Analytical method for mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor API
The detection of mutagenic impurities in WXSH0102 raw materials was solved by liquid phase-mass spectrometry, and the detection problems in the prior art were solved, and impurity analysis with high sensitivity and accuracy was achieved to ensure drug quality and safety.
Patent Information
- Application Number
- CN202510407100.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-02
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2045-04-02
AI Technical Summary
The prior art is difficult to effectively detect and quantify mutagenic impurities I and Impurities II in WXSH0102 raw materials, affecting the quality and safety of the drug.
The liquid phase-mass spectrometry combination method was used, and octadecylsilane bonded silica gel was used as the filler, gradient elution and reaction monitoring mode were selected, combined with electrospray ion source to detect mutagenic impurities I and Impurities II, and the quantitative ion mass-to-charge ratio was 232-234 and 203-205, respectively.
High sensitivity and high specificity impurity detection is achieved, with the quantitative limit and detection limits of 0.53 ng/ml and 0.27 ng/ml, 0.52 ng/ml and 0.26 ng/ml respectively, ensuring the accuracy and interference-free detection.
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Figure CN119915948B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to an analytical method for mutagenic impurities in the active pharmaceutical ingredient of a small molecule fungal inositolacyltransferase 1 inhibitor. Background Art
[0002] A small molecule fungal inositolacyltransferase 1 (Gwt1) inhibitor mainly inhibits the activity of Gwt1, preventing the synthesis of glycosylphosphatidylinositol-anchored proteins by inhibiting the synthesis of the glycosylphosphatidylinositol (GPI) precursor, thereby affecting the synthesis of GPI-anchored proteins (AP). This leads to the inability of mannose proteins on the fungal surface to crosslink to the cell wall, ultimately destroying the integrity of the fungal cell wall. At the same time, it has the ability to adhere to the host surface, has good antifungal broad-spectrum properties, and is not prone to cross-resistance with currently clinically used antifungal drugs. Therefore, it has potential broad clinical application prospects.
[0003] The active pharmaceutical ingredient of the Gwt1 inhibitor, designated as WXSH0102, has the following structural formula:
[0004] ;
[0005] In the preparation process of WXSH0102, potentially mutagenic impurities I and II are generated, and their structural formulas are as follows:
[0006] and ;
[0007] Whether these two mutagenic impurities can be effectively detected is crucial for the quality and safety of the active pharmaceutical ingredient of WXSH0102. The maximum daily dose of WXSH0102 does not exceed 600 mg. According to the calculation of the limit value stipulated in the guiding principle for the assessment and control of DNA-reactive (mutagenic) impurities in drugs to limit potential carcinogenic risks, the acceptable limits of mutagenic impurities I and II in the active pharmaceutical ingredient of WXSH0102 are not higher than 200 ppm (equivalent to 2×10 5 ng / ml). Therefore, establishing an analytical method with good specificity, high sensitivity, and the ability to accurately quantify mutagenic impurities is of great significance for ensuring the product quality and safety of the active pharmaceutical ingredient of WXSH0102. Summary of the Invention
[0008] The present invention aims to solve at least one of the technical problems existing in the related art. For this purpose, the object of the present invention is to provide an analytical method for mutagenic impurities in the raw material drug of a small molecule fungal inositol acyltransferase 1 inhibitor.
[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:
[0010] An analytical method for mutagenic impurities in the raw material drug of a small molecule fungal inositol acyltransferase 1 inhibitor, the structural formula of the raw material drug is as follows:
[0011] ;
[0012] The mutagenic impurities include impurity I and impurity II, and the structural formula of impurity I is as follows:
[0013] ;
[0014] The structural formula of impurity II is as follows:
[0015] ;
[0016] The mutagenic impurities are determined by liquid chromatography - mass spectrometry;
[0017] Among them, the liquid chromatography includes the following conditions:
[0018] The chromatographic column is packed with octadecylsilyl silica gel;
[0019] The mobile phase includes mobile phase A and mobile phase B. The initial volume ratio of mobile phase A is 70% ± 5%, and the initial volume ratio of mobile phase B is 30% ± 5%. Mobile phase A is selected from acidic aqueous solutions, and mobile phase B is selected from organic solvents;
[0020] The elution method is gradient elution;
[0021] The flow rate is 0.8 mL / min ± 0.5 mL / min;
[0022] The mass spectrometry includes the following conditions:
[0023] The scanning mode is selected from the selected reaction monitoring mode, the ionization mode is the positive ion mode, the ion source is the electrospray ion source, the collision voltage is 22 V ± 5 V, the quantitative ion mass - to - charge ratio of impurity I is 232 - 234, and the quantitative ion mass - to - charge ratio of impurity II is 203 - 205.
[0024] Further, the volume ratios of mobile phase A and mobile phase B during gradient elution are as follows: constant from 0 min to the 10th min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changing uniformly to the 14th min, with mobile phase A being 10% and mobile phase B being 90%; constant from the 14th min to the 16th min, with mobile phase A maintained at 10% and mobile phase B maintained at 90%; from the 16th min to the 16.1th min, mobile phase A changes from 10% to 70%, and mobile phase B changes from 90% to 30%; constant from the 16.1th min to the 18th min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.
[0025] Further, the acidic aqueous solution is a 0.05% - 0.2% formic acid aqueous solution, preferably a 0.1% formic acid aqueous solution.
[0026] Further, the organic solvent is selected from acetonitrile or methanol.
[0027] Further, the conditions of the liquid phase also include the set range of column temperature, and the set range of column temperature is 35°C ± 5°C.
[0028] Further, the particle size of the filler is selected from 3μm and / or 5μm, the model of the chromatographic column is preferably ACE 3 C18 or Shimadzu ODS - 3 C18, the column length is selected from 150mm, and the inner diameter of the chromatographic column is selected from 4.6mm.
[0029] Further, the quantitative sub - ion mass - to - charge ratio of impurity I is 233.
[0030] Further, the quantitative sub - ion mass - to - charge ratio of impurity II is 204.
[0031] Further, the quantitative limit concentration of impurity I is not higher than 0.53 ng / ml, and the detection limit concentration of impurity I is not higher than 0.27 ng / ml.
[0032] Further, the quantitative limit concentration of impurity II is not higher than 0.52 ng / ml, and the detection limit concentration of impurity II is not higher than 0.26 ng / ml.
[0033] One or more of the above - mentioned technical solutions in the embodiments of the present invention have at least one of the following technical effects:
[0034] The analytical method for mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor API provided by the present invention can be used for the trace detection of potential mutagenic impurity I and impurity II in API WXSH0102. The analytical method provided by the present invention has high sensitivity. The limit of quantitation concentration of impurity I is not higher than 0.53 ng / ml, and the limit of detection concentration of impurity I is not higher than 0.27 ng / ml; the limit of quantitation concentration of impurity II is not higher than 0.52 ng / ml, and the limit of detection concentration of impurity II is not higher than 0.26 ng / ml; the chromatographic detection results show that the analytical method has strong specificity, and the blank solvent does not interfere with the detection of each impurity; the results of the recovery investigation show that the average recoveries are all between 70% and 130%, indicating that the analytical method provided by the present invention has good accuracy and small systematic error.
[0035] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. Description of the Drawings
[0036] Figure 1 is the chromatogram of the diluent provided in Example 1 of the present invention.
[0037] Figure 2 is the chromatogram of the reference solution provided in Example 1 of the present invention.
[0038] Figure 3 is the chromatogram of the test solution provided in Example 1 of the present invention.
[0039] Figure 4 is the chromatogram of the spiked test solution provided in Example 1 of the present invention.
[0040] Figure 5 is the chromatogram of the limit of quantitation solution provided in Example 2 of the present invention.
[0041] Figure 6 is the chromatogram of the limit of detection solution provided in Example 2 of the present invention.
[0042] Figure 7 is the graph of the relationship between the concentration and peak area of impurity I provided in Example 3 of the present invention.
[0043] Figure 8 is the graph of the relationship between the concentration and peak area of impurity II provided in Example 3 of the present invention.
[0044] Figure 9 is the chromatogram of the 50% level spiked solution provided in Example 4 of the present invention.
[0045] Figure 10 is the chromatogram of the 100% level spiked solution provided in Example 4 of the present invention.
[0046] Figure 11 It is the chromatogram of the 150% spiked solution provided in Example 4 of the present invention. Detailed implementation manners
[0047] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with specific embodiments. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. The following embodiments are used to illustrate the present invention but cannot be used to limit the scope of the present invention.
[0048] In the following embodiments, the experimental methods used are all conventional methods unless otherwise specified, and the materials, reagents, etc. used are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. Unless otherwise specified, they can all be obtained from commercial channels.
[0049] The liquid chromatography-mass spectrometry coupling conditions are shown in the following table:
[0050]
[0051] Example 1 Specificity investigation.
[0052] Preparation and detection of diluent: Measure 300 ml of water and add it to a reagent bottle, then add 300 ml of acetonitrile and 0.3 ml of formic acid, mix evenly to obtain the diluent. Take 10 μL of the diluent and inject it into the liquid chromatography-mass spectrometry instrument. The chromatogram is as Figure 1 shown.
[0053] Preparation of reference stock solution: Take about 4 mg each of the reference substances of impurity I and impurity II, weigh accurately, add them to a 10-ml volumetric flask, then add the diluent and shake to dissolve impurity I and impurity II, and dilute to the mark with the diluent and mix evenly to obtain the mother liquor I of the reference stock solution; Measure 0.1 ml of the mother liquor I of the reference stock solution and add it to a 10-ml volumetric flask, dilute to the mark with the diluent and mix evenly to obtain the mother liquor II of the reference stock solution; Measure 0.5 ml of the mother liquor II of the reference stock solution and add it to a 20-ml volumetric flask, dilute to the mark with the diluent and mix evenly to obtain a reference stock solution containing 0.1 μg each of impurity I and impurity II.
[0054] Preparation and detection of reference solution: Weigh 1.0 ml of the reference stock solution, add it to a 10-ml volumetric flask, add the diluent (9 ml), shake well to obtain the reference solution. Take 10 μL of the reference solution and inject it into the liquid chromatography-mass spectrometry instrument, and record the chromatogram. The result is as Figure 2 shown.
[0055] Preparation of test sample stock solution: Weigh approximately 5 mg of the WXSH0102 active pharmaceutical ingredient, add it to a 10-ml volumetric flask, add diluent to the mark, shake well to obtain the test sample stock solution.
[0056] Preparation and detection of test sample solution: Pipette 1.0 ml of the test sample stock solution into a 5-ml volumetric flask, add diluent to the mark, shake well to obtain the test sample solution. Inject 10 μL of the test sample solution into the liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are as Figure 3 shown.
[0057] Preparation and detection of spiked test sample solution: Pipette 1.0 ml of the test sample stock solution and 0.5 ml of the reference stock solution into a 5-ml volumetric flask, then add diluent to the mark, shake well to obtain the spiked test sample solution. Inject 10 μL of the spiked test sample solution into the liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are as Figure 4 shown.
[0058] Figures 1 to 4 The test results show that the diluent has no interference on the determination of potential mutagenic impurities I and II. There are no adjacent peaks near impurities I and II in the test sample solution and the spiked test sample solution, meeting the requirements.
[0059] Example 2 Investigation of the limit of quantitation (LOQ) and limit of detection (LOD).
[0060] Limit of quantitation solution: Pipette 0.5 ml of the reference solution in Example 1 into a 10-ml volumetric flask, add diluent to the mark, shake well to obtain it.
[0061] Limit of detection solution: Pipette 0.25 ml of the reference solution in Example 1 into a 10-ml volumetric flask, add diluent to the mark, shake well to obtain it.
[0062] Pipette 10 μL of the limit of quantitation solution and the limit of detection solution respectively, inject them into the liquid chromatography-mass spectrometry instrument, record the chromatogram, and the results are as Figure 5 、 Figure 6 shown. The results of the limit of quantitation and limit of detection are as shown in the following table:
[0063]
[0064] As can be seen from the above table, using the analytical method provided by the present invention, the limit of detection concentration of potential mutagenic impurity I is 0.2632 ng / ml, and the limit of quantitation is 0.5264 ng / ml; the limit of detection concentration of mutagenic impurity II is 0.2563 ng / ml, and the limit of quantitation is 0.5127 ng / ml. Calculated according to the concentration of the test sample solution being 0.1 mg / ml, the sensitivity is relatively high, and all meet the detection requirements.
[0065] Example 3 Linearity investigation.
[0066] The linear solutions are prepared as follows:
[0067] Quantification limit solution: Prepared according to the preparation method of the quantification limit solution in Example 2;
[0068] 50% linear solution: Take the reference substance stock solution (0.5 ml) in Example 1 and diluent (9.5 ml) to prepare a linear solution (10 ml);
[0069] 100% linear solution: Take the reference substance stock solution (1.0 ml) in Example 1 and diluent (9.0 ml) to prepare a linear solution (10 ml);
[0070] 150% linear solution: Take the reference substance stock solution (1.5 ml) in Example 1 and diluent (8.5 ml) to prepare a linear solution (10 ml);
[0071] 300% linear solution: Take the reference substance stock solution (3.0 ml) in Example 1 and diluent (7.0 ml) to prepare a linear solution (10 ml);
[0072] Respectively take 10 μL of linear solutions at different levels and inject them into the liquid chromatography - mass spectrometry instrument, record the chromatogram, and the linear detection results are shown in the following table:
[0073]
[0074] As can be seen from the above table, for potential mutagenic impurity I, in the concentration range of 0.5264 - 31.5819 ng / ml, as Figure 7 shown, the concentration of impurity I has a linear relationship with the peak area, and the linear relationship is good. The linear regression equation is y = 626.9490x - 163.0934 (correlation coefficient r = 0.9998), where x is the concentration of impurity I and y is the peak area; for potential mutagenic impurity II, in the concentration range of 0.5127 - 30.7600 ng / ml, as Figure 8 shown, the concentration of impurity II has a linear relationship with the peak area, and the linear relationship is good. The linear regression equation is: y = 3768.1463x - 1248.6529 (correlation coefficient r = 0.9999), where x is the concentration of impurity II and y is the peak area.
[0075] Example 4 Recovery rate investigation.
[0076] Reference substance solution: Prepared according to the preparation method of the reference substance solution in Example 1.
[0077] Test solution: Prepared according to the preparation method of the test solution in Example 1.
[0078] 50% spiked solution: Weigh 1.0 ml of the test sample stock solution and 0.25 ml of the reference substance stock solution under Example 1, add them to a 5-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the 50% spiked solution.
[0079] 100% spiked solution: Weigh 1.0 ml of the test sample stock solution and 0.5 ml of the reference substance stock solution under Example 1, add them to a 5-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the 100% spiked solution.
[0080] 150% spiked solution: Weigh 1.0 ml of the test sample stock solution and 0.75 ml of the reference substance stock solution under Example 1, add them to a 5-ml volumetric flask, dilute to the mark with the diluent, and shake well to obtain the 150% spiked solution.
[0081] Measure 10 μL of the reference substance solution, the test sample solution and the spiked solutions at different levels respectively, inject them into the liquid chromatography-mass spectrometry instrument, record the chromatogram, and the test results are as Figure 9 、 Figure 10 and Figure 11 shown. Calculate the recovery rate, and the recovery rate results at each level are shown in the following table:
[0082]
[0083] It can be seen from the above table that for potential mutagenic impurity I and impurity II in the range of 50% - 150%, the average recovery rates are all between 70% - 130%. This result indicates that the analytical method provided by the present invention has good accuracy.
[0084] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims
1. An analytical method for mutagenic impurities in the raw material drug of a small molecule inhibitor of fungal inositol acyltransferase 1, characterized in that, The structural formula of the active pharmaceutical ingredient is as follows: ； The mutagenic impurities include Impurity I and Impurity II. The structural formula of Impurity I is as follows: ； The structural formula of Impurity II is as follows: ； The mutagenic impurities are determined by liquid chromatography - mass spectrometry; Among them, the liquid chromatography includes the following conditions: The chromatographic column is packed with octadecylsilyl silica gel; The mobile phase includes mobile phase A and mobile phase B. The initial volume ratio of mobile phase A is 70% ± 5%, and the initial volume ratio of mobile phase B is 30% ± 5%. Mobile phase A is selected from 0.05% - 0.2% formic acid aqueous solution, and mobile phase B is selected from acetonitrile or methanol; The elution method is gradient elution; The flow rate is 0.8 mL / min ± 0.5 mL / min; During the gradient elution process, the volume ratio of mobile phase A and mobile phase B is as follows: from 0 min to the 10th min, it is constant, mobile phase A remains at 70%, and mobile phase B remains at 30%; then it changes uniformly to the 14th min, mobile phase A is 10%, and mobile phase B is 90%; from the 14th min to the 16th min, it is constant, mobile phase A remains at 10%, and mobile phase B remains at 90%; from the 16th min to the 16.1th min, mobile phase A changes from 10% to 70%, and mobile phase B changes from 90% to 30%; from the 16.1th min to the 18th min, it is constant, mobile phase A remains at 70%, and mobile phase B remains at 30%; The mass spectrometry includes the following conditions: The scanning mode is selected from the selected reaction monitoring mode, the ionization mode is the positive ion mode, the ion source is the electrospray ionization source, the collision voltage is 22 V ± 5 V, the quantitative ion mass - to - charge ratio of Impurity I is 232 - 234, and the quantitative ion mass - to - charge ratio of Impurity II is 203 - 205.
2. The analytical method for mutagenic impurities in the raw material drug of the small molecule inhibitor of fungal inositol acyltransferase 1 according to claim 1, characterized in that, The conditions of the liquid chromatography also include the setting range of the column temperature, and the setting range of the column temperature is 35°C ± 5°C.
3. The analytical method for mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor API as described in claim 1, characterized in that, The particle size of the packing material is selected from 3 μm and / or 5 μm.
4. The analytical method for mutagenic impurities in the raw material drug of the small molecule fungal inositol acyltransferase 1 inhibitor as claimed in claim 1, characterized in that, The quantitative ion mass - to - charge ratio of Impurity I is 233.
5. The analytical method for mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor bulk drug as described in claim 1, characterized in that, The quantitative ion mass - to - charge ratio of Impurity II is 204.
6. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor bulk drug as claimed in claim 1, characterized in that, The limit of quantitation concentration of Impurity I is not higher than 0.53 ng / ml, and the limit of detection concentration of Impurity I is not higher than 0.27 ng / ml.
7. The analytical method for mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor bulk drug as described in claim 1, wherein The limit of quantitation concentration of Impurity II is not higher than 0.52 ng / ml, and the limit of detection concentration of Impurity II is not higher than 0.26 ng / ml.
Citation Information
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