A method for detecting dichloromethyl methyl carbonate as an impurity in mapacixavir raw materials

Abstract

The present invention provides a method for detecting dichloromethyl methyl carbonate, an impurity in mapasixavir API, and relates to the technical field of pharmaceutical analysis. The method comprises preparing a reference solution of dichloromethyl methyl carbonate and a test solution of the mapasixavir API; wherein the solvent in the reference solution and the test solution is selected from a DMSO reagent containing sulfuric acid; and testing the reference solution and the test solution using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method employs gas headspace-mass spectrometry (HS-GC-MS) to determine dichloromethyl methyl carbonate, and is characterized by rapidity, accuracy, good reproducibility, and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting dichloromethyl methyl carbonate as an impurity in a mapasixavir bulk drug. Background Art

[0002] Mapacisavir is an innovative Class 1 anti-influenza drug and a new cap-dependent endonuclease inhibitor that can effectively block viral replication and transmission. It has the characteristics of rapid onset, long-term viral inhibition, good tolerance, and oral administration is not affected by food. It can effectively inhibit influenza A and B viruses at the same time.

[0003] The main API (TG-1000) in mapasixavir is ([[1'-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiazol-11yl]-1',2',4',6'-tetrahydro-4',6'-dioxospiro[cyclopropane-1,3'-[3H]pyrido[1,2-b]pyridazine]-5'-yl]oxy]methyl carbonate), the structure of which is shown below:

[0004] .

[0005] Among them, dichloromethyl methyl carbonate is an impurity that may be introduced during the synthesis of mapasixavir API, as shown below:

[0006] .

[0007] Since the main raw material (TG-1000) ([[1'-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiophen-11yl]-1',2',4',6'-tetrahydro-4',6'-dioxospiro[cyclopropane-1,3'-[3H]pyrido[1,2-b]pyridazine]-5'-yl]oxy]methyl carbonate) and its impurity dichloromethyl methyl carbonate (TGB01) are both new compounds, there are currently no related literature and patent reports on detection.

[0008] Therefore, how to develop a quantitative detection method for the impurity dichloromethyl methyl carbonate in mapacixavir raw materials has become an urgent problem to be solved.

[0009] In view of this, the present invention is proposed. Summary of the Invention

[0010] The present invention aims to provide a method for detecting dichloromethyl methyl carbonate, an impurity in the mapacixavir API. This method utilizes gas phase headspace-mass spectrometry (HS-GC-MS) to determine dichloromethyl methyl carbonate, establishing a novel detection method characterized by rapidity, accuracy, good reproducibility, and high sensitivity.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0012] The present invention provides a method for detecting dichloromethyl methyl carbonate as an impurity in a mapacixavir bulk drug, the detection method comprising:

[0013] A reference solution of dichloromethyl methyl carbonate and a test solution of mapasixavir API are prepared; wherein the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid;

[0014] The reference solution and the test solution were detected by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate, an impurity in the mapasixavir raw material.

[0015] Furthermore, the concentration of dichloromethyl methyl carbonate in the reference solution is 25-75 μg / L.

[0016] Furthermore, the concentration of the mapasixavir raw material in the test solution is 150~400 mg / mL.

[0017] Furthermore, the concentration of sulfuric acid in the DMSO reagent containing sulfuric acid is 0.2-0.6 mol / L.

[0018] Furthermore, the detection chromatographic column includes a WAX polar chromatographic column.

[0019] Furthermore, the WAX ​​polar chromatography column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX.

[0020] Furthermore, the specifications of the Agilent DB-HeavyWAX are 30 m×0.32 mm×0.25 μm.

[0021] Furthermore, the column flow mode of the detection is a constant flow mode.

[0022] Furthermore, the column flow rate of the detection is 2.4~2.6 mL / min.

[0023] Furthermore, the injection volume of the detection is 1000~2000 μL.

[0024] Furthermore, the carrier gas for detection is helium.

[0025] Furthermore, the injection mode of the detection is split injection, and the split ratio is (5~20):1.

[0026] Furthermore, the temperature rising program of the detection is:

[0027] First, maintain at 55~65℃ for 1~5 min; then increase the temperature to 155~165℃ at a rate of 5~15℃ / min and maintain for 1~5 min; finally, increase the temperature to 235~245℃ at a rate of 25~35℃ / min and maintain for 1~10 min.

[0028] Furthermore, the headspace parameters during the detection process include:

[0029] The box temperature is 80~100℃; the quantitative loop temperature is 110~130℃; the transfer line temperature is 120~140℃; the equilibration time is 20~40 min; and the GC cycle time is 30~40 min.

[0030] Furthermore, the mass spectrometry parameters in the detection process include:

[0031] The ion source was EI; the ion source temperature was 240-260°C; the interface temperature was 240-260°C; the solvent delay time was 0-3 min; the scan time was 3-7.5 min; and the detector voltage was relative tuning voltage ±0.1 kV.

[0032] The scanning mode was selected ion monitoring mode, and the selected ions SIM were: m / z = 79, 83, 85, 113, 115; among them, 79 was the quantitative ion.

[0033] Furthermore, the detection includes:

[0034] Headspace gas chromatography-mass spectrometry was used to detect reference solutions of dichloromethyl methyl carbonate at different concentrations, and chromatograms of the reference solutions at different concentrations were obtained. Standard curves were then drawn based on the peak areas and the concentrations of the reference solutions.

[0035] The test solution of the mapasixavir bulk drug was detected by headspace gas chromatography-mass spectrometry to obtain a chromatogram of the test solution, and then the content of the impurity dichloromethyl methyl carbonate in the mapasixavir bulk drug was calculated using the standard curve.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The API (TG-1000) has a high boiling point and is difficult to volatilize. Direct injection of TG-1000 will enrich it in the injection port and chromatographic column, affecting the response of the impurity TGB01 and resulting in unstable accuracy of TGB01. In addition, the boiling point of TGB01 is relatively low, which is quite different from that of the API (TG-1000). The method of the present invention uses gas phase headspace-mass spectrometry (HS-GC-MS) with programmed temperature and selected ion monitoring mode for determination, avoiding the risk of high boiling point substances enriching and contaminating the injection port and having good repeatability.

[0038] (2) The API (TG-1000) is weakly alkaline and reacts with TGB01 at high temperatures, affecting the accuracy of detecting the impurity TGB01. The method of the present invention uses a dissolution method containing sulfuric acid in DMSO reagent to determine the impurity dichloromethyl methyl carbonate (TGB01) in the API (TG-1000), which has the advantages of simple pretreatment, easy operation, and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 The chromatogram of the reference solution (SST) obtained by the detection method provided in Example 1 is shown.

[0041] Figure 2 The chromatogram of the blank solution obtained by the detection method is provided for Example 1.

[0042] Figure 3 The chromatogram of the spiked test solution obtained by the detection method provided in Example 1.

[0043] Figure 4 The chromatogram of the quantitative limit solution obtained by the detection method provided in Example 1 is shown.

[0044] Figure 5 The linear spectrum plotted for test example 3 using the detection method of embodiment 1.

[0045] Figure 6 The chromatogram of the reference solution (SST) obtained by the detection method is provided for Comparative Example 1.

[0046] Figure 7 The chromatogram of the blank solution obtained by the detection method is provided for Comparative Example 1.

[0047] Figure 8 The chromatogram of the spiked test solution obtained by the detection method provided in Comparative Example 1 is shown.

[0048] Figure 9 The chromatogram of the quantitative limit solution obtained by the detection method is provided for Comparative Example 1.

[0049] Figure 10 The linear spectrum plotted for Test Example 11 using the detection method of Comparative Example 1.

[0050] Figure 11 The chromatogram of the reference solution (SST) obtained by the detection method is provided for Comparative Example 2.

[0051] Figure 12 The chromatogram of the blank solution obtained by the detection method is provided for Comparative Example 2.

[0052] Figure 13 The chromatogram of the spiked test solution obtained by the detection method is provided for Comparative Example 2.

[0053] Figure 14 The chromatogram of the quantitative limit solution obtained by the detection method is provided for Comparative Example 2.

[0054] Figure 15 The linear spectrum plotted for Test Example 15 using the detection method of Comparative Example 2.

[0055] Figure 16 The chromatogram of the reference solution (SST) obtained by the detection method is provided for Comparative Example 3.

[0056] Figure 17 The chromatogram of the blank solution obtained by the detection method is provided for Comparative Example 3.

[0057] Figure 18 The chromatogram of the spiked test solution obtained by the detection method is provided for Comparative Example 3.

[0058] Figure 19 The chromatogram of the quantitative limit solution obtained by the detection method is provided for Comparative Example 3.

[0059] Figure 20 The linear spectrum plotted for Test Example 19 using the detection method of Comparative Example 2. DETAILED DESCRIPTION

[0060] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0061] It should be noted that the following description sets forth specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways than those described herein, and those skilled in the art may make similar generalizations without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0062] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0063] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0064] The present invention provides a method for detecting dichloromethyl methyl carbonate as an impurity in a mapacixavir bulk drug, the detection method comprising:

[0065] A reference solution of dichloromethyl methyl carbonate and a test solution of mapasixavir API are prepared; wherein the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid;

[0066] The reference solution and the test solution were detected by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate, an impurity in the mapasixavir raw material.

[0067] Since the API (TG-1000) is weakly alkaline and unstable at high temperatures, the present invention uses DMSO reagent containing sulfuric acid (a non-volatile acid) as a solvent to prepare a reference solution of dichloromethyl methyl carbonate and a test solution of mapasixavir API to stabilize the API (TG-1000) and prevent direct heating degradation or side reactions that affect the response of the impurity dichloromethyl methyl carbonate (TGB01). This step is simple to operate and has a high impurity accuracy. Furthermore, because the mapacixavir API (TG-1000) has a high boiling point and the impurity dichloromethyl methyl carbonate (TGB01) has a lower boiling point, the present invention utilizes gas phase headspace-mass spectrometry (HS-GC-MS) in programmed temperature and selected ion monitoring mode to determine the presence of the dichloromethyl methyl carbonate impurity in both the reference and test solutions. Due to the temperature difference, the sample solution is heated, causing the dichloromethyl methyl carbonate impurity (TGB01) to evaporate from the sample matrix, reaching equilibrium in the gas-liquid phase. A fixed volume of the gas at the top is then directly extracted for chromatographic analysis. This simple operation effectively avoids the accumulation of the high-boiling-point API (TG-1000) in the inlet and chromatographic column, reducing contamination of the inlet and chromatographic column. This method, therefore, provides a rapid, accurate, reproducible, and highly sensitive method for detecting the dichloromethyl methyl carbonate impurity in the mapacixavir API.

[0068] As an optional embodiment, the method for detecting the impurity dichloromethyl methyl carbonate in the mapasixavir bulk drug comprises the following steps:

[0069] (A) Preparation of reference solution: Dissolve the reference substance dichloromethyl methyl carbonate (TGB01) in DMSO containing sulfuric acid to obtain a TGB01 reference stock solution. Dilute the reference solution with DMSO containing sulfuric acid to a series of reference solutions of different concentrations for later use.

[0070] (B) Prepare the test solution: Dissolve the mapasixavir API (TG-1000) in DMSO containing sulfuric acid to obtain the TG-1000 test solution, which is then set aside.

[0071] (C) The reference solution and the test solution were respectively transferred to a gas chromatography (headspace)-mass spectrometer, and a mass spectrum of TGB01 in the test sample was obtained using programmed temperature and selected ion scanning mode (SIM).

[0072] As an optional embodiment, the concentration of dichloromethyl methyl carbonate in the reference solution is 25~75 μg / L, for example, it can be 25 μg / L, 30 μg / L, 35 μg / L, 40 μg / L, 45 μg / L, 50 μg / L, 55 μg / L, 60 μg / L, 65 μg / L, 70 μg / L, 75 μg / L, etc.

[0073] As an optional embodiment, the concentration of the mapasixavir raw material in the test solution is 5-15 mg / mL, for example, it can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, etc.

[0074] As an optional embodiment, the concentration of sulfuric acid in the DMSO reagent containing sulfuric acid is 0.3~0.5 mol / L, for example, it can be 0.3 mol / L, 0.32 mol / L, 0.34 mol / L, 0.35 mol / L, 0.36 mol / L, 0.38 mol / L, 0.4 mol / L, 0.42 mol / L, 0.44 mol / L, 0.45 mol / L, 0.46 mol / L, 0.48 mol / L, 0.5 mol / L, etc.

[0075] As an optional embodiment, the detection chromatographic column includes a WAX polar chromatographic column.

[0076] As an optional embodiment, the WAX ​​polar chromatography column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX.

[0077] As an optional embodiment, the WAX ​​polar chromatography column has a specification of 30 m×0.32 mm×0.25 μm.

[0078] As an optional embodiment, the specifications of the Agilent DB-HeavyWAX are 30 m×0.32 mm×0.25 μm.

[0079] As an optional implementation, the column flow mode of the detection is a constant flow mode.

[0080] As an optional embodiment, the detection column flow rate is 2.4~2.6 mL / min, for example, it can be 2.4 mL / min, 2.45 mL / min, 2.5 mL / min, 2.55 mL / min, 2.6 mL / min, etc.

[0081] As an optional embodiment, the injection volume of the detection is 1000~2000 μL, for example, it can be 1000 μL, 1100 μL, 1200 μL, 1300 μL, 1400 μL, 1500 μL, 1600 μL, 1700 μL, 1800 μL, 1900 μL, 2000 μL, etc.

[0082] As an optional implementation, the carrier gas for detection is helium (He).

[0083] As an optional embodiment, the injection mode of the detection is split injection, and the split ratio is (5~20):1, for example, it can be 5:1, 6:1, 7:1, 8:1, 8.5:1, 9:1, 9.5:1, 10:1, 10.5:1, 11:1, 11.5:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, etc.

[0084] As an optional embodiment, the temperature rising program of the detection is:

[0085] First, maintain the temperature at 55-65°C (for example, 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, 65°C, etc.) for 1-5 minutes (for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, etc.);

[0086] Then, the temperature is raised to 155-165°C (for example, 155°C, 156°C, 158°C, 160°C, 162°C, 164°C, 165°C, etc.) at a rate of 5-15°C / min (for example, 5°C / min, 6°C / min, 8°C / min, 10°C / min, 12°C / min, 14°C / min, 15°C / min, etc.), and maintained for 1-5 min (for example, 1 min, 2 min, 3 min, 4 min, 5 min, etc.);

[0087] Finally, the temperature is raised to 235-245°C (for example, 23°C, 236°C, 238°C, 240°C, 242°C, 244°C, 345°C, etc.) at a rate of 25-35°C / min (for example, 25°C / min, 26°C / min, 28°C / min, 30°C / min, 32°C / min, 34°C / min, 35°C / min, etc.) and retained for 1-10 min (for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.).

[0088] As an optional embodiment, the temperature rising program of the detection is specifically as follows:

[0089] First, keep at 60℃ for 2 min;

[0090] Then, the temperature was raised to 160°C at a rate of 10°C / min and kept at that temperature for 2 min;

[0091] Finally, the temperature was raised to 240 °C at a rate of 30 °C / min and maintained for 5 min.

[0092] As an optional embodiment, the headspace parameters in the detection process include:

[0093] The box temperature is 80~100℃, for example, it can be 80℃, 82℃, 84℃, 85℃, 86℃, 88℃, 90℃, 92℃, 94℃, 95℃, 96℃, 98℃, 100℃, etc.

[0094] The quantitative loop temperature is 110-130°C, for example, 110°C, 112°C, 114°C, 115°C, 116°C, 118°C, 120°C, 122°C, 124°C, 125°C, 126°C, 128°C, 130°C, etc.

[0095] The transmission line temperature is 120-140°C, for example, 120°C, 122°C, 124°C, 125°C, 126°C, 128°C, 130°C, 132°C, 134°C, 135°C, 136°C, 138°C, 140°C, etc.

[0096] The equilibration time is 20 to 40 min, for example, 20 min, 22 min, 24 min, 25 min, 26 min, 28 min, 30 min, 32 min, 34 min, 35 min, 36 min, 38 min, 40 min, etc.

[0097] The GC cycle time is 30 to 40 min, for example, it can be 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, 36 min, 37 min, 38 min, 39 min, 40 min, etc.

[0098] As an optional embodiment, the headspace parameters in the detection process are specifically:

[0099] The chamber temperature was 90°C; the quantitative loop temperature was 120°C; the transfer line temperature was 130°C; the equilibration time was 30 min; and the GC cycle time was 32 min.

[0100] As an optional embodiment, the mass spectrometry parameters in the detection process include:

[0101] The ion source was EI.

[0102] The ion source temperature is 240-260°C, for example, 240°C, 242°C, 244°C, 245°C, 246°C, 248°C, 250°C, 252°C, 254°C, 255°C, 256°C, 258°C, 260°C, etc.

[0103] The interface temperature is 240~260℃, for example, it can be 240℃, 242℃, 244℃, 245℃, 246℃, 248℃, 250℃, 252℃, 254℃, 255℃, 256℃, 258℃, 260℃, etc.

[0104] The solvent delay time is 0~3 min, for example, it can be 0 min, 0.5 min, 1 min, 2 min, 3 min, etc. (can be adjusted according to the actual situation of the chromatographic column).

[0105] The scanning time is 3 to 7.5 min, for example, it can be 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min, 6 min, 6.5 min, 7 min, 7.5 min, etc. (can be adjusted according to the actual situation of the chromatographic column).

[0106] The detector voltage is ±0.1 kV relative to the tuning voltage.

[0107] The scanning mode was selected ion monitoring mode, and the selected ions SIM were: m / z = 79, 83, 85, 113, 115; among them, 79 was the quantitative ion.

[0108] As an optional embodiment, the mass spectrometry parameters in the detection process are specifically:

[0109] The ion source was EI; the ion source temperature was 250°C; the interface temperature was 250°C; the solvent delay time was 3 min; the scan time was 3–7.5 min; and the detector voltage was relative tuning voltage ±0.1 kV.

[0110] The scanning mode was selected ion monitoring mode, and the selected ions SIM were: m / z = 79, 83, 85, 113, 115; among them, 79 was the quantitative ion.

[0111] As an optional embodiment, the detection includes:

[0112] Headspace gas chromatography-mass spectrometry was used to detect reference solutions of dichloromethyl methyl carbonate at different concentrations, and chromatograms of the reference solutions at different concentrations were obtained. Standard curves were then drawn based on the peak areas and the concentrations of the reference solutions.

[0113] The test solution of the mapasixavir bulk drug was detected by headspace gas chromatography-mass spectrometry to obtain a chromatogram of the test solution, and then the content of the impurity dichloromethyl methyl carbonate in the mapasixavir bulk drug was calculated using the standard curve.

[0114] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0115] In the following examples, the main API of mapasixavir API is referred to as TG-1000; the impurity dichloromethyl methyl carbonate is referred to as TGB01.

[0116] Example 1

[0117] This embodiment provides a method for detecting dichloromethyl methyl carbonate as an impurity in mapacixavir raw materials, and the detection method specifically comprises the following steps:

[0118] (I) Preparation of solution:

[0119] (I-1) Diluent: Take 6.075 mL of 70% sulfuric acid solution and slowly add it to 250 mL of pre-cooled DMSO reagent. Shake well to obtain the diluent.

[0120] (I-2) Blank solution: Take 1 mL of the above diluent, place it in a 20 mL headspace bottle, and seal it.

[0121] (I-3) TGB01 reference substance stock solution ①: Take 21.14 mg of TGB01 reference substance and place it in a 10 mL volumetric flask. Add the above diluent to dissolve it, dilute to the scale, and shake well to obtain the solution (concentration is 1773 μg / mL).

[0122] (I-4) TGB01 reference substance stock solution ②: Take 2820 μL of the above TGB01 reference substance stock solution ① and place it in a 10 mL volumetric flask. Dissolve it in the above diluent and dilute to the mark. Shake well to obtain the solution (concentration is 500.0 μg / mL).

[0123] (I-5) Limit of Detection (LOD), Limit of Quantitation (LOQ), Linearity Solutions (L1-L6), and Reference Standard Solution (SST): Accurately measure an appropriate amount of TGB01 reference standard stock solution ② according to Table 1 below, place it in different volumetric flasks, dilute to the mark with the above diluents, shake well, accurately measure 1 mL of each solution, place it in a 20 mL headspace vial, and seal.

[0124] Table 1

[0125]

[0126] (I-6) Test solution: Take about 200 mg of the test sample mapasixavir raw material and place it in a 20 mL headspace bottle. Accurately add 1 mL of the above diluent to dissolve it (ultrasound can be used). Seal the bottle and shake well to obtain the solution.

[0127] (I-7) Spiked test solution: Take about 200 mg of the test sample mapacixavir raw material, place it in a 20 mL headspace bottle, accurately add 1 mL of the above-mentioned reference solution, seal, and shake to obtain the solution.

[0128] (II) Headspace gas chromatography-mass spectrometry detection:

[0129] The reference solution and the test solution were detected by headspace gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material. The method conditions are as follows:

[0130] Chromatographic column: Agilent DB-HeavyWAX (30 m × 0.32 mm × 0.25 μm).

[0131] Column flow rate: constant flow mode, flow rate 2.5 mL / min.

[0132] Injection volume: 1000 μL.

[0133] Carrier gas: He.

[0134] Injection mode: split mode, split ratio is 10:1.

[0135] Heating program: first maintain at 60℃ for 2 min; then increase the temperature to 160℃ at a rate of 10℃ / min and maintain for 2 min; finally increase the temperature to 240℃ at a rate of 30℃ / min and maintain for 5 min.

[0136] Headspace parameters: chamber temperature, 90°C; quantitative loop temperature, 120°C; transfer line temperature, 130°C; equilibration time, 30 min; GC cycle time, 32 min.

[0137] Mass spectrometry parameters: EI ion source; ion source temperature, 250°C; interface temperature, 250°C; solvent delay time, 3 min*; scan time, 3–7.5 min*; detector voltage, relative tuning voltage ±0.1 kV; scan mode, selected ion monitoring mode, selected ion SIM: m / z = 79 # , 83, 85, 113, 115; among them, 79 is the quantitative ion.

[0138] Note: The * can be adjusted according to the actual situation of the chromatographic column; the # is the quantitative ion.

[0139] (III) Test results:

[0140] The specific typical test results are as follows Figures 1 to 4 As shown, and the specific peak parameters are shown in Table 2 below:

[0141] Table 2

[0142]

[0143] Test Example 1

[0144] This test example provides verification of the system applicability and specificity of the detection method of Example 1.

[0145] (I) Preparation of solution:

[0146] Blank solution: Prepare the same blank solution as in (I-2) in Example 1 above.

[0147] Limit of quantitation solution: Prepared in the same manner as the LOQ solution in (I-5) in Example 1 (numbered L1 / LOQ in Table 1 above).

[0148] Reference solution: Prepared in the same manner as the SST in (I-5) in Example 1 (numbered L4 / SST in Table 1 above).

[0149] The spiked test solution was prepared in the same manner as the spiked test solution in (I-7) in Example 1 above.

[0150] (II) Headspace gas chromatography-mass spectrometry detection:

[0151] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, limit of quantitation solution, reference solution, and spiked test solution were injected as shown in Table 3 below, and chromatograms were recorded.

[0152] Table 3

[0153]

[0154] (III) Test results:

[0155] No TGB01 peak was detected in the blank solution. Six injections of the reference solution revealed a TGB01 peak area with an RSD of 0.89%, less than 15%. The RSDs for the TGB01 peak areas measured in the return solution and those in the first six injections ranged from 1.43% to 6.96%, both less than 15%. The retention times of the TGB01 peaks in the reference solution and the spiked test solution were essentially identical, demonstrating the excellent system suitability and specificity of the detection method described herein.

[0156] Among them, the system suitability test results are shown in Table 4 below:

[0157] Table 4

[0158]

[0159] The system suitability back-needle test results are shown in Table 5 below:

[0160] Table 5

[0161]

[0162] Among them, the specificity test results are shown in Table 6 below:

[0163] Table 6

[0164]

[0165] Test Example 2

[0166] This test example provides verification of the quantitative limit and detection limit of the detection method in Example 1.

[0167] (I) Preparation of solution:

[0168] Blank solution: Prepare the same blank solution as in (I-2) in Example 1 above.

[0169] Detection limit solution: (prepared in the same manner as the LOD in (I-5) in Example 1 above (numbered LOD in Table 1 above).

[0170] Limit of quantitation solution: Prepared in the same manner as the LOQ solution in (I-5) in Example 1 (numbered L1 / LOQ in Table 1 above).

[0171] (II) Headspace gas chromatography-mass spectrometry detection:

[0172] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, quantification limit solution, and detection limit solution were injected as shown in Table 7 below, and chromatograms were recorded.

[0173] Table 7

[0174]

[0175] (III) Test results:

[0176] When the TGB01 concentration was 5.000 μg / mL (equivalent to a test sample content of 0.0025%), the solution was injected continuously for 6 times. The signal-to-noise ratio (S / N) of the TGB01 peak was 16-20, all greater than 10; the RSD of the peak area was 4.44%, less than 15%; and the RSD of the retention time was 0.02%, less than 2%, which was the limit of quantification.

[0177] When the TGB01 concentration was 2.500 μg / mL (equivalent to a test sample content of 0.00125%), the signal-to-noise ratio (S / N) of the TGB01 peak was 12-13, all greater than 3, which was the detection limit.

[0178] Among them, the quantitative limit peak area results are shown in Table 8 below:

[0179] Table 8

[0180]

[0181] Among them, the quantitative limit retention time results are shown in Table 9 below:

[0182] Table 9

[0183]

[0184] Among them, the signal-to-noise ratio results of the quantification limit and detection limit are shown in Table 10 below:

[0185] Table 10

[0186]

[0187] Test Example 3

[0188] This test example provides verification of the linearity and range of the detection method of Example 1.

[0189] (I) Preparation of solution:

[0190] Blank solution: Prepare the same blank solution as in (I-2) in Example 1 above.

[0191] Linear solutions (L1-L6): prepared in the same manner as the linear solutions in (I-5) in Example 1 (numbered L1-L6 in Table 1 above); the linearity of TGB01 in concentration and peak area was examined within the limit concentration range of 10% to 200%.

[0192] (II) Headspace gas chromatography-mass spectrometry detection:

[0193] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, a blank solution and linear solutions L1 to L6 were injected as shown in Table 11 below, and chromatograms were recorded.

[0194] Table 11

[0195]

[0196] (III) Test results:

[0197] TGB01 showed good linearity within the limit concentration range of 10%~200%, with a linear correlation coefficient r of 0.998, which was greater than 0.990. The ratio of the absolute value of the Y-axis intercept to the 100% limit concentration response value was 5%, which was less than 25%, and the residual sum of squares (RSS) was 5.69E+06.

[0198] Among them, the linear test results are as follows Figure 5 As shown in Table 12 below:

[0199] Table 12

[0200]

[0201] Test Example 4

[0202] This test example provides accuracy verification of the detection method of Example 1.

[0203] (I) Preparation of solution:

[0204] Test solution: Prepare the same solution as in (I-6) in Example 1 above. Prepare two replicates. 30% spiked test solution (ACCL): Place approximately 200 mg of the product in a 20 mL headspace vial. Accurately add 1 mL of the L2 linearization solution, seal, and shake to obtain the desired solution. Prepare three replicates.

[0205] 100% spiked test solution (ACCM): Place approximately 200 mg of the product in a 20 mL headspace vial. Accurately add 1 mL of the L4 linearity solution (reference solution), seal, and shake to obtain the solution. Prepare three replicates.

[0206] 150% spiked test solution (ACCH): Place approximately 200 mg of the product in a 20 mL headspace vial. Accurately add 1 mL of the L5 linearity solution, seal, and shake to obtain the solution. Prepare three replicates.

[0207] (II) Headspace gas chromatography-mass spectrometry detection:

[0208] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, test solution, and each spiked test solution were injected as shown in Table 13 below, and chromatograms were recorded.

[0209] Table 13

[0210]

[0211] (III) Test results:

[0212] At the 30%, 100%, and 150% concentration limits, the recoveries of TGB01 ranged from 90% to 109%, all within the 70% to 125% range. The RSDs for the recoveries at each concentration ranged from 0.00% to 1.94%, all less than 15%. This demonstrates the excellent accuracy of the detection method described herein.

[0213] Among them, the test results of the test products are shown in Table 14 below:

[0214] Table 14

[0215]

[0216] The test results of the test products are shown in Table 15 below:

[0217] Table 15

[0218]

[0219] Test Example 5

[0220] This test example provides verification of the precision (repeatability and intermediate precision) of the detection method of Example 1.

[0221] (1) Repeatability:

[0222] (I) Preparation of solution:

[0223] Spiked test solution (ACCM): Prepare in the same manner as the 100% spiked test solution (ACCM) in Test Example 4. Prepare three replicates in parallel, and share the remaining three replicates with the 100% spiked test solution (ACCM) in Test Example 3, for a total of six replicates.

[0224] (II) Headspace gas chromatography-mass spectrometry detection:

[0225] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, test solution, and each spiked test solution were injected as shown in Table 16 below, and chromatograms were recorded.

[0226] Table 16

[0227]

[0228] (III) Test results:

[0229] Analyst 1 measured the RSD of TGB01 content in 6 spiked samples to be 2.06%, which is less than 15%.

[0230] The repeatability test results are shown in Table 17 below:

[0231] Table 17

[0232]

[0233] (2) Intermediate precision:

[0234] (I) Preparation of solution:

[0235] TGB01 reference substance stock solution ①: prepared in the same manner as the TGB01 reference substance stock solution ① provided in Example 1, except that the reference substance sample weight is 28.09 mg;

[0236] TGB01 reference substance stock solution ②: Prepare the same as the TGB01 reference substance stock solution ② provided in Example 1, and transfer the volume of the reference substance stock solution ① of this example to 2123 μL.

[0237] Reference solution (STD), limit of quantification (LOQ), and linear solutions (L1 to L6): prepared as described in Example 1 (I-5).

[0238] Test solution: Prepare the same solution (I-5) as (I-6) provided in Example 1. Prepare two portions in parallel.

[0239] Spiked test solution (ACCM): Prepare the solution in the same manner as the 100% spiked test solution (ACCM) in Test Example 4. Prepare six replicates.

[0240] (II) Headspace gas chromatography-mass spectrometry detection:

[0241] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, reference solution, linearization solution, test solution, and each spiked test solution were injected as shown in Table 18 below, and chromatograms were recorded.

[0242] Table 18

[0243]

[0244] (III) Test results:

[0245] Analyst 2 determined the RSD for TGB01 in six spiked samples to be 1.97%, less than 15%. Combined with the repeatability data, the RSD for TGB01 in 12 spiked samples was 2.63%, less than 20%. This method exhibits good precision.

[0246] Among them, the intermediate precision test results are shown in Table 19 below:

[0247] Table 19

[0248]

[0249] Test Example 6

[0250] This test example provides a solution stability verification for the detection method of Example 1.

[0251] (I) Preparation of solution:

[0252] Reference solution: Used together with the reference solution in Test Example 1.

[0253] Spiked test solution: Prepare the same method as the spiked test solution in Test Example 1. Prepare four replicates in parallel, one of which will be shared with the ACCM-6 spiked test solution in Test Example 5 (I) Repeatability, and one will be shared with the spiked test solution in Test Example 1.

[0254] (II) Headspace gas chromatography-mass spectrometry detection:

[0255] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, the reference and spiked test solutions were injected and analyzed at room temperature at different times. Chromatograms were recorded to examine the stability of the reference and spiked test solutions at different time points.

[0256] (III) Test results:

[0257] When the reference solution was stored at room temperature for 32.5 hours, the ratio of the TGB01 concentration measured to the concentration measured at 0 hours was 83% to 97%, both ranging from 70% to 125%. The reference solution was stable at room temperature for at least 32.5 hours.

[0258] After the spiked test solution was left at room temperature for 29 hours, the ratio of the TGB01 concentration to the concentration at 0 hours was 83% to 94%, both ranging from 70% to 125%. The spiked test solution was stable at room temperature for at least 29 hours.

[0259] Among them, the results of the stability test of the reference solution are shown in Table 20 below:

[0260] Table 20

[0261]

[0262] Among them, the stability test results of the spiked test solution are shown in Table 21 below:

[0263] Table 21

[0264]

[0265] Test Example 7

[0266] This test example provides a method durability verification for the detection method of Example 1.

[0267] (I) Preparation of solution:

[0268] Reference solution: Prepared with the same SST as in (I-5) in Example 1 (number L1 / LOQ in Table 1 above).

[0269] Spiked test solution: Prepare the same spiked test solution as in (I-7) in Example 1. Prepare six replicates in parallel, two of which will be shared with the first two spiked test solutions prepared in Test Example 5-(II) Intermediate Precision.

[0270] (II) Headspace gas chromatography-mass spectrometry detection:

[0271] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for step (II) in Example 1. More specifically, blank solution, reference solution, and spiked test solution were injected under different conditions as shown in Table 22 below, and chromatograms were recorded.

[0272] Table 22

[0273]

[0274] (III) Test results:

[0275] The spiked test solution was measured at flow rates of 2.4 ml / min, 2.5 ml / min, and 2.6 ml / min, respectively. The RSD of the TGB01 content in the spiked test sample measured under each condition was 1.97%, which was less than 20%. This method has good durability.

[0276] Among them, the durability investigation results are shown in Table 23 below:

[0277] Table 23

[0278]

[0279] In summary, the system suitability and specificity, limit of quantification and limit of detection, linearity and range, accuracy, precision (repeatability and intermediate precision), solution stability and method robustness studies provided in Test Examples 1 to 7 above demonstrate that the established gas chromatography (headspace) mass spectrometry method is suitable for the detection of dichloromethyl methyl carbonate, an impurity in mapasixavir API.

[0280] Comparative Example 1

[0281] This comparative example provides a method for detecting dichloromethyl methyl carbonate, an impurity in mapacixavir raw materials, which specifically comprises the following steps:

[0282] (I) Solution preparation: The difference from Example 1 is that the solvent in the reference solution and the test solution is only DMSO reagent (without sulfuric acid), which is more specifically shown below.

[0283] (I-1) Blank solution: Take 1 mL of DMSO, place it in a 20 mL headspace bottle, and seal it.

[0284] (I-2) TGB01 reference substance stock solution ①: Take 17.45 mg of TGB01 reference substance and place it in a 10 mL volumetric flask. Add the above DMSO reagent to dissolve it, dilute to the scale, and shake well to obtain the solution (concentration is 1467 μg / mL).

[0285] (I-3) TGB01 reference substance stock solution ②: Take 3405 μL of the above TGB01 reference substance stock solution ① and place it in a 10 mL volumetric flask. Add the above DMSO reagent to dissolve it, dilute to the scale, and shake well to obtain the solution (concentration is 499.5 μg / mL).

[0286] (I-4) Limit of Quantitation Solution (LOQ), Linearity Solutions (L1-L6), and Reference Standard Solution (SST): Accurately measure an appropriate amount of TGB01 reference standard stock solution ② according to Table 24 below, place it in different volumetric flasks, add the above-mentioned DMSO reagent to dilute to the scale, shake well, accurately measure 1 mL of each solution, place it in a 20 mL headspace vial, and seal.

[0287] Table 24

[0288]

[0289] (I-5) Detection limit solution: Take 300 μL of TGB01 reference solution and place it in a 5 mL volumetric flask. Add DMSO to dilute to the mark, shake well, accurately measure 1 mL and place it in a 20 mL headspace vial. Seal the bottle to obtain the solution (concentration is 2.997 μg / mL).

[0290] (I-6) Test solution: Take about 200 mg of the test sample mapacixavir raw material and place it in a 20 mL headspace bottle. Accurately add 1 mL of the above-mentioned DMSO reagent to dissolve it. Seal the bottle and shake well to obtain the solution.

[0291] (I-7) Spiked test solution:

[0292] 10% spiked test solution: Take about 200 mg of the test sample and place it in a 20 mL headspace vial. Accurately add 1 mL of L1 / LOQ to dissolve it. Seal the bottle and shake well.

[0293] 100% spiked test solution: Take about 200 mg of the test sample and place it in a 20 mL headspace bottle. Accurately add 1 mL of L4 / SST to dissolve it. Seal the bottle and shake well to obtain the solution.

[0294] 150% spiked test solution: Take about 200 mg of the test sample and place it in a 20 mL headspace bottle. Accurately add 1 mL of L5 to dissolve it, seal it, and shake it well.

[0295] (II) Headspace gas chromatography-mass spectrometry detection:

[0296] The reference solution and the test solution were detected by headspace gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material. The method conditions are as follows:

[0297] Chromatographic column: Agilent DB-HeavyWAX (30 m × 0.32 mm × 0.25 μm).

[0298] Column flow rate: constant flow mode, flow rate 2.5 mL / min.

[0299] Injection volume: 1000 μL.

[0300] Carrier gas: He.

[0301] Injection mode: split mode, split ratio is 10:1.

[0302] Heating program: first maintain at 60℃ for 2 min; then increase the temperature to 160℃ at a rate of 10℃ / min and maintain for 2 min; finally increase the temperature to 230℃ at a rate of 30℃ / min and maintain for 5 min.

[0303] Headspace parameters: chamber temperature, 110°C; quantitative loop temperature, 120°C; transfer line temperature, 130°C; equilibration time, 20 min; GC cycle time, 30 min.

[0304] Mass spectrometry parameters: EI ion source; ion source temperature, 250°C; interface temperature, 250°C; solvent delay time, 3 min*; scan time, 3–8 min*; detector voltage, relative tuning voltage ±0.1 kV; scan mode, selected ion monitoring mode; selected ion SIM: m / z = 79, 83, 85, 113, 98, 59 # ; Among them, 59 is the quantitative ion.

[0305] (III) Test results:

[0306] The specific typical test results are as follows Figures 6 to 9 As shown, and the specific peak parameters are shown in Table 25 below:

[0307] Table 25

[0308]

[0309] Test Example 9

[0310] This test example provides system applicability and specificity verification of the detection method of Comparative Example 1.

[0311] (I) Preparation of solution:

[0312] The blank solution, reference solution and spiked test solution were prepared according to the method provided in Comparative Example 1 above.

[0313] (II) Headspace gas chromatography-mass spectrometry detection:

[0314] The reference solution and test solution were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 1. More specifically, a blank solution, a quantitation limit solution, a reference solution, and a spiked test solution were injected as shown in Table 26 below, and chromatograms were recorded.

[0315] Table 26

[0316]

[0317] (III) Test results:

[0318] No TGB01 peak was detected in the blank solution. Three injections of the reference solution revealed a TGB01 peak area with an RSD of 1.30%, less than 15%. The RSDs for the TGB01 peak areas measured in the return solution and those in the first six solutions ranged from 2.16% to 5.45%, all less than 15%. The retention times of the TGB01 peaks in the reference solution and the spiked test solution were essentially identical.

[0319] Among them, the system suitability test results are shown in Table 27 below:

[0320] Table 27

[0321]

[0322] The system suitability back-needle test results are shown in Table 28 below:

[0323] Table 28

[0324]

[0325] Among them, the specificity test results are shown in Table 29 below:

[0326] Table 29

[0327]

[0328] Test Example 10

[0329] This test example provides verification of the quantitative limit and detection limit of the detection method of Comparative Example 1.

[0330] (I) Preparation of solution:

[0331] The blank solution, quantitative limit solution, and detection limit solution were prepared according to the method provided in the above comparative example 1.

[0332] (II) Headspace gas chromatography-mass spectrometry detection:

[0333] The reference solution and the test solution were assayed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 1. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 30 below, and the chromatograms were recorded.

[0334] Table 30

[0335]

[0336] (III) Test results:

[0337] When the TGB01 concentration was 4.995 μg / mL (equivalent to a test sample content of 0.0025%), the solution was injected continuously for 6 times. The signal-to-noise ratio (S / N) of the TGB01 peak was 57-125, all greater than 10. The RSD of the peak area was 8.20%, less than 15%, which was the limit of quantification.

[0338] When the TGB01 concentration was 2.997 μg / mL (equivalent to a test sample content of 0.0015%), the signal-to-noise ratio (S / N) of the TGB01 peak was 62-72, all greater than 3, which was the detection limit.

[0339] Among them, the quantitative limit peak area results are shown in Table 31 below:

[0340] Table 31

[0341]

[0342] Among them, the signal-to-noise ratio results of the quantification limit and detection limit are shown in Table 32 below:

[0343] Table 32

[0344]

[0345] Test Example 11

[0346] This test example provides verification of the linearity and range of the detection method of Comparative Example 1.

[0347] (I) Solution preparation: A linear solution was prepared according to the method provided in Comparative Example 1 above, and the linearity of concentration and peak area of ​​TGB01 within the limit concentration range of 10% to 200% was examined.

[0348] (II) Headspace gas chromatography-mass spectrometry detection:

[0349] The reference and test solutions were analyzed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 1. More specifically, a blank solution and linear solutions L1 to L6 were injected as shown in Table 33 below, and chromatograms were recorded.

[0350] Table 33

[0351]

[0352] (III) Test results:

[0353] TGB01 has a good linear relationship within the limit concentration range of 10% to 200%, and the linear correlation coefficient r is 0.999, which is greater than 0.990. Among them, the linear test results are as follows Figure 10 shown.

[0354] Test Example 12

[0355] This test example provides accuracy verification of the detection method of Comparative Example 1.

[0356] (I) Preparation of solution:

[0357] The blank solution, test solution, and each spiked test solution were prepared according to the method provided in the above comparative example 1.

[0358] (II) Headspace gas chromatography-mass spectrometry detection:

[0359] The reference solution and the test solution were assayed using headspace gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 1. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 34 below, and the chromatograms were recorded.

[0360] Table 34

[0361]

[0362] (III) Test results:

[0363] At the 10%, 100%, and 150% limit concentrations, the recoveries of TGB01 ranged from 0% to 37.4%, all less than 70%. This method has poor accuracy.

[0364] Among them, the test results of the test products are shown in Table 35 below:

[0365] Table 35

[0366]

[0367] Among them, the accuracy results are shown in Table 36 below:

[0368] Table 36

[0369]

[0370] In summary, the system applicability and specificity, limit of quantification and limit of detection, linearity and range, and accuracy studies provided in Test Examples 9 to 12 above indicate that the method provided in Comparative Example 1 has poor accuracy, demonstrating that the established gas chromatography (headspace) mass spectrometry method is not suitable for the detection of dichloromethyl methyl carbonate, an impurity in mapasixavir API, when sulfuric acid is not added to the solvent.

[0371] Comparative Example 2

[0372] This comparative example provides a method for detecting dichloromethyl methyl carbonate, an impurity in mapacixavir raw materials. The detection method adopts liquid sampling and specifically comprises the following steps:

[0373] (I) Preparation of solution:

[0374] (I-1) TGB01 reference substance stock solution ①: Take 28.50 mg of TGB01 reference substance and place it in a 10 mL volumetric flask. Add acetonitrile to dissolve and dilute to the scale. Shake well to obtain the solution (concentration is 2390 μg / mL).

[0375] (I-2) TGB01 reference substance stock solution ②: Take 418 μL of TGB01 reference substance stock solution ① and place it in a 20 mL volumetric flask. Add acetonitrile to dilute to the mark and shake well to obtain the solution (concentration is 49.95 μg / mL).

[0376] (I-3) Limit of Detection (LOD) / Limit of Quantitation (LOQ), Linearity Solutions (L1-L5), and Reference Standard Solution (SST): Accurately measure an appropriate amount of TGB01 reference standard stock solution ② according to Table 37 below, place it in different volumetric flasks, dilute to the mark with acetonitrile, shake well, and accurately measure 1 mL of each solution into a 20 mL headspace vial. Seal the bottle.

[0377] Table 37

[0378]

[0379] (I-4) Test solution: Dissolve approximately 12 mg of the test sample in 1 mL of acetonitrile. Seal the container and shake thoroughly. Prepare two replicates.

[0380] (I-5) Spiked test solution:

[0381] 10% spiked test solution: Dissolve approximately 12 mg of the test sample in 1 mL of L1, seal, and shake to obtain a 10% spiked test solution. Prepare three replicates.

[0382] 50% spiked test solution: Dissolve approximately 12 mg of the test sample in 1 mL of L2, seal, and shake to obtain a 50% spiked test solution. Prepare three replicates.

[0383] 100% spiked test solution: Dissolve approximately 12 mg of the test sample in 1 mL of L3, seal, and shake to obtain a 100% spiked test solution. Prepare three replicates.

[0384] 150% spiked test solution: Dissolve approximately 12 mg of the test sample in 1 mL of L4, seal, and shake to obtain a 150% spiked test solution. Prepare three replicates.

[0385] (II) Gas chromatography-mass spectrometry detection:

[0386] The reference solution and the test solution were detected by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material. The method conditions are as follows:

[0387] Chromatographic column: Agilent DB-HeavyWAX (30 m × 0.32 mm × 0.25 μm).

[0388] Column flow rate: constant flow mode, flow rate 2.0 mL / min.

[0389] Injection volume: 1 μL.

[0390] Carrier gas: He.

[0391] Inlet temperature: 250℃.

[0392] Injection mode: splitless.

[0393] Heating program: first maintain at 50 °C for 3 min, then increase the temperature to 270 °C at a rate of 25 °C / min and maintain for 3 min.

[0394] Mass spectrometry parameters: EI ion source; ion source temperature: 250°C; interface temperature: 250°C; solvent delay time: 4 min; scan time: 4-8 min*; detector voltage: relative tuning voltage ±0.1 kV; scan mode: selected ion monitoring mode, selected ion SIM: m / z = m / z: 79, 83, 85, 59 # ; Among them, 59 is the quantitative ion.

[0395] (III) Test results:

[0396] The specific typical test results are as follows Figures 11 to 14 As shown, and the specific peak parameters are shown in Table 38 below:

[0397] Table 38

[0398]

[0399] Test Example 13

[0400] This test example provides system applicability and specificity verification of the detection method of Comparative Example 2.

[0401] (I) Preparation of solution:

[0402] The blank solution, reference solution and spiked test solution were prepared according to the method provided in Comparative Example 2 above.

[0403] (II) Gas chromatography-mass spectrometry detection:

[0404] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 2. More specifically, a blank solution, a quantitation limit solution, a reference solution, and a spiked test solution were injected as shown in Table 39 below, and chromatograms were recorded.

[0405] Table 39

[0406]

[0407] (III) Test results:

[0408] No TGB01 peak was detected in the blank solution. Six injections of the reference solution revealed a TGB01 peak area with an RSD of 1.41%, less than 15%. The RSDs for the TGB01 peak areas measured in the return solution and those in the first six injections ranged from 2.19% to 3.66%, both less than 15%. The retention times of the TGB01 peaks in the reference solution and the spiked test solution were essentially identical.

[0409] The system suitability test results are shown in Table 40 below:

[0410] Table 40

[0411]

[0412] The system suitability back-needle test results are shown in Table 41 below:

[0413] Table 41

[0414]

[0415] Among them, the specificity test results are shown in Table 42 below:

[0416] Table 42

[0417]

[0418] Test Example 14

[0419] This test example provides verification of the quantitative limit and detection limit of the detection method of Comparative Example 2.

[0420] (I) Preparation of solution:

[0421] The blank solution, quantitative limit solution, and detection limit solution were prepared according to the method provided in the above comparative example 2.

[0422] (II) Gas chromatography-mass spectrometry detection:

[0423] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 2. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 43 below, and the chromatograms were recorded.

[0424] Table 43

[0425]

[0426] (III) Test results:

[0427] When the TGB01 concentration was 0.2997 μg / mL (equivalent to a test sample content of 0.0025%), the solution was injected continuously for 6 injections. The signal-to-noise ratio (S / N) of the TGB01 peak was 44, which was greater than 10 and was the limit of quantification.

[0428] When the TGB01 concentration was 0.1498 μg / mL (equivalent to a test sample content of 0.00125%), the signal-to-noise ratio (S / N) of the TGB01 peak was 16, which was greater than 3 and was the detection limit.

[0429] Among them, the signal-to-noise ratio results of the quantification limit and detection limit are shown in Table 44 below:

[0430] Table 44

[0431]

[0432] Test Example 15

[0433] This test example provides verification of the linearity and range of the detection method of Comparative Example 2.

[0434] (I) Solution Preparation: A linear solution was prepared according to the method provided in Comparative Example 2 above, and the linearity of concentration and peak area of ​​TGB01 within the limit concentration range of 10% to 200% was examined.

[0435] (II) Gas chromatography-mass spectrometry detection:

[0436] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 2. More specifically, a blank solution and the linear solutions L1 to L6 were injected as shown in Table 45 below, and the chromatograms were recorded.

[0437] Table 45

[0438]

[0439] (III) Test results:

[0440] TGB01 has a good linear relationship within the 10%~200% limit concentration range, and the linear correlation coefficient r is 0.996, which is greater than 0.990. The linear test results are as follows Figure 15 shown.

[0441] Test Case 16

[0442] This test example provides accuracy verification of the detection method of Comparative Example 2.

[0443] (I) Preparation of solution:

[0444] The blank solution, test solution, and each spiked test solution were prepared according to the method provided in the above comparative example 2.

[0445] (II) Gas chromatography-mass spectrometry detection:

[0446] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 2. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 46 below, and the chromatograms were recorded.

[0447] Table 46

[0448]

[0449] (III) Test results:

[0450] At the limit concentration levels of 10%, 50%, 100%, and 150%, the recovery of TGB01 was 67.7%~108.2%, the accuracy of 10% spike was less than 70%, and the accuracy of other spike concentrations was between 70% and 120%.

[0451] Among them, the test results of the test products are shown in Table 47 below:

[0452] Table 47

[0453]

[0454] The accuracy test results of each target object are shown in Table 48 below:

[0455] Table 48

[0456]

[0457] In summary, through the system suitability and specificity, quantification limit and detection limit, linearity and range, and accuracy studies provided in the above Test Examples 13 to 16, in the method provided in Comparative Example 2, TGB01 presents a double peak, the response values ​​between the double peaks are mutually converted, the single peak area is unstable and irregular, and combined control is required. During the combined calibration, the system suitability and specificity, quantification limit and detection limit, linearity and range all meet the requirements, but the accuracy is low and the recovery is poor, which proves that the established gas chromatography-mass spectrometry-liquid injection method for detecting the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material on a polar chromatographic column has defects.

[0458] Comparative Example 3

[0459] This comparative example provides a method for detecting dichloromethyl methyl carbonate, an impurity in mapacixavir raw materials, which specifically comprises the following steps:

[0460] (I) Preparation of solution:

[0461] (I-1) Diluent: Take 100 mL each of dichloromethane and ethyl acetate, mix well, and obtain the product.

[0462] (I-2) TGB01 reference substance stock solution ①: Take 12.70 mg of TGB01 reference substance and place it in a 10 mL volumetric flask. Add diluent to dissolve and dilute to the scale. Shake well to obtain the solution (concentration is 1068 μg / mL).

[0463] (I-3) TGB01 reference substance stock solution ②: Take 468 μL of TGB01 reference substance stock solution ① and place it in a 20 mL volumetric flask. Add diluent to dilute to the mark and shake well to obtain the solution (concentration is 19.99 μg / mL).

[0464] (I-4) Limit of Detection (LOD) / Limit of Quantitation (LOQ), Linearity Solutions (L1-L5), and Reference Standard Solution (SST): Accurately measure an appropriate amount of TGB01 reference standard stock solution ② according to Table 49 below, place in different volumetric flasks, dilute to the mark with diluent, and shake well.

[0465] Table 49

[0466]

[0467] (I-5) Test solution: Dissolve the test sample, mapacixavir API, in 1 mL of diluent, seal the container, and shake well. Prepare two replicates.

[0468] (I-6) Spiked test solution:

[0469] 10% spiked test solution: Dissolve approximately 20 mg of the test sample in 1 mL of L1, seal, and shake to obtain a 10% spiked test solution. Prepare three replicates.

[0470] 100% spiked test solution: Dissolve approximately 20 mg of the test sample in 1 mL of L3, seal, and shake to obtain a 100% spiked test solution. Prepare three replicates.

[0471] 150% spiked test solution: Dissolve approximately 20 mg of the test sample in 1 mL of L4, seal, and shake to obtain a 150% spiked test solution. Prepare three replicates.

[0472] (II) Gas chromatography-mass spectrometry detection:

[0473] The reference solution and the test solution were detected by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material. The method conditions are as follows:

[0474] Chromatographic column: Agilent HP-5 (30 m × 0.32 mm × 0.25 μm).

[0475] Column flow rate: constant flow mode, flow rate 2.0 mL / min.

[0476] Injection volume: 1 μL.

[0477] Carrier gas: He.

[0478] Inlet temperature: 280℃.

[0479] Injection mode: splitless.

[0480] Heating program: first maintain at 40 °C for 2 min, then increase the temperature to 60 °C at a rate of 5 °C / min and maintain for 2 min, and finally increase the temperature to 300 °C at a rate of 30 °C / min and maintain for 15 min.

[0481] Mass spectrometry parameters: EI ion source; ion source temperature: 280°C; interface temperature: 280°C; solvent delay time: 3.5 min; scan time: 3.5-10 min*; scan mode: selected ion monitoring mode, selected ion SIM: m / z: 59, 79, 83 # , 85, 99, 105, 113; among them, 83 is the quantitative ion.

[0482] (III) Test results: The target peak TGB01 has severe tailing. The specific typical test results are as follows Figures 16 to 19 Shown: Among them Figure 16 is the chromatogram of the reference solution (SST), Figure 17 is the blank solution chromatogram, Figure 18 The chromatogram of 100% spiked test solution is shown in Figure 2. Figure 19 The chromatogram is the quantification limit solution.

[0483] Test Case 17

[0484] This test example provides system applicability and specificity verification of the detection method of Comparative Example 3.

[0485] (I) Preparation of solution:

[0486] The blank solution, reference solution and spiked test solution were prepared according to the method provided in Comparative Example 3 above.

[0487] (II) Gas chromatography-mass spectrometry detection:

[0488] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 3. More specifically, a blank solution, a quantitation limit solution, a reference solution, and a spiked test solution were injected as shown in the table below. Chromatograms were recorded.

[0489] Table 50

[0490]

[0491] (III) Test results:

[0492] No TGB01 peak was detected in the blank solution. Six injections of the reference solution revealed a TGB01 peak area with an RSD of 2.09%, less than 15%. The RSDs for the TGB01 peak areas measured in the return solution and those in the first six injections ranged from 2.05% to 5.53%, both less than 15%. The retention times of the TGB01 peaks in the reference solution and the spiked test solution were essentially identical.

[0493] Among them, the system suitability test results are shown in Table 51 below:

[0494] Table 51

[0495]

[0496] The system suitability back-needle test results are shown in Table 52 below:

[0497] Table 52

[0498]

[0499] Among them, the specificity test results are shown in Table 53 below:

[0500] Table 53

[0501]

[0502] Test Example 18

[0503] This test example provides verification of the quantitative limit and detection limit of the detection method of Comparative Example 3.

[0504] (I) Preparation of solution:

[0505] The blank solution, quantitative limit solution, and detection limit solution were prepared according to the method provided in the above comparative example 3.

[0506] (II) Gas chromatography-mass spectrometry detection:

[0507] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 3. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 54 below, and the chromatograms were recorded.

[0508] Table 54

[0509]

[0510] (III) Test results:

[0511] When the concentration of TGB01 was 0.25 μg / mL (equivalent to the test sample content of 0.0025%), the solution was injected continuously for 6 times, and the signal-to-noise ratio (S / N) of the TGB01 peak was 62-81, all greater than 10, which was the limit of quantification;

[0512] When the TGB01 concentration was 0.2 μg / mL (equivalent to a test sample content of 0.0010%), the signal-to-noise ratio (S / N) of the TGB01 peak was 22-26, all greater than 3, which was the detection limit.

[0513] Among them, the signal-to-noise ratio results of the quantification limit and detection limit are shown in Table 55 below:

[0514] Table 55

[0515]

[0516] Test Case 19

[0517] This test example provides verification of the linearity and range of the detection method of Comparative Example 3.

[0518] (I) Solution preparation: A linear solution was prepared according to the method provided in Comparative Example 3 above, and the linearity of concentration and peak area of ​​TGB01 within the limit concentration range of 10% to 200% was examined.

[0519] (II) Gas chromatography-mass spectrometry detection:

[0520] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 3. More specifically, a blank solution and linear solutions L1 to L6 were injected as shown in Table 56 below, and the chromatograms were recorded.

[0521] Table 56

[0522]

[0523] (III) Test results:

[0524] TGB01 showed good linear relationship within the limit concentration range of 10%~200%, and the linear correlation coefficient r was 0.999, which was greater than 0.990.

[0525] The linear spectrum is shown below Figure 20 ; The linear test results are shown in Table 57 below:

[0526] Table 57

[0527]

[0528] Test Example 20

[0529] This test example provides accuracy verification of the detection method of Comparative Example 3.

[0530] (I) Preparation of solution:

[0531] The blank solution, test solution, and each spiked test solution were prepared according to the method provided in the above comparative example 3.

[0532] (II) Gas chromatography-mass spectrometry detection:

[0533] The reference solution and the test solution were assayed using gas chromatography-mass spectrometry to determine the content of the dichloromethyl methyl carbonate impurity in the mapasixavir API. The method conditions were identical to those for (II) in Comparative Example 3. More specifically, a blank solution, a quantification limit solution, and a detection limit solution were injected as shown in Table 58 below, and the chromatograms were recorded.

[0534] Table 58

[0535]

[0536] (III) Test results:

[0537] At the limit concentration levels of 10%, 100%, and 150%, the recoveries of TGB01 were 96%~112%, all ranging from 70% to 120%.

[0538] Among them, the test results of the test products are shown in Table 59 below:

[0539] Table 59

[0540]

[0541] Among them, the accuracy results are shown in Table 60 below:

[0542] Table 60

[0543]

[0544] In summary, through the system suitability and specificity, quantification limit and detection limit, linearity and range, and accuracy studies provided in the above-mentioned Test Examples 17 to 20, the method provided in Comparative Example 3, although the system suitability and specificity, quantification limit and detection limit, linearity and range, and accuracy all meet the requirements, the target peak TGB01 has severe tailing, with a tailing factor greater than 2. When a new chromatographic column is used, the tailing factor is not significantly improved. Moreover, as the number of injections increases, the raw material (TG-1000) is enriched at the injection port, and the signal-to-noise ratio of the quantification limit solution gradually decreases, which cannot meet the requirements. This proves that the gas chromatography-mass spectrometry-liquid injection method established on a weakly polar chromatographic column for detecting the content of the impurity dichloromethyl methyl carbonate in the mapasixavir raw material has defects.

[0545] 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 above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting dichloromethyl methyl carbonate as an impurity in mapacixavir bulk drug, characterized in that: The detection method comprises: A reference solution of dichloromethyl methyl carbonate and a test solution of mapasixavir API are prepared; wherein the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid; The reference solution and the test solution were detected by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate, an impurity in the mapasixavir raw material; The detection chromatographic column includes a WAX polar chromatographic column; The injection mode of the detection is split injection, and the split ratio is (5-20):1; The temperature rise program of the detection is: First, maintain at 55-65°C for 1-5 minutes; then increase the temperature to 155-165°C at a rate of 5-15°C / min and maintain for 1-5 minutes; finally, increase the temperature to 235-245°C at a rate of 25-35°C / min and maintain for 1-10 minutes; The mass spectrometry parameters in the detection process include: The ion source was EI; the ion source temperature was 240-260°C; the interface temperature was 240-260°C; the solvent delay time was 0-3 min; the scan time was 3-7.5 min; and the detector voltage was relative tuning voltage ±0.1 kV. The scanning mode was selected ion monitoring mode, and the selected ions SIM were: m / z = 79, 83, 85, 113, 115; among them, 79 was the quantitative ion.

2. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, wherein: The concentration of dichloromethyl methyl carbonate in the reference solution is 25-75 μg / L; And / or, the concentration of mapasixavir API in the test solution is 150-400 mg / mL.

3. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapasixavir bulk drug according to claim 1, wherein: The concentration of sulfuric acid in the DMSO reagent containing sulfuric acid is 0.2-0.6 mol / L.

4. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, wherein: The WAX ​​polar chromatography column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX.

5. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 4, wherein: The specifications of the Agilent DB-HeavyWAX are 30 m×0.32 mm×0.25 μm.

6. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, characterized in that: The column flow mode of the detection is a constant flow mode; and / or, the detection column flow rate is 2.4-2.6 mL / min; And / or, the injection volume of the detection is 1000~2000 μL.

7. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, characterized in that: The carrier gas used in the detection is helium.

8. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, wherein: The headspace parameters during the detection process include: The box temperature is 80~100℃; the quantitative loop temperature is 110~130℃; the transfer line temperature is 120~140℃; the equilibration time is 20~40 min; and the GC cycle time is 30~40 min.

9. The method for detecting dichloromethyl methyl carbonate as an impurity in the mapacixavir bulk drug according to claim 1, characterized in that: The detection includes: Headspace gas chromatography-mass spectrometry was used to detect reference solutions of dichloromethyl methyl carbonate at different concentrations, and chromatograms of the reference solutions at different concentrations were obtained. Standard curves were then drawn based on the peak areas and the concentrations of the reference solutions. The test solution of the mapasixavir bulk drug was detected by headspace gas chromatography-mass spectrometry to obtain a chromatogram of the test solution, and then the content of the impurity dichloromethyl methyl carbonate in the mapasixavir bulk drug was calculated using the standard curve.

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