Method for determining contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for determining the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride by utilizing HPLC (High Performance Liquid Chromatography), and belongs to the technical field of pharmaceutical analysis. The method comprises the following steps: 1) preparing a test solution and a contrast solution; (2) setting liquid chromatography detection conditions: carrying out gradient elution by adopting a chromatographic column taking octadecylsilane chemically bonded silica as a filling agent; (3) respectively and precisely sucking the test solution and the contrast solution, injecting the test solution and the contrast solution into a liquid chromatograph, starting to detect and recording a chromatogram; and 4) drawing a standard curve to obtain a standard curve equation, and calculating the content of benzyl chloride and benzaldehyde in nicardipine hydrochloride. The method disclosed by the invention is good in specificity, capable of directly performing trace detection on benzyl chloride and benzaldehyde by using a liquid phase, good in linearity, reliable in accuracy, high in sensitivity and applicable to most pharmaceutical enterprises.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and more specifically relates to a method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by using HPLC. Background Art

[0002] Genotoxic impurities refer to impurities that can cause genotoxicity, including mutagenic impurities and other types of non-mutagenic impurities. They mainly come from the production process of APIs, such as starting materials, reactants, catalysts, reagents, solvents, intermediates, by-products, degradation products, etc. Potential genotoxic impurities refer to impurities that contain chemical structures similar to the reactivity of genotoxic impurities, i.e. warning structures, and are usually also evaluated as genotoxic impurities. In 2009, the report of relevant EU agencies included more than 30 genotoxic warning structures such as chlorinated hydrocarbons, aldehydes, hydrazines, acyl halides, etc.

[0003] According to the process route analysis, the preparation of nicardipine hydrochloride requires benzyl chloride, m-nitrobenzaldehyde, etc. as starting materials, among which m-nitrobenzaldehyde is prepared by nitration of benzaldehyde and nitric acid under the catalysis of sulfuric acid. The chemical structures of benzyl chloride and benzaldehyde belong to genotoxicity warning structures. Although compounds with warning structures are not necessarily genotoxic, they can indicate that the compounds may have safety risks and point out the direction for further impurity safety evaluation and control.

[0004] According to the ICHQ3 genotoxicity control TTC (acceptable risk intake) method and the maximum daily dose requirement, the limit standards of benzyl chloride and benzaldehyde are calculated to be ≤12.5ppm. At present, there are very few literature reports on the use of liquid phase to determine the trace amount of benzyl chloride and benzaldehyde. 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 raw materials. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by using HPLC. The liquid chromatography method can quickly, effectively, accurately and reliably separate and detect benzyl chloride and benzaldehyde in nicardipine hydrochloride, which is beneficial to improving the product quality of the raw material drug nicardipine hydrochloride.

[0006] In order to solve the above 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 by HPLC comprises the following steps:

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

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

[0010] Accurately measure 250 mg of benzaldehyde and 250 mg of benzyl chloride respectively, accurately weigh them, place them in different 100 mL volumetric bottles, add methanol to dissolve and dilute to the scale, as each impurity reference substance reserve solution I; accurately measure 1 mL of each reference substance reserve solution I, place it in the same 100 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance reserve solution II; accurately measure 0.5 mL of the reference substance reserve solution II, place it in a 25 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance solution;

[0011] 2) Setting liquid chromatography detection conditions: using a chromatographic column with octadecylsilane bonded silica gel as a filler, the mobile phase consists of phase A and phase B, wherein phase A is a perchloric acid solution, phase B is a mixed solution of methanol and acetonitrile, the solvent is methanol, and gradient elution is performed;

[0012] 3) Accurately pipette the test solution and the control solution respectively, inject them into the liquid chromatograph, the detection wavelength of the liquid chromatograph is 210nm, start detection and record the chromatogram;

[0013] 4) Draw a 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 Welch XS-C18, the specifications of the chromatographic column are 250mm×4.6mm, the filler particle size is 5μm, and the column temperature is 35℃.

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

[0016] Preferably, in step 2), the gradient elution conditions are: 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] Preferably, the test solution has impurities with the same retention time as the control solution in the chromatogram, and the benzyl chloride content is not more than 0.00125%, and the benzaldehyde content is not more than 0.00125% calculated by peak area according to the external standard method.

[0019] Preferably, in the step 4), a linear regression equation is made according to the chromatogram with the peak area A as the ordinate and the concentration C as the abscissa. The linear equation of benzaldehyde is A=87.557C-0.3437, and the linear equation of 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 quantification is 0.1000 μg / mL, and the detection limit 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 quantification is 0.1021 μg / mL, and the detection limit is 0.0306 μg / mL.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

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

[0023] 2) Currently, there is no relevant literature reporting that nicardipine hydrochloride may contain genotoxic impurities. The liquid chromatography method used in the present invention can quickly, effectively, accurately and reliably separate and detect related substances in nicardipine hydrochloride, which is beneficial to improving the product quality of the raw material nicardipine hydrochloride and clearly identifying and controlling the risk of genotoxic impurities in the nicardipine hydrochloride process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a chromatogram for detecting the content of benzyl chloride and benzaldehyde in the raw material drug nicardipine hydrochloride; Figure 1 a is the blank chromatogram of the method of the present invention, Figure 1 b is the solution spectrum of the method-system applicability of the present invention, Figure 1 c is the spectrum of the method of the present invention-reference substance solution, Figure 1 d is the spectrum of the solution for localization of impurity A in the method of the present invention-EP, Figure 1 e is the solution spectrum of the method of the present invention -EP impurity B location; Figure 1 f is the spectrum of the solution for localization of impurity C in the method of the present invention-EP;

[0025] Figure 2 To detect the content-sensitivity chromatogram of benzyl chloride and benzaldehyde in nicardipine hydrochloride; Figure 2a is the quantitative limit spectrum of benzyl chloride and benzaldehyde; Figure 2 b is the detection limit spectrum of benzyl chloride and benzaldehyde;

[0026] Figure 3 is the standard curve of benzaldehyde;

[0027] Figure 4 This is the standard curve of benzyl chloride. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0029] In the following examples, the instruments, reagents, reference substances and sample information used are as follows:

[0030] The model of the high performance liquid chromatograph was Agilent 1260 series (VWD);

[0031] Methanol and acetonitrile are both chromatographic grade commercial products; perchloric acid is an analytical grade commercial product;

[0032] The benzaldehyde in the reference substance is a commercial product with a content of 99.873%, purchased from Aladdin, batch number 1902030;

[0033] The benzyl chloride in the reference substance is a commercial product with a content of 99.92%, purchased from Aladdin, with batch number K1703021;

[0034] The batch numbers of nicardipine hydrochloride in the samples are 201911001, 201911002, and 201911003, respectively, and they come from Jiangsu Cixin Pharmaceutical Co., Ltd.

[0035] Example 1

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

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

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

[0039] The preparation method of the reference solution is as follows: accurately measure 250 mg of benzaldehyde and 250 mg of benzyl chloride, respectively, accurately weigh them, place them in different 100 mL volumetric bottles, add methanol to dissolve and dilute to the scale, as each impurity reference substance reserve solution I; accurately measure 1 mL of each reference substance reserve solution I, place it in the same 100 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance reserve solution II; accurately measure 0.5 mL of the reference substance reserve solution II, place it in a 25 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance solution;

[0040] 2) Setting up the liquid chromatography detection conditions: Welch chromatography with octadecylsilane bonded silica gel as filler XS-C18 chromatographic column, the specification of the chromatographic column is 250mm×4.6mm, the filler particle size is 5μm, the column temperature is 35℃; the mobile phase is composed of phase A and phase B, wherein phase A is 0.086% perchloric acid solution (V / V); phase B is a mixed solution of acetonitrile and methanol, and the volume ratio of acetonitrile to methanol is 75:25; the injection volume is 20μL;

[0041] The solvent is methanol, and gradient elution is performed; the conditions of the gradient elution are: from 0 to 20 minutes, the volume percentage of the mobile phase A decreases from 80% to 65%, and the volume percentage of the mobile phase B increases from 20% to 35%; from 20 to 30 minutes, the volume percentage of the mobile phase A decreases from 65% to 55%, and the volume percentage of the mobile phase B increases from 35% to 45%; from 40 to 45 minutes, the volume percentage of the mobile phase A decreases from 50% to 45%, and the volume percentage of the mobile phase B increases from 50% to 55%;

[0042] 3) Accurately pipette the test solution and the control solution respectively, inject them into the liquid chromatograph, the detection wavelength of the liquid chromatograph is 210nm, detect and record the chromatogram;

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

[0044] Example 2

[0045] The genotoxic impurities contained in the production process of nicardipine hydrochloride are benzyl chloride and benzaldehyde. When calculating the acceptable intake according to TTC, the risk of a mutagenic impurity is considered to be negligible when it is consumed by each person per day at 1.5 μg. The maximum daily dose of nicardipine hydrochloride is 120 mg, and the concentration limit is 12.5 ppm (0.00125%).

[0046] 1. Exclusivity

[0047] Take 250 mg of benzaldehyde and 250 mg of benzyl chloride respectively, weigh accurately, place in different 100 mL volumetric bottles, add methanol to dissolve and dilute to the scale, as the impurity reference substance stock solution I;

[0048] Accurately measure 1 mL of each reference substance stock solution I, place it in the same 100 mL volumetric flask, and dilute to the mark with methanol to serve as reference substance stock solution II.

[0049] Accurately measure 0.5 mL of reference substance stock solution II, place it in a 25 mL volumetric flask, and add methanol to dilute to the mark as the reference substance solution;

[0050] System adaptability solution: Take 1 g of nicardipine hydrochloride, weigh it accurately, put it in a 25 mL volumetric flask, dissolve it in methanol by ultrasonication, accurately add 0.5 mL of the reference substance stock solution, and dilute it to the scale with methanol as 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, add methanol to dissolve and dilute to the scale, as the impurity location solutions.

[0052] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, and record the chromatogram. Investigate the peak time and separation of each component. See Table 1 for impurity information and Table 2 for results. Figure 1 .

[0053] Table 1 Impurity information

[0054]

[0055]

[0056] Table 2 Impurity location and separation

[0057]

[0058] From Table 1 and Figure 1It can be seen that the blank solvent does not interfere with the determination of impurities, and the process impurities and degradation impurities in nicardipine hydrochloride do not interfere with the determination of benzyl chloride and benzaldehyde content; in the system suitability solution chromatogram, the separation degree between benzaldehyde and adjacent peaks is greater than 1.5, and the separation degree between benzyl chloride and adjacent peaks is greater than 1.5. This method has good specificity, and the process impurities and degradation impurities that may be contained in nicardipine hydrochloride do not interfere with the content detection of benzyl chloride and benzaldehyde.

[0059] 2. Limit of quantification and limit of detection

[0060] Take appropriate amount of benzaldehyde and benzyl chloride reference substances, dissolve them in methanol and dilute them to a solution with a signal-to-noise ratio of 10 to 30 for each peak, as the quantitative limit solution, inject 6 needles continuously, and the relative standard deviation of the peak area shall not exceed 10.0%. Dilute to a solution with a signal-to-noise ratio of 3 to 9, as the detection limit solution. The concentration and signal-to-noise ratio of each compound are shown in Table 3 and Table 4.

[0061] Table 3 Detection limit test results

[0062]

[0063] Table 4 Quantitation limit test results

[0064]

[0065] 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 the quantitative limit solution of benzyl chloride and benzaldehyde is not less than 10, the relative standard deviation RSD (n=6) of the peak area is no more than 10.0%, and the quantitative limit concentration is approximately equivalent to 20% of the limit, which is less than the threshold of 30%, and can meet the requirements of quantitative detection.

[0066] 3. Linearity and range

[0067] Take 250 mg of each of benzaldehyde and benzyl chloride reference substances, weigh them accurately, place them in the same 100 mL volumetric flask, dissolve them ultrasonically with methanol and dilute them to the mark, as reference substance reserve solution I for each compound; accurately measure 1 mL of reference substance reserve solution I and place it in a 100 mL volumetric flask, add methanol to dilute to the mark, as reference substance reserve solution II; accurately measure 2 mL of reference substance reserve solution II and place it in a 50 mL volumetric flask, add methanol to dilute to make a solution containing 1 μg / mL of each compound as a linear pair reserve solution. Accurately measure an appropriate amount of linear reserve solution, add methanol to dilute to make a series of linear solutions (take 7 concentration points from 20% to 200% of the concentration of the limiting solution); accurately measure 20 μL of the linear solution, inject it into the liquid chromatograph, record the chromatogram, and perform linear regression on the concentration using the peak area. The results are shown in Tables 5 and 6.

[0068] Table 5 Benzaldehyde linear test results

[0069]

[0070] Table 6 Benzyl chloride linear test results

[0071]

[0072] It can be seen from Tables 5 and 6 that the concentration of benzaldehyde is in the range of 0.1000 μg / mL to 1.0003 μg / mL, and the linear equation is: A=87.557C-0.3437, and the correlation coefficient is r=0.9992; the concentration of benzyl chloride is in the range of 10.1021 μg / mL to 1.0212 μg / mL, and the linear equation is: A=57.58C+0.7348, and the correlation coefficient is r=0.9995.

[0073] 4. Accuracy

[0074] Take appropriate amount of benzyl chloride and benzaldehyde reference substances, add them to the nicardipine hydrochloride raw material at a ratio of 80% to 120% of the impurity limit, and the preparation process is as follows:

[0075] Take 250 mg of each of benzaldehyde and benzyl chloride reference substances, accurately weigh them, place them in different 100 mL volumetric bottles, ultrasonically dissolve them with methanol and dilute them to the mark, as benzaldehyde and benzyl chloride reference substance stock solution I; accurately measure 1 mL of reference substance stock solution I, place it in a 100 mL volumetric bottle, add diluent and dilute to the mark, as reference substance stock solution II;

[0076] Accurately measure 0.5 mL of reference substance stock solution II, place it in a 25 mL volumetric flask, and add diluent to dilute to the mark as the reference substance solution;

[0077] Take 1g of this product, weigh it accurately, put it in a 25mL volumetric flask, add appropriate amount of methanol to dissolve it by ultrasonic, accurately add 0.4mL of reference substance stock solution II, and dilute it to the scale with methanol to prepare the 80% recovery solution;

[0078] Take 1g of this product, weigh it accurately, put it in a 25mL volumetric flask, add appropriate amount of methanol to dissolve it by ultrasonic, accurately add 0.5mL of reference substance stock solution II, and dilute to the scale with methanol to serve as the 100% recovery solution;

[0079] Take 1g of this product, weigh it accurately, put it in a 25mL volumetric flask, add appropriate amount of methanol to dissolve it by ultrasonic, accurately add 0.6mL of reference substance stock solution II, add methanol to dilute to the scale, as the 120% recovery rate solution;

[0080] Take 1 g of this product, weigh it accurately, place it in a 25 mL volumetric flask, add methanol to dissolve it by ultrasonic and dilute it to the scale, which is used as the unspecified test solution.

[0081] Take 20 μL of reference solution, unspiked test solution and recovery solution, respectively, and inject them into the liquid chromatograph, record the chromatogram, calculate the peak area according to the external standard method, no benzaldehyde and benzyl chloride are detected in the unspiked test solution, compare the measured value of the external standard method with the theoretical value, calculate the recovery, and calculate the relative standard deviation of the recovery to examine the accuracy of the method. The recovery and accuracy results are shown in Tables 7 and 8.

[0082] Table 7 Benzaldehyde Accuracy Test

[0083]

[0084] Table 8 Benzyl chloride accuracy test

[0085]

[0086]

[0087] It can be seen from Tables 7 and 8 that the recoveries of benzyl chloride and benzaldehyde at three concentration levels of high (120% of the limit), medium (100% of the limit), and low (80% of the limit) are all within the range of 80.0% to 120.0%, and the relative standard deviation RSD (n=9) of the recoveries is less than 10.0%, indicating that the accuracy of this method is good.

[0088] 5. Repeatability

[0089] Take 250 mg of each of benzaldehyde and benzyl chloride reference substances, accurately weigh them, place them in different 100 mL volumetric bottles, ultrasonically dissolve them with methanol and dilute them to the mark, as benzaldehyde and benzyl chloride reference substance stock solution I; accurately measure 1 mL of reference substance stock solution I, place it in a 100 mL volumetric bottle, add diluent and dilute to the mark, as reference substance stock solution II;

[0090] Accurately measure 0.5 mL of reference substance stock solution II, place it in a 25 mL volumetric flask, and add diluent to dilute to the mark as the reference substance solution;

[0091] Take 1 g of this product, weigh it accurately, put it in a 25 mL volumetric flask, add appropriate amount of methanol and ultrasonically dissolve and dilute to the scale as the test solution; inject the above solution into the liquid chromatograph, record the chromatogram, calculate the content of each impurity in the test solution by peak area according to the external standard method, and calculate the change of each impurity content (in terms of relative standard deviation) to examine the repeatability of the method. The results are shown in Table 9.

[0092] Table 9 Repeatability test results

[0093] Impurities Repeatability -1 Repeatability -2 Repeatability - 3 Repeatability - 4 Repeatability -5 Repeatability - 6 RSD Benzaldehyde Not detected Not detected Not detected Not detected Not detected Not detected —— Benzyl chloride Not detected Not detected Not detected Not detected Not detected Not detected ——

[0094] As shown in Table 9, the same batch of nicardipine hydrochloride bulk drug was measured 6 times repeatedly, and no benzaldehyde and benzyl chloride were detected. The repeatability of the method of the present invention is good.

[0095] 6. Intermediate precision

[0096] On different dates, different experimenters prepared the reference solution and test solution according to the method of preparation under "5. Repeatability". The impurity content was calculated by peak area using the external standard method. The intermediate precision of the method was examined based on the change in impurity content. The difference in the measurement results between the two experimenters was statistically analyzed to examine the precision of the method. The results are shown in Tables 10 and 11.

[0097] Table 10 Intermediate precision test results

[0098]

[0099]

[0100] Table 11 Statistical results of each impurity content in precision

[0101] Impurities Repeatability average content Intermediate precision mean content RSD Benzaldehyde Not detected Not detected —— Benzyl chloride Not detected Not detected ——

[0102] It can be seen from Tables 10 and 11 that 6 test solutions were prepared in parallel with the same repeatability method at different times and by different personnel, and no benzaldehyde and benzyl chloride were detected. There was no significant difference in the results, and the precision of the method of the present invention was good.

[0103] 7. Solution stability

[0104] Take 20 μl of the reference solution under the test item “4. Accuracy” and inject it into the liquid chromatograph at different time periods. The changes in the peak areas of benzaldehyde and benzyl chloride are used to examine the stability of the solution at different times. The results are shown in Table 11.

[0105] Table 11 Stability results of reference solution

[0106]

[0107] As shown in Table 12, when the reference solution was placed at room temperature for 32h40min, the relative standard deviation (RSD) of the peak area of ​​each compound was no more than 6.0%, indicating that the reference solution was stable when placed at room temperature for 32h40min.

[0108] 8. Durability

[0109] The reference solution, test solution and system suitability solution were prepared according to the method under the test item "1. Specificity". The above solutions were sampled and analyzed under the chromatographic conditions of Table 12. The durability of the method was investigated by the change of 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.

[0110] Table 12 Chromatographic condition variation parameters

[0111]

[0112]

[0113] Table 13 Effect of slight changes in chromatographic conditions on the suitability of the chromatographic system

[0114] Chromatographic conditions Benzaldehyde Precision Benzyl chloride precision Separation Normal conditions 3.5％ 4.6％ 51.7 1.1mL / min 1.8％ 2.7％ 48.8 1.3mL / min 2.7％ 3.5％ 50.6 Column temperature 32℃ 0.85％ 2.9％ 45.2 Column temperature 38℃ 0.76％ 2.2％ 46.4 Mobile phase 2 0.82％ 3.8％ 45.6 Mobile phase 3 4.8％ 2.2％ 51.9 pHenomenex Columns 2.7％ 2.0％ 49.7

[0115] Table 14 Results of impurity content test in the test solution after changing the chromatographic conditions

[0116]

[0117] It can be seen from Tables 13 and 14 that the relative standard deviation of the impurity peak area in the reference solution under the conditions of flow rate (±0.1 mL / min), column temperature (±3°C), mobile phase B (±5), and changing to different brands (pHenomenex) of chromatographic columns 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 durability.

[0118] Example 3

[0119] Take 250 mg of each of benzaldehyde and benzyl chloride reference substances, accurately weigh them, place them in different 100 mL volumetric bottles, ultrasonically dissolve them with methanol and dilute them to the mark, as benzaldehyde and benzyl chloride reference substance stock solution I; accurately measure 1 mL of reference substance stock solution I, place it in a 100 mL volumetric bottle, add diluent and dilute to the mark, as reference substance stock solution II;

[0120] Accurately measure 0.5 mL of reference substance stock solution II, place it in a 25 mL volumetric flask, and add diluent to dilute to the mark as the reference substance solution;

[0121] Take 1g of this product, weigh it accurately, put it in a 25mL volumetric flask, add methanol to dissolve it by ultrasonic and dilute it to the scale as the test solution. Take 3 batches of nicardipine hydrochloride raw materials to test the content of benzyl chloride and benzaldehyde. The results are shown in Table 15.

[0122] Table 15 Results of benzyl chloride and benzaldehyde content testing of nicardipine hydrochloride bulk drug

[0123] batch number 201911001 201911002 201911003 Benzaldehyde 0.00009％ 0.00008％ 0.00005％ Benzyl chloride Not detected Not detected Not detected

[0124] As shown in Table 15, this method is suitable for the detection of trace amounts of benzyl chloride and benzaldehyde in nicardipine hydrochloride bulk drug.

[0125] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments. The embodiments in this application and the features in the embodiments can be combined with each other in any way without conflict. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. In addition, the technical details not described in detail in this specification belong to the common technology of those skilled in the art, so this specification will not be repeated.

Claims

1. A method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC, characterized in that: The following steps are involved: 1) Prepare test solution and control solution; Accurately weigh 1g of nicardipine hydrochloride, place it in a 25mL volumetric flask, dissolve it in methanol and dilute it to the mark, shake well as the test solution; Accurately measure 250 mg of benzaldehyde and 250 mg of benzyl chloride respectively, accurately weigh them, place them in different 100 mL volumetric bottles, add methanol to dissolve and dilute to the scale, as each impurity reference substance reserve solution I; accurately measure 1 mL of each reference substance reserve solution I, place it in the same 100 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance reserve solution II; accurately measure 0.5 mL of the reference substance reserve solution II, place it in a 25 mL volumetric bottle, add methanol to dilute to the scale, as the reference substance solution; 2) Setting liquid chromatography detection conditions: using a chromatographic column with octadecylsilane bonded silica gel as a filler, the mobile phase consists of phase A and phase B, wherein phase A is a perchloric acid solution, phase B is a mixed solution of methanol and acetonitrile, the solvent is methanol, and gradient elution is performed; 3) Accurately pipette the test solution and the control solution respectively, inject them into the liquid chromatograph, the detection wavelength of the liquid chromatograph is 210nm, start detection and record the chromatogram; 4) Draw a 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 contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC according to claim 1, characterized in that: In the step 2), the chromatographic column is Welch XS-C18, the specifications of the chromatographic column are 250mm×4.6mm, the filler particle size is 5μm, and the column temperature is 35℃.

3. The method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC according to claim 1, characterized in that: In the step 2), the concentration of the perchloric acid solution is 0.086% (V / V); in the mixed solution, the volume ratio of acetonitrile to methanol is 75:25; and the injection volume is 20 μL.

4. The method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC according to claim 1, characterized in that: In the step 2), the gradient elution conditions are: from 0 to 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%; from 20 to 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%; At 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%.

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

6. The method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC according to claim 1, characterized in that: There are impurities in the chromatogram of the test solution with the same retention time as the control solution. Calculated by peak area using the external standard method, the benzyl chloride content is not more than 0.00125%, and the benzaldehyde content is not more than 0.00125%.

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

8. The method for determining the contents of benzyl chloride and benzaldehyde in nicardipine hydrochloride by HPLC according to claim 1, characterized in that: The linear concentration range of benzaldehyde is 0.1000μg / mL~1.0003μg / mL, the limit of quantification is 0.1000μg / mL, the limit of detection is 0.03μg / mL; the linear concentration range of benzyl chloride is 0.11021μg / mL~1.0212μg / mL, the limit of quantification is 0.1021μg / mL, and the limit of detection is 0.0306μg / mL.

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