Method for detecting related substances in isosorbide mononitrate tablet

Abstract

The invention discloses a method for detecting a related substance in an isosorbide mononitrate tablet, which comprises the following steps of: detecting the related substance isosorbide acetate in the isosorbide mononitrate tablet by adopting a high performance liquid chromatography, and eluting by taking octadecylsilane chemically bonded silica as a chromatographic column of a filler, buffer salt or water as a mobile phase A and methanol as a mobile phase B; the detection wavelength is 210 to 220 nm; and the sample size is 10-50 [mu] l. By utilizing the method, the separation degrees of peaks of the related substances isosorbide acetate and isosorbide mononitrate both reach 2.0 or above, and the recovery rates of impurities both meet the requirements of 80.0%-120.0%; the method has the advantages of high specificity and high sensitivity, and realizes better quality control of the isosorbide mononitrate tablets.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting related substances in isosorbide mononitrate tablets. Background Technology

[0002] Isosorbide mononitrate can be divided into 5-isosorbide mononitrate and 2-isosorbide mononitrate. Currently, 5-isosorbide mononitrate is more commonly used and is also listed in the pharmacopoeia. Its chemical name is 3,6-didehydro-D-sorbitol-5-nitrate. Isosorbide mononitrate is a new generation of nitrate antianginal drugs and is one of the metabolites of isosorbide nitrate. This drug produces nitric oxide (NO), a vasodilator, which relaxes the smooth muscle of arteries, dilates corresponding blood vessels, alleviates symptoms of atherosclerosis and vascular stenosis, reduces ventricular wall tension, increases coronary perfusion, and increases the amount of blood flowing through the coronary arteries, thus increasing the amount of oxygen carried and relieving myocardial hypoxia. This drug can also reduce the cardiac load and myocardial oxygen consumption, directing arterial blood flow to ischemic areas of the myocardium, adjusting blood oxygen balance, and thereby improving myocardial damage.

[0003] The synthetic routes of isosorbide mononitrate usually use isosorbide as the starting material and are mainly divided into direct nitration, selective reduction and indirect nitration.

[0004] 1) Direct Nitration Method: This route uses isosorbide as a raw material. A nitro group is introduced through a nitration reaction under nitrating conditions (such as nitric acid / sulfuric acid, nitric acid / acetic anhydride, etc.). The target product is then obtained through separation and purification steps. The resulting product is a mixture, and post-processing methods include column chromatography. However, this route has low yields, uses fuming nitric acid during nitration which releases toxic gases, and relies on column chromatography for post-processing, making it unsuitable for industrial production. The synthetic route is shown below:

[0005]

[0006] 2) Selective Reduction Method: This route first prepares isosorbide nitrate, which is then selectively reduced to prepare isosorbide 5-mononitrate. The drawback of this route is that the selective reduction is not complete, and 2-mononitrate and isosorbide nitrate remain mixed in the product, making purification difficult. Furthermore, the selective reducing agent and catalyst are expensive, resulting in high overall costs. The synthetic route is shown below:

[0007]

[0008] 3) Indirect nitration method: This route can be summarized as starting with isosorbide, protecting the 2-position by acetylation, then nitrating the 5-position, and finally hydrolyzing the ethyl ester group at the 2-position to obtain isosorbide 5-mononitrate. However, this route has a long synthetic process, and the use of catalysts in some parts leads to excessively high costs. Furthermore, the selective acylation process conditions are difficult to control, and the generated byproducts are difficult to remove, resulting in difficult product purification and low yield. The synthetic route is shown below:

[0009]

[0010] No methods for detecting isosorbide acetate in isosorbide mononitrate tablets have been reported. According to ICH guidelines, research and strict control of organic impurities are necessary. Summary of the Invention

[0011] The purpose of this invention is to provide a method for detecting related substances in isosorbide mononitrate tablets. This method can detect the potential impurity isosorbide acetate in isosorbide mononitrate tablets with good accuracy and high recovery rate. The peaks of isosorbide mononitrate and the impurity to be tested do not interfere with each other, thereby achieving better quality control of isosorbide mononitrate tablets.

[0012] To achieve the above objectives, the present invention provides the following technical solution:

[0013] A method for detecting related substances in isosorbide mononitrate tablets, employing high-performance liquid chromatography (HPLC) to detect isosorbide acetate in the formulation. The method uses octadecylsilane-bonded silica gel as the stationary phase, and elution with a 5-30 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 2.5-4.0 with phosphoric acid) as mobile phase A and methanol as mobile phase B; gradient elution is performed; the detection wavelength is 210-220 nm; the injection volume is 10-50 μl; the flow rate is adjusted to maintain the column pressure between 2000 psi and 3000 psi; the theoretical plate number, calculated based on the isosorbide mononitrate peak, should be no less than 5000.

[0014] The test solution is prepared by grinding a quantity of isosorbide mononitrate tablets into a fine powder, accurately weighing an appropriate amount of the powder, dissolving and diluting it with a diluent to prepare a solution containing 1-10 mg per ml, shaking well, filtering, and collecting the filtrate.

[0015] Reference solution: Weigh an appropriate amount of isosorbide acetate reference standard accurately, dissolve and dilute it with a diluent to prepare a solution containing 10-15 μg of isosorbide acetate per 1 ml, shake well, and the solution is ready.

[0016] According to a further description of the present invention, preferably, the pH value of the mobile phase A is 2.5 to 3.5.

[0017] According to a further description of the present invention, preferably, the concentration of the potassium dihydrogen phosphate solution is 10-20 mmol / L.

[0018] According to a further description of the present invention, preferably, the diluent is a mixed solution of mobile phase A and mobile phase B with a volume ratio of 75:25.

[0019] According to a further description of the present invention, preferably, the elution program is as follows: 0-3 min, 95% A; 3-10 min, 95% → 75% A; 10-25 min, 75% A; 25-35 min, 75% → 95% A; 35-40 min, 95% A.

[0020] According to a further description of the present invention, preferably, the concentration of isosorbide mononitrate in the test solution is 5.0 mg / ml; the concentration of isosorbide mononitrate in the reference solution is 12.5 μg / ml, and the solution is shaken well to obtain the final product.

[0021] According to a further description of the present invention, preferably, the chromatographic column is an Agilent ZORBAX ODS series or a Waters Symmetry series C column. 18 The column; the chromatographic column packing material has a particle size ≤5μm, a length ≥25cm, and an inner diameter ≤4.6mm.

[0022] According to a further description of the present invention, preferably, the detection wavelength is 210 nm; the column temperature is 25℃~35℃; the injection volume is 10 μl~20 μl; and the flow rate is adjusted to 2000 psi~3000 psi by adjusting the column pressure.

[0023] According to a further description of the present invention, preferably, the isosorbide mononitrate tablets are isosorbide mononitrate regular tablets or isosorbide mononitrate sustained-release tablets.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention studies isosorbide acetate as a potential impurity, ensuring product quality more comprehensively and reducing product risk. By adjusting the elution ratio of mobile phase A and mobile phase B, this invention effectively separates isosorbide acetate, other impurities, and isosorbide mononitrate, achieving high recovery rates. The recovery rates of all impurities meet the requirements of 80.0%–120.0%. The detection method for isosorbide acetate in this invention exhibits good specificity, with sharp and symmetrical peaks and high sensitivity to the analyte. The limit of quantitation for isosorbide acetate is approximately 0.2 μg / ml, providing a reliable analytical basis for the quality testing of isosorbide mononitrate preparations. Attached Figure Description

[0026] Figure 1This is a chromatogram of the system solution in gradient elution procedure 1 of Example 1;

[0027] Figure 2 This is a chromatogram of the system solution in gradient elution procedure 2 of Example 1;

[0028] Figure 3 This is a chromatogram of the system solution in gradient elution procedure 3 of Example 1;

[0029] Figure 4 This is the chromatogram of the blank excipient solution in Example 1;

[0030] Figure 5 This is a chromatogram of the system suitability solution in Example 1;

[0031] Figure 6 This is the chromatogram of the reference solution in Example 1;

[0032] Figure 7 This is a chromatogram of the test solution in Example 1. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments, but this does not limit the invention to the scope of the embodiments described. Experimental methods not specifically described in the following embodiments were performed according to conventional methods and conditions, or according to the product instructions; in the chromatograms of the embodiments and comparative examples, the unit of peak time is minutes (min).

[0034] Example 1:

[0035] 1. Instruments and reagents

[0036] A Waters e2695 high-performance liquid chromatograph equipped with a Waters 2489 ultraviolet detector (Waters Corporation, USA); an analytical balance (Mettler-Toledo, Switzerland); and a pH meter S210 (Mettler-Toledo, Switzerland).

[0037] Isosorbide mononitrate reference standard (Source: National Institutes for Food and Drug Control 100694-202104, content 99.9%);

[0038] Isosorbide acetate reference standard (Source: Aozeal, batch number ESI-063-08, content 95.36%);

[0039] 2-Acetyl-5-nitrate isosorbide dinitrate reference standard (Source: Aozeal, batch number ESI-103-15, content 96.48%);

[0040] Isosorbide 2-nitrate-5-acetic acid (Source: Aozeal, batch number ESI-083-15, purity 96.54%);

[0041] 2-Isosorbide mononitrate (Source: National Institutes for Food and Drug Control, batch number 100695-201703, content 100.0%);

[0042] Isosorbide dinitrate (Source: National Institutes for Food and Drug Control, batch number 100250-202006, content 99.8%);

[0043] Isosorbide mononitrate tablets (20mg, batch numbers: 241001PR, 241002PR, 241003PR);

[0044] Acetonitrile, methanol, and phosphoric acid were of chromatographic grade; water was ultrapure water; and potassium dihydrogen phosphate was chemically pure.

[0045] The synthetic routes for isosorbide mononitrate typically use isosorbide as the starting material and are mainly divided into direct nitration, selective reduction, and indirect nitration methods. Each of these three synthetic routes has its advantages and disadvantages, but all must involve a nitration step. Because the isosorbide molecule contains two differently active hydroxyl groups, the reaction may produce multiple nitration products. The specific structure and proportion of these products depend on the reaction conditions (such as temperature and nitrifying agent concentration). This step requires strict control of reaction conditions to avoid over-nitration or side reactions. Theoretically, the nitration reaction can produce isosorbide acetate, 2-acetic-5-nitrate isosorbide, or 2-nitrate-5-acetic acid isosorbide. These have similar structures and polarities, making their separation somewhat difficult. The theoretical impurity analysis for the nitration step is shown below:

[0046]

[0047] Byproducts of the nitration reaction include isosorbide acetate, isosorbide 2-aceto-5-nitrate, or isosorbide 2-nitrate-5-acetoate. Existing literature reports that related substances to isosorbide mononitrate also include isosorbide 2-mononitrate and isosorbide nitrate. Information on possible impurities present in this invention is detailed in Table 1.

[0048] Table 1 Impurity Information Table

[0049]

[0050] 2. System adaptability testing

[0051] Preliminary experiments show that in common C 18When using commonly used systems such as methanol-water solution, methanol-phosphate solution, acetonitrile-water solution, and acetonitrile-phosphate solution on the chromatographic column, poor separation results were observed, including unstable baselines and poor peak shapes. The optimal separation was achieved when mobile phase A was selected as 5–30 mmol / L potassium dihydrogen phosphate solution, with the pH adjusted to 2.5–4.0 using phosphoric acid, and mobile phase B as methanol. However, conventional isocratic elution mobile phase A:mobile phase B ratios such as 70:30, 75:25, and 80:20 could not effectively separate the aforementioned impurities, despite optimization experiments of gradient elution conditions.

[0052] System adaptability experiments were conducted, and the high-performance liquid chromatography (HPLC) detection conditions were as follows:

[0053] High Performance Liquid Chromatography (HPLC):

[0054] Detector: UV detector;

[0055] Column: Agilent ZORBAX ODS;

[0056] Mobile phase A: 10–20 mmol / L potassium dihydrogen phosphate solution, pH adjusted to 2.5–3.5 with phosphoric acid;

[0057] Mobile phase B: Methanol;

[0058] Column temperature: 25℃~35℃;

[0059] Flow rate: Adjust the flow rate until the column pressure is approximately 2500 psi;

[0060] Detection wavelength: 210–220 nm;

[0061] Injection volume: 10–50 μl.

[0062] Diluent: A mixed solution of mobile phase A and mobile phase B in a volume ratio of 75:25.

[0063] 1) System suitability solution: Weigh appropriate amounts of isosorbide mononitrate and isosorbide 2-mononitrate reference standards accurately, dissolve and dilute with diluent to prepare a mixed solution containing approximately 5 μg of each per ml.

[0064] 2) Test solution: Take 20 tablets of isosorbide mononitrate, grind them into fine powder, take an appropriate amount of the powder, accurately weigh it, add an appropriate amount of diluent, sonicate for about 10 minutes to dissolve, dissolve and dilute with diluent to prepare a solution containing about 5 mg per ml, shake well, filter, and take the filtrate to obtain the test solution.

[0065] 3) Isosorbide acetate reference solution: Take appropriate amounts of isosorbide acetate reference standards, accurately weigh them, dissolve and dilute them with diluent to prepare a solution containing about 12.5 μg of isosorbide acetate per 1 ml, shake well, and the solution is ready.

[0066] 4) Isosorbide acetate impurity localization solution: Take appropriate amounts of isosorbide acetate reference standards, accurately weigh them, dissolve and dilute them separately with diluent to prepare a solution containing approximately 50 μg of impurities per 1 ml, and shake well to obtain the solution.

[0067] 5) Blank excipient solution: Take an appropriate amount of blank excipient, weigh it accurately, place it in a suitable volumetric bottle, dissolve it with diluent and make up to the mark, shake well, filter, and take the filtrate to obtain the solution.

[0068] 6) Preparation of system solutions: Weigh approximately 10 mg each of the impurities isosorbide acetate, isosorbide 2-aceto-5-nitrate, isosorbide 2-nitrate-5-aceto-acetate, isosorbide 2-mononitrate, and isosorbide nitrate. Place them in 50 ml volumetric flasks, add diluent to dissolve and dilute to the mark, shake well, and prepare impurity stock solutions (200 μg / ml).

[0069] Accurately add 5 ml each of the impurity stock solutions of isosorbide acetate, isosorbide 2-aceto-5-nitrate, isosorbide 2-nitrate-5-aceto-1-acetate, isosorbide 2-mononitrate, and isosorbide nitrate to a 50 ml volumetric flask, dilute to the mark with diluent, and mix well to prepare a mixed impurity stock solution (20 μg / ml). Accurately weigh approximately 12 mg of isosorbide mononitrate active pharmaceutical ingredient, place it in a 10 ml volumetric flask, and then accurately add 1 ml of the mixed impurity stock solution. Dilute with diluent to prepare a mixed solution containing 1 mg / ml isosorbide mononitrate and 2 μg / ml (0.2%) of each impurity as the system solution.

[0070] Six samples were injected into a high-performance liquid chromatograph for analysis, and gradient elution was performed using the following three gradient elution methods:

[0071] Washing procedure 1:

[0072] 0~3min, 95%A; 3~10min, 95%→75%A; 10~25min, 75%A; 25~35min, 75%→95%A; 35~40min, 95%A.

[0073] Washing procedure 2:

[0074] 0~3min, 95%A; 3~4min, 95%→60%A; 4~10min, 60%A; 10~11min, 60%→95%A; 11~30min, 95%A.

[0075] Washing procedure 3:

[0076] 0~3min, 95%A; 3~4min, 95%→60%A; 4~25min, 60%A; 25~26min, 60%→95%A; 26~35min, 95%A.

[0077] Record the chromatograms of each sample to be tested. The detection chromatograms of each sample are shown below. Figures 1 to 7 ,Depend on Figure 2 It can be seen that the blank excipient solution does not interfere with the analyte. From Figure 7 It can be seen that the analyte in the test solution maintains baseline separation from the main peak and other impurities. The separation results are shown in Table 2.

[0078] Table 2 Retention time and resolution of impurities in the test solution

[0079] Name RT(min) Resolution Isosorbide mononitrate 6.501 - Isosorbide dinitrate 13.188 14.0

[0080] Depend on Figure 1 The chromatogram of the system solution shows that the peak separation between the analyte and the main peak is good when using gradient elution program 1. The separation results are shown in Table 3.

[0081] Table 3 System suitability under gradient elution program 1: Impurity retention time and resolution in solution.

[0082] Name RT(min) Resolution Tailing factor Theoretical plates 2-Isosorbide mononitrate 5.866 - 1.1 10332 Isosorbide mononitrate 6.630 3.1 1.0 11336 Isosorbide dinitrate 13.204 15.8 1.2 9929 2-Isosorbide 5-nitrate acetate 19.565 9.3 1.2 10677 2-Isosorbide 5-acetate nitrate 23.663 4.8 1.1 11508 Isosorbide dinitrate 35.470 10.7 1.1 12338

[0083] The method using gradient elution procedure 1 showed that the blank excipient solution had no interference, and the separation degree between isosorbide mononitrate and impurities was greater than 1.5, which met the standard. Under this method, each impurity can be effectively separated, and the main component peak is not affected by the blank solvent and known impurities, indicating strong method specificity.

[0084] Depend on Figure 2 The chromatogram of the system solution shows that, using gradient elution procedure 2, the peak resolution between the analyte peak and the main peak in the test solution is less than 2.0, and baseline separation is not achieved, which does not meet the requirements. The resolution is shown in Table 4.

[0085] Table 4. System suitability under gradient elution program 2: Impurity retention time and resolution in solution.

[0086] Name RT(min) Resolution Tailing factor Theoretical plates 2-Isosorbide mononitrate 5.096 - 1.1 10543 Isosorbide mononitrate 5.501 1.6 1.1 10230 Isosorbide dinitrate 9.411 9.4 1.2 9855 2-Isosorbide 5-nitrate acetate 14.335 7.2 1.2 11203 2-Isosorbide 5-acetate nitrate 18.052 4.3 1.1 11174 Isosorbide dinitrate 25.446 6.7 1.2 13063

[0087] Depend on Figure 3 The chromatogram of the system solution shows that, using gradient elution program 3, the peak resolution between the analyte peak and the main peak in the test solution is less than 2.0, which affects the elution of the isosorbide nitrate peak. The resolution is shown in Table 5.

[0088] Table 5. System suitability under gradient elution program 3: Impurity retention time and resolution in solution.

[0089] Name RT(min) Resolution Tailing factor Theoretical plates 2-Isosorbide mononitrate 5.226 - 1.1 11032 Isosorbide mononitrate 5.702 1.9 1.0 10036 Isosorbide dinitrate 11.172 13.0 1.1 9812 2-Isosorbide 5-nitrate acetate 16.859 8.3 1.2 11277 2-Isosorbide 5-acetate nitrate 21.274 5.2 1.1 12008 Isosorbide dinitrate 33.862 11.4 1.3 13038

[0090] Conclusion: Gradient elution was performed using mobile phase A: 10–20 mmol / L potassium dihydrogen phosphate solution, with the pH adjusted to 2.5–3.5 by phosphoric acid; mobile phase B: methanol. The results showed that elution program 1 was the optimal gradient elution condition: 0–3 min, 95% A; 3–10 min, 95% → 75% A; 10–25 min, 75% A; 25–35 min, 75% → 95% A; 35–40 min, 95% A. The final determined gradient elution conditions are detailed in Table 6.

[0091] Table 6 Optimal gradient elution conditions

[0092] Time Mobile phase A proportion Mobile phase B proportion 0 min 95％ 5％ 3 min 95％ 5％ 10 min 75％ 25％ 25 min 75％ 25％ 35 min 95％ 5％ 40 min 95％ 5％

[0093] Testing of multiple batches and formulations of isosorbide mononitrate from various manufacturers confirmed the presence of isosorbide acetate impurities in the chromatographic peaks, indicating that isosorbide acetate impurities are present in the current synthetic route, consistent with theoretical findings. Although the optimal detection method in this experiment can achieve good separation of impurities such as isosorbide acetate, 2-aceto-5-nitrate isosorbide, 2-nitrate-5-acetosorbide, 2-mononitrate isosorbide, and nitrate isosorbide, as well as the main peak, and the separation between isosorbide mononitrate and the impurities is greater than 2.0, in the detection of isosorbide acetate, when the impurity is at the limit concentration of 0.2%, the concentration of the test solution needs to be increased to meet the detection requirements. Moreover, the correction factor for isosorbide acetate is greater than 5, and it needs to be calculated according to the external standard method. Therefore, the detection method of this application only targets the related substance isosorbide acetate for detection; the related substance detection of impurities such as 2-aceto-5-nitrate isosorbide, 2-nitrate-5-acetosorbide, 2-mononitrate isosorbide, and nitrate isosorbide is controlled separately under other methods.

[0094] 3. System Applicability

[0095] 1) Limit of Quantification

[0096] Determination: The impurity mixture solution is serially diluted, and the mixture is injected sequentially starting at a concentration of 2 μg / ml or less. The injection concentration with an S / N of 10 to 20 is used as the limit of quantitation concentration, and the limit of quantitation is calculated.

[0097] Table 7 Results of Limits of Quantification (S / N = 10–20)

[0098]

[0099] Acceptable criteria: For isosorbide mononitrate and known impurity peaks with a signal-to-noise ratio (S / N) of 10–20, the injection volume is the limit of quantitation. For a solution at the limit of quantitation concentration, six injections are required. The retention time RSD should not exceed 2%, and the peak area RSD should not exceed 5%. Conclusion: Meets the criteria.

[0100] 2) Detection limit

[0101] Determination: The detection limits for isosorbide acetate and isosorbide mononitrate were determined by gradually decreasing the concentration and performing injection analysis. The detection limits were calculated using the injection concentrations with S / N = 3–9 as the detection limit depths.

[0102] Table 8 Results of detection limits (S / N = 3–9)

[0103] Name S / N value Isosorbide dinitrate 4 Isosorbide mononitrate 8

[0104] Conclusion: The detection limit for isosorbide acetate, an impurity of this invention, is 0.5 μg / ml, which is equivalent to detecting an impurity at a concentration level of 0.02% of the test sample; the detection limit for isosorbide mononitrate is 0.2 μg / ml, which meets the requirements for the detection of related substances.

[0105] 3) Linear

[0106] Determination: Prepare mixed linear stock solutions of various concentration levels (LOQ to 200% limit concentration) and inject them sequentially.

[0107] Results: The linear equation for isosorbide acetate was y = 548.6856x + 36.2341, with a correlation coefficient r of 0.9998; the linear equation for isosorbide mononitrate was y = 13787.1313x - 37.3126, with a correlation coefficient r of 1.0000.

[0108] Conclusion: Isosorbide acetate showed a good linear relationship between concentration and peak area within the concentration range from the limit of quantitation to 25 μg / ml (200%), with a correlation coefficient of 0.9998, greater than 0.990. The results indicate that isosorbide mononitrate exhibits good linearity within the concentration range from the limit of quantitation to 25 μg / ml.

[0109] 4) Accuracy (Recovery Rate)

[0110] Test solution: Weigh 20 tablets of this product accurately, grind them into a fine powder, weigh an appropriate amount of the powder, place it in a suitable volumetric flask, add an appropriate amount of diluent, sonicate for about 10 minutes to dissolve, dilute with diluent to prepare a solution containing about 5.0 mg of isosorbide mononitrate per ml, shake well, filter, and collect the filtrate.

[0111] 50%–150% spiked test solution: Take 20 tablets of this product, accurately weigh them, grind them into a fine powder, accurately weigh an appropriate amount of the fine powder and isosorbide acetate reference standard, place them in a suitable volumetric flask, add an appropriate amount of diluent, sonicate for about 10 minutes to dissolve, dilute with diluent to prepare a mixed solution containing approximately 5.0 mg of isosorbide mononitrate and 6.25 μg–18.75 μg of isosorbide acetate per ml, shake well, filter, and collect the filtrate.

[0112] Determination: Accuracy was evaluated by assessing the recovery rate of the impurity by preparing three different concentrations of test solutions with varying accuracy levels, namely, impurity reference solutions at concentrations of 50%, 100%, and 150% of the impurity limit concentration, to the isosorbide mononitrate test solution. The accuracy of the impurity was determined by adding a known amount of the impurity and then measuring the ratio (recovery rate) between the measured result and the theoretical value of the known impurity in the spiked sample.

[0113] Table 9. Results of Impurity Recovery Rate

[0114]

[0115] Conclusion: From Figure 4 It can be seen that the blank solution does not interfere with the analyte. From Figures 5 to 7 It can be seen that the chromatographic peak of the analyte and the peak of isosorbide mononitrate were well separated. The recoveries of this impurity at impurity limit concentrations of 50%, 100%, and 150% were all within the range of 98.8% to 102.5%, and the RSDs of the nine recoveries at each concentration level were 1.4%, all lower than the limit requirements, indicating that the method has high accuracy.

[0116] 5) Repeatability

[0117] Determination: Take 6 parallel-prepared isosorbide mononitrate test solution, self-control solution, reference solution, and 6 parallel-prepared repeatable test spike solution (impurity limit concentration of 0.25%) prepared in the same manner as the isosorbide mononitrate test solution with 100% accuracy; inject them into the liquid chromatograph.

[0118] Table 10. Repeatability results of isosorbide mononitrate test solution

[0119]

[0120] Table 11 Repeatability results of the spiked solution of the test sample

[0121]

[0122] Conclusion: Other single impurities were calculated using the self-comparison method, and isosorbide acetate was calculated using the external standard method. The maximum difference in the amount of impurities detected in the six isosorbide mononitrate test solutions was 0.01%, not exceeding 0.03%. The RSD% of the amount of impurities detected in the six spiked test solutions was 1.7% to 1.9%, less than 2%, which meets the requirements.

[0123] 6) Solution stability

[0124] Procedure: Take one sample of the test solution and one sample of the spiked solution under the repeatability test item, and inject them at 0, 4, 8, 12, 18 and 24 hours respectively. Record the chromatogram and count the peak area at each time point.

[0125] Acceptable standard: The peak area RSD of each impurity measured at each time point is not greater than 5.0%.

[0126] Table 12 Stability results of the test solution

[0127]

[0128] Table 13 Stability results of the spiked solution of the test sample

[0129]

[0130] Conclusion: The RSD of the peak area of ​​impurities in the test solution measured at each time point was 1.2%. The RSDs of the peak areas of each impurity in the spiked test solution measured at each time point were 1.2% and 1.3%, respectively, neither exceeding 2.0%.

[0131] 7) Durability

[0132] Based on the established chromatographic conditions, we investigated the extent to which minor changes in column temperature, flow rate, and mobile phase pH, as well as column replacement, did not affect the system's suitability or the results of impurity content determination.

[0133] Column 1: Agilent C 18 Column 4.6×250mm, 5μm;

[0134] Column 2: Kromasil C 18 Column 4.6×250mm, 5μm;

[0135] Column 3: Waters Symmetry C 18 , 4.6×250mm, 5μm.

[0136] Conclusion: Using three different brands of C, namely Agilent, Kromasil, and Waters Symmetry... 18The chromatographic columns were tested using system suitability solutions. Results showed that using three different columns, the resolution of isosorbide mononitrate and the impurity isosorbide acetate was greater than 2.0, and the main component peak and its adjacent impurity peak were effectively separated. Even with minor changes to the mobile phase composition, flow rate, column temperature, and pH, effective separation of the main component peak and its adjacent impurity peak was still achieved.

[0137] The above are merely illustrative examples of embodiments of the present invention. Any simple modifications or equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting related substances in isosorbide mononitrate tablets, characterized in that, High-performance liquid chromatography (HPLC) was used to detect isosorbide acetate, a related substance in the formulation. Octadecylsilane-bonded silica gel was used as the stationary phase. Elution was performed using a 5–30 mmol / L potassium dihydrogen phosphate solution (pH adjusted to 2.5–4.0 with phosphoric acid) as mobile phase A and methanol as mobile phase B. Gradient elution was employed. Detection wavelength: 210–220 nm; injection volume: 10–50 μL; flow rate: adjusted to a column pressure between 2000 psi and 3000 psi; theoretical plate number, calculated based on the isosorbide mononitrate peak, should be no less than 5000. The test solution is prepared by grinding a quantity of isosorbide mononitrate tablets into a fine powder, accurately weighing an appropriate amount of the powder, dissolving and diluting it with a diluent to prepare a solution containing 1-10 mg per ml, shaking well, filtering, and collecting the filtrate. Reference solution: Weigh an appropriate amount of isosorbide acetate reference standard accurately, dissolve and dilute it with a diluent to prepare a solution containing 10-15 μg of isosorbide acetate per 1 ml, shake well, and the solution is ready.

2. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The pH value of the mobile phase A is 2.5 to 3.

5.

3. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The concentration of the potassium dihydrogen phosphate solution is 10–20 mmol / L.

4. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The diluent is a mixed solution of mobile phase A and mobile phase B with a volume ratio of 75:

25.

5. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The elution program is as follows: 0–3 min, 95% A; 3–10 min, 95% → 75% A; 10–25 min, 75% A; 25–35 min, 75% → 95% A; 35–40 min, 95% A.

6. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The concentration of isosorbide mononitrate in the test solution is 5.0 mg / ml; the concentration of isosorbide mononitrate in the reference solution is 12.5 μg / ml. Shake well to obtain the solution.

7. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The chromatographic column is either an Agilent ZORBAX ODS series or a Waters Symmetry series C column. 18 The column; the chromatographic column packing material has a particle size ≤5μm, a length ≥25cm, and an inner diameter ≤4.6mm.

8. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, Detection wavelength: 210 nm; column temperature: 25℃~35℃; injection volume: 10μl~20μl; flow rate: adjust column pressure to 2000psi~3000psi.

9. The method for detecting related substances in isosorbide mononitrate tablets according to claim 1, characterized in that, The isosorbide mononitrate tablets are isosorbide mononitrate regular tablets or isosorbide mononitrate sustained-release tablets.

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