HPLC (High Performance Liquid Chromatography) analysis method for Nicodil related substances

Abstract

According to the analysis method for the Nicodil related substances, a brand-new buffer system is adopted, trifluoroacetic acid with high toxicity and high corrosivity is abandoned, a safe and environment-friendly acetic acid-ammonium acetate system is adopted, the proportion of volatile salt is adjusted, the collection time is shortened, and the cost of the analysis process is reduced. The method is rapid, simple, accurate, good in repeatability and suitable for control and stability research of the related substances, and a foundation is established for formulating quality standards of the related substances.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a HPLC analysis method for nicorandil-related substances. Background Art

[0002] Nicorandil, also known as nicotinamide and nitronicotinamide, is a nitrate compound developed by the Japanese Chugai Pharmaceutical Company. It is the only cardiovascular system drug currently on the market that has a dual-action mechanism for treating coronary heart disease and angina pectoris. It has the effects of nitrate drugs in dilating coronary arteries, continuously increasing coronary blood flow, and preventing coronary artery spasm, and can also prevent the free release of intracellular calcium ions and increase the permeability of cell membranes to potassium ions. In addition, it also has the effect of inhibiting platelet aggregation and preventing the formation of blood clots. Its structural formula is as follows:

[0003]

[0004] The presence of related substances is directly related to the quality and safety of drugs. Related substances include impurities brought in by drug raw materials, related substances generated during the process, and related substances related to degradation, which will directly affect the purity and quality of the drug, and may even cause serious adverse reactions, affecting the effectiveness and safety of clinical medication. In order to improve product quality and ensure the safety and effectiveness of clinical medication, it is necessary to fully study the situation of related substances and develop appropriate separation and detection methods to control impurities within the safety limit, achieve controllable drug quality, and ensure the safety and effectiveness of clinical medication. In accordance with relevant requirements and regulations, it is required to provide appropriate methods for the accurate determination of related substances in the export release and stability test of Nicorandil finished products.

[0005] CN116359394B discloses a high performance liquid chromatography method for efficiently separating and detecting nicorandil and its related substances in nicorandil tablets, wherein the mobile phase consists of phase A and phase B, wherein phase A is trifluoroacetic acid-triethylamine-water-acetonitrile, and the volume ratio of trifluoroacetic acid:triethylamine:water:acetonitrile is 1.3-1.7:1.8-2.2:965:18-22, and phase B is acetonitrile.

[0006] CN106770737A A HPLC analysis method for nicorandil-related substances, wherein the mobile phase is a mixed solution of a buffer solution and methanol and tetrahydrofuran, wherein the buffer solution is composed of trifluoroacetic acid and triethylamine, and the pH range is adjusted to 5.8-6.2 by triethylamine, wherein the volume fraction of trifluoroacetic acid is 0.01%; the volume fraction of methanol is 0.5%; the volume fraction of tetrahydrofuran is 1.0%; and the volume fraction of the buffer solution is 98.5%.

[0007] CN109406706A discloses a method for determining related substances in nicorandil tablets using an HPLC correction factor method. The method uses a C18 chromatographic column and a trifluoroacetic acid-triethylamine-tetrahydrofuran-water mixture as a mobile phase.

[0008] CN113121425A discloses a nicorandil impurity compound and a preparation method and a detection method thereof. The mobile phase is a mixed solution of trifluoroacetic acid, triethylamine, tetrahydrofuran and water, with a ratio of 3 ml of trifluoroacetic acid, 5 ml of triethylamine, and 10 ml of tetrahydrofuran. The mixture is diluted with water to 1000 ml, mixed, and trifluoroacetic acid or triethylamine is added to adjust the pH to 2.35.

[0009] CN113552234A discloses a method for determining the content of nicorandil nitrogen oxide in nicorandil raw material medicine, and the chromatographic conditions are: chromatographic column Agilent Poroshell 120EC-C18 4.6×100mm 2.7μm, mobile phase A: 0.1% formic acid water, mobile phase B: methanol.

[0010] CN116973483A discloses a method for detecting nicorandil trimer impurities, wherein the mobile phase is divided into mobile phase A and mobile phase B, trifluoroacetic acid solution-methanol-acetonitrile (960:25:15) is used as mobile phase A, and methanol is used as mobile phase B. The pH value of the trifluoroacetic acid solution in the mobile phase A is 2.4-2.8.

[0011] CN117571856A discloses a method for analyzing related substances of nicorandil for injection, wherein mobile phase A is 0.02 mol / L diammonium phosphate buffer salt-acetonitrile with a volume ratio of 98:5, and the pH value is adjusted to 2.5 with phosphoric acid; mobile phase B is 0.02 mol / L diammonium phosphate buffer salt-acetonitrile with a volume ratio of 40:60, and the pH value is adjusted to 2.5 with phosphoric acid.

[0012] CN118130647A discloses an HPLC analysis method for nicorandil for injection, wherein mobile phase A is composed of triethylamine aqueous solution and trifluoroacetic acid, mobile phase B is methanol, and mobile phase A is adjusted to a pH value of 2.1-2.3 using trifluoroacetic acid.

[0013] The prior art mostly uses a trifluoroacetic acid-triethylamine buffer system, which uses trifluoroacetic acid with high toxicity, and has a low pH value of the mobile phase, which is highly corrosive to the entire pipeline. The diammonium phosphate buffer system has poor separation of some impurities. Summary of the invention

[0014] In view of the deficiencies in the prior art, the present invention provides a HPLC analysis method for nicorandil-related substances.

[0015] The technical scheme of the present invention is as follows: a method for analyzing nicorandil-related substances, the method adopts high performance liquid chromatography, a chromatographic column with octadecylsilane bonded silica gel as a filler, and a mobile phase consisting of a phase A and a phase B, wherein the phase A is a water-acetic acid-ammonium acetate-acetonitrile solution, the volume ratio of water: acetic acid: ammonium acetate: acetonitrile is 975:3:2:20, and the phase B is acetonitrile, and gradient elution is performed according to the following procedure,

[0016]

[0017] And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

[0018] In some embodiments, the chromatographic column is YMC-Pack ODS-AQ, with specifications of 4.6×250 mm and 5 μm.

[0019] In some embodiments, the flow rate of the mobile phase is 1.3 ml to 1.7 ml / min, preferably 1.5 ml / min.

[0020] In some embodiments, the column temperature of the chromatographic column is 28-32°C, preferably 30°C.

[0021] In some embodiments, the injection volume is 20 μl.

[0022] In some typical embodiments, the present invention provides a method for analyzing nicorandil-related substances, comprising the following steps:

[0023] Preparation of sample solution: Take an appropriate amount of Nicorandil sample, weigh accurately, dissolve in acetonitrile and quantitatively dilute to make a solution containing about 2 mg per 1 ml, then filter with a 0.45 μm filter membrane to prepare the Nicorandil sample solution;

[0024] Control solution: Accurately measure 1 ml of the test solution, put it into a 500 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well, and obtain a solution containing about 4 μg per 1 ml;

[0025] Sample determination: The chromatographic column is YMC-Pack ODS-AQ, with specifications of 4.6×250mm, 5μm. The mobile phase consists of phase A and phase B, wherein phase A is water-acetic acid-ammonium acetate-acetonitrile solution, with a volume ratio of water: acetic acid: ammonium acetate: acetonitrile of 975:3:2:20, and phase B is acetonitrile. Gradient elution is performed as shown in the table below; the flow rate is 1.5ml per minute; the column temperature is 30℃; the detection wavelength is 262nm; the injection volume is 20μl; and the chromatogram is recorded;

[0026]

[0027] And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

[0028] The analytical method of the present invention is used for efficiently separating and detecting nicorandil and its related substances in nicorandil raw materials or preparations.

[0029] Beneficial effects of the present invention: The present invention provides an analytical method for nicorandil-related substances, which adopts a new buffer system, abandons trifluoroacetic acid with high toxicity and strong corrosiveness, adopts a safe and environmentally friendly acetic acid-acetamide system, reduces the proportion of volatile salts, reduces the collection time, and reduces the cost of the analysis process. The method is fast, simple, accurate, and has good repeatability, is suitable for the control and stability research of related substances, and lays a foundation for the formulation of quality standards for related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a typical schematic diagram of the chromatographic analysis of Example 1 of the present invention.

[0031] Figure 2 It is a typical schematic diagram of the chromatographic analysis of Example 2 of the present invention.

[0032] Figure 3 It is a typical schematic diagram of the chromatographic analysis of Example 3 of the present invention.

[0033] Figure 4 It is a typical schematic diagram of the chromatographic analysis of Example 4 of the present invention.

[0034] Figure 5 It is a typical schematic diagram of the chromatographic analysis of Example 5 of the present invention.

[0035] Figure 6 It is a typical schematic diagram of the chromatographic analysis of Example 8 of the present invention.

[0036] Figure 7 It is a typical schematic diagram of the chromatographic analysis of Example 9 of the present invention. DETAILED DESCRIPTION

[0037] The following embodiments can enable those skilled in the art to more fully understand the present invention, but the present invention is not limited to the scope of the embodiments.

[0038] The impurities involved in the present invention include but are not limited to the following impurities:

[0039]

[0040] The HPLC was Ultimate3000 (Thermo Fisher Scientific), the workstation was Chameleon chromatography workstation, the chromatographic column was C18 (YMC-Pack ODS-AQ 4.6mm×250mm, 5um), the electronic analytical balance was (Mettler, 1 / 100,000), the ultrapure water system was (Euprotech), tetrahydrofuran (McLean, chromatography grade), and methanol (Merck, chromatography grade).

[0041] Example 1

[0042] 1.1 Sample solution preparation: Take an appropriate amount of this product, weigh it accurately, dissolve it with solvent and dilute it quantitatively to make a solution containing about 2 mg per 1 ml. Then filter it with a 0.45 μm filter membrane and use it as the Nicorandil sample solution. Control solution: Accurately measure 1 ml of the test solution, put it in a 500 ml volumetric flask, dissolve it with solvent and dilute it quantitatively to the scale, shake well, and you will get a solution containing about 4 μg per 1 ml.

[0043] 1.2 Chromatographic conditions: The chromatographic column was a C18 silica gel column (YMC-Pack ODS-AQ 4.6 mm × 250 mm, 5 μm), with water-acetic acid-ammonium acetate-acetonitrile solution (975:3:2:20) as mobile phase A and acetonitrile as mobile phase B, and gradient elution was performed according to the following table; the flow rate was 1.5 ml per minute; the column temperature was 30 °C; the detection wavelength was 262 nm; the injection volume was 20 μl;

[0044]

[0045] 1.3 Injection: Take the sample solution in 1.1 and inject it according to the chromatographic conditions in 1.2.

[0046] 1.4 Results: Chromatogram of sample solution. Results The various related substances of Nicorandil were baseline separated, and the peaks were well eluted. The obtained chromatogram had a moderate analysis time (the main peak of Nicorandil was about 16 minutes), which met the analysis requirements of Nicorandil related substances. The content of related substances in the sample solution was obtained by the self-control method.

[0047] Example 2

[0048] 2.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0049] 2.2 Chromatographic conditions: The flow rate was adjusted to 1.3 ml per minute; other conditions were the same as in Example 1.

[0050] 2.3 Injection: Take the sample solution in 2.1 and inject it according to the chromatographic conditions in 2.2.

[0051] 2.4 Results: After elution, a chromatogram of the sample solution was obtained. Results The various related substances of Nicorandil were baseline separated, the peak shape was good, the analysis time was prolonged, and the analysis requirements of Nicorandil related substances were met. The content of the related substances in the sample solution was obtained by the self-control method.

[0052] Example 3

[0053] 3.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0054] 3.2 Chromatographic conditions: The flow rate was adjusted to 1.7 ml per minute; other conditions were the same as in Example 1.

[0055] 3.3 Injection: Take the sample solution in 3.1 and inject it according to the chromatographic conditions in 3.2.

[0056] 3.4 Results: After elution, a chromatogram of the sample solution was obtained. Results The various related substances of Nicorandil achieved baseline separation, good peak shape, and a shorter analysis time, meeting the analytical standards for Nicorandil related substances, and the content of related substances in the sample solution was obtained by the self-control method. However, the flow rate of 1.7 ml / min has high requirements for the chromatographic column filler, and the service life of the chromatographic column will be seriously reduced.

[0057] Example 4

[0058] 4.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0059] 4.2 Chromatographic conditions: The column temperature was adjusted to 28°C; other conditions were the same as in Example 1.

[0060] 4.3 Injection: Take the sample solution in 4.1 and inject it according to the chromatographic conditions in 4.2.

[0061] 4.4 Results: After elution, a chromatogram of the sample solution was obtained. Results The related substances of Nicorandil were baseline separated, with good peak shape, meeting the analytical standards for related substances of Nicorandil, and the content of related substances in the sample solution was obtained by the self-control method.

[0062] Example 5

[0063] 5.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0064] 5.2 Chromatographic conditions: The column temperature was adjusted to 32°C; other conditions were the same as in Example 1.

[0065] 5.3 Injection: Take the sample solution in 5.1 and inject it according to the chromatographic conditions in 5.2.

[0066] 5.4 Results: After elution, a chromatogram of the sample solution was obtained. Results The related substances of Nicorandil were baseline separated, with good peak shape, meeting the analytical standards for the related substances of Nicorandil, and the content of the related substances in the sample solution was obtained by the self-control method.

[0067] Example 6

[0068] 6.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0069] 6.2 Chromatographic conditions: The chromatographic column is a C18 silica gel column (YMC-Pack ODS-AQ 4.6mm×250mm, 5um), with water-acetic acid-ammonium acetate-acetonitrile solution (970:8:2:20) as mobile phase A, pH value is 2.8; acetonitrile is used as mobile phase B, and gradient elution is performed according to the table below; the flow rate is 1.5ml per minute; the column temperature is 30℃; the detection wavelength is 262nm; the injection volume is 20μl.

[0070] 6.3 Injection: Take the sample solution in 6.1 and inject it according to the chromatographic conditions in 6.2.

[0071] 6.4 Results: After elution, some substances of Nicorandil could not be effectively separated.

[0072] Example 7

[0073] 7.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0074] 7.2 Chromatographic conditions: The chromatographic column is a C18 silica gel column (YMC-Pack ODS-AQ 4.6mm×250mm, 5um), with water-acetic acid-ammonium acetate-acetonitrile solution (970:3:7:20) as mobile phase A, and the pH value is adjusted to 6.2; acetonitrile is used as mobile phase B, and gradient elution is performed according to the table below; the flow rate is 1.5ml per minute; the column temperature is 30℃; the detection wavelength is 262nm; the injection volume is 20μl.

[0075] 7.3 Injection: Take the sample solution in 7.1 and inject it according to the chromatographic conditions in 7.2.

[0076] 7.4 Results: After elution, some substances of Nicorandil could not be effectively separated.

[0077] Example 8

[0078] 8.1 Sample solution preparation: the same as the sample solution preparation in Example 1.

[0079] 8.2 Chromatographic conditions: Adjust the detection wavelength to 284 nm; injection volume to 20 μl.

[0080] 8.3 Injection: Take the sample solution in 8.1 and inject it according to the chromatographic conditions in 8.2.

[0081] 8.4 Results: After elution, a chromatogram of the sample solution was obtained. Results The various related substances of Nicorandil were baseline separated, with good peak shapes, meeting the analytical standards for related substances of Nicorandil, and the content of related substances in the sample solution was obtained by the self-control method.

[0082] Example 9

[0083] 9.1 Sample solution preparation: Prepare the sample solution in the same manner as in Example 1, and prepare 6 portions of Nicorandil sample solutions and their own control solutions in parallel.

[0084] 9.2 Chromatographic conditions: Same as those in Example 1.

[0085] 9.3 Results: After injection and elution, 6 chromatograms of sample solutions were obtained. Results The various related substances of Nicorandil were baseline separated, with good peak shape, and met the analytical standards for related substances of Nicorandil. The content of related substances in the sample solution was obtained by self-control method, and the RSD (relative standard deviation) of impurity content was less than 2%, indicating that the method had good reproducibility.

[0086] In summary, the scheme of Example 1 is a preferred scheme, and the various related substances of Nicorandil achieve baseline separation, good peak shape, moderate analysis time, good separation, and meet the analysis requirements of Nicorandil related substances. This method is fast, simple, accurate, and has good reproducibility. It can effectively analyze the related substances of Nicorandil, and provides possibilities for quality control of drug related substances and drug safety.

Claims

1. A method for analyzing nicorandil-related substances, the method adopts high performance liquid chromatography, a chromatographic column with octadecylsilane bonded silica as a filler, and a mobile phase consisting of a phase A and a phase B, wherein the phase A is a water-acetic acid-ammonium acetate-acetonitrile solution, the volume ratio of water: acetic acid: ammonium acetate: acetonitrile is 975:3:2:20, and the phase B is acetonitrile, and gradient elution is performed according to the following procedure: And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

2. The analysis method according to claim 1, characterized in that The chromatographic column is YMC-Pack ODS-AQ, with specifications of 4.6×250 mm and 5 μm.

3. The analysis method according to claim 1, characterized in that The flow rate of the mobile phase is 1.3 ml to 1.7 ml / min, preferably 1.5 ml / min.

4. The analysis method according to claim 1, characterized in that The column temperature of the chromatographic column is 28-32°C, and the column temperature is preferably 30°C.

5. The analysis method according to claim 1, characterized in that The injection volume was 20 μl.

6. The analysis method according to claim 1, characterized in that The following steps are involved: Preparation of sample solution: Take an appropriate amount of Nicorandil sample, weigh accurately, dissolve in acetonitrile and quantitatively dilute to make a solution containing about 2 mg per 1 ml, then filter with a 0.45 μm filter membrane to prepare the Nicorandil sample solution; Control solution: Accurately measure 1 ml of the test solution, put it into a 500 ml volumetric flask, add solvent to dissolve and quantitatively dilute to the scale, shake well, and obtain a solution containing about 4 μg per 1 ml; Sample determination: The chromatographic column is YMC-Pack ODS-AQ, with specifications of 4.6×250mm, 5μm. The mobile phase consists of phase A and phase B, wherein phase A is water-acetic acid-ammonium acetate-acetonitrile solution, with a volume ratio of water: acetic acid: ammonium acetate: acetonitrile of 975:3:2:20, and phase B is acetonitrile. Gradient elution is performed as shown in the table below; the flow rate is 1.5ml per minute; the column temperature is 30℃; the detection wavelength is 262nm; the injection volume is 20μl; and the chromatogram is recorded; And in the process of gradient elution, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; the proportion of mobile phase A refers to the percentage of the volume of mobile phase A in the total volume of eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of eluent.

7. Use of the analytical method according to any one of claims 1 to 6 for separating and detecting nicorandil and its related substances in nicorandil bulk drug or preparation.

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