A method for determining N-methylpiperazine in clozapine

Abstract

The application discloses a detection method for N-methyl piperazine in clozapine, and belongs to the technical field of drug detection. The detection method comprises solution preparation and detection. The solution preparation uses clozapine to prepare a test sample solution and uses N-methyl piperazine to prepare a control sample solution. The detection adopts liquid chromatography-mass spectrometry to determine the test sample solution and the control sample solution, records a chromatogram, and calculates the content of N-methyl piperazine. In the detection, the conditions of the liquid chromatography are as follows: a mobile phase is composed of mobile phase A and mobile phase B, the mobile phase A is a 0.048-0.052% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile. The application can quantitatively detect the genotoxic impurity N-methyl piperazine in clozapine bulk drug and preparations, is convenient for monitoring the content of the impurity in the production and storage process of clozapine, and is high in selectivity, good in sensitivity, and low in quantitative limit to 0.165 ppm.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection, more particularly, to a detection method for determining N-methyl piperazine in clozapine. BACKGROUND

[0002] Clozapine is mainly used for treating various subtypes of acute and chronic schizophrenia, and is effective for hallucinatory and delusional types and can also reduce emotional symptoms associated with schizophrenia.

[0003] N-methyl piperazine (NMP) is a genotoxic impurity detected in clozapine bulk drug and its preparations. This impurity is a starting material in the synthesis of clozapine bulk drug and is introduced in the second half of the synthesis step, and is a potential process impurity and degradation impurity. It is reported that N-methyl piperazine is positive in the NITE database (reverse mutation test of 1-methyl piperazine in bacteria) in Japan, and is a genotoxic impurity.

[0004] N-methyl piperazine is a starting material used in the synthesis process of clozapine, and is a potential process impurity and genotoxic impurity. The molecular structural formula is as follows:

[0005]

[0006] According to the ICH M7 requirement, the impurities calculated by the PDE method need to be strictly controlled. According to the PDE method, the recommended toxicology threshold (TTC) of N-methyl piperazine is 1.5 μg / day, and the maximum daily dose of clozapine tablets is 0.9 g / person / day, so the control limit of this impurity should be no more than 1.67 ppm, and the sensitivity of the conventional detection method cannot be reached.

[0007] At present, there is no published method for detecting N-methyl piperazine in clozapine bulk drug and preparations. In order to achieve better quality control effect and prevent N-methyl piperazine from exceeding the limit of drug outflow, it is urgent to develop a detection method for N-methyl piperazine in clozapine to solve the above problems. SUMMARY

[0008] The present application aims to provide a detection method for determining N-methyl piperazine in clozapine, which can quantitatively detect the genotoxic impurity N-methyl piperazine in clozapine bulk drug and preparations, and facilitate monitoring the content of the impurity in the production and storage process of clozapine.

[0009] To solve the above technical problems, the technical solutions adopted by the present application are as follows:

[0010] A detection method for determining N-methyl piperazine in clozapine, comprising preparing a solution and detecting;

[0011] The preparation solution, using clozapine to prepare the test sample solution, using N-methyl piperazine to prepare the control sample solution;

[0012] Further, in the preparation solution, the preparation method of the test sample solution is: accurately weighing the clozapine, adding the solvent to ultrasonically dissolve and quantitatively dilute, filtering, and preparing the test sample solution;

[0013] In the preparation of the test sample solution, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 4:1;

[0014] In the preparation of the test sample solution, the filtering accuracy of the filter membrane used in the filtering is 0.22μm;

[0015] The concentration of clozapine in the test sample solution is 2.8-3.2mg / mL;

[0016] Further, in the preparation solution, the preparation method of the control sample solution is: accurately weighing 24.5-25.5mg of N-methyl piperazine control sample, adding into a centrifugal tube, adding 9.8-10.2mL of methanol, ultrasonically dissolving; taking 49-51μL therefrom and transferring into a centrifugal tube, adding 49-51mL of solvent and mixing uniformly; taking 98-102μL therefrom and transferring into a centrifugal tube, adding 49-51mL of solvent and mixing uniformly, to prepare the control sample solution;

[0017] In the preparation of the control sample solution, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 4:1;

[0018] The concentration of N-methyl piperazine in the control sample solution is 4.98-5.02ng / mL;

[0019] The detection, using liquid chromatography-mass spectrometry to determine the test sample solution and the control sample solution, recording the chromatogram, and calculating the content of N-methyl piperazine;

[0020] In the detection, the liquid mass spectrometer used is a triple quadrupole liquid mass spectrometer;

[0021] In the detection, the chromatographic column uses octadecylsilane bonded silica gel as the filler, with the specification of GeminiRNX-C18, 3μm, 150*4.6mm;

[0022] In the detection, the injection amount of the test sample solution and the control sample solution is 5μL respectively;

[0023] In the detection, the conditions of the liquid chromatography are: the mobile phase is composed of mobile phase A and mobile phase B, the mobile phase A is 0.048-0.052% trifluoroacetic acid aqueous solution; the mobile phase B is acetonitrile;

[0024] In the detection, the conditions of liquid chromatography are as follows: flow rate is 0.49-0.51 mL / min; injection volume is 4.8-5.2 μL; column temperature is 39.5-40.5 ℃;

[0025] In the detection, gradient elution is adopted, and the total volume of mobile phase is 100%, wherein the volume of the mobile phase A is 79-81% and the volume of the mobile phase B is 19-21% at 0-2 min; the volume of the mobile phase A decreases from 79-81% to 19-21% and the volume of the mobile phase B increases from 19-21% to 79-81% at 2-6 min; the volume of the mobile phase A decreases from 19-21% to 9-11% and the volume of the mobile phase B increases from 79-81% to 89-91% at 6-7 min; the volume of the mobile phase A is 9-11% and the volume of the mobile phase B is 89-91% at 7-15 min; the volume of the mobile phase A increases from 9-11% to 79-81% and the volume of the mobile phase B decreases from 89-91% to 19-21% at 15-16 min; the volume of the mobile phase A is 79-81% and the volume of the mobile phase B is 19-21% at 16-20 min;

[0026] In the detection, the conditions of mass spectrometry are as follows: ion source type is Heated ESI+;

[0027] Positive ion spray voltage: 3500 V;

[0028] Ion transmission tube temperature: 300 ℃;

[0029] Evaporation temperature: 350 ℃;

[0030] Sheath gas flow: 30 Arb;

[0031] Auxiliary gas flow: 15 Arb;

[0032] Parent ion (M / Z): 101.1; quantitative ion (M / Z): 58.1, collision voltage: 16 V, lens voltage: 43 V;

[0033] The sheath gas and the auxiliary gas are both high-purity nitrogen;

[0034] The collision gas adopted is high-purity argon;

[0035] In the detection, the flow switching is as follows: the switching valve is opened at 0-2.2 min, the chromatographic column effluent enters the waste liquid, the mass spectrometry is collected at 2.2-3.4 min, and the chromatographic column effluent enters the waste liquid after 3.4 min.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] (1) The detection method for detecting N-methylpiperazine in clozapine provided by the present application adopts a triple quadrupole liquid chromatograph mass spectrometer, according to the characteristics of the product and the properties of the compound, gradient elution is adopted to ensure that the blank solvent does not interfere with the detection of N-methylpiperazine, and through the optimization of the conditions, the sensitivity and accuracy of N-methylpiperazine are further improved, which can also meet the limit requirements, and the detection cost is also reduced.

[0038] (2) The present application adopts the combined use of ultra-high pressure high performance liquid chromatograph and triple quadrupole mass spectrometer technology, and the detection of N-methylpiperazine in clozapine can be realized on a low-resolution triple quadrupole mass spectrometer, which has high selectivity, good sensitivity, and a low quantitative limit of 0.165ppm.

[0039] (3) The detection method for detecting N-methylpiperazine in clozapine provided by the present application has a sample collection time of 20min, and the sample pretreatment operation is simple, and the detection of N-methylpiperazine in the sample to be detected can be completed within 30min, which is conducive to the popularization and promotion of the N-methylpiperazine detection method. BRIEF DESCRIPTION OF DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the present application or prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings described below are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0041] Figure 1 The liquid chromatogram of the blank solution in Example 1;

[0042] Figure 2 The liquid chromatogram of the control solution in Example 1;

[0043] Figure 3 The liquid chromatogram of the repeatability solution in Example 4;

[0044] Figure 4 The standard curve and linear equation of the N-methylpiperazine control solution with different concentrations obtained in Example 6. DETAILED DESCRIPTION

[0045] In order to more clearly illustrate the above-mentioned purposes, features and advantages of the present application, the following will further describe the present application in combination with the drawings and specific embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0046] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in the following description. In other instances, well-known methods have not been described in detail in order to avoid unnecessarily obscuring the present application.

[0047] Example 1

[0048] The present embodiment provides a method for detecting N-methylpiperazine in clozapine, specifically comprising:

[0049] 1. Instrument:

[0050] The detection is performed by using a triple quadrupole liquid chromatograph-mass spectrometer, and the chromatographic column uses octadecylsilane-bonded silica gel as the filler, with the specification being GeminiR NX-C18, 3 μm, 150*4.6 mm;

[0051] 2. Reagents:

[0052] The methanol used is mass spectrometry pure, the acetonitrile used is mass spectrometry pure, the water used is ultrapure water, and the trifluoroacetic acid used is chromatography pure;

[0053] The N-methylpiperazine control product used is from TCI, with the purity being 100%;

[0054] The clozapine used is from Shouguang Fukang Pharmaceutical Co., Ltd., with the purity being 99.6%.

[0055] 3. Preparation of solutions:

[0056] Preparation of the test sample solution: after accurately weighing the clozapine, the solvent is ultrasonically dissolved and quantitatively diluted, and a filter membrane with the filtration precision being 0.22 μm is used for filtration, so as to prepare a test sample solution containing 3 mg of clozapine per 1 mL;

[0057] The solvent is acetonitrile-water (the volume ratio of acetonitrile to water being 4:1).

[0058] Preparation of the control sample solution: accurately weigh 25 mg of the N-methylpiperazine control sample, add into a centrifuge tube with the specification being 15 mL, add 10 mL of methanol, and ultrasonically dissolve; from the solution, take 50 μL into a centrifuge tube with the specification being 50 mL, add 50 mL of the solvent, and mix uniformly; from the solution, take 100 μL into a centrifuge tube with the specification being 50 mL, add 50 mL of the solvent, and mix uniformly, so as to prepare a control sample solution containing 5 ng of N-methylpiperazine per 1 mL;

[0059] The solvent is acetonitrile-water (the volume ratio of acetonitrile to water being 4:1).

[0060] 4. The conditions of the liquid chromatograph and the mass spectrometer are as follows:

[0061] The flow rate is 0.5 mL / min;

[0062] Injection volume: 5 μL;

[0063] Column temperature: 40 °C;

[0064] Mobile phase A: 0.05% trifluoroacetic acid in water;

[0065] Mobile phase B: acetonitrile;

[0066] The gradient elution was used, and the total volume of the mobile phase was 100% at 0-2 min, the volume of the mobile phase A was 80%, and the volume of the mobile phase B was 20%;

[0067] At 2-6 min, the volume of the mobile phase A was decreased from 80% to 20%, and the volume of the mobile phase B was increased from 20% to 80%;

[0068] At 6-7 min, the volume of the mobile phase A was decreased from 20% to 10%, and the volume of the mobile phase B was increased from 80% to 90%;

[0069] At 7-15 min, the volume of the mobile phase A was 10%, and the volume of the mobile phase B was 90%;

[0070] At 15-16 min, the volume of the mobile phase A was increased from 10% to 80%, and the volume of the mobile phase B was decreased from 90% to 20%;

[0071] At 16-20 min, the volume of the mobile phase A was 80%, and the volume of the mobile phase B was 20%.

[0072] Ion source type Heated ESI+;

[0073] Positive ion spray voltage: 3500 V;

[0074] Ion transfer tube temperature: 300 °C;

[0075] Evaporation temperature: 350 °C;

[0076] Sheath gas flow: 30 Arb;

[0077] Auxiliary gas flow: 15 Arb;

[0078] Parent ion (M / Z): 101.1; Quantitative ion (M / Z): 58.1, collision voltage: 16 V, lens voltage: 43 V.

[0079] The sheath gas and the auxiliary gas were both high-purity nitrogen.

[0080] The collision gas used was high-purity argon.

[0081] Flow switching: 0-2.2 min switching valve open, column effluent goes to waste, 2.2-3.4 min mass spectrometer acquisition, 3.4 min later column effluent goes to waste.

[0082] 5. Detection: precisely pipette 5 μL of the test sample solution and the control solution respectively into the triple quadrupole liquid chromatograph mass spectrometer, record the chromatogram, according to the chromatogram, calculate the content of N-methyl piperazine according to the external standard method.

[0083] Example 2 Specificity test

[0084] Blank solution: acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0085] Preparation of the control solution: precisely weigh 25 mg of N-methyl piperazine control sample into a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, ultrasonic dissolution; from which, take 50 μL into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix uniformly; from which, take 100 μL into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix uniformly, to make a control solution containing 5 ng of N-methyl piperazine per 1 mL;

[0086] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0087] Take the blank solution and the control solution, detect according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, and record the liquid chromatogram; the obtained liquid chromatogram of the blank solution is shown in Figure 1 , and the obtained liquid chromatogram of the control solution is shown in Figure 2 .

[0088] Verification result: the blank solution has no interference with the determination of N-methyl piperazine.

[0089] Example 3 System suitability test

[0090] Preparation of the control solution: precisely weigh 25 mg of N-methyl piperazine control sample into a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, ultrasonic dissolution; from which, take 50 μL into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix uniformly; from which, take 100 μL into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix uniformly, to make a control solution containing 5 ng of N-methyl piperazine per 1 mL;

[0091] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0092] Take the control solution, continuously sample for 6 times according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, and record the liquid chromatogram.

[0093] Verification result: the RSD of peak area of impurity control solution for 6 times of continuous injection was 1.99%.

[0094] Example 4 Reproducibility test

[0095] Preparation of control stock solution: 25 mg of N-methyl piperazine control sample was accurately weighed into a centrifuge tube with a size of 15 mL, 10 mL of methanol was added, and it was ultrasonically dissolved; according to the preparation method of the control solution in Example 1, the solvent was diluted to prepare a control stock solution containing 2.5 μg of N-methyl piperazine per 1 mL;

[0096] The solvent was acetonitrile-water (volume ratio of acetonitrile to water was 4:1).

[0097] Preparation of reproducibility solution: 30 mg of clozapine sample was accurately weighed into a centrifuge tube with a size of 15 mL, 20 μL of control stock solution was added, 10 mL of solvent was added, and it was ultrasonically dissolved and filtered, and the filtrate was taken to obtain 6 reproducibility solutions;

[0098] The solvent was acetonitrile-water (volume ratio of acetonitrile to water was 4:1).

[0099] Determination: 6 reproducibility solutions were taken, and the liquid chromatogram was recorded according to the liquid chromatography and mass spectrometry conditions, detection method in Example 1, and the liquid chromatogram in the system suitability test in Example 3 was referred to, and the content of N-methyl piperazine was calculated according to the external standard method. The liquid chromatogram of the first reproducibility solution is shown in Figure 3 .

[0100] Verification result: the RSD of the content of the 6 samples was 2.64%.

[0101] Example 5 Intermediate precision test

[0102] On the different day of Example 4 reproducibility test, another analyst prepared 6 reproducibility solutions according to the preparation method of reproducibility solution in Example 4 reproducibility test.

[0103] Determination: 6 reproducibility solutions were taken, and the liquid chromatogram was recorded according to the liquid chromatography and mass spectrometry conditions, detection method in Example 1, and the liquid chromatogram in the system suitability test in Example 3 was referred to, and the content of N-methyl piperazine was calculated according to the external standard method.

[0104] Verification result: the RSD of the content of the 6 samples was 5.26%; the RSD of the content of the cumulative 12 samples in Example 4 reproducibility test and Example 5 intermediate precision test was 4.97%.

[0105] Example 6 Linearity test

[0106] Accurately weigh 25 mg of N-methylpiperazine reference substance into a centrifuge tube of 15 mL, add 10 mL of methanol, and ultrasonically dissolve; dilute with solvent to prepare linear No. 1-No. 5 solutions with concentrations of 1.5 ng / mL, 2.5 ng / mL, 5.0 ng / mL, 7.5 ng / mL, and 10.0 ng / mL, respectively, according to the method for preparing the reference substance solution in Example 1;

[0107] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0108] Determination: Take the linear No. 1-No. 5 solutions, and record the chromatogram according to the conditions for liquid chromatography and mass spectrometry and the detection method in Example 1.

[0109] Verification result: The linear equation Y=61.945X-36.608 is obtained in the concentration range of 1.5 ng / mL-10 ng / mL, and the correlation coefficient R is 0.9941. The standard curve of N-methylpiperazine reference substance at different concentrations and the linear equation are shown in Figure 4 .

[0110] Example 7 Accuracy test

[0111] Preparation of reference substance stock solution: accurately weigh 25 mg of N-methylpiperazine reference substance into a centrifuge tube of 15 mL, add 10 mL of methanol, and ultrasonically dissolve; dilute with solvent to prepare a reference substance stock solution containing 2.5 μg of N-methylpiperazine per 1 mL, according to the method for preparing the reference substance solution in Example 1;

[0112] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0113] Preparation of reference substance solution: accurately weigh 25 mg of N-methylpiperazine reference substance into a centrifuge tube of 15 mL, add 10 mL of methanol, and ultrasonically dissolve; take 50 μL therefrom and transfer into a centrifuge tube of 50 mL, add 50 mL of solvent, and mix well; take 100 μL therefrom and transfer into a centrifuge tube of 50 mL, add 50 mL of solvent, and mix well, to prepare a reference substance solution containing 5 ng of N-methylpiperazine per 1 mL;

[0114] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0115] Preparation of sample blank solution: accurately weigh 30 mg of clozapine sample into a centrifuge tube of 15 mL, add 10 mL of solvent, and ultrasonically dissolve, filter, and take the filtrate to obtain the sample blank solution.

[0116] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0117] Accuracy test solution No. 1 (limit of quantification concentration): precisely weigh 150 mg of clozapine sample into a centrifuge tube with a specification of 50 mL, add 30 μL of N-methyl piperazine control sample stock solution with a concentration of 2.5 μg / mL, add 50 mL of solvent to ultrasonic dissolution, filter, and take the filtrate to obtain. A total of 3 portions are prepared;

[0118] The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0119] Accuracy test solution No. 2: precisely weigh 150 mg of clozapine sample into a centrifuge tube with a specification of 50 mL, add 100 μL of N-methyl piperazine control sample stock solution with a concentration of 2.5 μg / mL, add 50 mL of solvent to ultrasonic dissolution, filter, and take the filtrate to obtain. A total of 3 portions are prepared;

[0120] The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0121] Accuracy test solution No. 3: precisely weigh 150 mg of clozapine sample into a centrifuge tube with a specification of 50 mL, add 200 μL of N-methyl piperazine control sample stock solution with a concentration of 2.5 μg / mL, add 50 mL of solvent to ultrasonic dissolution, filter, and take the filtrate to obtain. A total of 3 portions are prepared;

[0122] The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0123] Determination: take the control sample solution, sample blank solution, accuracy test solution No. 1-No. 3, and record the chromatogram according to the liquid chromatography and mass spectrometry conditions, detection method in Example 1.

[0124] Verification result: the average recovery rate is 116.059%.

[0125] Example 8: limit of detection and limit of quantification

[0126] Take the control sample solution in the system suitability test of Example 3, dilute with solvent to obtain a limit of quantification solution with a concentration of 0.496 ppm, and continuously detect the limit of quantification solution 6 times according to the liquid chromatography and mass spectrometry conditions, detection method in Example 1. The RSD of the peak area is 7.88%.

[0127] Take the limit of quantification solution, dilute with solvent to obtain a limit of detection solution with a concentration of 0.165 ppm.

[0128]

[0129] Example 9: durability test

[0130] Preparation of the control stock solution: 25 mg of N-methylpiperazine control sample was accurately weighed into a centrifuge tube with a capacity of 15 mL, 10 mL of methanol was added, and it was ultrasonically dissolved; the control stock solution containing 2.5 μg of N-methylpiperazine per 1 mL was prepared by diluting with the solvent according to the preparation method of the control solution in Example 1.

[0131] The solvent was acetonitrile-water (volume ratio of acetonitrile to water was 4:1).

[0132] Preparation of the control solution: 100 μL of the control stock solution was transferred into a centrifuge tube with a capacity of 50 mL, 50 mL of the solvent was added, and it was mixed uniformly to prepare a control solution containing 5 ng of N-methylpiperazine per 1 mL;

[0133] The solvent was acetonitrile-water (volume ratio of acetonitrile to water was 4:1).

[0134] Preparation of the robust solution: 30 mg of clozapine sample was accurately weighed into a centrifuge tube with a capacity of 15 mL, 20 μL of the control stock solution of N-methylpiperazine with a concentration of 2.5 μg / mL was added, and 10 mL of the solvent was added and ultrasonically dissolved, and then it was filtered to obtain the filtrate.

[0135] The solvent was acetonitrile-water (volume ratio of acetonitrile to water was 4:1).

[0136] Determination: The control solution and the robust solution were detected according to the liquid chromatography and mass spectrometry conditions and the detection method in Example 1, and then the column temperature was changed to 38°C and 42°C, respectively, and then the detection was performed again, the chromatograms were recorded, and the content of N-methylpiperazine was calculated according to the external standard method.

[0137] Determination: The control solution and the robust solution were detected according to the original chromatography conditions, the flow rate was changed to 0.45 mL / min and 0.55 mL / min, respectively, and then the detection was performed again, the chromatograms were recorded, and the content of N-methylpiperazine was calculated according to the external standard method.

[0138] Verification result: The ratio of the content of the robust solution measured after changing the parameters to the content measured under normal conditions was between 0.8 and 1.2.

[0139] Example 10 Stability test of the solution

[0140] Preparation of the control stock solution: 25 mg of N-methylpiperazine control sample was accurately weighed into a centrifuge tube with a capacity of 15 mL, 10 mL of methanol was added, and it was ultrasonically dissolved; the control stock solution containing 2.5 μg of N-methylpiperazine per 1 mL was prepared by diluting with the solvent according to the preparation method of the control solution in Example 1.

[0141] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0142] Preparation of the control solution: 100 μL of the control stock solution was transferred into a centrifuge tube with a specification of 50 mL, 50 mL of the solvent was added and mixed uniformly to prepare a control solution containing 5 ng of N-methylpiperazine per 1 mL;

[0143] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0144] Preparation of the sample solution: 30 mg of the clozapine sample was accurately weighed and added into a centrifuge tube with a specification of 15 mL, 20 μL of the control stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine was added, 10 mL of the solvent was added and ultrasonically dissolved, filtered, and the filtrate was obtained;

[0145] The solvent is acetonitrile-water (volume ratio of acetonitrile to water is 4:1).

[0146] Determination: the control solution and the sample solution were injected at different time periods, and the chromatograms were recorded.

[0147] Verification results: the RSD of the peak area of the control solution within 10 hours was 4.05%, and the RSD of the peak area of the sample solution within 10 hours was 5.60%.

[0148] The above only describes the preferred embodiments of the present application and is not used to limit the present application. Various modifications and changes can be made by those skilled in the art based on the principles and technical solutions of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for determining N-methylpiperazine in clozapine, characterized in that, This includes solution preparation and detection; The preparation of the solution involves using clozapine to prepare the test solution and using N-methylpiperazine to prepare the reference solution. The preparation method of the test solution in the prepared solution is as follows: after accurately weighing clozapine, add solvent, sonicate to dissolve and quantitatively dilute, filter, and prepare the test solution; In the preparation of the test solution, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 4:1; In the preparation of the test solution, the filter membrane used in the filtration has a filtration accuracy of 0.22 μm; The concentration of clozapine in the test solution was 2.8-3.2 mg / mL; The preparation method of the reference solution in the prepared solution is as follows: accurately weigh 24.5-25.5 mg of N-methylpiperazine reference standard, add it to a centrifuge tube, add 9.8-10.2 mL of methanol, and sonicate to dissolve; transfer 49-51 μL of the solution to a centrifuge tube, add 49-51 mL of solvent and mix well; transfer 98-102 μL of the solution to a centrifuge tube, add 49-51 mL of solvent and mix well to prepare the reference solution; In the preparation of the reference solution, the solvent is acetonitrile-water, wherein the volume ratio of acetonitrile to water is 4:1; The concentration of N-methylpiperazine in the reference solution was 4.98-5.02 ng / mL; The detection method employed liquid chromatography-mass spectrometry to determine the test solution and reference solution, record the chromatograms, and calculate the content of N-methylpiperazine. The liquid chromatography-mass spectrometer used in the detection was a triple quadrupole liquid chromatography-mass spectrometer; In the aforementioned detection, the liquid chromatography conditions are as follows: the mobile phase consists of mobile phase A and mobile phase B, where mobile phase A is an aqueous solution of 0.048-0.052% trifluoroacetic acid; and mobile phase B is acetonitrile. In the detection process, gradient elution is employed. The total volume of the mobile phase is 100%. In the first 0-2 minutes, the volume of mobile phase A is 79-81%, and the volume of mobile phase B is 19-21%. In the second-to-sixth minute, the volume of mobile phase A decreases from 79-81% to 19-21%, while the volume of mobile phase B increases from 19-21% to 79-81%. In the sixth-to-seventh minute, the volume of mobile phase A decreases from 19-21% to 9-11%, and the volume of mobile phase B... The volume of mobile phase A increases from 79-81% to 89-91%; in the 7th-15th minute, the volume of mobile phase A is 9-11% and the volume of mobile phase B is 89-91%; in the 15th-16th minute, the volume of mobile phase A increases from 9-11% to 79-81% and the volume of mobile phase B decreases from 89-91% to 19-21%; in the 16th-20th minute, the volume of mobile phase A is 79-81% and the volume of mobile phase B is 19-21%. In the aforementioned detection, the chromatographic column used was packed with octadecylsilane-bonded silica gel, with specifications of GeminiR NX-C18, 3μm, 150*4.6mm.

2. The method for determining N-methylpiperazine in clozapine according to claim 1, characterized in that, In the aforementioned detection, the injection volumes of the test solution and the reference solution are 5 μL, respectively.

3. The method for determining N-methylpiperazine in clozapine according to claim 1, characterized in that, In the detection, the liquid chromatography conditions are as follows: flow rate of 0.49-0.51 mL / min; injection volume of 4.8-5.2 μL; column temperature of 39.5-40.5℃.

4. The method for determining N-methylpiperazine in clozapine according to claim 1, characterized in that, In the aforementioned detection, the mass spectrometry conditions are: ion source type Heated ESI+; Positive ion spray voltage: 3500V; Ion transfer tube temperature: 300℃; Evaporation temperature: 350℃; Sheath gas flow rate: 30 Arb; Auxiliary gas flow rate: 15 Arb; Precursor ion (M / Z): 101.1; Quantitative ion (M / Z): 58.1; Collision voltage: 16V; Lens voltage: 43V; Both the sheath gas and the auxiliary gas are high-purity nitrogen. The collision gas used is high-purity argon.

5. The method for determining N-methylpiperazine in clozapine according to claim 1, characterized in that, During the detection process, the flow path is switched as follows: the switching valve opens from 0 to 2.2 minutes, and the column effluent enters the waste liquid; mass spectrometry is performed from 2.2 to 3.4 minutes; after 3.4 minutes, the column effluent enters the waste liquid.

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