CN116008442A

CN116008442A - Impurity detection method for synthesis intermediate of alpha 5-GABAA receptor modulator

Info

Publication number: CN116008442A
Application number: CN202310301792.5A
Authority: CN
Inventors: 高昂; 谢麟
Original assignee: Shanghai Simr Biotechnology Co ltd
Current assignee: Shanghai Simr Biotechnology Co ltd
Priority date: 2023-03-27
Filing date: 2023-03-27
Publication date: 2023-04-25
Anticipated expiration: 2043-03-27
Also published as: CN116008442B

Abstract

The present application relates to a method for detecting impurities of a synthetic intermediate of an α5-GABAA receptor modulator, the method comprising the steps of: taking a synthetic intermediate to be detected, and dissolving the synthetic intermediate in a first solvent to prepare a detection solution; performing high performance liquid chromatography detection on the detection solution, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, and the mobile phase B is acetonitrile for gradient elution. According to the impurity detection method for the synthetic intermediate of the alpha 5-GABAA receptor regulator, on the basis of high performance liquid chromatography, by adopting a proper mobile phase and an elution program, effective separation and accurate detection of each impurity in the synthetic intermediate can be realized, so that the quality control of the key synthetic intermediate is realized, and the method has important significance for production and research of raw materials.

Description

Impurity detection method for synthesis intermediate of alpha 5-GABAA receptor modulator

Technical Field

The application relates to the technical field of quality detection, in particular to an impurity detection method for a synthetic intermediate of an alpha 5-GABAA receptor modulator.

Background

N-isopropyl-6- (((3- (5- (methoxymethyl) isoxazol-3-yl) - [1,2,4] triazol [3,4-a ] phthalazin-6-yl) oxy) methyl) nicotinamide is a known α5-containing GABAA receptor (α5-GABAA receptor) α5-GABAA receptor modulator. The alpha 5-GABAA receptor has been shown to have a specific profile in the hippocampal tissue of the mammalian brain and alpha 5-GABAA receptor modulators have been shown to be useful in the treatment of pain, in particular Neuropathic Pain (NP).

The current synthetic route of the crude drug of N-isopropyl-6- (((3- (5- (methoxymethyl) isoxazol-3-yl) - [1,2,4] triazol [3,4-a ] phthalazin-6-yl) oxy) methyl) nicotinamide is shown as follows:

wherein the key intermediate compound V is prepared by first substituting and closing ring of the compound I and the compound II

) Then directly carrying out substitution reaction with the compound IV without post treatment. The synthesis route of the intermediate compound V is complex, and the generated impurities are more, so that the control of related substances in the bulk drug is more critical.

Based on this, it is necessary to detect impurities in the intermediate compound V.

Disclosure of Invention

Based on this, the present application provides a method that enables accurate detection of impurities in synthetic intermediates of α5-GABAA receptor modulators.

A method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator, the synthetic intermediate having structural characteristics represented by the following formula (V):

（V）

the impurity detection method comprises the following steps:

taking a synthetic intermediate to be detected, and dissolving the synthetic intermediate in a first solvent to prepare a detection solution;

performing high performance liquid chromatography detection on the detection solution, wherein the conditions of the high performance liquid chromatography detection comprise: the mobile phase A is phosphoric acid aqueous solution with volume concentration of 0.08% -0.12%, the mobile phase B is acetonitrile, and gradient elution is carried out, wherein the gradient elution program comprises the following steps:

0-0.5 min, and keeping the volume percentage of the mobile phase A to be 85%;

0.5 min-5 min, wherein the volume percentage of the mobile phase A is reduced from 85% to 65%;

5 min-13 min, and keeping the volume percentage of the mobile phase A to be 65%;

13-18 min, wherein the volume percentage of the mobile phase A is reduced from 65% to 50%;

18-21 min, wherein the volume percentage of the mobile phase A is reduced from 50% to 10%;

21-24 min, and keeping the volume percentage of the mobile phase A to be 10%.

In one embodiment, the conditions for high performance liquid chromatography detection further include one or more of the following conditions:

(1) The packing of the chromatographic column is silica gel bonded with octadecyl;

(2) The column temperature is 25-35 ℃;

(3) The flow rate is 0.8-1.2 mL/min;

(4) The detection wavelength is 215 nm-225 nm;

(5) The sample injection amount is 15-25 mu L.

In one embodiment, the first solvent is a mixture of acetonitrile and water.

In one embodiment, the first solvent is a mixed solution of acetonitrile and water in a volume ratio of 1 (0.8-1.2).

In one embodiment, the concentration of the detection solution is 0.1-0.5 mg/mL.

In one embodiment, the impurities include one or more of PA3, PA3-1, PA5-1, and PA5-2, the structures of PA3, PA3-1, PA5-1, and PA5-2 are as follows:

PA3

PA3-1

PA5-1/>

PA5-2。

in one embodiment, the impurities include PA3, PA3-1, PA5-1, and PA5-2.

In one embodiment, the method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator further comprises the steps of:

taking standard substances of the impurities and/or the synthetic intermediates, and dissolving the standard substances with the solvent to prepare a standard substance solution;

performing high performance liquid chromatography detection on the standard solution, and constructing a standard curve of the impurity and/or the synthetic intermediate according to a high performance liquid chromatography detection result of the standard solution;

substituting the high performance liquid chromatography detection result of the detection solution into a standard curve of the impurity and/or the synthesis intermediate, and calculating the content of the impurity and/or the synthesis intermediate in the detection solution.

In one embodiment, the standard curve for the impurity or synthetic intermediate includes one or more of the following:

(1) Standard curve for PA 3: y=75698.5568x+299.1079;

(2) Standard curve for PA 3-1: y=93755.4372x+1153.2835;

(3) Standard curve for PA 5-1: y=27189.8126x+523.1737;

(4) Standard curve for PA 5-2: y=34292.0147x+209.5764;

(5) Standard curve for synthetic intermediates: y= 87331.5656 x+ 2431.2546;

where x represents the concentration and y represents the response value of the peak area.

In one embodiment, the method of calculation is an area normalization method.

According to the impurity detection method for the synthetic intermediate of the alpha 5-GABAA receptor regulator, on the basis of high performance liquid chromatography, by adopting a proper mobile phase and an elution program, effective separation and accurate detection of each impurity in the synthetic intermediate can be realized, so that the quality control of the key synthetic intermediate is realized, and the method has important significance for production and research of raw materials.

Drawings

FIG. 1 is a diagram of a result of a specificity investigation in an embodiment;

FIG. 2 is a graph of linear survey results in one embodiment;

FIG. 3 is a detection chromatogram of a synthetic intermediate V to be detected in an embodiment;

FIG. 4 is a detection chromatogram of the sample solution in the comparative example.

Detailed Description

The method for detecting impurities in synthetic intermediates of α5-GABAA receptor modulators of the present application is described in further detail below with reference to specific examples. This application may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

Herein, "one or more" refers to any one, any two, or any two or more of the listed items.

In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.

In the present application, reference is made to numerical intervals, where the numerical intervals are considered to be continuous unless specifically stated, and include the minimum and maximum values of the range, and each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The percentage content referred to in the present application refers to mass percent for both solid-liquid and solid-solid phase mixing and volume percent for liquid-liquid phase mixing unless otherwise specified.

The percentage concentrations referred to in this application, unless otherwise indicated, refer to the final concentrations. The final concentration refers to the ratio of the additive component in the system after the component is added.

The temperature parameter in the present application is not particularly limited, and may be a constant temperature treatment or a treatment within a predetermined temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.

An example of the present application provides a method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator, the synthetic intermediate having structural characteristics represented by the following formula (V):

（V）

the impurity detection method comprises the following steps:

0-0.5 min, and keeping the volume percentage of the mobile phase A to be 85%;

21-24 min, and keeping the volume percentage of the mobile phase A to be 10%.

It will be appreciated that the sum of the volume percentages of mobile phase a and mobile phase B is 100%.

It will be appreciated that after the gradient elution procedure is completed, a recovery procedure is also included, including:

24-24.5 min, wherein the volume percentage of the mobile phase A is increased from 10% to 85%;

24.5 min-30 min, and keeping the volume percentage of the mobile phase A to be 85%.

In one example, the packing of the chromatographic column for high performance liquid chromatography detection is octadecyl bonded silica gel. Further, the chromatographic column is Waters X-Bridge C18.

In one example, the column temperature detected by the high performance liquid chromatography is 25-35 ℃. Specifically, the column temperature includes, but is not limited to: 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃.

In one example, the flow rate detected by the high performance liquid chromatography is 0.8-1.2 mL/min. In particular, the flow rates include, but are not limited to: 0.8mL/min, 0.9mL/min, 1mL/min, 1.1mL/min, 1.2mL/min.

In one example, the detection wavelength of the high performance liquid chromatography detection is 215 nm-225 nm. Specifically, the detection wavelengths include, but are not limited to: 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, 221nm, 222nm, 223nm, 224nm, 225nm.

In one example, the sample injection amount of the high performance liquid chromatography detection is 15-25 mu L. Specifically, the sample injection amount includes, but is not limited to: 15 μL, 16 μL, 17 μL, 18 μL, 19 μL, 20 μL, 21 μL, 22 μL, 23 μL, 24 μL, 25 μL.

Without limitation, the chromatograph used for the HPLC detection is a waters HPLC ACQuity Arc 2998 or a comparable chromatography system.

In one example, the first solvent is a mixture of acetonitrile and water. Further, the first solvent is a mixed solution of acetonitrile and water in a volume ratio of 1 (0.8-1.2).

In one example, the concentration of the detection solution is 0.1-0.5 mg/mL. Specifically, the concentration of the detection solution includes, but is not limited to: 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL.

In one example, the impurities include one or more of PA3, PA3-1, PA5-1, and PA5-2, the structures of PA3, PA3-1, PA5-1, and PA5-2 are shown in table 1 below:

TABLE 1

In one example, the impurities include PA3, PA3-1, PA5-1, and PA5-2. The impurity detection method can realize synchronous detection of the four impurities, and has high detection efficiency.

In one example, the method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator further comprises the steps of:

taking a standard substance of the impurity and/or the synthetic intermediate, and dissolving the standard substance by a second solvent to prepare a standard substance solution;

It is understood that the second solvent is a solvent that can dissolve the impurity or a standard of a synthetic intermediate. The mixed solution of acetonitrile and water is used, the volume ratio of the mixed solution of ammonia water and water can be 1 (0.8-1.2), and the volume concentration of ammonia water can be 0.05% -0.15%.

In one example, the standard curve for the impurity and/or synthetic intermediate includes one or more of the following:

(1) Standard curve for PA 3: y=75698.5568x+299.1079;

(2) Standard curve for PA 3-1: y=93755.4372x+1153.2835;

(3) Standard curve for PA 5-1: y=27189.8126x+523.1737;

(4) Standard curve for PA 5-2: y=34292.0147x+209.5764;

(5) Standard curve for synthetic intermediates: y= 87331.5656 x+ 2431.2546;

In one example, the method of calculation is an area normalization method.

The following are specific examples.

Example 1

This example is a method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator.

1. Detection method

1. Instrument and consumable

waters high performance liquid chromatograph acquality Arc 2998;

analytical balance: METTLER TOLEDO XPE105, XS204DR;

and (3) a water purifier: merck Milli-Q water purifier;

2. the reagents are shown in table 2 below:

TABLE 2

3. Chromatographic conditions

Chromatographic column: waters X-Bridge C18, 250X 4.6 mm, 5 μm;

mobile phase a: phosphoric acid with volume concentration of 0.1%;

mobile phase B: acetonitrile;

detection wavelength: 220nm;

flow rate: 1.0mL/min;

sample injection amount: 20. Mu.L;

column temperature: 30 ℃;

run time: 30min;

gradient elution procedure is as follows table 3:

TABLE 3 Table 3

4. Solution preparation

4.1 diluents: water acetonitrile=1:1 (v/v);

4.2 test solution (0.2 mg/mL): weighing about 20mg of a synthetic intermediate V sample, placing the sample in a 100mL volumetric flask, adding a proper amount of diluent for dissolution, adding the diluent for constant volume, and uniformly mixing;

4.3 blank solution: a diluent.

5. System applicability

In the chromatogram, the number of theoretical plates of the synthetic intermediate V is not less than 5000

6. Calculation of

Calculated by an area normalization method.

2. Sample information is shown in table 4 below:

TABLE 4 Table 4

The above reference substances are all obtained from Shanghai Sesamero biotechnology limited company, and can be synthesized by referring to the synthetic route disclosed in Chinese patent application CN114773352A or obtained by enrichment of byproducts.

The intermediate V to be detected is obtained from Shanghai Sesamero biotechnology Co., ltd; batch production was carried out with reference to the synthetic route disclosed in chinese patent application CN114773352 a.

3. Method verification

1. Specialization of

(1) Purpose of experiment

The blank solution is examined to detect whether the synthetic intermediate V and impurities PA3, PA3-1, PA5-1 and PA5-2 are interfered.

(2) Acceptance criteria

The blank solution should not interfere at the peak of the synthesized intermediate V, if any, its peak area should not exceed 0.05% of the peak area of the main component, the separation degree between the impurities and the main peak should not be less than 2.0, and the separation degree between the impurities should not be less than 1.5.

(3) Experimental details

Preparing blank solution, synthetic intermediate V reference solution, known impurity reference stock solution and known impurity positioning solution.

Blank solution: 1000mL of acetonitrile and 1000mL of water were mixed.

0.1% ammonia: 1mL of ammonia water is taken to 1000mL of water and evenly mixed.

Synthesizing an intermediate V reference substance solution: taking 20mg of synthetic intermediate V reference substance, precisely weighing, placing into a 100mL volumetric flask, adding diluent for dissolution, fixing the volume, and uniformly mixing.

Impurities PA3, PA3-1, PA5-1 and PA5-2 control stock solutions are known: taking 20mg of each known impurity reference substance, precisely weighing, respectively placing into 100mL volumetric flasks, adding diluent for dissolution, fixing the volume, and uniformly mixing.

PA3 localization solution: 1mL of the PA3 impurity stock solution was removed, dissolved in a 100mL volumetric flask with diluent and fixed in volume, and mixed well.

PA3-1 localization solution: transferring 3mLPA3-1 impurity stock solution into a 100mL volumetric flask, adding diluent for dissolution, fixing the volume, and uniformly mixing.

PA5-1 localization solution: transferring 1.5mLPA5-1 impurity stock solution into a 100mL volumetric flask, adding diluent to dissolve and fix volume, and mixing well.

PA5-2 localization solution: transferring 1.5mLPA5-2 impurity stock solution into a 100mL volumetric flask, adding diluent to dissolve and fix volume, and mixing well.

Adding a standard sample solution: 20mg of the synthetic intermediate V control is weighed into a 100mL volumetric flask, transferred into a 1mL LPA3 impurity stock solution, a 3mL PA3-1 impurity stock solution, a 1.5mL PA5-1 impurity stock solution and a 1.5mL PA5-2 impurity stock solution, and added with a diluent to be dissolved and fixed in volume, and uniformly mixed.

(4) The test was performed according to the test method item, and the results are shown in FIG. 1 and Table 5

TABLE 5 detection method for intermediate V-related substance synthesized to verify specificity

(5) Conclusion(s)

The blank solvent has no interference at the peak positions of the synthetic intermediate V and impurities PA3, PA3-1, PA5-1 and PA5-2. Impurities and main peaks, and baseline separation between impurities is achieved.

2. Linearity and range

(1) Purpose of experiment

It was examined whether the synthetic intermediate V and the known impurity response values were well-linear with their corresponding concentrations in the range of 0.1. Mu.g/mL to 12. Mu.g/mL (corresponding to the 0.05% to 6.0% level of the sample concentration).

(2) Acceptable standard

Within the above concentration range, the response value should be well linear with the concentration, and its linear correlation coefficient (r) should be not less than 0.998. The Y-axis intercept value must not be greater than ±50% of the 0.05% horizontal response value.

(3) Experimental details

Linear stock solution 1 (10 μg/mL): taking PA5-1 and PA5-2 impurity stock solutions under the 'special' item, respectively transferring 5.0mL of each solution into a same 100mL measuring flask, adding a diluent to fix the volume, and uniformly mixing to obtain stock solution 1. Each horizontal linear solution was prepared according to table 6.

TABLE 6 preparation of linear solutions of substances relevant to the detection method of Synthesis of intermediate V1

Linear stock solution 2 (PA 3-1 12. Mu.g/mL): taking a synthetic intermediate V reference substance solution and a PA3-1 reference substance stock solution under the 'special' item, respectively transferring 3.0mL of the synthetic intermediate V reference substance solution and 3.0mL of the PA3-1 solution into the same 50 mL measuring flask, adding a diluent to a constant volume, and uniformly mixing to obtain a stock solution 2. Each horizontal linear solution was prepared according to table 7.

TABLE 7 preparation of linear solutions of substances relevant to the detection method of Synthesis of intermediate V2

Linear stock solution 3 (PA 34 μg/mL): taking the PA3 reference substance stock solution under the 'special' item, transferring 1.0mL of the solution into a 50 mL measuring flask, adding a diluent to fix the volume, and uniformly mixing to obtain the stock solution 3. The PA3 horizontal linear solutions were prepared according to table 8.

TABLE 8 preparation of linear solutions of substances relevant to the detection method of Synthesis of intermediate V3

(4) The detection is carried out according to the detection method item, and the results are shown in fig. 2 and tables 9-13:

TABLE 9 PA3 Linear detection results

TABLE 10 PA3-1 Linear detection results

TABLE 11 Synthesis of intermediate V Linear detection results

TABLE 12 PA5-1 Linear detection results

TABLE 13 PA5-2 Linear detection results

(5) Conclusion(s)

The linear regression equations of peak areas versus concentrations of the synthetic intermediates V, PA3, PA3-1, PA5-1 and PA5-2 are y= 87331.5656x +2431.2546, y= 75698.5568x +299.1079, y= 93755.4372x +1153.2835, y= 27189.8126x +523.1737 and y= 34292.0147x +209.5764, respectively, the linear correlation coefficients r are all greater than 0.998, and the y-axis intercept are all less than ±50% of the response values at the respective 0.05% level.

The results show that the synthetic intermediates V, PA, PA3-1, PA5-1 and PA5-2 of the method respectively have good linearity in response values and concentrations in the concentration ranges of 0.0909-10.9045 mug/mL, 0.1020-4.0786 mug/mL, 0.0963-11.5576 mug/mL, 0.1009-6.056 mug/mL and 0.1009-6.054 mug/mL, and the linear range meets the requirement. The correction factors for PA3, PA3-1, PA5-1 and PA5-2 are 1.1, 0.9, 3.0 and 2.4, respectively.

3. Quantitative limit and detection limit

(1) Purpose of experiment

The quantitative limits and detection limits of the synthetic intermediates V, PA3, PA3-1, PA5-1 and PA5-2 were examined, and the detection ability of the method for impurities was evaluated.

(2) Acceptable standard

The quantitative limiting signal-to-noise ratio must not be lower than 10, and the RSD of the peak area must not exceed 5.0%; the detection limit signal-to-noise ratio must not be lower than 3.

(3) Experimental details

Quantitative limiting solution: a0.05% horizontal solution was prepared under the term "linearity and range". The signal to noise ratio and peak area Relative Standard Deviation (RSD) were calculated by 6 consecutive needles.

Detection limit solution: taking 3.0mL of quantitative limiting solution in a 10mL measuring flask, adding a diluent to fix the volume, and uniformly mixing.

(4) The detection is carried out according to the detection method item, and the results are shown in tables 14-15.

Table 14 limit of detection results

TABLE 15 quantitative limit results

(5) Conclusion(s)

The quantitative level-limiting sample is continuously injected into 6 needles, the signal to noise ratio of the synthesized intermediates V, PA3, PA3-1, PA5-1 and PA5-2 is not less than 10, and the peak areas RSD of the synthesized intermediates V, PA3, PA3-1, PA5-1 and PA5-2 are respectively 3.7%, 3.6%, 3.7%, 3.1% and 2.7%, which meet the requirements. The detection limit level, and the signal to noise ratio of the synthesized intermediates V, PA3, PA3-1, PA5-1 and PA5-2 are all larger than 3.

The detection limit concentrations of the synthesized intermediate V, PA, PA3-1, PA5-1 and PA5-2 are respectively 0.0299 mug/mL, 0.0306 mug/mL, 0.0289 mug/mL, 0.03028 mug/mL and 0.03027 mug/mL, which are respectively 0.015% of the sample concentration level, and the quantitative limit concentrations are respectively 0.0997 mug/mL, 0.1020 mug/mL, 0.0963 mug/mL, 0.1009 mug/mL and 0.1009 mug/mL, which are respectively 0.05% of the sample concentration level.

4. Precision of

(1) Purpose of experiment

The same analyst prepared 6 samples at 100% concentration level in parallel according to the method using the same equipment, added a limited amount of known impurities, calculated the impurity content in the sample solution, and calculated the Relative Standard Deviation (RSD). The proximity of the impurity measurement was examined.

(2) Acceptable standard

The quantity X of single impurities is less than or equal to 0.10 percent, and the RSD is less than or equal to 20 percent; 0.10 percent of < X is less than or equal to 0.25 percent, and RSD is less than or equal to 15 percent; x is more than 0.25%, and RSD is less than or equal to 5%.

(3) Experimental details

Adding a standard sample solution: 20mg of the synthetic intermediate V reference substance is weighed into a 100mL volumetric flask, 1mL of the PA3 impurity stock solution, 3mL of the PA3-1 impurity stock solution, 1.5mL of the PA5-1 and 1.5mL of the PA5-2 impurity stock solution under the 'special' item are transferred into the 100mL volumetric flask, and the mixture is added with a diluent to be dissolved and fixed in volume, and then evenly mixed.

6 portions of the sample solution were prepared in parallel.

(4) The test was performed according to the test method item, and the results are shown in Table 16.

Table 16 precision results of synthetic intermediate V detection method

Wherein RRT0.23, RRT0.31, RRT0.53, RRT0.56, RRT0.65, RRT0.70, RRT0.78, RRT1.21, RRT1.23 and RRT1.36 represent unknown impurities.

(5) Conclusion(s)

In 6 labeled samples, the RSD of all impurities met the requirements.

4. Sample detection

Taking the synthesized intermediate V to be detected, preparing a solution of 0.2mg/mL by using a diluent according to the method of '4.2 test sample solution', and detecting. As a result, as shown in FIG. 3, it was revealed from FIG. 3 that the synthetic intermediate V contained 0.11% of PA3-1, 1.32% of unknown impurity of RRT0.57, 0.13% of PA3, 0.24% of unknown impurity of RRT0.51, 0.08% of unknown impurity of RRT0.78, 0.06% of unknown impurity of RRT1.13 and 0.93% of unknown impurity of RRT 1.22.

Comparative example

This comparative example examined different elution procedures.

The detection of the sample solutions (taking appropriate amounts of the reference substances of PA3, PA3-1, PA5-2 and synthetic intermediate V, respectively, adding a diluent for dissolution, and then taking appropriate amounts for mixing, to prepare sample solutions with the concentrations of the reference substances of PA3, PA3-1, PA5-1 and PA5-2 of 2. Mu.g/mL, 6. Mu.g/mL, 3. Mu.g/mL and 0.2mg/mL, respectively) was carried out according to the detection method in the examples, and the elution procedure was adjusted to:

0-0.5 min, and keeping the volume percentage of the mobile phase A to be 95%;

0.5 min-13 min, wherein the volume percentage of the mobile phase A is reduced from 95% to 75%;

13-19 min, wherein the volume percentage of the mobile phase A is reduced from 75% to 53%;

19-24 min, and keeping the volume percentage of the mobile phase A to be 53%;

24-26 min, wherein the volume percentage of the mobile phase A is reduced from 53% to 10%;

26-28 min, and keeping the volume percentage of the mobile phase A to be 10%.

28 min-28.1 min, wherein the volume percentage of the mobile phase A is increased from 10% to 95%;

28.1 min-35 min, and keeping the volume percentage of the mobile phase A to be 95%.

The detection results are shown in FIG. 4. As can be seen, PA5-1 and PA5-2 are not separated.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples merely represent a few embodiments of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but are not to be construed as limiting the scope of the claims. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. It should be understood that those skilled in the art, based on the technical solutions provided in the present application, can obtain technical solutions through logical analysis, reasoning or limited experiments, all fall within the protection scope of the claims attached in the present application. The scope of the patent application is therefore intended to be limited by the content of the appended claims, the description and drawings being presented to the extent that the claims are defined.

Claims

1. A method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator, the synthetic intermediate having a structural feature represented by the following formula (V):

（V）

the impurity detection method comprises the following steps:

0-0.5 min, and keeping the volume percentage of the mobile phase A to be 85%;

21-24 min, and keeping the volume percentage of the mobile phase A to be 10%.

2. The method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 1, wherein the conditions for the detection by high performance liquid chromatography further comprise one or more of the following conditions:

(2) The column temperature is 25-35 ℃;

(3) The flow rate is 0.8-1.2 mL/min;

(4) The detection wavelength is 215 nm-225 nm;

(5) The sample injection amount is 15-25 mu L.

3. The method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 1, wherein the first solvent is a mixture of acetonitrile and water.

4. The method for detecting an impurity in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 3, wherein the first solvent is a mixed solution of acetonitrile and water in a volume ratio of 1 (0.8 to 1.2).

5. The method for detecting an impurity in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 1, wherein the concentration of the detection solution is 0.1 to 0.5mg/mL.

6. The method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator according to any one of claims 1 to 5, wherein the impurities include one or more of PA3, PA3-1, PA5-1 and PA5-2, and the structures of PA3, PA3-1, PA5-1 and PA5-2 are as follows:

PA3/>

PA3-1

PA5-1

PA5-2。

7. the method for detecting impurities of a synthetic intermediate of an α5-GABAA receptor modulator according to claim 6, wherein the impurities comprise PA3, PA3-1, PA5-1 and PA5-2.

8. The method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 6, further comprising the steps of:

9. The method for detecting impurities of a synthetic intermediate for an alpha 5-GABAA receptor modulator according to claim 8, wherein the standard curve of said impurities or synthetic intermediate comprises one or more of the following:

(1) Standard curve for PA 3: y=75698.5568x+299.1079;

(2) Standard curve for PA 3-1: y=93755.4372x+1153.2835;

(3) Standard curve for PA 5-1: y=27189.8126x+523.1737;

(4) Standard curve for PA 5-2: y=34292.0147x+209.5764;

(5) Standard curve for synthetic intermediates: y= 87331.5656 x+ 2431.2546;

10. The method for detecting impurities in a synthetic intermediate of an α5-GABAA receptor modulator according to claim 8, wherein the method for calculation is an area normalization method.