Analysis method of mutagenic impurities in small molecule fungal inositol acyltransferase 1 inhibitor bulk drug

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor raw material medicine, the analysis method can be used for trace detection of potential mutagenic impurities I and II in the raw material medicine, the limit of quantitation concentration of the impurity I is not higher than 0.53 ng / ml, and the limit of quantitation concentration of the impurity II is not higher than 0.53 ng / ml. The detection limit concentration of the impurity I is not higher than 0.27 ng / ml; the quantitation limit concentration of the impurity II is not higher than 0.52 ng / ml, and the detection limit concentration of the impurity II is not higher than 0.26 ng / ml. The analysis method suitable for the mutagenic impurities in the small-molecule fungal inositol acyltransferase 1 inhibitor bulk drug is high in sensitivity and high in specificity, and the blank solvent does not interfere with detection of the impurities.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor raw material drug. Background Art

[0002] A small molecule fungal inositolacyltransferase 1 (Gwt1) inhibitor mainly inhibits the activity of Gwt1, preventing the synthesis of glycosylphosphatidylinositol (GPI) precursors, thereby affecting the synthesis of GPI-anchored proteins (AP), resulting in the inability of fungal surface mannoproteins to cross-link on 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 a good broad-spectrum antifungal activity, and is not prone to cross-resistance with current clinically used antifungal drugs. Therefore, it has potential for broad clinical application.

[0003] The Gwt1 inhibitor API is referred to as WXSH0102, and its structural formula is as follows: ; In the preparation process of WXSH0102, potential mutagenic impurities Ⅰ and Ⅱ are produced, and their structural formulas are as follows: and ; Whether these two mutagenic impurities can be effectively detected is related to the quality and safety of WXSH0102 API. The maximum daily dose of WXSH0102 does not exceed 600 mg. According to the limit values ​​specified in the guidelines for evaluating and controlling DNA-reactive (mutagenic) impurities in drugs to limit potential carcinogenic risks, the acceptable limits of mutagenic impurities I and II in WXSH0102 API 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 to ensuring the product quality and safety of WXSH0102 API. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the object of the present invention is to provide a method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor raw material.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor API, the structural formula of the API is as follows: ; The mutagenic impurities include impurity I and impurity II, and the structural formula of impurity I is as follows: ; The structural formula of impurity II is shown below: ; The mutagenic impurities are determined by liquid chromatography-mass spectrometry; Among them, the liquid phase includes the following conditions: The chromatographic column is filled with octadecylsilane bonded silica gel; The mobile phase includes mobile phase A and mobile phase B, the initial volume of mobile phase A accounts for 70%±5%, the initial volume of mobile phase B accounts for 30%±5%, the mobile phase A is selected from an acidic aqueous solution, and the mobile phase B is selected from an organic solvent; The elution mode is gradient elution; Flow rate: 0.8 mL / min ± 0.5 mL / min; Mass spectrometry includes the following conditions: The scanning mode was selected from the selective reaction monitoring mode, the ionization mode was the positive ion mode, the ion source was the electrospray ion source, the collision voltage was 22V±5V, the mass-to-charge ratio of the quantitative daughter ion of impurity I was 232-234, and the mass-to-charge ratio of the quantitative daughter ion of impurity II was 203-205.

[0006] Furthermore, the volume proportions of mobile phase A and mobile phase B during gradient elution are as follows: constant from 0 min to 10 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changing uniformly until the 14th min, with mobile phase A at 10% and mobile phase B at 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 16.1 min, mobile phase A changed from 10% to 70%, and mobile phase B changed from 90% to 30%; constant from the 16.1 min to the 18th min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

[0007] Furthermore, the acidic aqueous solution is a 0.05% to 0.2% formic acid aqueous solution, preferably a 0.1% formic acid aqueous solution.

[0008] Furthermore, the organic solvent is selected from acetonitrile or methanol.

[0009] Furthermore, the liquid phase conditions also include a setting range of column temperature, and the setting range of column temperature is 35°C±5°C.

[0010] Furthermore, the particle size of the filler is selected from 3 μm and / or 5 μm, the model of the chromatographic column is preferably ACE 3 C18 or Shimadzu ODS-3 C18, the column length is selected from 150 mm, and the inner diameter of the chromatographic column is selected from 4.6 mm.

[0011] Furthermore, the mass-to-charge ratio of the quantitative daughter ion of impurity I is 233.

[0012] Furthermore, the mass-to-charge ratio of the quantitative daughter ion of impurity II is 204.

[0013] Furthermore, 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.

[0014] Furthermore, 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.

[0015] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor raw material drug, which can be used for trace detection of potential mutagenic impurities I and II in WXSH0102 raw material drug. The analytical method provided by the present invention has high sensitivity, the quantitative limit concentration of impurity I is not higher than 0.53ng / ml, and the detection limit concentration of impurity I is not higher than 0.27ng / ml; the quantitative limit concentration of impurity II is not higher than 0.52ng / ml, and the detection limit concentration of impurity II is not higher than 0.26ng / 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 recovery rate investigation results show that the average recovery rate is between 70% and 130%, which shows that the analytical method provided by the present invention has good accuracy and small systematic error.

[0016] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is a chromatogram of the diluent provided in Example 1 of the present invention.

[0018] Figure 2 It is a chromatogram of the reference solution provided in Example 1 of the present invention.

[0019] Figure 3 It is a chromatogram of the test solution provided in Example 1 of the present invention.

[0020] Figure 4 It is a chromatogram of the test sample spiked solution provided in Example 1 of the present invention.

[0021] Figure 5 It is a chromatogram of the quantitative limit solution provided in Example 2 of the present invention.

[0022] Figure 6 It is a chromatogram of the detection limit solution provided in Example 2 of the present invention.

[0023] Figure 7 This is a graph showing the relationship between the concentration and peak area of ​​impurity I provided in Example 3 of the present invention.

[0024] Figure 8 This is a graph showing the relationship between the concentration and peak area of ​​impurity II provided in Example 3 of the present invention.

[0025] Fig. 9 It is a chromatogram of the 50% level spiked solution provided in Example 4 of the present invention.

[0026] Fig.10 It is a chromatogram of the 100% level spiked solution provided in Example 4 of the present invention.

[0027] Fig.11 It is a chromatogram of the 150% level spiked solution provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection 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.

[0029] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used are all commercially available unless otherwise specified.

[0030] The HPLC-MS conditions are shown in the following table: Example 1 Specificity investigation.

[0031] Preparation and detection of diluent: Measure 300ml of water and add it to the reagent bottle, then add 300ml of acetonitrile and 0.3ml of formic acid, mix well to obtain diluent, take 10μL of diluent to the LC-MS instrument, the chromatogram is as follows Figure 1 shown.

[0032] Preparation of reference substance stock solution: Take about 4 mg of each impurity I and impurity II reference substances, weigh accurately, add to a 10 ml volumetric flask, then add diluent and shake to dissolve impurities I and impurity II, dilute to the scale with diluent, mix evenly, and use as mother solution I of reference substance stock solution; take mother solution I (0.1 ml) of reference substance stock solution and add to a 10 ml volumetric flask, dilute to the scale with diluent, mix evenly, and use as mother solution II of reference substance stock solution; take mother solution II (0.5 ml) of reference substance stock solution and add to a 20 ml volumetric flask, dilute to the scale with diluent, mix evenly, and obtain reference substance stock solution containing 0.1 µg of impurity I and impurity II respectively.

[0033] Preparation and detection of reference solution: Weigh 1.0 ml of reference stock solution, add it to a 10 ml volumetric flask, add diluent (9 ml), shake well to obtain reference solution, take 10 μL of reference solution and inject it into the LC-MS instrument, record the chromatogram, and the result is as follows: Figure 2 shown.

[0034] Preparation of test sample stock solution: Weigh about 5 mg of WXSH0102 raw material, add it into a 10 ml volumetric flask, add diluent to the scale, shake well, and obtain the test sample stock solution.

[0035] Preparation and detection of test solution: Weigh 1.0 ml of the test stock solution, add it to a 5 ml volumetric flask, dilute to the mark with diluent, shake well to obtain the test solution, take 10 μL of the test solution and inject it into the LC-MS instrument, record the chromatogram, and the result is as follows: Figure 3 shown.

[0036] Preparation and detection of test sample spiked solution: Weigh 1.0 ml of test sample stock solution and 0.5 ml of reference sample stock solution, add them into a 5 ml volumetric flask, then add diluent to the mark, shake well to obtain test sample spiked solution, take 10 μL of test sample spiked solution and inject it into the LC-MS instrument, record the chromatogram, and the result is as follows: Figure 4 shown.

[0037] Figures 1 to 4 The test results show that the diluent has no interference with the determination of potential mutagenic impurities Ⅰ and Ⅱ, and there are no adjacent peaks near impurities Ⅰ and Ⅱ in the test solution and the test spiked solution, which meets the requirements.

[0038] Example 2 Investigation of limit of quantitation and limit of detection.

[0039] Quantitative limit solution: Weigh 0.5 ml of the reference solution in Example 1, add it into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0040] Detection limit solution: Weigh 0.25 ml of the reference solution in Example 1, add it into a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain.

[0041] Take 10 μL of the quantitative limit solution and the detection limit solution respectively, inject them into the LC-MS instrument, and record the chromatogram. The results are as follows: Figure 5 , Figure 6 The results of the limit of quantification and limit of detection are shown in the following table:

[0042] As can be seen from the above table, the detection limit concentration of the potential mutagenic impurity I detected by the analytical method provided by the present invention is 0.2632 ng / ml, and the quantification limit is 0.5264 ng / ml; the detection limit concentration of the mutagenic impurity II is 0.2563 ng / ml, and the quantification limit is 0.5127 ng / ml. According to the test solution concentration of 0.1 mg / ml, the sensitivity is relatively high and both meet the detection requirements.

[0043] Example 3 Linear investigation.

[0044] The linearization solution was prepared as follows: Quantitative limit solution: prepared according to the preparation method of the quantitative limit solution in Example 2; 50% linear solution: Prepare a linear solution (10 ml) by taking the reference substance stock solution (0.5 ml) and diluent (9.5 ml) in Example 1; 100% linear solution: Prepare a linear solution (10 ml) by taking the reference substance stock solution (1.0 ml) and diluent (9.0 ml) in Example 1; 150% linear solution: Prepare a linear solution (10 ml) by taking the reference substance stock solution (1.5 ml) and the diluent (8.5 ml) in Example 1; 300% linear solution: Prepare a linear solution (10 ml) by taking the reference substance stock solution (3.0 ml) and diluent (7.0 ml) in Example 1; 10 μL of linear solutions at different levels were measured and injected into the LC-MS instrument, and the chromatograms were recorded. The linear detection results are shown in the following table: From the above table, we can see that the concentration of potential mutagenic impurity I is in the range of 0.5264 to 31.5819 ng / ml. Figure 7As shown, the concentration of impurity Ⅰ is linearly related to 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; the concentration of potential mutagenic impurity Ⅱ is in the range of 0.5127~30.7600ng / ml, as shown in Figure 8 As shown, the concentration of impurity II is linearly related to 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.

[0045] Example 4 Recovery rate investigation.

[0046] Reference solution: Prepare according to the preparation method of the reference solution in Example 1.

[0047] Test solution: Prepare according to the preparation method of the test solution in Example 1.

[0048] 50% level spike solution: weigh 1.0 ml of the test sample stock solution and 0.25 ml of the reference sample stock solution in Example 1, add them into a 5 ml volumetric flask, add diluent to dilute to the scale, shake well, and use this as the 50% level spike solution.

[0049] 100% level spike solution: weigh 1.0 ml of the test sample stock solution and 0.5 ml of the reference sample stock solution in Example 1, add them into a 5 ml volumetric flask, add diluent to dilute to the scale, shake well, and use this as the 100% level spike solution.

[0050] 150% level spike solution: weigh 1.0 ml of the test sample stock solution and 0.75 ml of the reference sample stock solution in Example 1, add them into a 5 ml volumetric flask, add diluent to dilute to the scale, shake well, and use this as the 150% level spike solution.

[0051] Take 10 μL of reference solution, test solution and different levels of spiked solution respectively, inject them into the LC-MS instrument, record the chromatogram, and the test results are as follows: Fig. 9 , Fig.10 and Fig.11 As shown, the recovery rate is calculated, and the recovery rate results at each level are shown in the following table: It can be seen from the above table that the potential mutagenic impurities I and II are in the range of 50% to 150%, and the average recovery rates are between 70% and 130%. This result shows that the analytical method provided by the present invention has good accuracy.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing mutagenic impurities in a small molecule fungal inositol acyltransferase 1 inhibitor raw material, characterized in that: The structural formula of the raw material is as follows: ； The mutagenic impurities include impurity I and impurity II, and the structural formula of impurity I is as follows: ； The structural formula of impurity II is shown below: ； The mutagenic impurities are determined by liquid chromatography-mass spectrometry; Among them, the liquid phase includes the following conditions: The chromatographic column is filled with octadecylsilane bonded silica gel; The mobile phase includes mobile phase A and mobile phase B, the initial volume of mobile phase A accounts for 70%±5%, the initial volume of mobile phase B accounts for 30%±5%, the mobile phase A is selected from an acidic aqueous solution, and the mobile phase B is selected from an organic solvent; The elution mode is gradient elution; Flow rate: 0.8 mL / min ± 0.5 mL / min; Mass spectrometry includes the following conditions: The scanning mode was selected from the selective reaction monitoring mode, the ionization mode was the positive ion mode, the ion source was the electrospray ion source, the collision voltage was 22V±5V, the mass-to-charge ratio of the quantitative daughter ion of impurity I was 232-234, and the mass-to-charge ratio of the quantitative daughter ion of impurity II was 203-205.

2. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The volume proportions of mobile phase A and mobile phase B during gradient elution are as follows: constant from 0 min to 10 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%; then changing at a constant speed until the 14th min, with mobile phase A at 10% and mobile phase B at 90%; constant from 14 min to 16 min, with mobile phase A maintained at 10% and mobile phase B maintained at 90%; from 16 min to 16.1 min, mobile phase A changed from 10% to 70%, and mobile phase B changed from 90% to 30%; constant from 16.1 min to 18 min, with mobile phase A maintained at 70% and mobile phase B maintained at 30%.

3. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The acidic aqueous solution is a 0.05% to 0.2% formic acid aqueous solution.

4. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The organic solvent is selected from acetonitrile or methanol.

5. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The liquid phase conditions also include the setting range of the column temperature, and the setting range of the column temperature is 35°C ± 5°C.

6. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The particle size of the filler is selected from 3 μm and / or 5 μm.

7. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The mass-to-charge ratio of the quantitative daughter ion of impurity Ⅰ is 233.

8. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor bulk drug according to claim 1, characterized in that: The mass-to-charge ratio of the quantitative daughter ion of impurity II is 204.

9. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor raw material drug according to claim 1, characterized in that: The quantitative limit concentration of impurity Ⅰ is not higher than 0.53ng / ml, and the detection limit concentration of impurity Ⅰ is not higher than 0.27ng / ml.

10. The method for analyzing mutagenic impurities in the small molecule fungal inositol acyltransferase 1 inhibitor bulk drug according to claim 1, characterized in that: The quantitative limit concentration of impurity II is not higher than 0.52ng / ml, and the detection limit concentration of impurity II is not higher than 0.26ng / ml.

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