Analysis method of mutagenic impurities in new drug bulk drug

Abstract

The present application relates to the technical field of pharmaceutical analysis, and particularly relates to a method for analyzing mutagenic impurities in new drug raw materials. The new drug raw material belongs to a small molecule fungal inositol acyltransferase 1 inhibitor, and is a new drug for treating fungal infections. Impurity A and impurity B are potential mutagenic impurities. The potential mutagenic impurities in the new drug raw material are detected and analyzed by using gas chromatography-mass spectrometry. By limiting the gas chromatography conditions and the mass spectrometry conditions, trace detection of impurity A and impurity B can be realized. The analysis method provided by the present application has strong specificity, and the blank solvent does not interfere with the detection of various impurities. The analysis method has high sensitivity and good accuracy, the average recovery rate of each impurity is within the range of 70% to 130%, and significant detection performance advantages are shown.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for analyzing mutagenic impurities in a new drug active pharmaceutical ingredient. Background Technology

[0002] New drug raw materials (Referring to WXSH0102) is a small molecule fungal inositol acyltransferase 1 (Gwtl) inhibitor, which is a new type of drug for the treatment of fungal infections.

[0003] In the preparation process of WXSH0102 active pharmaceutical ingredient, potentially mutagenic impurities A and B may be generated, with the following structural formulas:

[0004] , .

[0005] Impurity A is an impurity generated during the synthesis process from the reaction of the WXSH0102 intermediate with thionyl chloride, and may remain in the final product. This impurity belongs to the chloroalkane class of compounds and has a warning structure. Based on quantitative structure-activity relationship (QSAR) software evaluation, it is classified as Category 2 in ICH M7. According to the guidance principle of "Assessing and controlling DNA-reactive (mutagenic) impurities in drugs to limit potential carcinogenic risks (ICH M7)," it needs to be studied as a mutagenic impurity.

[0006] Impurity B is an unreacted raw material from the synthesis process and may remain in the final product. This impurity belongs to the chloroalkane class and has a warning structure; according to QSAR software evaluation, it is classified as Category 3 in ICH M7. In accordance with ICH M7 guidelines, research on impurity control data and limits should be conducted with strict adherence to regulations.

[0007] For the WXSH0102 active pharmaceutical ingredient (API), the ability to efficiently and accurately detect these two potentially mutagenic impurities directly impacts its safety and quality, and is therefore of great significance. Based on a daily dose not exceeding 600 mg, according to ICH M7 guidelines, the threshold of toxicological concern (TTC) for administration periods of less than one month is 120 μg / day. Considering this limit, to ensure clinical safety, the acceptable limits for impurities A and B are tightened to no more than 20 ppm (corresponding to a detection solution concentration of 400 ng / mL).

[0008] Therefore, it is essential to establish an analytical method that is specific, sensitive, and capable of accurately quantifying mutagenic impurities. Summary of the Invention

[0009] The present invention aims to at least solve one of the technical problems existing in related technologies. Therefore, the object of the present invention is to provide an analytical method for mutagenic impurities in a new pharmaceutical raw material.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] A method for analyzing mutagenic impurities in a new drug active pharmaceutical ingredient (API) using gas chromatography-mass spectrometry (GC-MS) to detect and analyze mutagenic impurities in the API.

[0012] The structural formula of the new drug active pharmaceutical ingredient is shown below:

[0013] ;

[0014] The mutagenic impurity includes impurity A and impurity B, and the structural formula of impurity A is shown below:

[0015] ;

[0016] The structural formula of impurity B is shown below:

[0017] ;

[0018] Gas chromatography-mass spectrometry (GC-MS) includes gas chromatography conditions and mass spectrometry conditions. Among them, gas chromatography conditions include at least the selection of the chromatographic column and the setting of the column temperature.

[0019] The chromatographic column is selected from polysiloxane chromatographic columns, and the stationary phase has polysiloxane as the main chain and phenyl and methyl side chains.

[0020] The initial column temperature was 59–61℃, the final temperature was 290–310℃, and the heating rate was 24–26℃ / min.

[0021] Mass spectrometry conditions include at least the setting of the scan mode and the selection of the ion source;

[0022] The scanning mode was ion monitoring. The quantitative ion-to-mass ratio for impurity A was 106–108 and 120–122, and the quantitative ion-to-mass ratio for impurity B was 197–199 and 232–234.

[0023] The ion source is an electron-bombardment ion source.

[0024] Furthermore, the molar ratio of phenyl to methyl in the side chain of the stationary phase is 1:18 to 1:20.

[0025] Furthermore, during the detection and analysis process, the new drug active pharmaceutical ingredient (API) is injected in the form of an API solution, and the preparation process of the API solution is as follows:

[0026] S100. Dissolve the new drug raw material in an aqueous sodium hydroxide solution with a concentration of 0.05-0.15 mol / L to obtain a mother liquor with a concentration of 15-25 mg / mL;

[0027] S200. Add an equal volume of ethyl acetate to the mother liquor, stir, let stand to separate the layers, and take the upper layer solution to obtain the raw material solution.

[0028] Furthermore, the gas chromatography conditions also include the setting of the injection mode, wherein the injection mode is split injection.

[0029] Furthermore, the gas chromatography conditions also include the setting of the flow rate, which is 0.8 mL / min to 1.2 mL / min.

[0030] Furthermore, the gas chromatography conditions also include setting the injection port temperature, which is 140–160°C.

[0031] Furthermore, the temperature of the ion source is 225–235°C.

[0032] Furthermore, the quantitative ion-mass ratios for impurity A were 107 and 121, and the quantitative ion-mass ratios for impurity B were 198 and 233.

[0033] Furthermore, the detection limit concentration of impurity A is not higher than 40 ng / mL, and the quantitation limit concentration is not higher than 79.5 ng / mL.

[0034] Furthermore, the detection limit concentration of impurity B is not higher than 42 ng / mL, and the quantitation limit concentration is not higher than 84 ng / mL.

[0035] The above-described one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects:

[0036] This invention provides a new drug raw material. The analytical method for mutagenic impurities in the drug substance was analyzed using gas chromatography-mass spectrometry. and By performing detection and analysis, and by limiting the gas chromatography and mass spectrometry conditions, trace detection of impurities A and B can be achieved.

[0037] The analytical method provided by this invention has high specificity, and the blank solvent does not interfere with the detection of various impurities; it has high sensitivity, with the detection limit concentration of impurity A not exceeding 40 ng / mL and the quantitation limit concentration not exceeding 79.5 ng / mL; the detection limit concentration of impurity B not exceeding 42 ng / mL and the quantitation limit concentration not exceeding 84 ng / mL; and it has good accuracy, with the average recovery rate of each impurity in the range of 70% to 130%, showing significant advantages in detection performance.

[0038] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0039] Figure 1 This is a chromatogram of the blank solution provided in Example 1 of the present invention.

[0040] Figure 2 This is a chromatogram of the reference solution provided in Example 1 of the present invention.

[0041] Figure 3 This is a chromatogram of the test solution provided in Example 1 of the present invention.

[0042] Figure 4 This is a chromatogram of the spiked solution of the test sample provided in Example 1 of the present invention.

[0043] Figure 5 This is a chromatogram of the limit of quantitation solution provided in Example 2 of the present invention.

[0044] Figure 6 This is a chromatogram of the detection limit solution provided in Embodiment 2 of the present invention.

[0045] Figure 7 This is a linear relationship graph between the concentration of impurity A and the peak area provided in Embodiment 3 of the present invention.

[0046] Figure 8 This is a linear relationship graph between the concentration of impurity B and the peak area provided in Embodiment 3 of the present invention.

[0047] Figure 9 This is a chromatogram of the 50% level spiked test solution provided in Example 4 of the present invention.

[0048] Figure 10 This is a chromatogram of the 100% level spiked test solution provided in Example 4 of the present invention.

[0049] Figure 11 This is a chromatogram of the 150% level spiked test solution provided in Example 4 of the present invention. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The following embodiments are used to illustrate this invention, but cannot be used to limit the scope of this invention.

[0051] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available, unless otherwise specified, and are carried out in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0052] A method for analyzing mutagenic impurities in a new drug active pharmaceutical ingredient (API) using gas chromatography-mass spectrometry (GC-MS) to detect and analyze mutagenic impurities in the API.

[0053] The structural formula of the new drug active pharmaceutical ingredient (referred to as WXSH0102) is shown below:

[0054] ;

[0055] The mutagenic impurity includes impurity A and impurity B, and the structural formula of impurity A is shown below:

[0056] ;

[0057] The structural formula of impurity B is shown below:

[0058] .

[0059] The gas chromatography and mass spectrometry conditions in gas chromatography-mass spectrometry are shown in Table 1.

[0060]

[0061] In the table above, 5%-phenyl-95% dimethyl polysiloxane means: polysiloxane as the main chain, with 5% molar percentage of phenyl and 95% molar percentage of methyl in the side chain.

[0062] Example 1: Examination of specificity.

[0063] The preparation of 0.1 mol / L sodium hydroxide solution is as follows: Add sodium hydroxide (about 800 mg) to a volumetric flask, then add water (200 mL), sonicate to dissolve, and shake well to obtain 0.1 mol / L sodium hydroxide solution.

[0064] The preparation and detection of the blank solution were as follows: 2.0 mL of 0.1 mol / L sodium hydroxide solution was placed in a centrifuge tube, and an equal volume of 2.0 mL of ethyl acetate was added. The tube was vortexed for 30 seconds, allowed to stand for separation, and the upper layer was collected as the blank solution to be tested. 1 μL of the blank solution was injected into a gas chromatograph-mass spectrometer (GC-MS), and the detection was performed according to the conditions set in Table 1. The results are as follows: Figure 1 As shown.

[0065] The preparation of the reference stock solution is as follows: Dissolve impurity A (40 mg) in acetonitrile (100 mL) to obtain impurity A stock solution with a concentration of 0.4 mg / mL; dissolve impurity B (40 mg) in acetonitrile (100 mL) to obtain impurity B stock solution with a concentration of 0.4 mg / mL; add impurity A stock solution (0.1 mL) and impurity B stock solution (0.1 mL) to ethyl acetate (10 mL), shake well, and the reference stock solution is obtained.

[0066] The preparation and detection of the reference solution are as follows: Measure 1 mL of the above reference stock solution and place it in a 10 mL volumetric flask. Dilute to the mark with ethyl acetate, shake well, and then measure 2.0 mL into a centrifuge tube. Add 2.0 mL of 0.1 mol / L sodium hydroxide solution, vortex for 30 seconds, allow to stand for layering, and take the upper layer solution, which is the reference solution. Inject 1 μL of the reference solution into a GC-MS and perform detection according to the conditions set in Table 1. The results are as follows: Figure 2 As shown.

[0067] The preparation and detection of the test solution are as follows: Weigh 40 mg of WXSH0102 into a centrifuge tube, add 2.0 mL of 0.1 mol / L sodium hydroxide solution to dissolve it, then add an equal volume of 2.0 mL of ethyl acetate, vortex for 30 s, allow to stand and separate into layers, and take the upper layer solution, which is the test solution; inject 1 μL of the test solution into a GC-MS, and perform detection according to the conditions set in Table 1. The results are as follows. Figure 3 As shown.

[0068] The preparation of 100% spiked stock solution is as follows: Accurately measure 1 mL of the above reference stock solution, place it in a 10 mL volumetric flask, dilute to the mark with ethyl acetate, and shake well to obtain 100% spiked stock solution.

[0069] The preparation and detection of the spiked solution for the test sample are as follows: Weigh 40 mg of WXSH0102 into a centrifuge tube, add 2.0 mL of 0.1 mol / L sodium hydroxide solution to dissolve it, then add 2.0 mL of 100% spiking stock solution, vortex for 30 s, allow to stand and separate into layers, and take the upper layer solution, which is the spiked solution for the test sample; inject 1 μL of the spiked solution for the test sample into a GC-MS, and perform detection according to the conditions set in Table 1. The results are as follows. Figure 4 As shown.

[0070] Figures 1 to 4 The test results show that the blank solution does not interfere with the determination of impurities A and B, and the test solution and the spiked test solution have no adjacent peaks near impurities A and B, which meets the requirements.

[0071] Example 2: Investigation of detection limit and quantitation limit.

[0072] The preparation and detection of the limit of quantitation solution are as follows: Take 0.2 mL of the reference stock solution from Example 1 and add it to a 10 mL volumetric flask. Dilute to the mark with ethyl acetate, take 2.0 mL and place it in a centrifuge tube. Then add 2.0 mL of 0.1 mol / L sodium hydroxide solution, vortex for 30 s, let stand to separate the layers, and take the upper layer solution, which is the limit of quantitation solution.

[0073] The preparation and detection of the detection limit solution are as follows: Take 0.1 mL of the reference stock solution from Example 1 and add it to a 10 mL volumetric flask. Then, add ethyl acetate to dilute to the mark. Take 2.0 mL and place it in a centrifuge tube. Add 2.0 mL of 0.1 mol / L sodium hydroxide solution, vortex for 30 s, let it stand to separate into layers, and take the upper layer solution, which is the detection limit solution.

[0074] 1 μL of the limit of quantitation (LOQ) solution and limit of detection (LOD) solution were injected into the GC-MS, and the detection was performed according to the conditions set in Table 1. The spectral results are shown below. Figure 5 and Figure 6 The results for the limits of quantitation and detection are shown in the table below:

[0075]

[0076] Example 3: Linear investigation.

[0077] Limit of Quantitation Solution: Prepared according to the preparation process of the limit of quantitation solution in Example 2.

[0078] The preparation of the 50% linear solution is as follows: Take 0.5 mL of the reference stock solution from Example 1 and add it to a 10 mL volumetric flask. Dilute to the mark with ethyl acetate and shake well to obtain the 50% linear solution.

[0079] The preparation of the 100% linear solution is the same as that of the 50% linear solution, except that the amount of the reference stock solution added is replaced with 1.0 mL.

[0080] 150% linear solution: Except for replacing the amount of the reference stock solution added with 1.5 mL, the rest of the process is the same as the preparation of the 50% linear solution.

[0081] 200% linear solution: Except for replacing the amount of the reference stock solution added with 2.0 mL, the rest of the process is the same as the preparation of the 50% linear solution.

[0082] Before injection, 2.0 mL of each solution should be placed in a centrifuge tube, 2.0 mL of 0.1 mol / L sodium hydroxide solution should be added, the mixture should be vortexed for 30 s, allowed to stand and separate into layers, and the upper layer solution should be taken as the test solution. 1 μL of each test solution should be injected into a GC-MS and detected according to the conditions set in Table 1. The results are shown in the table below:

[0083]

[0084] The data provided in the table above shows that impurity A, within the concentration range of 79.04–790.36 ng / mL, such as… Figure 7 As shown, the concentration of impurity A has a linear relationship with the peak area, and the linear relationship is good.

[0085] Impurity B, within the concentration range of 83.78–837.84 ng / mL, such as Figure 8 As shown, the concentration of impurity B has a linear relationship with the peak area, and the linear relationship is good.

[0086] Example 4: Recovery rate investigation.

[0087] Reference solution: Prepared according to the preparation process of the reference solution in Example 1.

[0088] Test solution: Prepared according to the test solution preparation process in Example 1.

[0089] 50% spiked stock solution: Prepared according to the preparation process of the 50% linear solution in Example 3.

[0090] 100% spiked stock solution: Prepared according to the preparation process of the 100% linear solution in Example 3.

[0091] 150% spiked stock solution: Prepared according to the preparation process of the 150% linear solution in Example 3.

[0092] The preparation of the 50% level spiked test solution is as follows: Weigh 40 mg of WXSH0102 and place it in a centrifuge tube. Add 2.0 mL of 0.1 mol / L sodium hydroxide solution to dissolve it. Then add 2.0 mL of 50% spiking stock solution. Vortex for 30 seconds, allow to stand and separate into layers, and take the upper layer solution, which is the 50% level spiked test solution. Prepare 3 parallel aliquots.

[0093] The preparation of the 100% spiked test solution is as follows: Weigh 40 mg of WXSH0102 and place it in a centrifuge tube. Dissolve it in 2.0 mL of 0.1 mol / L sodium hydroxide solution. Then add 2.0 mL of 100% spiked stock solution, vortex for 30 seconds, allow to stand and separate into layers, and take the upper layer solution, which is the 100% spiked test solution. Prepare 3 parallel solutions.

[0094] The preparation of the 150% level spiked test solution is as follows: Weigh 40 mg of WXSH0102 active pharmaceutical ingredient and place it in a centrifuge tube. Add 2.0 mL of 0.1 mol / L sodium hydroxide solution to dissolve it, then add 2.0 mL of 150% spiking stock solution. Vortex for 30 seconds, allow to stand and separate into layers, and take the upper layer solution, which is the 150% level spiked test solution. Prepare 3 parallel solutions.

[0095] 1 μL each of the reference solution, test solution, and spiked solutions at different levels were injected into a GC-MS. Detection was performed according to the conditions set in Table 1. The detection results for the 50%, 100%, and 150% spiked test solutions are shown below. Figure 9 , Figure 10 and Figure 11 As shown, the recovery rates were calculated, and the results for each level are shown in the table below:

[0096]

[0097] The data provided in the table above show that the average recovery rates of impurities A and B are between 70% and 130% within the concentration range of 50% to 150%, and the RSD values ​​of the recovery rates are all less than 20%. This result indicates that the analytical method provided by this invention has good accuracy.

[0098] RSD is an abbreviation for Relative Standard Deviation.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing a mutagenic impurity in a new drug substance, characterized by, The mutagenic impurity in the new drug raw material is detected and analyzed by gas chromatography-mass spectrometry; The structural formula of the new drug raw material is as follows: ； The mutagenic impurity includes impurity A and impurity B, and the structural formula of impurity A is as follows: ； The structural formula of impurity B is as follows: ； The gas chromatography-mass spectrometry includes gas chromatography conditions and mass spectrometry conditions, wherein the gas chromatography conditions at least include selection of a chromatographic column and setting of column temperature; The chromatographic column is selected from a polysiloxane chromatographic column, and the stationary phase has a polysiloxane main chain and a side chain containing a benzene group and a methyl group; The initial temperature of the column temperature is 59-61 DEG C, the terminal temperature is 290-310 DEG C, and the temperature rising rate is 24-26 DEG C / min; The mass spectrometry conditions at least include setting of a scanning mode and selection of an ion source; The scanning mode is ion monitoring, the monitoring quantitative ion mass-to-charge ratio of impurity A is 106-108 and 120-122, and the monitoring quantitative ion mass-to-charge ratio of impurity B is 197-199 and 232-234; The ion source is an electron impact ion source.

2. The method for analyzing a mutagenic impurity in a new drug substance according to claim 1, wherein The molar ratio of the benzene group to the methyl group in the side chain of the stationary phase is 1:18-1:

20.

3. The analytical method for mutagenic impurities in a new drug active pharmaceutical ingredient as described in claim 1, characterized in that, In the detection and analysis process, the new drug raw material is injected in the form of a raw material solution; The preparation process of the raw material solution is as follows: S100, the new drug raw material is dissolved in a 0.05-0.15 mol / L sodium hydroxide aqueous solution to obtain a 15-25 mg / mL mother liquor; S200, an equal volume of ethyl acetate is added to the mother liquor, stirred, and separated into layers, and the upper layer solution is obtained, that is, the raw material solution.

4. The method of claim 1, wherein the new drug substance is a pharmaceutical drug substance. The gas chromatography conditions further include setting of an injection mode, and the injection mode is split injection.

5. The analytical method for mutagenic impurities in a new drug active pharmaceutical ingredient as described in claim 1, characterized in that, The gas chromatography conditions further include setting of a flow rate, and the flow rate is 0.8 mL / min-1.2 mL / min.

6. The analytical method for mutagenic impurities in a new drug active pharmaceutical ingredient as described in claim 1, characterized in that, The gas chromatography conditions further include setting of an injection port temperature, and the injection port temperature is 140-160 DEG C.

7. The method of claim 1, wherein the new drug substance is a pharmaceutical drug substance. The temperature of the ion source is 225-235 DEG C.

8. The method for analyzing a mutagenic impurity in a new drug substance according to any one of claims 1 to 7, wherein The monitoring quantitative ion mass-to-charge ratio of impurity A is 107 and 121, and the monitoring quantitative ion mass-to-charge ratio of impurity B is 198 and 233.

9. The method of claim 1 to 7, wherein the new drug substance is a pharmaceutical drug substance. The detection limit concentration of impurity A is not higher than 40 ng / mL, and the quantitative limit concentration is not higher than 79.5 ng / mL.

10. The method for analyzing a mutagenic impurity in a new drug substance according to any one of claims 1 to 7, wherein The detection limit concentration of impurity B is not higher than 42 ng / mL, and the quantitative limit concentration is not higher than 84 ng / mL.

Images