Detection method of benidipine hydrochloride intermediate I related substances

Abstract

The invention belongs to the technical field of medicine quality control, and particularly relates to a method for detecting related substances of a benidipine hydrochloride intermediate I. The invention finds that an impurity R, namely 6-methyl-2, 4-bis (3-nitrophenyl)-1, 2, 3, 4-tetrahydropyrimidine-5-carboxylic acid methyl ester, exists in a benidipine hydrochloride intermediate I for the first time. Furthermore, the invention provides a method for detecting the quality of the benidipine hydrochloride intermediate I through high performance liquid chromatography, and the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and a phosphate solution with the pH value of 5.5-7.5 and acetonitrile are proportioned to serve as a mobile phase A or a mobile phase B. The invention further provides a method for detecting the quality of the benidipine hydrochloride intermediate I through high performance liquid chromatography. Within the range of the detection method provided by the invention, various impurities in the benidipine hydrochloride intermediate I can be accurately detected through the synergistic effect of detection conditions, the high-efficiency control of the medicine quality can be realized, the effectiveness and safety of the medicine are improved, and the application prospect is very good.

Description

Technical Field

[0001] The invention belongs to the technical field of drug quality control, and particularly relates to a method for detecting related substances of benidipine hydrochloride intermediate I. Background Art

[0002] Benidipine Hydrochloride has a chemical name: (R,R)-(+ / -)-2,6-dimethyl-4-(3-nitrobenzene)-1,4-dihydro-3,5-pyridinedicarboxylic acid-methyl-(R)-1-benzyl-3-piperidinyl ester hydrochloride. Benidipine Hydrochloride is mainly used in the treatment of essential hypertension and angina pectoris in clinical practice. It can stabilize blood pressure and improve arterial compliance without causing adverse reactions. In the preparation process of drugs, it is inevitable that a variety of impurities will be produced. The type and content of drug impurities will affect the safety of medication. The more impurities in the finished drug, the greater the possible medication risk. Therefore, it is necessary to effectively separate and detect the impurities of benidipine hydrochloride, which is of great significance to the production and quality control of benidipine hydrochloride.

[0003] Benidipine hydrochloride intermediate I is an important intermediate for the synthesis of benidipine hydrochloride. Its chemical name is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester. There are many impurities in benidipine hydrochloride intermediate I. During the research process, our company found that some of the impurities are as follows:

[0004]

[0005]

[0006] The related substances of benidipine hydrochloride intermediate I, especially the impurity SM-1 and the newly discovered impurity R (6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester) of the present invention and other nitro compounds, all have genotoxic impurity warning structures. They are likely to generate potential genotoxic compounds in further chemical synthesis, which may affect the quality of the final product benidipine hydrochloride. Therefore, it is very important to establish a complete quality control method for the related substances of benidipine hydrochloride intermediate I. For quality control, how to detect the related substances of benidipine hydrochloride intermediate I and effectively separate them from benidipine hydrochloride intermediate I is a crucial link. Summary of the invention

[0007] In view of the problems of the prior art, the present invention provides a method for detecting related substances of benidipine hydrochloride intermediate I, aiming to provide an accurate detection method for related substances of benidipine hydrochloride intermediate I and a newly discovered impurity R.

[0008] A benidipine hydrochloride intermediate I composition comprises the following components in percentage by weight: 90.0-93.3% of benidipine hydrochloride intermediate I, and impurity R not higher than 10.0%.

[0009] in,

[0010] The benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester;

[0011] The weight percentage of the impurity R is not 0, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester.

[0012] The present invention provides a use of an impurity R as a reference substance in a quality detection method for a benidipine hydrochloride intermediate I, wherein the benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester.

[0013] The present invention also provides a method for detecting related substances of benidipine hydrochloride intermediate I, wherein the benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, and high performance liquid chromatography is used to detect the benidipine hydrochloride intermediate I.

[0014] The chromatographic conditions are:

[0015] Octadecylsilane bonded silica gel is used as a filler, and a phosphate solution with a pH value of 5.5-7.5 and acetonitrile are mixed as a mobile phase A or a mobile phase B, wherein the mobile phase A is a solution in which the volume ratio of the phosphate solution to the acetonitrile is (60-65):(35-40); and the mobile phase B is a solution in which the volume ratio of the phosphate solution to the acetonitrile is (20-25):(75-80).

[0016] Preferably, a phosphate solution with a pH of 6.5-7.2 and acetonitrile are mixed as mobile phase A or mobile phase B.

[0017] Preferably, the concentration of phosphate in the phosphate solution is 0.01-0.02 mol / L, and / or the phosphate solution is prepared using anhydrous dipotassium hydrogen phosphate or anhydrous disodium hydrogen phosphate.

[0018] Preferably, the chromatographic conditions further include: the elution mode is gradient elution, and the elution conditions are:

[0019] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 8-13 75-85 15-25 16-25 55-76 24-45 28-32 28-32 68-72 34-36 30-33 67-70 37-42 90-100 0-10 44-48 100 0

[0020] Injection volume: 10 μL; flow rate: 0.8-1.2 mL / min; column temperature: 25°C-35°C, detection wavelength: 230-238 nm.

[0021] Preferably, the chromatographic conditions further include: the elution mode is gradient elution, and the elution conditions are:

[0022] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 9-12 80-85 15-20 20-22 55-65 35-45 28-30 28-30 70-72 35-36 30-33 67-70 37-42 95-100 0-5 45-48 100 0

[0023] Injection volume: 10 μL; flow rate: 0.9-1.1 mL / min; column temperature: 28°C-35°C, detection wavelength: 233-238 nm.

[0024] Preferably, the chromatographic column used in the liquid chromatography method is: Phenomenex Gemini, 250 mm×4.6 mm×5 μm.

[0025] Preferably, acetonitrile is used as a solvent in the liquid chromatography method, and the benidipine hydrochloride intermediate I sample is prepared into a test solution, wherein the concentration of the benidipine hydrochloride intermediate I in the test solution is 0.3-0.4 mg / mL.

[0026] Preferably, the chromatogram obtained by the high performance liquid chromatography detection includes a chromatographic peak of impurity R, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester, and the relative retention time of the impurity R is 0.95.

[0027] The present invention first discovered that the benidipine hydrochloride intermediate I contains an impurity R, the chemical name of the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester, and the structural formula is:

[0028]

[0029] Furthermore, the present invention realizes the quality detection of benidipine hydrochloride intermediate I by high performance liquid chromatography by optimizing the chromatographic conditions. The quality detection method of the present invention has a separation degree greater than 1.5 for the impurity R and other known impurities in the sample, and the impurity separation is good, and the related substances of benidipine hydrochloride intermediate I can be efficiently detected. Therefore, the quality detection method provided by the present invention has high detection accuracy, good specificity, high separation degree, and low detection limit for each impurity, which can realize efficient control of drug quality and improve the effectiveness and safety of drugs; and the detection method is easy to operate and has strong practicality. Therefore, the present invention has a good application prospect.

[0030] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0031] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the sample solution spectrum of Example 1;

[0033] Figure 2 This is the sample solution spectrum of Example 2;

[0034] Figure 3 This is the sample solution spectrum of Example 3;

[0035] Figure 4 This is the sample solution spectrum of Example 4;

[0036] Figure 5 This is the sample solution spectrum of Example 5;

[0037] Figure 6 This is the solution spectrum of the system suitability of Comparative Example 1;

[0038] Figure 7 This is the solution spectrum of the system suitability of Comparative Example 2;

[0039] Figure 8 This is the sample solution spectrum of Comparative Example 2;

[0040] Fig. 9 This is the solution spectrum of the system applicability of Comparative Example 3;

[0041] Fig.10 This is the solution spectrum of the system applicability of Comparative Example 4;

[0042] Fig.11 is the H NMR spectrum of impurity R;

[0043] Fig.12 is the carbon NMR spectrum of impurity R;

[0044] Fig.13 is the infrared spectrum of impurity R.

[0045] In the spectrum: the impurity with a relative retention time of 0.95 is impurity R. DETAILED DESCRIPTION

[0046] In the following examples, the reagents and materials used unless otherwise specified are commercially available.

[0047] The implementation results of the present invention are judged according to the following description:

[0048] If there are impurity peaks in the chromatogram of the test solution, impurity F shall be calculated according to the corrected peak area (multiplied by the correction factor 1.3) and shall not be greater than 0.1 times (0.10%) the main peak area of ​​the control solution; impurity R shall be calculated according to the corrected peak area (multiplied by the correction factor 3.1) and shall not be greater than 10 times (10.0%) the main peak area of ​​the control solution; the area of ​​other single impurity peaks shall not be greater than 0.4 times (0.4%) the main peak area of ​​the control solution; the sum of the peak areas of other impurities (except impurity R) shall not be greater than 1.5 times (1.5%) the main peak area of ​​the control solution, and peaks less than 0.03 times the main peak area of ​​the control solution shall be ignored.

[0049] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0050] Calculation formula:

[0051] Single impurity = impurity peak area / reference solution main peak area × F × 1%

[0052] The total amount of other impurities (excluding impurity R) = ∑ single impurity (excluding impurity R), where F is the correction factor for the impurity, impurity F is multiplied by the correction factor 1.3, and the correction factor for unknown impurities is 1.0.

[0053] In the following examples, the impurity R reference substance used was synthesized as follows:

[0054] Weigh 100.12g of m-nitrobenzaldehyde, 85.07g of methyl acetoacetate, 67ml of ammonia water and 200ml of methanol, heat to 60℃, reflux and stir for 4h. After the reaction is completed, the reaction solution is filtered, and the filter cake is added with 500ml of tetrahydrofuran and refluxed and stirred for 30min. After the reflux is completed, filter while hot, and add 500ml of tetrahydrofuran to the filter cake and reflux and stir for 30min. After the reflux is completed, filter while hot, and add 200ml of tetrahydrofuran to the filter cake and reflux and stir for 30min. After the reflux is completed, filter while hot, and the filter cake is dried under reduced pressure to obtain 22.40g of impurity R, with a content of 96.2%.

[0055] Figure 11-13 The hydrogen nuclear magnetic resonance spectrum, carbon nuclear magnetic resonance spectrum and infrared spectrum of impurity R are shown respectively, and it can be seen that impurity R is successfully synthesized.

[0056] Example 1

[0057] High performance liquid chromatography was used to detect related substances in benidipine hydrochloride intermediate I, specifically:

[0058] 1. Chromatographic conditions:

[0059] Chromatographic column: octadecylsilane bonded silica gel as filler (Phenomenex Gemini, 250 mm × 4.6 mm × 5 μm);

[0060] Mobile phase A: anhydrous potassium hydrogen phosphate buffer (1.74 g of anhydrous potassium hydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH value was adjusted to 6.5 with phosphoric acid)-acetonitrile = 65:35;

[0061] Mobile phase B: anhydrous potassium hydrogen phosphate buffer (take 1.74 g of anhydrous potassium hydrogen phosphate, dissolve it in water and dilute it to 1000 ml, and adjust the pH value to 6.5 with phosphoric acid)-acetonitrile = 25:75;

[0062] Injection volume: 10 μl; flow rate: 0.8 ml / min; column temperature: 25°C; detection wavelength: 230 nm; gradient elution conditions are as follows:

[0063] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 9 80 20 20 55 45 28 28 72 35 30 70 37 95 5 45 100 0

[0064] 2. Solution Preparation

[0065] Blank solution: acetonitrile.

[0066] Impurity F stock solution: Weigh about 50 mg of the impurity F reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, (concentration 2.5 mg / ml).

[0067] Impurity F 50 times limit solution: Accurately measure 1 ml of "Impurity F stock solution", place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain (concentration 25 μg / ml).

[0068] System suitability solution: Take 15 mg of intermediate I reference substance, weigh accurately, and accurately measure 1 ml of "impurity F 50 times limit solution", put it in the same 50 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and the solution is ready. (Intermediate I 0.3 mg / ml, impurity F 0.5 μg / ml).

[0069] Test solution: Grind the benidipine hydrochloride intermediate I test sample, weigh 15 mg, accurately weigh, place in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0070] 3. Determination

[0071] Take blank, system suitability solution and test solution respectively, inject into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. Figure 1As shown, it can be seen that the separation of each impurity is good and the peak shape is standard, which shows that the method of this embodiment can achieve accurate detection of these impurities. Taking benidipine hydrochloride intermediate I as the reference peak, the impurity with a relative retention time of 0.95 is impurity R.

[0072] Example 2

[0073] The related substances of benidipine hydrochloride intermediate I were detected by high performance liquid chromatography, and the conditions were basically the same as those in Example 1, except that:

[0074] 1. Chromatographic conditions:

[0075] Mobile phase A: anhydrous potassium hydrogen phosphate buffer (take 2.61 g of anhydrous potassium hydrogen phosphate, dissolve in water and dilute to 1000 ml, adjust the pH value to 7.2 with phosphoric acid)-acetonitrile = 60:40;

[0076] Mobile phase B: anhydrous potassium hydrogen phosphate buffer (take 2.61 g of anhydrous potassium hydrogen phosphate, dissolve in water and dilute to 1000 ml, adjust the pH value to 7.2 with phosphoric acid)-acetonitrile = 20:80;

[0077] Injection volume: 10 μl; flow rate: 0.9 ml / min; column temperature: 28 °C; detection wavelength: 233 nm;

[0078] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 12 85 15 22 65 35 30 30 70 36 33 67 42 100 0 48 100 0

[0079] 2. Solution Preparation

[0080] Blank solution: acetonitrile.

[0081] Impurity F stock solution: Weigh about 70 mg of the impurity F reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, (concentration 2.5 mg / ml).

[0082] Impurity F 50 times limit solution: Accurately measure 1 ml of "Impurity F stock solution", place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain (concentration 25 μg / ml).

[0083] System suitability solution: Take 25 mg of intermediate I reference substance, weigh accurately, and accurately measure 1 ml of "impurity F 50 times limit solution", put it in the same 50 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and the solution is ready. (Intermediate I 0.5 mg / ml, impurity F 0.7 μg / ml).

[0084] Test solution: Grind the product into powder, weigh 17.5 mg, accurately weigh, place in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well.

[0085] 3. Determination

[0086] Take blank, system suitability solution and test solution respectively, inject into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. Figure 2 As shown, it can be seen that the separation degree of each impurity is good and the peak shape is standard, which indicates that the method of this embodiment can achieve accurate detection of these impurities.

[0087] Example 3

[0088] The related substances of benidipine hydrochloride intermediate I were detected by high performance liquid chromatography, and the conditions were basically the same as those in Example 1, except that:

[0089] 1. Chromatographic conditions:

[0090] Mobile phase A: anhydrous potassium hydrogen phosphate buffer (1.74 g of anhydrous potassium hydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH value was adjusted to 7.0 with phosphoric acid)-acetonitrile = 63:37;

[0091] Mobile phase B: anhydrous potassium hydrogen phosphate buffer (take 1.74 g of anhydrous potassium hydrogen phosphate, dissolve it in water and dilute it to 1000 ml, and adjust the pH value to 7.0 with phosphoric acid)-acetonitrile = 20:80;

[0092] Injection volume: 10 μl; flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 237 nm; gradient elution conditions are as follows:

[0093]

[0094]

[0095] 2. Solution Preparation

[0096] Blank solution: acetonitrile.

[0097] Impurity F stock solution: Weigh about 60 mg of the impurity F reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, (concentration 3 mg / ml).

[0098] Impurity F 50 times limit solution: Accurately measure 1 ml of "Impurity F stock solution", place it in a 100 ml volumetric flask, add acetonitrile to dilute to the scale, shake well, and obtain (concentration 30 μg / ml).

[0099] System suitability solution: Take 20 mg of intermediate I reference substance, weigh accurately, and accurately measure 1 ml of "impurity F 50 times limit solution", put it in the same 50 ml volumetric bottle, add acetonitrile to dilute to the scale, shake well, and the solution is ready. (Intermediate I 0.4 mg / ml, impurity F 0.6 μg / ml).

[0100] Test solution: Grind the product into powder, weigh 20 mg, accurately weigh, place in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well.

[0101] 3. Determination

[0102] Take blank, system suitability solution and test solution respectively, inject into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. Figure 3 As shown, it can be seen that the separation degree of each impurity is good and the peak shape is standard, which indicates that the method of this embodiment can achieve accurate detection of these impurities.

[0103] Example 4

[0104] The related substances of benidipine hydrochloride intermediate I were detected by high performance liquid chromatography, and the conditions were basically the same as those in Example 1, except that:

[0105] 1. Chromatographic conditions:

[0106] Mobile phase A: anhydrous potassium hydrogen phosphate buffer (1.74 g of anhydrous potassium hydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH value was adjusted to 5.5 with phosphoric acid)-acetonitrile = 62:38;

[0107] Mobile phase B: anhydrous potassium hydrogen phosphate buffer (1.74 g of anhydrous potassium hydrogen phosphate was dissolved in water and diluted to 1000 ml, and the pH value was adjusted to 5.5 with phosphoric acid)-acetonitrile = 22:78;

[0108] Injection volume: 10 μl; flow rate: 1.2 ml / min; column temperature: 35°C; detection wavelength: 238 nm; gradient elution conditions are as follows:

[0109] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 13 75 25 25 55 45 32 32 68 36 30 70 42 100 0 48 100 0

[0110] 2. Determination

[0111] Take blank, system suitability solution and test solution respectively, inject into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. Figure 4 As shown, it can be seen that the separation degree of each impurity is good and the peak shape is standard, which indicates that the method of this embodiment can achieve accurate detection of these impurities.

[0112] Example 5

[0113] The related substances of benidipine hydrochloride intermediate I were detected by high performance liquid chromatography, and the conditions were basically the same as those in Example 4, except that:

[0114] 1. Chromatographic conditions:

[0115] Mobile phase A: anhydrous potassium hydrogen phosphate buffer (take 3.48 g of anhydrous potassium hydrogen phosphate, dissolve in water and dilute to 1000 ml, adjust the pH value to 7.5 with phosphoric acid)-acetonitrile = 62:38;

[0116] Mobile phase B: anhydrous potassium hydrogen phosphate buffer (take 3.48 g of anhydrous potassium hydrogen phosphate, dissolve in water and dilute to 1000 ml, adjust the pH value to 7.5 with phosphoric acid)-acetonitrile = 22:78;

[0117] Injection volume: 10 μl; flow rate: 1.1 ml / min; column temperature: 35 °C; detection wavelength: 238 nm;

[0118] The gradient elution conditions are as follows:

[0119]

[0120]

[0121] 2. Determination

[0122] Take blank, system suitability solution and test solution respectively, inject into the chromatographic system according to the above chromatographic conditions, and record the chromatogram. Figure 5 As shown, it can be seen that the separation degree of each impurity is good and the peak shape is standard, which indicates that the method of this embodiment can achieve accurate detection of these impurities.

[0123] Experimental Example 1 Methodological Verification

[0124] The method shown in the above Example 3 was verified by methodology:

[0125] 1. System suitability verification

[0126] The above blank solution and system suitability solution were taken to verify the system suitability. The results are shown in the following table:

[0127]

[0128] The test results showed that the blank baseline was stable and free of interference; in the system suitability solution, impurity F, impurity R, and intermediate I peaked in sequence, and the separation degree of each peak was not less than 1.5, which met the verification requirements.

[0129] 2. Exclusivity Verification

[0130] Take 10 μL of blank solution, each impurity location solution, test solution, control solution, and sample interference solution respectively, inject them into the chromatography system, and record the chromatogram to determine the interference of all potential impurities.

[0131] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0132] Impurity R positioning solution: The concentration of the impurity R reference substance is 0.5 mg / mL.

[0133] SM-1 positioning solution: The concentration of SM-1 reference substance is 4 μg / mL.

[0134] Impurity F localization solution: The concentration of the impurity F reference substance is 0.6 μg / mL.

[0135] Impurity J localization solution: The concentration of the impurity J reference substance is 4 μg / mL.

[0136] Impurity K localization solution: The concentration of impurity K reference substance is 4 μg / mL.

[0137] Impurity L positioning solution: The concentration of the impurity L reference substance is 4 μg / mL.

[0138] Impurity N localization solution: The concentration of the impurity N reference substance is 4 μg / mL.

[0139] Impurity P localization solution: The concentration of the impurity P reference substance is 4 μg / mL.

[0140] Impurity Q positioning solution: The concentration of the impurity Q reference substance is 4 μg / mL.

[0141] Sample interference solution: Weigh about 20 mg each of SM-1, impurity J, impurity K, impurity L, impurity N, impurity P, and impurity Q reference substances, accurately weigh, then accurately measure 1 ml of "impurity F stock solution", place it in the same 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, (impurity F is about 30 μg / ml, other impurity concentrations are about 0.2 mg / ml, limit 50 times), to obtain impurity mixed stock solution; grind this product, weigh 20 mg, accurately weigh, place it in a 50 ml volumetric flask, accurately measure 1 ml of "impurity mixed stock solution", place it in the same 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and obtain.

[0142] Impurity F stock solution: Weigh about 60 mg of the impurity F reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, (concentration is about 3 mg / ml).

[0143] The retention time of the positioning solution is shown in the following table:

[0144]

[0145]

[0146] The component separation of the sample interference solution and the test solution is shown in the following table:

[0147] name Sample Interference Solution Test solution Components Separation Separation Impurity J / Not detected Unknown impurity 1 8.4 / SM-1 8.8 5.3 Impurity K 17.0 11.3 Impurity F 6.4 6.1 Impurity L 4.5 Not detected Impurity R 2.9 6.8 Main Peak 3.5 3.5 Unknown impurity 2 9.7 9.6 Unknown impurity 3 6.0 6.0 Impurity N 3.7 Not detected Impurity Q 17.4 Not detected Impurity P 3.7 Not detected

[0148] The baseline of the blank solution is stable and free of interference; each positioning solution does not interfere with the detection; the separation between the main peak and the adjacent impurities in the sample interference solution, and between each known impurity and the adjacent impurities is not less than 1.5, and the peak purity factor of the main peak is 1000≥990 after detection by the DAD detector; the separation between the main peak and the adjacent impurities in the test solution, and between each known impurity and the adjacent impurities is not less than 1.5, and the peak purity factor of the main peak is 1000≥990 after detection by the DAD detector. In the control solution, the theoretical plate number is 64690≥5000 based on the main peak of the control solution, which meets the verification requirements.

[0149] 3. Detection Limit

[0150] Take the quantitative limit solution and dilute it to a low concentration sample, measure the signal and the signal at the blank (baseline noise), and calculate the lowest concentration or percentage that can be reliably detected. The measured S / N is required to be no less than 3.

[0151] In all tables, “ / ” indicates not applicable.

[0152] The statistical results of the detection limit test are shown in the following table:

[0153]

[0154] The signal-to-noise ratio (S / N) value of each component in each detection limit solution should be no less than 3, meeting the verification requirements.

[0155] 4. Limit of Quantitation

[0156] Prepare a known concentration of impurity stock solution and dilute it to a low concentration sample. Inject 6 injections at this concentration, measure the signal and the signal at the blank (baseline noise), calculate the lowest concentration or percentage that can be reliably detected, and take the average value. It is required that the S / N of 6 injections should not be less than 10 and the RSD of the peak area of ​​each component in the 6 injections of quantitative limit solution should not exceed 15%, and the quantitative limit concentration should not be greater than 50% of the limit concentration.

[0157] The statistical results of the quantitative limit test are shown in the following table:

[0158]

[0159] The signal-to-noise ratio (S / N) value of the 6-shot quantitative limit solution should be greater than 10; the RSD of the peak area of ​​each component in the 6-shot quantitative limit solution should not exceed 15%; the quantitative limit concentration should not be greater than 50% of the limit concentration, which meets the verification requirements.

[0160] 5. Accuracy Verification

[0161] The accuracy is determined by adding 25%, 100% and 150% of the impurity limit concentration to the test solution, and the recovery rate between the relevant measurement results and the theoretical value at different concentrations is achieved.

[0162] In the accuracy verification, the accuracy recovery results of the known impurities and impurity R were both 80%-120%, and the RSD was no more than 10.0%.

[0163] The above experimental results show that the method of the present invention has high detection accuracy, good specificity, high separation and low detection limit, and can therefore be used for the accurate detection of various impurities in benidipine hydrochloride intermediate I.

[0164] The following are some changes to the chromatographic conditions of the present invention and comparison of the detection effects.

[0165] Comparative Example 1

[0166] Compared with Example 3, the difference of Comparative Example 1 is:

[0167] Octadecylsilane bonded silica gel is used as filler (ZORBAX SB-CN 4.6*250mm, 5um);

[0168] Mobile phase A: 0.05 mol / L KH2PO4 (pH 3.0)-acetonitrile = 67:33

[0169] Mobile phase B: 0.05 mol / L KH2PO4 (pH 3.0)-acetonitrile = 67:33

[0170] Column temperature: 25℃.

[0171] Solution preparation

[0172] Blank solution: 40% acetonitrile.

[0173] Figure 6 The results of the spectrum test of Comparative Example 1 show that Bndp-1-ZZ-191014 is impurity F. It can be seen that the analysis method is isocratic elution, but the baseline is significantly elevated at about RT8min-30min; there are about 0.59% impurities at retention time 39min and about 0.49% impurities at retention time 71.2min, and the peak shape is relatively wide. The analysis time is too long (80min), and the test solution is unstable.

[0174] Comparative Example 2

[0175] Compared with Example 3, the difference of Comparative Example 1 is:

[0176] Chromatographic column: ZORBAX SB-CN 4.6*250mm, 5um 118#;

[0177] Mobile phase B: acetonitrile;

[0178] Mobile phase A: 0.01 mol / L KH2PO4 (pH 7.0)

[0179] Elution gradient:

[0180] time min B％ A％ 0 28 72 30 45 55 40 45 55 41 28 72 50 28 72

[0181] Depend on Figure 7 and Figure 8 It can be seen that the SM-1 peak is not at the forefront, and the separation degree from the adjacent impurity peak is 2.2; about 1.7% of impurities are detected after the main peak. However, the separation degree between the main peak and the adjacent impurity (content of about 1.7%) is 2.7, but it does not reach the baseline separation; SM-1 is unstable. At the same time, when the injection plate is controlled at 5°C, crystals precipitate in the sample bottle.

[0182] Comparative Example 3

[0183] Compared with Example 1, the difference of Comparative Example 1 is:

[0184] Mobile phase B: acetonitrile;

[0185] Mobile phase A: 0.01 mol / L KH2PO4 (pH 7.0)

[0186] Elution gradient

[0187] time min B％ A％ 0 37 63 12 47 53 25 47 53 35 55 45 40 55 45 41 37 63 50 37 63

[0188] Fig. 9 It is a system suitability solution. It can be seen that the SM-1 peak is at the forefront, but the separation degree from the adjacent impurities is good, and the separation degree from the adjacent impurities is 6.0 and 3.0; the main peak has a good separation degree from the adjacent impurities, with a separation degree of about 4, reaching baseline separation; however, compared with Examples 1 and 2, the impurity F in the system suitability solution is poorly separated from the adjacent impurities, with a separation degree of about 1.

[0189] Comparative Example 4

[0190] Compared with Example 1, the difference of Comparative Example 1 is:

[0191] Chromatographic column: Agilent ZORBAX Bonus-RP4.6*250mm, 5um; Mobile phase B: 0.01mol / L KH2PO4 (pH 7.0)-acetonitrile = 20:80; Mobile phase A: 0.01mol / L KH2PO4 (pH 7.0)-acetonitrile = 65:35;

[0192] Gradient elution:

[0193] time min A％ B％ 0 97 3 12 70 30 30 60 40 35 60 33 36 97 3 45 97 3

[0194] Fig.10 This is the spectrum of the system suitability solution. It can be seen that the separation degree near the main peak is 3 and 4, and the separation degree is good; however, in the system suitability solution, there is a baseline elevation phenomenon at SM-1; after 28 minutes, the impurity separation degrees are less than or equal to 1, and the separation degree is poor.

[0195] It can be seen from the above embodiments and comparative examples that within the scope of the detection method of the present invention, through the synergistic effect of detection conditions, the present invention can efficiently detect related substances of benidipine hydrochloride intermediate I (including various impurities such as impurity R), provide a basis for improving the effectiveness and safety of drugs, and further realize efficient control of drug quality.

Claims

1. A benidipine hydrochloride intermediate I composition, characterized in that: The invention comprises the following components in percentage by weight: 90.0-93.3% of benidipine hydrochloride intermediate I, and impurity R not higher than 10.0%. in, The benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester; The weight percentage of the impurity R is not 0, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester.

2. Impurity R is used as a reference substance in the quality detection method of benidipine hydrochloride intermediate I, wherein the benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester.

3. A method for detecting related substances of benidipine hydrochloride intermediate I, wherein the benidipine hydrochloride intermediate I is 1,4-dihydro-2,6-dimethyl-4-(m-nitrophenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, characterized in that: The benidipine hydrochloride intermediate I and its related substances are detected by high performance liquid chromatography. The chromatographic conditions are: Octadecylsilane bonded silica gel is used as a filler, and a phosphate solution with a pH value of 5.5-7.5 and acetonitrile are mixed as a mobile phase A or a mobile phase B, wherein the mobile phase A is a solution in which the volume ratio of the phosphate solution to the acetonitrile is (60-65):(35-40); and the mobile phase B is a solution in which the volume ratio of the phosphate solution to the acetonitrile is (20-25):(75-80).

4. The detection method according to claim 3, characterized in that: Phosphate solution with a pH of 6.5-7.2 and acetonitrile were mixed as mobile phase A or mobile phase B.

5. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 3, characterized in that: The concentration of phosphate in the phosphate solution is 0.01-0.02 mol / L, and / or the phosphate solution is prepared using anhydrous dipotassium hydrogen phosphate or anhydrous disodium hydrogen phosphate.

6. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 3, characterized in that: The chromatographic conditions also include: the elution mode is gradient elution, and the elution conditions are: Injection volume: 10 μL; flow rate: 0.8-1.2 mL / min; column temperature: 25°C-35°C, detection wavelength: 230-238 nm.

7. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 6, characterized in that: The chromatographic conditions also include: the elution mode is gradient elution, and the elution conditions are: Injection volume: 10 μL; flow rate: 0.9-1.1 mL / min; column temperature: 28°C-35°C, detection wavelength: 233-238 nm.

8. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 3, characterized in that: The chromatographic columns used in the liquid chromatography method are: Phenomenex Gemini, 250mm×4.6mm×5μm.

9. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 3, characterized in that: In the liquid chromatography method, acetonitrile is used as a solvent, and a sample of the benidipine hydrochloride intermediate I is prepared into a test solution, wherein the concentration of the benidipine hydrochloride intermediate I in the test solution is 0.3-0.4 mg / mL.

10. The method for detecting related substances of benidipine hydrochloride intermediate I according to claim 3, characterized in that: The chromatogram obtained by the high performance liquid chromatography method includes a chromatographic peak of impurity R, and the impurity R is 6-methyl-2,4-bis(3-nitrophenyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylic acid methyl ester. The relative retention time of the impurity R is 0.95.

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