A method for detecting impurities in finerenone intermediates

Abstract

The present invention relates to the technical field of analysis and detection, and specifically discloses a method for detecting impurities in a finerenone intermediate. The present invention uses high performance liquid chromatography to detect the impurity 4-dimethylaminopyridine in the finerenone intermediate, and the detection conditions are as follows: the chromatographic column is a C18 chromatographic column; mobile phase A: an aqueous solution containing phosphoric acid; mobile phase B: methanol; elution is performed in an isocratic elution mode, and the detection wavelength is 273-283nm. The detection method provided by the present invention has the advantages of strong specificity, high sensitivity, rapid detection, accurate and reliable test results, and good precision, stability and durability. The present invention effectively solves the problems of short elution time, interference by solvent peaks, poor peak shape, and even tailing in the detection method for 4-dimethylaminopyridine in the prior art, and there is no problem of the method for detecting 4-dimethylaminopyridine in the finerenone intermediate, which can be used as the basis for quality monitoring of finerenone.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and specifically discloses a method for detecting impurities in a finerenone intermediate. Background Art

[0002] Finerenone, chemically named (4S)-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, is a new type of non-steroidal mineralocorticoid receptor (MR) antagonist. It has better affinity and selectivity than traditional steroidal mineralocorticoid receptor antagonists. At the same time, the plasma half-life of finerenone is only about 2h-3h, which can greatly reduce the hyperkalemia caused by steroidal mineralocorticoid receptor antagonists. It is mainly used to treat chronic kidney disease in adults caused by type II diabetes.

[0003] Finerenone has a chiral center, in which the S-configured finerenone has an affinity of 18 nM for MR, playing a major antagonistic role. The prior art has reported a synthetic route for the S-configured finerenone, in which 4-dimethylaminopyridine (DMAP) is one of the key catalysts for catalyzing the synthesis of finerenone intermediate 5, namely, the finerenone racemate 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide. DMAP is a super-strong nucleophilic acylation catalyst that can significantly catalyze the acylation reaction of highly sterically hindered, low-reactive alcohols and amines, and its activity is about 104-106 times that of pyridine. However, 4-dimethylaminopyridine is a genotoxic impurity with a warning structure. After metabolism in the body, it will produce a metabolite called 4-dimethylaminopyridine-N-oxide, which can bind to proteins in cells, causing changes in the structure and function of proteins, thereby affecting the normal metabolism and physiological functions of cells. In addition, 4-dimethylaminopyridine-N-oxide can also bind to DNA, causing DNA damage and mutation, thereby increasing the risk of cell canceration. Therefore, in the process of finerenone synthesis, the residual 4-dimethylaminopyridine in finerenone intermediate 5 needs to be strictly controlled to ensure the quality of finerenone raw materials.

[0004]

[0005] At present, the detection method for 4-dimethylaminopyridine is prone to problems such as short elution time, interference from solvent peaks, poor peak shape, and even tailing. In addition, there is no method for detecting 4-dimethylaminopyridine in finerenone intermediates in the prior art. Based on this, developing a method specifically for detecting 4-dimethylaminopyridine in finerenone intermediates is of great significance for controlling the quality of finerenone. Summary of the invention

[0006] Aiming at the problems that the detection methods for 4-dimethylaminopyridine in the prior art are prone to short peak time, interference by solvent peaks, poor peak shape and even tailing, and there is no method for detecting 4-dimethylaminopyridine in finerenone intermediates, the present invention provides a method for detecting impurities in finerenone intermediates.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is 4-dimethylaminopyridine. The detection method is a high performance liquid chromatography method, which specifically comprises the following steps:

[0009] Step 1: Preparation of test solution and reference solution:

[0010] Preparation of the test solution: Dissolve the 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample in an organic alcohol to obtain a test solution;

[0011] Preparation of reference solution: dissolve 4-dimethylaminopyridine reference in organic alcohol to obtain reference solution;

[0012] Step 2: Take the reference solution and the test solution and perform injection testing according to the following chromatographic conditions:

[0013] The chromatographic conditions were as follows: the chromatographic column was a C18 column;

[0014] Mobile phase A: aqueous solution containing phosphoric acid;

[0015] Mobile phase B: methanol;

[0016] Elution mode: isocratic elution;

[0017] Detection wavelength: 275-285nm.

[0018] Compared with the prior art, the present invention provides a method for detecting 4-dimethylaminopyridine in a finerenone intermediate 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide by high performance liquid chromatography. The method can complete the detection of 4-dimethylaminopyridine in a finerenone intermediate in a short time, and the effect of separating the 4-dimethylaminopyridine detected by the method from the main component is excellent, and the detection efficiency is high. At the same time, the method has the advantages of simple operation, strong specificity, high sensitivity, etc., and the content of 4-dimethylaminopyridine in a finerenone intermediate detected by the method has a good linear relationship, good precision and durability, and the verification of various methods can meet the requirements. The detection method provided by the present invention can accurately detect the content of 4-dimethylaminopyridine in the finerenone intermediate 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, can be used as a basis for quality monitoring of the finerenone intermediate, and is beneficial to the quality control of finerenone.

[0019] Further preferably, the detection wavelength is 280 nm.

[0020] Preferably, the volume percentage of phosphoric acid in the mobile phase A is 0.08%-0.12%.

[0021] Further preferably, the volume percentage of phosphoric acid in the mobile phase A is 0.1%.

[0022] Preferably, the volume ratio of the mobile phase A to the mobile phase B is 38:62-42:58.

[0023] Further preferably, the volume ratio of the mobile phase A to the mobile phase B is 40:60.

[0024] Preferably, the organic alcohol is methanol.

[0025] Preferably, the specification of the chromatographic column is 4.6×250 mm, and the filler diameter is 5 μm.

[0026] Further preferably, the chromatographic column is Ultimate AQ-C18.

[0027] Preferably, in step 1, the concentration of the reference solution is 0.04-0.06 μg / mL.

[0028] Further preferably, in step 1, the concentration of the reference solution is 0.05 μg / mL.

[0029] Preferably, in step 1, the concentration of the test solution is 0.48 mg / mL-0.52 mg / mL.

[0030] Further preferably, in step 1, the concentration of the test solution is 0.5 mg / mL.

[0031] Preferably, the chromatographic conditions also include a column temperature of 20-30°C.

[0032] More preferably, the column temperature is 25°C.

[0033] Preferably, the chromatographic conditions also include an injection volume of 10 μL.

[0034] Preferably, the chromatographic conditions also include a flow rate of 0.95-1.05 mL / min.

[0035] Further preferably, the flow rate is 1.0 mL / min.

[0036] In summary, the present invention provides a method for detecting impurities in finerenone intermediates, which has the advantages of strong specificity, high sensitivity, rapid detection, accurate and reliable test results, etc. At the same time, the detection method provided by the present invention has good precision, stability and durability, and the verification of various methods can meet the requirements, and is suitable for the quantitative detection of 4-dimethylaminopyridine in finerenone intermediates. The detection method of the present invention effectively solves the problems of the detection methods for 4-dimethylaminopyridine in the prior art that are prone to short peak time, interference by solvent peaks, poor peak shape, and even tailing, and there is no problem of the method for detecting 4-dimethylaminopyridine in finerenone intermediates, and can be used as the basis for finerenone quality monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a high performance liquid chromatogram of the system suitability solution in Example 1 of the present invention;

[0038] Figure 2 is a high performance liquid chromatogram of the test solution in Example 1 of the present invention;

[0039] Figure 3 is the HPLC chromatogram of the blank solution in Example 1 of the present invention;

[0040] Figure 4 This is a linear test graph of 4-dimethylaminopyridine in Example 1 of the present invention. DETAILED DESCRIPTION

[0041] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] This embodiment provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance, specifically comprising the following contents:

[0044] 1.1 Solution preparation

[0045] Blank solution: methanol.

[0046] Reference solution: Weigh 0.15 mg of 4-dimethylaminopyridine reference substance and dissolve it in 3000 mL of methanol to obtain a reference solution with a concentration of 0.05 μg / mL.

[0047] Test solution: Weigh 50 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample and dissolve it in 100 mL of methanol to obtain a test solution with a concentration of 0.5 mg / mL.

[0048] System suitability solution: Weigh 0.025 mg of 4-dimethylaminopyridine reference substance and 0.25 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, mix well, and dissolve in 500 mL of methanol to obtain a system suitability solution.

[0049] 1.2 HPLC detection conditions

[0050] HPLC conditions:

[0051] Chromatographic column: Ultimate AQ-C18 column, size 4.6×250mm, packing diameter 5μm;

[0052] Mobile phase: Aqueous solution containing 0.1% phosphoric acid was used as mobile phase A, and methanol was used as mobile phase B;

[0053] Flow rate: 1.0 mL / min;

[0054] Column temperature: 25°C;

[0055] Injection volume: 10 μL;

[0056] Elution method: isocratic elution according to the following program: 0-10min, 40% mobile phase A, 60% mobile phase B;

[0057] Detection wavelength: 280nm.

[0058] Methodological validation:

[0059] 2.1 Specificity test:

[0060] Take the blank solution, system suitability solution and test solution described in Example 1 and perform HPLC detection according to the above HPLC conditions, and record the chromatogram. The test results are shown in Table 1, and the chromatogram is shown in Figure 1-3 .

[0061] The test results showed that the elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak was about 3.5min, and the elution time of 4-dimethylaminopyridine was about 2.5min. The separation degree between the two was 3.41, which was greater than 1.5, and the separation degree was good. The peak shapes of the two were good. Among them, the tailing factor of the 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak was 1.03, and the tailing factor of the 4-dimethylaminopyridine peak was 0.96, both of which met the range requirement of tailing factor 0.95-1.05. The blank solution and the test solution had no interference with the detection of 4-dimethylaminopyridine, and the method had good specificity.

[0062] Table 1 Specificity test results

[0063]

[0064] 2.2 Limit of Quantitation and Limit of Detection Test

[0065] Limit of quantitation: 4-dimethylaminopyridine reference substance and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance were taken separately, and methanol was added to prepare 4-dimethylaminopyridine reference substance solution with a concentration of 0.05 μg / mL and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance solution with a concentration of 0.5 μg / mL, respectively, and diluted stepwise with methanol, and high performance liquid chromatography was performed according to the conditions proposed in Example 1, and the spectrum was recorded. The concentration when the signal-to-noise ratio was greater than or equal to 10 was the limit of quantitation concentration. The results are shown in Table 2. Six portions of the limit of quantitation solution were prepared in parallel, and high performance liquid chromatography was performed according to the conditions proposed in Example 1, and the spectrum was recorded. The repeatability was examined, and the results are shown in Table 3.

[0066] Detection limit: Take 4-dimethylaminopyridine reference substance and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance respectively, add methanol to prepare 4-dimethylaminopyridine reference substance solution with a concentration of each component of 0.05 μg / mL and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance solution with a concentration of 0.5 μg / mL, dilute stepwise with methanol, and perform high performance liquid chromatography detection according to the conditions set out in Example 1. The concentration diluted to a signal-to-noise ratio greater than or equal to 3 is the detection limit concentration. The results are shown in Table 2.

[0067] Table 2 Detection limit and quantification limit test results

[0068]

[0069] Table 3 Quantitative limit precision test results

[0070]

[0071] It can be seen from Tables 2 and 3 that the detection limit, quantification limit and quantification limit repeatability tests can all meet the detection requirements, and the detection method has excellent sensitivity.

[0072] 2.3 Linearity test

[0073] Take an appropriate amount of 4-dimethylaminopyridine reference substance, accurately weigh it, and use methanol to prepare a series of linear solutions containing about 0.010μg, 0.031μg, 0.042μg, 0.052μg, 0.105μg, and 0.157μg of 4-dimethylaminopyridine per 1mL. Perform high performance liquid chromatography detection under the conditions proposed in Example 1 and record the spectrum. A linear regression curve is drawn with 4-dimethylaminopyridine concentration (μg / mL) as the abscissa and peak area as the ordinate. The results are shown in Table 4, and the linear graph is shown in Figure 4 .

[0074] Table 4 4-dimethylaminopyridine linear test results

[0075]

[0076] The test results showed that 4-dimethylaminopyridine had a good linear relationship within the concentration range of 0.010-0.157 μg / mL.

[0077] 2.4 Repeatability test

[0078] Take 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance, add methanol to prepare 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide test solution with a concentration of 0.5 μg / mL, prepare 6 portions in parallel, perform high performance liquid chromatography detection according to the conditions proposed in Example 1, and record the spectrum. The test results are shown in Table 5.

[0079] Table 5 4-dimethylaminopyridine repeatability test results

[0080]

[0081] The test results showed that 4-dimethylaminopyridine was not detected in 6 test sample solutions, and the method had good repeatability.

[0082] 2.5 Accuracy Test

[0083] The accuracy of 4-dimethylaminopyridine is expressed in terms of recovery (%), where recovery (%) = (measured amount - original amount) / added amount × 100%.

[0084] Take appropriate amounts of 4-dimethylaminopyridine reference substance and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance, respectively, and add methanol to prepare 0.05 μg / mL 4-dimethylaminopyridine reference substance solution and 0.5 μg / mL 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance solution;

[0085] Take 3 portions of 0.5mL, 1mL and 2mL of 4-dimethylaminopyridine reference solution and place them in 9 10mL volumetric flasks respectively, then add 1mL of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference solution respectively, make up to volume with methanol, and use them as low concentration recovery solution, medium concentration recovery solution and high concentration recovery solution in turn.

[0086] A low-concentration recovery solution, a medium-concentration recovery solution and a high-concentration recovery solution were taken and subjected to HPLC detection according to the conditions set out in Example 1. The spectra were recorded. The recovery results are shown in Table 6.

[0087] Table 6 Recovery test results

[0088]

[0089]

[0090] The results showed that the average recovery rate of 4-dimethylaminopyridine was 99.6%, the RSD value was 0.97%, and the method had good accuracy.

[0091] 2.6 Durability Test

[0092] Take appropriate amounts of 4-dimethylaminopyridine reference substance and 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance, respectively, and add methanol to prepare 0.05 μg / mL 4-dimethylaminopyridine reference substance solution and 0.5 μg / mL 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance solution;

[0093] Fine-tune the conditions proposed in Example 1 to perform HPLC detection and record the spectrum.

[0094] By fine-tuning the chromatographic conditions: changing the mobile phase ratio to 38-42:58-62, column temperature to 25±5℃, total flow rate

[0095] The durability of 4-dimethylaminopyridine was investigated at 1.0±0.05mL / min and detection wavelength of 280±5nm. The changes in retention time and separation degree of each component were recorded. The test results are shown in Table 7.

[0096] Table 7 4-dimethylaminopyridine durability test results

[0097]

[0098]

[0099] The results showed that fine-tuning the chromatographic conditions had no effect on the detection of 4-dimethylaminopyridine, and the separation between the peaks of each component was greater than 1.5, indicating that the detection method of the present invention has good durability.

[0100] 2.7 Intermediate precision test

[0101] Repeated tests were performed by different personnel on different dates. Take an appropriate amount of 4-dimethylaminopyridine reference substance, accurately weigh it, and prepare a solution containing about 0.05 μg of 4-dimethylaminopyridine per 1 mL as the reference substance solution;

[0102] Take an appropriate amount of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance, add methanol to prepare a 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide test solution with a concentration of 0.5 mg / mL, prepare 6 portions in parallel, perform high performance liquid chromatography detection according to the conditions proposed in Example 1, and record the spectrum. The results are shown in Table 8.

[0103] Table 8 Intermediate precision test results

[0104]

[0105] The results showed that 4-dimethylaminopyridine was not detected in 6 tests, and the intermediate precision of this method was good.

[0106] 2.8 Stability Test

[0107] Take 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide reference substance, add methanol to prepare a 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide test solution with a concentration of 0.5 mg / mL, and place it for 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. Then, the high performance liquid chromatography conditions described in Example 1 were used for detection. The test results are shown in Table 9 below.

[0108] Table 9 Stability test results

[0109]

[0110]

[0111] As shown in Table 9, when the test solution was placed at room temperature for 0 h, 2 h, 4 h, 6 h, and 8 h, 4-dimethylaminopyridine was not detected, indicating that the test solution had good stability.

[0112] 2.9 Various sample tests

[0113] Five batches of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide samples produced with the same production specification were taken, namely, test samples 1-5, and were tested according to the high performance liquid chromatography detection method described in Example 1. The test results are shown in Table 10.

[0114] Table 10 Multi-sample test results

[0115] name 4-Dimethylaminopyridine Test sample 1 Not detected Test sample 2 Not detected Test sample 3 Not detected Test sample 4 Not detected Test sample 5 Not detected

[0116] According to Table 10, 4-dimethylaminopyridine was not detected in any of the samples 1-5, proving that the samples met the requirements.

[0117] Example 2

[0118] This embodiment provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance. The difference from Example 1 is that the mobile phase ratio is different: isocratic elution is performed according to the following procedure: 0-10 min, 60% mobile phase A, 40% mobile phase B, and other parameters and processes remain unchanged, which will not be repeated here.

[0119] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 6 minutes, and the elution time of 4-dimethylaminopyridine is about 3 minutes. The separation degree between the two is 2.98, which is greater than 1.5, and the separation degree is good; the peak shapes of the two are good, among which the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.12, and the tailing factor of 4-dimethylaminopyridine peak is 0.92, slightly exceeding the range requirement of tailing factor of 0.95-1.05.

[0120] Example 3

[0121] This embodiment provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance. The difference from Example 1 is that the elution method is different: isocratic elution is performed according to the following procedure: 0-20 min, 70% mobile phase A, 30% mobile phase B, and other parameters and processes remain unchanged, which will not be repeated here.

[0122] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 12 minutes, and the elution time of 4-dimethylaminopyridine is about 3 minutes. The separation degree between the two is 3.68, which is greater than 1.5, and the separation degree is good; the peak shapes of the two are good, among which the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.27, and the tailing factor of 4-dimethylaminopyridine peak is 0.93, slightly exceeding the range requirement of tailing factor 0.95-1.05.

[0123] Comparative Example 1

[0124] This comparative example provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance, specifically comprising the following contents:

[0125] 1.1 Solution preparation

[0126] Blank solution: methanol;

[0127] Reference solution: weigh 0.15 mg of 4-dimethylaminopyridine reference substance and dissolve it in 3000 mL of methanol to obtain a reference solution with a concentration of 0.05 μg / mL;

[0128] Test solution: Weigh 50 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample and dissolve it in 100 mL of methanol to obtain a test solution with a concentration of 0.5 mg / mL;

[0129] System suitability solution: weigh 0.025 mg of 4-dimethylaminopyridine reference substance and 0.25 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, mix well, and dissolve in 500 mL of methanol to obtain a system suitability solution;

[0130] 1.2 HPLC detection conditions

[0131] HPLC conditions:

[0132] Chromatographic column: Aglient ZORBAX Eclipse XDB-C8 column, size 3.5×150mm, packing diameter 3.5μm;

[0133] Mobile phase: methanol as mobile phase A, water as mobile phase B;

[0134] Total flow rate: 1.0 mL / min;

[0135] Column temperature: 25°C;

[0136] Injection volume: 10 μL;

[0137] Elution method: Isocratic elution according to the following program: 0-10 min, 80% mobile phase A, 20% mobile phase B.

[0138] Detection wavelength: 254nm.

[0139] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 1.7min, and the elution time of 4-dimethylaminopyridine peak is about 2.7min. The separation degree between the two is 1.64, which is greater than 1.5, and the separation degree is general; the response value is low, and it is impossible to achieve accurate quantitative calculation of 4-dimethylaminopyridine; the peak shapes of the two are poor, among which the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.69, and the tailing factor of 4-dimethylaminopyridine peak is 0.89, which obviously exceeds the range requirement of tailing factor of 0.95-1.05.

[0140] Comparative Example 2

[0141] This comparative example provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance, specifically comprising the following contents:

[0142] 1.1 Solution preparation

[0143] Blank solution: methanol;

[0144] Reference solution: weigh 0.15 mg of 4-dimethylaminopyridine reference substance and dissolve it in 3000 mL of methanol to obtain a reference solution with a concentration of 0.05 μg / mL;

[0145] Test solution: Weigh 50 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample and dissolve it in 100 mL of methanol to obtain a test solution with a concentration of 0.5 mg / mL;

[0146] System suitability solution: weigh 0.025 mg of 4-dimethylaminopyridine reference substance and 0.25 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, mix well, and dissolve in 500 mL of methanol to obtain a system suitability solution;

[0147] 1.2 HPLC detection conditions

[0148] HPLC conditions:

[0149] Chromatographic column: Aglient ZORBAX Eclipse XDB-C8 column, size 3.5×150mm, packing diameter 3.5μm;

[0150] Mobile phase: 0.2% formic acid aqueous solution as mobile phase A, acetonitrile as mobile phase B;

[0151] Total flow rate: 1.0 mL / min;

[0152] Column temperature: 25°C;

[0153] Injection volume: 10 μL;

[0154] Elution method: Gradient elution according to the following procedure:

[0155] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 12 40 60 16 40 60 16.1 100 0

[0156] Detection wavelength: 280nm.

[0157] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 1.2min, and the elution time of 4-dimethylaminopyridine is about 3.1min. The separation degree between the two is 2.85, which is greater than 1.5, and the separation degree is average. The peak shapes of the two are poor. Among them, the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.58, and the tailing factor of 4-dimethylaminopyridine peak is 0.84, which obviously exceeds the range requirement of tailing factor of 0.95-1.05.

[0158] Comparative Example 3

[0159] This comparative example provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance. The only difference from Comparative Example 2 is that the mobile phase A is replaced with a 0.1% phosphoric acid aqueous solution, and the other parameters and operations remain unchanged.

[0160] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 1.2min, and the elution time of 4-dimethylaminopyridine peak is about 2.2min. The separation degree between the two is 1.77, which is greater than 1.5, and the separation degree is average. The peak shapes of the two are poor. Among them, the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.59, and the tailing factor of 4-dimethylaminopyridine peak is 0.88, which obviously exceeds the range requirement of tailing factor of 0.95-1.05.

[0161] Comparative Example 4

[0162] This comparative example provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance, specifically comprising the following contents:

[0163] 1.1 Solution preparation

[0164] Blank solution: methanol;

[0165] Reference solution: weigh 0.15 mg of 4-dimethylaminopyridine reference substance and dissolve it in 3000 mL of methanol to obtain a reference solution with a concentration of 0.05 μg / mL;

[0166] Test solution: Weigh 50 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample and dissolve it in 100 mL of methanol to obtain a test solution with a concentration of 0.5 mg / mL;

[0167] System suitability solution: weigh 0.025 mg of 4-dimethylaminopyridine reference substance and 0.25 mg of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, mix well, and dissolve in 500 mL of methanol to obtain a system suitability solution;

[0168] 1.2 HPLC detection conditions

[0169] HPLC conditions:

[0170] Chromatographic column: Aglient ZORBAX Eclipse XDB-C8 column, size 3.5×150mm, packing diameter 3.5μm;

[0171] Mobile phase: 0.1% formic acid aqueous solution as mobile phase A, methanol as mobile phase B;

[0172] Total flow rate: 1.0 mL / min;

[0173] Column temperature: 25°C;

[0174] Injection volume: 10 μL;

[0175] Elution method: Gradient elution according to the following procedure:

[0176]

[0177] Detection wavelength: 280nm.

[0178] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 1.7min, and the elution time of 4-dimethylaminopyridine is about 1.3min. The separation degree of the two is 1.46, which is less than 1.5, and the separation degree is poor. The peak shapes of the two are poor. Among them, the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.39, and the tailing factor of 4-dimethylaminopyridine peak is 0.82, which obviously exceeds the range requirement of tailing factor of 0.95-1.05.

[0179] Comparative Example 5

[0180] This comparative example provides a method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is a 4-dimethylaminopyridine reference substance. The difference from Example 1 is that the chromatographic column used is different: chromatographic column: Aglient ZORBAX Eclipse XDB-C8 chromatographic column, with a specification of 3.5×150 mm and a filler diameter of 3.5 μm. Other parameters and processes remain unchanged and will not be repeated here.

[0181] Test results: The elution time of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is about 1.7min, and the elution time of 4-dimethylaminopyridine is about 1.8min. The separation degree of the two is 1.31, which is less than 1.5, and the separation degree is poor. The peak shapes of the two are poor. Among them, the tailing factor of 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide peak is 1.46, and the tailing factor of 4-dimethylaminopyridine peak is 0.79, which obviously exceeds the range requirement of tailing factor of 0.95-1.05.

[0182] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting impurities in a finerenone intermediate, wherein the finerenone intermediate is 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide, and the impurity is 4-dimethylaminopyridine, characterized in that: The detection method is a high performance liquid chromatography method, which specifically comprises the following steps: Step 1: Preparation of test solution and reference solution: Preparation of the test solution: Dissolve the 4-(4-cyano-2-methoxyphenyl)-5-ethoxy-2,8-dimethyl-1,4-dihydro-1,6-naphthyridine-3-carboxamide sample in an organic alcohol to obtain a test solution; Preparation of reference solution: dissolve 4-dimethylaminopyridine reference in organic alcohol to obtain reference solution; Step 2: Take the reference solution and the test solution and perform injection testing according to the following chromatographic conditions: The chromatographic conditions were as follows: the chromatographic column was a C18 column; Mobile phase A: aqueous solution containing phosphoric acid; Mobile phase B: methanol; Elution mode: isocratic elution; Detection wavelength: 275-285nm; The chromatographic column is Ultimate AQ-C18.

2. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: The volume percentage of phosphoric acid in the mobile phase A is 0.08%-0.12%.

3. The method for detecting impurities in a finerenone intermediate according to claim 1 or 2, characterized in that: The volume ratio of the mobile phase A to the mobile phase B is 38:62-42:

58.

4. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: The organic alcohol is methanol.

5. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: In step 1, the concentration of the reference solution is 0.04-0.06 μg / mL; and / or In step 1, the concentration of the test solution is 0.48-0.52 mg / mL.

6. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: The chromatographic conditions also include a column temperature of 20-30°C.

7. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: The chromatographic conditions also include an injection volume of 10 μL.

8. The method for detecting impurities in a finerenone intermediate according to claim 1, characterized in that: The chromatographic conditions also include a flow rate of 0.95-1.05 mL / min.

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