CN114235975A - Method for determining simvastatin related substances - Google Patents

Abstract

The invention relates to a method for determining substances related to simvastatin bulk drugs. Dissolving simvastatin raw material medicine with a solvent (0.01% dibutyl hydroxy toluene/acetonitrile-water (pH7.0)) in water bath at 15 ℃ to prepare 1ml of solution containing 1.5 mg; in addition, 9 characteristic impurities possibly existing in the bulk drugs are additionally added for positioning and system applicability investigation, high performance liquid chromatography is adopted for detection, the result can more scientifically and accurately reflect the impurity level of the product, and a more strict control limit is formulated according to the international pharmacopoeia and the actual water of the product; the method lays a good research foundation for the research of impurities in the preparation prepared from the bulk drugs, and also provides guarantee for the safety of clinical application of the product preparation.

Description

Method for determining simvastatin related substances

Technical Field

The invention belongs to the field of drug impurity detection, and relates to a method for determining simvastatin related substances.

Background

Simvastatin bulk drug is provided by Zhejiang Shangyu Yujing new pharmaceutical company, a qualified supplier of the company, and the dosage form developed by the company for clinical use is tablet, clinical indications are hyperlipidemia, coronary heart disease with hypercholesterolemia and children patients with heterozygous familial hypercholesterolemia, and the product can be used for reducing total cholesterol, low-density lipoprotein cholesterol, apolipoprotein B and triglyceride by combining diet control.

The determination method of related substances in Chinese pharmacopoeia is as follows:

related substances are as follows: measuring by high performance liquid chromatography (general rule 0512).

Solvent: acetonitrile-0.01 mol/L potassium dihydrogen phosphate solution (pH adjusted to 4.0 with phosphoric acid) (60: 40). Test solution: the appropriate amount of this product was taken, dissolved in a solvent and diluted to make a solution containing about 0.8mg per 1ml (measured within 3 hours).

Control solution: a suitable amount of the sample solution was precisely measured and quantitatively diluted with a solvent to give a solution containing about 4. mu.g of the sample solution per 1ml (corresponding to 0.5% of impurities).

System applicability solution: taking 20mg of simvastatin reference substance, placing the simvastatin reference substance in a 50ml measuring flask, adding 5ml of a mixed solution of 0.2mol/L sodium hydroxide solution and acetonitrile (1:1), shaking to dissolve the simvastatin reference substance, placing the mixture for 5 minutes, adding diluted hydrochloric acid to neutralize the mixture, and diluting the mixture to a scale with a solvent to obtain a simvastatin acid solution containing the open-loop degradation product; taking about 2mg of lovastatin and simvastatin respectively, placing the lovastatin and the simvastatin in the same 100ml measuring flask, adding 5ml of simvastatin acid solution, dissolving and diluting the lovastatin and the simvastatin acid solution to the scale with a solvent, and shaking up the mixture.

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a packing material (4.6 mm. times.33 mm,3 μm or equivalent performance column); acetonitrile-0.1% phosphoric acid solution (50:50) is used as a mobile phase A; 0.1% phosphoric acid in acetonitrile as mobile phase B; gradient elution was performed as in table 1 below; the flow rate was 3.0ml per minute; the detection wavelength is 238 nm; the injection volume was 10. mu.l.

TABLE 1

System applicability requirements: in the system applicability solution chromatogram, the separation degree between the simvastatin acid peak and the lovastatin peak is required, and the figure is shown in figure 1.

The separation between the lovastatin peak and the simvastatin peak should be greater than 4.0. See fig. 2.

The determination method comprises the following steps: precisely measuring the test solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatogram.

Limitation: if a chromatographic peak caused by the retention time of a lovastatin peak exists in a chromatogram of a test solution, the peak area of the chromatographic peak is not larger than the main peak area (0.5%) of a control solution, the peak area of other single impurities is not larger than 0.8 times (0.4%) of the main peak area of the control solution, the sum of the peak areas of other impurities is not larger than 2 times (1.0%) of the main peak area of the control solution, and chromatographic peaks smaller than 0.05 times of the main peak area of the control solution are ignored.

The existing detection method has the defects that:

(1) the stability time limit of the test solution is only 3 hours, and the operability in a laboratory is not ideal;

(2) only lovastatin (called as impurity C for short) and simvastatin acid (called as impurity B for short) are positioned in a system applicability solution, and lovastatin diol lactone (called as impurity A for short), epilovastatin (called as impurity D for short), methylene simvastatin (called as impurity E for short), methyl ester rivastatin (called as impurity F for short), acetyl simvastatin (called as impurity G for short), anhydrous simvastatin (called as impurity H for short) and simvastatin dimer (called as impurity I for short) which are also present in the raw material medicines can not be effectively positioned and identified.

(3) Separating impurities, performing gradient elution according to the above chromatographic conditions, maintaining the main component peak for about 2min, separating impurities, and performing gradient transformation for too short time to balance the instrument, wherein the RT recurrence difference of each component in the same system is shown in FIGS. 4, 5 and 6.

(4) Control of impurity type and limits: currently, the standard control is that lovastatin (C) is not more than 0.5%, other impurities are not more than 0.4%, the sum of other impurities is not more than 1.0%, only two known impurities (B, C) are controlled, and other known impurities (A, D, E, F, G, H, I) existing in the bulk drug are not positioned and are not specifically limited; and under the system, the separation of lovastatin (C) and epilovastatin (D) cannot be completed, and the result accuracy of lovastatin (C) is not high. See fig. 1.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for measuring simvastatin related substances, which can more scientifically and accurately detect the impurity level of the simvastatin related substances,

the technical scheme adopted by the invention is as follows:

a method for measuring simvastatin related substances, a reagent and a solution are prepared, and the method comprises the following steps:

(1) solvent: water-acetonitrile is prepared according to the volume ratio of 2 (2.5-3.5);

(2) test solution: and taking simvastatin raw material medicine, precisely weighing, placing in a volumetric flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain a test solution.

Preferably, the temperature for dissolving the solvent is 12-18 ℃ during the preparation of the test solution.

Further preferably, the temperature for dissolving the test solution is 15 ℃.

Preferably, the pH of the water in the solvent is adjusted to 7.0 using 1mol/L potassium hydroxide solution.

Preferably, the solvent contains 0.005-0.015% of dibutyl hydroxy toluene.

Further preferably, the solvent contains 0.01% of dibutylhydroxytoluene.

Preferably, the water-acetonitrile is prepared according to the volume ratio of 2: 3;

test solution: taking simvastatin raw material medicine, precisely weighing 75mg, placing the simvastatin raw material medicine in a 50ml volumetric flask, adding a solvent to dissolve and dilute the simvastatin raw material medicine to a scale, and shaking up to obtain a test solution with the concentration of 1.5 mg/ml.

Preferably, the control solution, the sensitivity solution and the system applicability solution used in the method are prepared by the following steps:

control solution: precisely measuring 1.0ml of the test solution, placing the test solution into a 100ml measuring flask, adding a solvent to dilute the test solution to a scale, and shaking the test solution uniformly to obtain the test solution with the concentration of 15 mu g/ml;

sensitivity solution: precisely measuring 1ml of the control solution, putting the control solution into a 50ml measuring flask, adding a solvent to dilute the control solution to a scale, and shaking the control solution uniformly to obtain the compound (the concentration is 0.3 mu g/ml, which is equivalent to 0.02 percent of impurity content);

system applicability solution: weighing simvastatin 75mg, impurities A3 mg, B6 mg, C3 mg, D3 mg, E3 mg, F3 mg, G3 mg, H3 mg and I3 mg in the same 50ml measuring flask, adding a solvent, performing ultrasonic treatment to dissolve and dilute the mixture to a scale, and shaking up to obtain the simvastatin.

Preferably, the method employs chromatographic conditions and a system suitability test: chromatography column Agilent ZORBAX Eclipse XDB-C18, 4.6mm × 150mm, 5 μm; mixing acetonitrile: 0.1% phosphoric acid solution 45:55 as mobile phase a; acetonitrile: 0.1% phosphoric acid solution 90:10 as mobile phase B; the detection wavelength is 238 nm; the column temperature is 30 ℃, the temperature of the sample feeding disc is controlled at 10 ℃, and the flow rate is 2.0 ml/min.

Preferably, the chromatographic run gradient is as follows in table 2:

TABLE 2

The operation is as follows: precisely measuring the above system applicability solution and sensitivity solution 20 μ l each, injecting into liquid chromatograph, measuring according to the following chromatographic conditions, recording chromatogram,

the separation degree of a simvastatin peak and a methyl ester ivastatin peak in the spectrum of the system applicability chart (figure 3) is not less than 1.5; the retention time of a simvastatin peak in a map is about 30 min;

the RRT and correction factors for each impurity peak are referenced in table 2 below:

TABLE 3

Serial number	Code of impurity	Name (R)	RRT	Correction factor	Limit of
						1	A	Lovastatin diol lactone	0.02	1.03	≤0.1％
2	B	Simvastatin acid	0.43	0.98	≤0.2％
						3	C	Lovastatin	0.63	1.01	≤0.2％
4	D	Eplovastatin	0.67	1.06	≤0.2％
						5	E	Methylene simvastatin	0.81	1.00	≤0.2％
6	F	Methyl ester atorvastatin	1.13	--	--
						7	G	Acetyl simvastatin	1.55	1.03	≤0.2％
8	H	Anhydrous simvastatin	1.59	0.96	≤0.2％
						9	I	Simvastatin dimer	2.31	0.98	≤0.2％

The signal-to-noise ratio of simvastatin peak in the sensitive solution map is not lower than 10.

Preferably, the method is specifically determined as follows,

and (3) determination: under the condition that the applicability of the system meets the requirements, precisely measuring a reference solution and a test solution, injecting the reference solution and the test solution into a liquid chromatograph, and recording a chromatogram;

limitation: if an impurity peak exists in a chromatogram of a test solution, the content of the impurity A, B, C, D, E, G, H, I is calculated according to a self-comparison method of a main component added with a correction factor except a solvent peak, the content of each impurity should be less than the limit of the list, and any unknown single impurity peak area should be less than 0.1 time of the area of a main peak of a comparison solution; the sum of all impurity peak areas should not exceed 1.0 times of the main peak area of the control solution; with the exception of impurity C, D, E, less than 0.02% of the impurities were ignored.

The invention has the beneficial effects that:

the invention solves two problems existing in the consistent measurement of related substances of the product:

on one hand, the product is extremely unstable under acid, alkali, oxidation and damp heat conditions to cause inaccurate and unrepeatable measurement results of related substances, and the following method is adopted for control:

(1) the concentration of the test solution is increased from 0.8mg/ml to 1.5mg/ml, the detection capability of impurities is enhanced, the reproducibility of detection results is improved, the retention time of the main component in a new system is about 30min, the separation of the impurities is not influenced by the increase of the concentration of the test solution, and the method is actually better than that of the original system, and is shown in figure 3.

(2) Solvent for test solution preparation "water-acetonitrile (2:3) solution of 0.01% dibutylhydroxytoluene (BHT): (water uses 1mol/L potassium hydroxide pH7.0) to neutral, controls the acid and alkali degradation in the solution preparation and determination process, and can avoid the trouble that the result is inaccurate and can not be reproduced due to the degradation of simvastatin acid (B); and the measurable time of placing is prolonged, and the reliability of test operation is improved.

Wherein the simvastatin acid degradation mechanism is as follows: under acidic and basic conditions, the lactone will hydrolyze to form B.

The test data are shown in Table 6, and the stability of the test solution preparation solvent water-acetonitrile (2:3) and water (pH7.0) -acetonitrile (2:3) is compared and studied.

(3) Solvent for test solution preparation "water-acetonitrile (2:3) solution of 0.01% dibutylhydroxytoluene (BHT): (water with 1mol/L potassium hydroxide pH 7.0). The oxidative degradation of simvastatin in a sample solution containing 0.01% dibutylhydroxytoluene in a solvent, especially the oxidative degradation is accelerated when the sample is injected into an instrument and contacts with metal. The test data are shown in Table 7 for comparative studies of the stability of the test solutions in the presence and absence of 0.01% BHT.

(4) The test solution is prepared in a water bath with the temperature controlled about 15 ℃, so that the trouble that the result is inaccurate and cannot be reproduced due to the degradation of anhydrous simvastatin (H) and simvastatin dimer (I) generated by high temperature in simvastatin can be avoided; and the measurable time of placing is prolonged, and the reliability of test operation is improved.

The degradation mechanism of impurity H: dehydration of the tertiary hydroxyl group in simvastatin under high temperature conditions

The mechanism of impurity I degradation: 2 molecules of simvastatin generate impurity I by ester exchange under the high temperature condition

The test data are shown in Table 8 for the preparation of the test solutions and the stability study of temperature control and non-temperature control.

(5) The stability of the original test solution is only 3 hours, and the stability of the solution is prolonged to 34 hours by the improvement.

Secondly, the product has more impurities, and the impurities can not be completely separated under the condition of primary color spectrum, so the following improvements are carried out:

the gradient running time adopted by the primary color spectrum condition is only 13min, the main component peak retention time is 2min, and impurities in the sample cannot be completely separated; the gradient is optimized to ensure that the running time reaches 100min, and the impurities with various characteristics are completely separated.

The original chromatographic condition can not be completely separated because of each impurity, the impurities in the standard only control 2 characteristic impurities, namely simvastatin acid (impurity B) and lovastatin (impurity C), the optimized chromatographic condition completely separates each impurity, and the positions and the limits of other 8 characteristic impurities are more scientifically attributed,

(6) the concentration of the sensitive solution in the newly developed determination method is only 0.3 mug/ml, the detection sensitivity is higher, the neglect limit of impurities is only 0.02 percent (the original system is 0.05 percent), and the standard is stricter.

(7) The newly developed determination method carries out complete attribute research on characteristic impurities in the product, establishes impurity control requirements which are more in line with technical guidelines for chemical drug impurity research, improves the standard, improves the product quality, lays a solid foundation for the safety of clinical medication, and the impurity control and limit of the original new determination method are shown in a table 9.

(8) The company has carried out comprehensive verification on the newly developed simvastatin related substance determination method, the quantitative limit, linearity and accuracy results of each impurity in the system are shown in table 2, the determination method is operated in the actual detection of materials entering a factory, a quality agreement is achieved with the factory, and the newly developed simvastatin related substance determination method has high accuracy and good reproducibility of the determination results. The results of the specific tests are shown in Table 10.

(9) In order to embody the scientificity of a newly developed simvastatin related substance determination method, the strict impurity control and the technical foresight, a test result is taken for tabulation comparison and is shown in a table 11; see FIGS. 4-9.

Drawings

FIG. 1 is a systematic applicability map of the original assay, with a separation of 3.89 between the simvastatin acid peak and the lovastatin peak;

FIG. 2 is a systematic applicability map of the assay, with a separation of 4.13 between the lovastatin peak and the simvastatin peak;

FIG. 3 is a plot of the suitability of the system under the process of the present invention, simvastatin peak (32.033min) and methyl ester ivastatin (36.042min) peak separation 3.37;

FIG. 4 is a sample solution map of SVT-161102-2 under the original method system;

FIG. 5 is a sample solution map of SVT-B170701-1 under the original method system;

FIG. 6 is a sample solution map of SVT-B181201-2 under the original method system;

FIG. 7 is a sample solution map of SVT-161102-2 under the method of the present invention;

FIG. 8 is a diagram of a test solution of SVT-B170701-1 under the method of the present invention;

FIG. 9 is a sample solution map of SVT-B181201-2 under the method of the present invention;

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

A method for measuring simvastatin related substances comprises the following steps of:

(1) solvent: preparing water-acetonitrile according to the volume ratio of 2: 3;

(2) test solution: taking 75mg of simvastatin raw material medicine, precisely weighing, placing in a 50ml volumetric flask, adding a solvent to dissolve (dissolving a sample in a water bath with the temperature controlled at about 15 ℃) and diluting to a scale, and shaking uniformly to obtain the simvastatin with the concentration of 1.5 mg/ml;

(3) control solution: precisely measuring 1.0ml of the test solution, placing the test solution into a 100ml measuring flask, adding a solvent to dilute the test solution to a scale, and shaking the test solution uniformly to obtain the test solution with the concentration of 15 mu g/ml;

(4) sensitivity solution: precisely measuring 1ml of the control solution, putting the control solution into a 50ml measuring flask, adding a solvent to dilute the control solution to a scale, and shaking the control solution uniformly to obtain the compound (the concentration is 0.3 mu g/ml, which is equivalent to 0.02 percent of impurity content);

(5) system applicability solution: weighing simvastatin 75mg, impurities A3 mg, B6 mg, C3 mg, D3 mg, E3 mg, F3 mg, G3 mg, H3 mg and I3 mg in the same 50ml measuring flask, adding a solvent, performing ultrasonic treatment to dissolve and dilute the mixture to a scale, and shaking up to obtain the simvastatin.

In the process of preparing the test solution, the temperature for dissolving the test solution by adding the solvent is 15 ℃.

The pH of the water in the solvent was adjusted to 7.0 with 1mol/L potassium hydroxide solution.

The solvent contained 0.01% dibutylhydroxytoluene.

Preferably, the method employs chromatographic conditions and a system suitability test: chromatography column Agilent ZORBAX Eclipse XDB-C18, 4.6mm × 150mm, 5 μm; mixing acetonitrile: 0.1% phosphoric acid solution 45:55 as mobile phase a; acetonitrile: 0.1% phosphoric acid solution 90:10 as mobile phase B; the detection wavelength is 238 nm; the column temperature is 30 ℃, the temperature of the sample feeding disc is controlled to be 10 ℃, the flow rate is 2.0ml/min, and the chromatographic operation gradient is shown in the following table 4:

TABLE 4

Time (min)	Mobile phase A (%) (V/V)	Mobile phase B (%) (V/V)
			0	100	0
38	100	0
			40	65	35
50	65	35
			70	0	100
90	0	100
			92	100	0
100	100	0

；

The operation is as follows: precisely measuring 20 mul of each of the system applicability solution and the sensitivity solution, injecting into a liquid chromatograph, performing measurement according to the following chromatographic conditions, and recording a chromatogram, wherein the separation degree of a simvastatin peak and a methyl ester ivastatin peak in the system applicability spectrum is not lower than 1.5; the retention time of a simvastatin peak in a map is about 30 min;

system applicability fig. 3;

the RRT and correction factors for each impurity peak are referenced in table 5 below:

TABLE 5

Serial number	Code of impurity	Name (R)	RRT	Correction factor	Limit of
						1	A	Lovastatin diol lactone	0.02	1.03	≤0.1％
2	B	Simvastatin acid	0.43	0.98	≤0.2％
						3	C	Lovastatin	0.63	1.01	≤0.2％
4	D	Eplovastatin	0.67	1.06	≤0.2％
						5	E	Methylene simvastatin	0.81	1.00	≤0.2％
6	F	Methyl ester atorvastatin	1.13	--	--
						7	G	Acetyl simvastatin	1.55	1.03	≤0.2％
8	H	Anhydrous simvastatin	1.59	0.96	≤0.2％
						9	I	Simvastatin dimer	2.31	0.98	≤0.2％

The signal-to-noise ratio of simvastatin peak in the sensitive solution map is not lower than 10.

Preferably, the method is specifically determined as follows,

and (3) determination: under the condition that the applicability of the system meets the requirements, precisely measuring 20 mu l of reference solution and sample solution, injecting the reference solution and the sample solution into a liquid chromatograph, and recording a chromatogram;

limitation: if an impurity peak exists in a chromatogram of a test solution, the content of the impurity A, B, C, D, E, G, H, I is calculated according to a self-comparison method of a main component added with a correction factor except a solvent peak, the content of each impurity should be less than the limit of the list, and any unknown single impurity peak area should be less than 0.1 time of the area of a main peak of a comparison solution; the sum of all impurity peak areas should not exceed 1.0 times of the main peak area of the control solution; with the exception of impurity C, D, E, less than 0.02% of the impurities were ignored.

TABLE 6 stability study of test solutions for preparing solvent Water-acetonitrile (2:3) and solvent Water (pH7.0) -acetonitrile (2:3)

As can be seen from Table 6, the degradation rate of impurity B (simvastatin acid) is significantly slowed when the pH of the formulated solvent bar for the test article is 7.0 (neutral).

TABLE 7 comparative study of stability of test solutions prepared with and without 0.01% BHT

As can be seen from Table 7, the antioxidant BHT was added to the formulation solvent for the test article: the degradation rates of impurity B (simvastatin acid), impurity H (anhydrous simvastatin) and impurity I (simvastatin dimer) are obviously slowed down.

TABLE 8 preparation of test solutions and temperature-controlled and temperature-uncontrolled stability study

As can be seen from table 8, (1) the sample solution was prepared at room temperature (without temperature control), and the amount of impurities B (simvastatin acid), H (anhydrous simvastatin), and I (simvastatin dimer) was larger than that at 15 ℃. (2) When the test solution is detected (temperature is not controlled), the impurity B (simvastatin acid) is operated for 15 hours, the impurity increment is close to the report limit (0.05%) of the API specified in the ICH, the impurity H (anhydrous simvastatin) is operated for 23 hours, the impurity increment already exceeds the report limit (0.05%) of the API specified in the ICH, the impurity I (simvastatin dimer) is operated for 23 hours, the impurity increment reaches the report limit (0.05%) of the API specified in the ICH, and the stability of the temperature-uncontrolled test solution is 15 hours. (3) When the test sample solution is detected (temperature is controlled to be 10 ℃), the impurity B (simvastatin acid) is operated for 34 hours, the impurity increment reaches the report limit (0.05%) of the API specified in the ICH, the impurity H (anhydrous simvastatin) is operated for 34 hours, the impurity increment is close to the report limit (0.05%) of the API specified in the ICH, the impurity I (simvastatin dimer) is operated for 34 hours, the impurity increment is only 0.02%, and the stability of the temperature-controlled test sample solution is proved to be at least 34 hours.

TABLE 9 comparison of impurity control and limits of original assay and assay of the invention

As can be seen from the impurities in Table 9, the RRT of each impurity in the two determination methods shows that the separation degree of each impurity in the determination method of the invention is better, which indicates that the chromatographic system of the novel invention is better; the characteristic impurity control in the measuring method of the invention is 9, the original measuring method is only 2, the impurity limit in the measuring method of the invention is lower, which shows that the impurity research of the measuring method of the invention is more comprehensive and reasonable, and the quality requirement of the product is more strict.

TABLE 10 validation of the assay of the invention

As can be seen from Table 10, the determination method of the present invention is verified by comprehensive methodology, and all indexes can meet the related substance determination of the product, and the method is reliable.

TABLE 11 comparison of the results of the original assay with the assay of the invention

As can be seen from table 11, the above two methods are used to detect 3 batches of the same samples, because the original method does not solve the problem of solution stability of the test sample, and 3 batches of the samples are simultaneously prepared, the impurity B content of the 3 batches of the samples is greater than the result of the determination method of the present invention, and 3 batches of the samples are simultaneously prepared for detection, the impurity B content is higher and higher along with the extension of the placing time, the placing time of the 3 rd batch is longest, and the impurity B content reaches 0.43% (which should not be more than 0.4%), and is out of limit; this gave a false result, and the acceptable sample was detected as being unacceptable.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims (10)

1. The method for measuring simvastatin related substances is characterized in that a reagent and a solution are prepared, and the reagent and the solution comprise:

(1) solvent: water-acetonitrile is prepared according to the volume ratio of 2 (2.5-3.5);

(2) test solution: and taking simvastatin raw material medicine, precisely weighing, placing in a volumetric flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain a test solution.

2. The method for assaying simvastatin related substances according to claim 1, wherein the dissolution temperature of the solubilizing agent during the preparation of the sample solution is 12 to 18 ℃.

3. The method for assaying simvastatin related substances according to claim 2, wherein the dissolution temperature of the solubilizing agent during the preparation of the sample solution is 15 ℃.

4. The method for assaying simvastatin related substances according to claim 1, wherein the pH of water in the solvent is adjusted to 7.0 using 1mol/L potassium hydroxide solution.

5. The method for assaying simvastatin related substances according to claim 1, wherein the solvent contains 0.005 to 0.015% of dibutylhydroxytoluene.

6. The method for assaying simvastatin related substances according to claim 1, wherein the water-acetonitrile is prepared in a volume ratio of 2: 3;

test solution: taking simvastatin raw material medicine, precisely weighing 75mg, placing the simvastatin raw material medicine in a 50ml volumetric flask, adding a solvent to dissolve and dilute the simvastatin raw material medicine to a scale, and shaking up to obtain a test solution with the concentration of 1.5 mg/ml.

7. The method for assaying simvastatin related substances according to claim 1, wherein the control solution, the sensitivity solution and the system suitability solution are prepared by:

control solution: precisely measuring 1.0ml of the test solution, placing the test solution into a 100ml measuring flask, adding a solvent to dilute the test solution to a scale, and shaking the test solution uniformly to obtain the test solution with the concentration of 15 mu g/ml;

sensitivity solution: precisely measuring 1ml of the control solution, putting the control solution into a 50ml measuring flask, adding a solvent to dilute the control solution to a scale, and shaking the control solution uniformly to obtain the compound (the concentration is 0.3 mu g/ml, which is equivalent to 0.02 percent of impurity content);

system applicability solution: weighing simvastatin 75mg, impurities A3 mg, B6 mg, C3 mg, D3 mg, E3 mg, F3 mg, G3 mg, H3 mg and I3 mg in the same 50ml measuring flask, adding a solvent, performing ultrasonic treatment to dissolve and dilute the mixture to a scale, and shaking up to obtain the simvastatin.

8. The method for assaying simvastatin-related substances according to claim 1, wherein the chromatographic conditions and the system suitability test are as follows: chromatography column Agilent ZORBAX Eclipse XDB-C18, 4.6mm × 150mm, 5 μm; mixing acetonitrile: 0.1% phosphoric acid solution 45:55 as mobile phase a; acetonitrile: 0.1% phosphoric acid solution 90:10 as mobile phase B; the detection wavelength is 238 nm; the column temperature is 30 ℃, the temperature of the sample feeding disc is controlled at 10 ℃, and the flow rate is 2.0 ml/min.

9. The method for assaying simvastatin related substances according to claim 1, wherein the gradient of the chromatographic run is as shown in table 1 below:

TABLE 1

Time (min) Mobile phase A (%) (V/V) Mobile phase B (%) (V/V) 0 100 0 38 100 0 40 65 35 50 65 35 70 0 100 90 0 100 92 100 0 100 100 0

；

The operation is as follows: precisely measuring the above system applicability solution and sensitivity solution 20 μ l each, injecting into liquid chromatograph, measuring according to the following chromatographic conditions, recording chromatogram,

the separation degree of a simvastatin peak and a methyl ester ivastatin peak in a system applicability map is not lower than 1.5; the retention time of a simvastatin peak in a map is about 30 min;

the RRT and correction factors for each impurity peak are referenced in table 2 below:

TABLE 2

Serial number Code of impurity Name (R) RRT Correction factor Limit of 1 A Lovastatin diol lactone 0.02 1.03 ≤0.1％ 2 B Simvastatin acid 0.43 0.98 ≤0.2％ 3 C Lovastatin 0.63 1.01 ≤0.2％ 4 D Eplovastatin 0.67 1.06 ≤0.2％ 5 E Methylene simvastatin 0.81 1.00 ≤0.2％ 6 F Methyl ester atorvastatin 1.13 -- -- 7 G Acetyl simvastatin 1.55 1.03 ≤0.2％ 8 H Anhydrous simvastatin 1.59 0.96 ≤0.2％ 9 I Simvastatin dimer 2.31 0.98 ≤0.2％

The signal-to-noise ratio of simvastatin peak in the sensitive solution map is not lower than 10.

10. The method for assaying simvastatin related substances according to claim 1, wherein the method is specifically assayed as follows,

and (3) determination: under the condition that the applicability of the system meets the requirements, precisely measuring a reference solution and a test solution, injecting the reference solution and the test solution into a liquid chromatograph, and recording a chromatogram;

limitation: if an impurity peak exists in a chromatogram of a test solution, the content of the impurity A, B, C, D, E, G, H, I is calculated according to a self-comparison method of a main component added with a correction factor except a solvent peak, the content of each impurity should be less than the limit of the list, and any unknown single impurity peak area should be less than 0.1 time of the area of a main peak of a comparison solution; the sum of all impurity peak areas should not exceed 1.0 times of the main peak area of the control solution; with the exception of impurity C, D, E, less than 0.02% of the impurities were ignored.

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