HPLC Detection Method and Application of Trimetazidine Hydrochloride

Abstract

The present invention relates to the field of detection technology, and specifically discloses a HPLC detection method and application of trimetazidine hydrochloride. The HPLC detection method of trimetazidine hydrochloride of the present invention, its mobile phase A is composed of 0.287% sodium heptane sulfonate solution and methanol in a volume ratio of (53-55): (45-47), and mobile phase B is methanol; the pH value of the mobile phase A is 4.0-4.2 or 2.0-3.0; during detection, the solvent of the sample to be tested is a mixed solvent of methanol and water in a volume ratio of 1: (0.5-1.5). The method of the present invention can effectively distinguish multiple impurities or can effectively analyze and distinguish impurity I and newly discovered impurities, is convenient for determining the content of impurities, provides effective process monitoring means for preparing qualified samples for synthetic preparation processes, can guide process optimization, and improve product quality control standards.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to an HPLC detection method and application of trimetazidine hydrochloride. Background Art

[0002] Trimetazidine hydrochloride is a white crystalline powder used for the treatment of cardiovascular and cerebrovascular diseases, the preventive treatment of angina attacks, and the auxiliary symptomatic treatment of vertigo and tinnitus.

[0003] Trimetazidine hydrochloride is prepared from 2,3,4-trimethoxybenzaldehyde and 1-formylpiperazine via a two-step reaction and a one-step salt formation. First, 1-formylpiperazine and 2,3,4-trimethoxybenzaldehyde undergo a Leuckart-Wallach reaction for reductive amination to prepare the intermediate formylated trimetazidine, which is then hydrolyzed with potassium hydroxide to prepare trimetazidine. Finally, the product reacts with concentrated hydrochloric acid to prepare trimetazidine hydrochloride.

[0004] In commercial batch production, the preparation of trimetazidine hydrochloride will produce a variety of impurities. The existing analytical methods can effectively control all known impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity I) included in the current EP10.0 trimetazidine hydrochloride standard, but cannot effectively control all objectively existing impurities. Therefore, further research is needed on the impurities in the production of trimetazidine hydrochloride and their control methods. Summary of the Invention

[0005] In view of the problems of the prior art, the object of the present invention is to provide a new quality control method for trimetazidine hydrochloride.

[0006] The present invention has found that the types of impurities detected by the existing trimetazidine hydrochloride detection method are still very limited, so a new detection method (related substance I) has been developed. In addition to being able to detect all the known impurities (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity I) recorded in the EP10.0 trimetazidine hydrochloride standard, it can also detect known impurities J, impurity K, impurity L, and impurity M. In addition, the present invention also found that the content of impurity I detected in trimetazidine hydrochloride in commercial production batches exceeded the proposed limit (the impurity actually present in the production process), but according to the detection of the laboratory research batch, impurity I met the requirements and could be controlled within the limit. Therefore, it was analyzed that a mixed substance of unknown impurities and impurity I existed in the production of trimetazidine hydrochloride. In response to this problem, the present invention further adjusted certain conditions of the above-mentioned detection method and established a corresponding detection method (related substance II), which can distinguish the unknown impurities from impurity I, provide an effective means for monitoring the unknown impurities, and provide an accurate detection method for further optimization of the process.

[0007] The information of each impurity is shown in Table 1 below.

[0008] Table 1 Organic impurity information of trimetazidine hydrochloride

[0009]

[0010]

[0011] In order to achieve this object, the technical solution of the present invention is as follows:

[0012] A HPLC detection method for trimetazidine hydrochloride comprises mobile phase A consisting of a 0.287% sodium heptanesulfonate solution and methanol in a volume ratio of (53-55):(45-47), preferably 55:45; mobile phase B is methanol; the pH value of mobile phase A is 4.0-4.2 or 2.0-3.0; and during detection, the solvent for the sample to be tested is a mixed solvent of methanol and water in a volume ratio of 1:(0.5-1.5), preferably 1:1.

[0013] The detection method of the present invention uses a specific mobile phase and detection solvent, and with different pH values of mobile phase A, it can achieve different focuses on the quality detection of trimetazidine hydrochloride. When the pH value of mobile phase A is 4.0-4.2 (related substance I method), multiple impurities can be detected and distinguished simultaneously. When the pH value of mobile phase A is 2.0-3.0 (related substance II method), impurity I and unknown impurities that cannot be detected by conventional technology can be detected and distinguished. The method of the present invention can control the quality of trimetazidine hydrochloride from multiple angles and effectively control the objectively existing multiple impurities.

[0014] Specifically, the method of the related substance I of the present invention not only detects the known impurities of trimetazidine hydrochloride that have been included in EP10.0, but also allows more impurities J, impurity K, impurity L, and impurity M to be detected. And under the premise that new unknown impurities were found in the production of trimetazidine hydrochloride, the method of the related substance I was further adjusted to form the method of the related substance II, so that the new impurity can be separated from the known impurity I, thereby better and more effectively controlling the quality of the trimetazidine hydrochloride product, laying the foundation for further optimization of the production process of the product.

[0015] When detecting newly discovered unknown impurities, the present invention found that the separation between the unknown impurities and the known impurity I was mainly affected by the pH value of the mobile phase. The pH range of the mobile phase is very critical. If the pH is lower than 2.0, the unknown impurities will not be found. If the pH is higher than 3.0, the separation degree between impurity I and the unknown impurities is poor.

[0016] In the present invention, during actual testing, the system suitability solution also uses the solvent of the sample to be tested during the above-mentioned testing.

[0017] In the detection method of the present invention, when the pH value of the mobile phase A is 4.0-4.2, the gradient elution conditions are as follows:

[0018]

[0019] When the pH value of the mobile phase A is 2.0-3.0, the gradient elution conditions are as follows:

[0020]

[0021] In the detection method of the present invention, the chromatographic column is Agilent ZORBAX SB-C18; the detector is an ultraviolet detector; the detection wavelength is 230nm to 240nm; the column temperature is 25°C to 40°C;

[0022] When the pH value of the mobile phase A is 4.0-4.2, the flow rate is 0.8 ml / min-1.2 ml / min; the injection volume is 10 μl;

[0023] When the pH value of the mobile phase A is 2.0-3.0, the flow rate is 1 ml / min-2 ml / min; the injection volume is 20 μl.

[0024] Preferably, when performing multiple impurity detection, in the detection method of the present invention, the pH value of the mobile phase A is 4.0; the detection wavelength is 231 nm; the flow rate is 1.0 ml / min; and the column temperature is 35°C.

[0025] Preferably, when distinguishing unknown impurities from impurity I, in the detection method of the present invention, the pH value of the mobile phase A is 3.0; the detection wavelength is 231 nm; the flow rate is 1.5 ml / min; the column temperature is 30° C.; and the gradient elution conditions are as follows:

[0026]

[0027] According to the different pH values of mobile phase A (different detection focuses), the present invention also explores other corresponding detection conditions, and the optimal combination of detection conditions can achieve better results in separating and detecting impurities.

[0028] The present invention also provides application of the above detection method in quality control of trimetazidine hydrochloride.

[0029] The detection method of the present invention can be used for trimetazidine hydrochloride raw materials, commercially available preparations, and crude products of the raw materials before refining.

[0030] The present invention also provides application of the above detection method in optimizing the production process of trimetazidine hydrochloride.

[0031] The present invention also provides application of the above detection method in distinguishing impurities generated in the production of trimetazidine hydrochloride.

[0032] The present invention also provides application of the above detection method in quantitatively detecting impurities generated in the production of trimetazidine hydrochloride.

[0033] The beneficial effects of the present invention are at least:

[0034] The method of the present invention can detect more impurities (impurity J, impurity K, impurity L, impurity M), and can improve the quality control standard of the product.

[0035] The present invention also found that when trimetazidine hydrochloride is produced, an unknown impurity is generated in the trimetazidine intermediate, and the impurity exceeds the proposed limit. Therefore, the present invention further adjusts the detection conditions of trimetazidine hydrochloride so that it can effectively analyze and distinguish impurity I from the newly discovered impurities, providing an effective process monitoring means for preparing qualified samples in the synthetic preparation process, and guiding the process optimization. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a system suitability chromatogram of the method for trimetazidine hydrochloride related substance I in Example 1. The order of peaks in the figure is: impurity D, impurity L, impurity J, impurity K, impurity C, impurity M, impurity A, impurity I, impurity H, impurity E, main peak, impurity F, impurity B.

[0037] Figure 2 The chromatogram is the detection result of the method for related substance I of the test solution in Example 1.

[0038] Figure 3 This is a chromatogram of the related substance II method detected by the crude product of trimetazidine hydrochloride in Example 3.

[0039] Figure 4 This is a qualitative chromatogram of impurity I detected by the method for trimetazidine hydrochloride related substance II in Example 3.

[0040] Figure 5 This is the overlapping spectrum obtained by the detection method of Comparative Example 2.

[0041] Figure 6 This is the overlapping spectrum obtained by the detection method of Comparative Example 3.

[0042] Figure 7 This is the overlapping spectrum obtained by the detection method of Comparative Example 4.

[0043] Figure 8 This is the overlapping spectrum obtained by the detection method of Comparative Example 5.

[0044] Figure 9 This is the overlapping spectrum obtained in Comparative Example 6.

[0045] Figure 10 This is the system suitability solution spectrum obtained by reproducing the experiment of Comparative Example 6.

[0046] Figure 11 This is the system suitability solution spectrum obtained by the first method in Comparative Example 7.

[0047] Figure 12 This is the system suitability solution spectrum obtained by the second method in Comparative Example 7.

[0048] Figure 13 This is the system suitability solution spectrum obtained by the third method in Comparative Example 7.

[0049] Figure 14 This is the system suitability solution spectrum obtained by the fourth method in Comparative Example 7.

[0050] Figure 15 This is the detection spectrum of Comparative Example 8. DETAILED DESCRIPTION

[0051] The preferred embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0052] Example 1

[0053] This embodiment provides a method for detecting trimetazidine hydrochloride (related substance I detection method), which is as follows:

[0054] Chromatographic conditions include:

[0055] Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm);

[0056] Mobile phase: Mobile phase A was 0.287% sodium heptanesulfonate solution-methanol (55:45), pH adjusted to 4.0 with 10% phosphoric acid; mobile phase B was methanol; gradient elution conditions are shown in Table 2.

[0057] Table 2

[0058]

[0059]

[0060] Detector: UV detector; detection wavelength: 231 nm; flow rate: 1.0 ml / min; column temperature: 35°C; injection volume: 10 μl.

[0061] The preparation of the solution includes the following steps:

[0062] Stock solution of each impurity reference substance: Take an appropriate amount of each known impurity (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity I, impurity J, impurity K, impurity L and impurity M) reference substance, dissolve and dilute with methanol to make a solution containing approximately 1 mg / ml of each impurity, as the stock solution of each impurity reference substance.

[0063] Impurity mixed stock solution: Take 1 ml of each known impurity reference stock solution, place it in the same 25 ml volumetric flask, and dilute it with methanol-water (50:50) to make a solution containing approximately 40 μg / ml of each impurity, as the impurity mixed stock solution.

[0064] System suitability solution: Take about 40 mg of trimetazidine hydrochloride reference substance and place it in a 10 ml volumetric flask. Add 1 ml of impurity mixed stock solution, add methanol-water (50:50) to dissolve and dilute to make a solution containing about 4 mg / ml of trimetazidine hydrochloride and 4 μg / ml of each impurity. Shake well and use it as the system suitability solution.

[0065] Test solution: Take an appropriate amount of this product (trimetazidine hydrochloride raw material), accurately weigh it, add methanol-water (50:50) to dissolve and dilute it to make a solution containing about 4 mg / ml of trimetazidine hydrochloride, shake well, and use it as the test solution.

[0066] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with methanol-water (50:50), shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with methanol-water (50:50), shake well, and use it as the control solution.

[0067] Accurately measure 10 μl of each of the system suitability solution, test solution, and control solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 1 , the chromatogram of the test solution is shown in Figure 2 .

[0068] Example 2

[0069] This example verifies the detection method for related substance I proposed in Example 1. The verification content includes: solution stability, injection precision, repeatability, and intermediate precision.

[0070] 1. Solution stability

[0071] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in methanol-water (50:50), and dilute it to make a solution containing about 4 mg / ml of trimetazidine hydrochloride, which is used as the test solution.

[0072] Accurately measure 10 μl of the above solution and place it at room temperature at 0, 2.5, 4.5, 7, 11.5, and 23 hours. Inject it into a liquid chromatograph and record the chromatogram. Calculate the content of each impurity by peak area normalization. The results are shown in Table 3.

[0073] Table 3 Solution stability test results

[0074]

[0075]

[0076] The results showed that when the test solution was injected at 0, 2.5, 4.5, 7, 11.5 and 23 hours after being placed at room temperature, there was no significant change in the number of impurities and the content of each impurity, indicating that the solution had good stability.

[0077] 2. Injection precision

[0078] Stock solutions of each impurity: Take approximately 10 mg of each impurity (impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M), accurately weigh, place in 10 ml volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use as stock solutions of the above impurities.

[0079] Impurity D stock solution: Take about 20 mg of impurity D, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute it to the scale, shake it well, and use it as the stock solution of impurity D.

[0080] Impurity localization solutions: Accurately measure an appropriate amount of each impurity stock solution, add methanol-water (50:50) to dilute to make a solution containing approximately 40 μg / ml of each impurity, shake well, and use as the localization solution for each impurity.

[0081] Trimetazidine hydrochloride reference substance stock solution: Take an appropriate amount of trimetazidine hydrochloride reference substance, accurately weigh it, and dilute it with methanol-water (50:50) to make a solution containing approximately 100 μg / ml of trimetazidine hydrochloride, which is used as the trimetazidine hydrochloride reference substance stock solution.

[0082] Linearity stock solution: Accurately measure an appropriate amount of each impurity and trimetazidine hydrochloride reference stock solution, dissolve and dilute with methanol-water (50:50) to prepare a solution containing approximately 40 μg / ml of each impurity and 40 μg / ml of trimetazidine hydrochloride, which is used as the linearity stock solution.

[0083] Reference substance mixed solution: Accurately measure 1 ml of the linear stock solution, place it in a 10 ml volumetric flask, dilute it to the scale with methanol-water (50:50), shake well, and use it as the reference substance mixed solution.

[0084] Take 10 μl of the reference solution and inject it into the liquid chromatograph. Record the chromatogram. Repeat the injection 6 times. The results are shown in Tables 4 and 5.

[0085] Table 4 Related substance I inspection - injection precision test results -1

[0086]

[0087]

[0088] Table 5 Related substance I inspection - injection precision test results -2

[0089]

[0090] The results showed that the RSD values of the retention time of each peak were less than 2% and the RSD values of the peak area were less than 5% when the reference solution was injected six times continuously, indicating good injection precision.

[0091] 3. Repeatability

[0092] Reference solution: Accurately weigh an appropriate amount of each impurity reference solution and dissolve and dilute with methanol-water (50:50) to prepare a solution containing approximately 4 μg / ml of each impurity (except impurity I, which has a concentration of 8 μg / ml). Prepare two parallel aliquots.

[0093] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in methanol-water (50:50), and dilute it to a solution containing approximately 4 mg / ml of trimetazidine hydrochloride. This will be the test solution. Prepare 6 replicates.

[0094] Control solution: Accurately measure 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with methanol-water (50:50), and shake well. Accurately measure 1 ml into a 10 ml volumetric flask, dilute to the mark with methanol-water (50:50), and shake well. This is used as the control solution. Prepare 6 replicates.

[0095] According to the related substance I inspection method of Example 1, 10 μl of each of the reference solution, test solution and control solution was accurately measured and injected into the liquid chromatograph. The chromatogram was recorded and the contents of single impurities and total impurities were calculated by the self-control method with correction factors and the external standard method. The results are shown in Table 6.

[0096] Table 6 Related substance I inspection - repeatability test results

[0097]

[0098]

[0099] The results showed that the number of impurity peaks was basically the same when 6 portions of the test solution were measured in parallel, and there was no significant difference in the content of each impurity; the impurity contents calculated by the external standard method and the self-control method with correction factors were basically consistent, with good repeatability.

[0100] 4. Intermediate precision

[0101] To examine the impact of random variation on precision, a second experimenter independently set up the system and re-prepared six test solutions and six control solutions as described under repeatability. The tests were performed using a different instrument and on different days. The precision and repeatability were required to ensure that the number and content of impurities were essentially consistent across 12 measurements. The results are shown in Tables 7 and 8 (the data obtained by the first experimenter represent the repeatability results).

[0102] Table 7 Related substance I inspection - intermediate precision test results -1

[0103]

[0104]

[0105] Table 8 Related substance I inspection - intermediate precision test results - 2

[0106]

[0107] The results showed that there was no significant difference in the number of impurity peaks and the content of each impurity in the 12 test solutions, which were measured by two people each in parallel, and the intermediate precision was good.

[0108] Example 3

[0109] This embodiment provides a method for detecting trimetazidine hydrochloride (related substance II detection method), which is as follows:

[0110] Chromatographic conditions include:

[0111] Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm);

[0112] Mobile phase: Mobile phase A was 0.287% sodium heptanesulfonate solution-methanol (55:45), pH adjusted to 3.0 with 10% phosphoric acid; mobile phase B was methanol; gradient elution conditions are shown in Table 9.

[0113] Table 9

[0114] Time, minutes Mobile phase A, % Mobile phase B, % 0 100 0 45 100 0 60 75 25 65 75 25 70 100 0 75 100 0

[0115] Detector: UV detector; detection wavelength: 231 nm; flow rate: 1.5 ml / min; column temperature: 30°C; injection volume: 20 μl.

[0116] The preparation of the solution includes the following steps:

[0117] Impurity I reference substance stock solution: Take an appropriate amount of Impurity I reference substance, dissolve and dilute with methanol to make a solution containing approximately 10 μg / ml of Impurity I, which is used as the Impurity I reference substance stock solution.

[0118] Test solution: Take an appropriate amount of this product (crude trimetazidine hydrochloride), accurately weigh it, add methanol-water (50:50) to dissolve and dilute it to make a solution containing about 4 mg / ml of trimetazidine hydrochloride, shake well, and use it as the test solution.

[0119] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute it to the scale with methanol-water (50:50), shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the scale with methanol-water (50:50), shake well, and use it as the control solution.

[0120] Accurately measure 10 μl of impurity I reference stock solution, test solution and reference solution, respectively, and inject them into the liquid chromatograph to record the chromatogram. Figure 3 , the qualitative chromatogram of impurity I is shown in Figure 4 .

[0121] The unknown impurity did not overlap with the known impurity I. The unknown impurity was further analyzed by LC-MS, and the analytical molecular weight was 252.5, which was different from the molecular weight of other known impurities. The presence of the unknown impurity was further confirmed, which supported the fact that the content of impurity I in the commercial production batch of trimetazidine hydrochloride discovered by the present invention exceeded the proposed limit. However, according to the detection of the laboratory research batch, impurity I met the requirements. Therefore, it was analyzed that there was an inference that a mixture of unknown impurities and impurity I existed in the production of trimetazidine hydrochloride.

[0122] Example 4

[0123] This example verifies the detection method for related substance II proposed in Example 3. The verification content includes: solution stability, repeatability, and intermediate precision.

[0124] 1. Solution stability

[0125] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in methanol-water (50:50), and dilute it to make a solution containing about 4 mg / ml of trimetazidine hydrochloride, which is used as the test solution.

[0126] Accurately measure 10 μl of the above solution and place it at room temperature for 0, 2, 4, 6, 8, and 24 hours. Inject it into a liquid chromatograph and record the chromatogram. Calculate the content of impurity I and unknown impurities using the principal component self-reference method. The results are shown in Table 10.

[0127] Table 10 Solution stability test results

[0128]

[0129] The results showed that when the test solution was injected at 0, 2, 4, 6, 8 and 24 hours after being placed at room temperature, there was no significant change in the content of impurity I and unknown impurities, and the solution had good stability.

[0130] 2. Repeatability

[0131] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve it in methanol-water (50:50), and dilute it to a solution containing approximately 4 mg / ml of trimetazidine hydrochloride. This will be the test solution. Prepare 6 replicates.

[0132] Control solution: Accurately measure 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with methanol-water (50:50), and shake well. Accurately measure 1 ml into a 10 ml volumetric flask, dilute to the mark with methanol-water (50:50), and shake well. This is used as the control solution. Prepare 6 replicates.

[0133] According to the related substance II inspection method of Example 3, 10 μl of the test solution and the control solution were accurately measured and injected into the liquid chromatograph. The chromatogram was recorded and the content of the single impurity was calculated by the self-control method. The results are shown in Table 11.

[0134] Table 11 Related Substance II Inspection - Repeatability Test Results

[0135]

[0136]

[0137] The results showed that the test solution was measured in parallel for 6 times, and the detection of impurity I was basically consistent, and no unknown impurities were detected, indicating good repeatability.

[0138] 3. Intermediate precision

[0139] To examine the impact of random variation on precision, a second experimenter independently set up the system and re-prepared six test solutions and six control solutions as described under repeatability. The tests were performed using a different instrument and on different days. The precision and repeatability tests were required to ensure that the number and content of impurities were essentially consistent across 12 measurements. The results are shown in Table 12 (the data obtained by the first experimenter represent the repeatability results).

[0140] Table 12 Related Substance II Inspection - Intermediate Precision Test Results

[0141]

[0142] The results showed that there was no significant difference in the content of impurity I and unknown impurities in the test solution, which was measured by two people in parallel, 6 times each, and a total of 12 test solutions, and the intermediate precision was good.

[0143] Comparative Example 1

[0144] This comparative example provides a method for detecting trimetazidine hydrochloride, and the chromatographic conditions are set with reference to the EP10.0 standard.

[0145] Chromatographic conditions:

[0146] Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm).

[0147] Mobile phase A: 0.287% sodium heptanesulfonate solution-methanol (643:357), adjusted to pH 3.0 with 10% (v / v) phosphoric acid.

[0148] Mobile phase B: methanol; flow rate: 1.0 ml / min; column temperature: 30°C; detection wavelength: 240 nm; injection volume: 10 μl.

[0149] The gradient elution program is shown in Table 13.

[0150] Table 13

[0151] Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 50 75 25 52 95 5 60 95 5

[0152] Solution preparation:

[0153] Stock solutions of each impurity: Take approximately 10 mg of each impurity (impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity I, impurity J), accurately weigh, place in 10 ml volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use as stock solutions of the above impurities.

[0154] Impurity H stock solution: Take about 20 mg of impurity H, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute it to the scale, shake it well, and use it as the stock solution of impurity H.

[0155] Impurity localization solution: Take 1 ml of each impurity localization solution, dilute with water to make a solution containing approximately 40 μg / ml of each impurity, shake well, and use as the impurity localization solution.

[0156] Test solution: Take about 40 mg of trimetazidine hydrochloride raw material, accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the scale with water, shake well, and use it as the test solution.

[0157] Accurately measure 10 μl of each of the test solution and each impurity location solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 14.

[0158] Table 14 Comparative Example 1 - System Suitability Test Results

[0159]

[0160] The results showed that the main peak appeared too late, and impurities F and B did not appear, indicating that this method was not good.

[0161] Comparative Example 2

[0162] This comparative example provides a method for detecting trimetazidine hydrochloride. The method is the same as that of Comparative Example 1, except that mobile phase A is changed to 0.287% sodium heptanesulfonate solution-methanol (357:643), and the pH is adjusted to 3.0 with 10% (v / v) phosphoric acid.

[0163] Accurately measure 10 μl of the test solution and each impurity location solution under Comparative Example 1, inject them into the liquid chromatograph, and record the chromatogram. Overlapping spectra are shown in Figure 5 The elution curves of the chromatographic peaks in the figure are from bottom to top: 1. test solution, 2. impurity A positioning, 3. impurity B positioning, 4. impurity C positioning, 5. impurity D positioning, 6. impurity E positioning, 7. impurity F positioning, 8. impurity H positioning, 9. impurity I positioning, and 10. impurity J positioning.

[0164] The results showed that when the ratio of mobile phase A was 357:643, trimetazidine hydrochloride and its impurities eluted earlier, and baseline separation could not be achieved between the impurities or between the impurities and the main peak.

[0165] Comparative Example 3

[0166] This comparative example provides a method for detecting trimetazidine. The method is identical to that of Comparative Example 1, except that mobile phase A is changed to a 0.287% sodium heptanesulfonate solution in methanol (50:50), and the pH is adjusted to 3.0 with 10% (v / v) phosphoric acid. The wavelength is changed to 231 nm.

[0167] In addition, the following solution was prepared based on the solution prepared in Comparative Example 1:

[0168] Impurity L stock solution: Take about 10 mg of impurity L, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute it to the scale, shake well, and use it as the stock solution of impurity L.

[0169] Impurity L localization solution: Take 1 ml of the Impurity L stock solution, dilute it with water to make a solution containing approximately 40 μg / ml of Impurity L, shake well, and use it as the Impurity L localization solution.

[0170] Mixed solution: Take about 40 mg of trimetazidine hydrochloride raw material, accurately weigh, place in a 10 ml volumetric flask, add 0.4 ml of each impurity location solution (see Comparative Example 1, except impurities J and L), dilute with water to the scale, shake well, and prepare the mixed solution.

[0171] Accurately measure 10 μl of each of the mixed solution, the impurity L localization solution, and the impurity localization solutions of Comparative Example 1, inject them into a liquid chromatograph, and record the chromatogram. The statistical results are shown in Table 15.

[0172] Overlapping maps are shown in Figure 6 The elution curves of the chromatographic peaks in the figure are from bottom to top: 1. mixed solution, 2. impurity B positioning, 3. impurity A positioning, 4. impurity C positioning, 5. impurity D positioning, 6. impurity E positioning, 7. impurity F positioning, 8. impurity H positioning, 9. impurity I positioning, 10. impurity J positioning, and 11. impurity L positioning.

[0173] Table 15 Comparative Example 3 - System Suitability Test Results

[0174]

[0175] The results showed that when the ratio of mobile phase A was 50:50, trimetazidine hydrochloride peaked at about 15 minutes, and the retention time was appropriate, but impurities A, E and I could not be completely separated. There were only two chromatographic peaks of impurities A, E and I in the mixed solution.

[0176] Comparative Example 4

[0177] This comparative example provides a method for detecting trimetazidine. The method is the same as that of Comparative Example 3, except that mobile phase A is changed to 0.287% sodium heptanesulfonate solution-methanol (55:45), the pH is adjusted to 3.0 with 10% (v / v) phosphoric acid, and the chromatographic column is changed to an Agilent Eclipse XDB-C18 (4.6 mm × 250 mm, 5 μm).

[0178] Each impurity localization solution: see Comparative Example 1 and Comparative Example 3.

[0179] Mixed solution (same as Comparative Example 3): Take about 40 mg of trimetazidine hydrochloride raw material, accurately weigh, place in a 10 ml volumetric flask, add 0.4 ml of each impurity location solution, dilute with water to the scale, shake well, and prepare the mixed solution.

[0180] Accurately measure 10 μl of each of the mixed solution, impurity L localization solution, and each impurity localization solution under Comparative Example 1, inject them into the liquid chromatograph, and record the chromatogram. Overlapping spectra are shown in Figure 7 The elution curves of the chromatographic peaks in the figure are from bottom to top: 1. mixed solution, 2. impurity B positioning, 3. impurity A positioning, 4. impurity C positioning, 5. impurity D positioning, 6. impurity E positioning, 7. impurity F positioning, 8. impurity H positioning, 9. impurity I positioning, 10. impurity J positioning, and 11. impurity L positioning.

[0181] The results showed that when the ratio of mobile phase A was 55:45, trimetazidine hydrochloride peaked at about 25 minutes, and the retention time was appropriate, but impurities A, E and I could not be completely separated. In the mixed solution, impurities A, E and I only had one chromatographic peak.

[0182] Comparative Example 5

[0183] This comparative example provides a method for detecting trimetazidine hydrochloride. The method is the same as that of Comparative Example 3, except that mobile phase A is changed to 0.287% sodium heptanesulfonate solution-methanol (55:45), and the pH is adjusted to 3.0 with 10% (v / v) phosphoric acid. The chromatographic column is changed to an Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm), and the column temperature is 35°C.

[0184] In addition, the following solution was prepared based on the solution prepared in Comparative Example 3:

[0185] Impurity M localization solution: Take an appropriate amount of impurity M reference substance, dissolve it in methanol and dilute it to make a solution containing approximately 100 μg / ml of impurity M, which is used as the impurity M localization solution.

[0186] Each impurity localization solution: see Comparative Example 1 and Comparative Example 3.

[0187] Mixed solution: Take an appropriate amount of trimetazidine hydrochloride reference substance, add an appropriate amount of each impurity location solution, dissolve and dilute with water to prepare a solution containing approximately 4 mg / ml of trimetazidine hydrochloride and approximately 4 μg / ml of each impurity, as the mixed solution.

[0188] Impurity reference substance mixed solution (except impurity M): Take 0.4 ml of each impurity location solution (except impurity M), place it in a 10 ml volumetric flask, dilute to the scale with water, shake well, and use it as the impurity reference substance mixed solution.

[0189] Accurately measure 10 μl of each of the mixed solution, impurity reference solution, impurity M localization solution, and each impurity localization solution, inject them into the liquid chromatograph, and record the chromatogram. Overlapping spectra are shown in Figure 8The elution curves of the chromatographic peaks in the figure are from bottom to top: 1. impurity reference mixed solution, 2. mixed solution, 3. impurity A positioning, 4. impurity B positioning, 5. impurity C positioning, 6. impurity D positioning, 7. impurity E positioning, 8. impurity F positioning, 9. impurity H positioning, 10. impurity I positioning, 11. impurity J positioning, 12 impurity L positioning, and 13 impurity M positioning.

[0190] The results showed that the separation of impurities A, E, and I was improved, but impurities M and I overlapped and could not be completely separated. Therefore, the chromatographic column was temporarily selected as Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm) and the method was further explored.

[0191] Comparative Example 6

[0192] This comparative example provides four methods for detecting trimetazidine hydrochloride. Method 1 Chromatographic conditions:

[0193] Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm).

[0194] Mobile phase A: 0.287% sodium heptanesulfonate solution-methanol (55:45), adjusted to pH 3.0 with 10% (v / v) phosphoric acid.

[0195] Mobile phase B: methanol; flow rate: 1.0 ml / min; column temperature: 35°C; detection wavelength: 231 nm; injection volume: 10 μl.

[0196] The gradient elution program is shown in Table 16.

[0197] Table 16

[0198]

[0199]

[0200] The chromatographic conditions of Method 2, Method 3 and Method 4 are the same as those of Method 1. The only difference is that, based on Method 1, other conditions remain unchanged and the pH value of the mobile phase is adjusted to 2.5 (Method 2), 3.5 (Method 3) and 4.0 (Method 4), respectively.

[0201] Each impurity localization solution: see Comparative Example 1 and Comparative Example 3.

[0202] Impurity M localization solution: see Comparative Example 5.

[0203] Mixed solution: Take an appropriate amount of trimetazidine hydrochloride reference substance, add an appropriate amount of each impurity location solution, dissolve and dilute with water to prepare a solution containing approximately 4 mg / ml of trimetazidine hydrochloride and approximately 4 μg / ml of each impurity, as the mixed solution.

[0204] Impurity reference substance mixed solution: Take 0.4 ml of each impurity location solution, place it in a 10 ml volumetric flask, dilute to the scale with water, shake well, and use it as the impurity reference substance mixed solution.

[0205] Accurately measure 10 μl of the mixed solution, inject it into the liquid chromatograph, and record the chromatogram. The overlapping spectrum is shown below. Figure 9 The chromatographic peak elution curves in the figure are 1. Method 4, 2. Method 3, 3. Method 2, and 4. Method 1 from bottom to top.

[0206] The results showed that pH had a significant effect, with good separation of the impurities at pH 4.0. Method 4 was further investigated, with each impurity location solution used to locate the impurities. The results are shown in Tables 17 and 18.

[0207] Table 17 Method 4 - System suitability test results -1

[0208]

[0209] Table 18 Method 4-System suitability test results-2

[0210]

[0211]

[0212] The results showed that under this condition, the separation between each peak met the requirements and could be used to detect impurities A, B, C, D, E, F, H, I, J, L, and M in trimetazidine hydrochloride. However, the method reproducibility was not ideal after adding impurity K.

[0213] The specific process of method reproduction is as follows:

[0214] Chromatographic conditions:

[0215] Chromatographic column: Agilent ZORBAX SB-C18, 250 mm × 4.6 mm, 5 μm.

[0216] Mobile phase A: 0.287% sodium heptanesulfonate solution-methanol (55:45), adjusted to pH 4.0 with 10% (v / v) phosphoric acid; mobile phase B: methanol.

[0217] Flow rate: 1.0 ml / min; column temperature: 35°C; detection wavelength: 231 nm; injection volume: 10 μl.

[0218] The gradient elution program is shown in Table 19.

[0219] Table 19

[0220]

[0221] Solution preparation:

[0222] Stock solutions of each impurity: Take approximately 10 mg of each impurity (impurity A, impurity B, impurity C, impurity E, impurity F, impurity H, impurity I, impurity J, impurity K, impurity L, impurity M), accurately weigh, place in 10 ml volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use as stock solutions of the above impurities.

[0223] Impurity D stock solution: Take about 20 mg of impurity D, accurately weigh it, place it in a 10 ml volumetric flask, add methanol to dissolve it and dilute it to the scale, shake it well, and use it as the stock solution of impurity D.

[0224] Preparation of impurity localization solution: Accurately measure 0.1 ml of each impurity stock solution, place it in a 10 ml volumetric flask, and dilute to the scale with methanol.

[0225] System suitability solution: Take approximately 40 mg of trimetazidine hydrochloride raw material, accurately weigh it, and place it in a 10 ml volumetric flask. Add 1 ml of each impurity location solution, and dilute it with water to make a solution containing approximately 4 mg / ml of trimetazidine hydrochloride and approximately 40 μg / ml of each impurity. Shake well and use it as the system suitability solution.

[0226] Accurately measure 10 μl of the system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The system suitability solution chromatogram is shown in Figure 10 , the statistical results are shown in Table 20.

[0227] Table 20 Method reproducibility-system suitability test results

[0228]

[0229]

[0230] The results showed that the separation between impurity D and impurity L in the system suitability solution was about 1.374, and the separation between impurity J and impurity K was about 1.023, indicating that the method was not ideal.

[0231] Comparative Example 7

[0232] This comparative example provides four methods for detecting trimetazidine hydrochloride.

[0233] First method:

[0234] Chromatographic conditions: Based on the chromatographic conditions reproduced by the method in Comparative Example 6, other conditions remained unchanged and the gradient elution program was changed as shown in Table 21 below.

[0235] Table 21

[0236]

[0237] Accurately measure 10 μl of the system suitability solution in Comparative Example 6, inject it into the liquid chromatograph, and record the chromatogram. Figure 11 , the statistical results are shown in Table 22.

[0238] Table 22 Method 1 - System suitability test results

[0239]

[0240]

[0241] The results showed that the separation between impurity D and impurity L in the system suitability solution was about 1.652, the separation between impurity J and impurity K was about 1.367, and the separation between impurity I and impurity H was 0.945, which was poor.

[0242] Second method:

[0243] Chromatographic conditions:

[0244] Based on the chromatographic conditions of the first method, other conditions remained unchanged, and mobile phase A was changed to 0.287% sodium heptanesulfonate solution-methanol (60:40), and the pH was adjusted to 4.0 with 10% (v / v) phosphoric acid.

[0245] Accurately measure 10 μl of the system suitability solution of Comparative Example 6, inject it into the liquid chromatograph, and record the chromatogram. Figure 12 , the statistical results are shown in Table 23.

[0246] Table 23 Second method - system suitability test results

[0247]

[0248] The results showed that compared with the first method, the separation between impurity D and impurity L in the system suitability solution was about 1.801, the separation between impurity I and impurity H was 1.929, and the separation between impurity J, impurity K and impurity C was less than 1.5. When the proportion of mobile phase A was changed by 5%, the retention time of each peak changed significantly. Impurity B did not appear within 60 minutes, and the detection effect was poor.

[0249] The third method:

[0250] Chromatographic conditions:

[0251] Based on the chromatographic conditions of the first method, other conditions remain unchanged and the gradient elution program is changed as shown in Table 24.

[0252] Table 24

[0253]

[0254] Accurately measure 10 μl of the system suitability solution of Comparative Example 6, inject it into the liquid chromatograph, and record the chromatogram. Figure 13 , the statistical results are shown in Table 25.

[0255] Table 25 The third method - system suitability test results

[0256]

[0257] The results showed that the separation degree between impurity I and impurity H in the system applicability solution of the third method was 1.743; the separation degree between impurity D and impurity L was about 1.623, and the separation degree between impurity J and impurity K was 1.319. The separation degree of each impurity was still not ideal.

[0258] Fourth method:

[0259] Chromatographic conditions:

[0260] Based on the chromatographic conditions of the first method, other conditions remain unchanged and the gradient elution program is changed as shown in Table 26.

[0261] Table 26

[0262]

[0263] Accurately measure 10 μl of the system suitability solution of Comparative Example 6, inject it into the liquid chromatograph, and record the chromatogram. Figure 14 , the statistical results are shown in Table 27.

[0264] Table 27 Fourth method - system suitability test results

[0265]

[0266] The results showed that the separation between impurity I and impurity H in the system applicability solution of the fourth method was 1.675, and the separation between impurity D and impurity L was about 1.793; but the separation between impurity J, impurity K and impurity C were all less than 1.5, indicating poor separation.

[0267] Example 5

[0268] In this example, different batches of samples were tested using the related substance I detection method described in Example 1 and the related substance II detection method described in Example 3.

[0269] When using the detection method for related substance II for detection, since the unknown impurity identified in Example 3 could not be prepared and was not commercially available, impurity I was used to prepare a qualitative solution. The RRT of the unknown impurity relative to impurity I was further determined to be 0.86 based on the relative retention time in the chromatogram in Example 3. Based on this, the unknown impurity was qualitatively determined and quantitatively determined using the main component self-reference peak. The preparation of the solution included the following steps:

[0270] Preparation of qualitative solution: Take an appropriate amount of trimetazidine hydrochloride impurity I reference substance, accurately weigh it, and dissolve it in a suitable solvent to prepare a solution of appropriate concentration.

[0271] Preparation of test solution: Take an appropriate amount of test sample and dissolve it in a suitable solvent to make a solution of appropriate concentration.

[0272] Self-reference solution: Take an appropriate amount of the test solution and dilute it 1000 times with a suitable solvent.

[0273] The specific solution involved in this embodiment is:

[0274] Qualitative solution: Take an appropriate amount of impurity I reference substance, dissolve it in water-methanol (1:1) and dilute it to make a solution containing 0.1 mg per 1 ml, which will serve as the impurity positioning solution.

[0275] Test solution: Take an appropriate amount of the test sample and dilute it with methanol-water (50:50) to a solution containing approximately 5 mg of trimetazidine hydrochloride per 1 ml.

[0276] Control solution: Accurately measure an appropriate amount of the test solution and dilute it with methanol-water (50:50) to a solution containing approximately 5 μg of trimetazidine hydrochloride per 1 ml.

[0277] During the test, the above-mentioned qualitative solution, control solution and test solution were accurately measured and injected into the liquid chromatograph. The chromatographic conditions of Example 1 and Example 3 were respectively measured, the chromatograms were recorded, and the contents of impurity I and unknown impurities in the test sample were calculated.

[0278] The results are shown in Tables 28 and 29, where commercially available 01-03 comes from Hebei Mojin Biotechnology Co., Ltd., Henan Tianfu Chemical Co., Ltd., and Guizhou Longshengda Biotechnology Co., Ltd., respectively; research batches and commercial batches (20 kg) are prepared by two-step reaction and one-step salt formation of 2,3,4-trimethoxybenzaldehyde (TH-A) and 1-formylpiperazine (TH-1). Specifically, TH-1 and 2,3,4-trimethoxybenzaldehyde (TH-A) are first subjected to Leuckart-Wallach Reaction (Liukart-Wallach Reaction) reductive amination to prepare intermediate TH-2, intermediate TH-2 is hydrolyzed with potassium hydroxide to prepare intermediate TH-3, and finally intermediate TH-3 is reacted with concentrated hydrochloric acid to prepare the raw material trimetazidine hydrochloride method.

[0279] Table 28 Inspection results of related substances I method for samples from different sources

[0280]

[0281] Table 29 Inspection results of related substances method II for samples from different sources

[0282]

[0283] The results showed that different batches of the test sample contained varying amounts of impurity I and unknown impurities. Commercially available trimetazidine hydrochloride raw materials from different sources contained varying amounts of impurity I and unknown impurities. Using the detection method for related substance I in Example 1 carries the risk of exceeding the combined control limit. However, using the detection method for related substance II in Example 3 met the strictest requirements of current standards. It is generally recommended to continue optimizing the process with the goal of removing unknown impurities.

[0284] This example also shows that the impurity I and unknown impurities in the homemade product show an increasing trend as the batch size is increased, suggesting that the batch size-up effect directly affects the content of impurity I and unknown impurities in the product.

[0285] To improve yield and assess quality, this example investigated the removal of unknown impurities and impurity I using the method described for related substance II in a commercial batch (20 kg). The results are shown in Tables 30 and 31. As can be seen, while ensuring quality, two purification steps (using 95% ethanol as the refining solvent, dissolving the test product, crystallizing for 3 hours, crystallizing at 10-15°C, and drying with forced air at 45-50°C for 3-5 hours) achieved a reasonable yield, meeting production requirements.

[0286] Table 30

[0287]

[0288] Table 31

[0289] batch Yield of purification once Refining 2nd time yield Commercialization Batch 04 20kg 81％ 59％ Commercialization Batch 05 20kg 77％ 56％ Commercialization Batch 06 20kg 75％ 53％

[0290] Comparative Example 8

[0291] This comparative example provides an HPLC detection method for trimetazidine hydrochloride, which is the same as Example 1, except that the gradient elution conditions refer to claim 2 of Chinese patent CN109307725A.

[0292] Test results are shown in Figure 15 , from which we can see that the known impurities A, E, I, and M cannot be effectively separated.

[0293] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A HPLC detection method for trimetazidine hydrochloride, characterized in that: The chromatographic column was an Agilent ZORBAX SB-C18; the mobile phase A consisted of a 0.287% sodium heptanesulfonate solution and methanol in a volume ratio of (53-55):(45-47); the mobile phase B was methanol; the pH value of the mobile phase A was 4.0-4.2; the solvent for the sample to be tested was a mixed solvent of methanol and water in a volume ratio of 1:(0.5-1.5); the detector was an ultraviolet detector; the detection wavelength was 230 nm-240 nm; and the column temperature was 25°C-40°C. Gradient elution conditions were as follows: ； The impurities detected by the HPLC detection method of trimetazidine hydrochloride include impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity H, impurity I, impurity J, impurity K, impurity L and impurity M; The chemical structural formula of the impurity A is: , the chemical structural formula of the impurity B is: , the chemical structural formula of the impurity C is: , the chemical structural formula of the impurity D is: , the chemical structural formula of the impurity E is: , the chemical structural formula of the impurity F is: , the chemical structural formula of the impurity H is: , the chemical structural formula of the impurity I is: , the chemical structural formula of the impurity J is: , the chemical structural formula of the impurity K is: , the chemical structural formula of the impurity L is: , the chemical structural formula of the impurity M is: .

2. The detection method according to claim 1, wherein Flow rate: 0.8ml / min~1.2ml / min; injection volume: 10μl.

3. The detection method according to claim 2, characterized in that The pH value of the mobile phase A was 4.0; the detection wavelength was 231 nm; the flow rate was 1.0 ml / min; and the column temperature was 35° C.

4. The detection method according to any one of claims 1 to 3, characterized in that When the content of impurity I exceeds the proposed limit, further testing is performed. The specific method includes: the chromatographic column is Agilent ZORBAX SB-C18; the mobile phase A is composed of 0.287% sodium heptane sulfonate solution and methanol in a volume ratio of (53-55): (45-47); the mobile phase B is methanol; the pH value of the mobile phase A is 2.0-3.0; the solvent for the test sample is a mixed solvent of methanol and water in a volume ratio of 1: (0.5-1.5); the detector is a UV detector; the detection wavelength is 230nm-240nm; the column temperature is 25℃-40℃; the gradient elution conditions are as follows: 。 5. The detection method according to claim 4, characterized in that Flow rate: 1ml / min~2ml / min; injection volume: 20μl.

6. The detection method according to claim 5, characterized in that The pH value of the mobile phase A was 3.0; the detection wavelength was 231 nm; the flow rate was 1.5 ml / min; the column temperature was 30° C.; and the gradient elution conditions were as follows: 。 7. Use of the detection method according to any one of claims 1 to 6 in the quality control of trimetazidine hydrochloride.

8. Use of the detection method according to any one of claims 1 to 6 in optimizing the production process of trimetazidine hydrochloride.

9. Use of the detection method according to any one of claims 1 to 6 in distinguishing impurities generated in the production of trimetazidine hydrochloride.

10. Use of the detection method according to any one of claims 1 to 6 in the quantitative detection of impurities generated in the production of trimetazidine hydrochloride.

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