Method for separating and determining benserazide hydrochloride SM2 and related substances thereof

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for separating and determining benserazide hydrochloride SM2 and related substances thereof. According to the self-built high performance liquid chromatography, octadecylsilane chemically bonded silica is used as a chromatographic column filler, a phosphoric acid aqueous solution is used as a mobile phase A, and methanol is used as a mobile phase B; the impurity BES-SM2a, the impurity BES-SM2c, the impurity BES-SM2d, the benserazide hydrochloride SM2 and / or the impurity BES-SM2e are sequentially separated through gradient elution; and then detecting in a detector with the wavelength of 215nm to obtain a chromatogram, and calculating the content of each component according to the peak area by adopting a correction factor-added principal component self-contrast method. The analysis method provided by the invention can be used for determining benserazide hydrochloride SM2 and related substances thereof within 50 minutes, and has the characteristics of strong specificity, high sensitivity and good reproducibility.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for separating and determining benserazide hydrochloride SM2 and related substances thereof. Background Art

[0002] Benserazide hydrochloride, a peripheral decarboxylase inhibitor, is often combined with levodopa to form a compound preparation, dopamine, for the treatment of Parkinson's disease and post-encephalitis, arteriosclerotic or toxic Parkinson's syndrome.

[0003] Benserazide hydrochloride SM2 (BES-SM2) is the key starting material of benserazide hydrochloride, with the chemical name of 2,3,4-trihydroxybenzaldehyde and the structural formula as shown in Formula I. According to the manufacturer's process route of benserazide hydrochloride SM2, it may contain the impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d 、Impurity BES-SM 2e 4 impurities.

[0004]

[0005] At present, there is no regular analytical method to detect the related substances of benserazide hydrochloride SM2, and the peak shapes of the impurities in the literature methods are not good, and the impurities cannot be effectively separated. Some existing patent technologies have detected related substances in dobase hydrazine compound preparations. For example, the CN116482257A patent discloses an analytical method for dissolution detection of dobase hydrazine preparations, and uses high-performance liquid chromatography to detect the dissolution amount of the active ingredient levodopa dissolved in the dobase hydrazine preparation and the dissolution amount of benserazide hydrochloride; the CN111812227A patent discloses a method for detecting the content of benserazide impurity A in dobase hydrazine compound preparations, which uses high-performance liquid chromatography to detect benserazide impurity A (2RS)-2-amino-3-hydroxypropionyl hydrazide in dobase hydrazine compound preparations. However, the above-mentioned existing technologies cannot achieve the separation and detection of the above-mentioned four impurities.

[0006] Therefore, it is necessary to construct a new method to determine benserazide hydrochloride SM2 and its related substances, further control the impurity content and improve product quality. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating benserazide hydrochloride SM2 and its related substances using high performance liquid chromatography. The method has high sensitivity and good durability, and can effectively separate benserazide hydrochloride SM2 and its related substances, providing technical support for the subsequent content determination of benserazide hydrochloride SM2 and its related substances.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] Method for separating benserazide hydrochloride SM2 and its related substances by high performance liquid chromatography, wherein the related substances include impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d and impurity BES-SM 2e Any one or more of; the structural formula of benserazide hydrochloride SM2 is as shown in Formula I, the impurity BES-SM 2a The structural formula is shown in Formula II, and the impurity BES-SM 2c The structural formula is shown in Formula III, and the impurity BES-SM 2d The structural formula is shown in Formula IV, and the impurity BES-SM 2e The structural formula is shown in Formula V;

[0010] In the high performance liquid chromatography method, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a phosphoric acid aqueous solution and the mobile phase B is methanol; the impurities BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d , benserazide hydrochloride SM2 and / or impurity BES-SM 2e separation;

[0011]

[0012] The components can be characterized according to their separation order.

[0013] Further, the gradient elution procedure is as follows:

[0014] At 0 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0015] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0016] At 28 min-32 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0017] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0018] At 41 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0019] At 50 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3.

[0020] Furthermore, the gradient elution procedure is preferably:

[0021] At 0 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0022] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0023] At 30 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0024] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0025] At 41 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0026] At 50 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5.

[0027] Furthermore, the mobile phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%.

[0028] Furthermore, the flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min, preferably 1.0 ml / min; the column temperature of the chromatographic column is 33° C.-37° C., preferably 35° C.

[0029] Furthermore, the injection volume was 20 μl.

[0030] The second object of the present invention is to provide a method for identifying benserazide hydrochloride SM2 and its related substances, which can achieve qualitative identification of benserazide hydrochloride SM2 and its related substances.

[0031] To achieve the above object, the present invention adopts the following technical solutions:

[0032] The method for identifying benserazide hydrochloride SM2 and its related substances is to separate benserazide hydrochloride SM2 and its related substances by the aforementioned separation method, and to detect them by a detector; the detection wavelength of the detector is 215nm.

[0033] Furthermore, the retention time of each component from short to long is impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d , benserazide hydrochloride SM2 and / or impurity BES-SM 2e .

[0034] Each component can be identified based on its retention time.

[0035] As a preferred implementation method, the chromatographic column has a specification of 4.6 mm×250 mm, 5 μm; the mobile phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%, and the mobile phase B is methanol; the mobile phase flow rate is 1.0 ml / min; the column temperature of the chromatographic column is 35° C.; and the detection wavelength is 215 nm;

[0036] The components were eluted using a gradient elution program to obtain a chromatographic column, and the gradient elution program was as follows:

[0037] At 0 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0038] At 5 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0039] At 30 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0040] At 40 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65;

[0041] At 41 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0042] At 50 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5.

[0043] Furthermore, the impurity BES-SM was found to have a retention time of 8.1±0.5 min. 2a The impurity BES-SM is at a retention time of 9.5±0.5min 2c The impurity BES-SM is at a retention time of 16.4±0.5min 2d The retention time is 17.8±0.5min for benserazide hydrochloride SM2, and the retention time is 19.6±0.5min for impurity BES-SM 2e .

[0044] The third object of the present invention is to provide a method for detecting the content of benserazide hydrochloride SM2 and its related substances, which can achieve the determination of benserazide hydrochloride SM2 and its related substances within 50 minutes or more than 50 minutes.

[0045] To achieve the above object, the present invention adopts the following technical solutions:

[0046] The method for detecting the content of benserazide hydrochloride SM2 and its related substances comprises the following steps:

[0047] (1) Separation: Using the above separation method to separate benserazide hydrochloride SM2 and its related substances;

[0048] (2) Detection: benserazide hydrochloride SM2 and its related substances are detected by the aforementioned identification method to obtain a chromatogram;

[0049] (3) Content calculation: Based on the chromatogram obtained in step (2), the content of each component is calculated by peak area using the principal component self-comparison method with the addition of a correction factor.

[0050] Furthermore, before separation, a solvent is used to prepare a system suitability solution, a control solution and a sample solution to be tested; the solvent is a mixed solution of 0.1% aqueous phosphoric acid solution and acetonitrile, wherein the volume ratio of the 0.1% aqueous phosphoric acid solution to the acetonitrile is 90:10.

[0051] Preferably, the method for detecting the content of benserazide hydrochloride SM2 and its related substances is as follows:

[0052] 1. Prepare the sample / test solution: Take an appropriate amount of the product, weigh it accurately, add a solvent and ultrasonically dissolve it and dilute it to a solution with a concentration of about 1 mg / ml.

[0053] 2. Prepare control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; accurately measure 1 ml of the above solution, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0054] 3. Prepare system suitability solution: Take benserazide hydrochloride SM2 system suitability reference substance (containing benserazide hydrochloride SM2, impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d With impurity BES-SM 2e ) and dissolve and dilute with sonication to produce a solution containing about 1 mg per 1 ml.

[0055] 4. Take the sample to be tested, the control solution and the system suitability solution prepared in steps 1, 2 and 3, such as 20 μl, and inject them separately to obtain the chromatogram; according to the chromatogram, calculate the content of each component by the peak area using the main component self-reference method with the correction factor added.

[0056] Calculation formula: C a =(A a ×C b ) / A b ×f

[0057] Where F is the correction factor;

[0058] C a is the concentration of the analyte;

[0059] A a is the peak area of ​​the analyte;

[0060] C b is the concentration of the reference substance;

[0061] A b is the peak area of ​​the reference substance.

[0062] The beneficial effects of the present invention are:

[0063] The high performance liquid chromatography provided by the present invention can remove the impurity BES-SM within 50 minutes. 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d 、Impurity BES-SM 2e It is well separated from 2,3,4-trihydroxybenzaldehyde (BES-SM2) and accurately quantified, solving the problem of separation and determination of known impurities in BES-SM2 that cannot be solved in the prior art. The analytical method of the invention has high sensitivity, strong specificity, good reproducibility, and is simple and feasible to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 is the chromatogram of the blank solution;

[0065] Figure 2 is the chromatogram of the test solution;

[0066] Figure 3 is the chromatogram of the mixed solution;

[0067] Figure 4 is the chromatogram of the detection limit solution;

[0068] Figure 5 Robustness (mixed solution) - Chromatogram under normal conditions;

[0069] Figure 6 Durability (mixed solution) - Chromatogram at flow rate 0.9 ml / min;

[0070] Figure 7 Durability (mixed solution) - Chromatogram at flow rate 1.1 ml / min;

[0071] Figure 8 Durability (mixed solution) - Chromatogram at column temperature 33°C;

[0072] Fig. 9 Durability (mixed solution) - Chromatogram at column temperature 37°C;

[0073] Fig.10 Durability (mixed solution) - chromatogram with initial mobile phase ratio A:B = 93:7;

[0074] Fig.11 Durability (mixed solution) - chromatogram with initial mobile phase ratio A:B = 97:3;

[0075] Fig.12 Durability (mixed solution) - chromatogram of gradient 5→28min;

[0076] Fig.13 Durability (mixed solution) - chromatogram of gradient 5→32min;

[0077] Fig.14 Robustness (mixed solution) - Chromatograms from different column batches. DETAILED DESCRIPTION

[0078] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0079] The embodiments of the present invention are determined strictly in accordance with the high performance liquid chromatography method (General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0080] In the embodiment of the present invention, the solvent is a mixed solution of 0.1% phosphoric acid aqueous solution and acetonitrile, wherein the volume ratio of 0.1% phosphoric acid aqueous solution to acetonitrile is 90:10.

[0081] In the embodiment of the present invention, the preparation method of 0.1% phosphoric acid aqueous solution is as follows: 1000 ml of water is measured, and 1 ml of phosphoric acid is added to obtain the solution.

[0082] In the embodiment of the present invention, the analysis method is as follows:

[0083] (1) Preparation of solution

[0084] Test solution: Take an appropriate amount of the product, weigh it accurately, add solvent and sonicate to dissolve and dilute it to make a solution with a concentration of about 1 mg / ml.

[0085] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; Accurately measure 1 ml of the above solution, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well.

[0086] System suitability solution: Take benserazide hydrochloride SM2 system suitability reference substance (containing benserazide hydrochloride SM2, impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d With impurity BES-SM 2e ) and dissolve and dilute with sonication to produce a solution containing about 1 mg per 1 ml.

[0087] (2) Chromatographic conditions

[0088] Octadecylsilane bonded silica gel was used as filler (Shim-pack GIST C18, 4.6 mm × 250 mm, 5 μm, or a chromatographic column with equivalent performance), 0.1% by volume phosphoric acid aqueous solution was used as mobile phase A, methanol was used as mobile phase B, and linear gradient elution was performed according to Table 1; the flow rate was 1.0 ml / min; the column temperature was 35°C; the detection wavelength was 215 nm; and the injection volume was 20 μl.

[0089] Table 1. Gradient elution program

[0090] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 5 95 5 30 35 65 40 35 65 41 95 5 50 95 5

[0091] (3) Determination

[0092] System suitability requirements: Impurities in system suitability solution BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d , Benserazide hydrochloride SM2 and impurity SM 2e The peaks should be extracted in sequence, and the separation degree between the main peak and adjacent impurities should meet the requirements.

[0093] Determination method: Accurately measure the test solution and control solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0094] Limits: In the chromatogram of the test solution, except for the solvent peak and the gradient elution peak, the impurity content calculated by the main component self-reference method with the correction factor should comply with the provisions of Table 2 below, and the total amount of impurities shall not exceed 2.0%. Chromatographic peaks with a content of less than 0.05% are ignored.

[0095] Table 2. Impurity content regulations

[0096]

[0097]

[0098] Example 1. Specificity

[0099] The key starting material SM2 may contain the following four impurities: Impurity BES-SM2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d and impurity BES-SM 2e In this example, the impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d 、Impurity BES-SM 2e The separation between the main components was investigated.

[0100] (1) Prepare the test solution

[0101] Impurity BES-SM 2a Stock solution: Take impurity BES-SM 2a 30 mg of reference substance was accurately weighed and placed in a 50 ml volumetric flask. MeOH was added to dissolve and dilute to the mark, and then shaken to mix well.

[0102] Impurity BES-SM 2c Stock solution: Take impurity BES-SM 2c 30 mg of reference substance was accurately weighed and placed in a 50 ml volumetric flask. MeOH was added to dissolve and dilute to the mark, and then shaken to mix well.

[0103] Impurity BES-SM 2d Stock solution: Take impurity BES-SM 2d 30 mg of reference substance was accurately weighed and placed in a 50 ml volumetric flask. MeOH was added to dissolve and dilute to the mark, and then shaken to mix well.

[0104] Impurity BES-SM 2e Stock solution: Take impurity BES-SM 2e 30 mg of reference substance was accurately weighed and placed in a 50 ml volumetric flask. MeOH was added to dissolve and dilute to the mark, and then shaken to mix well.

[0105] Impurity BES-SM 2a Positioning solution: measuring impurities BES-SM 2a Place 0.5 ml of the stock solution in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0106] Impurity BES-SM 2c Positioning solution: measuring impurities BES-SM 2c Place 0.5 ml of the stock solution in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0107] Impurity BES-SM 2d Positioning solution: measuring impurities BES-SM 2d Place 0.5 ml of the stock solution in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0108] Impurity BES-SM 2e Positioning solution: measuring impurities BES-SM 2e Place 0.5 ml of the stock solution in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0109] Mixed impurity stock solution: Precisely measure impurities BES-SM 2a Stock solution, impurities BES-SM 2c Stock solution, impurities BES-SM 2d Stock solution and impurities BES-SM 2e Place 2.5 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0110] Mixed solution: Take 25 mg of the test sample, weigh it accurately, place it in a 25 ml volumetric flask, add solvent and sonicate to dissolve it, then measure 2.5 ml of the mixed impurity stock solution, place it in the same volumetric flask, dilute to the scale with solvent, and shake well.

[0111] (2) Detection

[0112] Take 20 μl of blank solvent, each impurity location solution, test solution and mixed solution for injection and record the chromatogram.

[0113] The results are shown in Tables 3-5. Figure 1-Figure 3 As shown, the results show that the blank solution and the test solution do not interfere with the impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d 、Impurity BES-SM 2e Detection of the main peak BES-SM2 and impurity BES-SM in the mixed solution 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d 、Impurity BES-SM 2e The separation between adjacent chromatographic peaks is greater than 1.5. The specificity of the method of the present invention meets the requirements.

[0114] Table 3. Specificity test results

[0115]

[0116] Table 4. Figure 2 The integral results table

[0117]

[0118] Table 5. Figure 3 The integral results table

[0119]

[0120] Example 2. Detection Limit

[0121] (1) Solution preparation

[0122] BES-SM2 stock solution: Take 30 mg of the test sample, weigh accurately, put it in a 50 ml volumetric flask, add solvent and ultrasonically dissolve it and dilute it to the scale, and shake well.

[0123] Impurity BES-SM 2a Stock solution, impurities BES-SM 2c Stock solution, impurities BES-SM 2d Stock solution and impurities BES-SM 2e Stock solutions: All were prepared according to the preparation method of each impurity stock solution under "Example 1, Specificity".

[0124] Detection limit stock solution: Accurately measure BES-SM2 linear stock solution, impurity BES-SM 2a Stock solution, impurities BES-SM 2c Stock solution, impurities BES-SM 2d Stock solution and impurities BES-SM 2e 2.5 ml of each stock solution is placed in a 100 ml volumetric flask, diluted to the mark with solvent, and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0125] Detection limit solution: Accurately measure 3.0 ml of the detection limit stock solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0126] (2) Detection

[0127] Take 20 μl of the detection limit solution and inject it three times in succession. Inject the samples in sequence, record the chromatogram, and calculate the ratio of the peak height of each impurity peak to the noise (signal-to-noise ratio).

[0128] The test results are shown in Table 6-Table 7. Figure 4 As shown, impurity BES-SM 2a The detection limit concentration is 0.0916μg / ml, the S / N average is 22.9, and the concentration in the sample is expressed as 0.009%; impurity BES-SM 2c The detection limit concentration is 0.0905μg / ml, the S / N average is 49.6, and the concentration in the sample is expressed as 0.009%; impurity BES-SM 2d The detection limit concentration is 0.0907μg / ml, the average S / N is 39.5, and the concentration in the sample is 0.009%; the detection limit concentration of impurity BES-SM2 is 0.0908μg / ml, the average S / N is 38.0, and the concentration in the sample is 0.009%; the impurity BES-SM2e The detection limit concentration is 0.0872 μg / ml, the S / N average value is 9.8, and the concentration in the sample is 0.009%, which meets the detection limit requirements.

[0129] Table 6. Detection limit determination results

[0130]

[0131]

[0132] Table 7. Figure 4 The integral results table

[0133]

[0134] Example 3. Chromatographic Conditions Robustness

[0135] Take the mixed solution under the "Specificity" item of Example 1, use the normal mobile phase, different column temperatures, column flow rates, different mobile phase ratios, different gradient change times and different batches of chromatographic columns for the predetermined test, test them respectively after the instrument system is stable, record the chromatograms, and examine the changes in the separation between the peaks of each component.

[0136] Results: As shown in Table 8-9, Figure 5-Figure 14 As shown, when there are slight changes in the chromatographic conditions flow rate, column temperature, the starting ratio of the mobile phase organic phase to the phosphoric acid aqueous solution, and the gradient time, and under different chromatographic column conditions, the separation between the impurities and between the impurities and the main components controlled by this analytical method is greater than 1.5; the method has good durability.

[0137] Table 8. Results of the durability test for changes in chromatographic conditions (resolution)

[0138]

[0139] Table 9. Chromatographic condition change durability test results (retention time)

[0140]

Claims

1. A method for separating benserazide hydrochloride SM2 and its related substances by high performance liquid chromatography, characterized in that: The related substances include the impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d and impurity BES-SM 2e Any one or more of; the structural formula of benserazide hydrochloride SM2 is as shown in Formula I, the impurity BES-SM 2a The structural formula is shown in Formula II, and the impurity BES-SM 2c The structural formula is shown in Formula III, and the impurity BES-SM 2d The structural formula is shown in Formula IV, and the impurity BES-SM 2e The structural formula is shown in Formula V; In the high performance liquid chromatography method, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes mobile phase A and mobile phase B, wherein the mobile phase A is a phosphoric acid aqueous solution, and the mobile phase B is methanol; The impurity BES-SM was removed by gradient elution. 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d , benserazide hydrochloride SM2 and / or impurity BES-SM 2e separation; 2. The method according to claim 1, characterized in that: The gradient elution procedure is as follows: At 0 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 5 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 28 min-32 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65; At 40 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65; At 41 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 50 min, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3.

3. The method according to claim 1, characterized in that The mobile phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%.

4. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.9 ml / min-1.1 ml / min; The column temperature of the chromatographic column is 33°C-37°C.

5. A method for identifying benserazide hydrochloride SM2 and its related substances, characterized in that: Benserazide hydrochloride SM2 and its related substances are separated by the method described in any one of claims 1 to 4, and detected by a detector; the detection wavelength of the detector is 215 nm.

6. The method according to claim 5, characterized in that The retention time of each component from short to long is impurity BES-SM 2a 、Impurity BES-SM 2c 、Impurity BES-SM 2d , benserazide hydrochloride SM2 and / or impurity BES-SM 2e .

7. The method according to claim 5, characterized in that The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm; the mobile phase A is a phosphoric acid aqueous solution with a volume fraction of 0.1%, and the mobile phase B is methanol; the mobile phase flow rate is 1.0 ml / min; the column temperature of the chromatographic column is 35° C.; and the detection wavelength is 215 nm; The components were eluted using a gradient elution program to obtain a chromatographic column, and the gradient elution program was as follows: At 0 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 5 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 30 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65; At 40 min, the volume ratio of the mobile phase A to the mobile phase B was set to 35:65; At 41 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 50 min, the volume ratio of the mobile phase A to the mobile phase B was set to 95:

5.

8. The method according to claim 7, characterized in that The impurity BES-SM is at a retention time of 8.1±0.5min 2a The impurity BES-SM is at a retention time of 9.5±0.5min 2c The impurity BES-SM is at a retention time of 16.4±0.5min 2d The retention time is 17.8±0.5min for benserazide hydrochloride SM2, and the retention time is 19.6±0.5min for impurity BES-SM 2e .

9. A method for detecting the content of benserazide hydrochloride SM2 and its related substances, characterized in that: The following steps are involved: (1) Separation: separating benserazide hydrochloride SM2 and its related substances by the method described in any one of claims 1 to 4; (2) Detection: benserazide hydrochloride SM2 and its related substances are detected by the method described in any one of claims 5 to 8 to obtain a chromatogram; (3) Content calculation: Based on the chromatogram obtained in step (2), the content of each component is calculated by peak area using the principal component self-comparison method with the addition of a correction factor.

10. The method according to claim 9, characterized in that Before separation, a system suitability solution, a control solution and a sample solution to be tested are prepared with a solvent; the solvent is a mixed solution of 0.1% phosphoric acid aqueous solution and acetonitrile, wherein the volume ratio of the 0.1% phosphoric acid aqueous solution to the acetonitrile is 90:10.

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