A method for detecting related substances of nicardipine hydrochloride injection

Abstract

The application provides a detection method for related substances of nicardipine hydrochloride injection, which adopts high performance liquid chromatography for detection, and detection conditions comprise the following steps: step S1, preparing mobile phase A and mobile phase B; step S2, preparing a test solution, wherein the test solution comprises blank solvent, system suitability solution, nicardipine hydrochloride reference solution and test sample solution; step S3, gradient elution is performed on the test solution in the following order: blank solvent, system suitability solution, nicardipine hydrochloride reference solution and test sample solution by using the mobile phase A and the mobile phase B, so as to obtain a high performance liquid chromatogram; and step S4, analyzing related substances in the nicardipine hydrochloride injection based on the high performance liquid chromatogram obtained in step S3.

Description

Technical Field

[0001] This invention relates to the field of drug detection, specifically to a method for detecting related substances in nicardipine hydrochloride injection. Background Technology

[0002] Nicardipine hydrochloride injection is a common antihypertensive drug. Various impurities (such as degradation products and process impurities) may be generated during its production and storage, directly affecting the drug's safety and efficacy. Therefore, the detection of related substances is crucial in drug quality control.

[0003] The impurities in nicardipine hydrochloride injection are mainly introduced and generated by the degradation of the active pharmaceutical ingredient nicardipine hydrochloride. Referring to USP quality standards and import registration standards for reference preparations, nine known impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, and impurity I) were quantitatively studied as the main impurities. Since the related substances method cannot effectively detect impurity E, a separate method was developed to detect impurity E and it was tested as a separate item. Therefore, the related substances detection method of this product is applicable to the detection of impurities A, B, C, D, F, G, H, and I (i.e., the related substances detection method of this invention is also for impurities other than impurity E). This point will be further explained in the specificity verification section below, the structure of which is as follows: Impurity A: 2,6-Dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid-2-(N-benzyl-N-methyl)-ethyl ester methyl ester

[0004] Impurity B: bis{2-(benzyl(methyl)amino)ethyl}2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate

[0005] Impurity C: Dimethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid ester

[0006] Impurity D: 5-Methoxycarbonyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid

[0007] Impurity F: Methyl 3-methyl-3'-nitro-5-oxo-1,2,5,6-tetrahydro-[1,1'-biphenyl]-2-carboxylic acid ester

[0008] Impurity G: Methyl 5-methyl-3'-nitro-3-oxo-1,2,3,6-tetrahydro-[1,1'-biphenyl]-2-carboxylic acid

[0009] Impurity H: Methyl 2-acetyl-3-(3-nitrophenyl)-5-oxohexanoate and its isomers Impurity I: 5-((2-(benzyl(methyl)amino)ethoxy)carbonyl)-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid

[0010] The standard limits for the above impurities in each pharmacopoeia are shown in Table 1 below:

[0011] According to our research, the relevant substance detection methods for this product are included in ChP2020 (Chinese Pharmacopoeia 2020), JP18 (Japanese Pharmacopoeia), USPNF (Official as of 20-Sep-2023) (United States Pharmacopoeia) and the reference preparation import drug registration standard (standard number: JX20170097), but are not included in BP and EP.

[0012] In existing technologies, the ChP and imported reference formulation methods suffer from insufficient separation due to isocratic elution, interfering with the detection of the test sample. The USP and JP methods cannot be directly transferred to the ChP system due to differences in mobile phase and detection conditions. The imported reference formulation drug registration standard (standard number: JX20170097) method reproducibility shows that the solvent peak interferes with the detection of impurities H, D, F, and G; and impurities D, F, G, and H are not completely separated in the mixed sample. Therefore, there is an urgent need for a detection method that can meet the requirements for separation of related substances in nicardipine hydrochloride injection while also possessing high sensitivity and good stability. Summary of the Invention

[0013] To address the shortcomings of existing technologies, this invention provides a method for detecting related substances in nicardipine hydrochloride injection. The method of this invention not only meets the requirements for detection sensitivity but is also unaffected by blank excipients, exhibits high separation of impurities, good stability, and high repeatability.

[0014] This invention provides a method for detecting related substances in nicardipine hydrochloride injection, employing high-performance liquid chromatography (HPLC). The detection conditions include using an octadecylsilane-bonded silica gel column and a ghost peak trapping column. The detection method comprises the following steps: Step S1, preparing mobile phase A and mobile phase B; Step S2, preparing a test solution, comprising: blank solvent, system suitability solution, nicardipine hydrochloride reference solution, and test solution; Step S3, performing gradient elution of the test solution with mobile phase A and mobile phase B in the following order: blank solvent, system suitability solution, nicardipine hydrochloride reference solution, and test solution, thereby obtaining an HPLC chromatogram; Step S4, analyzing the related substances in the nicardipine hydrochloride injection based on the HPLC chromatogram obtained in step S3.

[0015] Furthermore, the ghost peak trapping column is installed downstream of the chromatograph's injection pump and upstream of the injector.

[0016] Furthermore, the related substances of the nicardipine hydrochloride injection include impurities A, B, C, D, F, G, H, and I.

[0017] Further, the gradient elution conditions in step S3 are as follows: 0 min~15 min, 100% mobile phase A; 15 min~20 min, 100% mobile phase A linearly changes to 75% mobile phase A and 25% mobile phase B; 20 min~25 min, 75% mobile phase A and 25% mobile phase B; 25 min~30 min, 75% mobile phase A and 25% mobile phase B linearly change to 50% mobile phase A and 50% mobile phase B; 30 min~45 min, 50% mobile phase A and 50% mobile phase B; 45 min~55 min, 50% mobile phase A and 50% mobile phase B linearly change to 100% mobile phase B; 55 min~70 min, 100% mobile phase B; 70 min~70.1 min, 100% mobile phase B linearly changes to 100% mobile phase A; 70.1 min~80 min... min, 100% mobile phase A.

[0018] Further, the mobile phase A is a mixture formed by mixing a 0.01 mol / L potassium dihydrogen phosphate solution with methanol, wherein the mass ratio of the 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 60:40. The mobile phase B is a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of the 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 20:80.

[0019] Further, the preparation process of the test solution is as follows: take 5 vials of nicardipine hydrochloride injection, mix well, accurately weigh 5 mL and place it in a 10 mL volumetric flask, dilute to the mark with the mobile phase A and shake well, thereby obtaining the test solution.

[0020] Furthermore, the preparation process of the system suitability solution is as follows: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I and nicardipine hydrochloride standard, dissolve and dilute them with solvent to prepare a mixed solution containing 0.5 mg of nicardipine hydrochloride standard and 5 μg of each impurity per 1 ml of solvent, which is the system suitability solution.

[0021] Furthermore, the preparation process of the nicardipine hydrochloride reference solution is as follows: take the nicardipine hydrochloride reference standard, dissolve it in mobile phase A and quantitatively dilute it to a solution containing 5 μg of nicardipine hydrochloride reference standard per 1 mL of solution, which is the nicardipine hydrochloride reference solution; and the blank solvent has the same composition as the mobile phase A.

[0022] Further, in step S4-1, record the peak area of ​​each peak in the spectrum; Step S4-2: Calculate the content of each impurity based on the peak area of ​​nicardipine hydrochloride and the content of nicardipine hydrochloride in the spectrum.

[0023] Furthermore, the detection conditions include: a detection wavelength of 236 nm, a flow rate of 1.0 mL / min, an injection volume of 20 μL, and a column temperature of 28 °C.

[0024] The present invention provides a method for detecting related substances in nicardipine hydrochloride injection. Gradient elution of related substances in nicardipine hydrochloride injection completely separates impurities F and G, as well as nicardipine and impurity C, from the mixed sample, and effectively separates other impurities. The present invention incorporates ghost peak trapping column filtration, optimizes the specific composition of the mobile phase and test conditions, achieving effective impurity separation. The detection method of the present invention overcomes the instrument pump sticking problem caused by the mobile phase in various pharmacopoeias, improving detection sensitivity and lowering the detection limit while increasing instrument durability and improving the stability of the detection method. Therefore, the method provided by the present invention has high precision, low detection limit, strong stability, good repeatability, and high accuracy. Attached Figure Description

[0025] Figure 1A This is a high-performance liquid chromatogram of a blank solvent detected by the method according to the present invention.

[0026] Figure 1B High-performance liquid chromatogram of a system suitability solution detected by the method according to the present invention.

[0027] Figure 1C The image shows a high-performance liquid chromatogram of the first group of nicardipine hydrochloride reference solutions detected by the method according to the present invention.

[0028] Figure 1D The image shows a high-performance liquid chromatogram of the second set of nicardipine hydrochloride reference solutions detected by the method according to the present invention.

[0029] Figure 1E The image shows a high-performance liquid chromatogram of the third group of nicardipine hydrochloride reference solutions detected by the method according to the present invention.

[0030] Figure 1F This is a high-performance liquid chromatogram of the test solution detected by the method according to the present invention.

[0031] Figure 2 Linearity plot for related substance method validation - linearity test of impurity A.

[0032] Figure 3 Linearity plot for related substance method validation - impurity B linearity test.

[0033] Figure 4 Linearity plot for related substance method validation - impurity C linearity test.

[0034] Figure 5 Linearity plot for related substance method validation - impurity D linearity test.

[0035] Figure 6 Linearity plot for related substance method validation - impurity F linearity test.

[0036] Figure 7 Linearity plot for related substance method validation - impurity G linearity test.

[0037] Figure 8 Linearity plot for related substance method validation - impurity H linearity test.

[0038] Figure 9 Linearity plot for related substances method validation - impurity I linearity test.

[0039] Figure 10 Linearity graph for the linearity test of nicardipine hydrochloride injection for related substances method validation.

[0040] Figure 11 Spectra for related substance method validation - specificity test. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. The principles and features of the present invention are described below with reference to the accompanying drawings. It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The embodiments given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0042] It should be noted that, in this application, the related substances for nicardipine hydrochloride injection refer to the collective name of the above-mentioned impurities A, B, C, D, F, G, H and I (the reason why impurity E is not included in the detection method of this invention will be described in detail below, and will not be repeated here).

[0043] Furthermore, the reference standard (i.e., standard article) mentioned in this invention is a standard measure provided by the Drug Standard Reference Material Research Center, serving as a standard content in content determination, rather than a specific limitation.

[0044] Furthermore, it should be noted that, unless otherwise specified, the so-called "preparation of a solution" mentioned or not mentioned in the following content of this invention refers to the preparation using mobile phase A as the solvent. For example, in the verification of the limit of quantitation and limit of detection, the phrase "preparing a solution of a certain concentration using nicardipine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I reference standard, and then gradually diluting it..." refers to "preparing a solution of a certain concentration using mobile phase A as the solvent using nicardipine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I reference standard, and then gradually diluting it using mobile phase A...".

[0045] This invention provides a method for detecting related substances in nicardipine hydrochloride injection, using high-performance liquid chromatography (HPLC). The related substances detected in nicardipine hydrochloride injection include impurities A, B, C, D, F, G, H, and I, as well as some unknown impurities. Their structures and related descriptions are as described above and will not be repeated here. The detection conditions of this invention include the use of an octadecylsilane-bonded silica gel column and a ghost peak trapping column. Specifically, the ghost peak trapping column is installed downstream of the chromatograph's injection pump and upstream of the injector. Furthermore, the detection conditions provided in this application include: a detection wavelength of 236 nm, a flow rate of 1.0 mL / min, an injection volume of 20 μL, and a column temperature of 28°C.

[0046] Example 1 The detection method provided by this invention has the following steps: Step S1: Preparation of mobile phase A: Mobile phase A is a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 60:40.

[0047] Preparation of mobile phase B: Mobile phase B is a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 20:80.

[0048] It should be noted that the potassium dihydrogen phosphate solution is a 0.01 mol / L potassium dihydrogen phosphate solution prepared with ultrapure water.

[0049] Step S2: Prepare the test solution, which includes: Preparation of blank solvent: The blank reagent has the same composition as the mobile phase A, that is, a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 60:40.

[0050] Preparation of system suitability solution: The preparation process of system suitability solution is as follows: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I and nicardipine hydrochloride standard, dissolve and dilute them with solvent to prepare a mixed solution containing 0.5 mg of nicardipine hydrochloride standard and 5 μg of each impurity per 1 ml of solvent. This is the system suitability solution.

[0051] Preparation of nicardipine hydrochloride reference solution: Dissolve and quantitatively dilute nicardipine hydrochloride reference standard with mobile phase A to obtain a solution containing 5 μg of nicardipine hydrochloride reference standard per 1 mL. Preparation of the test solution: Take 5 vials of nicardipine hydrochloride injection, mix well, accurately weigh 5 mL, place in a 10 mL volumetric flask, dilute to the mark with the mobile phase A and shake well to obtain the test solution.

[0052] The blank solvent has the same composition as mobile phase A.

[0053] Step S3: The test solution is subjected to gradient elution with mobile phase A and mobile phase B in the following order: blank solvent, system suitability solution, reference solution, and test solution, thereby obtaining a high-performance liquid chromatogram.

[0054] In addition, it should be noted that the blank solvent mentioned above is used for baseline calibration.

[0055] The gradient elution conditions are shown in Table 2 below: Table 2: Gradient elution conditions for the detection of related substances in nicardipine hydrochloride injection

[0056] The chromatographic conditions were as follows: the column used was a YMC-pack Pro C18, 4.6 mm × 150 mm, 3 μm column; the ghost peak collection column used was a Welch Ghost-Buster column, 4.6 mm × 50 mm; the detection wavelength was 236 nm, the flow rate was 1.0 ml / min, the injection volume was 20 μL, and the column temperature was 28℃.

[0057] Specifically, the above-mentioned test solutions were injected into the liquid chromatograph in the order described above, and the high-performance liquid chromatogram was recorded. The chromatogram is shown in Figure 1.

[0058] Step S4: Analyze the relevant substances in the nicardipine hydrochloride injection based on the high performance liquid chromatogram obtained in step S3.

[0059] Specifically, the analysis of relevant substances in nicardipine hydrochloride injection in step S4 includes: Step S4-1: Record the peak area of ​​each peak in the spectrum; after injection, the system records and can directly calculate the peak area of ​​each peak. In some cases, the retention time can also be recorded, which can be used for subsequent calculation of the content of related substances.

[0060] Step S4-2: Calculate the content of each impurity based on the peak area of ​​nicardipine hydrochloride and the content of nicardipine hydrochloride in the spectrum. The specific calculation is performed using the following formula: The F-value of nicardipine hydrochloride = concentration of nicardipine hydrochloride reference solution / average peak area of ​​nicardipine hydrochloride reference solution Other impurity content = F value of nicardipine hydrochloride × peak area of ​​other impurities / concentration of test solution.

[0061] The high-performance liquid chromatogram obtained by gradient elution of the test solution with mobile phase A and mobile phase B in the above-described order using the method of the present invention is shown below. Figures 1A to 1F As shown, where Figure 1A High-performance liquid chromatogram of blank solvent; Figure 1B High-performance liquid chromatogram of the system suitability solution; Figures 1C to 1E The image shows the high-performance liquid chromatograms of three sets of nicardipine hydrochloride reference solutions. To calculate the impurity content, three sets of nicardipine hydrochloride reference solutions are usually tested, and the final calculation is based on the average value. Figure 1F This is the high-performance liquid chromatogram of the test solution.

[0062] The blank solvent was used as a baseline control spectrum.

[0063] Table 3 below shows the peak area correspondence of the high performance liquid chromatograms of the system-adaptive solutions.

[0064] Table 3. Peak area correspondence of high performance liquid chromatograms for system-adaptive solutions.

[0065] from Figure 1B As can be seen from the chromatogram and Table 3 above, the system suitability solution prepared by the method of the present invention has good impurity separation, the test sample has good impurity separation, and the instrument has good durability.

[0066] Figure 1F The test sample solution was prepared by mixing five vials of nicardipine hydrochloride injection from a randomly selected batch, weighing 5 mL tightly, and placing it in a 10 mL volumetric flask. The solution was diluted to the mark with mobile phase A and shaken well. The labeled concentration of this batch of nicardipine hydrochloride injection is 1.0 mg / mL. Since the volume taken was 5 mL and the dilution factor was 2, the concentration of the test solution was 0.5 mg / mL.

[0067] The highest peak in the figure is the peak of nicardipine hydrochloride, and the remaining two smaller peaks are impurity peaks. Tables 4 to 6 below are the peak area correspondence tables of the high performance liquid chromatograms of the nicardipine hydrochloride reference solution, and Table 7 is the peak area correspondence table of the high performance liquid chromatograms of the test solution.

[0068] Table 4. Peak area correspondence of the first group of nicardipine hydrochloride reference solutions.

[0069] Table 5. Peak area correspondence of nicardipine hydrochloride reference solution in Group 2.

[0070] Table 6. Peak Area Correspondence of Nicardipine Hydrochloride Reference Solution in Group 3

[0071] Table 7. Peak Area Correspondence of Test Sample Solutions

[0072] Based on the data in Tables 4 to 7 above, the peak area homogeneity of the nicardipine hydrochloride reference solution was calculated to be 4.751, and its concentration was 0.005 mg / mL (5 μg / mL). According to the calculation formula above, the F-value of nicardipine hydrochloride is = 0.005 / 4.751 = 1.052 × 10⁻⁶. -3 ; Taking impurity A as an example, by comparing the spectrum of the test solution with that of the system suitability solution, it can be determined that the impurity contained in this batch of test solution is impurity A, and the other impurity peak is an unknown impurity. The content of impurity A is calculated using the above formula as 1.052 × 10⁻⁶. -3 ×0.135 / 0.5=2.84×10 -4 ; It should be noted that, in addition to the known impurities mentioned above, nicardipine hydrochloride injection also contains some unknown impurities. However, since the impact of these unknown impurities on the evaluation criteria of nicardipine hydrochloride injection is negligible, the pharmacopoeia does not require special testing for these unknown impurities. According to the method of this invention, the content of these unknown impurities can also be determined: the content of the unknown impurity = 1.052 × 10⁻⁶. -3 ×0.332 / 0.5=6.99×10 -4 .

[0073] The method of this invention can not only accurately detect known impurities, but also calculate the content of unknown impurities based on the spectrum. When the content of unknown impurities is higher than the limit, the batch of nicardipine hydrochloride injection will be recovered or otherwise processed. Thus, the method provided by this invention can better separate various impurities, thereby achieving accurate detection of related substances in nicardipine hydrochloride injection.

[0074] The method provided by this invention achieves excellent separation of impurities, enabling the calculation of other impurity contents based on the spectrum. It exhibits high impurity separation and overcomes the technical problem of sample sticking to the pump. In practical operation, the above formulas can be entered into a computer, allowing the calculated impurity contents to be directly obtained from the generation of the spectrum. Therefore, the detection method provided by this invention is fast and accurate.

[0075] Example 2 The above-described method of the present invention is subjected to methodological verification to further determine the feasibility of the method of the present invention.

[0076] Solution preparation: Using the chromatographic conditions described in Example 1, and following the method described in Example 1, mobile phase A, mobile phase B, and reference solution were prepared. Additionally, the following test solutions were prepared: Sensitivity solution: Take nicardipine hydrochloride reference standard, add it to mobile phase A to dissolve and quantitatively dilute to prepare a solution containing 0.25 μg of nicardipine hydrochloride reference standard per 1 mL.

[0077] It should be noted that, unless otherwise specified, the solutions prepared above shall be used in the following methodological verification experiments.

[0078] 1. System applicability verification Take the above mobile phase A, reference solution, system suitability solution and sensitivity solution, where mobile phase A is used as blank solvent and is injected once continuously. Prepare one part of system suitability solution and inject it once continuously. Prepare one part of sensitivity solution and inject it once continuously. Prepare one part of reference solution and inject it five times continuously (the results are recorded as reference solution-1 to reference solution-5).

[0079] Elution was then carried out under the same elution conditions as in Example 1.

[0080] The acceptable criteria for the applicability verification of this system are as follows: In the system suitability solution chromatogram, the resolution between the main peak and the adjacent impurity peak should be greater than 1.5, and the resolution between each impurity should not be less than 1.2. In a sensitivity solution chromatogram, the signal-to-noise ratio of the peak height of nicardi hydrochloride should be greater than 10. In the chromatogram of the reference solution, the peak area and RSD of nicardipine hydrochloride and impurity A should not exceed 2.0% (n=5), and the retention time RSD should not exceed 1.0% (n=5). Table 8 below shows the chromatographic data of the system suitability test results. Table 8: Chromatographic data of system suitability test results

[0081] As can be seen from the data in Table 8 above: In the system suitability solution chromatogram, the resolution between the main peak and the adjacent impurity peak is greater than 1.5, and the resolution between each impurity is greater than 1.2. In the sensitivity solution chromatogram, the signal-to-noise ratio of the peak height of nicardi is 195, which is greater than 10. In the chromatogram of the reference solution, the peak area RSD of nicardipine and impurity A was 0.01%, which is less than 2.0% (n=5), and the retention time RSD was 0.01%, which is less than 1.0% (n=5), meeting the validation requirements.

[0082] 2. Specificity verification 1) Impurity localization and blank interference test Validation method: Inject blank solvent, blank excipient, and mixed sample solution once each. The preparation methods for each solution are as follows: Blank solvent: 0.01 mol / L potassium dihydrogen phosphate solution - methanol (60:40); Blank excipient: Accurately measure 5 ml of blank excipient and place it in a 10 ml volumetric flask. Dilute to the mark with solvent.

[0083] Mixed sample solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I and nicardipine hydrochloride reference standard, and dilute with solvent to prepare a mixed solution containing approximately 0.5 mg of nicardipine hydrochloride, 200 μg of impurity E and 5 μg of other impurities per 1 ml.

[0084] For each known impurity localization solution: Take appropriate amounts of reference standards for impurities A, B, C, D, F, G, H, and I, dissolve and dilute them with solvents to prepare solutions containing approximately 5 μg of each known impurity per ml; take an appropriate amount of impurity E, dissolve and dilute it with solvents to prepare solutions containing approximately 200 μg of the known impurity per ml; take an appropriate amount of nicardipine hydrochloride, dissolve and dilute it with solvents to prepare solutions containing approximately 0.5 mg of nicardipine hydrochloride per ml.

[0085] Acceptable standards: The blank solvent and blank excipient should not cause any interference at the main peak and the peaks of known impurities; For system suitability, the resolution between the main peak and adjacent known impurity peaks in the solution should be greater than 1.5, and the resolution between each impurity should not be less than 1.2.

[0086] Table 9 below lists the results of the specificity test.

[0087] Table 9 List of Specificity Test Results

[0088] From the table above and Figure 11 It can be seen that the blank solvent and blank excipients do not interfere with the main peak and the peaks of each known impurity; the resolution between the main peak and the adjacent impurity peak in the system suitability solution is greater than 1.5, and the resolution between each impurity is greater than 1.2, which meets the requirements.

[0089] Under the same chromatographic conditions, impurity E elutes earlier, and its peak position easily overlaps with other peaks that appear earlier in the localization solutions of known impurities, making accurate differentiation difficult. Therefore, impurity E is tested as a separate item to eliminate instrument interference and ensure the specificity and accuracy of related substance detection. 2) Forced degradation test Forced degradation tests are conducted in solid / liquid states and employ methods such as strong light irradiation, high temperature, oxidative decomposition, acid / alkali hydrolysis, and water bath. The amount of degradation products is adjusted by the exposure time, temperature, and concentration to achieve a main peak degradation rate of 5%-10%.

[0090] The specific experimental method is as follows: Blank solvent: 0.01 mol / L potassium dihydrogen phosphate solution - methanol (60:40).

[0091] Blank excipient: Measure 5.00 ml of blank excipient, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0092] System suitability solution: Prepared using the method described above.

[0093] Undamaged sample solution: Measure 5.00 ml of nicardipine hydrochloride injection into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0094] Acid-induced degradation of blank excipient: Measure 5.00 ml of blank excipient into a 10 ml volumetric flask, add 0.5 ml of 5 mol / L hydrochloric acid, place in a water bath at 80°C for 2 hours, cool to room temperature, add 0.5 ml of 5 mol / L sodium hydroxide aqueous solution for neutralization, dilute to the mark with mobile phase A, and shake well.

[0095] Acid destruction: Measure 5.00 ml of nicardipine hydrochloride injection into a 10 ml volumetric flask, add 0.5 ml of 5 mol / L hydrochloric acid, place in a water bath at 80℃ for 2 hours, cool to room temperature, add 0.5 ml of 5 mol / L sodium hydroxide aqueous solution for neutralization, dilute to the mark with solvent, and shake well.

[0096] Alkali-induced degradation of blank excipient: Measure 5.00 ml of blank excipient into a 10 ml volumetric flask, add 0.5 ml of 5 mol / L sodium hydroxide aqueous solution, place in a water bath at 60℃ for 3 hours, cool to room temperature, add 0.5 ml of 5 mol / L hydrochloric acid for neutralization, dilute to the mark with solvent, and shake well.

[0097] Alkali destruction: Measure 5.00 ml of nicardipine hydrochloride injection into a 10 ml volumetric flask, add 0.5 ml of 5 mol / L sodium hydroxide aqueous solution, place in a water bath at 60℃ for 3 hours, cool to room temperature, add 0.5 ml of 5 mol / L hydrochloric acid for neutralization, dilute to the mark with solvent, and shake well.

[0098] Oxidative destruction of blank excipients: Measure 5.00 ml of blank excipients into a 10 ml volumetric flask, add 1.0 ml of 3% hydrogen peroxide, place in a water bath at 60°C for 1 hour, dilute to the mark with solvent, and shake well.

[0099] Oxidative damage: Measure 5.00 ml of nicardipine hydrochloride injection into a 10 ml volumetric flask, add 1.0 ml of 3% hydrogen peroxide, place in a 60℃ water bath for 1 hour, dilute to the mark with solvent, and shake well.

[0100] Water bath destruction of blank excipients: Measure 5.00 ml of blank excipients into a 10 ml volumetric flask, place it in a 95°C water bath for 5 hours, cool to room temperature, dilute to the mark with solvent, and shake well.

[0101] Water bath destruction: Measure 5.00 ml of nicardipine hydrochloride injection into a 10 ml volumetric flask, place it in a 95°C water bath for 5 hours, cool to room temperature, dilute to the mark with solvent, and shake well.

[0102] High temperature destruction of blank excipients: Take 5.00 ml of blank excipients that have been placed at 60℃ for 10 days and place them in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0103] High temperature destruction: Take 5.00 ml of nicardipine hydrochloride injection that has been stored at 60℃ for 10 days and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0104] Light exposure to destroy blank excipients (remove labels and packaging): Take 5.00 ml of blank excipients that have been stored at 5000 lx for 10 days and place them in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0105] Light damage (removal of label and packaging): Take 5.00 ml of nicardipine hydrochloride injection that has been stored at 5000 lx for 10 days and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0106] Light-induced damage to blank excipients (transparent ampoules): Take 5.00 ml of blank excipients that have been placed under a fluorescent lamp for 48 hours and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0107] Light damage (transparent ampoule): Take 5.00 ml of nicardipine hydrochloride injection that has been placed under a fluorescent lamp for 48 h and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0108] Acceptable standards: Under the forced degradation condition, the separation degree between the main peak and the adjacent impurity peak should be greater than 1.5, the material balance should be in the range of 90-110%, and the purity of the main peak should meet the requirements.

[0109] Table 10 below lists the results of the forced degradation test.

[0110] Table 10 List of Forced Degradation Test Results

[0111] Note: ND indicates not detected, N / A indicates not applicable.

[0112] As shown in the table above: Under forced degradation, the resolution between the main peak and adjacent impurity peaks is greater than 1.5. Compared with the undestructed chromatogram, this product is relatively stable under water bath degradation and light irradiation (transparent ampoule) conditions. Light irradiation (removing outer packaging and label), alkali degradation, and high-temperature degradation cause slight degradation, while oxidative degradation significantly leads to impurity production. Light irradiation (removing outer packaging and label) causes slight degradation, with impurity A increasing from 0.154% to 0.656%. Acid degradation also causes slight degradation, with impurity A... The percentage of impurity A increased from 0.154% to 0.258%, impurity H increased from 0.018% to 0.166%, and the percentage of unknown impurity (tR 36.104 min) increased from undetectable to 0.240%. Alkali degradation conditions resulted in slight degradation, with impurity A increasing from 0.154% to 0.416%, impurity H from 0.018% to 0.116%, and the percentage of unknown impurity (tR 54.888 min) from undetectable to 0.117%. High-temperature degradation conditions also resulted in slight degradation, with the main degradation impurities being impurity A, impurity F, and impurity H. Impurity A increased from 0.154% to 0.327%, impurity H from 0.018% to 0.077%, and impurity F from undetectable to 0.283%. Oxidative degradation resulted in significant degradation, with the largest degradation impurity being impurity A, increasing from 0.154% to 6.027%. Under the specified chromatographic conditions, the degradation product peaks generated by the destructive test can be effectively detected, with a recovery rate of 95.6% to 100.8%. Material balance is maintained, and the purity of the main peak is greater than 990, which meets the validation requirements.

[0113] 3) Limit of quantitation and limit of detection Test method: Solution preparation: Blank solvent: Take mobile phase A as blank solvent and inject it once.

[0114] Limit of quantitation solution: Prepare a solution of a certain concentration using reference standards of nicardipine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I (see Table 11 for specific concentrations). Through stepwise dilution, the concentration at which the signal-to-noise ratio is approximately 10 can be used as the limit of quantitation concentration. Inject the solution 6 times consecutively.

[0115] Detection limit solution: Prepare a solution of nicardipine hydrochloride, impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I reference standard with a certain concentration (see Table 12 for specific concentrations). Through stepwise dilution, the concentration at which the signal-to-noise ratio is about 3 can be used as the detection limit. Inject once.

[0116] Acceptable standards When the signal-to-noise ratio is approximately 3, the corresponding concentration is the limit of detection; when the signal-to-noise ratio is approximately 10, the corresponding concentration is the limit of quantitation.

[0117] The peak area RSD of each component in 6 repeated determinations at the limit of quantitation should not exceed 20%, and the retention time RSD should not exceed 2%.

[0118] Tables 11 and 12 below list the results of the limit of quantitation test and the limit of detection test, respectively.

[0119] Table 11 Results of Limit of Quantitation Test

[0120] Table 12 Detection Limit Test Results

[0121] The data results from Tables 11 and 12 above show that: Limit of quantitation: The S / N of the limits of quantitation for nicardipine hydrochloride and all known impurities is greater than 10; after six consecutive injections of the limit of quantitation solution, the RSD of the peak area of ​​all known impurities and nicardipine is less than 20%, and the RSD of the retention time is less than 2%, which meets the validation requirements.

[0122] Detection limits: The S / N ratios of the detection limits for nicardipine hydrochloride and all known impurities are greater than 3, which meets the validation requirements.

[0123] 4) Linearity and Range Experimental Method: Within the limit of quantitation to a certain concentration range (twice the impurity limit concentration), prepare 6 mixed solutions with different concentrations (see Table 13 below for specific concentrations). Measure the peak area of ​​each component. Plot concentration on the x-axis (X) and average peak area on the y-axis (Y) for linear regression analysis. Solution preparation method is as follows: Blank solvent: Use mobile phase A as the blank solvent and inject once.

[0124] Prepare one system suitability solution and inject it into one syringe.

[0125] Prepare one dose of the sensitivity solution and inject it into one syringe.

[0126] Reference solution: The reference solution was prepared according to the method in Example 1.

[0127] Reference stock solution: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H, impurity I and nicardipine hydrochloride reference standard, add them to mobile phase A to dissolve and dilute to prepare a mixed solution containing approximately 10 μg each of impurity B, impurity C, impurity D, impurity I and nicardipine hydrochloride, 25 μg of impurity A, 15 μg of impurity H, and 30 μg each of impurity F and impurity G per ml.

[0128] The preparation methods for each concentration of reference solution are as follows: (prepare one part for each concentration, and inject each part of the sample twice).

[0129] Table 13 below lists the linearity and range results for each impurity.

[0130] Table 13 Linearity and Range Results for Each Impurity

[0131] Acceptable criteria: linear correlation coefficient r ≥ 0.990, |Y-intercept| / 100% response value ≤ 25%.

[0132] Tables 14 to 22 below list the linearity test results of each impurity with nicardipine hydrochloride injection. The above results correspond to the attached table. Figures 2 to 10 Linear relationship graph.

[0133] Table 14 Linearity test results for impurity A

[0134] Table 15 Linearity test results for impurity B

[0135] Table 16 Linearity test results for impurity C

[0136] Table 17 Linearity test results for impurity D

[0137] Table 18. Linearity test results for impurity F

[0138] Table 19 Linearity test results for impurity G

[0139] Table 20 Linearity test results for impurity H

[0140] Table 21 Linearity test results for impurity I

[0141] Table 22 Linearity test results of nicardipine hydrochloride injection

[0142] Based on the data in Tables 14 to 22 above, and Figures 2 to 10 It can be seen that, in the process of detecting related substances in nicardipine hydrochloride injection using the method of the present invention, the correlation coefficient r is greater than 0.990 and the 100% y-intercept response value is less than 25%, which all meet the requirements.

[0143] 5) Repeatability test Experimental method: Prepare the following solution, prepare 6 parallel batches, and calculate the RSD of the results.

[0144] Blank solvent: Use mobile phase A as blank solvent and inject once.

[0145] Prepare one system suitability solution and inject it once.

[0146] Sensitivity solution: Prepare the sensitivity solution according to the method in Example 1. Prepare one part and inject it once.

[0147] Reference solution: Prepare the reference solution according to the method in Example 1. Prepare one portion and inject it three times.

[0148] Test solution: Prepare the test solution according to the method in Example 1. Prepare 6 portions and inject once.

[0149] Acceptable standards: In the 6 test sample solutions, the RSD of impurities with a content of 0.01% to <0.1% should be ≤10%, and the RSD of impurities with a content of 0.1% to <0.5% should be ≤3%.

[0150] Table 23 below lists the results of the repeatability test.

[0151] Table 23 List of Repeatability Test Results

[0152] As can be seen from the data in the table above, in the determination results of the 6 test solutions, the RSD of impurities with a content of 0.01% ≤ content < 0.1% is ≤ 10%, and the RSD of impurities with a content of 0.1% ≤ content < 0.5% is ≤ 3%; which meets the verification requirements and indicates that the method of the present invention has good repeatability.

[0153] 6) Intermediate precision Using the method described in the repeatability test above, six additional test solutions were measured, and two experimenters, A and B, conducted the experiments using different instruments. It should be noted that, to avoid repeated testing, the data from the repeatability test above can be used as the experimental results for experimenter A (therefore, the first row of Table 23 is written as "Repeatability-1"). Thus, Table 24 below lists the data results obtained by experimenter B using another instrument (similarly, the first row of Table 24 is written as "Repeatability-2"), and Table 25 lists the results of the intermediate precision test.

[0154] Table 24 Precision test results of the 6 additional test solutions

[0155] Table 25 Comparison of test results for testers A and B using different instruments

[0156] As can be seen from the data results in Tables 23, 24 and 25 above, among the 12 spiked test solutions, the RSD of impurities with a content of 0.01% ≤ content < 0.1% is ≤ 15%, and the RSD of impurities with a content of 0.1% ≤ content < 0.5% is ≤ 6%; which meets the validation requirements and indicates that the intermediate precision of this method is good.

[0157] 7) Accuracy test Test method: Take an appropriate amount of nicardipine hydrochloride injection with known impurity content, add an appropriate amount of impurity reference stock solution, add mobile phase A to dissolve and dilute to prepare three spiked test solutions containing 50%, 100%, and 150% of the limit concentration of each impurity. Prepare three replicates for each concentration, for a total of nine. Take the above nine spiked test solutions and determine them according to the method in Example 1, and calculate the recovery rate of each impurity in the spiked test solutions.

[0158] Recovery rate (%) = (Total measured amount - Original amount of each impurity in the sample) / Amount added × 100% The solution preparation method is the same as that described in Example 1 regarding the preparation of blank solvent, system adaptability solution and sensitivity solution, wherein the blank solvent is injected once.

[0159] Prepare one system suitability solution and inject it once.

[0160] Prepare one dose of the sensitivity solution and inject it once.

[0161] In addition, prepare the following solutions: Reference stock solution: Take appropriate amounts of each of the reference standards A, B, C, D, F, G, H, and I, accurately weigh them, dissolve and dilute them in mobile phase A to prepare a mixed solution containing 5 μg each of impurities B, C, D, and I, 12.5 μg of impurity A, 7.5 μg of impurity H, and 15 μg each of impurities F and G per 1 ml.

[0162] Impurity reference solution: Accurately measure 2 ml of the above impurity stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and mix well. (Preferably, each 1 mL contains 1 μg each of impurity B, impurity C, impurity D, and impurity I, 2.5 μg of impurity A, 1.5 μg of impurity H, and 3 μg each of impurities F and G). Prepare one dose and inject three times.

[0163] Mix the test solution: Take 10 vials of this product and mix well.

[0164] Test solution: Accurately measure 5 ml of the mixed test solution and place it in a 10 ml volumetric flask. Dilute to the mark with mobile phase A and mix well. Prepare one dose and inject once.

[0165] 50% spiked test solution: Accurately measure 5 ml of the mixed test solution and place it in a 10 ml volumetric flask. Accurately add 1 ml of the reference stock solution and dilute to the mark with solvent. Prepare 3 aliquots and inject each once.

[0166] 100% Spiked Test Solution: Accurately measure 5 ml of the mixed test solution and place it in a 10 ml volumetric flask. Accurately add 2 ml of the reference stock solution and dilute to the mark with solvent. Prepare three aliquots and inject each once. 150% Spiked Test Solution: Accurately measure 5 ml of the mixed test solution and place it in a 10 ml volumetric flask. Accurately add 3 ml of the reference stock solution and dilute to the mark with solvent. Prepare three aliquots and inject each once.

[0167] Acceptable criteria: The recoveries of impurities A, B, C, D, F, G, H and I at each concentration should be between 90% and 108%, and the RSD should be ≤3%.

[0168] Table 26 below lists the results of the accuracy test. Table 26 Accuracy Test Results

[0169] As can be seen from the results in Table 26, the recovery rates of each known impurity were between 90% and 108% at the nine concentration levels, and the RSDs were all less than 3%, which met the verification requirements and indicated that the method of the present invention had good accuracy.

[0170] 8) Solution stability test Test methods: Take the reference solution (prepared as in Example 1), place it at room temperature, and inject it at regular intervals (e.g., 0, 3, 6, 12, 18, 24, 36, 48 hours) to examine the stability of the solution. Take the test solution (prepared as in Example 1), place it at room temperature, and inject it at regular intervals (e.g., 0, 3, 6, 12, 18, 24, 36, 48 hours) to examine the stability of the solution. One copy of the reference solution is prepared and injected once at the specified sampling time. One copy of the test solution is prepared and injected once at the specified sampling time.

[0171] Solution preparation method is as follows: Blank solvent: Take mobile phase A as blank solvent and inject once.

[0172] Acceptable standards: For the reference solution: compared to 0 h, the peak area change rate of nicardipine hydrochloride at each time point should not exceed 5.0%; for the test solution: compared to 0 h, the absolute difference in impurity content at each time point should be within 0.05%, and the absolute difference in total impurity content should be within 0.1%. No new impurities exceeding the reporting limit should appear. Tables 27 and 28 list the results of the solution stability tests. Table 27 Results of stability test data for reference solution

[0173] Table 28 Results of the stability test of the test sample solution

[0174] As can be seen from the data in Tables 27 and 28 above: Within 40 hours of storage at room temperature, the peak area change rate of the reference solution compared to 0 hours was between 0.07% and 0.51%. Within 36 hours of storage at room temperature, compared to 0 hours, impurities C, D, and I were not detected in the test solution. The maximum content difference for impurity A was 0.002%, for impurity B it was 0.002%, for impurity F and G it was 0.001%, for impurity H it was 0.002%, and for other individual impurities it was 0.000%, all less than 0.05%. The maximum content difference for total impurities was 0.002%, less than 0.1%. No new impurities exceeding the reporting limit were observed, meeting the requirements. Therefore, the test solution was stable after 36 hours of storage at room temperature.

[0175] 9) Durability test Test methods: Take 5 vials of nicardipine hydrochloride injection, mix well, accurately measure 5 ml, place in a 10 ml volumetric flask, dilute to the mark with mobile phase A, shake well, and use as the test solution; take an appropriate amount of nicardipine hydrochloride reference standard, accurately weigh it, dissolve it in mobile phase A, and quantitatively dilute it to prepare a mixed solution containing 5 μg of nicardipine hydrochloride per 1 ml, as the reference solution; take an appropriate amount of nicardipine hydrochloride reference standard, accurately weigh it, dissolve it in solvent, and quantitatively dilute it to prepare a solution containing approximately 0.25 μg of nicardipine hydrochloride per 1 ml, as the sensitivity solution; take appropriate amounts of impurities A, B, C, D, F, G, H, and I, and nicardipine hydrochloride reference standard, dissolve them in mobile phase A, and dilute them to prepare a mixed solution containing 0.5 mg of nicardipine hydrochloride and 5 μg of each impurity per 1 ml, as the system suitability solution. Based on the established chromatographic conditions, the effects of column temperature ±2℃, wavelength ±2nm, phosphate concentration ±2mmol / L, organic phase ratio ±2%, and different batches of columns were investigated. Tables 29 and 30 below list the robustness test results.

[0176] Table 29 Durability Test Data Results

[0177] Table 30 Durability Test Data Results

[0178] As can be seen from the data in Tables 29 and 30 above... Despite minor variations in chromatographic conditions (column temperature -2℃ / +1℃, wavelength ±2 nm, phosphate concentration ±2 mmol / L, organic phase ratio -2% / +1%, different batches of columns, etc.), the resolution between the main peak and adjacent impurity peaks in the system suitability solution was greater than 1.5 under all conditions, and the resolution between each impurity was not less than 1.2. In the sensitivity chromatograms, the signal-to-noise ratio of the nicardipine hydrochloride main peak was greater than 10. Compared with the baseline conditions, impurities C, D, and I were not detected in the sample. The maximum absolute difference in the content of impurity A was 0.009%, impurity B was 0.004%, impurities F+G were 0.008%, impurity H was 0.006%, and the maximum absolute difference in the content of the unknown largest single impurity was 0.015%, all within 0.05%. The maximum absolute difference in the total impurity content was 0.042%, within 0.1%, meeting the requirements. This indicates that the method has good robustness.

[0179] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. The specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

Claims

1. A method for detecting related substances in nicardipine hydrochloride injection, comprising high performance liquid chromatography, characterized in that, The detection conditions include: using an octadecylsilane-bonded silica gel column and a ghost peak trapping column for detection; the detection method includes the following steps: Step S1: Prepare mobile phase A and mobile phase B; Step S2: Prepare the test solution, which includes: blank solvent, system suitability solution, nicardipine hydrochloride reference solution and test solution; Step S3: The test solution is subjected to gradient elution with mobile phase A and mobile phase B in the following order: blank solvent, system suitability solution, nicardipine hydrochloride reference solution and test solution, thereby obtaining a high performance liquid chromatogram; Step S4: Analyze the relevant substances in the nicardipine hydrochloride injection based on the high performance liquid chromatogram obtained in step S3.

2. The method for detecting related substances in nicardipine hydrochloride injection according to claim 1, characterized in that, The ghost peak trapping column is installed downstream of the chromatograph's injection pump and upstream of the injector.

3. The method for detecting related substances in nicardipine hydrochloride injection according to claim 1, characterized in that, The related substances of the nicardipine hydrochloride injection include impurities A, B, C, D, F, G, H and I.

4. The method for detecting related substances in nicardipine hydrochloride injection according to claim 1, characterized in that, The gradient elution conditions in step S3 are as follows: 0 min ~ 15 min, 100% mobile phase A; 15 min ~ 20 min, 100% mobile phase A linearly changes to 75% mobile phase A and 25% mobile phase B; 20 min ~ 25 min, 75% mobile phase A and 25% mobile phase B; 25 min ~ 30 min, 75% mobile phase A and 25% mobile phase B linearly change to 50% mobile phase A and 50% mobile phase B; 30 min ~ 45 min, 50% mobile phase A and 50% mobile phase B; 45 min ~ 55 min, 50% mobile phase A and 50% mobile phase B linearly change to 100% mobile phase B; 55 min ~ 70 min, 100% mobile phase B; 70 min ~ 70.1 min, 100% mobile phase B linearly changes to 100% mobile phase A. 70.1 min ~ 80 min, 100% mobile phase A.

5. The method for detecting related substances in nicardipine hydrochloride injection according to claim 4, characterized in that, The mobile phase A is a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 60:40; the mobile phase B is a mixture of 0.01 mol / L potassium dihydrogen phosphate solution and methanol, wherein the mass ratio of 0.01 mol / L potassium dihydrogen phosphate solution to methanol is 20:

80.

6. The method for detecting related substances in nicardipine hydrochloride injection according to claim 5, characterized in that, The preparation process of the test solution is as follows: Take 5 vials of nicardipine hydrochloride injection, mix well, accurately weigh 5 mL and place it in a 10 mL volumetric flask, dilute to the mark with the mobile phase A and shake well to obtain the test solution.

7. The method for detecting related substances in nicardipine hydrochloride injection according to claim 5, characterized in that, The preparation process of the system suitability solution is as follows: Take appropriate amounts of impurity A, impurity B, impurity C, impurity D, impurity F, impurity G, impurity H and impurity I and nicardipine hydrochloride standard, dissolve and dilute them in solvent to prepare a mixed solution containing 0.5 mg of nicardipine hydrochloride standard and 5 μg of each impurity per 1 ml of solvent, which is the system suitability solution.

8. The method for detecting related substances in nicardipine hydrochloride injection according to claim 5, characterized in that, The preparation process of the nicardipine hydrochloride reference solution is as follows: take the nicardipine hydrochloride reference standard, dissolve it in mobile phase A and quantitatively dilute it to a solution containing 5 μg of nicardipine hydrochloride reference standard per 1 mL of solution, which is the nicardipine hydrochloride reference solution; and the blank solvent has the same composition as the mobile phase A.

9. The method for detecting related substances in nicardipine hydrochloride injection according to claim 1, characterized in that, The analysis of related substances in the nicardipine hydrochloride injection in step S4 includes: step S4-1, recording the peak area of ​​each peak in the spectrum; step S4-2, calculating the content of each impurity based on the peak area of ​​nicardipine hydrochloride in the spectrum and the content of nicardipine hydrochloride.

10. The method for detecting related substances in nicardipine hydrochloride injection according to any one of claims 1 to 9, characterized in that, The detection conditions include: a detection wavelength of 236 nm, a flow rate of 1.0 mL / min, an injection volume of 20 μL, and a column temperature of 28 °C.

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