Detection method for determining N-methyl piperazine in clozapine

Abstract

The invention discloses a detection method for determining N-methylpiperazine in clozapine, and belongs to the technical field of drug detection, the detection method comprises solution preparation and detection; in the step of preparing the solution, clozapine is used for preparing a test solution, and N-methyl piperazine is used for preparing a reference solution; the detection comprises the following steps: determining the test solution and the reference solution by adopting a liquid chromatography-mass spectrometry method, recording a chromatogram, and calculating the content of N-methyl piperazine; in the detection, the conditions of liquid chromatography are as follows: a mobile phase is composed of a mobile phase A and a mobile phase B, and the mobile phase A is an aqueous solution of 0.048-0.052% trifluoroacetic acid; the mobile phase B is acetonitrile; according to the method, the genotoxic impurity N-methylpiperazine in the clozapine raw material medicine and the preparation can be quantitatively detected, the content of the impurity in the clozapine production and storage process can be conveniently monitored, the selectivity is high, the sensitivity is good, and the limit of quantitation can be as low as 0.165 ppm.

Description

Technical Field

[0001] The present invention relates to the technical field of drug detection, and more specifically, to a detection method for determining N-methyl piperazine in clozapine. Background Art

[0002] Clozapine is mainly used for treating various subtypes of acute and chronic schizophrenia, has good effects on the hallucinatory delusional type and the hebephrenic type, and can also relieve the emotional symptoms related to schizophrenia.

[0003] N-methyl piperazine (NMP) is a genotoxic impurity detected in clozapine raw materials and its preparations. This impurity is the starting material in the synthesis of clozapine raw materials, is introduced in the latter 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 test in the Japanese NITE database (reverse mutation test of 1-methyl piperazine in bacteria), and is a genotoxic impurity.

[0004] N-methyl piperazine is the starting material used in the clozapine synthesis process and belongs to potential process impurities and genotoxic impurities. The molecular structural formula is as follows:

[0005] According to the requirements of ICH M7, it is necessary to strictly control the calculation of impurities by the PDE method. Calculated by the PDE method, the recommended toxicological concern 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. Therefore, the control limit of this impurity should not exceed 1.67 ppm, and the sensitivity of the conventional detection method cannot reach it.

[0006] At present, there is no publicly published method for detecting N-methyl piperazine in clozapine raw materials and preparations. In order to achieve better quality control effects and prevent the outflow of drugs with excessive N-methyl piperazine, it is urgent to develop a detection method for N-methyl piperazine in clozapine to solve the above problems. Summary of the Invention

[0007] The purpose of the present invention is to provide a detection method for determining N-methyl piperazine in clozapine, which can quantitatively detect the genotoxic impurity N-methyl piperazine in clozapine raw materials and preparations, and is convenient for monitoring the content of this impurity during the production and storage of clozapine.

[0008] To solve the above technical problems, the technical solutions adopted by the present invention are as follows: A detection method for determining N-methyl piperazine in clozapine, including preparing a solution and detecting; For the preparation of the solution, a test solution is prepared using clozapine, and a reference solution is prepared using N-methyl piperazine; Furthermore, in the preparation solution, the preparation method of the test solution is as follows: After accurately weighing clozapine, it is ultrasonically dissolved in a solvent and quantitatively diluted, filtered to prepare a 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 filtration accuracy of the filter membrane used in the filtration is 0.22 μm; The concentration of clozapine in the test solution is 2.8 - 3.2 mg / mL; Furthermore, in the preparation solution, the preparation method of the reference solution is as follows: Accurately weigh 24.5 - 25.5 mg of N - methylpiperazine reference substance, add it to a centrifuge tube, add 9.8 - 10.2 mL of methanol, and ultrasonically dissolve it; Take 49 - 51 μL from it and transfer it into a centrifuge tube, add 49 - 51 mL of solvent and mix evenly; Take 98 - 102 μL from it and transfer it into a centrifuge tube, add 49 - 51 mL of solvent and mix evenly to prepare a 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 is 4.98 - 5.02 ng / mL; For the detection, the test solution and the reference solution are determined by liquid chromatography - mass spectrometry, the chromatogram is recorded, and the content of N - methylpiperazine is calculated; In the detection, the liquid mass spectrometer used is a triple quadrupole liquid mass spectrometer; In the detection, the chromatographic column uses octadecylsilane chemically bonded silica gel as the filler, with the specification of Gemini RNX - C18, 3 μm, 150 * 4.6 mm; In the detection, the injection volumes of the test solution and the reference solution are 5 μL respectively; In the detection, the conditions of liquid chromatography are as follows: The mobile phase consists of mobile phase A and mobile phase B. Mobile phase A is an aqueous solution of 0.048 - 0.052% trifluoroacetic acid; Mobile phase B is acetonitrile; In the detection, the conditions of liquid chromatography are as follows: The flow rate is 0.49 - 0.51 mL / min; The injection volume is 4.8 - 5.2 μL; The column temperature is 39.5 - 40.5 °C; In the said detection, gradient elution is adopted. Calculated by 100% for the total volume of the mobile phase, from 0 to 2 min, the volume of the mobile phase A is 79 - 81%, and the volume of the mobile phase B is 19 - 21%; from 2 to 6 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%; from 6 to 7 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%; from 7 to 15 min, the volume of the mobile phase A is 9 - 11%, and the volume of the mobile phase B is 89 - 91%; from 15 to 16 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%; from 16 to 20 min, the volume of the mobile phase A is 79 - 81%, and the volume of the mobile phase B is 19 - 21%. In the said detection, the conditions of the mass spectrometry are: ion source type Heated ESI+; Positive ion spray voltage: 3500 V; Ion transfer tube temperature: 300 °C; Evaporation temperature: 350 °C; Sheath gas flow rate: 30 Arb; Auxiliary gas flow rate: 15 Arb; Parent ion (M / Z): 101.1; Quantitative ion (M / Z): 58.1, collision voltage: 16 V, lens voltage: 43 V; Both the sheath gas and the auxiliary gas are high-purity nitrogen; The collision gas used is high-purity argon; In the said detection, the flow path switching is as follows: the switching valve is opened from 0 to 2.2 min, the effluent from the chromatographic column enters the waste liquid, mass spectrometry is collected from 2.2 to 3.4 min, and the effluent from the chromatographic column enters the waste liquid after 3.4 min.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) For the detection method of N-methylpiperazine in clozapine of the present invention, a triple quadrupole liquid chromatography mass spectrometer is adopted. According to the characteristics of the product and the properties of the compound, gradient elution is adopted, which ensures that the blank solvent does not interfere with the detection of N-methylpiperazine. Through the optimization of the conditions, the sensitivity and accuracy of N-methylpiperazine are further improved, and its limit requirements can also be met. At the same time, the detection cost is also reduced.

[0010] (2) The present invention uses the combined technology of ultra-high pressure high performance liquid chromatography and triple quadrupole mass spectrometry, and can detect N-methylpiperazine in clozapine on a triple quadrupole mass spectrometer with low resolution. Moreover, it has high selectivity, good sensitivity, and a low quantification limit of up to 0.165 ppm.

[0011] (3) The detection method for determining N-methylpiperazine in clozapine of the present invention has a sample collection time of 20 min, and the sample pretreatment is simple. The detection of N-methylpiperazine in the sample to be tested can be completed within 30 min, which is conducive to the popularization and promotion of the detection method for N-methylpiperazine. Description of the Drawings

[0012] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0013] Figure 1 It is the liquid chromatography diagram of the blank solution in Example 1; Figure 2 It is the liquid chromatography diagram of the reference substance solution in Example 1; Figure 3 It is the liquid chromatography diagram of the repeatability solution in Example 4; Figure 4 It is the standard curve and linear formula of N-methylpiperazine reference substances with different concentrations obtained in Example 6. Specific Embodiments

[0014] In order to more clearly understand the above-mentioned objects, features, and advantages of the present invention, the following will further describe the present invention in detail with reference to the drawings and specific embodiments. It should be noted that, without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other.

[0015] Many specific details are set forth in the following description to facilitate a thorough understanding of the present invention. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited by the specific embodiments disclosed below.

[0016] Example 1 This example provides a detection method for determining N-methylpiperazine in clozapine, specifically as follows: 1. Instrument: Detection is carried out using a triple quadrupole liquid chromatography-mass spectrometer. The chromatographic column uses octadecylsilyl-bonded silica gel as the filler, with a specification of GeminiR NX-C18, 3 μm, 150 * 4.6 mm; 2. Reagents: The methanol used is of mass spectrometry purity, the acetonitrile used is of mass spectrometry purity, the water used is ultrapure water, and the trifluoroacetic acid used is of chromatographic purity; The manufacturer of the N-methylpiperazine reference substance used is TCI, and the purity is 100%; The manufacturer of clozapine used is Shouguang Fukang Pharmaceutical Co., Ltd., and the purity is 99.6%.

[0017] 3. Preparation of solutions: Preparation of the test solution: After accurately weighing clozapine, dissolve it by ultrasonic wave with the solvent and quantitatively dilute it, filter it with a filter membrane with a filtration accuracy of 0.22 μm to prepare a test solution containing 3 mg of clozapine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0018] Preparation of the reference solution: Accurately weigh 25 mg of the N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic wave; take 50 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of the solvent and mix evenly; take 100 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of the solvent and mix evenly to prepare a reference solution containing 5 ng of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0019] 4. The conditions of liquid chromatography and mass spectrometry are as follows: Flow rate: 0.5 mL / min; Injection volume: 5 μL; Column temperature: 40 °C; Mobile phase A: Aqueous solution of 0.05% trifluoroacetic acid; Mobile phase B: Acetonitrile; Gradient elution is adopted. The total volume of the mobile phase is calculated as 100%. At 0-2 min, the volume of the mobile phase A is 80%, and the volume of the mobile phase B is 20%; At 2-6 min, the volume of the mobile phase A decreases from 80% to 20%, and the volume of the mobile phase B increases from 20% to 80%; At 6-7 min, the volume of the mobile phase A decreases from 20% to 10%, and the volume of the mobile phase B increases from 80% to 90%; At 7-15 min, the volume of the mobile phase A is 10%, and the volume of the mobile phase B is 90%; From 15 - 16 min, the volume of mobile phase A increases from 10% to 80%, and the volume of mobile phase B decreases from 90% to 20%. From 16 - 20 min, the volume of mobile phase A is 80%, and the volume of mobile phase B is 20%.

[0020] Ion source type: Heated ESI+; Positive ion spray voltage: 3500 V; Ion transfer tube temperature: 300 °C; Evaporation temperature: 350 °C; Sheath gas flow rate: 30 Arb; Auxiliary gas flow rate: 15 Arb; Parent ion (M / Z): 101.1; Quantification ion (M / Z): 58.1, Collision voltage: 16 V, Lens voltage: 43 V.

[0021] Both the sheath gas and the auxiliary gas are high-purity nitrogen.

[0022] The collision gas used is high-purity argon.

[0023] Flow path switching: The switching valve is open from 0 - 2.2 min, the effluent from the chromatographic column enters the waste liquid, mass spectrometry is collected from 2.2 - 3.4 min, and the effluent from the chromatographic column enters the waste liquid after 3.4 min.

[0024] 5. Detection: Precisely pipette 5 μL each of the test solution and the reference solution, inject them into a triple quadrupole liquid chromatography - mass spectrometer, record the chromatogram, and calculate the content of N - methylpiperazine according to the external standard method based on the chromatogram.

[0025] Example 2 Specificity test Blank solution: Acetonitrile - water (volume ratio of acetonitrile to water is 4:1).

[0026] Preparation of reference solution: Accurately weigh 25 mg of N - methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; take 50 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly; take 100 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly to prepare a reference solution containing 5 ng of N - methylpiperazine per 1 mL. The solvent is acetonitrile - water (volume ratio of acetonitrile to water is 4:1).

[0027] Take the blank solution and the reference solution, and perform detection according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, and record the liquid chromatogram; the liquid chromatogram of the obtained blank solution is shown in Figure 1, the liquid chromatogram of the reference solution obtained is shown in Figure 2 .

[0028] Verification result: The blank solution does not interfere with the determination of N-methylpiperazine.

[0029] Example 3 System Suitability Test Preparation of reference solution: Weigh accurately 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; take 50 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly; take 100 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly to prepare a reference solution containing 5 ng of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0030] Take the reference solution, and according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, inject samples continuously for 6 times, and record the liquid chromatogram.

[0031] Verification result: The RSD of the peak areas of the impurity reference solution injected continuously for 6 times is 1.99%.

[0032] Example 4 Repeatability Test Preparation of reference stock solution: Weigh accurately 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; refer to the preparation method of the reference solution in Example 1, and dilute it with the solvent to prepare a reference stock solution containing 2.5 μg of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0033] Preparation of repeatability solution: Weigh accurately 30 mg of clozapine sample, add it to a centrifuge tube with a specification of 15 mL, add 20 μL of the reference stock solution, add 10 mL of solvent, dissolve it by ultrasonic treatment and filter, and take the subsequent filtrate to obtain 6 repeatability solutions in total; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0034] Determination: Take the 6 prepared repeatability solutions, and according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, record the chromatogram, refer to the liquid chromatogram in the system suitability test of Example 3, and calculate the content of N-methylpiperazine by the external standard method. The liquid chromatogram of the first repeatability solution is shown in Figure 3 .

[0035] Verification result: The RSD of the contents of the 6 samples is 2.64%.

[0036] Example 5 Intermediate Precision Test On a different day from the repeatability test in Example 4, another analyst continuously prepared 6 portions of the repeatability solution according to the preparation method of the repeatability solution in the repeatability test of Example 4.

[0037] Determination: Inject the 6 portions of the repeatability solution, record the chromatogram according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, refer to the liquid chromatogram in the system suitability test of Example 3, and calculate the content of N-methylpiperazine by the external standard method.

[0038] Verification result: The RSD of the contents of the 6 samples was 5.26%; the RSD of the contents of the cumulative 12 samples in the repeatability test of Example 4 and the intermediate precision test of Example 5 was 4.97%.

[0039] Example 6 Linearity Test Accurately weigh 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; refer to the preparation method of the reference solution in Example 1, and dilute it with the 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; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0040] Determination: Take the linear No. 1-No. 5 solutions, record the chromatogram according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1.

[0041] Verification result: The linear equation Y = 61.945X - 36.608 was obtained in the concentration range of 1.5 ng / mL - 10 ng / mL, and the correlation coefficient R was 0.9941. The standard curves and linear formulas of N-methylpiperazine reference substances at different concentrations are shown in Figure 4 .

[0042] Example 7 Accuracy Test Preparation of the reference stock solution: Accurately weigh 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; refer to the preparation method of the reference solution in Example 1, and dilute it with the solvent to prepare a reference stock solution containing 2.5 μg of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0043] Preparation of reference solution: Weigh accurately 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; take 50 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly; take 100 μL from it and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of solvent and mix evenly to prepare a reference solution containing 5 ng of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0044] Preparation of sample blank solution: Weigh accurately 30 mg of clozapine sample, add it to a 15 mL centrifuge tube, add 10 mL of solvent, dissolve it by ultrasonic treatment, filter, and take the subsequent filtrate to obtain it; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0045] Accuracy test solution No.1 (quantitation limit concentration): Weigh accurately 150 mg of clozapine sample, add it to a centrifuge tube with a specification of 50 mL, add 30 μL of a reference stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine, add 50 mL of solvent, dissolve it by ultrasonic treatment, filter, and take the subsequent filtrate to obtain it. A total of 3 portions are prepared; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0046] Accuracy test solution No.2: Weigh accurately 150 mg of clozapine sample, add it to a centrifuge tube with a specification of 50 mL, add 100 μL of a reference stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine, add 50 mL of solvent, dissolve it by ultrasonic treatment, filter, and take the subsequent filtrate to obtain it. A total of 3 portions are prepared; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0047] Accuracy test solution No.3: Weigh accurately 150 mg of clozapine sample, add it to a centrifuge tube with a specification of 50 mL, add 200 μL of a reference stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine, add 50 mL of solvent, dissolve it by ultrasonic treatment, filter, and take the subsequent filtrate to obtain it. A total of 3 portions are prepared; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0048] Determination: Take the reference solution, sample blank solution, and accuracy test solutions No.1 - No.3, and record the chromatograms according to the liquid chromatography and mass spectrometry conditions and detection methods in Example 1.

[0049] Verification result: The average recovery rate is 116.059%.

[0050] Example 8 Detection Limit and Quantitation Limit Take the reference solution in the system suitability test of Example 3, dilute it with the solvent to obtain a quantitation limit solution with a concentration of 0.496 ppm, and continuously detect the quantitation limit solution 6 times according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1. The RSD of the peak area is 7.88%.

[0051] Take the quantitation limit solution and dilute it with the solvent to obtain a detection limit solution with a concentration of 0.165 ppm.

[0052]

[0053] Example 9 Durability Test Preparation of reference stock solution: Weigh accurately 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; referring to the preparation method of the reference solution in Example 1, dilute it with the solvent to prepare a reference stock solution containing 2.5 μg of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0054] Preparation of reference solution: Measure 100 μL of the reference stock solution and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of the solvent and mix evenly to prepare a reference solution containing 5 ng of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0055] Preparation of durability solution: Weigh accurately 30 mg of clozapine sample, add it to a centrifuge tube with a specification of 15 mL, add 20 μL of the reference stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine, then add 10 mL of the solvent and dissolve it by ultrasonic treatment, filter, and take the subsequent filtrate to obtain; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0056] Determination: Detect the reference solution and the durability solution according to the liquid chromatography and mass spectrometry conditions and detection method in Example 1, then change the column temperature from 40 °C to 38 °C and 42 °C, and then detect them respectively, record the chromatogram, and calculate the content of N-methylpiperazine according to the external standard method.

[0057] Determination: Detect the reference solution and the durability solution according to the original chromatographic conditions, change the flow rate from 0.5 mL / min to 0.45 mL / min and 0.55 mL / min, and then detect them respectively, record the chromatogram, and calculate the content of N-methylpiperazine according to the external standard method.

[0058] Verification result: The ratio of the content of the durability solution measured after changing the parameters to the content measured under normal conditions is between 0.8 and 1.2.

[0059] Example 10 Solution Stability Test Preparation of reference stock solution: Weigh accurately 25 mg of N-methylpiperazine reference substance, add it to a centrifuge tube with a specification of 15 mL, add 10 mL of methanol, and dissolve it by ultrasonic treatment; referring to the preparation method of the reference solution in Example 1, dilute it with the solvent to prepare a reference stock solution containing 2.5 μg of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0060] Preparation of reference solution: Measure 100 μL of the reference stock solution and transfer it into a centrifuge tube with a specification of 50 mL, add 50 mL of the solvent and mix evenly to prepare a reference solution containing 5 ng of N-methylpiperazine per 1 mL; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0061] Preparation of sample solution: Weigh accurately 30 mg of clozapine sample, add it to a centrifuge tube with a specification of 15 mL, add 20 μL of the reference stock solution with a concentration of 2.5 μg / mL of N-methylpiperazine, then add 10 mL of the solvent and dissolve it by ultrasonic treatment, filter, and take the continuous filtrate to obtain; The solvent is acetonitrile-water (the volume ratio of acetonitrile to water is 4:1).

[0062] Determination: Inject the reference solution and the sample solution at different time intervals respectively, and record the chromatogram.

[0063] Verification result: The RSD of the peak area of the reference solution within 10 hours is 4.05%. The RSD of the peak area of the sample solution within 10 hours is 5.60%.

[0064] The above is only the preferred embodiment of the present invention and is not used to limit the present invention. For those skilled in the art, the present invention can also have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A detection method for determining N-methylpiperazine in clozapine, characterized in that, Including solution preparation and testing; The solution preparation includes using clozapine to prepare the test solution and using N-methylpiperazine to prepare the reference solution; The concentration of clozapine in the test solution is 2.8-3.2 mg / mL; The concentration of N-methylpiperazine in the reference solution is 4.98-5.02 ng / mL; The detection adopts liquid chromatography-mass spectrometry to determine the test solution and the reference solution, record the chromatogram, and calculate the content of N-methylpiperazine; In the detection, the liquid phase mass spectrometer used is a triple quadrupole liquid phase mass spectrometer; In the detection, the conditions of liquid chromatography are as follows: the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is an aqueous solution of 0.048-0.052% trifluoroacetic acid; mobile phase B is acetonitrile; In the detection, gradient elution is adopted, and the total volume of the mobile phase is calculated as 100%. At 0-2 minutes, the volume of the mobile phase A is 79-81%, and the volume of the mobile phase B is 19-21%; at 2-6 minutes, 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 6-7 minutes, the volume of the mobile phase A decreases from 19-21% to 9-11%, and the volume of the mobile phase B increases from 19-21% to 9-11%. The volume of the mobile phase A increases from 79-81% to 89-91%; at 7-15 minutes, the volume of the mobile phase A is 9-11%, and the volume of the mobile phase B is 89-91%; at 15-16 minutes, 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 16-20 minutes, the volume of the mobile phase A is 79-81%, and the volume of the mobile phase B is 19-21%.

2. The method for detecting N-methylpiperazine in clozapine according to claim 1, characterized in that: In the prepared solution, the preparation method of the test solution is: after accurately weighing clozapine, adding a solvent to ultrasonically dissolve and quantitatively dilute, filtering, and preparing 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 filtration accuracy of the filter membrane used in the filtration is 0.22 μm.

3. The detection method for determining N-methylpiperazine in clozapine according to claim 1, characterized in that, In the prepared solution, the preparation method of the reference solution is as follows: accurately weigh 24.5-25.5 mg of N-methylpiperazine reference substance, add it to a centrifuge tube, add 9.8-10.2 mL of methanol, and dissolve it by ultrasonication; measure 49-51 μL therefrom and transfer it to a centrifuge tube, add 49-51 mL of solvent and mix it evenly; measure 98-102 μL therefrom and transfer it to a centrifuge tube, add 49-51 mL of solvent and mix it evenly to prepare a 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.

4. The method for detecting N-methylpiperazine in clozapine according to claim 1, characterized in that: In the detection, the chromatographic column used uses octadecylsilane bonded silica gel as a filler, and the specifications are GeminiR NX-C18, 3μm, 150*4.6mm.

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

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

7. The method for detecting N-methylpiperazine in clozapine according to claim 1, characterized in that: In the detection, the mass spectrometry conditions were: ion source type Heated ESI+; Positive ion spray voltage: 3500V; Ion transfer tube temperature: 300°C; Evaporation temperature: 350℃; Sheath gas flow: 30Arb; Auxiliary gas flow: 15Arb; Parent ion (M / Z): 101.1; quantitative ion (M / Z): 58.1, collision voltage: 16 V, lens voltage: 43 V; The sheath gas and auxiliary gas are both high-purity nitrogen; The collision gas used was high-purity argon.

8. The method for detecting N-methylpiperazine in clozapine according to claim 1, characterized in that: In the detection, the flow path is switched: the switching valve is opened from 0 to 2.2 minutes, the effluent from the chromatographic column enters the waste liquid, the mass spectrum is collected from 2.2 to 3.4 minutes, and the effluent from the chromatographic column enters the waste liquid after 3.4 minutes.

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