Method for detecting impurity dichloromethyl methyl carbonate in macassvir raw material medicine

Abstract

The invention provides a method for detecting impurity dichloromethyl methyl carbonate in a macassvir raw material medicine, and relates to the technical field of medicine analysis. The detection method comprises the following steps: preparing a reference substance solution of dichloromethyl methyl carbonate and a test solution of a macassvir bulk drug; wherein a solvent in the reference solution and the test solution is selected from a DMSO reagent containing sulfuric acid; and detecting the reference substance solution and the test solution by headspace gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the macassavir bulk drug. According to the detection method disclosed by the invention, the dichloromethyl methyl carbonate is determined by adopting a gas phase headspace-mass spectrometry (HS-GC-MS) method, and the detection method has the characteristics of rapidness, accuracy, good repeatability and high sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting dichloromethyl methyl carbonate, an impurity in the raw material drug of mapracilavir. Background Art

[0002] Mapracilavir is a class 1 innovative anti-influenza drug, a novel cap-dependent endonuclease inhibitor, which can effectively block the replication and transmission of the virus, and has the characteristics of rapid onset, long virus inhibition time, good tolerance, and oral administration not affected by food, and can effectively inhibit both influenza A and B viruses.

[0003] The main raw material drug (TG-1000) in mapracilavir is ([[1'-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl]-1',2',4',6'-tetrahydro-4',6'-dioxospiro[cyclopropane-1,3'-[3H]pyrido[1,2-b]pyridazine]-5'-yl]oxy]methyl carbonate), and the structure is as follows: .

[0004] Among them, dichloromethyl methyl carbonate is an impurity that may be introduced in the synthesis process of the raw material drug of mapracilavir, and it is as follows: .

[0005] Since the main raw material drug (TG-1000) ([[1'-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl]-1',2',4',6'-tetrahydro-4',6'-dioxospiro[cyclopropane-1,3'-[3H]pyrido[1,2-b]pyridazine]-5'-yl]oxy]methyl carbonate) and the impurity dichloromethyl methyl carbonate (TGB01) therein are both novel compounds, there are currently no relevant detection literatures and patent reports.

[0006] Therefore, how to develop a quantitative detection method for dichloromethyl methyl carbonate, an impurity in the raw material drug of mapracilavir, has become an urgent problem to be solved.

[0007] In view of this, the present invention is specifically proposed. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for detecting dichloromethyl methyl carbonate, an impurity in the raw material drug of mapracilavir. The detection method uses headspace gas chromatography-mass spectrometry (HS-GC-MS) to determine dichloromethyl methyl carbonate, and a new detection method is established, which has the characteristics of being fast, accurate, good repeatability, and high sensitivity.

[0009] To achieve the above object of the present invention, the following technical solutions are specifically adopted: The present invention provides a method for detecting dichloromethyl methyl carbonate, an impurity in marpasivir raw material medicine, and the detection method includes: Preparing a reference solution of dichloromethyl methyl carbonate and a test solution of marpasivir raw material medicine; wherein, the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid; Detecting the reference solution and the test solution by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate, an impurity in marpasivir raw material medicine.

[0010] Further, the concentration of dichloromethyl methyl carbonate in the reference solution is 25 - 75 μg / L.

[0011] Further, the concentration of marpasivir raw material medicine in the test solution is 150 - 400 mg / mL.

[0012] Further, the concentration of sulfuric acid in the DMSO reagent containing sulfuric acid is 0.2 - 0.6 mol / L.

[0013] Further, the chromatographic column for detection includes a WAX polar chromatographic column.

[0014] Further, the WAX polar chromatographic column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX.

[0015] Further, the specification of Agilent DB-HeavyWAX is 30 m × 0.32 mm × 0.25 μm.

[0016] Further, the column flow mode for detection is a constant flow mode.

[0017] Further, the column flow rate for detection is 2.4 - 2.6 mL / min.

[0018] Further, the injection volume for detection is 1000 - 2000 μL.

[0019] Further, the carrier gas for detection is helium.

[0020] Further, the injection mode for detection is split injection, and the split ratio is (5 - 20):1.

[0021] Further, the temperature programming for detection is as follows: First, maintain at 55 - 65 °C for 1 - 5 min; then increase the temperature to 155 - 165 °C at a rate of 5 - 15 °C / min and hold for 1 - 5 min; finally, increase the temperature to 235 - 245 °C at a rate of 25 - 35 °C / min and hold for 1 - 10 min.

[0022] Furthermore, the headspace parameters during the detection process include: The oven temperature is 80 - 100 °C; the temperature of the quantitative loop is 110 - 130 °C; the temperature of the transfer line is 120 - 140 °C; the equilibration time is 20 - 40 min; the GC cycle time is 30 - 40 min.

[0023] Furthermore, the mass spectrometry parameters during the detection process include: The ion source is EI; the ion source temperature is 240 - 260 °C; the interface temperature is 240 - 260 °C; the solvent delay time is 0 - 3 min; the scan time is 3 - 7.5 min; the detector voltage is ±0.1 kv relative to the tuning voltage; The scan mode is selected ion monitoring mode, and the selected ions SIM: m / z = 79, 83, 85, 113, 115; among them, 79 is the quantitative ion.

[0024] Furthermore, the detection includes: Use headspace gas chromatography - mass spectrometry to detect the reference substance solutions of dichloromethyl methyl carbonate at different concentrations, respectively obtain the chromatograms of the reference substance solutions at different concentrations, and then draw a standard curve based on the peak area and the concentration of the reference substance solution; Use headspace gas chromatography - mass spectrometry to detect the test solution of mapacitabine raw material drug, obtain the chromatogram of the test solution, and then calculate using the standard curve to obtain the content of the impurity dichloromethyl methyl carbonate in the mapacitabine raw material drug.

[0025] Compared with the prior art, the present invention has the following beneficial effects: (1) The boiling point of the raw material drug (TG - 1000) is relatively high and it is difficult to volatilize. Direct injection of TG - 1000 will be enriched at the injection port and in the chromatographic column, affecting the response of the impurity TGB01, resulting in unstable accuracy of TGB01. Moreover, the boiling point of TGB01 is relatively low and there is a large difference in boiling point from the raw material drug (TG - 1000). The method of the present invention uses gas - phase headspace - mass spectrometry (HS - GC - MS) for determination through programmed temperature rise and selected ion monitoring mode, avoiding the risk of enrichment and contamination of the injection port by high - boiling substances, and having advantages such as good repeatability.

[0026] (2)The active pharmaceutical ingredient (TG-1000) is weakly basic and reacts with TGB01 at high temperatures, affecting the accuracy of detecting the impurity TGB01. The method of the present invention uses a DMSO reagent containing sulfuric acid for dissolution to determine the impurity dichloromethyl methyl carbonate (TGB01) in the marpocasvir active pharmaceutical ingredient (TG-1000), which has the advantages of simple and easy pretreatment and high accuracy. Description of the Drawings

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0028] Figure 1 Chromatogram of the reference solution (SST) obtained by the detection method provided for Example 1.

[0029] Figure 2 Chromatogram of the blank solution obtained by the detection method provided for Example 1.

[0030] Figure 3 Chromatogram of the spiked test solution obtained by the detection method provided for Example 1.

[0031] Figure 4 Chromatogram of the quantitation limit solution obtained by the detection method provided for Example 1.

[0032] Figure 5 Linear spectrum diagram drawn by using the detection method of Example 1 for detection provided in Test Example 3.

[0033] Figure 6 Chromatogram of the reference solution (SST) obtained by the detection method provided for Comparative Example 1.

[0034] Figure 7 Chromatogram of the blank solution obtained by the detection method provided for Comparative Example 1.

[0035] Figure 8 Chromatogram of the spiked test solution obtained by the detection method provided for Comparative Example 1.

[0036] Figure 9 Chromatogram of the quantitation limit solution obtained by the detection method provided for Comparative Example 1.

[0037] Figure 10 Linear spectrum diagram drawn by using the detection method of Comparative Example 1 for detection provided in Test Example 11.

[0038] Figure 11 Chromatogram of the reference solution (SST) obtained by the detection method provided for Comparative Example 2.

[0039] Figure 12 Chromatogram of the blank solution obtained by the detection method provided for Comparative Example 2.

[0040] Figure 13 Chromatogram of the spiked test sample solution obtained by the detection method provided for Comparative Example 2.

[0041] Figure 14 Chromatogram of the quantitation limit solution obtained by the detection method provided for Comparative Example 2.

[0042] Figure 15 Linear spectrum chart drawn by detecting using the detection method of Comparative Example 2 provided for Test Example 15.

[0043] Figure 16 Chromatogram of the reference solution (SST) obtained by the detection method provided for Comparative Example 3.

[0044] Figure 17 Chromatogram of the blank solution obtained by the detection method provided for Comparative Example 3.

[0045] Figure 18 Chromatogram of the spiked test sample solution obtained by the detection method provided for Comparative Example 3.

[0046] Figure 19 Chromatogram of the quantitation limit solution obtained by the detection method provided for Comparative Example 3.

[0047] Figure 20 Linear spectrum chart drawn by detecting using the detection method of Comparative Example 2 provided for Test Example 19. Detailed implementation manners

[0048] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms is non-restrictive.

[0049] It should be noted that specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific implementation manners disclosed below.

[0050] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0051] In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: The present invention provides a method for detecting dichloromethyl methyl carbonate as an impurity in marpocasavir raw material drug, and the detection method includes: Preparing a reference solution of dichloromethyl methyl carbonate and a test solution of marpocasavir raw material drug; wherein, the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid; Detecting the reference solution and the test solution by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate as an impurity in marpocasavir raw material drug.

[0052] Since the raw material drug (TG-1000) has weak basicity and is unstable at high temperature, the present invention uses DMSO reagent containing sulfuric acid (non-volatile acid) as a solvent to prepare a reference solution of dichloromethyl methyl carbonate and a test solution of marpocasavir raw material drug to stabilize the raw material drug (TG-1000), avoiding direct heating degradation or side reactions, thereby affecting the response of dichloromethyl methyl carbonate (TGB01) as an impurity. This step is simple to operate and has high impurity accuracy. At the same time, since the boiling point of marpocasavir raw material drug (TG-1000) is high and the boiling point of dichloromethyl methyl carbonate (TGB01) as an impurity is low, the present invention uses gas phase headspace-mass spectrometry (HS-GC-MS) to measure the reference solution and the test solution by programmed temperature rise and selected ion monitoring mode. Through the temperature difference, the sample solution is heated, and dichloromethyl methyl carbonate (TGB01) as an impurity volatilizes from the sample matrix and reaches equilibrium in the gas-liquid two phases. A fixed volume of the gas part at the top is directly extracted for chromatographic analysis. The operation is simple, and it well avoids the enrichment of the high-boiling raw material drug (TG-1000) at the injection port and in the chromatographic column, reducing the pollution of the injection port and the chromatographic column. Thus, a method for detecting dichloromethyl methyl carbonate as an impurity in marpocasavir raw material drug is established, and this method has the characteristics of being fast, accurate, good repeatability, and high sensitivity.

[0053] As an optional implementation manner, the method for detecting dichloromethyl methyl carbonate as an impurity in marpocasavir raw material drug includes the following steps: (A) Prepare the reference solution: Dissolve the reference dichloromethyl methyl carbonate (TGB01) in DMSO reagent containing sulfuric acid to obtain the TGB01 reference stock solution, and dilute the reference solution with DMSO reagent containing sulfuric acid to a series of reference solutions with different concentrations for standby; (B) Prepare the test solution: Dissolve the raw material of mapracilavir (TG-1000) in DMSO reagent containing sulfuric acid to obtain the TG-1000 test solution for standby; (C) Transfer the reference solution and the test solution to a gas chromatography (headspace)-mass spectrometry instrument respectively, and adopt the programmed temperature rise and selected ion monitoring mode (SIM) to obtain the mass spectrum of TGB01 in the test sample.

[0054] As an optional implementation method, 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.

[0055] As an optional implementation method, the concentration of the raw material of mapracilavir 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.

[0056] As an optional implementation method, 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.

[0057] As an optional implementation method, the chromatographic column for detection includes a WAX polar chromatographic column.

[0058] As an optional implementation method, the WAX polar chromatographic column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX.

[0059] As an alternative embodiment, the specifications of the WAX polar chromatographic column are 30 m × 0.32 mm × 0.25 μm.

[0060] As an alternative embodiment, the specifications of the Agilent DB-HeavyWAX are 30 m × 0.32 mm × 0.25 μm.

[0061] As an alternative embodiment, the column flow mode for the detection is a constant flow mode.

[0062] As an alternative embodiment, the column flow rate for the detection 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.

[0063] As an alternative embodiment, the injection volume for 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.

[0064] As an alternative embodiment, the carrier gas for the detection is helium (He).

[0065] As an alternative embodiment, the injection mode for 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.

[0066] As an alternative embodiment, the temperature programming for the detection is as follows: First, maintain at 55 - 65°C (for example, it can be 55°C, 56°C, 58°C, 60°C, 62°C, 64°C, 65°C, etc.) for 1 - 5 min (for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, etc.); Then heat it up at a rate of 5 to 15 °C / min (for example, it can be 5 °C / min, 6 °C / min, 8 °C / min, 10 °C / min, 12 °C / min, 14 °C / min, 15 °C / min, etc.) to 155 to 165 °C (for example, it can be 155 °C, 156 °C, 158 °C, 160 °C, 162 °C, 164 °C, 165 °C, etc.), and hold for 1 to 5 min (for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, etc.); Finally, heat it up at a rate of 25 to 35 °C / min (for example, it can be 25 °C / min, 26 °C / min, 28 °C / min, 30 °C / min, 32 °C / min, 34 °C / min, 35 °C / min, etc.) to 235 to 245 °C (for example, it can be 23 °C, 236 °C, 238 °C, 240 °C, 242 °C, 244 °C, 345 °C, etc.), and hold for 1 to 10 min (for example, it can be 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.).

[0067] As an optional implementation manner, the heating program for the detection is specifically: First, hold at 60 °C for 2 min; Then heat it up at a rate of 10 °C / min to 160 °C, and hold for 2 min; Finally, heat it up at a rate of 30 °C / min to 240 °C, and hold for 5 min.

[0068] As an optional implementation manner, the headspace parameters during the detection include: The oven temperature is 80 to 100 °C, for example, it can be 80 °C, 82 °C, 84 °C, 85 °C, 86 °C, 88 °C, 90 °C, 92 °C, 94 °C, 95 °C, 96 °C, 98 °C, 100 °C, etc.

[0069] The temperature of the quantitative loop is 110 to 130 °C, for example, it can be 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.

[0070] The temperature of the transfer line is 120 to 140 °C, for example, it can be 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.

[0071] The equilibration time is 20 to 40 min, for example, it can be 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.

[0072] 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.

[0073] As an optional embodiment, the headspace parameters in the detection process are specifically: The oven temperature is 90 °C; the temperature of the quantitative loop is 120 °C; the temperature of the transfer line is 130 °C; the equilibration time is 30 min; the GC cycle time is 32 min.

[0074] As an optional embodiment, the mass spectrometry parameters in the detection process include: The ion source is EI.

[0075] The ion source temperature is 240 to 260 °C, for example, it can be 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.

[0076] The interface temperature is 240 to 260 °C, for example, it can be 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.

[0077] The solvent delay time is 0 to 3 min, for example, it can be 0 min, 0.5 min, 1 min, 2 min, 3 min, etc. (which can be adjusted according to the actual situation of the chromatographic column).

[0078] The scan 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. (which can be adjusted according to the actual situation of the chromatographic column).

[0079] The detector voltage is the relative tuning voltage ±0.1 kv.

[0080] The scanning mode is selected ion monitoring mode, and the selected ions SIM are: m / z = 79, 83, 85, 113, 115; among them, 79 is the quantitative ion.

[0081] As an alternative implementation, the mass spectrometry parameters during the detection are specifically: The ion source is EI; the ion source temperature is 250 °C; the interface temperature is 250 °C; the solvent delay time is 3 min; the scanning time is 3 - 7.5 min; the detector voltage is ±0.1 kv relative to the tuning voltage; The scanning mode is selected ion monitoring mode, and the selected ions SIM are: m / z = 79, 83, 85, 113, 115; among them, 79 is the quantitative ion.

[0082] As an alternative implementation, the detection includes: Using headspace gas chromatography - mass spectrometry to detect the reference substance solutions of dichloromethyl methyl carbonate with different concentrations, obtaining the chromatograms of the reference substance solutions with different concentrations respectively, and then drawing a standard curve based on the peak area and the concentration of the reference substance solution; Using headspace gas chromatography - mass spectrometry to detect the test sample solution of marpociclib API, obtaining the chromatogram of the test sample solution, and then calculating using the standard curve to obtain the content of dichloromethyl methyl carbonate, an impurity in marpociclib API.

[0083] The present invention will be further described below through examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0084] In the following examples, the main API in marpociclib API is abbreviated as TG - 1000; among them, the impurity dichloromethyl methyl carbonate is abbreviated as TGB01.

[0085] Example 1 This example provides a method for detecting dichloromethyl methyl carbonate, an impurity in marpociclib API. The detection method specifically includes the following steps: (I) Preparation of solutions: (I - 1) Diluent: Take 6.075 mL of 70% sulfuric acid solution, slowly add it to 250 mL of pre - cooled DMSO reagent, and shake well to obtain.

[0086] (I - 2) Blank solution: Take 1 mL of the above diluent, place it in a 20 mL headspace vial, and seal it to obtain.

[0087] (I-3)Stock solution ① of reference substance TGB01: Take 21.14 mg of reference substance TGB01, place it in a 10 mL volumetric flask, add the above diluent to dissolve it, and dilute to the mark, shake well, then it is obtained (the concentration is 1773 μg / mL).

[0088] (I-4)Stock solution ② of reference substance TGB01: Take 2820 μL of the above stock solution ① of reference substance TGB01, place it in a 10 mL volumetric flask, add the above diluent to dissolve it, and dilute to the mark, shake well, then it is obtained (the concentration is 500.0 μg / mL).

[0089] (I-5)Limit of detection solution (LOD), limit of quantitation solution (LOQ), linear solutions (L1 - L6), reference solution (SST): Accurately measure an appropriate amount of stock solution ② of reference substance TGB01 according to Table 1 below, place it in different volumetric flasks, add the above diluent to dilute to the mark respectively, shake well, accurately measure 1 mL of each solution and place it in a 20 mL headspace vial, seal it, then it is obtained.

[0090] Table 1

[0091] (I-6)Test solution: Take about 200 mg of the test substance marpaviride API, place it in a 20 mL headspace vial, accurately add 1 mL of the above diluent to dissolve it (ultrasonic treatment can be used), seal it and shake well, then it is obtained.

[0092] (I-7)Spiked test solution: Take about 200 mg of the test substance marpaviride API, place it in a 20 mL headspace vial, accurately add 1 mL of the above reference solution, seal it and shake well, then it is obtained.

[0093] (II)Determination by headspace gas chromatography - mass spectrometry: Use headspace gas chromatography - mass spectrometry to detect the above reference solution and test solution, and obtain the content of impurity dichloromethyl methyl carbonate in marpaviride API; the method conditions are as follows: Chromatographic column: Agilent DB - HeavyWAX (30 m × 0.32 mm × 0.25 μm).

[0094] Column flow rate: Constant flow mode, flow rate 2.5 mL / min.

[0095] Injection volume: 1000 μL.

[0096] Carrier gas: He.

[0097] Injection mode: Split mode, split ratio is 10:1.

[0098] Temperature rising program: First, maintain at 60°C for 2 min; then, increase the temperature to 160°C at a rate of 10°C / min and hold for 2 min; finally, increase the temperature to 240°C at a rate of 30°C / min and hold for 5 min.

[0099] Headspace parameters: Oven temperature is 90°C; Loop temperature is 120°C; Transfer line temperature is 130°C; Equilibration time is 30 min; GC cycle time is 32 min.

[0100] Mass spectrometry parameters: Ion source is EI; Ion source temperature is 250°C; Interface temperature is 250°C; Solvent delay time is 3 min*; Scan time is 3 - 7.5 min*; Detector voltage is relative tuning voltage ±0.1 kV; Scan mode is selected ion monitoring mode, selected ions SIM: m / z = 79 # , 83, 85, 113, 115; among them, 79 is the quantitation ion.

[0101] Note: Those marked with * can be adjusted according to the actual situation of the chromatographic column; those marked with # are quantitation ions.

[0102] (III) Detection results: Specific typical detection results are as follows Figures 1 to 4 as shown, and the specific peak elution parameters are as shown in Table 2 below: Table 2

[0103] Test Example 1 This test example provides the system suitability and specificity verification of the detection method for Example 1.

[0104] (I) Preparation of solutions: Blank solution: Prepared in the same way as the blank solution in (I-2) of Example 1 above.

[0105] Quantitation limit solution: Prepared in the same way as the LOQ in (I-5) of Example 1 above (number L1 / LOQ in Table 1 above).

[0106] Reference solution: Prepared in the same way as the SST in (I-5) of Example 1 above (number L4 / SST in Table 1 above).

[0107] Spiked test sample solution: Prepared in the same way as the spiked test sample solution in (I-7) of Example 1 above.

[0108] (II) Detection by headspace gas chromatography - mass spectrometry: The headspace gas chromatography-mass spectrometry method was used to detect the reference solution and the test solution, and the content of the impurity dichloromethyl methyl carbonate in the mapracilvir API was obtained; the method conditions were exactly the same as the parameters in (II) of Example 1. More specifically, the blank solution, the quantitation limit solution, the reference solution, and the spiked test solution were injected according to Table 3 below, and the chromatograms were recorded.

[0109] Table 3

[0110] (III)Test results: The TGB01 peak was not detected in the blank solution; the RSD of the TGB01 peak area was 0.89% when the reference solution was injected continuously for 6 times, which was less than 15%; the RSD of the TGB01 peak area measured by re-injecting the solution and the corresponding TGB01 peak areas measured by the previous 6 solutions was 1.43% - 6.96%, all of which were less than 15%. The retention times of the TGB01 peak in the reference solution and the spiked test solution were basically the same, which fully demonstrated that the system suitability and specificity of the detection method described in the present invention were good.

[0111] Among them, the system suitability test results are shown in Table 4 below: Table 4

[0112] Among them, the system suitability re-injection test results are shown in Table 5 below: Table 5

[0113] Among them, the specificity test results are shown in Table 6 below: Table 6

[0114] Test Example 2 This test example provides the verification of the quantitation limit and detection limit of the detection method in Example 1.

[0115] (I)Preparation of solutions: Blank solution: Prepared in the same manner as the blank solution in (I-2) of Example 1 above.

[0116] Detection limit solution: (Prepared in the same manner as the LOD preparation in (I-5) of Example 1 above (number LOD in Table 1 above).

[0117] Quantitation limit solution: Prepared in the same manner as the LOQ preparation in (I-5) of Example 1 above (number L1 / LOQ in Table 1 above).

[0118] (II)Detection by headspace gas chromatography-mass spectrometry: The headspace gas chromatography - mass spectrometry method was used to detect the reference substance solution and the test substance solution, and the content of the impurity dichloromethyl methyl carbonate in the mapracilavir raw material was obtained; the method conditions were completely consistent with the parameters in (II) of Example 1. More specifically, the blank solution, the quantitation limit solution, and the detection limit solution were injected according to Table 7 below, and the chromatogram was recorded.

[0119] Table 7

[0120] (III)Detection results: When the concentration of TGB01 was 5.000 μg / mL (equivalent to the test substance content of 0.0025%), the solution was injected continuously for 6 needles. 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%; the RSD of the retention time was 0.02%, less than 2%, which was the quantitation limit. When the concentration of TGB01 was 2.500 μg / mL (equivalent to the test substance 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.

[0121] Among them, the peak area results of the quantitation limit are shown in Table 8 below: Table 8

[0122] Among them, the retention time results of the quantitation limit are shown in Table 9 below: Table 9

[0123] Among them, the signal - to - noise ratio results of the quantitation limit and the detection limit are shown in Table 10 below: Table 10

[0124] Test Example 3 This test example provides the verification of the linearity and range of the detection method in Example 1.

[0125] (I)Preparation of solutions: Blank solution: Prepared in the same way as the blank solution in (I - 2) of Example 1 above.

[0126] Linear solutions (L1 - L6): Prepared in the same way as the linear solutions in (I - 5) of Example 1 above (numbered L1 - L6 in Table 1 above); the linear relationship between the concentration and the peak area of TGB01 was investigated in the range of 10% - 200% of the limit concentration.

[0127] (II)Detection by headspace gas chromatography - mass spectrometry: The headspace gas chromatography-mass spectrometry method was used to detect the reference substance solution and the test substance solution, and the content of the impurity dichloromethyl methyl carbonate in the mapracilavir raw material was obtained; the method conditions were completely consistent with the parameters in (II) of Example 1. More specifically, the blank solution and the linear solutions L1-L6 were taken and injected according to Table 11 below, and the chromatograms were recorded.

[0128] Table 11

[0129] (III)Detection results: TGB01 had a good linear relationship within the limit concentration range of 10% - 200%, the linear correlation coefficient r was 0.998, greater than 0.990, the ratio of the absolute value of the Y-axis intercept to the response value at the 100% limit concentration was 5%, less than 25%, and the residual sum of squares (RSS) was 5.69E+06.

[0130] Among them, the results of the linear test were as follows Figure 5 and shown in Table 12 below: Table 12

[0131] Test Example 4 This test example provides a verification of the accuracy of the detection method in Example 1.

[0132] (I)Preparation of solutions: Test substance solution: Prepared in the same manner as the test substance solution in (I-6) of Example 1 above. Prepared in parallel in 2 portions. 30% spiked test substance solution (ACCL): Take about 200 mg of this product, place it in a 20 mL headspace vial, accurately add 1 mL of the L2 linear solution, seal, and shake well to obtain. Prepared in parallel in 3 portions.

[0133] 100% spiked test substance solution (ACCM): Take about 200 mg of this product, place it in a 20 mL headspace vial, accurately add 1 mL of the L4 linear solution (reference substance solution), seal, and shake well to obtain. Prepared in parallel in 3 portions.

[0134] 150% spiked test substance solution (ACCH): Take about 200 mg of this product, place it in a 20 mL headspace vial, accurately add 1 mL of the L5 linear solution, seal, and shake well to obtain. Prepared in parallel in 3 portions.

[0135] (II)Detection by headspace gas chromatography-mass spectrometry: The headspace gas chromatography-mass spectrometry method was used to detect the reference solution and the test solution, and the content of the impurity dichloromethyl methyl carbonate in the marpaviride raw material drug was obtained; the method conditions were exactly the same as the parameters in (II) of Example 1. More specifically, the blank solution, the test solution, and each spiked test solution were injected according to Table 13 below, and the chromatogram was recorded.

[0136] Table 13

[0137] (III)Detection results: At the 30%, 100%, and 150% limit concentration levels, the recovery rate of TGB01 was 90% - 109%, all within the range of 70% - 125%; the RSD of the recovery rates at each concentration level was 0.00% - 1.94%, all less than 15%. This fully shows that the accuracy of the detection method described in the present invention is good.

[0138] Among them, the test results of the test sample are shown in Table 14 below: Table 14

[0139] Among them, the test results of the test sample are shown in Table 15 below: Table 15

[0140] Test Example 5 This test example provides the verification of the precision (repeatability and intermediate precision) of the detection method in Example 1.

[0141] (I)Repeatability: (I)Preparation of solutions: Spiked test solution (ACCM): Prepared in the same manner as the 100% spiked test solution (ACCM) in Test Example 4. Prepare 3 portions in parallel, and another 3 portions of the solution are shared with the 100% spiked test solution (ACCM) in Test Example 3, for a total of 6 portions.

[0142] (II)Detection by headspace gas chromatography-mass spectrometry: The headspace gas chromatography-mass spectrometry method was used to detect the reference solution and the test solution, and the content of the impurity dichloromethyl methyl carbonate in the marpaviride raw material drug was obtained; the method conditions were exactly the same as the parameters in (II) of Example 1. More specifically, the blank solution, the test solution, and each spiked test solution were injected according to Table 16 below, and the chromatogram was recorded.

[0143] Table 16

[0144] (III)Test Results: The RSD of the content of TGB01 in 6 spiked test samples measured by Analyst 1 was 2.06%, which was less than 15%.

[0145] Among them, the results of the repeatability test are shown in Table 17 below: Table 17

[0146] (II)Intermediate Precision: (I)Preparation of Solutions: Stock solution ① of TGB01 reference substance: Prepared in the same manner as the stock solution ① of TGB01 reference substance provided in Example 1, except that the weighed amount of the reference substance was 28.09 mg; Stock solution ② of TGB01 reference substance: Prepared in the same manner as the stock solution ② of TGB01 reference substance provided in Example 1, and the volume of the stock solution ① of the reference substance in this example taken was 2123 μL.

[0147] Reference solution (STD), limit of quantitation (LOQ), linear solutions (L1 - L6): Prepared in the same manner as (I - 5) provided in Example 1.

[0148] Test sample solution: Prepared in the same manner as the test sample solution (I - 6) provided in Example 1 according to (I - 5). Prepared in parallel for 2 portions.

[0149] Spiked test sample solution (ACCM): Prepared in the same manner as the 100% spiked test sample solution (ACCM) in Test Example 4. Prepared in parallel for 6 portions.

[0150] (II)Detection by Headspace Gas Chromatography - Mass Spectrometry: The reference solution and the test sample solution were detected by headspace gas chromatography - mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracorat raw material; the method conditions were completely consistent with the parameters in (II) of Example 1. More specifically, the blank solution, reference solution, linear solutions, test sample solution, and each spiked test sample solution were injected according to Table 18 below, and the chromatograms were recorded.

[0151] Table 18

[0152] (III)Test Results: The RSD of the content of TGB01 in 6 spiked test samples measured by Analyst 2 was 1.97%, which was less than 15%. Calculated jointly with the repeatability data, the RSD of the content of TGB01 in 12 spiked test samples was 2.63%, which was less than 20%. The precision of this method was good.

[0153] Among them, the results of the intermediate precision test are shown in Table 19 below: Table 19

[0154] Test Example 6 This test example provides verification of the solution stability of the detection method in Example 1.

[0155] (I) Preparation of the solution: Reference solution: Shared with the reference solution in Test Example 1.

[0156] Spiked test sample solution: Prepared in the same way as the spiked test sample solution in Test Example 1. Prepared in parallel for 4 portions, one of which is shared with the ACCM-6 spiked test sample solution in (I) Repeatability of Test Example 5, and one is shared with the spiked test sample solution in Test Example 1.

[0157] (II) Detection by headspace gas chromatography-mass spectrometry: The reference solution and the test sample solution are detected by headspace gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracorat raw material; the method conditions are exactly the same as the parameters in (II) of Example 1. More specifically, the reference solution and the spiked test sample solution are respectively injected for analysis at different times at room temperature, the chromatograms are recorded, and the stability of the reference solution and the spiked test sample solution at different time points is investigated.

[0158] (III) Detection results: The reference solution was placed at room temperature for 32.5 h, and the ratio of the measured amount by TGB01 to the measured amount at 0 h was 83% - 97%, all within the range of 70% - 125%. The reference solution is stable at room temperature for at least 32.5 h.

[0159] The spiked test sample solution was placed at room temperature for 29 h, and the ratio of the measured amount by TGB01 to the measured amount at 0 h was 83% - 94%, all within the range of 70% - 125%. The spiked test sample solution is stable at room temperature for at least 29 h.

[0160] Among them, the stability investigation results of the reference solution are shown in Table 20 below: Table 20

[0161] Among them, the stability investigation results of the spiked test sample solution are shown in Table 21 below: Table 21

[0162] Test Example 7 This test example provides verification of the method durability of the detection method in Example 1.

[0163] (I) Preparation of solutions: Reference solution: Prepared with SST as in (I-5) of Example 1 above (number L1 / LOQ in Table 1 above).

[0164] Spiked test solution: Prepared as in the spiked test solution in (I-7) of Example 1 above. Prepared in parallel in 6 portions, and 2 of them are shared with the first two spiked test solutions prepared in the intermediate precision in Test Example 5-(II).

[0165] (II) Detection by headspace gas chromatography-mass spectrometry: The reference solution and the test solution are detected by headspace gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the marpociclib API; the method conditions are exactly the same as the parameters in (II) of Example 1. More specifically, take the blank solution, reference solution, and spiked test solution, inject samples according to Table 22 below under different conditions, and record the chromatograms.

[0166] Table 22

[0167] (III) Detection results: The spiked test solution is measured respectively under the conditions of a flow rate of 2.4 ml / min, 2.5 ml / min, and 2.6 ml / min. The RSD of the content of TGB01 in the spiked test sample measured under each condition is 1.97%, which is less than 20%, indicating that the method has good durability.

[0168] Among them, the results of the durability study are shown in Table 23 below: Table 23

[0169] In summary, through the system suitability and specificity, limit of quantitation and limit of detection, linearity and range, accuracy, precision (repeatability and intermediate precision), solution stability, and method durability studies provided by the above Test Examples 1 to 7, it is proved that the established gas chromatography (headspace) mass spectrometry method is applicable to the detection of the impurity dichloromethyl methyl carbonate in the marpociclib API.

[0170] Comparative Example 1 This comparative example provides a method for detecting the impurity dichloromethyl methyl carbonate in the marpociclib API. The detection method specifically includes the following steps: (I) Preparation of solutions: Different from Example 1 in that the solvents in the reference solution and the test solution are only DMSO reagent (without sulfuric acid), more specifically as follows.

[0171] Blank solution (I-1): Take 1 mL of DMSO and place it in a 20 mL headspace vial. Seal it to obtain the blank solution.

[0172] Reference stock solution ① of TGB01 (I-2): Take 17.45 mg of the TGB01 reference substance and place it in a 10 mL volumetric flask. Add the above DMSO reagent to dissolve it and dilute to the mark. Shake well to obtain the solution (with a concentration of 1467 μg / mL).

[0173] Reference stock solution ② of TGB01 (I-3): Take 3405 μL of the above reference stock solution ① of TGB01 and place it in a 10 mL volumetric flask. Add the above DMSO reagent to dissolve it and dilute to the mark. Shake well to obtain the solution (with a concentration of 499.5 μg / mL).

[0174] Limit of quantitation solution (LOQ), linear solutions (L1 - L6), and reference solution (SST) (I-4): Accurately measure an appropriate amount of the reference stock solution ② of TGB01 according to Table 24 below, place it in different volumetric flasks, add the above DMSO reagent to dilute to the mark respectively, shake well. Accurately measure 1 mL of each solution and place it in a 20 mL headspace vial. Seal it to obtain the solutions.

[0175] Table 24

[0176] Limit of detection solution (I-5): Take 300 μL of the TGB01 reference solution, place it in a 5 mL volumetric flask, dilute to the mark with DMSO, shake well. Accurately measure 1 mL and place it in a 20 mL headspace vial. Seal it to obtain the solution (with a concentration of 2.997 μg / mL).

[0177] Test solution (I-6): Take about 200 mg of the test substance Marpocasavir raw material and place it in a 20 mL headspace vial. Accurately add 1 mL of the above DMSO reagent to dissolve it, seal and shake well to obtain the test solution.

[0178] Spiked test solution (I-7): 10% spiked test solution: Take about 200 mg of the test substance and place it in a 20 mL headspace vial. Accurately add 1 mL of L1 / LOQ to dissolve it, seal and shake well to obtain the 10% spiked test solution.

[0179] 100% spiked test solution: Take about 200 mg of the test substance and place it in a 20 mL headspace vial. Accurately add 1 mL of L4 / SST to dissolve it, seal and shake well to obtain the 100% spiked test solution.

[0180] 150% spiked test solution: Take about 200 mg of the test substance and place it in a 20 mL headspace vial. Accurately add 1 mL of L5 to dissolve it, seal and shake well to obtain the 150% spiked test solution.

[0181] (II) Headspace gas chromatography - mass spectrometry detection: The reference solution and the test solution were detected by headspace gas chromatography - mass spectrometry to obtain the content of dichloromethyl methyl carbonate in the Mapracorat raw material; the method conditions are as follows: Chromatographic column: Agilent DB - HeavyWAX (30 m × 0.32 mm × 0.25 μm).

[0182] Column flow rate: Constant flow mode, flow rate 2.5 mL / min.

[0183] Sample injection volume: 1000 μL.

[0184] Carrier gas: He.

[0185] Injection mode: Split mode, split ratio 10:1.

[0186] Temperature programming: First hold at 60 °C for 2 min; then increase the temperature to 160 °C at a rate of 10 °C / min and hold for 2 min; finally increase the temperature to 230 °C at a rate of 30 °C / min and hold for 5 min.

[0187] Headspace parameters: Oven temperature 110 °C; Quantitative loop temperature 120 °C; Transfer line temperature 130 °C; Equilibration time 20 min; GC cycle time 30 min.

[0188] Mass spectrometry parameters: Ion source is EI; Ion source temperature 250 °C; Interface temperature 250 °C; Solvent delay time 3 min*; Scan time 3 - 8 min*; Detector voltage is relative tuning voltage ±0.1 kv; Scan mode is selected ion monitoring mode, selected ions SIM: m / z = 79, 83, 85, 113, 98, 59 # ; among them, 59 is the quantitative ion.

[0189] (III) Detection results: Specific typical detection results are as follows Figures 6 to 9 shown, and the specific peak - out parameters are as shown in Table 25 below: Table 25

[0190] Test Example 9 This test example provides the system suitability and specificity verification of the detection method of Comparative Example 1.

[0191] (I) Preparation of solutions: The blank solution, reference solution and spiked test solution were prepared according to the method provided in Comparative Example 1 above.

[0192] (II) Detection by headspace gas chromatography - mass spectrometry: 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 marpociclib API; the method conditions were exactly the same as the parameters in (II) of Comparative Example 1. More specifically, the blank solution, the quantitation limit solution, the reference solution, and the spiked test solution were injected according to Table 26 below, and the chromatograms were recorded.

[0193] Table 26

[0194] (III) Detection results: The TGB01 peak was not detected in the blank solution; the RSD of the peak area of TGB01 was 1.30% for 3 consecutive injections of the reference solution, which was less than 15%; the RSD of the peak area of TGB01 measured by re - injecting the solution and the corresponding peak areas of TGB01 measured from the previous 6 solutions was 2.16 - 5.45%, all of which were less than 15%. The retention times of the TGB01 peak in the reference solution and the spiked test solution were basically the same.

[0195] Among them, the results of the system suitability test are shown in Table 27 below: Table 27

[0196] Among them, the results of the system suitability re - injection test are shown in Table 28 below: Table 28

[0197] Among them, the results of the specificity test are shown in Table 29 below: Table 29

[0198] Test Example 10 This test example provides the verification of the quantitation limit and detection limit of the detection method in Comparative Example 1.

[0199] (I) Preparation of solutions: The blank solution, the quantitation limit solution, and the detection limit solution were prepared according to the method provided in Comparative Example 1 above.

[0200] (II) Detection by headspace gas chromatography - mass spectrometry: 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 marpociclib API; the method conditions were exactly the same as the parameters in (II) of Comparative Example 1. More specifically, the blank solution, the quantitation limit solution, and the detection limit solution were injected according to Table 30 below, and the chromatograms were recorded.

[0201] Table 30

[0202] (III)Test results: When the concentration of TGB01 was 4.995 μg / mL (equivalent to 0.0025% of the test sample content), the solution was continuously injected 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 quantitation; When the concentration of TGB01 was 2.997 μg / mL (equivalent to 0.0015% of the test sample content), the signal-to-noise ratio (S / N) of the TGB01 peak was 62 - 72, all greater than 3, which was the limit of detection.

[0203] Among them, the peak area results of the limit of quantitation are shown in Table 31 below: Table 31

[0204] Among them, the signal-to-noise ratio results of the limit of quantitation and the limit of detection are shown in Table 32 below: Table 32

[0205] Test Example 11 This test example provides the verification of the linearity and range of the detection method of Comparative Example 1.

[0206] (I)Preparation of solution: The linear solution was prepared according to the method provided in Comparative Example 1 above, and the linear relationship between the concentration and the peak area of TGB01 was investigated within the limit concentration range of 10% - 200%.

[0207] (II)Detection by headspace gas chromatography - mass spectrometry: 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 mapracorat raw material; the method conditions were exactly the same as the parameters in (II) of Comparative Example 1. More specifically, the blank solution and the linear solutions L1 - L6 were injected according to Table 33 below, and the chromatograms were recorded.

[0208] Table 33

[0209] (III)Test results: TGB01 had a good linear relationship within the limit concentration range of 10% - 200%, and the linear correlation coefficient r was 0.999, greater than 0.990. Among them, the linear test results are as follows Figure 10 shown.

[0210] Test Example 12 This test example provides an accuracy verification of the detection method in Comparative Example 1.

[0211] (I) Preparation of solutions: The blank solution, test solution, and each spiked test solution were prepared according to the method provided in the above Comparative Example 1.

[0212] (II) Detection by headspace gas chromatography - mass spectrometry: 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 marpocasvir raw material. The method conditions were exactly the same as the parameters in (II) of Comparative Example 1. More specifically, the blank solution, the quantitation limit solution, and the detection limit solution were injected according to Table 34 below, and the chromatograms were recorded.

[0213] Table 34

[0214] (III) Detection results: At the 10%, 100%, and 150% limit concentration levels, the recovery rate of TGB01 was 0% - 37.4%, all less than 70%. The accuracy of this method was poor.

[0215] Among them, the test results of the test samples are shown in Table 35 below: Table 35

[0216] Among them, the accuracy results are shown in Table 36 below: Table 36

[0217] In summary, through the system suitability, specificity, quantitation limit and detection limit, linearity and range, and accuracy studies provided by the above Test Examples 9 - 12, it shows that the method provided in Comparative Example 1 has poor accuracy, proving that the established gas chromatography (headspace) mass spectrometry method is not applicable to the detection of the impurity dichloromethyl methyl carbonate in the marpocasvir raw material without adding sulfuric acid in the solvent.

[0218] Comparative Example 2 This comparative example provides a method for detecting the impurity dichloromethyl methyl carbonate in the marpocasvir raw material. The detection method uses liquid injection and specifically includes the following steps: (I) Preparation of solutions: (I-1)Stock solution ① of reference substance TGB01: Take 28.50 mg of reference substance TGB01, place it in a 10 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well, and you will get it (the concentration is 2390 μg / mL).

[0219] (I-2)Stock solution ② of reference substance TGB01: Take 418 μL of stock solution ① of reference substance TGB01, place it in a 20 mL volumetric flask, dilute to the mark with acetonitrile, shake well, and you will get it (the concentration is 49.95 μg / mL).

[0220] (I-3)Limit of detection solution (LOD) / Limit of quantitation solution (LOQ), linear solutions (L1~L5), reference solution (SST): Accurately measure an appropriate amount of stock solution ② of reference substance TGB01 according to Table 37 below, place it in different volumetric flasks, dilute to the mark with acetonitrile, shake well, accurately measure 1 mL of each solution and place it in a 20 mL headspace vial, seal it, and you will get it.

[0221] Table 37

[0222] (I-4)Test solution: Take about 12 mg of the test sample, accurately add 1 mL of acetonitrile to dissolve it, seal it and shake well, and you will get it. Prepare 2 portions in parallel.

[0223] (I-5)Spiked test solution: 10% spiked test solution: Take about 12 mg of the test sample, accurately add 1 mL of L1 to dissolve it, seal it and shake well, and you will get it. Prepare 3 portions in parallel.

[0224] 50% spiked test solution: Take about 12 mg of the test sample, accurately add 1 mL of L2 to dissolve it, seal it and shake well, and you will get it. Prepare 3 portions in parallel.

[0225] 100% spiked test solution: Take about 12 mg of the test sample, accurately add 1 mL of L3 to dissolve it, seal it and shake well, and you will get it. Prepare 3 portions in parallel.

[0226] 150% spiked test solution: Take about 12 mg of the test sample, accurately add 1 mL of L4 to dissolve it, seal it and shake well, and you will get it. Prepare 3 portions in parallel.

[0227] (II)Detection by gas chromatography - mass spectrometry: Detect the reference solution and the test solution by gas chromatography - mass spectrometry to obtain the content of dichloromethyl methyl carbonate in the mapracorat raw material; the method conditions are as follows: Chromatographic column: Agilent DB - HeavyWAX (30m×0.32mm×0.25μm).

[0228] Column flow rate: Constant flow mode, flow rate 2.0 mL / min.

[0229] Sample injection volume: 1 μL.

[0230] Carrier gas: He.

[0231] Injector temperature: 250 °C.

[0232] Injection mode: Splitless.

[0233] Temperature programming: First hold at 50 °C for 3 min, then increase the temperature to 270 °C at a rate of 25 °C / min and hold for 3 min.

[0234] Mass spectrometry parameters: Ion source is EI; ion source temperature is 250 °C; interface temperature is 250 °C; solvent delay time is 4 min; scan time is 4 - 8 min*; detector voltage is relative tuning voltage ±0.1 kv; scan mode is selected ion monitoring mode, selected ions SIM: m / z = m / z: 79, 83, 85, 59 # ; among which, 59 is the quantitative ion.

[0235] (III) Detection results: The specific typical detection results are as follows Figures 11 to 14 as shown, and the specific peak parameters are as shown in Table 38 below: Table 38

[0236] Test Example 13 This test example provides the system suitability and specificity verification of the detection method for Comparative Example 2.

[0237] (I) Preparation of solutions: The blank solution, reference solution, and spiked test sample solution are prepared according to the method provided in Comparative Example 2 above.

[0238] (II) Detection by gas chromatography - mass spectrometry: The reference solution and the test sample solution are detected by gas chromatography - mass spectrometry to obtain the content of dichloromethyl methyl carbonate in the mapracilavir API; the method conditions are exactly the same as the parameters in (II) of Comparative Example 2. More specifically, take the blank solution, limit of quantification solution, reference solution, and spiked test sample solution, inject them according to Table 39 below respectively, and record the chromatogram.

[0239] Table 39

[0240] (III) Detection results: The peak of TGB01 was not detected in the blank solution; the control solution was injected continuously for 6 times, and the RSD of the peak area of TGB01 was 1.41%, less than 15%; the RSD of the peak area of TGB01 measured by re-injecting the solution and the corresponding peak area of TGB01 measured in the previous 6 solutions was 2.19% - 3.66%, all less than 15%. The retention times of the peak of TGB01 in the control solution and the spiked test solution were basically the same.

[0241] Among them, the results of the system suitability test are shown in Table 40 below: Table 40

[0242] Among them, the results of the system suitability re-injection test are shown in Table 41 below: Table 41

[0243] Among them, the results of the specificity test are shown in Table 42 below: Table 42

[0244] Test Example 14 This test example provides the verification of the quantitative limit and detection limit of the detection method in Comparative Example 2.

[0245] (I) Preparation of solutions: The blank solution, the quantitative limit solution, and the detection limit solution were prepared according to the method provided in Comparative Example 2 above.

[0246] (II) Detection by gas chromatography - mass spectrometry: The control solution and the test solution were detected by gas chromatography - mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracilavir raw material; the method conditions were exactly the same as the parameters in (II) of Comparative Example 2. More specifically, the blank solution, the quantitative limit solution, and the detection limit solution were injected respectively according to Table 43 below, and the chromatograms were recorded.

[0247] Table 43

[0248] (III) Detection results: When the concentration of TGB01 was 0.2997 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 peak of TGB01 was 44, greater than 10, which was the quantitative limit; When the concentration of TGB01 was 0.1498 μg / mL (equivalent to the test sample content of 0.00125%), the signal - to - noise ratio (S / N) of the peak of TGB01 was 16, greater than 3, which was the detection limit.

[0249] Among them, the signal-to-noise ratio results of the quantitation limit and detection limit are shown in Table 44 below: Table 44

[0250] Test Example 15 This test example provides the verification of the linearity and range of the detection method for Comparative Example 2.

[0251] (I) Preparation of solutions: Prepare the linear solutions according to the method provided in Comparative Example 2 above, and examine the linear relationship between the concentration and peak area of TGB01 within the limit concentration range of 10% to 200%.

[0252] (II) Detection by gas chromatography-mass spectrometry: Detect the reference substance solution and the test sample solution by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the marpaxavir raw material; the method conditions are exactly the same as the parameters in (II) of Comparative Example 2. More specifically, take the blank solution and the L1-L6 linear solutions, inject samples according to Table 45 below, and record the chromatograms.

[0253] Table 45

[0254] (III) Detection results: TGB01 has a good linear relationship within the limit concentration range of 10% to 200%, and the linear correlation coefficient r is 0.996, which is greater than 0.990. Among them, the linear test results are as follows Figure 15 shown.

[0255] Test Example 16 This test example provides the accuracy verification of the detection method for Comparative Example 2.

[0256] (I) Preparation of solutions: Prepare the blank solution, the test sample solution, and each spiked test sample solution with reference to the method provided in Comparative Example 2 above.

[0257] (II) Detection by gas chromatography-mass spectrometry: Detect the reference substance solution and the test sample solution by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the marpaxavir raw material; the method conditions are exactly the same as the parameters in (II) of Comparative Example 2. More specifically, take the blank solution, the quantitation limit solution, and the detection limit solution, inject samples according to Table 46 below, and record the chromatograms.

[0258] Table 46

[0259] (III) Test Results: At the concentration levels of 10%, 50%, 100%, and 150%, the recovery rate of TGB01 was 67.7% - 108.2%. The accuracy at 10% spiked standard was less than 70%, and the accuracy at other spiked concentrations was between 70% - 120%.

[0260] Among them, the test results of the test samples are shown in Table 47 below: Table 47

[0261] Among them, the accuracy test results of each target are shown in Table 48 below: Table 48

[0262] In summary, through the system suitability, specificity, limit of quantitation and limit of detection, linearity and range, and accuracy studies provided by the above Test Examples 13 - 16, in the method provided in Comparative Example 2, TGB01 presented double peaks, and the response values between the double peaks were mutually transformed. The area of a single peak was unstable and irregular, and combination control was required. When combined calibration was performed, the system suitability, specificity, limit of quantitation and limit of detection, linearity and range, etc. all met the requirements, but the accuracy was at a low level and the recovery was poor, proving that there were defects in the method for detecting the content of dichloromethyl methyl carbonate in Mapracorat raw material drug by gas chromatography - mass spectrometry with liquid injection on a polar chromatographic column.

[0263] Comparative Example 3 This comparative example provides a method for detecting dichloromethyl methyl carbonate in Mapracorat raw material drug. The detection method specifically includes the following steps: (I) Preparation of solutions: (I - 1) Diluent: Take 100 mL each of dichloromethane and ethyl acetate, mix well, and obtain.

[0264] (I - 2) Stock solution ① of TGB01 reference substance: Take 12.70 mg of TGB01 reference substance, place it in a 10 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well to obtain (concentration: 1068 μg / mL).

[0265] (I - 3) Stock solution ② of TGB01 reference substance: Take 468 μL of stock solution ① of TGB01 reference substance, place it in a 20 mL volumetric flask, add diluent to dilute to the mark, and shake well to obtain (concentration: 19.99 μg / mL).

[0266] (I-4) Detection Limit Solution (LOD) / Quantitation Limit Solution (LOQ), Linear Solutions (L1-L5), Reference Solution (SST): Accurately pipette an appropriate amount of the reference stock solution ② of TGB01 according to Table 49 below, place it in different volumetric flasks, dilute to the mark with the diluent, and shake well to obtain.

[0267] Table 49

[0268] (I-5) Test Solution: Take the test substance Mapacilavir API, accurately add 1 mL of the diluent to dissolve it, seal and shake well to obtain. Prepare 2 portions in parallel.

[0269] (I-6) Spiked Test Solution: 10% Spiked Test Solution: Take about 20 mg of the test substance, accurately add 1 mL of L1 to dissolve it, seal and shake well to obtain. Prepare 3 portions in parallel.

[0270] 100% Spiked Test Solution: Take about 20 mg of the test substance, accurately add 1 mL of L3 to dissolve it, seal and shake well to obtain. Prepare 3 portions in parallel.

[0271] 150% Spiked Test Solution: Take about 20 mg of the test substance, accurately add 1 mL of L4 to dissolve it, seal and shake well to obtain. Prepare 3 portions in parallel.

[0272] (II) Detection by Gas Chromatography-Mass Spectrometry: Use gas chromatography-mass spectrometry to detect the reference solution and the test solution to obtain the content of the impurity dichloromethyl methyl carbonate in the Mapacilavir API; the method conditions are as follows: Chromatographic column: Agilent HP-5 (30 m × 0.32 mm × 0.25 μm).

[0273] Column flow rate: Constant flow mode, flow rate 2.0 mL / min.

[0274] Injection volume: 1 μL.

[0275] Carrier gas: He.

[0276] Injection port temperature: 280 °C.

[0277] Injection mode: Splitless.

[0278] Temperature programming: First hold at 40 °C for 2 min, then increase the temperature at a rate of 5 °C / min to 60 °C and hold for 2 min, and finally increase the temperature at a rate of 30 °C / min to 300 °C and hold for 15 min.

[0279] Mass spectrometry parameters: The ion source is EI; the ion source temperature is 280 °C; the interface temperature is 280 °C; the solvent delay time is 3.5 min; the scanning time is 3.5 - 10 min*; the scanning mode is selected ion monitoring mode, and the selected ions SIM: m / z: 59, 79, 83 # , 85, 99, 105, 113; among them, 83 is the quantitation ion.

[0280] (III) Test results: The target peak TGB01 has serious tailing, and 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 chromatogram of the blank solution, Figure 18 is the chromatogram of the 100% spiked test sample solution, Figure 19 is the chromatogram of the quantitation limit solution.

[0281] Test Example 17 This test example provides the system suitability and specificity verification of the detection method for Comparative Example 3.

[0282] (I) Preparation of solutions: The blank solution, reference solution and spiked test sample solution are prepared according to the method provided in Comparative Example 3 above.

[0283] (II) Detection by gas chromatography - mass spectrometry: The reference solution and the test sample solution are detected by gas chromatography - mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracilavir API; the method conditions are exactly the same as the parameters in (II) of Comparative Example 3. More specifically, the blank solution, quantitation limit solution, reference solution, and spiked test sample solution are injected according to Table 50 below, and the chromatograms are recorded.

[0284] Table 50

[0285] (III) Test results: The TGB01 peak was not detected in the blank solution; the reference solution was injected continuously for 6 injections, and the RSD of the TGB01 peak area was 2.09%, less than 15%; the RSD of the TGB01 peak area measured by the re - injected solution and the corresponding TGB01 peak areas measured by the previous 6 solutions was 2.05% - 5.53%, all less than 15%. The retention times of the TGB01 peaks in the reference solution and the spiked test sample solution are basically the same.

[0286] Among them, the system suitability test results are shown in Table 51 below: Table 51

[0287] Among them, the results of the system suitability back needle test are shown in Table 52 below: Table 52

[0288] Among them, the results of the specificity test are shown in Table 53 below: Table 53

[0289] Test Example 18 This test example provides the verification of the quantitative limit and detection limit of the detection method for Comparative Example 3.

[0290] (I) Preparation of solutions: The blank solution, the quantitative limit solution, and the detection limit solution are prepared according to the method provided in the above Comparative Example 3.

[0291] (II) Detection by gas chromatography-mass spectrometry: The reference solution and the test solution are detected by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracilvir raw material; the method conditions are exactly the same as the parameters in (II) of Comparative Example 3. More specifically, the blank solution, the quantitative limit solution, and the detection limit solution are injected respectively according to Table 54 below, and the chromatograms are recorded.

[0292] Table 54

[0293] (III) Detection results: When the concentration of TGB01 is 0.25 μg / mL (equivalent to the test sample content of 0.0025%), the solution is injected continuously for 6 needles, and the signal-to-noise ratio (S / N) of the TGB01 peak is 62 - 81, all greater than 10, which is the quantitative limit; When the concentration of TGB01 is 0.2 μg / mL (equivalent to the test sample content of 0.0010%), the signal-to-noise ratio (S / N) of the TGB01 peak is 22 - 26, all greater than 3, which is the detection limit.

[0294] Among them, the signal-to-noise ratio results of the quantitative limit and detection limit are shown in Table 55 below: Table 55

[0295] Test Example 19 This test example provides the verification of the linearity and range of the detection method for Comparative Example 3.

[0296] (I) Preparation of solutions: Prepare the linear solutions according to the method provided in Comparative Example 3 above, and examine the linear relationship between the concentration and the peak area of TGB01 within the limit concentration range of 10% to 200%.

[0297] (II) Detection by gas chromatography-mass spectrometry: Detect the reference solution and the test solution by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracorat raw material; the method conditions are exactly the same as the parameters in (II) of Comparative Example 3. More specifically, take the blank solution and the linear solutions of L1-L6, inject samples according to Table 56 below, and record the chromatograms.

[0298] Table 56

[0299] (III) Detection results: 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.

[0300] Among them, the linear chromatogram is as follows Figure 20 ; the results of the linear test are shown in Table 57 below: Table 57

[0301] Test Example 20 This test example provides an accuracy verification of the detection method in Comparative Example 3.

[0302] (I) Preparation of solutions: Prepare the blank solution, the test solution, and each spiked test solution with reference to the method provided in Comparative Example 3 above.

[0303] (II) Detection by gas chromatography-mass spectrometry: Detect the reference solution and the test solution by gas chromatography-mass spectrometry to obtain the content of the impurity dichloromethyl methyl carbonate in the mapracorat raw material; the method conditions are exactly the same as the parameters in (II) of Comparative Example 3. More specifically, take the blank solution, the quantitation limit solution, and the detection limit solution, inject samples according to Table 58 below, and record the chromatograms.

[0304] Table 58

[0305] (III) Detection results: At the limit concentration levels of 10%, 100%, and 150%, the recovery rate of TGB01 is 96% to 112%, all within the range of 70% to 120%.

[0306] Among them, the test results of the test samples are shown in Table 59 below: Table 59

[0307] Among them, the accuracy results are shown in Table 60 below: Table 60

[0308] In summary, through the system suitability, specificity, limit of quantitation and limit of detection, linearity and range, and accuracy studies provided by the above Test Examples 17 to 20, in the method provided by Comparative Example 3, although the system suitability, specificity, limit of quantitation and limit of detection, linearity and range, accuracy, etc. all meet the requirements, the target peak TGB01 has serious tailing, the tailing factor is greater than 2. Using a brand-new chromatographic column, the tailing factor is not significantly improved. Moreover, as the number of injections increases, the API (TG-1000) accumulates at the injection port, and the signal-to-noise ratio of the limit of quantitation solution gradually decreases, unable to meet the requirements; it is proved that there are defects in the established gas chromatography-mass spectrometry-liquid injection method for detecting the content of dichloromethyl methyl carbonate in Mapracorat API on a weakly polar chromatographic column.

[0309] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting dichloromethyl methyl carbonate, an impurity in marapavirine raw material drug, which is characterized in that, The detection method includes: Preparing a reference solution of dichloromethyl methyl carbonate and a test solution of mapracorat raw material; wherein, the solvent in the reference solution and the test solution is selected from DMSO reagent containing sulfuric acid; Detecting the reference solution and the test solution by headspace gas chromatography-mass spectrometry to obtain the content of dichloromethyl methyl carbonate in the mapracorat raw material.

2. The detection method of dichloromethyl methyl carbonate, an impurity in marapavirine API, according to claim 1, is characterized in that, The concentration of dichloromethyl methyl carbonate in the reference solution is 25 - 75 μg / L; And / or, the concentration of mapracorat raw material in the test solution is 150 - 400 mg / mL.

3. The detection method of dichloromethyl methyl carbonate, an impurity in marpasivir API according to claim 1, is characterized in that The concentration of sulfuric acid in the DMSO reagent containing sulfuric acid is 0.2 - 0.6 mol / L.

4. The detection method of dichloromethyl methyl carbonate, an impurity in marapavirine API according to claim 1, is characterized in that, The chromatographic column for the detection includes a WAX polar chromatographic column; And / or, the WAX polar chromatographic column includes any one of Agilent DB-HeavyWAX, Agilent DB-WAX, Agilent HP-INNOWAX or SH-WAX; And / or, the specification of the Agilent DB-HeavyWAX is 30 m × 0.32 mm × 0.25 μm.

5. The detection method of dichloromethyl methyl carbonate as an impurity in Mapacitabine API according to claim 1, wherein The column flow mode of the detection is a constant flow mode; And / or, the column flow rate of the detection is 2.4 - 2.6 mL / min; And / or, the injection volume of the detection is 1000 - 2000 μL.

6. The detection method of dichloromethyl methyl carbonate, an impurity in mapracorat raw material drug, according to claim 1, is characterized in that, The carrier gas for the detection is helium; And / or, the injection mode of the detection is split injection, and the split ratio is (5 - 20):

1.

7. The detection method of dichloromethyl methyl carbonate as an impurity in marpavirine raw material drug according to claim 1, wherein, The temperature programming of the detection is as follows: First, hold at 55 - 65°C for 1 - 5 min; then, increase the temperature to 155 - 165°C at a rate of 5 - 15°C / min and hold for 1 - 5 min; finally, increase the temperature to 235 - 245°C at a rate of 25 - 35°C / min and hold for 1 - 10 min.

8. The detection method of dichloromethyl methyl carbonate as an impurity in marpasivir API according to claim 1, wherein The headspace parameters during the detection include: The oven temperature is 80 - 100°C; the quantitative loop temperature is 110 - 130°C; the transfer line temperature is 120 - 140°C; the equilibration time is 20 - 40 min; the GC cycle time is 30 - 40 min.

9. The detection method of dichloromethyl methyl carbonate, an impurity in marpasivir raw material drug, according to claim 1, is characterized in that, The mass spectrometry parameters during the detection include: The ion source is EI; the ion source temperature is 240 - 260°C; the interface temperature is 240 - 260°C; the solvent delay time is 0 - 3 min; the scan time is 3 - 7.5 min; the detector voltage is ±0.1 kv relative to the tuning voltage; The scan mode is selected ion monitoring mode, and the selected ions SIM: m / z = 79, 83, 85, 113, 115; where 79 is the quantitative ion.

10. The detection method of dichloromethyl methyl carbonate as an impurity in mapracorat raw material drug according to claim 1, characterized in that, The detection includes: Detecting reference solutions of dichloromethyl methyl carbonate with different concentrations by headspace gas chromatography-mass spectrometry, respectively obtaining chromatograms of different concentration reference solutions, and then plotting a standard curve based on the peak area and the concentration of the reference solution; The headspace gas chromatography-mass spectrometry method is used to detect the test solution of the mapracilavir raw material drug, and the chromatogram of the test solution is obtained. Then, the content of the impurity dichloromethyl methyl carbonate in the mapracilavir raw material drug is obtained by calculation using the standard curve.

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