than the limit concentration and a signal-to-noise ratio greater than 10 as the quantitation limit solution; dilute the quantitation limit solution by 2 times as the detection limit solution, with a signal-to-noise ratio greater than 3; the quantitation limit and detection limit of N,N-nitrosodimethylamine are shown in Table 1 below:

[0135] Table 1

[0136]

[0137]

[0138] (3) Linearity and range

[0139] Take an appropriate amount of N,N-dimethylnitrosamine, dissolve it in methanol and dilute to prepare a solution with a concentration of 100 ng / ml as the linear stock solution. Measure an appropriate amount of the linear stock solution and prepare a series of linear solutions with concentrations of 2 ng / ml, 5 ng / ml, 10 ng / ml, 15 ng / ml, and 20 ng / ml. N,N-dimethylnitrosamine shows a good linear relationship in the concentration range of 1 (quantification limit) to 20 ng / ml, with a correlation coefficient r greater than 0.9980, meeting the requirements;

[0140] (4) Repeatability

[0141] Using the same batch of test samples (neostigmine methylsulfate), prepared in parallel 6 portions of test sample solutions by the same analyst. Use the spiked solution (accurately measure this product and the N,N-dimethylnitrosamine reference substance (standard product), dissolve and dilute with methanol to prepare a solution containing 0.45 mg of neostigmine methylsulfate and 10 ng of N,N-dimethylnitrosamine per ml) to investigate the precision and evaluate the precision of the method. The results show that for the 6 parallel solutions of the method of the present invention, the RSD of the detected amount of NDMA is ≤10%, meeting the verification requirements;

[0142] (5) Intermediate precision

[0143] On different dates, by different personnel, conduct tests according to the repeatability item. For the total 12 samples under the repeatability item, summarize the results of the 12 samples respectively. The results show that for the 12 spiked solutions (accurately measure this product and the N,N-dimethylnitrosamine reference substance (standard product), dissolve and dilute with methanol to prepare a solution containing 0.45 mg of neostigmine methylsulfate and 10 ng of N,N-dimethylnitrosamine per ml), the RSD of the detected amount of NDMA is ≤20%, meeting the verification requirements;

[0144] (6) Accuracy

[0145] Verify through the spiked recovery test: Investigate the recovery rates of known impurities at 3 concentration levels: respectively investigate the 50% limit concentration, 100% limit concentration, and 150% limit concentration. Prepare 3 portions in parallel for each level. Among the 9 samples at the 3 concentration levels, the spiked recovery rates of NDMA are all between 80% and 120%, and the RSD is less than 10%, meeting the verification requirements;

[0146] (7) Solution stability

[0147] For the reference substance solution and the spiked solution, place them at 4°C for 22 hours. The ratio of the peak area of NDMA to that at 0 h is between 80% and 120%, indicating that the reference substance solution and the spiked solution are stable when placed at 4°C for 22 hours;

[0148] (8) Robustness

[0149] Based on the established chromatographic conditions, the mobile phase ratio, flow rate, and column temperature of the chromatographic system were investigated within the range of "Summary of the Invention (1)", and the detection results were all within the range of 80% - 120%, indicating good durability under the above conditions.

[0150] As Figure 1 shown, it is the detection chromatogram of the N,N-dimethylnitrosamine reference substance solution in Example 1 of the present invention; as Figure 2 shown, it is the detection chromatogram of the applicability solution in Example 1 of the present invention.

[0151] The method for detecting N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection of the present invention creatively develops a set of scientific and reasonable chromatographic conditions, suitable sample preparation solvents and sample concentrations for the test samples and reference substances by using ultra-high performance liquid chromatography - mass spectrometry, and conducts methodological verification on the developed analytical method, proving that it can be used for the detection of N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, and the limit of quantification can reach 2 ppm. This method for detecting N,N-dimethylnitrosamine has strong specificity, high sensitivity, good precision, stable solution, rapid and sensitive detection, and accurate and reliable results.

[0152] The above is only the preferred specific implementation manner of the present invention, and is not intended to limit the protection scope of the present invention. Any person skilled in the art can make various changes and modifications according to the technical solution of the present invention without departing from the spirit and scope of the present invention, and it still belongs to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be defined by the claims.

Claims

1. A method for detecting N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection, the steps are as follows: (1) Chromatographic conditions of ultra-high performance liquid chromatography: Chromatographic column: Kinetex F5; Flow rate: 0.1 ml / min to 0.8 ml / min; Column temperature: 10°C to 40°C; Injector temperature: 2°C to 6°C; Mobile phase: Mobile phase A: 2 mmol / L to 10 mmol / L ammonium formate solution; Mobile phase B: Methanol solution with 0.1% to 0.5% formic acid; Mobile phase ratio: Mobile phase A: Mobile phase B = 30:70 to 95:5; (2) Mass spectrometry conditions: Corona needle current: 3 to 8 μA; Desolvation gas temperature: 400°C to 600°C; Desolvation gas: 600 L / Hr to 900 L / Hr; Cone gas: 40 L / Hr to 60 L / Hr; Ionization mode: APCI+; Acquired ion pair: 75>43; (3) Preparation of test solution: Take neostigmine methylsulfate or neostigmine methylsulfate injection, dissolve it with methanol and dilute it to a solution of 0.3 to 0.8 mg / ml; (4) Preparation of reference solution: Take an appropriate amount of N,N-dimethylnitrosamine reference substance, dissolve it with methanol and dilute it to a reference solution of 5 ng / ml to 15 ng / ml; (5) Applicability solution: Measure an appropriate amount of neostigmine methylsulfate or neostigmine methylsulfate injection and N,N-dimethylnitrosamine reference substance, dissolve and dilute with methanol, mix and make a solution containing neostigmine methylsulfate: 0.45 mg / ml, N,N-dimethylnitrosamine: 10 ng / ml as the applicability solution; (6) Test measurement: Measure 10 to 50 μl of the applicability solution, reference solution, and test solution respectively, inject them into the chromatograph, record the chromatogram, and calculate by the external standard method.

2. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In step (1), the chromatographic column is a pentafluorophenyl column, the column temperature is 35°C ± 5°C, 5 mmol / L ammonium formate solution is used as mobile phase A, and methanol solution with 0.1% formic acid is used as mobile phase B, and the mobile phase ratio is: 40:60, and the flow rate is 0.4 ml / min.

3. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In steps (1) and (2), the ultra-high performance liquid chromatography-mass spectrometer is an ultra-high performance liquid chromatography triple quadrupole mass spectrometer of the brands Waters, SCIEX, Shimadzu or Agilent.

4. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In step (2), for the mass spectrometry conditions, the corona needle current: 5 μA; the desolvation gas temperature: 500°C; the desolvation gas: 800 L / Hr; The cone gas: 50 L / Hr; the ionization mode: APCI+; the acquired ion pair: 75>43.

5. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In step (3), the concentration of the test solution is 0.5 mg / ml.

6. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In step (4), the concentration of the reference solution is 10 ng / ml.

7. The detection method of neostigmine methylsulfate and N,N-dimethylnitrosamine in the injection according to claim 1, wherein: In step (5), the applicability solution contains neostigmine methylsulfate: 0.45 mg / ml, N,N-dimethylnitrosamine: 10 ng / ml.

8. The detection method of N,N-dimethylnitrosamine in neostigmine methylsulfate and its injection according to claim 1, wherein: In step (6), the injection volume of the applicability solution, reference solution, and test solution is 50 μl.

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