CN116539765B

CN116539765B - Method for detecting related substances of teprenone capsules

Info

Publication number: CN116539765B
Application number: CN202310629397.XA
Authority: CN
Inventors: 张文杰; 李耀湘; 斯智萍; 李会; 刘佳伟; 黄雨佳
Original assignee: Zhejiang Heli Xinjian Pharmaceutical Co ltd
Current assignee: Zhejiang Heli Xinjian Pharmaceutical Co ltd
Priority date: 2023-05-30
Filing date: 2023-05-30
Publication date: 2023-10-20
Anticipated expiration: 2043-05-30
Also published as: CN116539765A

Abstract

The invention provides a method for detecting related substances of a teprenone capsule, which belongs to the technical field of medicine analysis, and adopts a high performance liquid chromatography, acetonitrile and water are used as mobile phases to detect two related substances which are not detected and controlled by the method at present in the teprenone capsule, wherein the related substances are teprenone single cis-isomer 5E,9E,13Z and teprenone single cis-isomer 5E,9Z,13E; then the total amount of the components is calculated and regulated by an area normalization method and is not more than 0.2%, so that the quality standard of the product is perfected, the quality of the product is controllable, and the safety and the effectiveness of the product are ensured.

Description

Method for detecting related substances of teprenone capsules

Technical Field

The invention relates to the technical field of medicine analysis, in particular to a method for detecting related substances of a teprenone capsule.

Background

The teprenone capsule has effects of resisting ulcer, increasing gastric mucus, increasing gastric mucosa prostaglandin, increasing and improving gastric mucosa blood flow, protecting gastric mucosa, maintaining stability of gastric mucosa cell proliferation region, inhibiting lipid peroxidation, and inducing cytoprotection caused by heat shock protein generation, and can be used for treating acute gastritis, chronic gastritis, and acute gastric mucosa lesion (erosion, hemorrhage, flushing, edema) improvement and gastric ulcer.

The teprenone compound is named as 6,10,14, 18-tetramethyl-5, 9,13, 17-nonatetralin-2-one, is a mixture of a single cis isomer (5Z, 9E, 13E) and an all trans isomer (5E, 9E, 13E), and has chemical structures shown in a formula I and a formula II respectively:

as is clear from the chemical structure, the product has 8 geometric isomers, the other 6 isomers except the main components 5Z,9E,13E and 5E,9E,13E are all impurities, and the chemical structure is shown in Table 1.

The above related substances may be produced during the process of synthesizing teprenone as a raw material and during the process of producing and storing teprenone capsules, and the conditions of the related substances of isomers in teprenone capsules are shown in the following table 1:

table 1 list of substances related to the teprenone isomers

Note 1: related substances of national drug standards (standard numbers JX20050119 and YBH 05822009) can detect and control tri-cis isomer impurities (relative retention time 0.7) and bi-cis isomer impurities (relative retention time 0.8) and control the total amount to be 1.0%;

and (2) injection: items of substances related to the Japanese pharmacopoeia standards (JP 17, JP18: teprenone) are detectable and control the content of the bisisomer impurity (relative retention time 0.8), controlling the total amount to 0.5%.

The detection methods of the related substances of the teprenone are carried by the national drug standards JX20050119 and YBH05822009, the detection methods are consistent, gas Chromatography (GC) and the same chromatographic conditions are adopted for detection, the total amount of the tri-cis isomer impurity at the relative retention time 0.7 and the total amount of the bi-cis isomer impurity at the relative retention time 0.8 are controlled to be 1.0%, but the response of the FID detectors is similar due to the similarity of the inter-isomer properties, so that the detection method of the national drug standards can only separate and detect the 4 related substances (tri-cis isomer ZZZ and bi-cis isomer ZEZ, EZZ, ZZE), but the other two single cis-isomer related substances (5E, 9E, 13Z) and (5E, 9Z, 13E) are overlapped with the main peaks (5Z, 9E, 13E), and the method cannot effectively separate the two single cis-isomer related substances and cannot detect and control the two single cis-isomer related substances.

The Japanese pharmacopoeia standards (JP 17, JP18: teprenone) also use gas chromatography, which is basically consistent with the above national standards, and also cannot separate and detect these two single cis-isomer related substances which coincide with the main peak.

At present, no suitable method is available at home and abroad for separating and detecting related substances of the two single cis-isomers, so that the quality control of the product has defects and needs to be improved.

Disclosure of Invention

The invention aims to provide a method for detecting related substances of a teprenone capsule, which can accurately and efficiently separate two related substances (5E, 9E, 13Z) and (5E, 9Z, 13E) from main components under a liquid phase condition: under the condition of the liquid phase, the impurities (5E, 9E, 13Z) and (5E, 9Z, 13E) are overlapped to form a peak, and the separation degree from the main peak of the API is good; therefore, the total amount of the two single cis-isomer impurities is not more than 0.2% by combining and detecting the two single cis-isomer impurities, the quality control of the product is perfected, the quality of the product is ensured, the quantitative limit of the detection method is 0.02%, the detection limit is 0.01%, and the method has high sensitivity.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a detection method of related substances in a teprenone capsule, which adopts a high performance liquid chromatography to detect the related substances in the teprenone capsule, and calculates the content of the related substances in the teprenone capsule according to an area normalization method;

the related substances are a teprenone single cis isomer (5E, 9E, 13Z) and a teprenone single cis isomer (5E, 9Z, 13E);

the mobile phase in the high performance liquid chromatography is a mixed solution of acetonitrile and water.

Preferably, the ratio of acetonitrile to water is 60-70:30-40.

Preferably, the flow rate of the mobile phase is 0.8-1.2 mL/min.

Preferably, the chromatographic column in the high performance liquid chromatography uses silica gel with amylose-tris- (3, 5-dimethylphenylcarbamate) coated on the surface as a filler.

Preferably, the column model is Chiralpak AD-RH, 4.6X250 mm,5 μm.

Preferably, the temperature of the chromatographic column is 25-35 ℃.

Preferably, the detector in the high performance liquid chromatography is an ultraviolet detector, and the detection wavelength of the ultraviolet detector is 208-212 nm.

Preferably, the test solution in the high performance liquid chromatography is a solution obtained by mixing teprenone capsule fine powder and acetonitrile; the concentration of the teprenone in the test solution is 0.8-1.2 mg/mL.

Preferably, the system applicability solution in the high performance liquid chromatography is a mixed solution obtained by mixing a mixture of 4 isomer reference substances of teprenone and acetonitrile;

the 4 isomer reference mixture of teprenone comprises a teprenone tri-cis isomer impurity ZZZ, a bi-cis isomer impurity ZEZ, a bi-cis isomer impurity EZZ and a mono-cis isomer impurity EEZ.

Preferably, the concentration of the teprenone in the mixed solution is 0.8-1.2 mg/mL, and the concentration of the teprenone 4 isomer reference substance mixture is 8-12 mug/mL.

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

the invention provides a method for detecting related substances of a teprenone capsule, which adopts a high performance liquid chromatography method, takes acetonitrile and water as mobile phases, performs combined detection on two single cis-isomer impurities which are not detected and controlled by the method at present in the teprenone capsule, controls the total quantity of the single cis-isomer impurities to be not more than 0.2%, improves the quality control of products, ensures the controllable quality of the products, and ensures the safety and effectiveness of the products.

The invention adopts the detection method of the impurity reference substance contrast, the separation degree, the specificity, the quantitative limit, the detection limit, the linearity, the precision and the like of the detection method are verified in detail, and all verification results meet the requirements of relevant regulations and guidelines, and the actual detection effect is good.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is an HPLC chart of a blank adjuvant solution for specificity of example 1;

FIG. 2 is an HPLC chart of a mixed solution of 2 isomers in the specificity of example 1; wherein the impurities represented by the peaks are respectively: 1-ZZE;2-EZE;

FIG. 3 is an HPLC chart of a mixture of 4 isomers in the specificity of example 1; wherein the impurities represented by the peaks are respectively: 1-ZZZ;2-ZEZ;3-EZZ;4-EEZ;

FIG. 4 is an HPLC chart of 8 isomer mixtures in the specificity of example 1; wherein each peak represents an isomer of: 1-ZZZ;2-ZEZ;3-ZZE and EZZ; 4-target impurities EZE and EEZ;5-ZEE (major component); 6-EEE (principal component);

FIG. 5 is an HPLC chart of the impurity localization and separation degree investigation solution in example 1; wherein each peak represents an isomer of: 1-ZZZ;2-ZEZ;3-ZZE and EZZ; 4-target impurities EZE and EEZ;5-ZEE (major component); 6-EEE (principal component);

FIG. 6 is an HPLC chart of the detection limit in example 2; wherein each peak represents an isomer of: 1-ZEE (major component); 2-EEE (principal component);

FIG. 7 is an HPLC chart of the limit of quantitation in example 2; wherein each peak represents an isomer of: 1-ZEE (major component); 2-EEE (principal component);

FIG. 8 is a graph of the linear relationship of teprenone in example 2;

FIG. 9 is a GC diagram of a solution for investigating the separation degree of relevant substances in the detection method of the Teprenone capsules according to the Chinese medicine standard (standard number YBH 05822009) of comparative example 1; the isomers represented by each peak were: 1-ZZZ;2-ZEZ and ZZE;3-EZZ; 4-main peak and target impurity three-peak are combined: ZEE (principal component), EZE and EEZ;5-EEE (principal component).

Detailed Description

The invention provides a method for detecting related substances of a teprenone capsule, which adopts a high performance liquid chromatography to detect the related substances of the teprenone capsule and calculates the content of the related substances in the teprenone capsule according to an area normalization method.

In the present invention, the related substances are teprenone single cis isomer (5E, 9E, 13Z) and teprenone single cis isomer (5E, 9Z, 13E);

in the present invention, a mixed solution of acetonitrile and water is used as a mobile phase, and the volume ratio of acetonitrile to water in the mixed solution is preferably 70:30 to 60:40, more preferably 62 to 68:32 to 38, and even more preferably 65:35.

In the present invention, the column is preferably silica gel coated with amylose-tris- (3, 5-dimethylphenylcarbamate) as a filler.

In the present invention, the column model is preferably Chiralpak AD-RH, 4.6X108 mm,5 μm.

In the present invention, the temperature of the column is preferably 25 to 35 ℃, more preferably 28 to 32 ℃, and even more preferably 30 ℃.

In the present invention, the flow rate of the mobile phase is preferably 0.8 to 1.2mL/min, more preferably 1.0 to 1.2mL/min, and still more preferably 1.0mL/min.

In the present invention, the column operation time is preferably 100 to 200min, more preferably 150 to 200min, and still more preferably 150min.

In the present invention, the detector in the high performance liquid chromatography is preferably an ultraviolet detector.

In the present invention, the detection wavelength of the ultraviolet detector is preferably 208 to 212nm, more preferably 210 to 212nm, and still more preferably 210nm.

In the invention, the test solution in the high performance liquid chromatography is a solution obtained by mixing teprenone capsule fine powder and acetonitrile; the concentration of teprenone in the test solution is 0.8 to 1.2mg/mL, more preferably 1.0 to 1.2mg/mL, and still more preferably 1.0mg/mL.

In the invention, the system applicability solution in the high performance liquid chromatography is a mixed solution obtained by mixing a mixture of 4 isomer reference substances of teprenone, a teprenone reference substance and acetonitrile;

In the present invention, the concentration of teprenone in the mixed solution is preferably 0.8 to 1.2mg/mL, more preferably 1.0 to 1.2mg/mL, and even more preferably 1.0mg/mL.

In the present invention, the concentration of the 4 isomer control mixture of teprenone in the mixed solution is preferably 8 to 12. Mu.g/mL, more preferably 10 to 12. Mu.g/mL, and still more preferably 10. Mu.g/mL.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Specialization of

Specific information of the reagent instrument used in the embodiment of the invention is as follows:

1. control information:

teprenone 4 isomer control mixture: tri-cis isomer impurity ZZZ, bi-cis isomer EZZ, bi-cis isomer impurity ZEZ, mono-cis isomer impurity EEZ;

teprenone 2 isomer control mixture: a dual cis-isomer impurity ZZE, a single cis-isomer impurity EZE;

teprenone 8 isomer control mixture: tri-cis isomer (ZZZ), bi-cis isomer (ZZE, EZZ, ZEZ), mono-cis isomer (ZEE, EZE, EEZ), all-trans isomer (EEE).

2. Solution preparation

Impurity localization solution:

2 isomer control mixtures: weighing a proper amount of a mixture consisting of 2 isomer reference substances of the teprenone, and diluting the teprenone with acetonitrile to prepare a solution with the concentration of the mixture of about 1 mg/mL;

4 isomer control mixtures: weighing a proper amount of a mixture consisting of 4 isomer reference substances of the teprenone, and diluting the teprenone with acetonitrile to prepare a solution with the concentration of the mixture of about 1 mg/mL;

8 isomer control mixtures: and weighing a proper amount of a mixture consisting of 8 isomer reference substances of the teprenone, and diluting the teprenone with acetonitrile to prepare a solution with the concentration of the mixture of about 1 mg/mL.

Degree of separation solution:

and (3) placing a proper amount of a mixture consisting of the teprenone reference substance and the 8 isomer reference substances into the same measuring flask, and diluting with acetonitrile to prepare a solution containing teprenone with the concentration of about 1mg/mL and a mixture containing the 8 isomer reference substances with the concentration of about 10 mug/mL.

Blank auxiliary material solution: taking a proper amount of blank auxiliary materials (including silicon dioxide, corn starch and the like), and adding acetonitrile to dilute the blank auxiliary materials to prepare a blank auxiliary material solution.

3. High performance liquid chromatography conditions:

instrument: SHIMADZU high performance liquid chromatograph, waters high performance liquid chromatograph;

chromatographic column: silica gel with amylose-tris- (3, 5-dimethylphenylcarbamate) coated on the surface is used as a filler, and the model of the chromatographic column is Chiralpak AD-RH, 4.6X250 mm and 5 mu m;

mobile phase: a mixed solution of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 65:35;

flow rate: 1.0mL/min;

wavelength: 210nm;

run time: 180min;

column temperature: 30 ℃;

sample injection amount: 20. Mu.L;

according to the chromatographic conditions, precisely measuring 20 mu L of each of the blank auxiliary material solution, the impurity positioning solution and the separation degree solution, injecting into a liquid chromatograph, and recording the chromatograms, wherein the specific results are shown in Table 2 and figures 1-5.

TABLE 2 specific results

As is clear from Table 2 and FIGS. 1 to 5, the peaks of the target impurities (EZE, EEZ) are hardly separated from each other and substantially overlap with each other to form one peak, so that the two target impurities are combined and calculated to control the total amount thereof to be within 0.2%. The target impurity can be completely separated from the single cis isomer (ZEE) and other impurities in the main component, and the specificity is good.

Example 2

Quantitative limit and detection limit

Chromatographic conditions

The chromatographic conditions for high performance liquid chromatography were the same as in example 1.

The 3 single cis-isomers of teprenone have similar properties and are correspondingly basically consistent, so that the sensitivity is examined by single cis (ZEE) with lower response in the main component of teprenone (a mixture of EEE and ZEE).

Taking a proper amount of teprenone reference substance, precisely weighing, adding acetonitrile for dissolving and gradually diluting to a proper concentration, and when the signal-to-noise ratio of single cis isomer (ZEE) of teprenone is about 10:1 and 3:1, obtaining the quantitative limit and the detection limit concentration.

20. Mu.L of the solution was precisely measured and injected into a liquid chromatograph under the chromatographic conditions described in example 1, and the chromatogram was recorded. The results are shown in Table 3, FIGS. 6-7.

TABLE 3 quantitative limit and detection limit results

Project	Concentration (μg/mL)	Corresponding to the main component	S/N
				Quantitative limit	0.20	0.02％	14.76
Detection limit	0.10	0.01％	8.38

As a result of the detection, the limit of detection of the single cis isomer of teprenone (ZEE) was 0.10. Mu.g/mL, and the limit of quantification was 0.20. Mu.g/mL.

Example 3

Linearity and range

Chromatographic conditions

The single cis isomer impurity in this example is consistent with the main component response, so that the principal component of teprenone is examined linearly, the linear concentration range is examined from quantitative limit to concentration of 1mg/mL, the limit concentration is 200 mug/mL in this example, the linear range is equivalent to 0.1% -500% of the range of the limit concentration, wherein 0.1% is the quantitative limit, 1mg/mL is 500% of the limit concentration, and the examination results are shown in the linear relation diagrams of Table 4 and FIG. 8.

Stock solution: accurately weighing a proper amount of teprenone reference substance, adding acetonitrile for dissolving and diluting to prepare a solution with the concentration of about 1mg/mL as stock solution.

And precisely measuring a proper amount of stock solution, and respectively adding acetonitrile to dilute the stock solution to prepare a series of concentrations which are equivalent to 0.1%, 0.25%, 1%, 2.5%, 5%, 50%, 100%, 250% and 500% of the limit concentration of the teprenone as linear solutions. Each 20 μl of the above solution was precisely measured and injected into a liquid chromatograph, and the chromatogram was recorded.

Table 4 results of linear investigation

The results show that: the teprenone main component has good linear relation within the concentration range of 0.20-988.53 mug/mL, the linear regression equation is y=57, 688.4650x-33,378.9716, and the correlation coefficient r is 0.9999.

Example 4

1. Precision of

Chromatographic conditions

Control stock solution: and taking a proper amount of a mixture consisting of 8 isomer reference substances, adding acetonitrile for dissolving and diluting to prepare a solution with the mixture concentration of about 0.1 mg/mL.

Test solution: taking a proper amount of teprenone fine powder (about equivalent to 50mg of teprenone), placing into a 50mL measuring flask, precisely adding 5mL of reference stock solution, adding acetonitrile, ultrasonically shaking to dissolve and dilute the teprenone fine powder to a scale, shaking uniformly, filtering, and taking a subsequent filtrate. 6 parts of the mixture were prepared in the same manner.

20 μl of the sample solution was measured precisely and injected into the liquid chromatograph, the chromatogram was recorded, and the single cis-isomer (EZE, EEZ) content was calculated by area normalization method, and the results are shown in Table 5 below.

TABLE 5 repeatability test results

The results showed that the isomer measurement was performed on 6 samples, and the single cis-isomer (EZE, EEZ) content was calculated by area normalization method, and the single cis-isomer (EZE, EEZ) RSD in 6 samples was 1.8%, indicating good reproducibility of the method.

2. Intermediate precision

The various laboratory workers, at various times, performed intermediate precision tests with liquid chromatography, measured the single cis-isomer (EZE, EEZ) content as described above, and analyzed and compared with the results of the repeatability test measurements, the results are given in table 6 below.

TABLE 6 results of intermediate precision test

The results show that the single cis-isomer (EZE, EEZ) content is calculated according to the area normalization method by taking the same sample from different persons at different times, and the single cis-isomer (EZE, EEZ) RSD in 12 samples is 2.2%, which indicates that the method has good intermediate precision.

Example 5

Inspection of finished products

Chromatographic conditions

The chromatographic conditions were the same as in example 1 using high performance liquid chromatography.

Test solution: and taking a proper amount of fine powder (about 50mg equivalent to teprenone) of three batches of teprenone capsule contents produced in a production workshop, placing the three batches of teprenone capsule contents in a 50mL measuring flask, adding acetonitrile, carrying out ultrasonic vibration to dissolve and dilute the teprenone fine powder to a scale, shaking uniformly, filtering, and taking a subsequent filtrate, wherein the teprenone concentration in the test solution is about 1.0mg/mL.

System applicability solution: taking a mixture of 4 isomer controls of teprenone and a proper amount of teprenone controls, adding acetonitrile to dissolve and dilute the teprenone controls to prepare a solution with the concentration of about 10 mug/mL in the mixture of 4 isomer controls of teprenone, wherein the concentration of teprenone is about 1.0mg/mL.

And respectively precisely measuring 20 mu l of system applicability solution and 20 mu l of sample solution to be tested, injecting into a liquid chromatograph, recording a chromatogram, and calculating target impurities according to an area normalization method: total amount of single cis-isomer (EZE, EEZ). The results are shown in Table 7 below.

TABLE 7 results of finished product inspection

Lot number	Total amount of single cis isomer (EZE, EEZ) related substances
		200401	0.12％
200501	0.09％
		200502	0.14％

The results show that the impurity content of single cis-isomer (EZE, EEZ) of 3 batches of finished products is less than 0.2%, and the results meet the requirements.

In summary, examples 1-5 show that the method for detecting the single cis-isomer in the teprenone capsule provided by the invention has the characteristics of simplicity and convenience in operation, high precision and good separation degree, and is suitable for detecting the single cis-isomer in the teprenone capsule.

Comparative example 1

According to the detection method of the related substances of the teprenone by adopting a gas chromatography method according to the national drug standard (standard number JX 20050119), taking a teprenone system applicability reference substance (4 isomer mixtures ZZZ, EZZ, ZEZ, EEZ), 2 isomer mixture reference substances (ZZE, EZE), 8 isomer mixture reference substances (ZZZ, EZZ, ZEZ, EEZ, ZZE, EZE, ZEE, EEE), an isomer (ZZE), a main component reference substance (ZEE) and a main component reference substance (EEE), respectively diluting with normal hexane to prepare a solution with the concentration of about 1mg/mL to serve as an impurity positioning solution, taking a teprenone and a proper amount of each impurity positioning solution, and diluting with normal hexane to prepare a solution with the concentration of about 2.5mg/mL of teprenone and the impurity concentration of about 100 mug/mL to serve as a separation degree investigation solution. The results are shown in tables 8-9 and FIG. 9:

table 8 positioning solution retention time in comparative example 1

Table 9 retention time and relative retention time for the separation investigation in comparative example 1

As can be seen from tables 8-9 and fig. 9, the detection method under the related substances of the national drug standard JX20050119 teprenone, the single cis isomer impurity (EZE and EEZ) of the target impurity and the ZEE isomer of the main component are overlapped to form a peak, and cannot be separated, so that the detection and control of the single cis isomer target impurity cannot be performed.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The method for detecting the relevant substances of the teprenone capsules is characterized by detecting the relevant substances of the teprenone capsules by adopting a high performance liquid chromatography, and calculating the content of the relevant substances in the teprenone capsules by adopting an area normalization method;

the related substances are teprenone single cis isomer 5E,9E,13Z and teprenone single cis isomer 5E,9Z,13E;

the conditions of the high performance liquid chromatography are as follows:

flow rate: 1.0mL/min;

the detector is an ultraviolet detector, the wavelength: 210nm;

run time: 180min;

column temperature: 30 ℃;

sample injection amount: 20. Mu.L.

2. The detection method according to claim 1, wherein the test solution in the high performance liquid chromatography is a solution obtained by mixing fine powder of a teprenone capsule with acetonitrile; the concentration of the teprenone in the test solution is 0.8-1.2 mg/mL.

3. The detection method according to claim 1, wherein the system applicability solution in the high performance liquid chromatography is a mixture of 4 isomer control substances of teprenone, and a mixed solution obtained by mixing the teprenone control substances with acetonitrile;

4. The method according to claim 3, wherein the concentration of teprenone in the mixed solution is 0.8-1.2 mg/mL; the concentration of the teprenone 4 isomer reference substance mixture is 8-12 mug/mL.