Method for detecting nicardipine hydrochloride related substances

Abstract

The invention relates to a method for detecting nicardipine hydrochloride related substances, and relates to the technical field of medicine quality detection. According to the method, high performance liquid chromatography is used for detection, and detection conditions of the high performance liquid chromatography are as follows: a mobile phase is composed of a mobile phase A and a mobile phase B, the mobile phase A is 0.01 M monopotassium phosphate buffer solution and 0.3% triethylamine (phosphoric acid is used for adjusting pH to 5.0), and the mobile phase B is methanol. According to the method, triethylamine is used as a mobile phase, the peak shape is effectively improved, the symmetry of a main peak is good, effective separation is achieved, and related substances in nicardipine hydrochloride can be rapidly, effectively and accurately monitored, the method is high in specificity, sensitivity and recovery rate, impurities are quantitatively analyzed through a main component external standard method with correction factors, and the detection accuracy is high. The detection accuracy of the related substances is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting nicardipine hydrochloride related substances. Background Art

[0002] Related substances are starting materials, intermediates, side reaction products and degradation impurities introduced during the drug synthesis production process. Testing of related substances can control the quality and safety of drugs.

[0003] Nicardipine hydrochloride, molecular formula is C 26 H 29 N 3 O 6 HCl is a light yellow powder or yellow crystalline powder; odorless, almost tasteless. Nicardipine hydrochloride tablets: used for hypertension and exertional angina pectoris; Nicardipine hydrochloride sustained-release capsules: used for essential hypertension; Nicardipine hydrochloride injection: 1. used for emergency treatment of abnormally high blood pressure during surgery; 2. used for hypertensive emergencies.

[0004] At present, regarding the detection of related substances of nicardipine hydrochloride, it is known from the literature that the European Pharmacopoeia (EP), the United States Pharmacopoeia (USP), and the nicardipine hydrochloride standards monitor impurities including nicardipine pyridine analogs, while the Chinese Pharmacopoeia only controls nicardipine pyridine analogs. Chinese patent CN116381121A discloses a separation and detection method using liquid chromatography that can effectively separate related substances in nicardipine hydrochloride raw materials, but the patent document can only partially separate and determine nicardipine hydrochloride and its impurities. 3-(2-(benzyl(methyl)amino)ethyl)5-methyl 2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate is an impurity that needs to be monitored, and there are easily miscellaneous peaks near it, which makes separation difficult.

[0005] Table Comparison of standard chromatographic analysis methods for some drugs of Nicardipine Hydrochloride at home and abroad

[0006]

[0007]

[0008]

[0009] The currently provided analytical methods are unable to fully separate the various related substances or the related substances from the elution peaks of nicardipine hydrochloride. Therefore, it is of great significance to provide a detection method for the related substances of nicardipine hydrochloride. The high performance liquid chromatography method for determining the related substances in the nicardipine hydrochloride raw material provided by the present invention can effectively separate the impurities in nicardipine hydrochloride, including degradation impurities and process impurities. The method of the present invention adopts a gradient elution method for analysis, which has good reproducibility, strong specificity, high sensitivity and simple operation. Summary of the invention

[0010] The object of the present invention is to provide a high performance liquid chromatography method for determining related substances in nicardipine hydrochloride raw material medicine, using a chromatographic column with octadecylsilane bonded silica gel as a filler.

[0011] To achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] A high performance liquid chromatography method for determining related substances in nicardipine hydrochloride raw material medicine comprises the following steps:

[0013] (1) Diluent: Mobile phase

[0014] (2) Test solution: dissolve the nicardipine hydrochloride raw material in a diluent to obtain a test solution;

[0015] (3) Reference solution: dissolve nicardipine hydrochloride reference substance in a diluent to prepare the reference solution;

[0016] (4) System suitability solution: Take each impurity of nicardipine hydrochloride, dilute with solvent, and prepare system suitability solution;

[0017] (5) Detection was performed using a high performance liquid chromatograph, and the chromatographic conditions were:

[0018] Chromatographic column: octadecylsilane bonded silica gel filled chromatographic column;

[0019] Detection wavelength: 236nm;

[0020] Flow rate: 1.0ml / min;

[0021] Injection volume: 10 μl;

[0022] Mobile phase A: potassium dihydrogen phosphate-triethylamine aqueous solution;

[0023] Mobile phase B: methanol;

[0024] (6) Detection:

[0025] Separately inject the test solution, reference solution, and system suitability solution into the liquid chromatograph, record the chromatogram, and calculate according to the main component external standard method;

[0026] Furthermore, the concentration of the test solution in step (2) is 0.5 mg / ml;

[0027] Furthermore, the concentration of the reference solution in step (3) is 0.5 μg / ml;

[0028] Furthermore, the main component concentration of the system suitability solution in step (4) is 0.5 mg / ml, and the impurity concentration is 0.5 μg / ml.

[0029] Furthermore, in the step (5), the mobile phase A is potassium dihydrogen phosphate-triethylamine aqueous solution, which is 0.01M potassium dihydrogen phosphate containing 0.3% triethylamine aqueous solution, and the pH is adjusted to 5.0±0.2 with phosphoric acid.

[0030] Furthermore, the specification of the chromatographic column is Agilent (5) HC-C18 4.6*250mm, 5μm.

[0031] Furthermore, the elution method used in step (6) is: gradient elution;

[0032]

[0033] The present invention has the following beneficial effects compared with the prior art:

[0034] (1) The present invention traces the origin of the related substances of nicardipine hydrochloride for the first time and identifies 7 related substances, providing a reliable impurity spectrum reference for the study of related substances of nicardipine hydrochloride, which has a great positive progress effect and practical application value;

[0035] (2) The present invention optimizes the chromatographic conditions and controls the analysis time to 45 minutes, thereby improving the test efficiency and saving experimental time and cost;

[0036] (3) This method has higher sensitivity, and a concentration of 0.5 mg / ml can meet the sensitivity requirement; by optimizing the mobile phase composition and gradient time, the main peak shape is ensured to be free of tailing, thereby increasing the number of theoretical plates;

[0037] (4) The determination method of the present invention comprehensively analyzes the impurity spectrum in nicardipine hydrochloride according to the synthesis process of nicardipine hydrochloride raw material, and can effectively separate nicardipine hydrochloride from various impurities, thereby ensuring the accuracy of the test results;

[0038] (5) The present invention has conducted a comprehensive evaluation of the effectiveness and detection ability of the method, and the evaluation results show that the detection method has high accuracy, strong specificity and good durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the liquid chromatogram of the system suitability solution of method 3 in Example 1;

[0040] Figure 2 It is a Nika horizon sex relationship diagram;

[0041] Figure 3 is the linear relationship graph of nicardipine impurity A;

[0042] Figure 4 is the linear relationship graph of nicardipine impurity B;

[0043] Figure 5 is the linear relationship graph of nicardipine impurity C;

[0044] Figure 6 is the linear relationship graph of impurity D of nicardipine;

[0045] Figure 7 It is the linear relationship graph of impurity E of nicardipine;

[0046] Figure 8 is the linear relationship graph of impurity F of nicardipine;

[0047] Fig. 9 This is the linear relationship graph of impurity G of nicardipine. DETAILED DESCRIPTION

[0048] In the embodiments of the present invention, the structural information of nicardipine hydrochloride and seven impurities is shown in the table.

[0049] Known impurities and structural information

[0050]

[0051]

[0052] The method for detecting nicardipine hydrochloride related substances provided by the present invention can achieve sufficient separation of nicardipine hydrochloride from known related compounds with high separation degree by selecting appropriate liquid chromatography detection conditions.

[0053] The following will be described clearly and completely in conjunction with the technical solutions of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Example 1

[0055] Method exploration process, the specific exploration plan is shown in Table 2

[0056]

[0057]

[0058]

[0059]

[0060] Example 2: Related substance method validation

[0061] 1. Exclusivity

[0062] The instrument and chromatographic conditions were the same as those in Example 1.

[0063] System suitability experiment

[0064] Accurately weigh nicardipine hydrochloride reference substance, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G, dissolve them in methanol, and then dilute them with solvent to contain 0.5 mg / ml of nicardipine hydrochloride and 0.5 μg / ml each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, and impurity G. Take 10 μl and inject it into high performance liquid chromatograph. The test results are shown in the table.

[0065]

[0066] The blank solvent does not affect the inspection of related substances of this product. The separation degrees between the nicardipine hydrochloride peak and the adjacent impurity peaks are 8.46 and 6.06, respectively. The separation degrees between each impurity peak are greater than 2.0, and the theoretical plate numbers are greater than 5000. The test results show that this method has good system applicability.

[0067] 2. Limit of detection and limit of quantification

[0068] The impurity reference stock solution was diluted stepwise. The quantification limit was determined when the signal-to-noise ratio was approximately 10:1, and the detection limit was determined when the signal-to-noise ratio was approximately 3:1. The results are shown in the table.

[0069]

[0070] Conclusion: The quantitative limits of each impurity are between 0.1038μg / ml and 0.1354μg / ml, which is 0.02076% to 0.02708% relative to the concentration of the test solution. The results are good and meet the detection sensitivity requirements.

[0071] 3. Linearity and correction factors

[0072] Take the impurity reference stock solution to prepare different concentration linear relationship solutions, calculate the linear relationship equation, and use the slope value to calculate the impurity correction factor (f = K 主 / K 杂 ), and the results are shown in the table.

[0073] Linear relationship and correction factor test results

[0074]

[0075]

[0076] Conclusion: The test results show that the components have good linear relationships within the range of quantitative limit concentration to 150% of the limit concentration, and the correlation coefficients are all greater than 0.995; except for the correction factor of impurity D of 0.70, the correction factor of impurity C of 0.72, and the correction factor of impurity B of 2.22, which are beyond the range of 0.9 to 1.1, the correction factors of other impurities are all within the range of 0.9 to 1.1. In order to make the test simpler and faster and reduce the inspection cost, the main component external standard method with correction factors is used to calculate the content.

[0077] 4. System precision test

[0078] The impurity reference stock solution was diluted to prepare a mixed solution containing 0.5 μg per 1 ml, and the sample was injected 6 times continuously. The peak area and retention time of each component were counted, and the RSD (%) was calculated. The test results are shown in the table.

[0079] System precision test results

[0080]

[0081]

[0082] Conclusion: The mixed solution was injected 6 times continuously, and the RSD (%) of peak area and retention time were less than 2%. The experimental results showed that the system precision of this method was good.

[0083] 5. Accuracy Experiment

[0084] Take the impurity reference stock solution and prepare 50% limit concentration, 100% limit concentration and 150% limit concentration test solution respectively. Calculate the measured amount and added amount of 3 recovery solutions at each concentration by the principal component external standard method with correction factor, calculate the original amount of impurities in the test solution, calculate the recovery rate according to the added amount, original amount and measured amount, and calculate the mean of 3 recoveries at the quantitative limit concentration, the mean of 9 recoveries at other concentrations and the relative standard deviation (RSD). The test results are shown in the table.

[0085] Accuracy test results

[0086]

[0087]

[0088]

[0089]

[0090] Conclusion: The recovery rate test was carried out using the method of the present invention. The recovery rates of various impurities at different concentrations were all in the range of 80% to 110%, and the RSD (%) of the recovery rates of various impurities were all within 10%. The test results showed that this method had good accuracy, and the impurity detection results were accurate and reliable.

[0091] 6. Durability test

[0092] The flow rate, wavelength, and pH value of the mobile phase in the chromatographic conditions of this method were fine-tuned respectively, and the separation degree of each component in the mixed solution was used as an indicator. The test results are shown in the table.

[0093]

[0094]

[0095] Conclusion: The separation degrees between the components in the mixed solution were all greater than 1.5, and the separation degrees between nicardipine hydrochloride and adjacent impurities were all greater than 2.5. The experimental results showed that this method had good durability.

[0096] In summary, the detection method provided by the present invention has strong specificity and good durability. Changing the chromatographic conditions such as flow rate and mobile phase pH value within a small range will not affect the detection of nicardipine hydrochloride related substances; the method provided by the present invention can accurately detect the relevant substances in nicardipine hydrochloride, and the separation between the impurities and between the main component and the impurities is good. The detection result is accurate and reliable, and the quality standard of nicardipine hydrochloride can be controlled more scientifically. The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting related substances of nicardipine hydrochloride, characterized in that: It includes the following steps: (1) Preparation of test solution and control solution: a. Diluent: mobile phase; b. Test solution: Take an appropriate amount of nicardipine hydrochloride bulk drug, dissolve it in methanol and dilute it with a diluent to prepare the test solution; c. Reference solution: Take an appropriate amount of nicardipine hydrochloride reference substance, dissolve it in methanol and dilute it with a diluent to obtain the reference solution; d. System suitability solution: Take appropriate amount of each impurity reference substance of nicardipine hydrochloride, dilute with solvent, and use as system suitability solution (1) Chromatographic conditions Chromatographic column: octadecylsilane bonded silica gel as filler; Mobile phase A: 0.01 M potassium dihydrogen phosphate buffer; Mobile phase B: methanol; Detection wavelength: 236nm; Flow rate: 1.0ml / min; Injection volume: 10 μl (2) Determination method Separately inject the test solution, reference solution and system suitability solution into the liquid chromatograph, record the chromatogram, and calculate according to the principal component external standard method with correction factor added.

2. The high performance liquid chromatography method for determining related substances in nicardipine hydrochloride bulk drug according to claim 1, characterized in that: Mobile phase A: 0.01 M potassium dihydrogen phosphate buffer plus 0.3% triethylamine (pH adjusted to 5.0 with phosphoric acid).

3. A high performance liquid chromatography method for determining related substances in nicardipine hydrochloride bulk drug according to claim 1, characterized in that: The flow rate of the detection method is 1.0 ml / min; the injection volume is 10 μl; and the detection wavelength is 236 nm.

4. The high performance liquid chromatography method for determining related substances in nicardipine hydrochloride bulk drug according to claim 1, characterized in that: The chromatographic column specification is Agilent (5) HC-C18 4.6*250mm,5μm.

5. The high performance liquid chromatography method for determining related substances in nicardipine hydrochloride bulk drug according to claim 1, characterized in that: Elution method used: gradient elution; 6. The method for testing related substances of nicardipine hydrochloride according to claim 1, characterized in that: The concentration of the test solution in step (1) is 0.5 mg / ml; the concentration of the reference solution is 0.5 μg / ml; the concentration of the main component of the system suitability solution is 0.5 mg / ml, and the concentration of the impurity is 0.5 μg / ml.

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