A method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC

Abstract

The application discloses a method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride by using HPLC, and belongs to the technical field of drug analysis. The method comprises the following steps: 1) preparing a test sample solution and a control solution; 2) setting liquid chromatography detection conditions: using a chromatographic column with octadecylsilane bonded silica gel as a filler, and performing gradient elution; 3) precisely taking the test sample solution and the control solution respectively, injecting the solutions into a liquid chromatograph, starting detection, and recording a chromatogram; 4) drawing a standard curve, obtaining a standard curve equation, and calculating the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride. The method has good specificity, can directly detect trace benzyl chloride and benzaldehyde by using liquid phase, has good linearity, reliable accuracy, high sensitivity, and can be suitable for most pharmaceutical enterprises.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and more specifically, relates to a method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC. Background Technology

[0002] Genotoxic impurities refer to impurities that can cause genotoxicity, including mutagenic impurities and other types of non-mutagenic impurities. They mainly originate from the production process of active pharmaceutical ingredients (APIs), such as starting materials, reactants, catalysts, reagents, solvents, intermediates, byproducts, and degradation products. Potential genotoxic impurities refer to those containing chemical structures with similar reactivity to genotoxic impurities; these are warning structures and are also typically assessed as genotoxic impurities. In 2009, a report from relevant EU agencies included more than thirty genotoxic warning structures, such as chlorinated hydrocarbons, aldehydes, hydrazines, and acyl halides.

[0003] Analysis of the process route shows that the preparation of nicardipine hydrochloride requires benzyl chloride and m-nitrobenzaldehyde as starting materials. m-nitrobenzaldehyde is prepared by nitration of benzaldehyde and nitric acid under sulfuric acid catalysis. The chemical structures of benzyl chloride and benzaldehyde are genotoxicity warning structures. Although compounds with warning structures are not necessarily genotoxic, they can indicate potential safety risks, providing direction for further impurity safety evaluation and control.

[0004] Based on the ICH Q3 genotoxicity control TTC (acceptable risk intake) method and maximum daily dose requirements, the limits for both benzyl chloride and benzaldehyde were calculated to be ≤12.5 ppm. Currently, there are very few reports in the literature on the determination of trace amounts of benzyl chloride and benzaldehyde using liquid chromatography. Therefore, providing a method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride is of great significance for the production and storage of active pharmaceutical ingredients. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC. Using this liquid chromatography method, benzyl chloride and benzaldehyde in nicardipine hydrochloride can be separated and detected rapidly, effectively, accurately and reliably, which is beneficial to improving the product quality of the active pharmaceutical ingredient nicardipine hydrochloride.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC includes the following steps:

[0008] 1) Prepare the test solution and the control solution;

[0009] Accurately weigh 1g of nicardipine hydrochloride, place it in a 25mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the test solution.

[0010] Accurately weigh 250 mg of benzaldehyde and 250 mg of benzyl chloride, respectively, and place them in different 100 mL volumetric flasks. Dissolve and dilute to the mark with methanol to prepare stock solutions I for each impurity. Accurately weigh 1 mL of each stock solution I and place it in the same 100 mL volumetric flask. Dilute to the mark with methanol to prepare stock solution II. Accurately weigh 0.5 mL of stock solution II and place it in a 25 mL volumetric flask. Dilute to the mark with methanol to prepare the reference solution.

[0011] 2) Setting up liquid chromatography detection conditions: A chromatographic column packed with octadecylsilane-bonded silica gel was used. The mobile phase consisted of phase A and phase B, where phase A was a perchloric acid solution and phase B was a mixed solution of methanol and acetonitrile. Methanol was used as the solvent for gradient elution.

[0012] 3) Accurately pipette the test solution and the control solution separately and inject them into the liquid chromatograph. The detection wavelength of the liquid chromatograph is 210 nm. Start the detection and record the chromatogram.

[0013] 4) Plot the standard curve, obtain the standard curve equation, and calculate the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride.

[0014] Preferably, in step 2), the chromatographic column is a Welch Ultimate® XS-C18 with dimensions of 250 mm × 4.6 mm, a packing particle size of 5 μm, and a column temperature of 35 °C.

[0015] Preferably, in step 2), the concentration of the perchloric acid solution is 0.086% (V / V); the volume ratio of acetonitrile to methanol in the mixed solution is 75:25; and the injection volume is 20µL.

[0016] Preferably, in step 2), the gradient elution conditions are as follows: 0-20 min, the volume percentage of mobile phase A decreases from 80% to 65%, and the volume percentage of mobile phase B increases from 20% to 35%; 20-30 min, the volume percentage of mobile phase A decreases from 65% to 55%, and the volume percentage of mobile phase B increases from 35% to 45%; 40-45 min, the volume percentage of mobile phase A decreases from 50% to 45%, and the volume percentage of mobile phase B increases from 50% to 55%.

[0017] Preferably, in step 2), the gradient elution flow rate is 1.2 mL / min.

[0018] As a preferred embodiment, the chromatogram of the test solution contains impurities with retention times consistent with those of the control solution, and the content of benzyl chloride is not greater than 0.00125% and the content of benzaldehyde is not greater than 0.00125% when calculated by peak area using the external standard method.

[0019] Preferably, in step 4), based on the chromatogram, a linear regression equation is plotted with peak area A as the ordinate and concentration C as the abscissa. The linear equation for benzaldehyde is A = 87.557C - 0.3437, and the linear equation for benzyl chloride is A = 57.58C + 0.7348.

[0020] Preferably, the linear concentration range of benzaldehyde is 0.1000 µg / mL to 1.0003 µg / mL, the limit of quantitation is 0.1000 µg / mL, and the limit of detection is 0.03 µg / mL; the linear concentration range of benzyl chloride is 0.11021 µg / mL to 1.0212 µg / mL, the limit of quantitation is 0.1021 µg / mL, and the limit of detection is 0.0306 µg / mL.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1) The method of the present invention has good specificity, can be used for direct liquid chromatography to detect trace amounts of benzyl chloride and benzaldehyde, has good linearity, reliable accuracy, and high sensitivity, and can be applied to most pharmaceutical companies;

[0023] 2) Currently, there are no relevant literature reports that nicardipine hydrochloride may contain genotoxic impurities. The liquid chromatography method used in this invention can quickly, effectively, accurately and reliably separate and detect relevant substances in nicardipine hydrochloride, which is beneficial to improving the product quality of the active pharmaceutical ingredient nicardipine hydrochloride and enabling the clear identification and control of the risk of genotoxic impurities in the nicardipine hydrochloride process. Attached Figure Description

[0024] Figure 1 The chromatogram shows the content of benzyl chloride and benzaldehyde in the active pharmaceutical ingredient nicardipine hydrochloride; among which, Figure 1 a is a blank chromatogram of the method of this invention. Figure 1 b is a solution spectrum illustrating the applicability of the method-system of this invention. Figure 1 c is the chromatogram of the reference solution of the method of the present invention. Figure 1 d is the spectrum of the solution for EP impurity A localization in the method of this invention. Figure 1 e is the spectrum of the solution for the localization of impurity B in the EP method of this invention; Figure 1 f is the spectrum of the EP impurity C localization solution obtained by the method of the present invention;

[0025] Figure 2 Sensitivity chromatogram for the detection of benzyl chloride and benzaldehyde in nicardipine hydrochloride; where, Figure 2a is the limit spectrum for the quantitative determination of benzyl chloride and benzaldehyde; Figure 2 b is the detection limit spectrum for benzyl chloride and benzaldehyde;

[0026] Figure 3 This is a standard curve for benzaldehyde.

[0027] Figure 4 This is the standard curve for benzyl chloride. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0029] The instruments, reagents, reference standards, and sample information used in the following examples are as follows:

[0030] The high performance liquid chromatograph is an Agilent 1260 series (VWD).

[0031] Methanol and acetonitrile are both commercially available chromatographic grade products; perchloric acid is a commercially available analytical grade product.

[0032] The reference standard was a commercially available product with a purity of 99.873%, purchased from Aladdin, batch number 1902030;

[0033] The reference standard was a commercially available product with a purity of 99.92%, purchased from Aladdin, batch number K1703021;

[0034] The batch numbers of nicardipine hydrochloride in the samples were 201911001, 201911002, and 201911003, respectively, and the source was Jiangsu Cixing Pharmaceutical Co., Ltd.

[0035] Example 1

[0036] A method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC includes the following steps:

[0037] 1) Prepare the test solution and control solution:

[0038] The test solution is prepared as follows: accurately weigh 1g of nicardipine hydrochloride, place it in a 25mL volumetric flask, dissolve and dilute it to the mark with methanol, and shake well to obtain the test solution;

[0039] The preparation method of the reference solutions is as follows: accurately weigh 250 mg of benzaldehyde and 250 mg of benzyl chloride, place them in different 100 mL volumetric flasks, dissolve them in methanol and dilute to the mark to prepare reference stock solutions I for each impurity; accurately weigh 1 mL of each reference stock solution I, place it in the same 100 mL volumetric flask, and dilute to the mark with methanol to prepare reference stock solution II; accurately weigh 0.5 mL of reference stock solution II, place it in a 25 mL volumetric flask, and dilute to the mark with methanol to prepare the reference solution.

[0040] 2) Setting up liquid chromatography detection conditions: A Welch Ultimate® XS-C18 column with octadecylsilane-bonded silica gel as the packing material was used. The column dimensions were 250 mm × 4.6 mm, the packing particle size was 5 μm, and the column temperature was 35 °C. The mobile phase consisted of phase A and phase B. Phase A was a 0.086% perchloric acid solution (V / V), and phase B was a mixed solution of acetonitrile and methanol with a volume ratio of acetonitrile to methanol of 75:25. The injection volume was 20 µL.

[0041] Methanol was used as the solvent, and gradient elution was performed. The gradient elution conditions were as follows: 0-20 min, the volume percentage of mobile phase A decreased from 80% to 65%, and the volume percentage of mobile phase B increased from 20% to 35%; 20-30 min, the volume percentage of mobile phase A decreased from 65% to 55%, and the volume percentage of mobile phase B increased from 35% to 45%; 40-45 min, the volume percentage of mobile phase A decreased from 50% to 45%, and the volume percentage of mobile phase B increased from 50% to 55%.

[0042] 3) Accurately pipette the test solution and the control solution separately and inject them into the liquid chromatograph. The detection wavelength of the liquid chromatograph is 210 nm. Detect and record the chromatograms.

[0043] 4) Based on the chromatograms, a linear regression equation was plotted with peak area A as the ordinate and concentration C as the abscissa. The linear equation for benzaldehyde was A = 87.557C - 0.3437, and the linear equation for benzyl chloride was A = 57.58C + 0.7348. The linear concentration range for benzaldehyde was 0.1000µg / mL to 1.0003µg / mL, with a limit of quantitation of 0.1000µg / mL and a limit of detection of 0.03µg / mL. The linear concentration range for benzyl chloride was 0.11021µg / mL to 1.0212µg / mL, with a limit of quantitation of 0.1021µg / mL and a limit of detection of 0.0306µg / mL.

[0044] Example 2

[0045] The genotoxic impurities present in the manufacturing process of nicardipine hydrochloride are benzyl chloride and benzaldehyde. When calculating acceptable intake based on the TTC, the risk of a mutagenic impurity is considered negligible at a daily intake of 1.5 μg per person. The maximum daily dose of nicardipine hydrochloride is 120 mg, with a concentration limit of 12.5 ppm (0.00125%).

[0046] 1. Exclusivity

[0047] Accurately weigh 250 mg of benzaldehyde and 250 mg of benzyl chloride, place them in different 100 mL volumetric flasks, dissolve them in methanol and dilute to the mark, and prepare stock solutions of each impurity reference standard. ;

[0048] Accurately measure the stock solutions of each reference standard. 1 mL was placed in the same 100 mL volumetric flask and diluted to the mark with methanol to prepare the reference stock solution. ;

[0049] Accurately measure the reference standard stock solution 0.5 mL was placed in a 25 mL volumetric flask and diluted to the mark with methanol to prepare the reference solution.

[0050] System adaptability solution: Accurately weigh 1 g of nicardipine hydrochloride, place it in a 25 mL volumetric flask, dissolve it by sonication with methanol, accurately add 0.5 mL of the reference stock solution, and dilute to the mark with methanol to obtain the system adaptability solution.

[0051] Take 20 mg each of EP impurity A, EP impurity B and EP impurity C, place them in different 20 mL volumetric flasks, dissolve them in methanol and dilute to the mark to prepare the positioning solutions for each impurity.

[0052] Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. Examine the peak times and separation of each component. Impurity information is shown in Table 1, and results are shown in Table 2. See details below. Figure 1 .

[0053] Table 1 Impurity Information

[0054]

[0055] Table 2 Impurity Location and Separation

[0056]

[0057] From Table 1 and Figure 1It is evident that the blank solvent does not interfere with the determination of impurities, and process impurities and degradation impurities in nicardipine hydrochloride do not interfere with the determination of benzyl chloride and benzaldehyde content. In the system suitability chromatogram, the resolution between benzaldehyde and adjacent peaks is greater than 1.5, and the resolution between benzyl chloride and adjacent peaks is greater than 1.5. This method has good specificity; process impurities and degradation impurities that may be present in nicardipine hydrochloride do not interfere with the detection of benzyl chloride and benzaldehyde content.

[0058] 2. Limit of Quantitation and Limit of Detection

[0059] Take appropriate amounts of benzaldehyde and benzyl chloride reference standards, dissolve and dilute them in methanol to a signal-to-noise ratio (SNR) of 10–30 for each peak, and use this solution as the limit of quantitation (LOQ) solution. Inject six consecutive times, and the relative standard deviation of the peak area should not exceed 10.0%. Dilute this solution to a SNR of 3–9 to use as the limit of detection (LOD) solution. The concentrations and SNRs of each compound are shown in Tables 3 and 4.

[0060] Table 3 Results of the detection limit test

[0061]

[0062] Table 4 Results of Limit of Quantitation Test

[0063]

[0064] From Table 3, Table 4 and Figure 2 It can be seen that the signal-to-noise ratio of each compound in the chromatogram of benzyl chloride and benzaldehyde limit solutions is not less than 10, the relative standard deviation of peak area (RSD) (n=6) is not more than 10.0%, the limit concentration is about 20% of the limit and less than 30% of the threshold, which can meet the requirements of quantitative detection.

[0065] 3. Linearity and Range

[0066] Accurately weigh 250 mg each of benzaldehyde and benzyl chloride reference standards and place them in the same 100 mL volumetric flask. Dissolve and dilute to the mark with methanol using sonication to prepare stock solutions I for each compound. Accurately measure 1 mL of stock solution I and place it in a 100 mL volumetric flask. Dilute to the mark with methanol to prepare stock solution II. Accurately measure 2 mL of stock solution II and place it in a 50 mL volumetric flask. Dilute with methanol to prepare a solution containing 1 µg / mL of each compound as a linear pair stock solution. Accurately measure an appropriate amount of the linear stock solution and dilute with methanol to prepare a series of linear solutions (7 concentration points from 20% to 200% of the limit solution concentration). Accurately measure 20 μL of the linear solution and inject it into the liquid chromatograph. Record the chromatogram and perform linear regression on the peak area against the concentration. The results are shown in Tables 5 and 6.

[0067] Table 5 Results of linearity test for benzaldehyde

[0068]

[0069] Table 6. Results of linearity test for benzyl chloride

[0070]

[0071] As shown in Tables 5 and 6, benzaldehyde showed good linearity with peak area in the range of 0.1000 µg / mL to 1.0003 µg / mL, with the linear equation being A = 87.557C - 0.3437 and a correlation coefficient r = 0.9992; benzyl chloride also showed good linearity with peak area in the range of 10.1021 µg / mL to 1.0212 µg / mL, with the linear equation being A = 57.58C + 0.7348 and a correlation coefficient r = 0.9995.

[0072] 4. Accuracy

[0073] Take appropriate amounts of benzyl chloride and benzaldehyde reference standards, and add them to the nicardipine hydrochloride raw material at a ratio of 80% to 120% of the limits for each impurity. The preparation process is as follows:

[0074] Accurately weigh 250 mg each of benzaldehyde and benzyl chloride reference standards, place them in different 100 mL volumetric flasks, dissolve them in methanol by sonication and dilute to the mark to prepare benzaldehyde and benzyl chloride reference standard stock solution I; accurately measure 1 mL of reference standard stock solution I, place it in a 100 mL volumetric flask, add diluent and dilute to the mark to prepare reference standard stock solution II.

[0075] Accurately measure 0.5 mL of reference stock solution II and place it in a 25 mL volumetric flask. Dilute to the mark with diluent to obtain the reference solution.

[0076] Weigh 1g of this product accurately, place it in a 25mL volumetric flask, add an appropriate amount of methanol and sonicate to dissolve it, accurately add 0.4mL of reference stock solution II, and dilute with methanol to the mark to obtain an 80% recovery solution.

[0077] Weigh 1g of this product accurately, place it in a 25mL volumetric flask, add an appropriate amount of methanol and sonicate to dissolve it, accurately add 0.5mL of reference stock solution II, and dilute with methanol to the mark to obtain a 100% recovery solution.

[0078] Weigh 1g of this product accurately, place it in a 25mL volumetric flask, add an appropriate amount of methanol and sonicate to dissolve it, accurately add 0.6mL of reference stock solution II, and dilute with methanol to the mark to obtain a 120% recovery solution.

[0079] Weigh 1g of this product accurately, place it in a 25mL volumetric flask, add methanol and sonicate to dissolve and dilute to the mark to obtain the unspecified test solution.

[0080] Take 20 μL each of the reference solution, the unspecified test solution, and the recovery solution, and inject them separately into the liquid chromatograph. Record the chromatograms and calculate the recovery rate based on peak area using the external standard method. Benzaldehyde and benzyl chloride were not detected in the unspecified test solution. Compare the measured values ​​with the theoretical values ​​using the external standard method to calculate the recovery rate, and calculate the relative standard deviation of the recovery rate to examine the accuracy of the method. The recovery rate and accuracy results are shown in Tables 7 and 8.

[0081] Table 7 Benzaldehyde Accuracy Test

[0082]

[0083] Table 8. Accuracy Test of Benzyl Chloride

[0084]

[0085] As shown in Tables 7 and 8, the recoveries of benzyl chloride and benzaldehyde at the high (120% limit), medium (100% limit), and low (80% limit) concentration levels are all within the range of 80.0% to 120.0%, and the relative standard deviations (RSD) of the recoveries (n=9) are all less than 10.0%, indicating that the accuracy of this method is good.

[0086] 5. Repeatability

[0087] Accurately weigh 250 mg each of benzaldehyde and benzyl chloride reference standards, place them in different 100 mL volumetric flasks, dissolve them in methanol by sonication and dilute to the mark to prepare benzaldehyde and benzyl chloride reference standard stock solution I; accurately measure 1 mL of reference standard stock solution I, place it in a 100 mL volumetric flask, add diluent and dilute to the mark to prepare reference standard stock solution II.

[0088] Accurately measure 0.5 mL of reference stock solution II and place it in a 25 mL volumetric flask. Dilute to the mark with diluent to obtain the reference solution.

[0089] Accurately weigh 1g of this product and place it in a 25mL volumetric flask. Add an appropriate amount of methanol and sonicate to dissolve and dilute to the mark to prepare the test solution. Inject the above solution into a liquid chromatograph, record the chromatogram, calculate the content of each impurity in the test solution by peak area using the external standard method, and calculate the change in the content of each impurity (in terms of relative standard deviation). Examine the repeatability of the method. The results are shown in Table 9.

[0090] Table 9 Results of Repeatability Tests

[0091]

[0092] As shown in Table 9, benzaldehyde and benzyl chloride were not detected in the same batch of nicardipine hydrochloride raw material after six repeated tests, indicating that the method of the present invention has good repeatability.

[0093] 6. Intermediate precision

[0094] On different dates, by different experimenters, the preparation methods for the reference solution and the test solution were prepared according to the "5. Repeatability" section. The content of each impurity was calculated by peak area using the external standard method. The intermediate precision of the method was examined by the changes in impurity content. The differences between the results measured by two experimenters were statistically analyzed to examine the precision of the method. The results are shown in Tables 10 and 11.

[0095] Table 10 Results of intermediate precision test

[0096]

[0097] Table 11 Statistical results of impurity content in precision

[0098]

[0099] As shown in Tables 10 and 11, when six test solutions were prepared in parallel using the same repeatability method at different times and by different personnel, benzaldehyde and benzyl chloride were not detected in any of them, and there was no significant difference in the results. The precision of the method of the present invention is good.

[0100] 7. Solution stability

[0101] Take 20 µl of the reference solution from the "4. Accuracy" test item and inject it into the liquid chromatograph at different time intervals. The stability of the solution at different time intervals is examined by the changes in the peak areas of benzaldehyde and benzyl chloride. The results are shown in Table A.

[0102] Table A shows the stability results of the reference solution.

[0103]

[0104] As shown in Table A, after the reference solution was placed at room temperature for 32 hours and 40 minutes, the relative standard deviation (RSD) of the peak area of ​​each compound was no greater than 6.0%, indicating that the reference solution was stable after being placed at room temperature for 32 hours and 40 minutes.

[0105] 8. Durability

[0106] Prepare the reference solution, test solution, and system suitability solution according to the method described in "1. Specificity" test section. Take the above solutions and analyze them under the chromatographic conditions in Table 12. The robustness of the method is examined by measuring the change in impurity content in the test solution and the relative standard deviation of the peak area of ​​each impurity in the system suitability solution. The results are shown in Tables 13 and 14.

[0107] Table 12 Chromatographic Condition Variation Parameters

[0108]

[0109] Table 13. Effects of minor changes in chromatographic conditions on the suitability of the chromatographic system

[0110]

[0111] Table 1. Detection results of impurity content in test sample solutions under chromatographic conditions.

[0112]

[0113] As shown in Tables 13 and 14, under the conditions of flow rate (±0.1 mL / min), column temperature (±3℃), mobile phase B (±5), and different brands (pHenomenex) chromatographic columns, the relative standard deviation of the impurity peak area in the reference solution was less than 6.0%, and benzaldehyde and benzyl chloride were not detected in the chromatogram of the test solution. The method of the present invention has good robustness.

[0114] Example 3

[0115] Accurately weigh 250 mg each of benzaldehyde and benzyl chloride reference standards, place them in different 100 mL volumetric flasks, dissolve them in methanol by sonication and dilute to the mark to prepare benzaldehyde and benzyl chloride reference standard stock solution I; accurately measure 1 mL of reference standard stock solution I, place it in a 100 mL volumetric flask, add diluent and dilute to the mark to prepare reference standard stock solution II.

[0116] Accurately measure 0.5 mL of reference stock solution II and place it in a 25 mL volumetric flask. Dilute to the mark with diluent to obtain the reference solution.

[0117] Accurately weigh 1g of this product and place it in a 25mL volumetric flask. Dissolve it in methanol by sonication and dilute to the mark to prepare the test solution. Three batches of nicardipine hydrochloride raw material were tested for benzyl chloride and benzaldehyde content. The results are shown in Table 15.

[0118] Table 15 Results of Detection of Benzyl Chloride and Benzaldehyde Content in Nicardipine Hydrochloride Raw Material

[0119]

[0120] As shown in Table 15, this method is applicable to the detection of trace amounts of benzyl chloride and benzaldehyde in nicardipine hydrochloride raw material.

[0121] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the described embodiments. The embodiments and features in the embodiments of this application can be arbitrarily combined with each other without conflict. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Furthermore, technical details not described in detail in this specification are well-known to those skilled in the art, and therefore will not be repeated here.

Claims

1. A method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC, characterized in that, Includes the following steps: 1) Prepare the test solution and the control solution; Accurately weigh 1g of nicardipine hydrochloride, place it in a 25mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the test solution. Accurately weigh 250 mg of benzaldehyde and 250 mg of benzyl chloride, respectively, and place them in different 100 mL volumetric flasks. Dissolve and dilute to the mark with methanol to prepare stock solutions I for each impurity. Accurately weigh 1 mL of each stock solution I and place it in the same 100 mL volumetric flask. Dilute to the mark with methanol to prepare stock solution II. Accurately weigh 0.5 mL of stock solution II and place it in a 25 mL volumetric flask. Dilute to the mark with methanol to prepare the reference solution. 2) Setting up liquid chromatography detection conditions: A column packed with octadecylsilane-bonded silica gel was used. The mobile phase consisted of phase A and phase B, where phase A was a perchloric acid solution and phase B was a mixed solution of methanol and acetonitrile, with methanol as the solvent, using gradient elution; the concentration of the perchloric acid solution was 0.086% V / V; the volume ratio of acetonitrile to methanol in the mixed solution was 75:25; the injection volume was 20 µL. The gradient elution conditions were as follows: 0-20 min, the volume percentage of mobile phase A decreased from 80% to 65%, and the volume percentage of mobile phase B increased from 20% to 35%; 20-30 min, the volume percentage of mobile phase A decreased from 65% to 55%, and the volume percentage of mobile phase B increased from 35% to 45%; 40-45 min, the volume percentage of mobile phase A decreased from 50% to 45%, and the volume percentage of mobile phase B increased from 50% to 55%. 3) Accurately pipette the test solution and the control solution separately and inject them into the liquid chromatograph. The detection wavelength of the liquid chromatograph is 210 nm. Start the detection and record the chromatogram. 4) Plot the standard curve, obtain the standard curve equation, and calculate the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride.

2. The method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC according to claim 1, characterized in that, In step 2), the chromatographic column is a Welch Ultimate® XS-C18 with dimensions of 250 mm × 4.6 mm, a packing particle size of 5 μm, and a column temperature of 35 °C.

3. The method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC according to claim 1, characterized in that, In step 2), the gradient elution flow rate is 1.2 mL / min.

4. The method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC according to claim 1, characterized in that, The chromatogram of the test solution contains impurities with retention times consistent with those of the control solution. Calculated by peak area using the external standard method, the content of benzyl chloride is not greater than 0.00125%, and the content of benzaldehyde is not greater than 0.00125%.

5. The method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride using HPLC according to claim 1, characterized in that, In step 4), based on the chromatogram, a linear regression equation is plotted with peak area A as the ordinate and concentration C as the abscissa. The linear equation for benzaldehyde is A = 87.557C - 0.3437, and the linear equation for benzyl chloride is A = 57.58C + 0.7348.

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