Method for separating related impurities in doprazide tablet and measuring content of each component

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating related impurities in a dopurazide tablet and determining the content of each component. According to the method, octadecyl silane bonded silica gel is adopted as a filling agent, a potassium hexafluorophosphate solution is adopted as a mobile phase A, methyl alcohol is adopted as a mobile phase B, and benserazide hydrochloride, a benserazide hydrochloride impurity C and a benserazide hydrochloride impurity E are sequentially separated through gradient elution; and then the sample enters a detector with the detection wavelength of 210 nm to be detected. And calculating the content of each component by adopting an external standard method according to the measured chromatogram. The self-established high performance liquid chromatography is not interfered by other impurities or auxiliary materials, and separation and determination of the benserazide hydrochloride, the benserazide hydrochloride impurity C and the benserazide hydrochloride impurity E can be realized within 35 minutes. The method is high in specificity, high in accuracy and good in precision and durability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating relevant impurities in dopamine hydrazine tablets and determining the content of each component. Background Art

[0002] Dobasehydrazide tablets are a compound preparation, mainly used for the treatment of Parkinson's disease, symptomatic Parkinson's syndrome caused by encephalitis, arteriosclerosis or poisoning. The main ingredients of Dobasehydrazide tablets are levodopa and benserazide hydrochloride, and the excipients include mannitol, anhydrous calcium hydrogen phosphate, microcrystalline cellulose, cross-linked polyvinylpyrrolidone, magnesium stearate, ethyl cellulose, pregelatinized starch, colloidal silicon dioxide, sodium docusate, red iron oxide, etc. The main degradation impurities of Dobasehydrazide tablets are benserazide hydrochloride impurity A and benserazide hydrochloride impurity B, but under high temperature conditions, benserazide hydrochloride impurity E will also increase slightly.

[0003] At present, Dopamine Tablets are only included in ChP2020 Part II, and only control is performed on Benserazide Hydrochloride Impurity A and Benserazide Hydrochloride Impurity B, and other impurities such as Benserazide Hydrochloride Impurity C, Benserazide Hydrochloride Impurity E, and Benserazide Hydrochloride Impurity F are not controlled. In addition, the known determination methods for related substances in Dopamine Tablets also make it difficult to effectively separate the degradation impurity Benserazide Hydrochloride Impurity E from the process impurity Benserazide Hydrochloride Impurity C, so it is necessary to establish another method to detect Benserazide Hydrochloride Impurity E.

[0004] Benserazide hydrochloride impurity A is benserazide hydrochloride, and its structural formula is shown in Formula V; benserazide hydrochloride impurity B is trihydroxybenzyl benserazide hydrochloride, and its structural formula is shown in Formula VI; the structural formulas of benserazide hydrochloride impurity C, benserazide hydrochloride impurity E, and benserazide hydrochloride impurity F are shown in Formulas II-IV, respectively.

[0005]

[0006]

[0007] At present, there are reports that publicly use high performance liquid chromatography to detect impurities in dobase hydrazine compound preparations. For example, the invention patent with publication number CN111812227A discloses an analysis method for benserazide impurity A in dobase hydrazine compound preparations. However, this method cannot achieve the separation and content detection of benserazide hydrochloride impurity E and benserazide hydrochloride impurity C in dobase hydrazine tablets. Summary of the invention

[0008] In view of this, one of the objects of the present invention is to provide a method for separating benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E by high performance liquid chromatography, which method has good accuracy, durability and repeatability, and is not interfered by other impurities or excipients, and provides technical support for the subsequent qualitative and quantitative analysis of each component.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for separating benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E by high performance liquid chromatography, wherein the separated components are benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E; the structural formula of the benserazide hydrochloride is shown in formula I, the structural formula of the benserazide hydrochloride impurity C is shown in formula II, and the structural formula of the benserazide hydrochloride impurity E is shown in formula III in sequence; the chromatographic column of the high performance liquid chromatography adopts octadecylsilane bonded silica gel as a filler, and the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a potassium hexafluorophosphate solution, and the mobile phase B is methanol; the benserazide hydrochloride, the benserazide hydrochloride impurity C and the benserazide hydrochloride impurity E are separated in sequence by gradient elution;

[0011]

[0012] Furthermore, the sample to be tested is dobenserazide tablets, which contain benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E.

[0013] Furthermore, when the method is used to simultaneously separate benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets, the separation time is 35 minutes.

[0014] Preferably, the procedure of the gradient elution is:

[0015] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0016] At 2 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0017] At 25 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 55:45;

[0018] At 26 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3;

[0019] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3.

[0020] More preferably, the procedure of the gradient elution is:

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

[0022] At 2 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0023] At 25 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 55:45;

[0024] At 26 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0025] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5.

[0026] Furthermore, the pH value of the mobile phase A is 2.5-4.5, preferably 3.4-3.6, and more preferably 3.5; the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1.0 ml / min; and the column temperature of the chromatographic column is 23-27°C, preferably 25°C.

[0027] Furthermore, the injection volume was 5 μl.

[0028] The second object of the present invention is to provide a method for qualitatively identifying benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine tablets based on the above-mentioned separation method. The method realizes the identification of benserazide hydrochloride impurity E in dopamine tablets, which is of great significance to the quality control of dopamine tablets.

[0029] To achieve the above object, the present invention adopts the following technical solutions:

[0030] A method for qualitatively identifying benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dobasehydrazide tablets based on the aforementioned separation method, wherein benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dobasehydrazide tablets are separated by the aforementioned method; after separation, detection is performed using a detector with a detection wavelength of 210 nm, and a chromatogram is obtained.

[0031] Furthermore, according to the retention time, the three components are ranked from short to long as follows: benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E.

[0032] As a preferred technical solution, the mobile phase A is a potassium hexafluorophosphate solution with a pH value of 3.5, and the mobile phase B is methanol; the flow rate of the mobile phase is 1.0 ml / min; the column temperature of the chromatographic column is 25°C; the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm;

[0033] Perform linear gradient elution according to the following gradient elution program and obtain the chromatogram:

[0034] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0035] At 2 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0036] At 25 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 55:45;

[0037] At 26 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5;

[0038] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5.

[0039] Further, the retention time of 7.1±0.5min is benserazide hydrochloride, the retention time of 11.4±0.5min is benserazide hydrochloride impurity C, and the retention time of 13.7±0.5min is benserazide hydrochloride impurity E.

[0040] The third object of the present invention is to provide a method for quantitatively detecting benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine tablets based on the above-mentioned identification method, which realizes the content detection of components such as benserazide hydrochloride impurity E in dopamine tablets.

[0041] To achieve the above object, the present invention adopts the following technical solutions:

[0042] The method for quantitatively detecting benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets based on the aforementioned identification method comprises the following steps:

[0043] (1) Separation: using the method described in any one of claims 1 to 4 to separate benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets;

[0044] (2) Detection: Detecting the separated components using the method described in any one of claims 5 to 8 to obtain a chromatogram;

[0045] (3) Content calculation: According to the chromatogram obtained in step (2), the content of each component is calculated by the peak area using the external standard method. Furthermore, before separation, a reference solution and a sample to be tested are prepared using a solvent; the solvent is methanol. The details are as follows:

[0046] (1) Preparation of sample solution: Take an appropriate amount of the product (equivalent to 100 mg of benserazide) and place it in a 100 ml volumetric flask. Add 80 ml of solvent and shake to dissolve. Dilute to the mark with methanol, shake well, filter, and take the filtrate as the sample solution.

[0047] (2) Preparation of reference solution: Take an appropriate amount of benserazide hydrochloride reference substance, weigh it accurately, add solvent to dissolve it and quantitatively dilute it to make a solution containing about 2.3 μg of benserazide hydrochloride per 1 ml, which is used as the reference solution.

[0048] (3) Detection: Accurately measure the test solution and the control solution, such as 5 μl, and inject them into the liquid chromatograph respectively, and record the chromatogram; then use the external standard method to calculate the content of each impurity based on the peak area.

[0049] Furthermore, the calculation formula of each component content is as follows:

[0050] formula:

[0051] Where, Ai is the peak area of ​​the impurity in the chromatogram of the test solution;

[0052] As is the peak area of ​​benserazide in the chromatogram of the reference solution;

[0053] Cs is the concentration of benserazide hydrochloride in the reference solution, mg / ml;

[0054] F1 is the conversion factor between benserazide and its hydrochloride;

[0055] F2 is the impurity correction factor;

[0056] C is the benserazide concentration of the test solution, mg / ml.

[0057] The beneficial effects of the present invention are:

[0058] 1. There is no legal method or literature to detect the impurity E of benserazide hydrochloride in benserazide hydrochloride bulk drug or dobenserazide tablets. The self-built high performance liquid chromatography method of the present invention can perform quality control on the impurity E of benserazide hydrochloride in dobenserazide tablets or benserazide hydrochloride bulk drug, and can separate the impurities related to levodopa and benserazide hydrochloride without interfering with the determination.

[0059] 2. The high performance liquid chromatography method built by the present invention is used to extract the impurity E of benserazide hydrochloride, remove the insoluble auxiliary materials, and the dissolved blank auxiliary materials do not interfere with the determination.

[0060] 3. The analysis method of related substances in dopamine tablets provided by the present invention successfully solves the problem that the degradation impurity benserazide hydrochloride impurity E and the process impurity benserazide hydrochloride impurity C are difficult to separate, and realizes the effective separation, qualitative and quantitative analysis of benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E, and has the characteristics of short separation time, strong specificity, high accuracy, good precision, good durability, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 is the chromatogram of the test solution;

[0062] Figure 2 is the chromatogram of the reference solution;

[0063] Figure 3 The chromatogram is the 100% spiked test solution;

[0064] Figure 4 is the chromatogram of the resolution solution;

[0065] Figure 5 This is the chromatogram of the separation solution at a flow rate of 0.9 ml / min in the chromatographic durability test;

[0066] Figure 6 This is the chromatogram of the separation solution at a flow rate of 1.1 ml / min in the chromatographic durability test;

[0067] Figure 7 This is the chromatogram of the separation solution under the condition of column temperature of 23°C in the chromatographic durability test;

[0068] Figure 8 This is the chromatogram of the separation solution under the condition of column temperature of 27℃ in the chromatographic durability test;

[0069] Fig. 9 This is the chromatogram of the separation solution under the condition of pH value of 3.4 in the chromatography durability test;

[0070] Fig.10 This is the chromatogram of the separation solution under the condition of mobility pH 3.6 in the chromatographic durability test;

[0071] Fig.11 This is the chromatogram of the separation solution under the condition of initial mobility ratio of 93:7 in the chromatographic durability test;

[0072] Fig.12 This is the chromatogram of the separation solution under the condition of initial mobility ratio of 97:3 in the chromatographic durability test;

[0073] Fig.13 This is the chromatogram of the separation solution under the conditions of chromatographic column LC887 in the chromatographic durability test. DETAILED DESCRIPTION

[0074] 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.

[0075] The invention adopts a potassium hexafluorophosphate solution with a concentration of 50mM as a mobile phase A, and keeps the mobile phase A in an acidic environment (screening the mobile phase pH value to be in the range of 2.5-4.5), adopts methanol as a mobile phase B, adjusts the linear gradient elution, and makes the benserazide hydrochloride impurity E peak at a suitable time; the flow rate is set to 1.0ml per minute; a chromatographic column with octadecylsilane bonded silica gel as a filler is used to test a mixed impurity solution (each 1ml contains about 20μg of levodopa, levodopa impurity A, levodopa impurity B, levodopa impurity C, benserazide hydrochloride, benserazide hydrochloride impurity A, benserazide hydrochloride impurity B, benserazide hydrochloride impurity C, benserazide hydrochloride impurity E and benserazide hydrochloride impurity F), and finally obtains the optimal chromatographic conditions.

[0076] The structural formulas of the above-mentioned levodopa, levodopa impurity A, levodopa impurity B, levodopa impurity C, benserazide hydrochloride, benserazide hydrochloride impurity A, benserazide hydrochloride impurity B, benserazide hydrochloride impurity C, benserazide hydrochloride impurity E and benserazide hydrochloride impurity F are shown in Table 1.

[0077] Table 1

[0078]

[0079]

[0080] In the embodiment of the present invention, methanol is used as a solvent / diluent; the sample preparation method is as follows:

[0081] Test solution: Take the product, grind it into powder, accurately weigh an appropriate amount (equivalent to 100 mg of benserazide), put it in a 100 ml volumetric flask, add 80 ml of methanol, shake to dissolve, dilute to the scale with methanol, shake well, filter, and take the filtrate as the test solution.

[0082] Reference solution: Take an appropriate amount of benserazide hydrochloride reference substance, weigh it accurately, add methanol to dissolve it and quantitatively dilute it to make a solution containing about 2.3 μg of benserazide hydrochloride per 1 ml.

[0083] System suitability solution / separation solution: Take an appropriate amount of benserazide hydrochloride system suitability reference substance (containing benserazide hydrochloride, benserazide hydrochloride impurity C, and benserazide hydrochloride impurity E), dissolve it in methanol and dilute it to make a solution of about 1.1 mg per 1 ml; or take an appropriate amount of benserazide hydrochloride, benserazide hydrochloride impurity C, and benserazide hydrochloride impurity E, dissolve them in methanol and dilute them to make a mixed solution containing about 1.1 mg of benserazide hydrochloride and 2 μg of each impurity per 1 ml.

[0084] Sensitivity solution: Accurately measure an appropriate amount of the reference solution and quantitatively dilute it with a solvent to make a solution containing approximately 0.57 μg of benserazide hydrochloride per 1 ml.

[0085] Blank excipient solution: weigh about 586 mg of blank excipient, place in a 100 ml volumetric flask, add an appropriate amount of methanol, shake for 5 minutes, dilute to the scale with methanol, shake well, filter, and obtain the filtrate.

[0086] In the embodiment of the present invention, the chromatographic conditions are as follows:

[0087] 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); potassium hexafluorophosphate solution was used as mobile phase A, methanol was used as mobile phase B, and linear gradient elution was performed according to Table 2 below; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 210 nm; and the injection volume was 5 μl.

[0088] Table 2. Gradient elution schedule

[0089] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 2 95 5 25 55 45 26 95 5 35 95 5

[0090] In the embodiment of the present invention, the preparation method of potassium hexafluorophosphate solution (fluidity A) is as follows: 9.2 g of potassium hexafluorophosphate is taken, 1000 ml of water is added to dissolve it, and the pH value is adjusted to 3.5 with phosphoric acid.

[0091] In the embodiments of the present invention, the quality standard stipulates that it is calculated based on benserazide, which can be converted through the salt conversion coefficient and benserazide hydrochloride.

[0092] Example 1. Specificity

[0093] Take 5 μl of blank solvent, blank excipient solution and system suitability solution respectively, inject according to the method and record the chromatogram.

[0094] The results are shown in Table 3. Figure 4 As shown, blank solvent, excipients, and benserazide hydrochloride impurity C do not interfere with the determination of benserazide hydrochloride impurity E under this chromatographic system, and the separation degree of the chromatographic peaks of benserazide hydrochloride impurity C and benserazide hydrochloride impurity E is 6.04, indicating that this method has good specificity for the determination of benserazide hydrochloride impurity E.

[0095] Table 3. Specificity determination results

[0096]

[0097] Example 2. Accuracy

[0098] 1. Preparation of solution

[0099] Benserazide hydrochloride impurity E stock solution: Accurately weigh about 10 mg of benserazide hydrochloride impurity E, place it in a 50 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well (200 ug / ml).

[0100] Spiked test solution: Take the product, grind it, accurately weigh an appropriate amount (equivalent to 100 mg of benserazide), put it in a 100 ml volumetric flask, add 80 ml of methanol, shake to dissolve, a total of 9 portions, add 80 ml of methanol, accurately add 0.8 ml, 1.0 ml, and 1.2 ml of the above-mentioned benserazide hydrochloride impurity E stock solution, ultrasonically and shake from time to time for 5 minutes to dissolve, cool, dilute to the scale with solvent, shake well, filter, and take the filtrate as 80%, 100% and 120% spiked test solutions (3 portions each).

[0101] Background test solution: Take the product, grind it into powder, accurately weigh an appropriate amount (equivalent to 100 mg of benserazide), put it in a 100 ml volumetric flask, add 80 ml of methanol, shake to dissolve, cool, dilute to the scale with solvent, shake well, filter, and take the filtrate.

[0102] Reference solution: accurately weigh 23 mg of benserazide hydrochloride reference substance, place in a 100 ml volumetric flask, add methanol to ultrasonically dissolve and dilute to the scale, shake well, then accurately measure 1 ml of the solution, place in a 100 ml volumetric flask, dilute to the scale with methanol, shake well, to make a solution containing approximately 2.3 μg of benserazide hydrochloride per 1 ml.

[0103] Impurity reference solution: Accurately measure 1 ml of benserazide hydrochloride impurity E stock solution into a 100 ml volumetric flask, dilute to the scale with methanol, and shake well.

[0104] 2. Accurately pipette 5 μl of the background test solution, spiked test solution, impurity reference solution, and reference solution, respectively, and inject them into the liquid chromatograph, record the chromatogram, and calculate the impurity recovery rate and RSD according to the main component external standard method with correction factor and the impurity external standard method. The results are shown in Table 4. Figure 1-Figure 3 As shown, Figure 1-Figure 3 The corresponding integral results are shown in Tables 5 to 7. The recovery rate of impurity E of benserazide hydrochloride is between 97.3% and 102.8%, the average recovery rate is 100.7%, and the RSD is 1.6%, which meets the accuracy requirements of the method; the accuracy of the correction factor is between 97.7% and 100.9%, and the average is 98.7%, which meets the verification requirements. The results show that the accuracy of this test method is good.

[0105] Table 4. Accuracy test results

[0106]

[0107] Table 5. Figure 1 The integral result table

[0108] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation 1 7.202 Benserazide 31360750 1470437 99.98 0.82 —— 2 13.791 Benserazide Hydrochloride Impurity E 6132 411 0.02 0.81 13.90 total 31366882 1470848 100.00

[0109] Table 6. Figure 2 The integral result table

[0110]

[0111]

[0112] Table 7. Figure 3 The integral results table

[0113] Peak Retention time (min) Peak name Peak area Peak height Peak area% Tailing Factor Separation 1 7.172 Benserazide 30343720 1401413 99.78 0.80 —— 2 13.755 Benserazide Hydrochloride Impurity E 65801 4042 0.22 0.83 13.46 total 30409522 1405455 100.00

[0114] Example 3. Precision

[0115] The results of the "accuracy" test in Example 2 were used to evaluate the RSD of the measured amounts at low, medium and high concentrations and the RSD of the recoveries of 9 samples. The calculation results are shown in Table 8. The RSD of the measured amounts of the spiked samples at low, medium and high concentrations were 0.6%, 2.8% and 1.4%, respectively, and the RSD of the recoveries of 9 samples was 2.0%, indicating good precision of the method.

[0116] Table 8. Results of precision test

[0117]

[0118] Example 4. Solution stability

[0119] Take the 100% spiked test solution and reference solution prepared under the "Accuracy" item of Example 2, place them in the injector at 5°C, accurately measure 5 μl at different time points, inject them into the chromatograph, record the chromatogram, calculate the RSD of the peak area, and calculate the relative deviation compared with 0 hours. The stability test results of the solution are shown in Table 9. Figure 2 , Figure 3 As shown, when the injector is placed at 5°C, the impurity E of benserazide hydrochloride in the spiked test solution is stable within 30 hours, and the peak area RSD is 3.4%. When the injector is placed at 5°C, the benserazide in the reference solution is stable within 30 hours, and the peak area RSD is 1.9%.

[0120] Table 9. Solution stability test results

[0121]

[0122]

[0123] Example 5. Chromatographic Durability

[0124] The system suitability solution and sensitivity solution were accurately measured, and the flow rate, column temperature, pH value of mobile phase buffer salt, and mobile phase ratio were slightly changed. The chromatographic columns of the same brand but different batches were used for testing. The separation between the peaks of benserazide hydrochloride impurities C and E in the system suitability solution, the relative retention time of benserazide hydrochloride impurity E, and the signal-to-noise ratio of the benserazide peak height in the sensitivity solution were recorded. The test results are shown in Table 10. Figure 4-Figure 13 As shown, when the flow rate, column temperature, mobile phase pH value, and mobile phase ratio are slightly changed or different chromatographic columns are used, the resolution solution, the resolution between the benserazide hydrochloride impurity C and the benserazide hydrochloride impurity E peaks are greater than 1.5, and the sensitivity solution, the benserazide peak signal-to-noise ratio is greater than 10; the chromatographic conditions are durable.

[0125] Table 10. Chromatographic durability test results

[0126]

Claims

1. A method for separating benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E by high performance liquid chromatography, characterized in that: The separated components are benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E; the structural formula of the benserazide hydrochloride is shown in Formula I, the structural formula of the benserazide hydrochloride impurity C is shown in Formula II, and the structural formula of the benserazide hydrochloride impurity E is shown in Formula III in sequence; the chromatographic column of the high performance liquid chromatography method uses octadecylsilane bonded silica gel as a filler, and the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a potassium hexafluorophosphate solution, and the mobile phase B is methanol; Separating benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in sequence by gradient elution; 2. The method according to claim 1, characterized in that The sample to be tested is dobenserazide tablets, which contain benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E.

3. The method according to claim 1, characterized in that: The procedure of the gradient elution is: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 2 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 25 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 55:45; At 26 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3; At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 93-97:7-3.

4. The method according to claim 1, characterized in that: The pH value of the mobile phase A is 2.5-4.5; the flow rate of the mobile phase is 0.9-1.1 ml / min; and the column temperature of the chromatographic column is 23-27°C.

5. A method for qualitatively identifying benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets based on the method described in any one of claims 1 to 4, characterized in that: The method according to any one of claims 1 to 4 is used to separate benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets; after separation, detection is performed using a detector with a detection wavelength of 210 nm to obtain a chromatogram.

6. The method according to claim 5, characterized in that According to the retention time, the three components are ranked from short to long as: benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E.

7. The method according to claim 5, characterized in that The mobile phase A is a potassium hexafluorophosphate solution with a pH value of 3.5, and the mobile phase B is methanol; the flow rate of the mobile phase is 1.0 ml / min; the column temperature of the chromatographic column is 25° C.; the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm; Perform linear gradient elution according to the following gradient elution program and obtain the chromatogram: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 2 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 25 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 55:45; At 26 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 95:5; At 35 minutes, 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 retention time of 7.1±0.5min is benserazide hydrochloride, the retention time of 11.4±0.5min is benserazide hydrochloride impurity C, and the retention time of 13.7±0.5min is benserazide hydrochloride impurity E.

9. A method for quantitatively detecting benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets based on the method described in any one of claims 5 to 8, characterized in that: The following steps are involved: (1) Separation: using the method described in any one of claims 1 to 4 to separate benserazide hydrochloride, benserazide hydrochloride impurity C and benserazide hydrochloride impurity E in dopamine hydrazide tablets; (2) Detection: Detecting the separated components using 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 was calculated by the peak area using the external standard method.

10. The method according to claim 9, characterized in that Before separation, a solvent is used to prepare a reference solution and a sample to be tested; the solvent is methanol.

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