Separation and determination method of Nicodil starting material SM1 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and determining a Nicodil starting material SM1 and impurities thereof. The impurities comprise any one or more of an impurity SM1a, an impurity SM1b, an impurity SM1c, an impurity SM1j, an impurity SM1l, an impurity SM1m, an impurity SM1n, an impurity SM1o, an impurity SM1f, an impurity SM1k, an impurity SM1i, an impurity SM1h and an impurity SM1g. The method comprises the following steps: carrying out linear gradient elution on a chromatographic column by taking octadecyl silane bonded silica gel as a filling agent, an ammonium acetate buffer solution as a mobile phase A and a mixed solution of methanol and acetonitrile as a mobile phase B; and then detecting in a detector, and carrying out qualitative and quantitative detection on each impurity according to the detected chromatogram. The method disclosed by the invention can be used for effectively separating and determining various impurities in the Nicodil starting material SM1, and has the characteristics of high sensitivity, strong practicability and accurate and reliable detection result.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for separating and determining a nicorandil starting material SM1 and its impurities. Background Art

[0002] Nicorandil is a nitrate compound, mainly used to treat angina pectoris. It has the functions of preventing the release of intracellular calcium ions, increasing the permeability of cell membranes to potassium ions, dilating coronary blood vessels, continuously increasing coronary blood flow, inhibiting coronary artery spasm, and inhibiting platelet aggregation to prevent thrombosis. In addition, when dilating coronary blood vessels, the drug does not affect blood pressure, heart rate, myocardial contractility, and myocardial oxygen consumption. Nicorandil requires strict dosage control during treatment. However, during its production process, many impurities are generated, and its impurities are bound to affect the content of the active ingredient of the main drug. Therefore, it is particularly important to detect its impurities during the production process.

[0003] Nicorandil starting material SM1 (abbreviated as SM1) is the key starting material in the synthesis of Nicorandil. Studies have found that Nicorandil starting material SM1 may introduce the following 13 impurities during the synthesis process: Impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g These impurities will affect the quality of SM1, and the impurities SM 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i Contains warning structures, so it needs to be detected and controlled during the production process.

[0004] High performance liquid chromatography is often used to detect various drug impurities, but there is currently no method that can simultaneously separate and determine the main component and related impurities in the starting material SM1 of Nicorandil. The pharmacopoeias of various countries have not found the quality standard of Nicorandil starting material SM1. The literature was consulted to find the method of related substances in the finished product, but the method of related substances in the finished product was used to determine Nicorandil starting material SM1 and its impurities. It was found that the peak shape of each impurity was poor, the peak was tailing, and multiple impurities could not be effectively separated. This method is not suitable for the detection of these impurities.

[0005] In the prior art, patent CN106770737A discloses an HPLC analysis method for nicorandil related substances, which uses a reverse phase column of carbon octadecyl bonded silica gel as the stationary phase, and a buffer solution and a mixed solution of methanol and tetrahydrofuran as the mobile phase to separate and determine nicorandil related substances; the patent does not disclose the specific related substances. Patent CN109406706A discloses a method for determining related substances in nicorandil tablets using an HPLC correction factor method, which uses a C18 chromatographic column and a trifluoroacetic acid-triethylamine-tetrahydrofuran-water mixed solution as the mobile phase, and the impurities that can be quantitatively determined simultaneously include impurity A, impurity B, impurity C, impurity D, impurity E and related unknown impurities.

[0006] However, the methods disclosed in the above prior art are not suitable for the separation and determination of Nicorandil starting material SM1 and its related impurities. The 13 impurities have similar structures and are difficult to separate. Therefore, it is necessary to establish a new method to control the impurities in Nicorandil starting material SM1 to improve the production efficiency and finished product qualification rate of Nicorandil starting material SM1 API and subsequent preparation products. Summary of the invention

[0007] In view of this, one of the objects of the present invention is to provide a method for separating the nicorandil starting material SM1 and its impurities based on high performance liquid chromatography, which can complete the effective separation of the nicorandil starting material SM1 and its impurities within 50 minutes or more.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] A method for separating a nicorandil starting material SM1 and its impurities based on high performance liquid chromatography, wherein the nicorandil starting material SM1 and the impurities together constitute a composition to be tested; the impurities include impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of the following; the method comprises: using octadecylsilane bonded silica gel as a filler in the chromatographic column, using ammonium acetate buffer as mobile phase A, and using a mixed solution of methanol and acetonitrile as mobile phase B, and performing linear gradient elution;

[0010] The structural formula of the Nicorandil starting material SM1 and its impurities is as follows:

[0011]

[0012] The separated substances can also be used in production.

[0013] Furthermore, the above impurities can be arranged and combined in various ways.

[0014] For example, combination 1: the impurities include impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1i Any one or more of .

[0015] Combination 2: The impurities include impurity SM 1j 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g Any one or more of .

[0016] Combination 3: The impurities include impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c Any one or more of .

[0017] Combination 4: the impurities include combination 1, combination 2 and / or combination 3.

[0018] This is not an exhaustive list of all permutations and combinations.

[0019] Theoretically, when the upper limit of the substances for separation, identification and / or content detection of the present method is 14 (including the main component and 13 impurities), any 1 to 14 substances therein can naturally be detected.

[0020] Further, the procedure of the linear gradient elution is as follows:

[0021] At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10;

[0022] At 6 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10;

[0023] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 35-55:65-45;

[0024] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 15-35:85-65;

[0025] At 44 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 15-35:85-65;

[0026] At 45 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10;

[0027] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 70-90:30-10.

[0028] Further, the procedure of the linear gradient elution is as follows:

[0029] At 0 minute, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0030] At 6 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0031] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 43-47:57-53;

[0032] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 23-27:77-73;

[0033] At 44 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 23-27:77-73;

[0034] At 45 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0035] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 78-82:22:18.

[0036] Preferably, the procedure of the linear gradient elution is as follows:

[0037] At 0 minute, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0038] At 6 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0039] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 45:55;

[0040] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 25:75;

[0041] At 44 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 25:75;

[0042] At 45 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 78-82:22:18;

[0043] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 78-82:22:18.

[0044] Most preferably, the procedure of the linear gradient elution is as follows:

[0045] At 0 minute, the volume ratio of the mobile phase A to the mobile phase B was set to 80:20;

[0046] At 6 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 80:20;

[0047] At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 45:55;

[0048] At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 25:75;

[0049] At 44 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 25:75;

[0050] At 45 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 80:20;

[0051] At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 80:20.

[0052] Furthermore, the mobile phase A is an ammonium acetate solution with a concentration of 0.005-0.015 mol / L and a pH of 6.5-7.5; in the mobile phase B, the volume ratio of methanol to acetonitrile is 45-55:55-45.

[0053] Preferably, the mobile phase A is an ammonium acetate solution with a concentration of 0.008 to 0.012 mol / L and a pH of 6.8 to 7.2.

[0054] Most preferably, the mobile phase A is an ammonium acetate solution with a concentration of 0.01 mol / L and a pH of 7.0.

[0055] Furthermore, the pH of mobile phase A was adjusted with aqueous ammonia.

[0056] Preferably, in the mobile phase B, the volume ratio of methanol to acetonitrile is 50:50.

[0057] Furthermore, the flow rate is 0.5 mL / min to 1.5 mL / min; and the column temperature is 20°C to 30°C.

[0058] Preferably, the flow rate is 0.9 mL / min to 1.1 mL / min, and most preferably 1.0 mL / min.

[0059] Preferably, the column temperature is 23°C to 27°C, most preferably 25°C.

[0060] Preferably, the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm; most preferably, it is YMC-Pack ODS-AQ, 4.6 mm×250 mm, 5 μm or a chromatographic column with equivalent performance.

[0061] Furthermore, a mobile phase impurity trapping column is added.

[0062] Preferably, the mobile phase impurity trapping column has a specification of 4.6 mm×50 mm; the most preferred is the Chromatograph Ghost-Sniper Column, 4.6 mm×50 mm or a trapping column with equivalent performance.

[0063] Furthermore, the injection volume was 10 μl.

[0064] Furthermore, the running time is preferably 50 minutes.

[0065] The second object of the present invention is to provide a method for identifying the nicorandil starting material SM1 and its impurities, which can complete the identification of multiple impurities in the nicorandil starting material SM1 in a relatively short time, and the detection limit concentration is as low as about 0.04 μg / ml to 0.12 μg / ml.

[0066] To achieve the above object, the present invention adopts the following technical solutions:

[0067] The method for identifying the nicorandil starting material SM1 and its impurities comprises separating the composition by the aforementioned separation method, and entering a detector for detection to obtain a chromatogram; and determining whether the test sample contains the nicorandil starting material SM1 and its impurities by comparing the chromatogram characteristics of the test sample with those of the reference sample.

[0068] Furthermore, the detection wavelength of the detector is 250±2nm.

[0069] Preferably, the detection wavelength of the detector is 250 nm.

[0070] As a preference, the components are separated in the following order from earliest to latest: impurity SM 1a 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1b , Nicorandil starting material SM1, impurity SM 1c 、Impurity SM 1f 、Impurity SM 1i 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g .

[0071] Preferably, the impurities include impurities SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of .

[0072] Furthermore, the impurity SM is present at a retention time of 2.5 ± 0.5 min. 1a ; The impurity SM is with a retention time of 8.0±0.5min 1j ; The impurity SM has a retention time of 9.2±0.5min 1l ; The impurity SM has a retention time of 11.3±0.5min 1m ; The impurity SM has a retention time of 14.7±0.5min 1n ; The impurity SM has a retention time of 16.1±0.5min 1o ; The impurity SM has a retention time of 16.4±0.5min 1b ; The retention time is 19.6±0.5min, which is the starting material of Nicorandil SM1; The retention time is 20.4±0.5min, which is the impurity SM 1c ; The retention time is 22.5±0.5min, which is impurity SM 1f ; The impurity SM has a retention time of 23.6±0.5min 1i ; The impurity SM has a retention time of 23.9±0.5min 1k ; The impurity SM has a retention time of 25.1±0.5min 1h ; The impurity SM is at a retention time of 41.0±0.5min 1g .

[0073] The components can be characterized based on the order in which they are separated, their relative retention times and / or their retention times.

[0074] The third object of the present invention is to provide a method for detecting whether the impurity content in the nicorandil starting material SM1 is qualified. The method can complete the identification of multiple impurities in the nicorandil starting material SM1 in a relatively short time, and the quantitative limit concentration is as low as about 0.1 μg / ml to 0.4 μg / ml.

[0075] To achieve the above object, the present invention adopts the following technical solutions:

[0076] The method for detecting whether the impurity content in the nicorandil starting material SM1 is qualified is to separate and identify the nicorandil starting material SM1 and its impurities by the above-mentioned identification method to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in each test product is qualified.

[0077] Preferably, the impurities include impurities SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of .

[0078] Furthermore, if the impurity SM in the test product 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h and / or impurities SM 1g The peak area of ​​the impurity in the test sample is greater than the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is unqualified; on the contrary, if the impurity SM in the test sample is 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h and / or impurities SM 1g The peak area of ​​the impurity is not greater than the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is qualified; the impurity SM 1j 、the impurity SM1o 、the impurity SM 1f 、the impurity SM 1k 、the impurity SM 1h and the impurity SM 1g The impurity limit is 0.10%, the impurity SM 1l The impurity limit is 0.02%, the impurity SM 1m 、the impurity SM 1n 、the impurity SM 1i The impurity limit is 0.0018%.

[0079] Furthermore, the sample preparation solvent is a mixed solution of methanol and phosphoric acid water.

[0080] Furthermore, in the solvent, the volume ratio of methanol to phosphoric acid water is 5:95.

[0081] Preferably, the concentration of phosphoric acid water in the solvent is 0.1%.

[0082] Most preferably, the solvent is prepared from methanol and 0.1% phosphoric acid water in a volume ratio of 5:95.

[0083] Furthermore, the concentration of the sample solution is 2 mg / mL, 4 mg / mL and / or 28 mg / mL.

[0084] Further, the method comprises the following steps:

[0085] (1) Take an appropriate amount of the product, weigh it accurately, dissolve it in a solvent, and quantitatively dilute it to make a solution containing about 2 mg per 1 ml, as sample solution 1;

[0086] (2) Take the impurity SM separately 1j 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to make a mixed solution containing about 2 μg per 1 ml, which is used as reference substance solution 1;

[0087] (3) Take an appropriate amount of the product, weigh it accurately, dissolve it in a solvent, and quantitatively dilute it to make a solution containing about 4 mg per 1 ml, as sample solution 2;

[0088] (4) Take the impurity SM 1l An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to make a mixed solution containing about 0.8 μg per 1 ml, which is used as reference substance solution 2;

[0089] (5) Take an appropriate amount of the product, weigh it accurately, dissolve it in a solvent, and quantitatively dilute it to make a solution containing about 28 mg per 1 ml, as sample solution 3;

[0090] (6) Take the impurity SM separately 1m 、Impurity SM 1n 、Impurity SM 1i An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to make a mixed solution containing about 0.5 μg per 1 ml, which is used as reference substance solution 3;

[0091] (7) Accurately measure the sample solution and reference solution, inject them into a liquid chromatograph, and obtain a chromatogram. Based on the chromatogram, use the limit method to detect whether the impurity content is qualified by using the peak area.

[0092] Further, the method is used to separate and determine the impurities in the Nicorandil starting material SM1 and its related substances; the related substances include the impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c Any one or more of; the impurities include impurities SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of .

[0093] The beneficial effects of the present invention are:

[0094] (1) The present invention provides for the first time a high performance liquid chromatography method for separating and determining the nicorandil starting material SM1 and its related impurities. The method can effectively separate the main component and 13 known impurities in the nicorandil starting material SM1 in a set of HPLC system: the impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h and impurity SM 1gThe separation degrees between other known impurities are all greater than 1.5, which has the advantages of simplicity, speed and high accuracy.

[0095] (2) Among the 13 impurities related to the nicorandil starting material SM1, individual impurities have similar structures and are difficult to separate. In order to simultaneously detect the nicorandil starting material SM1 and its 13 impurities using a method to save detection time and cost. The present invention improves the chromatographic condition parameters of the related substances, and by screening different chromatographic columns, adding buffer salts, adjusting the pH value of the mobile phase, adjusting the gradient elution program, etc., the nicorandil starting material SM1 and related impurities can be well separated, and then the method is used to perform qualitative and quantitative analysis on the main drug and its impurities.

[0096] (3) The method of the present invention has strong specificity, high durability, good reproducibility, and is not affected by impurities SM 1a 、Impurity SM 1b 、Impurity SM 1c , blank solvent and other unknown impurities, and the separation degrees between known impurities were greater than 1.3.

[0097] (4) This method is a self-developed high performance liquid chromatography method, which fills the gap in the detection of Nicorandil starting material SM1 and its related impurities.

[0098] (5) The analytical method provided by the present invention can effectively control the quality of the nicorandil starting material SM1, and obtain the nicorandil raw material that meets the quality standards, which is more conducive to the research and development and quality control of subsequent preparation products.

[0099] (6) This method has a short detection time, detects more impurities, and has a simple detection process, which effectively reduces the time cost and labor cost for production inspection work. In addition, the mobile phase preparation process of this method is simple and easy to operate.

[0100] (7) Some impurities in the nicorandil starting material SM1 have warning structures, and the limit is as low as 0.0018%. This method can simultaneously meet the requirements of the limits of 0.0018%, 0.02% and 0.1%, and has the characteristics of high sensitivity.

[0101] (8) The method of the present invention can separate 13 impurities in the nicorandil starting material SM1, and can perform qualitative and quantitative analysis on 10 of the impurities, and has the characteristics of high sensitivity, strong practicability, and accurate and reliable detection results. The method of the present invention is of great significance for achieving quality control of the nicorandil starting material SM1. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 is the chromatogram of the blank solution in Example 1;

[0103] Figure 2is the chromatogram of the mixed solution in Example 1;

[0104] Figure 3 is the chromatogram of the reference substance solution in Example 2;

[0105] Figure 4 is the chromatogram of the quantitative limit solution in Example 3;

[0106] Figure 5 is the chromatogram of the detection limit solution in Example 4;

[0107] Figure 6 The chromatogram of the test sample mixed solution in Example 5 at a flow rate of 0.9 ml / min;

[0108] Figure 7 The chromatogram of the test sample mixed solution in Example 5 at a flow rate of 1.1 ml / min;

[0109] Figure 8 The chromatogram of the test sample mixed solution in Example 5 when the column temperature is 23°C;

[0110] Fig. 9 The chromatogram of the test sample mixed solution in Example 5 when the column temperature is 27°C;

[0111] Fig.10 This is the chromatogram of the test sample mixed solution in Example 5 when the mobile phase pH is 6.8;

[0112] Fig.11 This is the chromatogram of the test sample mixed solution in Example 5 when the mobile phase pH is 7.2;

[0113] Fig.12 The chromatogram of the test sample mixed solution in Example 5 when the initial ratio of the mobile phase is A:B=82:18;

[0114] Fig.13 This is the chromatogram of the test sample mixed solution in Example 5 when the initial ratio of the mobile phase is A:B=78:22;

[0115] Fig.14 is the chromatogram of the blank solution in Example 6;

[0116] Fig.15 is the chromatogram of the mixed solution (concentration is 28 mg / ml) in Example 6;

[0117] Fig.16 is the chromatogram of the reference substance solution in Example 7;

[0118] Fig.17 is the chromatogram of the quantitative limit solution in Example 8;

[0119] Fig.18 This is the chromatogram of the detection limit solution in Example 9. DETAILED DESCRIPTION

[0120] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0121] In the present invention, the sample to be tested is a solution whose main component is the nicorandil starting material SM1, and the sample to be tested contains the nicorandil starting material SM1, the impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of .

[0122] The sample to be tested may also contain other unspecified compounds, which refer to other impurities that may be introduced when preparing the Nicorandil starting material SM1 using conventional methods known in the art.

[0123] Unless otherwise specified, all numerical values ​​and similar values ​​used in the present invention to express parameters are understood to be modified by the term "about". That is, these numerical values ​​are allowed to have certain reasonable deviations or error ranges in practical applications, and do not have to strictly conform to the exact numerical values ​​given. Unless otherwise indicated, the numerical parameters described in the present invention are approximate values ​​that can be changed according to the performance required to be obtained.

[0124] Unless otherwise stated, terms or phrases involved in the present invention should not be considered as uncertain or unclear if there is no special definition, but should be understood according to the ordinary meaning in the art.

[0125] In the present invention, the term "high performance liquid chromatography", also known as "high pressure liquid chromatography", "high speed liquid chromatography", "high resolution liquid chromatography", "modern column chromatography", etc., is a chromatographic analysis technology based on classical liquid chromatography and citing the theory of gas chromatography. High performance liquid chromatography (HPLC) uses liquid as the mobile phase and adopts a high-pressure infusion system to pump a single solvent with different polarities or a mixed solvent with different proportions, a buffer and other mobile phases into a chromatographic column equipped with a stationary phase. After the components in the column are separated, they enter the detector for detection, thereby realizing the analysis of the sample. This method can be used to separate, identify and quantitatively analyze various complex samples.

[0126] In the present invention, specificity, also known as selectivity or specificity, refers to the ability of the analytical method to accurately and selectively detect the component being measured when other components may be present in the sample. Specificity is a measure of mutual interference when the method is used for complex sample analysis. The present invention verifies whether the HPLC method has sufficient specificity to ensure accurate and unerring determination of the target analyte in complex samples.

[0127] In the present invention, the limit of detection (LOD) refers to the minimum concentration or amount of the target compound that the analytical method can reliably detect. The detection sensitivity of the HPLC method is determined, that is, the lowest concentration of the target analyte in the sample that can be identified. This helps to evaluate the reliability of the method at very low concentrations.

[0128] In the present invention, the limit of quantitation (LOQ) refers to the lowest concentration or content of the target compound that the analytical method can reliably quantify. The limit of quantitation reflects the ability of the analytical method to detect sensitive quantification and determines at what concentration the HPLC method can accurately and reliably quantify the target analyte.

[0129] In the present invention, robustness refers to the degree to which the measurement results are not affected when there are small changes in the measurement conditions. It is mainly used to detect the anti-interference ability of the analytical method to variable test factors.

[0130] In the present invention, information on Nicorandil starting material SM1 and its related impurities are shown in Table 1.

[0131] Table 1. Compound information

[0132]

[0133]

[0134] In the embodiments of the present invention, the determination was strictly carried out in accordance with the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512).

[0135] In the embodiment of the present invention, the preparation method of the test solution and the reference solution is as follows:

[0136] Solvent: a mixed solution of methanol and 0.1% phosphoric acid water, wherein the volume ratio of methanol to phosphoric acid water is 5:95.

[0137] Test solution 1: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 2 mg per 1 ml.

[0138] Reference solution 1: Take impurity SM separately 1j 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to produce a mixed solution containing approximately 2 μg per 1 ml.

[0139] Test solution 2: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 4 mg per 1 ml.

[0140] Reference solution 2: Take impurity SM 1l An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to produce a mixed solution containing approximately 0.8 μg per 1 ml.

[0141] Test solution 3: Take an appropriate amount of the product, weigh accurately, dissolve in solvent and quantitatively dilute to make a solution containing about 28 mg per 1 ml.

[0142] Reference solution 3: Take impurity SM separately 1m 、Impurity SM 1n 、Impurity SM 1i An appropriate amount of reference substance is accurately weighed, dissolved in solvent and quantitatively diluted to produce a mixed solution containing approximately 0.5 μg per 1 ml.

[0143] In the embodiment of the present invention, the chromatographic conditions include: using octadecylsilane bonded silica gel as a filler (YMC-PackODS-AQ, 4.6mm×250mm, 5μm or a chromatographic column with equivalent performance); using 0.01mol / L ammonium acetate solution (adjusting the pH to 7.0 with ammonia water) as mobile phase A, using methanol-acetonitrile (50:50) as mobile phase B, performing gradient elution according to Table 2, and adding a mobile phase impurity trapping column (Ghost-Sniper Column, 4.6mm×50mm or a trapping column with equivalent performance); the detection wavelength is 250nm; the flow rate is 1.0ml per minute; the column temperature is 25°C; and the injection volume is 10μl.

[0144] Table 2. Gradient elution schedule

[0145] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 6 80 20 20 45 55 35 25 75 44 25 75 45 80 20 50 80 20

[0146] In the embodiment of the present invention, the system suitability requirement is: in the chromatogram of the reference solution, the peak order is impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1i 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g , the peak separation of each component should meet the requirements.

[0147] In the embodiment of the present invention, the determination method is: accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0148] In the embodiment of the present invention, the impurity limit requirement is: if there is any impurity SM in the chromatogram of the test solution 1j 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g The peak area of ​​the chromatographic peak with the same retention time shall not be greater than the peak area of ​​the corresponding impurity in the reference solution (0.10%). 1l The peak area of ​​the chromatographic peak with the same retention time shall not be greater than the peak area of ​​the corresponding impurity in the reference solution (0.02%). 1m 、Impurity SM 1n 、Impurity SM 1i The peak area of ​​the chromatographic peak with the same retention time shall not be greater than the peak area of ​​the corresponding impurity in the reference solution (0.0018%).

[0149] In the embodiment of the present invention, due to the impurity SM 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g With impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1i The limits of the two tests are different, so the validation experiments such as specificity, system suitability, limit of quantitation, and limit of detection are performed separately. However, the methods are still the same, but the concentrations of the test products are different. The two sets of validation share the specificity and durability data.

[0150] In the embodiment of the present invention, the impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c In this system, only the specificity is proved to have no influence on the determination of impurities.

[0151] Example 1. Specificity experiment ①

[0152] Impurity SM in Nicorandil SM1 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h and impurity SM 1g Perform specific verification. The specific method is as follows:

[0153] 1. Prepare the test solution

[0154] Solvent: a mixed solution of methanol and 0.1% phosphoric acid water, wherein the volume ratio of methanol to phosphoric acid water is 5:95.

[0155] Impurity SM 1a Stock solution: Accurately weigh the impurity SM 1a Place 5.18 mg of the reference substance in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is ready.

[0156] Impurity SM 1b Stock solution: Accurately weigh the impurity SM 1b Place 10.16 mg of the reference substance in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is ready.

[0157] Impurity SM 1c Stock solution: Accurately weigh the impurity SM 1cPlace 12.61 mg of the reference substance in a 10 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is ready.

[0158] Impurity SM 1j Stock solution: Accurately weigh the impurity SM 1j Place 19.99 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0159] Impurity SM 1l Stock solution: Accurately weigh the impurity SM 1l Place 19.97 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0160] Impurity SM 1m Stock solution: Accurately weigh the impurity SM 1m Place 19.68 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0161] Impurity SM 1n Stock solution: Accurately weigh the impurity SM 1n Place 19.88 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0162] Impurity SM 1o Stock solution: Accurately weigh the impurity SM 1o Place 19.95 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0163] Impurity SM 1f Stock solution: Accurately weigh the impurity SM 1f Place 19.96 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0164] Impurity SM 1k Stock solution: Accurately weigh the impurity SM 1k Place 19.89 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0165] Impurity SM 1i Stock solution: Accurately weigh the impurity SM 1i Place 19.86 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0166] Impurity SM 1h Stock solution: Accurately weigh the impurity SM 1h Place 19.92 mg of reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain the product.

[0167] Impurity SM 1g Stock solution: Accurately weigh the impurity SM 1g Place 19.94 mg of the reference substance in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and the solution is obtained.

[0168] Impurity SM 1a Positioning solution: Precisely measure impurities SM 1a Place 4 ml of the stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0169] Impurity SM 1b Positioning solution: Precisely measure impurities SM 1b Place 2 ml of stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0170] Impurity SM 1c Positioning solution: Precisely measure impurities SM 1c Place 2 ml of stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0171] Other impurity location solutions (including impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g ): Accurately measure 1 ml of each impurity stock solution mentioned above, place it in different 10 ml volumetric bottles, dilute to the scale with solvent, shake well, and obtain.

[0172] Reference stock solution: Precisely pipette impurities SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Place 5 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0173] Test solution: Accurately weigh 52.15 mg of this product (batch number: ABEZ-240101), place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0174] Mixed solution: Accurately weigh 21.56 mg of this product and place it in a 10 ml volumetric flask. Accurately measure the impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c Dissolve 1 ml of the positioning solution and 2 ml of the reference stock solution in solvent and dilute to the scale. Shake well to obtain.

[0175] 2. Detection

[0176] Take 10 μl of blank solvent, each impurity location solution, test solution and mixed solution, inject them into liquid chromatograph respectively, and record the chromatogram.

[0177] The test results are shown in Tables 3 and 4. Figure 1-2 The blank solution, other impurities and other components in the test sample did not interfere with the detection. Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g The separation between adjacent peaks is greater than 1.3, and the specificity meets the requirements.

[0178] Table 3. Specificity test results

[0179]

[0180] Table 4. Figure 2 The integral results table

[0181]

[0182] Example 2. System suitability experiment ①

[0183] Impurity SM in Nicorandil SM1 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h and impurity SM 1g Conduct system suitability test. The specific method is as follows:

[0184] Reference substance solution: Accurately measure 5 ml of the reference substance stock solution under "Example 1. Specificity Experiment ①", place it in a 25 ml volumetric flask, dilute to the scale with solvent, shake well, and obtain.

[0185] Accurately measure 10 μl of the reference solution, inject it into the liquid chromatograph, measure it six times continuously, record the chromatogram, and calculate the retention time, theoretical plate number, tailing factor, peak area and peak area RSD of each impurity peak.

[0186] Test results such as Figure 3 , as shown in Table 5. The RSD of the peak area of ​​each impurity in the reference solution was less than 5.0%, the number of theoretical plates was greater than 5000, and the tailing factor was less than 2.0, which met the requirements.

[0187] Table 5. Figure 3 The integral result table

[0188]

[0189] Example 3. Quantitative limit experiment ①

[0190] Impurity SM in Nicorandil SM1 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h and impurity SM 1g Perform quantitation limit detection.

[0191] Quantitative limit solution: Accurately measure 2 ml of the reference solution under "Example 2. System suitability experiment ①", place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0192] Take the quantitative limit solution and measure it 6 times continuously, record the chromatogram, and calculate the ratio of each peak to noise (signal-to-noise ratio) and peak area RSD. The quantitative limit calculation formula is as follows:

[0193]

[0194] The test results are shown in Tables 6 to 7. Figure 4 SM 1j The limit of quantification concentration is 0.399μg / ml, which is expressed as 0.020% in the sample, with a peak area RSD of 1.1% and an S / N average of 35.9. 1o The quantitative limit concentration is 0.396μg / ml, which is expressed as 0.020% in the sample, with a peak area RSD of 4.1% and an S / N average of 15.1. The impurity SM 1fThe quantitative limit concentration is 0.391μg / ml, which is expressed as 0.020% in the sample, with a peak area RSD of 1.2% and an S / N average of 25.8. 1k The quantitative limit concentration is 0.388μg / ml, which is expressed as 0.019% in the sample, with a peak area RSD of 3.5% and an S / N average of 20.0; the impurity SM 1h The quantitative limit concentration is 0.397μg / ml, which is expressed as 0.020% in the sample, with a peak area RSD of 6.3% and an S / N average of 41.7. 1g The quantitative limit concentration is 0.394μg / ml, which is expressed as 0.020% in the sample, the peak area RSD is 2.4%, and the S / N average is 40.5. At the quantitative limit concentration of each impurity, the signal-to-noise ratio is greater than 10, and the peak area RSD is less than 10.0%, which meets the detection requirements of related substances, indicating that the impurities can be accurately quantified at this level.

[0195] Table 6. Quantitation limit test results

[0196]

[0197]

[0198] Table 7. Figure 4 The integral results table

[0199]

[0200] Example 4. Detection limit experiment ①

[0201] Impurity SM in Nicorandil SM1 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h and impurity SM 1g Detection limit determination was performed.

[0202] Detection limit solution: Accurately measure 3 ml of the quantification limit solution under "Example 3. Quantification limit experiment ①", place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0203] The detection limit solution was taken for continuous measurement three times, the chromatogram was recorded, and the ratio of each peak to noise (signal-to-noise ratio) was calculated.

[0204]

[0205] The test results are shown in Tables 8 and 9. Figure 5 SM 1jThe detection limit concentration is 0.120μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 9.1; the impurity SM 1o The detection limit concentration is 0.119μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 7.5; the impurity SM 1f The detection limit concentration is 0.117μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 7.8; the impurity SM 1k The detection limit concentration is 0.117μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 7.0; the impurity SM 1h The detection limit concentration is 0.119μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 11.8; the impurity SM 1g The detection limit concentration is 0.118μg / ml, which is expressed as 0.006% in the sample, and the average S / N is 12.6; when the impurities are at the detection limit concentration, the signal-to-noise ratio is greater than 3, which meets the detection requirements of relevant substances, indicating that the impurities can be effectively detected at this level.

[0206] Table 8. Detection limit test results

[0207]

[0208] Table 9. Figure 5 The integral result table

[0209]

[0210] Example 5. Durability test

[0211] Impurity SM in Nicorandil SM1 1j 、Impurity SM 1o 、Impurity SM1 f 、Impurity SM 1k 、Impurity SM 1h 、Impurity SM 1g 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i Durability verification was performed. The mixed solution under "Example 1. Specificity Experiment ①" was used as the test sample mixed solution for detection. Accurately measure 10 μl of the test sample mixed solution, adjust the experimental conditions (column flow rate ± 0.1 ml / min, column temperature ± 2 °C, mobile phase pH value ± 0.2, mobile phase initial ratio ± 2%), and test after the instrument system is stable. Investigate the relative retention time and separation of each peak.

[0212] The results are shown in Tables 10 to 19. Figure 2 , Figure 6 to Figure 13 When there are slight fluctuations in column temperature, flow rate, initial ratio of mobile phase and pH value, there is no significant difference in the relative retention time of each impurity, and the separation between the impurities and the adjacent peaks is greater than 1.3, which meets the requirements.

[0213] Table 10. Chromatographic system durability test results (relative retention time)

[0214]

[0215] Table 11. Chromatographic system durability test results (separation)

[0216]

[0217] Table 12. Figure 6 The integral results table

[0218]

[0219] Table 13. Figure 7 The integral results table

[0220]

[0221] Table 14. Figure 8 The integral results table

[0222]

[0223] Table 15. Fig. 9 The integral results table

[0224]

[0225] Table 16. Fig.10 The integral results table

[0226]

[0227] Table 17. Fig.11 The integral results table

[0228]

[0229] Table 18. Fig.12 The integral results table

[0230]

[0231] Table 19. Fig.13 The integral results table

[0232]

[0233] Example 6. Specificity experiment ②

[0234] Impurity SM in Nicorandil SM1 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i Perform specific verification. The specific method is as follows:

[0235] 1. Prepare the test solution

[0236] Solvent: a mixed solution of methanol and 0.1% phosphoric acid water, wherein the volume ratio of methanol to phosphoric acid water is 5:95.

[0237] Each impurity stock solution: use each impurity stock solution under "Example 1. Specificity Experiment ①".

[0238] Reference stock solution 1: Accurately pipette impurities SM 1l Place 1 ml of stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0239] Reference stock solution 2: Precisely pipette impurities SM 1m 、Impurity SM 1n 、Impurity SM 1i Place 1 ml of each stock solution in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0240] Test solution 1: Accurately weigh 40.24 mg of this product (batch number: ABEZ-240101), place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0241] Test solution 2: Accurately weigh 282.67 mg of this product (batch number: ABEZ-240101), place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0242] Mixed solution: accurately weigh 281.31 mg of this product, place it in a 10 ml volumetric bottle, add solvent to dissolve and dilute to the scale, shake well; accurately transfer 1 ml of the solution, place it in a sample injection bottle, and then add impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h、Impurity SM 1g Add 5 μl of each stock solution, shake well, and you are done.

[0243] 2. Detection

[0244] Take 10 μl of blank solvent, test solution and mixed solution respectively, inject them into liquid chromatograph and record the chromatogram.

[0245] Impurity localization solution spectra under "Example 1. Specificity experiment ①".

[0246] The test results are shown in Table 20 to Table 21. Figure 14-15 The blank solution, other impurities and other components in the test sample did not interfere with the detection. Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1i The separation between adjacent peaks is greater than 1.3, and the specificity meets the requirements.

[0247] Table 20. Specificity test results

[0248]

[0249] Table 21. Fig.15 The integral result table

[0250]

[0251] Example 7. System suitability experiment ②

[0252] Targeting the impurity SM in Nicorandil SM1 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i , system suitability experiments were carried out to ensure the reliability and effectiveness of the analytical method.

[0253] Reference substance solution: Accurately measure 4 ml of reference substance stock solution 1 and 2.5 ml of reference substance stock solution 2 under "Example 6. Specificity Experiment ②", place in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, and obtain.

[0254] Accurately measure 10 μl of the reference solution, inject it into the liquid chromatograph, measure it continuously for 6 times, record the chromatogram, and calculate the retention time, theoretical plate number, tailing factor, peak area and peak area RSD of each impurity peak.

[0255] Test results such as Fig.16 , as shown in Table 22. The RSD of the peak area of ​​each impurity in the reference solution is less than 5.0%, the number of theoretical plates is greater than 5000, and the tailing factor is less than 2.0, which meets the requirements.

[0256] Table 22. Fig.16 The integral result table

[0257]

[0258] Example 8. Quantitative limit experiment ②

[0259] Impurity SM in Nicorandil SM1 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i Carry out quantitative limit detection. The specific method is as follows:

[0260] Quantitative limit solution: Accurately measure 3 ml of the reference solution under "Example 7. System suitability experiment ②", place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0261] The quantitative limit solution was taken for continuous determination 6 times, the chromatogram was recorded, and the ratio of each peak to noise (signal-to-noise ratio) and the peak area RSD were calculated.

[0262]

[0263] The test results are shown in Table 23 to Table 24. Fig.17 SM 1l The quantitative limit concentration is 0.239μg / ml, which is expressed as 0.006% in the sample, with a peak area RSD of 4.2% and an S / N average of 27.2. The impurity SM 1m The quantitative limit concentration is 0.145μg / ml, which is expressed as 0.0005% in the sample, the peak area RSD is 3.2%, and the S / N average is 34.3; the impurity SM 1n The quantitative limit concentration is 0.145μg / ml, which is expressed as 0.0005% in the sample, the peak area RSD is 1.4%, and the S / N average is 39.9; the impurity SM 1i The quantitative limit concentration is 0.147μg / ml, which is expressed as 0.0005% in the sample, the peak area RSD is 2.1%, and the S / N average is 31.2. At the quantitative limit concentration of each impurity, the signal-to-noise ratio is greater than 10, and the peak area RSD is less than 10.0%, which meets the detection requirements of relevant substances, indicating that the impurities can be accurately quantified at this level.

[0264] Table 23. Quantitation limit experimental results

[0265]

[0266]

[0267] Table 24. Fig.17 The integral result table

[0268]

[0269] Example 9. Detection limit experiment ②

[0270] Impurity SM in Nicorandil SM1 1l 、Impurity SM 1m 、Impurity SM 1n and impurity SM 1i The specific method is as follows:

[0271] Detection limit solution: Accurately measure 3 ml of the quantification limit solution under "Example 8. Quantification limit experiment ②", place it in a 10 ml volumetric flask, add solvent to dilute to the scale, shake well, and obtain.

[0272] The detection limit solution was taken for continuous measurement three times, the chromatogram was recorded, and the ratio of each peak to noise (signal-to-noise ratio) was calculated.

[0273]

[0274] The test results are shown in Table 25 to Table 26. Fig.18 SM 1l The detection limit concentration is 0.072μg / ml, which is expressed as 0.002% in the sample, and the average S / N is 7.1; the impurity SM 1m The detection limit concentration is 0.043μg / ml, which is expressed as 0.00016% in the sample, and the average S / N is 8.4; the impurity SM 1n The detection limit concentration is 0.044μg / ml, which is expressed as 0.00016% in the sample, and the average S / N is 10.0; the impurity SM 1i The detection limit concentration is 0.044μg / ml, which is expressed as 0.00016 in the sample, and the average S / N is 8.4; when the impurities are at the detection limit concentration, the signal-to-noise ratio is greater than 3, which meets the detection requirements of relevant substances, indicating that the impurities can be effectively detected at this level.

[0275] Table 25. Detection limit experimental results

[0276]

[0277]

[0278] Table 26. Fig.18 The integral result table

[0279]

Claims

1. A method for separating nicorandil starting material SM1 and its impurities based on high performance liquid chromatography, characterized in that: The nicorandil starting material SM1 and the impurities together constitute the composition to be tested; the impurities include impurity SM 1a 、Impurity SM 1b 、Impurity SM 1c 、Impurity SM 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h 、Impurity SM 1g Any one or more of the following; the method comprises: using octadecylsilane bonded silica gel as a filler in the chromatographic column, using ammonium acetate buffer as mobile phase A, and using a mixed solution of methanol and acetonitrile as mobile phase B, and performing linear gradient elution; The structural formula of the Nicorandil starting material SM1 and its impurities is as follows:

2. The method according to claim 1, characterized in that The procedure of the linear gradient elution is as follows: At 0 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10; At 6 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10; At 20 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 35-55:65-45; At 35 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 15-35:85-65; At 44 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 15-35:85-65; At 45 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 70-90:30-10; At 50 minutes, the volume ratio of the mobile phase A to the mobile phase B was set to 70-90:30-10.

3. The method according to claim 1, characterized in that The mobile phase A is an ammonium acetate solution with a concentration of 0.005-0.015 mol / L and a pH of 6.5-7.5; in the mobile phase B, the volume ratio of methanol to acetonitrile is 45-55:55-45.

4. The method according to claim 1, characterized in that: The flow rate is 0.5-1.5 mL / min; the column temperature is 20℃-30℃.

5. A method for identifying Nicorandil starting material SM1 and its impurities, characterized in that: The composition is separated by the method described in any one of claims 1 to 4, and enters a detector for detection to obtain a chromatogram; by comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains the nicorandil starting material SM1 and its impurities.

6. The method according to claim 5, characterized in that The detection wavelength of the detector is 250±2 nm.

7. The method according to claim 5, characterized in that The impurity SM is retained at 2.5±0.5min 1a ; The impurity SM is with a retention time of 8.0±0.5min 1j ; The impurity SM has a retention time of 9.2±0.5min 1l ; The impurity SM has a retention time of 11.3±0.5min 1m ; The impurity SM has a retention time of 14.7±0.5min 1n ; The impurity SM has a retention time of 16.1±0.5min 1o ; The impurity SM has a retention time of 16.4±0.5min 1b ; The retention time is 19.6±0.5min, which is the starting material of Nicorandil SM1; The retention time is 20.4±0.5min, which is the impurity SM 1c ; The retention time is 22.5±0.5min, which is impurity SM 1f ; The impurity SM has a retention time of 23.6±0.5min 1i ; The impurity SM has a retention time of 23.9±0.5min 1k ; The impurity SM has a retention time of 25.1±0.5min 1h ; The impurity SM is at a retention time of 41.0±0.5min. 1g .

8. A method for detecting whether the impurity content in the Nicorandil starting material SM1 is qualified, characterized in that: The method according to any one of claims 5 to 7 is used to separate and identify the nicorandil starting material SM1 and its impurities to obtain a chromatogram; based on the obtained chromatogram, the limit method is used to determine whether the impurity content in each test product is qualified.

9. The method according to claim 8, characterized in that If the impurity SM in the test product 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h and / or impurities SM 1g The peak area of ​​the impurity in the test sample is greater than the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is unqualified; on the contrary, if the impurity SM in the test sample is 1j 、Impurity SM 1l 、Impurity SM 1m 、Impurity SM 1n 、Impurity SM 1o 、Impurity SM 1f 、Impurity SM 1k 、Impurity SM 1i 、Impurity SM 1h and / or impurities SM 1g The peak area of ​​the impurity is not greater than the peak area of ​​the corresponding impurity in the reference solution, indicating that the impurity content is qualified; the impurity SM 1j 、the impurity SM 1o 、the impurity SM 1f 、the impurity SM 1k 、the impurity SM 1h and the impurity SM 1g The impurity limit is 0.10%, the impurity SM 1l The impurity limit is 0.02%, the impurity SM 1m 、the impurity SM 1n 、the impurity SM 1i The impurity limit is 0.0018%.

10. The method according to claim 8, characterized in that The sample preparation solvent was a mixed solution of methanol and phosphoric acid water.

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