Method for detecting content of three potential genotoxic impurities in metoprolol tartrate

Abstract

The invention discloses a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate. The method comprises the following steps: detecting a sample to be detected by adopting gas chromatography-mass spectrometry (GC-MS); the parameter conditions of the gas chromatography are as follows: the temperature of a sample inlet is 210-230 DEG C; the heating procedure is as follows: the initial temperature is 35-45 DEG C, and the temperature is kept for 4-6 minutes; the temperature is raised to 235-245 DEG C at the temperature raising speed of 10-20 DEG C / min, and the temperature is kept for 1.5-2.5 min; headspace parameters are as follows: the temperature of a heating box is 85-95 DEG C; the temperature of a quantitative loop is 120-130 DEG C; the temperature of the transmission line is 125-135 DEG C; the balance time of the headspace bottle is 8-12 min; the sample introduction time is 0.3 to 0.8 min; and the GC circulation time is 25 to 30 minutes. The method disclosed by the invention is simple to operate and high in detection speed, and has the advantages of high sensitivity, high precision and high repeatability.

Description

Technical Field

[0001] This invention relates to the technical field of biomedical testing, specifically to a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate. Background Technology

[0002] Metoprolol tartrate, chemically named (±)-1-isopropylamino-3-[4-(2-methoxyethyl)phenoxy]-2-propanol L(+)-tartrate, has the molecular formula (C15H25NO3)2·C4H6O6, a molecular weight of 684.82, and CAS number 56392-17-7. Its structural formula is shown in formula (a) below.

[0003] Metoprolol tartrate is a selective β-1 adrenergic receptor blocker. On April 18, 1975, metoprolol tartrate tablets were launched in Sweden by AstraZeneca under the brand name Seloken, in strengths of 50 mg and 100 mg. It is mainly used to treat hypertension, angina pectoris, and myocardial infarction. The synthetic route of metoprolol tartrate is as follows... Figure 1 As shown, by Figure 1 It is known that the starting material epichlorohydrin and the impurities ethylene oxide and benzene that may be introduced from the starting material p-methoxyethylaniline during the synthesis process both contain genotoxic warning structures in their impurity structures, and therefore may be potential genotoxic impurities. According to ICH M7 guidelines, when calculating acceptable intake based on TTC, the risk of a single mutagenic impurity at a daily intake of 1.5 μg per person is considered negligible (the theoretical risk of cancer under lifetime exposure is less than one in 100,000). This value can be applied to most drugs as the default value for acceptable control limits. The maximum daily dose of metoprolol tartrate is 450 mg / day, therefore its theoretical limit standard = 1.5 μg / day / 450 mg / day = 3.3 ppm. Furthermore, benzene is listed as a Group 1 carcinogen. According to Pharmacopoeia General Chapter 0861, it is a Group 1 residual solvent with a limit of 0.0002%, or 2 ppm. Therefore, the limits for epichlorohydrin and ethylene oxide are set at no more than 3.3 ppm; the limit for benzene is no more than 2 ppm. The structures of the impurities are shown in the table below.

[0004]

[0005] To control the content of the above-mentioned impurities in metoprolol, ensure the quality of metoprolol, and ensure the safety of patients taking the medication, developing a new detection method for the three potential genotoxic impurities in metoprolol tartrate is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] In order to solve the above technical problems, the present application provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0007] The present application provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate, which uses gas chromatography-mass spectrometry (GC-MS) to detect the sample to be tested.

[0008] The parameters of the gas chromatography are as follows: the injection port temperature is 210-230 DEG C; the temperature rising program is as follows: the initial temperature is 35-45 DEG C, and is kept for 4-6 min; the temperature is raised to 235-245 DEG C at a rate of 10-20 DEG C / min, and is kept for 1.5-2.5 min; the post-run time is 20-21 min.

[0009] The headspace parameters are as follows: the heating box temperature is 85-95 DEG C; the quantitative ring temperature is 120-130 DEG C; the transfer line temperature is 125-135 DEG C; the headspace bottle equilibrium time is 8-12 min; the injection time is 0.3-0.8 min; the GC cycle time is 25-30 min; and the sample volume in the bottle is 1.8-2.2 ml.

[0010] The parameters of the mass spectrometry are as follows: the acquisition type is SIM; the ion source is EI; the transfer line temperature is 240-260 DEG C; the quadrupole rod temperature is 140-160 DEG C; and the ion source temperature is 220-240 DEG C.

[0011] The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate of the present application is gas chromatography-mass spectrometry (GC-MS), which uses gas chromatography as the separation system of the three potential genotoxic impurities in metoprolol tartrate, and mass spectrometry as the detection system of the three potential genotoxic impurities.

[0012] Gas chromatography-mass spectrometry combines the high separation ability of gas chromatography for the sample to be tested with the high selectivity, high sensitivity and the advantages of providing relative molecular mass and structure information of mass spectrometry.

[0013] Gas chromatography has the characteristics of strong separation degree for the sample to be tested, and by selecting appropriate chromatographic column, initial column temperature, temperature rising rate, column flow rate and other parameter conditions, the separation effect of the three potential genotoxic impurities and other substances can be effectively improved, which not only provides sufficient ions for mass spectrometry analysis, but also preliminarily separates out the impurities that interfere with mass spectrometry, avoids the influence of interfering impurities on subsequent mass spectrometry, and thus obtains good peak parameters, thereby effectively improving the accuracy and precision of the detection results.

[0014] The application provides a detection method for the content of three potential genotoxic impurities in metoprolol tartrate by screening and optimizing the related test conditions of gas chromatography and mass spectrometry in GC-MS, and the method has the advantages of low detection limit, high precision, high repeatability and high accuracy.

[0015] Preferably, the temperature rising procedure is: the initial temperature is 37-42 DEG C, and the temperature is kept for 5 min; the temperature is raised to 238-242 DEG C at a temperature rising rate of 12-18 DEG C / min, and the temperature is kept for 2 min; the post running time is 20.333 min.

[0016] Preferably, the chromatographic column of the gas chromatography condition is DB-624, and the specification is 30 m*0.250 mm*1.40 mu m.

[0017] Preferably, the parameter condition of the gas chromatography further comprises: the flow rate is 0.8-1.2 ml / min; the split mode is split, and the split ratio is 25-35:1; the control mode is constant flow; and the carrier gas is He.

[0018] Preferably, the parameter condition of the mass spectrometry is: the transmission line temperature is 245-255 DEG C; the quadrupole rod temperature is 145-155 DEG C; and the ion source temperature is 225-235 DEG C.

[0019] Preferably, the sampling mode of the mass spectrometry is:

[0020]

[0021] Preferably, the three potential genotoxic impurities are ethylene oxide, epichlorohydrin and benzene.

[0022] Preferably, the detection limit of the ethylene oxide in the metoprolol tartrate is 0.4992 ppm, and the quantification limit is 0.9985 ppm;

[0023] the detection limit of the epichlorohydrin in the metoprolol tartrate is 0.5326 ppm, and the quantification limit is 1.0651 ppm;

[0024] the detection limit of the benzene in the metoprolol tartrate is 0.3183 ppm, and the quantification limit is 0.6366 ppm.

[0025] Preferably, the sample to be detected comprises: a control solution, a sensitivity solution and a metoprolol tartrate sample solution.

[0026] Preferably, the preparation method of the metoprolol tartrate sample solution is as follows: 200 mg of metoprolol tartrate raw material sample is weighed into a 20 ml headspace bottle, 2 ml of solvent is removed, and then mixed and capped, and the metoprolol tartrate sample solution is obtained. Based on the above, the method is evaluated for system suitability, specificity, linearity and range, detection limit and quantification limit, precision, accuracy, solution stability and durability, etc. The results meet the requirements of the "9101 Drug Quality Standard Analysis Method Validation Guidelines" in the fourth volume of the Chinese Pharmacopoeia 2020 edition. Therefore, the method can be used to accurately detect the content of the above-mentioned three potential genotoxic impurities in metoprolol tartrate.

[0027] The content of the above-mentioned three potential genotoxic impurities in metoprolol tartrate is detected by gas chromatography-mass spectrometry technology. The present application provides a detection method with fast detection speed and simple operation method.

[0028] The present application provides a detection method for the content of three potential genotoxic impurities in metoprolol tartrate by screening and optimizing the related test conditions of gas chromatography and mass spectrometry in gas chromatography-mass spectrometry, which has the advantages of low detection limit, high precision, high repeatability and high accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The synthesis process flow chart of metoprolol tartrate is shown in the figure;

[0030] Figure 2 The specificity determination results of the blank solution in Example 1 are shown in the figure;

[0031] Figure 3 The specificity determination results of the control solution in Example 1 are shown in the figure;

[0032] Figure 4 The specificity determination results of the test solution in Example 1 are shown in the figure;

[0033] Figure 5 The specificity determination results of the 100% limit concentration spiked test solution in Example 1 are shown in the figure. DETAILED DESCRIPTION

[0034] The present application will be further described in detail below in combination with examples, comparative examples and performance test. These examples should not be understood as limiting the scope of the present application.

[0035] According to the limit requirements of the present application, the limit of the three potential genotoxic impurities in metoprolol tartrate, i.e. (RS) 2 [ (4 (2-methoxyethyl) phenoxy) methyl] oxirane and metoprolol USP impurity B, is 3.3 ppm. The present application establishes a GC-MS method for detecting the content of the three potential genotoxic impurities in metoprolol tartrate according to the properties of the raw material and target substance, and verifies the method.

[0036] Under the finally selected conditions, the system suitability, specificity, linearity and range, detection limit, quantification limit, accuracy, repeatability and stability of the method are verified to confirm that the method is suitable for determining the content of the three potential genotoxic impurities in metoprolol tartrate.

[0037] Example

[0038] Example 1

[0039] Example 1 provides a method for detecting the content of the three potential genotoxic impurities in metoprolol tartrate, which uses gas chromatography-mass spectrometry (GC-MS) to detect the sample to be tested;

[0040] 1. Analyze the method parameters, as shown in Table 1 below:

[0041] Table 1.

[0042]

[0043]

[0044] 2. Solution preparation:

[0045] Solvent: dimethyl sulfoxide

[0046] Control solution 1: accurately pipette 0.2 ml of oxirane control into a 10 ml volumetric flask containing a certain amount of solvent, dilute to the mark with solvent and mix well, and label it as control solution 1. (1000 μg / ml)

[0047] Control solution 2: accurately weigh 10 mg of epichlorohydrin control into a 10 ml volumetric flask containing a certain amount of solvent, dilute to the mark with solvent and mix well. Label it as control solution 2. (1000 μg / ml)

[0048] Control solution 3: accurately weigh 10 mg of benzene control into a 10 ml volumetric flask containing a certain amount of solvent, dilute to the mark with solvent and mix well. Label it as control solution 3. (1000 μg / ml)

[0049] Stock mixture control solution: Pipette 0.33 mL of Control Stock Solution 1 and 0.2 mL of Control Stock Solution 3 into a 100 mL volumetric flask containing a certain amount of solvent, dilute to the mark with solvent and mix. Label as Control Stock Mixture Solution (i.e. L-1000%). (Ethylene oxide: 3300 ng / mL; Epichlorohydrin: 3300 ng / mL; Benzene: 2000 ng / mL)

[0050] Control solution: Pipette 10.0 mL of Control Stock Mixture Solution into a 100 mL volumetric flask, dilute to the mark with solvent and mix. Label as STD (i.e. L-100%). (Ethylene oxide: 330 ng / mL; Epichlorohydrin: 330 ng / mL; Benzene: 200 ng / mL)

[0051] Sensitivity solution: Pipette 3 mL of Control Stock Mixture Solution into a 100 mL volumetric flask, dilute to the mark with solvent and mix. Label as LOQ. (Ethylene oxide: 99.85 ng / mL; Epichlorohydrin: 106.51 ng / mL; Benzene: 63.66 ng / mL); Pipette 1.5 mL of Control Stock Mixture Solution into a 100 mL volumetric flask, dilute to the mark with solvent and mix. Label as LOD. (Ethylene oxide: 49.92 ng / mL; Epichlorohydrin: 53.26 ng / mL; Benzene: 31.83 ng / mL)

[0052] Sample solution: Weigh accurately 200 mg of Metoprolol tartrate drug substance sample into a 20 mL headspace vial, pipette 2 mL of solvent and mix. Label as SS#1 and SS#2. (Prepare in duplicate, 100 mg / mL)

[0053] 1.1. Specificity solution

[0054] Prepare the Specificity solution as per the example in Table 2 below.

[0055] Table 2.

[0056] Name Procedure Blank Diluent DMSO STD Use L- 100% 100% Spiked Sample Solution Use 100% Sample - 01

[0057] 1.2. Linearity test solution

[0058] Prepare the Linearity test solution as per the examples in Table 3 and Table 4 below:

[0059] Prepare the L-1000% stock solution as per Table 3.

[0060] Table 3.

[0061]

[0062] Prepare 30% to 200% linear solutions from L-1000% stock solution according to Table 4, dilute with solvent.

[0063] Table 4.

[0064]

[0065] 1.3, LOD and LOQ test solutions

[0066] Prepare LOD and LOQ test solutions according to Table 5.

[0067] Table 5.

[0068]

[0069] 1.4, Accuracy test solutions

[0070] Prepare two sample solutions for accuracy, repeatability and intermediate precision tests according to Table 6, Table 7, Table 8 and Table 9.

[0071] Table 6. Sample solutions to be tested

[0072]

[0073] Prepare three replicates of spiked sample solutions at 50%, 100% and 150% levels according to Table 7 for accuracy tests.

[0074] Table 7.

[0075]

[0076] 1.5, Repeatability test solutions

[0077] Prepare six replicates of spiked sample solutions at 100% level according to Table 8 for repeatability tests. Three replicates of spiked sample solutions at 100% level prepared in accuracy tests can be used.

[0078] Table 8.

[0079]

[0080] 1.6, Intermediate precision test solutions

[0081] Prepare six replicates of spiked sample solutions at 100% level by different analysts at different times according to Table 9 for intermediate precision tests of the method.

[0082] Table 9.

[0083]

[0084] 1.7 Solution stability tests

[0085] Re-analyze 100% control solution stored under method conditions for 24 hours or other time period and one spiked sample solution at 100% level.

[0086] 1.8 Robustness Test

[0087] Prepare two spiked sample solutions at 100% limit for robustness test. The results are shown in Table 10.

[0088] Table 10.

[0089]

[0090]

[0091]

[0092] Take metoprolol tartrate sample, prepare one test solution according to the above method, and detect according to the parameters in Table 1, the results of the specificity test of the test solution are shown in Figure 4 .

[0093] Example 2

[0094] Example 2 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0095] Example 2 is different from Example 1 in that the contents of three potential genotoxic impurities are different, and the temperature rising program in the gas chromatography conditions is different, as shown below.

[0096] The gas chromatography conditions are as follows:

[0097] The chromatographic column is DB-624 with a specification of 30 m x 0.250 mm x 1.40 μm;

[0098] The parameter conditions of gas chromatography are as follows: injection port temperature: 210°C; temperature rising program: initial temperature of 35°C, holding for 4 min; rising to 235°C at a temperature rising rate of 10°C / min, holding for 1.5 min; post-run time: 20 min;

[0099] Flow rate: 0.8 ml / min; split mode: split, split ratio of 25:1; control mode: constant flow; carrier gas: He;

[0100] Headspace parameters: heating box temperature 85°C; quantification ring temperature 120°C; transmission line temperature: 125°C; headspace bottle equilibrium time: 8 min; injection time: 0.3 min; GC cycle time: 25 min; sample volume in bottle: 1.8 ml;

[0101] The parameter conditions of mass spectrometry are: acquisition type: SIM; ion source: EI; transfer line temperature: 240°C; quadrupole temperature: 140°C; ion source temperature: 220°C; transfer line temperature: 245°C; quadrupole temperature: 145°C; ion source temperature: 225°C.

[0102] The specificity test results are as follows:

[0103] The results show that the target peak is not displayed in the chromatogram of the blank solution, and there is no interference in detection; the target peak is displayed in the chromatogram of the test solution; the target peak is displayed in the chromatogram of the 100% limit concentration reference substance solution; the target peak is displayed in the chromatogram of the 100% limit concentration spiked test solution, and no adjacent peak with a peak area greater than LOQ is detected; the results meet the requirements, and the method has good specificity.

[0104] Example 3

[0105] Example 3 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0106] Example 3 is different from Example 1 in that the temperature rising program in the gas chromatography conditions is different, and is specifically as follows.

[0107] The gas chromatography conditions are as follows:

[0108] The chromatographic column of the gas chromatography is DB-624, with a specification of 30 m x 0.250 mm x 1.40 μm.

[0109] The parameter conditions of the gas chromatography are: injection port temperature: 230°C; temperature rising program: initial temperature of 45°C, holding for 6 min; rising to 245°C at a temperature rising rate of 20°C / min, holding for 2.5 min; post-run time: 21 min; flow rate: 1.2 ml / min; split mode: split, split ratio of 35:1; control mode: constant flow; carrier gas: He.

[0110] The headspace parameters are: heating box temperature 95°C; quantification ring temperature 130°C; transfer line temperature: 135°C; headspace bottle equilibrium time: 12 min; injection time: 0.8 min; GC cycle time: 30 min; sample volume in the bottle: 2.2 ml;

[0111] The parameter conditions of mass spectrometry are: acquisition type: SIM; ion source: EI; transfer line temperature: 260°C; quadrupole temperature: 160°C; ion source temperature: 240°C; transfer line temperature: 255°C; quadrupole temperature: 155°C; ion source temperature: 235°C.

[0112] The specificity test results are as follows:

[0113] The results show that the target peak is not displayed in the chromatogram of the blank solution, which does not interfere with the detection; the target peak is displayed in the chromatogram of the test solution; the target peak is displayed in the chromatogram of the 100% limit concentration reference substance solution; the target peak is displayed in the chromatogram of the 100% limit concentration spiked test solution, and no adjacent peak with a peak area greater than LOQ is detected; the results meet the requirements, and the method has good specificity.

[0114] Comparative Example 1

[0115] Comparative Example 1

[0116] Comparative Example 1 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0117] Comparative Example 1 and Example 1 differ in the gas chromatography conditions, as shown below.

[0118] DB-624, specification 30 m x 0.250 mm x 1.40 μm.

[0119] Inlet temperature: 200℃; temperature program: initial temperature 30℃, hold for 2 min; temperature rise rate 5℃ / min to 230℃, hold for 1.0 min; post-run time: 18 min; flow rate: 0.5 ml / min; split mode: split, split ratio 20:1; control mode: constant flow; carrier gas: He.

[0120] Specificity test results:

[0121] The results show that the test sample interferes with the detection of the target peak.

[0122] Comparative Example 2

[0123] Comparative Example 2 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0124] Comparative Example 2 and Example 1 differ in the gas chromatography parameter conditions, as shown below.

[0125] The gas chromatography parameter conditions are as follows: inlet temperature: 235℃; temperature program: initial temperature 50℃, hold for 8 min; temperature rise rate 25℃ / min to 250℃, hold for 3.0 min; post-run time: 22 min; flow rate: 1.5 ml / min; split mode: split, split ratio 40:1; control mode: constant flow; carrier gas: He.

[0126] Specificity test results:

[0127] The results show that the test sample interferes with the detection of the target peak.

[0128] Comparative Example 3

[0129] Comparative Example 3 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0130] Comparative Example 3 differs from Example 1 in that the headspace parameters are different, as shown below.

[0131] Headspace parameters: heating block temperature 80°C; loop temperature 135°C; transfer line temperature: 120°C; headspace vial equilibration time: 5 min; injection time: 1.0 min; GC cycle time: 35 min; sample volume in vial: 2.5 ml.

[0132] Specificity test results:

[0133] The results show that the test sample interferes with the detection of the target peaks.

[0134] Comparative Example 4

[0135] Comparative Example 4 provides a method for detecting the content of three potential genotoxic impurities in metoprolol tartrate.

[0136] Comparative Example 4 differs from Example 1 in that the mass spectrometry parameter conditions are different, as shown below.

[0137] Mass spectrometry parameter conditions: acquisition type: SIM; ion source: EI; transfer line temperature: 235°C; quadrupole temperature: 170°C; ion source temperature: 245°C.

[0138] Specificity test results:

[0139] The results show that no target peaks were detected.

[0140] Although the present application has been described in detail with general description and specific embodiments above, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A method for detecting the content of three potential genotoxic impurities in metoprolol tartrate, characterized in that, Gas chromatography-mass spectrometry (GC-MS) was used to detect the test samples; The parameters for the gas chromatography are as follows: injection port temperature: 210~230℃; temperature program: initial temperature 35~45℃, hold for 4~6 min; increase temperature to 235~245℃ at a rate of 10~20℃ / min, hold for 1.5~2.5 min; subsequent run time: 20~21 min. Headspace parameters: Heating chamber temperature 85~95 o C; Metering ring temperature 120~130 o C; Transmission line temperature: 125~135 o C; Headspace equilibration time: 8~12 min; Injection time: 0.3~0.8 min; GC cycle time: 25~30 min; Sample volume in vial: 1.8~2.2 ml; The parameters of the mass spectrometer are as follows: acquisition type: SIM; ion source: EI; transfer line temperature: 240~260℃; quadrupole temperature: 140~160℃; ion source temperature: 220~240℃.

2. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The heating program is as follows: initial temperature is 37~42℃, held for 5 min; heating rate is 12~18℃ / min to 238~242℃, held for 2 min; subsequent running time: 20.333 min.

3. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The gas chromatography column used was a DB-624 with dimensions of 30 m × 0.250 mm × 1.40 μm.

4. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The parameters and conditions of the gas chromatography are also Includes: Flow rate: 0.8~1.2 ml / min; Split mode: split, split ratio is 25~35:1; Control mode: constant flow; Carrier gas: He.

5. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The parameters of the mass spectrometer are as follows: transfer line temperature: 245~255℃; quadrupole temperature: 145~155℃; ion source temperature: 225~235℃.

6. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The sampling mode of the mass spectrometer is: 。 7. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 1, characterized in that, The three potential genotoxic impurities are ethylene oxide, epichlorohydrin, and benzene.

8. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 7, characterized in that, The detection limit for ethylene oxide in metoprolol tartrate is 0.4992 ppm, and the quantitation limit is 0.9985 ppm. The detection limit for epichlorohydrin in metoprolol tartrate is 0.5326 ppm, and the quantitation limit is 1.0651 ppm. The detection limit for benzene in metoprolol tartrate is 0.3183 ppm, and the quantitation limit is 0.6366 ppm.

9. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to any one of claims 1-8, characterized in that, The test samples include: reference solution, sensitivity solution and metoprolol tartrate sample solution.

10. The method for detecting the content of three potential genotoxic impurities in metoprolol tartrate according to claim 9, characterized in that, The preparation method of the metoprolol tartrate sample solution is as follows: weigh 200 mg of metoprolol tartrate raw material sample, place it in a 20 ml headspace vial, transfer 2 ml of solvent, mix well and cap, and the solution is obtained.

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