CN118067860A

CN118067860A - Detection method of related substances in epalrestat tablet

Info

Publication number: CN118067860A
Application number: CN202311549093.9A
Authority: CN
Inventors: 康宝玲; 李好好; 郝一江; 崔渊; 陈亚坤; 胡徐伟; 谷云鹏
Original assignee: BEIJING SL PHARMACEUTICAL CO LTD; Beijing Sl Lisheng Medical Technology Co ltd
Current assignee: BEIJING SL PHARMACEUTICAL CO LTD; Beijing Sl Lisheng Medical Technology Co ltd
Priority date: 2023-11-20
Filing date: 2023-11-20
Publication date: 2024-05-24

Abstract

The invention provides a detection method for determining related substances in epalrestat tablets by an HPLC method, which relates to the field of medicine analysis and comprises the following steps: preparing a system applicability solution by taking proper amounts of epalrestat impurity C, F, G, H and each reference substance of epalrestat, detecting the separation condition and the theoretical plate number by using the system applicability solution, wherein the separation degree of an impurity C peak and the epalrestat peak is not less than 1.5, and the theoretical plate number is not less than 5000 according to the epalrestat peak; taking a proper amount of epalrestat tablet fine powder to prepare a sample solution; taking a proper amount of sample solution, and preparing self-control solution; the chromatograms of the sample solution and the reference solution are recorded, and the four impurities in the sample are measured by a self-comparison method of adding correction factors, so that the separation and analysis of epalrestat and related substances thereof are realized, the separation degree is good, the specificity is high, the sensitivity is high, and the accuracy is high.

Description

Detection method of related substances in epalrestat tablet

Technical Field

The invention provides a detection method for detecting related substances in epalrestat tablets by high performance liquid chromatography, in particular relates to a detection method for related substances in epalrestat tablets, and belongs to the field of medicine analysis.

Background

The related substance inspection is one of main indexes of medicine quality control, wherein the research of impurities is an important content of medicine research and development. It includes selecting proper analysis method, accurately distinguishing and measuring impurity content and synthesizing results of pharmaceutical, toxicological and clinical research to determine reasonable limit of impurity. Therefore, the research of impurities is carried out regularly and is controlled within a safe and reasonable limit range, and the quality and the safety of medicines on the market are directly related.

Epalrestat is a reversible non-competitive inhibitor of aldose reductase and has selective inhibition effect on aldose reductase. Can specifically inhibit aldose reductase and inhibit accumulation of sorbitol in nerve, thereby improving subjective symptom and nerve dysfunction of diabetic peripheral nerve disorder. Can be used for improving diabetic peripheral neuropathy (such as tingling and pain), vibration sense abnormality, abnormal heart beat, and high glycosylated hemoglobin content.

The epalrestat tablet can degrade to generate impurities in the preparation process and the storage process to influence the quality of the product. The main degradation products that may be produced are: impurity C, impurity G, impurity H, impurity F. The impurities are related substances in pharmaceutical quality control, the related substances are not controlled in Japanese pharmacopoeia of the current epalrestat preparation, and only the impurity C is researched in the national standard, so that the product quality control is not facilitated, therefore, the physical and chemical properties of epalrestat and the main process of the preparation are considered, the impurity C, the impurity F, the impurity G and the impurity H are controlled by adopting a high performance liquid chromatography in combination with the synthesis process and the degradation (metabolism) way of epalrestat, and the control of other single impurities and total impurities is inspected, so that the determination method of the related substances in the epalrestat is formulated.

Disclosure of Invention

The invention provides a method for measuring related substances in epalrestat tablets. The method can effectively detect epalrestat and 4 impurities thereof simultaneously, has high separation degree, and effectively improves detection sensitivity by examining the concentration, pH and mixing proportion of the salt solution in the mobile phase A and different elution procedures.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the detection method of the related substances in the epalrestat tablet comprises the following steps:

step 1: preparing a system applicability solution: taking a proper amount of epalrestat impurity C, F, G, H and each reference substance of epalrestat, and diluting with a solvent to obtain a system applicability solution; detecting separation conditions and theoretical plate numbers by using a system applicability solution, wherein the separation degree of an impurity C peak and an epalrestat peak is not less than 1.5, and the theoretical plate numbers are not less than 5000 according to the epalrestat peak.

Step 2: preparing a test sample solution: taking a proper amount of epalrestat tablet fine powder, and diluting the epalrestat tablet fine powder with a solvent to obtain a sample solution;

step 3: preparing self-control solution: taking a proper amount of the solution obtained in the step2, and diluting with a solvent to serve as a self-control solution;

step 4: and obtaining chromatograms of the sample solution and the control solution under qualified separation conditions, and determining the impurity content in the epalrestat tablet by a self-contrast method of adding a correction factor.

In some embodiments of step 1 above, the formulation of the system-wide solution is: taking appropriate amounts of epalrestat reference substance, impurity C, impurity F, impurity G and impurity H, placing into a 20ml measuring flask, adding solvent to dissolve and dilute to scale, preparing into 1ml solution containing epalrestat 0.75mg and impurity 10 μg, shaking uniformly, and obtaining the system applicability solution.

In some embodiments of step 2 above, the test solution is formulated as: taking a proper amount of epalrestat tablet fine powder, precisely weighing, adding a proper amount of solvent, shaking for dissolving, and quantitatively diluting to prepare a solution containing about 0.75mg of epalrestat in each 1ml, wherein the solution is used as a test sample solution.

In some embodiments of step 3 above, the self-control solution is formulated as: the appropriate amount of the solution in the above step 2 was precisely measured, and 1.5. Mu.g of the solution containing epalrestat per 1ml was prepared by dilution with a solvent, and used as a self-control solution.

In some embodiments of the invention, the solvent described in step 1, step 2 and step 3 above is N, N-dimethylformamide.

Further, the steps 1, 2 and 3 need to be performed in a dark place.

Further, the step 2 needs to be newly manufactured.

In the above step 4, the chromatographic conditions of the liquid chromatography are as follows: the chromatographic column is octadecylsilane chemically bonded silica chromatographic column.

Further, in the step 4, the mobile phase detected by liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of disodium hydrogen phosphate solution and acetonitrile, the concentration of the disodium hydrogen phosphate solution is 0.01-0.03 mol/L, the pH of the disodium hydrogen phosphate solution is regulated to be 6.0+/-1.0 by phosphoric acid, and the volume ratio of the disodium hydrogen phosphate solution to the acetonitrile is (70:30) - (80:20); the mobile phase B is acetonitrile.

Further, in the step 4, the flow rate of the mobile phase detected by the liquid chromatography is 0.5-1.5 ml/min; the column temperature is 25-40 ℃;

the detection wavelength of the liquid chromatography detection is 260-300 nm;

Further, in the step 4, the elution procedure of the liquid chromatography detection is gradient elution, and the specific flow of the gradient elution is as follows:

And the volume fraction of the mobile phase A is changed from 100% to 85% at uniform speed and the volume fraction of the mobile phase B is changed from 0 to 15% at uniform speed within 0-15 min.

And the volume fraction of the mobile phase A is changed from 85% to 80% at the uniform speed and the volume fraction of the mobile phase B is changed from 15 to 20% at the uniform speed within 15-25 min.

25-30 Min, the volume fraction of the mobile phase A is changed from 80% to 20% at uniform speed, and the volume fraction of the mobile phase B is changed from 20% to 80% at uniform speed.

30-40 Min, the volume fraction of the mobile phase A is 20%, and the volume fraction of the mobile phase B is 80%.

The volume fraction of the mobile phase A is changed from 20% to 100% at the uniform speed and the volume fraction of the mobile phase B is changed from 80% to 0% at the uniform speed within 40-50 min.

50-51 Min, the volume fraction of the mobile phase A is 100%, and the volume fraction of the mobile phase B is 0%.

Compared with the prior art, the invention has the following beneficial effects:

the detection method of the related substances of the epalrestat tablet can detect degradation impurities possibly generated in the processes of raw material synthesis, preparation production and storage, has the advantages of strong specificity, high sensitivity, good precision and the like, ensures the quality of the epalrestat tablet, and improves the safety of clinical medication.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate certain embodiments of the present invention and should not be considered as limiting the scope of the present invention.

FIG. 1 is a chromatogram of a system-applicable solution in the method for measuring substances related to epalrestat tablet in example 1 of the present application.

FIG. 2 is a chromatogram of a sample solution in the method for measuring substances related to epalrestat tablet in example 1 of the present application.

FIG. 3 is a chromatogram of a self-control in the method for measuring an epalrestat tablet-related substance in example 1 of the present application.

FIG. 4 is a chromatogram for determining epalrestat linearity.

FIG. 5 is a chromatogram for determining the linearity of impurity C.

FIG. 6 is a chromatogram for determining the linearity of impurity F.

FIG. 7 is a chromatogram for determining the linearity of impurity G.

FIG. 8 is a chromatogram for determining the linearity of impurity H.

Detailed Description

The present invention is further described below in conjunction with embodiments, which are merely some, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art without making any inventive effort, are intended to be within the scope of the present invention.

Example 1:

Step 1: preparing a system applicability solution: taking a proper amount of epalrestat reference substance, impurity C, impurity F, impurity G and impurity H, placing into a20 ml measuring flask, adding N, N-dimethylformamide to dissolve and dilute to scale, and preparing a solution containing epalrestat 0.75mg and impurity 10 mug in 1ml, and shaking uniformly to obtain a system applicability solution. Detecting separation conditions and theoretical plate numbers by using a system applicability solution, wherein the separation degree of an impurity C peak and an epalrestat peak is not less than 1.5, and the theoretical plate numbers are not less than 5000 according to the epalrestat peak.

Step 2: preparing a test sample solution: taking a proper amount of epalrestat tablet fine powder, precisely weighing, adding a proper amount of N, N-dimethylformamide, shaking for dissolving, and quantitatively diluting to prepare a solution containing about 0.75mg of epalrestat in each 1ml serving as a test sample solution.

Step 3: preparing self-control solution: the solution obtained in the above step 2 was measured precisely and diluted with N, N-dimethylformamide to prepare a solution containing about 1.5. Mu.g of epalrestat per 1ml as a self-control solution.

The above steps 1,2 and 3 require light-shielding operation.

Step 2 above requires a new preparation.

Step 4: under the qualified separation conditions, the sample solution and the control solution are respectively subjected to liquid chromatography analysis, and the chromatographic conditions are as follows:

Chromatographic conditions:

Chromatographic column: WELCH ALK C ₁₈, 5 μm,4.6mm x 250mm; column temperature: 35 ℃; detection wavelength: 280nm; sample injection volume: 10 μl; flow rate: 1.0ml/min; mobile phase a:0.02% disodium hydrogen phosphate solution (pH adjusted to 6.0 with phosphoric acid) -acetonitrile (75:25), mobile phase B: acetonitrile; the gradient elution procedure was as follows:

table 1:

as a result, as shown in FIG. 1,1 to 5 peaks are in sequence impurity G, impurity F, impurity C, epalrestat and impurity H, and the contents of the related substances are calculated by combining with the self-comparison:

Table 2:

Related substances

Impurity C

Impurity F

Impurity G

Impurity H

Other single impurities

Total impurities

Content (%)

0.09

N.D

0.01

0.06

0.02

0.21

The specificity, linearity, accuracy, limit of detection and limit of quantification, and precision of the detection method of example 1 were tested.

(1) Specificity test:

Respectively taking an impurity C, an impurity F, an impurity G, an impurity H and epalrestat, respectively diluting with N, N-dimethylformamide, respectively, directly injecting samples in a sample injection vial according to chromatographic conditions, and recording a chromatogram; mixing the above materials, directly injecting sample according to chromatographic conditions, and recording chromatogram.

The result shows that the retention time of the impurity C, the impurity F, the impurity G, the impurity H and the epalrestat are not interfered with each other under the planned chromatographic condition, and the separation degree of the impurity F and the left and right adjacent impurities G and the impurity C after mixed sample injection is 15.023 and 8.523 respectively. The separation degree of epalrestat from the left and right adjacent impurities C and H is 4.258 and 69.309, and the retention time of epalrestat does not interfere with the impurities C, F, G and H, so that the method has good specificity.

(2) Linear test:

preparing a reference stock solution with the concentration of 10 times of impurities, precisely measuring 0.1ml, 0.5ml, 1.0ml, 5ml and 10ml of the reference stock solution, respectively placing the reference stock solution and the reference stock solution into a 100ml measuring flask, respectively diluting the reference stock solution to scale marks by using N, N-dimethylformamide, and shaking the reference stock solution uniformly to prepare a series of horizontal concentration solutions. And carrying out linear regression analysis on the peak area of each target object and the concentration of each target object to obtain a standard working curve of each target compound. The linear equation of epalrestat is shown in fig. 4, the linear equation of impurity C is shown in fig. 5, and the linear equation of impurity D is shown in fig. 6. The linear equation of the impurity F is shown in fig. 7, and the linear equation of the impurity H is shown in fig. 8.

Epalrestat is in the concentration range of 0.095-9.501 mug/ml, and the linear equation is y= 14808.34x-513.90; the correlation coefficient R is 0.9999 and is more than 0.990, which shows that the linear relation is good.

Impurity C is in the concentration range of 0.103-10.287 μg/ml, and the linear equation is y= 18947.84x-595.74; the correlation coefficient R is 0.9997 and is more than 0.999, which shows that the linear relation is good.

Impurity F is in the concentration range of 0.095-9.457 μg/ml, and the linear equation is y= 13185.36x-1298.58; the correlation coefficient R is 0.9995 and is more than 0.999, which shows that the linear relation is good.

Impurity G is in the concentration range of 0.113-11.342 μg/ml, and the linear equation is y= 15517.25x-1840.72; the correlation coefficient R is 0.9993, which is more than 0.999, indicating that the linear relationship is good.

Impurity H is in the concentration range of 0.102-10247 mug/ml, and the linear equation is y= 11673.24x-2169.56; the correlation coefficient R is 0.9991 which is larger than 0.999, which shows that the linear relation is good.

(3) Accuracy test:

Taking fine powder of the product, precisely weighing a proper amount (about 15mg of epalrestat in terms of C ₁₅H₁₃NO₃S₂), placing the fine powder into a 20ml measuring flask, respectively adding 0.1ml, 0.2ml and 0.3ml of mixed reference stock solution (about 150 mug/ml), adding a proper amount of N, N-dimethylformamide solution, ultrasonically dissolving and diluting to a scale, and preparing test sample solutions with the impurity limit of 50%, 100% and 150%, wherein 3 parts of samples are prepared for each concentration.

Precisely transferring 1ml of each sample solution, placing into a 500ml measuring flask, adding N, N-dimethylformamide for dissolution, diluting to constant volume, and respectively serving as corresponding self-control solutions.

Taking a proper amount of fine powder (about 15mg of epalrestat in terms of C ₁₅H₁₃NO₃S₂), precisely weighing, placing into a 20ml measuring flask, ultrasonically dissolving with N, N-dimethylformamide, diluting to a scale, and shaking uniformly to obtain a blank sample solution.

1Ml of the blank sample solution is precisely removed, placed in a 500ml measuring flask, dissolved by adding N, N-dimethylformamide and diluted to a constant volume, and used as a blank self-control solution.

Taking the solution, precisely measuring 10 μl, injecting into a liquid chromatograph, and recording the chromatogram. The amounts of the respective impurities in each sample were measured, and the recovery rates were calculated based on the amounts added and the measured amounts, and the results are shown in tables 3 to 6. The result shows that the recovery rate of each impurity is between 90% and 108%, and the RSD value is less than 2%, which shows that the method has good accuracy.

TABLE 3 results of impurity C accuracy test

TABLE 4 impurity F accuracy test results

TABLE 5 impurity G accuracy test results

TABLE 6 impurity H accuracy test results

(4) Detection limit and quantitative limit test:

Taking impurity C, impurity F, impurity G and impurity H, successively diluting with N, N-dimethylformamide, taking the concentration at a signal to noise ratio of 10:1 as a quantitative limit, and measuring the quantitative limits as follows: impurity C1.029ng, impurity F0.946 ng, impurity G1.134ng, impurity H1.025ng; taking the concentration at a signal-to-noise ratio of 3:1 as a detection limit, and respectively measuring the detection limit as follows: impurity C0.514 ng, impurity F0.315ng, impurity G0.567 ng, impurity H0.512 ng.

Through the analysis, the analysis method provided by the invention is accurate and reliable, can sensitively, accurately, qualitatively and quantitatively detect the contents of related substances impurity C, impurity F, impurity G and impurity H in the epalrestat tablet, and provides scientific basis for quality control of the epalrestat tablet.

(5) Precision test:

taking the reference substance solution to continuously sample for 6 times, and examining the sample injection precision of the method by using the peak area of each target object by using the reference substance solution. RSD of the sample injection precision of the impurity C, the impurity F, the impurity G and the impurity H is 0.68%, 0.82%, 0.22% and 0.24% respectively.

Example 2:

the present example provides a method for detecting related substances in epalrestat tablets, which comprises the following steps in the specific steps, except for the gradient elution procedure, and the rest steps are the same as those in example 1:

table 7:

example 3:

The present example provides a method for detecting related substances in epalrestat tablet, and the specific steps are the same as those in example 1 except that the pH of disodium hydrogen phosphate in mobile phase a is 6.0±1.0.

Example 4:

the present example provides a method for detecting related substances in epalrestat tablet, and the specific steps are the same as those in example 1 except that the concentration of disodium hydrogen phosphate in mobile phase A is 0.01-0.03 mol/L.

Example 5:

The present example provides a method for detecting related substances in epalrestat tablet, and the specific steps are the same as those of example 1 except that the volume ratio of disodium hydrogen phosphate to acetonitrile in mobile phase A is (70:30) - (80:20).

Example 6:

the present example provides a method for detecting related substances in epalrestat tablet, and the specific steps are the same as those of example 1 except that the detection wavelength is 260-300 nm.

Comparative example 1:

a detection method of related substances in epalrestat tablets comprises the following steps:

Step 1: preparing a system applicability solution: the preparation method is the same as in example 1;

step 2: preparing a test sample solution: the preparation method is the same as in example 1;

step 3: preparing self-control solution: the preparation method is the same as in example 1;

step 4: and (3) taking the solutions obtained in the steps 1-3, and respectively injecting the solutions into a liquid chromatograph for analysis and measurement.

In the above step 4, the chromatographic conditions of the liquid chromatograph are as follows:

Chromatographic column: thermo SCIENTIFIC HYPERSLL C ₁₈, 5 μm,4.6mm x 250mm; column temperature: 35 ℃; detection wavelength: 280nm; sample injection volume: 10 μl; flow rate: 1.0ml/min; mobile phase a:0.02% disodium hydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) -acetonitrile (60:40), mobile phase B: acetonitrile; the gradient elution procedure was as follows:

Table 8:

Recording the chromatograms generated in the liquid chromatograph, and calculating the contents of related substances impurity C, impurity F, impurity G and impurity H in the epalrestat tablet according to a main component self-comparison method of self-comparison and correction factors.

Comparative example 1 the baseline shift of the chromatogram of the test solution was severe and the interference peak had affected the calculation of the content of each impurity in epalrestat tablet. The impurity C is an isomer, the chemical structure is very similar to that of epalrestat, the system applicability solution prepared by each impurity reference substance is poor in impurity C and epalrestat peak separation degree by using the detection method of comparative example 1, the sensitivity of other three impurities is low, the detection limit is influenced, the detection methods of examples 1-6 are used, the detection effect is good, and meanwhile, the detection separation degree, sensitivity and accuracy are effectively improved, the detection limit is reduced, and the recovery rate is between 90-108% by controlling the detection wavelength, the elution program, the concentration of phosphate in the mobile phase, the pH value, the proportion of organic phase and the like.

The applicant states that what has been described above is merely a preferred embodiment of the invention, it being noted that it will be apparent to those skilled in the art that several improvements and modifications can be made without departing from the principle of the invention, which should also be considered as the scope of protection of the invention.

Claims

1. The detection method of the related substances in the epalrestat tablet is characterized by comprising the following steps of:

Step 1: and (3) preparing a system applicability solution by taking proper amounts of the epalrestat impurity C, F, G, H and each reference substance of epalrestat, and detecting the separation condition and the theoretical plate number by using the system applicability solution, wherein the separation degree of an impurity C peak and an epalrestat peak is not less than 1.5, and the theoretical plate number is not less than 5000 according to the epalrestat peak.

Step 2: taking a proper amount of epalrestat tablet fine powder and preparing the epalrestat tablet fine powder as a test sample solution.

Step 3: and (3) taking a proper amount of the solution obtained in the step (2) to prepare a self-control solution.

2. The method according to claim 1, wherein the chromatographic column for liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column.

3. The method for detecting related substances in epalrestat according to claim 1, wherein the mobile phase detected by liquid chromatography comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixed solution of disodium hydrogen phosphate solution and acetonitrile, the concentration of the disodium hydrogen phosphate solution is 0.01-0.03 mol/L, the pH of the disodium hydrogen phosphate solution is regulated to be 6.0+/-1.0 by phosphoric acid, and the volume ratio of the disodium hydrogen phosphate solution to the acetonitrile is (70:30) - (80:20); the mobile phase B is acetonitrile.

The concentration of the disodium hydrogen phosphate solution may be 0.01mol/L, 0.015mol/L, 0.02mol/L, 0.025mol/L, 0.03mol/L, etc., the pH of the disodium hydrogen phosphate solution may be adjusted to 5.0, 5.2, 5.4, 5.8, 6.0, 6.2, 6.4, 6.8, 7.0, etc., and the volume ratio of the disodium hydrogen phosphate solution to acetonitrile may be 70:30, 73:27, 75:25, 78:22, 80:20, etc., but the present invention is not limited to the above-listed values, and other non-listed values within the above-listed values are equally applicable.

4. The method according to claim 1, wherein the flow rate of the mobile phase detected by liquid chromatography is 0.7-1.3 ml/min and the column temperature is 25-40 ℃.

The flow rate of the mobile phase may be 0.7ml/min, 0.8ml/min, 0.9ml/min, 1.0ml/min, 1.1ml/min, 1.2ml/min, 1.3ml/min, etc., the column temperature may be 25 ℃, 28 ℃,30 ℃, 32 ℃, 35 ℃, 38 ℃, 40 ℃ or the like, but not limited to the above-listed values, and other values not listed in the above-listed value ranges are equally applicable.

5. The method according to claim 1, wherein the detection wavelength of the liquid chromatography detection is 260 to 300nm.

6. The method according to claim 5, wherein the detection wavelength of the liquid chromatography detection is 280nm.

The specific wavelength selection can further improve the detection accuracy.

7. The method according to claim 1, wherein the elution procedure of the liquid chromatography is gradient elution, and the specific procedure of the gradient elution is as follows:

8. The detection method according to any one of claims 1 to 7 can effectively distinguish various related substances, effectively improves the detection separation degree, and also provides application of the detection method in epalrestat tablet quality control.