Method for detecting impurities in Adenafil citrate intermediate V and application of Adenafil citrate intermediate V

Abstract

The invention provides a method for detecting impurities in an Adenafil citrate intermediate V and application of the method. The method comprises the following steps: mixing a sample to be detected with a solvent to obtain a test solution, then carrying out liquid chromatography detection, and determining the variety and / or content of the impurities in the sample to be detected according to a detection result. The method provided by the invention can be used for simultaneously detecting three specific impurities in the aldenafil citrate intermediate V, and has the advantages of strong specificity, high separation degree, high sensitivity, high accuracy, good repeatability and good durability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for detecting impurities in sildenafil citrate intermediate V and application thereof. BACKGROUND

[0002] Sildenafil citrate is a new derivative of sildenafil citrate, which is a 5-type phosphodiesterase (Phosphodieseterase 5, PDE5) inhibitor, and is used for treating male erectile dysfunction (Erectile Dysfunction, ED). Its chemical name is: 1-(3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxybenzenesulfonyl)-cis-3,5-dimethylpiperazine citrate (C23H32N6O4S·C6H8O7), and its structural formula is:

[0003]

[0004] Impurity analysis and detection is a key link in line with international quality control standards. In the current global production and trade environment, the quality control of various products (especially medical, chemical, food and other products closely related to human health and safety) has formed a strict and unified international standard system. These international quality control standards clearly require that the impurities that may exist in the product be accurately identified and quantitatively detected to ensure the quality stability, safety and effectiveness of the product, and to ensure that it can meet the market access requirements of different countries and regions, and avoid quality risks and safety hazards caused by impurities exceeding the standard. Therefore, the specific impurity analysis and detection fully meet the core requirements of international quality control standards, and is an important technical means indispensable in the product quality control process, and has a wide range of industry applications. The related substances (impurities) are a direct indicator of the purity of the drug, and controlling the content of related substances is the key to ensuring the quality of the drug. According to the synthesis process, the starting materials and other process impurities may exist in the sildenafil citrate intermediate, so in order to ensure the quality of the finished product, the intermediate needs to be strictly controlled, especially the quality of the crude intermediate.

[0005] The important intermediate of sildenafil citrate is 1-[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxybenzenesulfonyl]-cis-3,5-dimethylpiperazine (sildenafil citrate intermediate V), and its structural formula is:

[0006]

[0007] Three important known impurities A, impurity B, impurity C can be introduced in the synthesis process thereof.

[0008]

[0009] The three specific impurities involved in the present patent can be clearly expected by those skilled in the art to have significant toxicity risks, and there is a clear and urgent need for separation and detection. Specifically, regarding impurity A, according to the authoritative PubChem database, it has a clear and high probability of toxic effects, specifically: H315 (occurrence probability 97.6%): can cause skin irritation, belonging to the [warning: skin corrosion / irritation] category, meaning that after the impurity contacts the skin, it can easily cause skin redness, pain, itching and other irritating reactions, and in severe cases, it can cause damage to the skin barrier; H319 (occurrence probability 97.6%): can cause serious eye irritation, classified as [warning: serious eye damage / eye irritation] category, once the impurity contacts the eye, it will produce strong irritation to the eye tissue, which can cause eye congestion, tearing, pain, and even the risk of causing eye organic damage; H335 (occurrence probability 95.2%): can cause respiratory tract irritation, belonging to the [warning: specific target organ toxicity, single exposure] type, when the human body inhales gas or dust containing the impurity, it will cause irritation to the respiratory mucosa, which can cause coughing, wheezing, difficulty breathing and other respiratory discomfort symptoms, posing a threat to respiratory health.

[0010] In addition, for impurities B and C, professional toxicity evaluation software (such software is usually based on a large amount of toxicology data, chemical structure-activity relationship models and related algorithms, and can scientifically and systematically predict and evaluate the toxicity of substances) was used for identification and analysis, and the results showed that both of them also have clear toxicity risks. This indicates that impurities B and C, after contacting or entering the human body, can also have toxic effects on specific organs or systems of the human body, thereby endangering human health.

[0011] In summary, the above three specific impurities all have clear toxicity risks, and if they cannot be effectively separated and accurately detected, it may cause potential health hazards and safety risks to operators, users and the environment during the production, storage, use or flow of products containing these impurities. Therefore, it is of great practical necessity and urgency to develop a method that can effectively separate and accurately detect the three specific impurities.

[0012] Currently, there is no method for the separation and detection of the three known impurities in the existing literature reports, so it is urgent to provide a separation method for the determination of the three impurities in the sildenafil citrate intermediate.

[0013] The prior art mainly exists as follows: the detection of the related impurities in Sildenafil Citrate:

[0014] Document 1: "CN105334275A_A detection method of Sildenafil Citrate related substances", the chromatographic condition method adopts a C18 chromatographic column, the mobile phase A is 0.005mmol / l ammonium acetate (pH is adjusted to 7.0 by ammonia water), the mobile phase B is acetonitrile-methanol=1:1, seven larger process or degradation impurities in different source products are researched, wherein the impurity A is a starting material, which is a process impurity, but the other six known impurities are different from the impurities A, B and C.

[0015] Document 2: "CN112198243B_A detection method of Sildenafil Citrate impurities", wherein the liquid phase condition adopts a Waters Acquity UPLC BEH C18 chromatographic column (2.1mm*100mm, 1.7um), the mobile phase A is 20mmol / L potassium dihydrogen phosphate (pH is adjusted to 5.5)-acetonitrile (80:20), the mobile phase B is phosphate buffer (pH 5.5)-methanol-acetonitrile (20:25:55), the known impurities and the impurities A, B and C are completely different under the chromatographic method condition system, and the method adopts a salt system ultra-high performance chromatographic condition method, the column pressure is extremely high, and the normal operation cannot be reproduced. -1

[0016] Document 3: "Preparation research of Sildenafil Citrate oral disintegrating tablets_Wei Xiaolin", the method is to analyze and determine the related substances and content in the preparation product, the patent is to study the quality of three known impurities in Sildenafil Citrate intermediate V, and then control the quality of the intermediate V, but the chromatographic condition and method of the document are not suitable for the quality control detection of the intermediate.

[0017] Only document 1 introduces the HPLC method for detecting the related substances of Sildenafil Citrate, and records one impurity A, but there is still no effective detection means for impurities B and C, and the three known impurities cannot be accurately quantified. Therefore, how to provide a method for effectively detecting three impurities in Sildenafil Citrate intermediate V has become a problem to be solved. SUMMARY

[0018] In view of the deficiencies of the prior art, the purpose of the present application is to provide a detection method of impurities in Sildenafil Citrate intermediate V and application thereof. The method provided by the present application can simultaneously detect three specific impurities in Sildenafil Citrate intermediate V, and has the advantages of strong specificity, high separation degree, high sensitivity, high accuracy, good repeatability and good durability.

[0019] ​To achieve the object of the present application, the present application adopts the following technical solutions:

[0020] In one aspect, the present application provides a method for detecting impurities in Sildenafil citrate intermediate V, which comprises the following steps:

[0021] Mixing the sample to be tested with a solvent to obtain a test solution, and then performing liquid chromatography detection, and determining the types and / or contents of impurities in the sample to be tested according to the detection results;

[0022] The structure of the Sildenafil citrate intermediate V is as follows:

[0023] .

[0024] The above method can simultaneously detect three specific impurities in Sildenafil citrate intermediate V, and has the advantages of strong specificity, high separation degree (the minimum separation degree is greater than 1.5), high sensitivity (the quantitative limit and detection limit are significantly reduced, and the quantitative limit of part of the known impurities can be as low as 0.02 μg / mL, and the detection limit can be as low as 0.01 μg / mL), high accuracy, good repeatability (the determination results of each known impurity are basically consistent), and good durability (the absolute value of the detection result is less than 0.05%).

[0025] Preferably, the chromatographic column for liquid chromatography detection is an octadecylsilane silica gel bonded chromatographic column, including any one of Agilent Poroshell 120 EC-C18, Waters Xterra RP18, Agilent ZORBAX SB-C18 or YMC XB C8, and preferably Agilent ZORBAX SB-C18.

[0026] Preferably, the mobile phase for liquid chromatography detection includes mobile phase A and mobile phase B, the mobile phase A is an ammonium acetate aqueous solution, and the mobile phase B is a methanol-acetonitrile mixed solution.

[0027] Preferably, the concentration of the ammonium acetate aqueous solution is 0.03-0.07 mol / L, such as 0.03 mol / L, 0.04 mol / L, 0.05 mol / L, 0.06 mol / L or 0.07 mol / L, etc., but not limited to the above listed values, and other values not listed in the above range are also applicable.

[0028] Preferably, the pH of the ammonium acetate aqueous solution is 6.3-6.7, such as 6.3, 6.4, 6.5, 6.6 or 6.7, etc., but not limited to the above listed values, and other values not listed in the above range are also applicable.

[0029] Preferably, the volume ratio of methanol and acetonitrile in the methanol-acetonitrile mixed solution is (4-6):(6-4).

[0030] Preferably, the elution mode of the liquid chromatography detection is gradient elution, and the gradient elution process is as follows:

[0031] 0-25 min, the volume fraction of mobile phase A is uniformly changed from 47-53% to 5-7%, and the rest is mobile phase B;

[0032] 25-25.01 min, the volume fraction of mobile phase A is changed from 5-7% to 47-53%, and the rest is mobile phase B;

[0033] 25.01 min later, the volume fraction of mobile phase A is 47-53%, and the rest is mobile phase B.

[0034] Preferably, the column temperature of the liquid chromatography detection is 20-30℃, for example, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃, etc., but not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0035] Preferably, the flow rate of the mobile phase of the liquid chromatography detection is 0.8-1.2 mL / min, for example, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min or 1.2 mL / min, etc., but not limited to the above listed values, and other values not listed in the above value range are also applicable.

[0036] The above parameters and reagents can further improve the detection effect.

[0037] Preferably, the impurities include any one, a combination of two or three of the following structural compounds:

[0038]

[0039] Preferably, the solvent is a mixture of mobile phase A and mobile phase B, and the volume ratio of mobile phase A to mobile phase B is (4-6):(6-4).

[0040] In another aspect, the application also provides the use of the detection method as described above in the quality control of Sildenafil Citrate.

[0041] Compared with the prior art, the application has the following beneficial effects:

[0042] The application provides a method for detecting impurities in Vildenafil citrate intermediate, which can simultaneously detect three specific impurities in Vildenafil citrate intermediate, has the advantages of strong specificity, high resolution (the minimum resolution is greater than 1.5), high sensitivity (the quantitative limit and the detection limit are significantly reduced, the quantitative limit of part of the known impurities can be as low as 0.02 μg / mL, the detection limit can be as low as 0.01 μg / mL, high accuracy, good repeatability (the determination results of each known impurity are basically consistent), and good durability (the absolute value of the detection result is less than 0.05 %).

[0043] Specifically,

[0044] 1) By using the method, the resolution of the characteristic peaks of the three known impurities is greater than 4.5, and the resolution is very good;

[0045] 2) The quantitative limit and the detection limit of the three known impurities are significantly reduced, and the quantitative limit of the intermediate and impurities A-C is not higher than 0.03 μg / mL; the detection limit of the intermediate and impurities A-C is not higher than 0.01 μg / mL;

[0046] Among them, the quantitative limit can be as low as 0.02 μg / mL, and the quantitative limit concentration is only 0.004 % of the concentration of the test solution (0.004 % of the concentration of the test solution = 0.5 mg / mL * 0.004 % = 0.02 μg / mL), which is about one twenty-fifth of the limit concentration; the detection limit is as low as 0.01 μg / mL, and the detection limit concentration is only 0.002 % of the concentration of the test solution (0.002 % of the concentration of the test solution = 0.5 mg / mL * 0.002 % = 0.01 μg / mL), which is about one fiftieth of the limit concentration, and the detection sensitivity is very high;

[0047] 3) Within the limited concentration range, the characteristic absorption peak area of Vildenafil citrate intermediate and known impurities A-C and its corresponding concentration show a good linear relationship, and the linear correlation coefficient r of the linear equation is greater than 0.999, which is higher than the general standard linear correlation coefficient r of greater than 0.990, and the detection method has higher accuracy;

[0048] The test results show that the intermediate has a good linear relationship in the range of 0.05-10.11 μg / mL; impurity A has a good linear relationship in the range of 0.05-10.17 μg / mL; impurity B has a good linear relationship in the range of 0.05-10.71 μg / mL; and impurity C has a good linear relationship in the range of 0.05-10.26 μg / mL;

[0049] 4) The recovery rate of each known impurity is maintained between 90 % and 108 %, the maximum RSD of the recovery rate of 9 samples is 1.5 %, the RSD is less than 5 %, and the method has good accuracy;

[0050] 5) The detection method of the present application can detect parallel samples repeatedly, and the range of each known impurity detection result is less than 0.02%, which shows good repeatability;

[0051] 6) The detection method of the present application can achieve good and accurate detection effect within the above-mentioned condition range, and the detection result shows good and significant durability within the above-mentioned condition range. At the same time, there is no related control method for directly and simultaneously separating and detecting the three impurities in the sildenafil citrate intermediate, so the method is worth popularizing. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 The blank solvent chromatogram of the specificity test;

[0053] Figure 2 The system suitability chromatogram of the specificity test;

[0054] Figure 3 The test sample solution chromatogram of the specificity test. DETAILED DESCRIPTION

[0055] The technical solutions of the present application will be further described through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitation to the present application.

[0056] Example 1: Determination and analysis method of impurities in sildenafil citrate intermediate V:

[0057] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0058] Test sample solution: take the intermediate V sample to be tested, accurately weigh, dissolve and dilute to prepare a solution containing 0.5 mg per 1 mL.

[0059] Control solution: accurately take the test sample solution, dilute with solvent to prepare a solution containing 0.005 mg per 1 mL.

[0060] System suitability solution: take intermediate V, impurity A, impurity B, and impurity C controls, accurately weigh, dissolve and dilute to prepare a solution containing 0.5 mg, 0.5 μg, 0.5 μg, and 0.5 μg per 1 mL.

[0061]

[0062] Chromatographic conditions:

[0063] Chromatographic column: octadecylsilane bonded silica gel as filler (Agilent ZORBAX SB-C18 4.6x250mm, 5μm);

[0064] Mobile phase A: 0.05 mol / L ammonium acetate solution, pH adjusted to 6.5;

[0065] Mobile phase B: methanol-acetonitrile = 50:50;

[0066] Elution gradient:

[0067]

[0068] The column temperature was 25°C, the flow rate was 1.0 mL per minute, and the detection wavelength was 240 nm. The injection volume was 20 μL.

[0069] Specificity test of Example 2

[0070] 2-1 Solution preparation:

[0071] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0072] Test solution: accurately weigh an appropriate amount of the sample to be tested, dissolve and dilute to prepare a solution containing 0.5 mg per 1 mL.

[0073] Control solution: accurately measure 1 mL of the test solution into a 100 mL volumetric flask, dilute to the mark with solvent, and shake well.

[0074] Impurity control stock solution: accurately weigh an appropriate amount of impurity A, impurity B, and impurity C controls, respectively, dissolve and dilute to prepare a control stock solution containing 0.5 mg of impurity A, impurity B, and impurity C per 1 mL.

[0075] Impurity control positioning solution: accurately measure 1 mL of the impurity A, impurity B, and impurity C control stock solutions, respectively, into 100 mL volumetric flasks, dilute to the mark with solvent, and shake well.

[0076] System suitability solution: take 50 mg of intermediate, accurately add 1 mL of impurity A, impurity B, and impurity C control stock solutions, respectively, into a 100 mL volumetric flask, dissolve and dilute to prepare a mixed solution containing 0.5 mg of intermediate and 0.5 μg of impurity A, impurity B, and impurity C per 1 mL.

[0077] 2-2 Test conditions:

[0078] Instrument: high performance liquid chromatograph;

[0079] Column: Agilent ZORBAX SB-C18 4.6 x 250 mm, 5 μm;

[0080] Mobile phase A: 0.05 mol / L ammonium acetate solution, pH adjusted to 6.5;

[0081] Mobile phase B: methanol-acetonitrile = 50:50,

[0082] The elution mode is gradient elution, and the elution gradient is as follows:

[0083]

[0084] Column temperature: 25 DEG C;

[0085] Flow rate: 1.0 mL / min;

[0086] Detection wavelength: 240 nm;

[0087] Injection volume: 20 μL;

[0088] 2-3 Experimental steps and conclusions:

[0089] Precisely take 20 μL of the above solution, respectively inject into high performance liquid chromatograph, record chromatogram and results, results are shown in Table 1, see the related drawings Figure 1 、 Figure 2 And Figure 3

[0090] Table Specificity test results

[0091]

[0092] Conclusion: the above samples are separated by high performance liquid chromatograph, and the test results show that: the blank solvent does not interfere with the determination of the main peak and known impurities. In the mixed solution, the separation degree between the main peak and the adjacent known impurity chromatographic peak is greater than 4.0; the small separation degree between the known impurity peak and the main peak is good, and the theoretical plate number is not less than 3000.

[0093] From the above experimental test results, it can be seen that the determination method provided by the application can simultaneously separate three kinds of known impurities in the intermediate, and the chromatographic peaks of citric acid sildenafil main peak and three kinds of known impurities show good separation effect and good specificity.

[0094] Example 3 selection of chromatographic column

[0095] System suitability solution: same as example 1;

[0096] Referring to the test conditions of example 1, the type of chromatographic column is changed:

[0097] Replace the "chromatographic column is Agilent ZORBAX SB-C18, 5 μm" in example 1 with the following:

[0098] ​Method 1: The chromatographic column is Agilent Poroshell 120 EC-C18 4.6x250 mm;

[0099] Method 2: The chromatographic column is Waters Xterra RP18, 4.6x150 mm, 5 μm;

[0100] Method 3: The chromatographic column is Ultmate C8, 4.6x250 mm, 5.0 μm;

[0101] Other chromatographic conditions are the same as in Example 1, and 20 μL of the system suitability solution is accurately measured and analyzed for detection, and the test results are shown in Table 2.

[0102] Table Influence of chromatographic column type on system suitability solution test results

[0103]

[0104] Conclusion:

[0105] When the chromatographic column Agilent ZORBAX SB-C18, 5 μm of Example 1 of the present application is used to separate the system suitability solution, Figure 2 ), the separation degree between impurity A and impurity B is 4.6, the number of other impurity peaks after the main peak of the test sample is 5, and the separation degree of the impurity immediately adjacent to the main peak is greater than 1.5, achieving complete baseline separation, meeting the requirements for related substance determination, and being the best in terms of impurity detection number and separation degree effect, and being able to meet the separation of three known impurities and unknown impurities.

[0106] Using other different brands of chromatographic columns, such as Agilent Poroshell 120 EC-C18 4.6x250 mm in Example 3-Method 1, the separation degree between impurity A and impurity B is 1.3, the separation degree between the main peak and unknown impurities is 1.3, and the number of impurities is 4; using Waters Xterra RP18, 4.6x150 mm, 5 μm in Example 3-Method 2, the separation degree between impurity A and impurity B is 1.3, the separation degree between the main peak and unknown impurities is 1.3, and the number of impurities is 4; two chromatographic columns cannot completely achieve ideal separation effect for all impurities, and the separation effect is not as good as that of Agilent ZORBAX SB-C18, 5 μm.

[0107] In Example 3-Method 3, a chromatographic column different from C18 packing is used to determine each known impurity, which cannot completely separate all known impurities, and the separation effect is significantly different from that of C18, so it is not suitable to be used.

[0108] Selection of the concentration of mobile phase A in Example 4

[0109] System suitability solution: same as Example 1;

[0110] Referring to the test conditions of Example 1, the concentration of the mobile phase water phase is changed, and the "0.05 mol / L ammonium acetate solution, pH adjusted to 6.5, as mobile phase A, methanol-acetonitrile mixed solution (1:1) as mobile phase B" in Example 1 is replaced by the following:

[0111] Method 1: 0.02 mol / L ammonium acetate solution, (pH adjusted to 6.5) as mobile phase A, methanol-acetonitrile mixed solution (1:1) as mobile phase B;

[0112] Method 2: 0.04 mol / L ammonium acetate solution, (pH adjusted to 6.5) as mobile phase A, methanol-acetonitrile mixed solution (1:1) as mobile phase B;

[0113] Method 3: 0.08 mol / L ammonium acetate solution, (pH adjusted to 6.5) as mobile phase A, methanol-acetonitrile mixed solution (1:1) as mobile phase B;

[0114] Other chromatographic conditions are the same as Example 1, and 10 μL of the system suitability solution is accurately measured for analysis and detection, and the test results are shown in Table 3.

[0115] Table Effect of mobile phase concentration on system suitability solution test results

[0116]

[0117] Conclusion: The mobile phase chromatographic conditions of Example 1 of the present application are used, and the separation degree between impurity A and impurity B is 4.6 when determining the system suitability solution, the main peak of the test sample is good, and the number of other impurity peaks after the main peak is 5, and the separation degree between the main peak impurity and the next peak is greater than 1.5, which achieves complete baseline separation and meets the requirements of related substance determination.

[0118] The low concentration of the mobile phase water phase of Example 4 Method 1 and Method 2, the impurity A and impurity B of Method 1 do not achieve complete separation, and although Method 2 achieves complete baseline separation, the two methods are slightly worse than Example 1 in impurity detection ability, and the peak shape is also slightly worse, which is not the optimal chromatographic condition. Example 4 Method 3 is similar to Method 2, and under the same conditions, the mobile phase water phase of Example 1 is optimized.

[0119] Selection of mobile phase pH in Example 5

[0120] System suitability solution: same as Example 1;

[0121] Referring to the test conditions of Example 1, the pH of mobile phase A is changed:

[0122] Example 1: pH of mobile phase A is 6.5;

[0123] Method 1: pH of mobile phase A is 5.8;

[0124] Method 2: pH of mobile phase A is 6.0;

[0125] Method 3: pH of mobile phase A is 7.0;

[0126] Method 4: pH of mobile phase A is 7.2;

[0127] Other chromatographic conditions are the same as in Example 1, and 20 μL of the system suitability solution is accurately measured and analyzed for detection, and the test results are shown in Table 4 below.

[0128] Table pH change of mobile phase A and impurity separation

[0129]

[0130] Conclusion: When the chromatographic conditions in Example 5 are used, the separation degree between impurity A and impurity B is 1.4 when the pH of mobile phase A is between 5.8 and 6.0, the number of impurities detected is reduced, and the impurity detection capacity is not enough. When the pH value exceeds 7.0-7.2, the separation between impurity A and impurity B is reduced, the impurity detection capacity is poor, and the separation of multiple known impurities and unknown impurities cannot be achieved. Considering the optimal conditions, the pH of the water phase of mobile phase A is 6.5.

[0131] Example 6: destructive test

[0132] The forced degradation test is to accelerate the destruction of the sample under relatively severe conditions, such as strong light irradiation, high temperature, high humidity, acid and base destruction, and hydrolysis and oxidation destruction, etc. The purpose is to evaluate the effectiveness and applicability of the analysis method by investigating the separation of the degradation products, the main peak and the known impurities. At the same time, the peak purity is checked by using a photodiode array detector: when the peak purity angle of the impurities and the main peak in the spectrum obtained by the degradation experiment is less than the purity threshold, it is judged that the determination method meets the requirements of the determination.

[0133] 6-1. Solution preparation:

[0134] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0135] Undegraded solution: accurately weigh 25 mg of the product into a 50 mL volumetric flask, dissolve and dilute to the mark with the solvent, and shake well to obtain the solution;

[0136] Acid destruction: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, accurately add 1 mol / L hydrochloric acid solution 1 mL, shake, and place at room temperature for 24 hours. Accurately add 1 mol / L sodium hydroxide solution for neutralization, add solvent for dissolution and dilution to the mark, shake, and use as the acid destruction sample solution. Prepare the acid-base blank solvent in the same way.

[0137] Alkali destruction: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, accurately add 1 mol / L sodium hydroxide solution 1 mL, shake, and place at room temperature for 24 hours. Accurately add 1 mol / L hydrochloric acid solution for neutralization, add solvent for dissolution and dilution to the mark, shake, and use as the alkali destruction sample solution.

[0138] Oxidation destruction: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, accurately add 3% hydrogen peroxide solution 1 mL, shake, and place at room temperature for 48 hours. Dilute to the mark, shake, and use as the oxidation destruction sample solution. Prepare the oxidation blank solvent in the same way.

[0139] High temperature destruction: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, and placed at 105°C for 12 hours. Take it out, cool it down, add solvent for dissolution and dilution to the mark, shake, and use as the high temperature destruction sample solution.

[0140] Light destruction: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, and irradiated under the condition of 4500xl±500xl for 48 hours. Take it out, add solvent for dissolution and dilution to the mark, shake, and use as the light destruction sample solution.

[0141] Water degradation: take 25 mg of the product, accurately weighed, placed in a 50 mL volumetric flask, accurately add 1 mL of water, shake, and place at room temperature for 48 hours. Add solvent for dissolution and dilution to the mark, shake, and use as the water degradation sample solution.

[0142] 6-2. Experimental steps and conclusions:

[0143] Accurately take 10 μL of each of the above solutions, inject into a high performance liquid chromatograph, and record the chromatogram and results. The results are shown in Tables 5-6.

[0144] Table Destruction test results-1

[0145]

[0146] Conclusion: The results of the forced degradation test showed that the intermediate was stable under the conditions of acid, base, high temperature, light, and water. Under the condition of oxidation, it was not stable at room temperature for 48 hours, and the total impurities degraded by 3.04%. The degradation products were well separated from other impurities and the main peak. The purity of the main peak under each degradation condition was not less than 0.999, and the material conservation was between 99.8 and 101.9%, or between 90.0% and 110.0%, which met the acceptable standards.

[0147] The requirements for the determination of related substances of the product were met.

[0148] The above results showed that the analysis method was effective and applicable under each degradation condition.

[0149] The above content separated the three known impurities in Sildenafil Citrate Intermediate V. At the same time, the following content also quantitatively detected all the impurities involved, achieving the evaluation and calculation of the impurity content of Sildenafil Citrate.

[0150] Example 7 Linear Test

[0151] 7-1. Solution Preparation:

[0152] Solvent (diluent): mobile phase A - mobile phase B (50:50).

[0153] Intermediate V Control Solution 1: Take an appropriate amount of the product, accurately weigh, dissolve in solvent, and quantitatively dilute to prepare a solution containing 0.1 mg per 1 mL, shake well, and obtain (100 μg / mL);

[0154] Intermediate V Control Solution 2: Take 5 mL of Intermediate Control Solution 1, place it in a 50 mL volumetric flask, dilute to the mark with solvent, shake well, and obtain (10 μg / mL);

[0155] Impurity A Control Solution: Take an appropriate amount of Impurity A control, accurately weigh, dissolve in solvent, and quantitatively dilute to prepare a solution containing 0.1 mg per 1 mL, shake well, and use as Impurity A stock solution;

[0156] Impurity B Control Solution: Take an appropriate amount of Impurity B control, accurately weigh, dissolve in solvent, and quantitatively dilute to prepare a solution containing 0.1 mg per 1 mL, shake well, and use as Impurity B stock solution;

[0157] Impurity C Control Solution: Take an appropriate amount of Impurity C control, accurately weigh, dissolve in solvent, and quantitatively dilute to prepare a solution containing 0.1 mg per 1 mL, shake well, and use as Impurity C stock solution;

[0158] Preparation of linear solution: The linear solution of corresponding concentration was prepared according to the following table using the linear stock solution, and mixed uniformly.

[0159] Preparation of linear solution: The linear solution of corresponding concentration was prepared according to the following table using the linear stock solution, and mixed uniformly.

[0160] Table Preparation of linear solution for method validation of related substances of intermediate

[0161]

[0162] Table Preparation of linear solution for method validation of related substances of impurity control

[0163]

[0164] 7-2. Experimental procedure and conclusion:

[0165] Precisely pipette 20 μL of the above linear solution, and inject into the high performance liquid chromatograph, respectively. The chromatographic conditions are the same as those in Example 1. Record the chromatogram and results, and the results are shown in the following table.

[0166] Table Results of determination of linear range by related substances method- intermediate

[0167]

[0168] Table Results of determination of linear range by related substances method- impurity A

[0169]

[0170] Table Results of determination of linear range by related substances method- impurity B

[0171]

[0172] Table Results of determination of linear range by related substances method- impurity C

[0173]

[0174] Conclusion:

[0175] It was found through analysis of the above linear solution that:

[0176] The intermediate V was linearly regressed with the concentration as the abscissa and the peak area as the ordinate in the concentration range of 0.03 μg / mL to 5.05 μg / mL (equivalent to the concentration of the test solution of 0.006% to 1.00%), the linear regression equation was y = 55010x - 241.49, r = 1.0000, and the linear relationship was good.

[0177] The impurity A was linearly regressed with the concentration as the abscissa and the peak area as the ordinate in the concentration range of 0.03 μg / mL to 5.08 μg / mL (equivalent to the concentration of the test solution of 0.006% to 1.02%), the linear equation was y = 41306x - 151.5, r = 1.0000, greater than 0.990; the linear relationship was good.

[0178] The impurity B was linearly regressed with the concentration as the abscissa and the peak area as the ordinate in the concentration range of 0.02 μg / mL to 5.36 μg / mL (equivalent to the concentration of the test solution of 0.004% to 1.07%), the linear equation was y = 39030x + 388.54, r = 1.0000, greater than 0.990; the linear relationship was good.

[0179] The impurity C was linearly regressed with the concentration as the abscissa and the peak area as the ordinate in the concentration range of 0.03 μg / mL to 5.13 μg / mL (equivalent to the concentration of the test solution of 0.006% to 1.03%), the linear equation was y = 49317x - 111.66, r = 1.0000, greater than 0.990; the linear relationship was good.

[0180] The above results show that the linear correlation coefficients r of the intermediate V and the impurities A to C in the linear range are all greater than 0.990, the peak area and the concentration have good linear relationship. Among them, the linear correlation coefficient r is optimal to reach 1.0000; compared with the general standard requirement of the linear correlation coefficient r > 0.990, the accuracy of the determination is significantly improved.

[0181] Example 8 quantitative limit and detection limit test

[0182] 8-1. Solution preparation: the intermediate V and the impurity A, the impurity B and the impurity C control samples were prepared into control sample solutions, a step-by-step dilution method was used, 20 μl was precisely taken and injected into the liquid chromatograph, the chromatogram and the results were recorded, the signal-to-noise ratio was calculated, and the signal-to-noise ratio was about 10 times the baseline noise when it was the quantitative limit, and the signal-to-noise ratio was not less than 3 times the baseline noise when it was the detection limit.

[0183] 8-2. Experimental steps and conclusions:

[0184] Accurately pipette 10 μL of each of the above solutions into the HPLC, and record the chromatogram and results. The results are shown in the table below.

[0185] Table Method validation for related substances - limit of quantitation and limit of detection results - intermediate

[0186]

[0187] Table Method validation for related substances - limit of quantitation and limit of detection results - impurity A

[0188]

[0189] Table Method validation for related substances - limit of quantitation and limit of detection results - impurity B

[0190]

[0191] Table Method validation for related substances - limit of quantitation and limit of detection results - impurity C

[0192]

[0193] Conclusion: Using the above separation conditions, the limit of quantitation and limit of detection results of the method are as follows:

[0194] The limit of quantitation and limit of detection of the above three known impurities showed a significant downward trend. The limit of quantitation of intermediate V and impurities A-C was not higher than 0.03 μg / mL; 0.03, 0.02, 0.02, and 0.03 μg / mL, respectively. The limit of detection of intermediate and impurities A-C was not higher than 0.01 μg / mL; 0.01, 0.01, 0.01, and 0.01 μg / mL, respectively.

[0195] Among them, the limit of quantitation can be as low as 0.02 μg / mL, and the limit of quantitation concentration is only 0.004% of the test solution concentration (0.004% of the test solution concentration = 0.5 mg / mL * 0.004% = 0.02 μg / mL), which is about one twenty-fifth of the limit concentration; the detection limit is as low as 0.01 μg / mL, and the detection limit concentration is only 0.002% of the test solution concentration (0.002% of the test solution concentration = 0.5 mg / mL * 0.002% = 0.01 μg / mL), which is about one fifty-fifth of the limit concentration, and the detection sensitivity is very high.

[0196] The characteristic absorption peak area of Sildenafil Citrate intermediate V and known impurities A-C within a defined concentration range showed a good linear relationship between the characteristic absorption peak area and the corresponding concentration, the linear correlation coefficient r of the linear equation was greater than 0.999, wherein, the linear correlation coefficient of impurity A, impurity B and impurity C reached 1.0000; compared with the general standard linear correlation coefficient r of greater than 0.990, the detection method was more accurate;

[0197] The test results showed that the experimental method could meet the requirements of simultaneous separation and quality control of known impurities in Sildenafil Citrate intermediate, and the sensitivity was significantly improved.

[0198] Example 9 recovery rate test

[0199] 9-1. Solution preparation:

[0200] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0201] Preparation of background solution: take about 25 mg of the product, accurately weigh, place in a 50 mL volumetric flask, dissolve and dilute to the mark with solvent, shake well, and you get it;

[0202] Preparation of impurity control stock solution 1: take about 5 mg of impurity A, impurity B, impurity C control respectively, accurately weigh, respectively in 100 mL volumetric flask, with solvent to dissolve and dilute to the mark, shake well, as each impurity control stock solution (0.05 mg / mL). Parallel configuration of 2 copies.

[0203] Preparation of impurity control solution: take 1 mL of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G stock solution respectively, same in 100 mL volumetric flask, add solvent to dilute to the mark, shake well (0.5 μg / mL). Parallel configuration of 2 copies.

[0204] Accuracy solution: take about 25 mg of the product, accurately weigh, place in a 50 mL volumetric flask, parallel take 9 copies, average into 3 groups, respectively accurately add the above impurity control stock solution 0.25 mL, 0.5 mL, 0.75 mL, add solvent to dilute to the mark, shake well, respectively as 50%, 100%, 150% accuracy solution.

[0205] 9-2. Experimental steps and conclusions:

[0206] Precisely take 20 μL of each of the above accuracy test sample solution, respectively inject into high performance liquid chromatograph, the chromatographic conditions are the same as in example 1, record the chromatogram and the result, the result is shown in table 24. The relative standard deviation calculation formula is as follows:

[0207]

[0208] Table Results of the assay recovery test

[0209]

[0210] Conclusion: By determining the 50%, 100%, and 150% accuracy solutions (equivalent to the limit concentration), it was found that the intra-group average recovery rate and the inter-group average recovery rate of impurities A-C were between 98.9% and 100.9%, and the RSD of impurities A-C was less than 0.77%, which was much smaller than the standard detection requirement of each impurity (RSD < 5%). The test showed that the method was good in accuracy for detecting the above-mentioned impurities A-C.

[0211] Example 10 Precision test

[0212] Six sample solutions were repeatedly determined by different experimenters on different dates using different instruments, the repeatability test results of the determination of the six sample solutions were calculated, the intermediate precision test was determined in the same way, and the test results were calculated. The precision of the results of 12 data was statistically analyzed.

[0213] 10-1. Preparation of solutions:

[0214] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0215] Test solution: about 25 mg of the product was accurately weighed into a 50 mL volumetric flask, dissolved and diluted with the solvent to the mark, and shaken to obtain the test solution.

[0216] Control solution: 1 mL of the test solution was accurately measured into a 100 mL volumetric flask, diluted with the solvent to the mark, and shaken to obtain the control solution.

[0217] Impurity control stock solution: an appropriate amount of impurity A, impurity B, and impurity C control was accurately weighed into a 100 mL volumetric flask, respectively, and dissolved and diluted with the solvent to obtain a control stock solution containing 0.05 mg of impurity A, impurity B, and impurity C per 1 mL.

[0218] System suitability solution: 50 mg of intermediate V was taken into a 100 mL volumetric flask, 1 mL of impurity A, impurity B, and impurity C control stock solution was accurately added, respectively, and dissolved and diluted with the solvent to obtain a mixed solution containing 0.5 mg of intermediate and 0.5 μg of impurity A, impurity B, and impurity C per 1 mL.

[0219] 10-2. Experimental procedures and conclusions:

[0220] 20 μL of each of the above sample solutions was accurately measured and injected into the high performance liquid chromatograph, and the chromatogram and results were recorded under the same chromatographic conditions as in Example 1. The results are shown in the table below.

[0221] Table Results of repeatability test for related substances

[0222]

[0223] Table Results of intermediate precision test for related substances

[0224]

[0225] Table Results of precision test for related substances

[0226]

[0227] Conclusion: The RSD of total impurity content was less than 5% in the repeatability test of 6 samples; the RSD of each impurity content was less than 5% in the intermediate precision test of 6 samples; the determination results of known impurities were basically consistent; the determination results of 12 samples showed that the method had good precision.

[0228] Example 11 Solution stability test

[0229] About 12.5 mg of the product was precisely weighed into a 25 mL volumetric flask, dissolved and diluted to the mark with solvent, shaken well to serve as the test solution; 1 mL of the test solution was precisely measured into a 100 mL volumetric flask, diluted to the mark with solvent, shaken well to serve as the control solution. The test solution was placed at room temperature, and 20 μl was precisely measured at 0, 2, 4, 6, 8, 12, and 24 hours and injected into the liquid chromatograph to record the chromatogram. The results are shown in Table-84.

[0230] The test solution was prepared as described in Example 10 under the repeatability sample solution preparation, and the chromatographic conditions were the same as those in Example 1. The determination results are shown in the following table.

[0231] Table Results of solution stability test for related substances

[0232]

[0233] Conclusion: The determination results of each known impurity and total impurity were basically consistent when the test solution was placed at room temperature for 24 hours; the test results showed that the sample solution was stable at room temperature for 24 hours, and met the requirements of the related substance test.

[0234] Example 12 Durability test

[0235] The system suitability was investigated by changing the method parameters to examine the change in system suitability and the extent of the determination results not affected. The robustness of the product was investigated under the detection conditions of Example 1, changing the detection column temperature (±5℃), flow rate (±0.2 mL / min), pH value of mobile phase A (±0.2), and changing different batches of chromatographic column, etc. The solution preparation and other chromatographic conditions are the same as in Example 1, and the robustness results are shown below.

[0236] 12-1 Solution preparation:

[0237] Solvent (diluent): mobile phase A-mobile phase B (50:50).

[0238] Test solution: about 25 mg of the product was accurately weighed into a 50 mL volumetric flask, dissolved and diluted to the mark with the solvent, and shaken to obtain the test solution.

[0239] Control solution: 1 mL of the test solution was accurately measured into a 100 mL volumetric flask, diluted to the mark with the solvent, and shaken to obtain the control solution.

[0240] Impurity control stock solution: an appropriate amount of impurity A, impurity B, and impurity C control was accurately weighed into a 100 mL volumetric flask, respectively, and dissolved and diluted with the solvent to prepare a control stock solution containing 0.05 mg of impurity A, impurity B, and impurity C per 1 mL.

[0241] System suitability solution: 50 mg of the intermediate was placed in a 100 mL volumetric flask, and 1 mL of the impurity A, impurity B, and impurity C control stock solution was accurately added, respectively, and dissolved and diluted with the solvent to prepare a mixed solution containing 0.5 mg of the intermediate and 0.5 μg of impurity A, impurity B, and impurity C per 1 mL, to obtain the system suitability solution.

[0242] Table Table of parameter changes for method validation-robustness

[0243]

[0244] Table Test results of the robustness spiked test solution

[0245]

[0246] Conclusion: The results of the robustness test of changing different flow rates, column temperatures, and mobile phase pH values, and replacing different batches of chromatographic columns showed that the RSD of the content of each known single impurity and the maximum unknown single impurity in the test solution was 8.1%, which was less than 10.0%, and the RSD of the total impurity content was 3.6%, which was less than 5%. It was shown that the method of the present disclosure for detecting 3 impurities in the citric acid sildenafil intermediate had good robustness.

[0247] Comparative Example

[0248] The three impurities (impurity A, impurity B, and impurity C) in the test sample solution and the mixed impurity control solution of the embodiment of the application were separated and determined by referring to the detection method of the comparative document.

[0249] Comparative Document 1: CN105334275A, a detection method for related substances of sildenafil citrate.

[0250] Table Comparison of chromatographic conditions between the application and the comparative document

[0251]

[0252] The three impurities in Example 1 of the application were investigated by referring to the detection conditions and method of Comparative Document 1, and the three impurities (impurity A, impurity B, and impurity C) were analyzed and determined, and the results are shown in the following table.

[0253] Table Comparison of detection results between the application and the comparative document

[0254]

[0255] Conclusion: Under the detection conditions of Example 1 of the application, the three impurities (impurity A and impurity B) can be effectively separated and detected at the same time, the separation degree between the main peak and the adjacent known impurity peak is greater than 5.0, and the separation degree with unknown impurities is not less than 1.5. Each known impurity can be eluted under this condition, and can be effectively separated and detected.

[0256] Under the detection conditions of Comparative Document 1, impurity A and impurity B are coincident, impurity A, impurity B, and impurity C all have a small response, the main peak of the test sample has a poor tailing peak shape, and the tailing factor is greater than 3.5. There are two other unknown impurity peaks after the main peak of the test sample, and the separation degree is less than 1.5, so the chromatographic method and mobile phase condition system directly adopted in Comparative Document 1, i.e., the method and condition system in the literature, cannot accurately determine the three known impurities.

[0257] Summary: Compared with other detection methods, only the detection method of the application (for example, Example 1) can realize the detection of the main peak and the three known impurities (impurity A, impurity B, and impurity C), and the separation effect is good.

[0258] On the contrary, other detection methods (Comparative Document 1) with the most similar conditions cannot realize the quantitative detection of the main peak and the three known impurities.

[0259] The application is not limited to the above-mentioned embodiments, and does not mean that the application must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the application, equivalent replacement of each raw material of the product of the application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the application.

[0260] The preferred embodiments of the application are described in detail above, but the application is not limited to the specific details in the above-mentioned embodiments. Within the technical concept scope of the application, various simple modifications can be made to the technical solutions of the application, and these simple modifications all belong to the protection scope of the application.

[0261] In addition, it should be noted that each specific technical feature described in the above-mentioned specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the application will not further describe various possible combination manners.

Claims

1. A method for detecting impurities in Sildenafil citrate intermediate V, characterized in that, The method includes the following steps: The sample to be tested is mixed with a solvent to obtain a test solution, which is then detected by liquid chromatography. The type and / or content of impurities in the sample to be tested are determined based on the detection results. The structure of the citrate intermediate V is shown below: 。 2. The detection method according to claim 1, characterized in that, The chromatographic column used for liquid chromatography detection is an octadecyl silica bonded column, including any one of Agilent Poroshell 120 EC-C18, Waters Xterra RP18, Agilent ZORBAX SB-C18 or Yuexu XB C8, preferably Agilent ZORBAX SB-C18.

3. The detection method according to claim 1 or 2, characterized in that, The mobile phases used in the liquid chromatography detection include mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution of ammonium acetate and mobile phase B is a methanol-acetonitrile mixed solution.

4. The detection method according to claim 3, characterized in that, The concentration of the ammonium acetate aqueous solution is 0.03-0.07 mol / L; Preferably, the pH of the ammonium acetate aqueous solution is 6.3-6.

7.

5. The detection method according to claim 3 or 4, characterized in that, In the methanol-acetonitrile mixed solution, the volume ratio of methanol to acetonitrile is (4-6):(6-4).

6. The detection method according to any one of claims 3-5, characterized in that, The elution method for the liquid chromatography detection is gradient elution, and the gradient elution process is as follows: From 0 to 25 minutes, the volume fraction of mobile phase A changes uniformly from 47-53% to 5-7%, with the remainder being mobile phase B; Between 25 and 25.01 min, the volume fraction of mobile phase A changed from 5-7% to 47-53%, with the remainder being mobile phase B; After 25.01 min, the volume fraction of mobile phase A was 47-53%, with the remainder being mobile phase B.

7. The detection method according to any one of claims 1-6, characterized in that, The column temperature for the liquid chromatography detection is 20-30℃; Preferably, the flow rate of the mobile phase detected by the liquid chromatography is 0.8-1.2 mL / min.

8. The detection method according to any one of claims 1-7, characterized in that, The impurities include any one or a combination of at least two of the following structural compounds: 、 、 。 9. The detection method according to any one of claims 3-8, characterized in that, The solvent is a mixture of mobile phase A and mobile phase B, wherein the volume ratio of mobile phase A to mobile phase B is (4-6):(6-4).

10. The application of a detection method according to any one of claims 1-9 in the quality control of edenafil citrate.

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