CN116429937A

CN116429937A - Detection method for related substances in pirenzepine bulk drug and application thereof

Info

Publication number: CN116429937A
Application number: CN202310386938.0A
Authority: CN
Inventors: 颜世强; 侯慧杰; 曹焕英; 解春文; 王鹏; 于晓燕; 何淑旺
Original assignee: Beijing Dayin High Tech Children Medicine Research Institute Co ltd; Shandong Dyne Marine Biopharmaceutical Co Ltd
Current assignee: Beijing Dayin High Tech Children Medicine Research Institute Co ltd; Shandong Dyne Marine Biopharmaceutical Co Ltd
Priority date: 2023-04-04
Filing date: 2023-04-04
Publication date: 2023-07-14

Abstract

The invention relates to a detection method of related substances in a pirenzepine bulk drug and application thereof. Belongs to the technical field of medicine analysis. The invention adopts high performance liquid chromatography to detect the sample by a DAD detector. The chromatographic column with octadecylsilane chemically bonded silica as stuffing adopts ammonium acetate water solution and acetonitrile solution in certain proportion as mobile phase for gradient elution. Through comprehensive methodological verification, the method can effectively separate and measure related substances of the pirenzepine raw material medicine of the process, and further control the quality of the pirenzepine raw material.

Description

Detection method for related substances in pirenzepine bulk drug and application thereof

Technical Field

The invention belongs to the technical field of medicine quality control, and particularly relates to a detection method of related impurities in a pirenzepine raw material medicine and a pirenzepine raw material quality control method based on the detection method.

Background

The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.

Pirenzenenaphthalene (PER) is a third generation new type antiepileptic drug that exerts an antiepileptic effect mainly by acting on the alpha-aminomethylol oxazolopropionic acid (AMPA) receptor. The FDA approval PER in 2012 was used to treat focal epileptic seizure patients 12 years old and older, and the new indications obtained in 2021 in China were used to treat partial seizure patients in adults and children 4 years old and older.

At present, no detection method for related substances of the pirenzepine bulk drug is carried in the domestic and foreign pharmacopoeias. Patent CN109342603a discloses a method for detecting substances related to pirenzepine tablet; patent CN109799298A discloses a method for detecting related substances of a pirenzepine bulk drug.

Aiming at the current research situation, the current pirenzepine product process route of the inventor is different from the process, so that the impurity spectrum of the raw material medicine has large difference, and the existing detection method cannot meet the quality control requirement of the raw material medicine of the pirenzepine product.

Disclosure of Invention

The invention aims to provide a detection method suitable for a product of a pirenzenenaphthalene bulk drug, and the types of impurities mainly contained in the product of the pirenzenenaphthalene bulk drug are impurities introduced in a process and degradation impurities according to the determination, wherein the types of the impurities are shown in the following table 1:

TABLE 1 sample and impurity control information

In order to meet the detection analysis and quality control of the raw materials of the medicines, the invention provides the following technical scheme:

in a first aspect, a method for detecting related substances in a pirenzenenaphthalene bulk drug is provided, the method adopts liquid chromatography to quantitatively detect the pirenzenenaphthalene bulk drug, the detection target is pirenzenenaphthalene and impurity components related to table 2, the liquid chromatography adopts octadecylsilane chemically bonded silica gel as a stationary phase, mobile phase A is ammonium acetate solution with pH of 3-5 and 0.08-0.12%, and mobile phase B is acetonitrile, and the liquid chromatography is separated by gradient elution.

In a preferred embodiment, the gradient elution procedure is as follows:

the initial proportion, the mobile phase A is 88-92%, and the mobile phase B is 8-12%;

0-10 min, the mobile phase A is changed to 53-57% at constant speed, and the mobile phase B is 43-47%;

10-35 min, the flow phase ratio is kept unchanged;

the mobile phase A is changed to 18 to 22 percent at constant speed within 35 to 50 minutes, and the mobile phase B is 78 to 82 percent;

50-55 min, the flow phase ratio is kept unchanged;

55-56 min, the mobile phase A is changed to 88-92% at uniform speed, and the mobile phase B is 8-12%;

the flow ratio is kept unchanged for 56-60 min.

In one specific embodiment of the column, inertisl ODS-3V 250 mm. Times.4.6 mm,5 μm; other chromatographic columns of comparable performance can also be adapted by the person skilled in the art.

Further, the mobile phase A is 0.1% ammonium acetate solution, the pH is 4.0, and glacial acetic acid can be used for adjusting the pH of the mobile phase A.

In some other embodiments, the detection method further comprises setting other parameters of the liquid chromatograph, such as flow rate, column temperature, wavelength, etc.; in a specific example, the flow rate: 1.0ml/min; column temperature: 25 ℃; wavelength: 290nm.

The detection method also comprises the preparation of a reference substance and a test sample, and the detection method is obtained by taking acetonitrile-ammonium acetate solution as a solvent and fixing the volume. The solvents of the test sample and the reference sample can be prepared by adopting mobile phases or the two mobile phases are mixed, the mixing proportion can be adjusted according to the dissolution effect of impurities, and the solvents specifically comprise acetonitrile: the volume ratio of the 0.1% ammonium acetate solution is 60:40.

the preparation method of the reference substance is as follows: and (3) taking a sample to be measured, adding acetonitrile for dissolution, diluting by using a mobile phase A, and fixing the volume.

The preparation method of the test sample is as follows: and adding acetonitrile into a sample to be measured for dissolution, and then adding a solvent for dilution and volume fixing to obtain the sample.

The concentrations of the reference substance, the sample to be detected in the sample and the impurity can be adjusted according to the proportion of the medicine and the impurity in the sample, the detection system and other factors, and the method belongs to technical content which can be determined conventionally by a person skilled in the art. In the actual detection process, in order to ensure that the detection system can stably run under the above conditions, the preparation of a system applicability solution and a sensitivity solution may be involved, wherein the system applicability solution comprises pirrennet and one or a part of impurity reference substances, and the sensitivity solution is a diluted solution of the reference substances.

One specific embodiment of the present invention is as follows:

the solvent is acetonitrile-0.1% ammonium acetate solution (pH value is adjusted to 4.0 by glacial acetic acid) (60:40);

test solution: taking a proper amount of a sample to be measured, precisely weighing, adding 60% acetonitrile with a fixed volume, dissolving, and quantitatively diluting with a mobile phase A to prepare a solution containing about 1mg in each 1 ml;

control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with solvent, and shaking;

system applicability solution: taking a proper amount of each of the pirenzepine reference substance and one reference substance, dissolving in a solvent, and diluting to prepare a mixed solution containing about 1mg of the pirenzepine and 0.01mg of impurities in each 1 ml;

sensitivity solution: a proper amount of the control solution is precisely measured, and the control solution is quantitatively diluted by adding a solvent to prepare a solution with about 0.2 mug in each 1 ml.

According to the second aspect of the invention, a quality control method of the pirenzepine bulk drug is provided, a reference substance and a test substance are prepared according to the method in the first aspect, liquid phase detection is carried out on the reference substance and the test substance to obtain drug and impurity content information, and the main component self-reference method is adopted to calculate the impurity content.

The beneficial effects of the above technical scheme are:

the first aspect of the invention provides a method for detecting substances related to a pirenzepine bulk drug, which can be completed by liquid phase detection, and is simple to operate; through methodological verification, the detection method has good specificity, quantitative limit and detection limit, linearity, sample injection precision, accuracy, precision and durability, can meet the quality control requirement of the pirenzepine bulk drug, and has important significance for ensuring the drug quality of the pirenzepine.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

FIG. 1 is a blank solvent chromatogram;

FIG. 2 is a chromatogram of a test solution;

FIG. 3 is a chromatogram of a sample solution;

FIG. 4 is a graph of impurity J linear relationship;

FIG. 5 is a linear relationship diagram of impurity I;

FIG. 6 is a linear relationship diagram of impurity F;

FIG. 7 is a graph of impurity E in linear relationship;

FIG. 8 is a linear relationship of pirenzepine;

FIG. 9 is a graph of impurity G linear relationship;

FIG. 10 is a graph of impurity A in linear relationship;

FIG. 11 is a graph of impurity H in linear relationship;

FIG. 12 is a graph of impurity C in linear relationship;

fig. 13 is a linear relationship diagram of impurity B.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

As described in the background art, the current production process of pirenzenenaphthalene implemented by the inventor is different from the prior art, so that the impurity spectrum of crude raw medicines is greatly different, and the provision of a corresponding quality control method has important significance for the current research situation. In order to solve the technical problems, the invention provides a method for detecting a pirenzenenaphthalene bulk drug and related substances.

The specific information of the detection object related to the invention is as follows:

table 2 table of pirenzenepamil and the respective impurity information

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.

The main devices involved in the following examples are as follows:

1260, available from Agilent corporation;

DAD detector, purchased from agilent company;

electronic analytical balances, available from mertler company;

inertisl ODS-3V column (250X 4.6mm 5 μm), available from Shimadzu;

example 1

The methodological test conditions for the detection method of this example are as follows:

instrument: agilent 1260 liquid chromatograph

A detector: DAD (digital versatile disc)

Mobile phase a: with 0.1% ammonium acetate solution (pH adjusted to 4.0 with glacial acetic acid);

mobile phase B: acetonitrile;

flow rate: 1.0ml/min;

column temperature: 25 DEG C

Sample injection amount: 10 μl of

Chromatographic column: inertisl ODS-3V 250 mm. Times.4.6 mm,5 μm

Elution gradient:

1. specificity test

1.1 impurity localization and separation

(1) Solution preparation

A diluent: acetonitrile-0.1% ammonium acetate solution (pH adjusted to 4.0 with glacial acetic acid) (60:40)

The stock solutions of impurity A, impurity B, impurity C, impurity E, impurity F, impurity G, impurity H, impurity I and impurity J were each prepared as a 0.1mg/ml solution.

The positioning solutions of the impurity A, the impurity B, the impurity C, the impurity E, the impurity F, the impurity G, the impurity H, the impurity I and the impurity J are prepared into 0.001mg/ml solutions.

Test solution: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, diluted to a scale by using a mobile phase A, and shaken uniformly to obtain the product.

Sample addition test solution: about 10mg of the product is taken, put into a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 100 mu l of each of impurity C, impurity E, impurity F, impurity G, impurity H, impurity I and impurity J stock solutions, 50 mu l of each of impurity A and impurity B stock solutions, diluted to scale by using a mobile phase A, and shaken uniformly to obtain the product.

Control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with a diluent, and shaking uniformly.

System applicability solution: taking about 10mg of a pirenzepine reference substance, placing in a 10ml measuring flask, adding 6ml of acetonitrile for dissolution, adding 1ml of an impurity G stock solution, diluting to a scale with a mobile phase A, and shaking uniformly to obtain the pirenzepine reference substance.

Sensitivity solution: precisely measuring 1ml of control solution, placing in a 50ml measuring flask, diluting to a scale with a diluent, and shaking uniformly.

(2) Test results

TABLE 3 separation degree investigation test results

Conclusion: the retention time of the impurity A and the retention time of the impurity B are overlapped, and the impurity A and the impurity B are controlled according to one impurity; the separation degree between the impurities and the main peak meets the requirements.

1.2 destructive testing

(1) Sample preparation

(1) Acid damage: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with about 1ml of 4mol/L hydrochloric acid solution, shaken well, placed at 60 ℃ for 52 hours, then added with 6ml of acetonitrile for dissolution, diluted to scale by mobile phase A, and shaken well to serve as acid damage test sample solution.

The same method is used for preparing an acid damage blank solution: about 1ml of 4mol/L hydrochloric acid solution is taken, placed in a 10ml measuring flask, 6ml of acetonitrile is added, diluted to scale with mobile phase A, and shaken well.

(2) Alkali destruction: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with 6ml of acetonitrile for dissolution, added with 1ml of 1mol/L sodium hydroxide solution, shaken well, placed at 60 ℃ for 48 hours, diluted to scale by using mobile phase A, and shaken well to be used as a solution of a sample to be destroyed by alkali.

The alkali damage blank solution is prepared by the same method: about 1ml of 1mol/LNaOH solution was taken, placed in a 10ml measuring flask, 6ml of acetonitrile was added, diluted to scale with mobile phase A, and shaken well.

(3) Oxidative destruction: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with about 1ml of 30% hydrogen peroxide solution, shaken well, placed at room temperature for 52 hours, then added with 6ml of acetonitrile for dissolution, diluted to scale by mobile phase A, and shaken well to serve as a sample solution for oxidative damage.

Preparing an oxidation damage blank solution by the same method: about 1ml of 30% hydrogen peroxide solution is taken, placed in a 10ml measuring flask, 6ml of acetonitrile is added, diluted to scale with mobile phase A, and shaken well.

(4) High temperature damage: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with 1ml of water, placed in a 60 ℃ oven for 8 days, added with 6ml of acetonitrile for dissolution, diluted to a scale by using a mobile phase A, and shaken uniformly to be used as a solution of a sample to be destroyed at a high temperature.

(5) Light damage: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with 1ml of water, placed in an illumination box with the illuminance of 4500 lx+/-500 lx for 8 days, added with 6ml of acetonitrile for dissolution, diluted to a scale by using a mobile phase A, and shaken uniformly to be used as a light damage test sample solution.

(6) High temperature damage (solid state): about 10mg of the sample is taken, precisely weighed, placed in a 10ml measuring flask, placed in an oven at 60 ℃ for 8 days, added with 6ml of acetonitrile, dissolved, diluted to a scale by using a mobile phase A, and shaken uniformly to be used as a solution of a sample to be destroyed at a high temperature.

(7) Photodisruption (solid state): about 10mg of the sample is taken, precisely weighed, placed in a 10ml measuring flask, placed in an illumination box with the illuminance of 4500 lx+/-500 lx for 8 days, added with 6ml of acetonitrile, shaken to dissolve the sample, diluted to a scale by using a mobile phase A, and shaken uniformly to be used as a light damage test sample solution.

(8) Unbroken: about 10mg of the sample is taken, precisely weighed, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, diluted to a scale by using a mobile phase A, and shaken uniformly to obtain a sample solution without damage.

(2) Experimental results

TABLE 4 results of destructive testing

Conclusion: (1) the blank solvent has no interference to the measurement of the substance related to the pirenzenenaphthalene under each condition. (2) The degradation impurity peaks and main peaks generated under the conditions of acid, alkali, oxidation and illumination damage of the solution can be completely separated, and the separation degree among the degradation peaks meets the requirements. (3) Under the illumination condition, although the solid of the product has the generation of new impurities, the content of each impurity is lower and the separation degree of the solid of the product from the main peak meets the requirement. (4) Under each damage condition, the material balance is 95-105%, and the material is conserved. (5) The purity threshold of the main peak under each destruction condition is more than 995, and the purity of the peak meets the requirement. In conclusion, the method has good specificity and can effectively detect various impurities.

2 detection limit and quantitative limit

And diluting the impurity stock solution with diluent, wherein the solution can be used as a quantitative limiting solution when the detected signal/baseline noise (S/N) is more than or equal to 10, and can be used as a detection limiting solution when the detected signal/baseline noise (S/N) is more than or equal to 3.

TABLE 5 quantitative limit test results

TABLE 6 detection limit test results

Conclusion: the quantitative limit concentration of each impurity and the pirenzenenaphthalene is smaller than the reporting limit concentration, the signal to noise ratio is between 8 and 20, the peak area RSD of each impurity and the pirenzenenaphthalene of 6 repeated samples is not more than 15%, the retention time RSD is not more than 2%, and the quantitative limit meets the requirements; the signal to noise ratio of the detection limit also meets the requirements.

3 linearity and range

(1) Sample preparation

The stock solution of the pirenzepine reference substance is the same as the stock solution of the pirenzepine reference substance under the item of '2 detection limit and quantitative limit'.

Impurity a, impurity B, impurity C, impurity E, impurity F, impurity G, impurity H, impurity I, impurity J liquid stock solution: stock solutions of the respective impurities under the heading "1.1 separation degree" were examined.

Linear A1 test solution: precisely measuring 0.2ml of impurity A stock solution, 0.15ml of impurity C stock solution, 0.15ml of impurity E stock solution, 0.1ml of impurity F stock solution, 0.2ml of impurity G stock solution, 0.15ml of impurity H stock solution, 0.08ml of impurity J stock solution and 0.1ml of pirenzenep control stock solution, placing into a 100ml measuring flask, and adding a diluent for dilution to a scale.

Linear A2 test solution: precisely measuring 0.5ml of each of impurity A, impurity C, impurity E, impurity F, impurity G, impurity H, impurity J and pirenzenepamil stock solution, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking.

Linear A3 test solution: precisely measuring 1.0ml of each of impurity A, impurity C, impurity E, impurity F, impurity G, impurity H, impurity J and pirenzenepamil stock solution, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking.

Linear A4 test solution: precisely measuring 1.5ml of impurity A, impurity C, impurity E, impurity F, impurity G, impurity H, impurity J and pirenzenenapestrap stock solution respectively, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking.

Linear A5 test solution: precisely measuring 2.0ml of impurity A, impurity C, impurity E, impurity F, impurity G, impurity H, impurity J and pirenzenenapestrap stock solution respectively, placing into a 100ml measuring flask, diluting to scale with diluent, and shaking.

Linear B1 test solution: precisely measuring 0.15ml of impurity B stock solution and 0.08ml of impurity I stock solution, placing into a 100ml measuring flask, adding diluent, diluting to scale, and shaking.

Linear B2 test solution: precisely measuring 0.5ml of stock solution of the impurity B and the impurity I, placing into a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly.

Linear B3 test solution: accurately measuring 1.0ml of stock solution of the impurity B and the impurity I, placing into a 100ml measuring flask, adding a diluent to dilute to a scale, and shaking uniformly.

Linear B4 test solution: accurately measuring 1.5ml of stock solution of impurity B and impurity I, placing into a 100ml measuring flask, adding diluent, diluting to scale, and shaking.

Linear B5 test solution: accurately measuring 2.0ml of impurity B and impurity I stock solution, placing into a 100ml measuring flask, adding diluent, diluting to scale, and shaking.

(2) Test results

TABLE 7 impurity J Linear results

TABLE 8 impurity I Linear results

TABLE 9 Linear results for impurity F

TABLE 10 impurity E Linear results

Table 11 linear results of pirenzenepamine

TABLE 12 impurity G Linear results

TABLE 13 impurity A Linear results

TABLE 14 impurity H Linear results

TABLE 15 impurity C Linear results

TABLE 16 impurity B Linear results

Conclusion: the correlation coefficient (R) of each impurity and the pirenzenenaphthalene regression line is larger than 0.990, the Y-axis intercept is within 25% of the 100% response value, and the relative standard deviation of the response factor is not larger than 10%, so that the impurity and the pirenzenenaphthalene have good linearity within the concentration range of the quantitative limit to 200%.

Correction factor of 4

(1) Calculation method

Calculation of correction factors the standard curve method is chosen: namely, the ratio of the principal component regression line slope to the impurity regression line slope is expressed as follows:

wherein: slope of _Pirenpananel : slope of linear curve of peak area versus concentration of pirenzenenaphthalene

Slope of _Impurity(s) : linear curve slope of impurity peak area versus concentration

(2) Test results

TABLE 17 correction factors for impurities

Impurity(s)	Slope of	Correction factor
			Pirenpananel	35.0509	-
J	28.605	1.2
			I	28.6089	1.2
F	28.2944	1.2
			E	22.1478	1.6
G	32.226	1.1
			A	31.2358	1.1
H	31.8091	1.1
			C	29.2446	1.2
B	33.6362	1

5 accuracy degree

(1) Sample preparation

Impurity stock solution a: respectively taking about 10mg of each of the reference substances of the impurity A, the impurity C, the impurity E, the impurity F, the impurity G, the impurity H, the impurity I and the impurity J, putting the reference substances into a same 100ml measuring flask, dissolving and diluting the reference substances into scales by using acetonitrile, and shaking the mixture uniformly to obtain the finished product. 2 parts were prepared in parallel.

Impurity control solution a: accurately measuring 1ml of the impurity stock solution A, placing into a 10ml measuring flask, diluting to scale with diluent, and shaking.

50% level test solution a: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 50 μl of impurity stock solution A, diluted to scale with mobile phase A, and shaken well. Triplicate formulations were prepared.

100% level test solution a: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 100 mu l of impurity stock solution A, diluted to scale by mobile phase A, and shaken well. Triplicate formulations were prepared.

150% level test solution a: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 150 μl of impurity stock solution A, diluted to scale with mobile phase A, and shaken well. Triplicate formulations were prepared.

Impurity stock solution B: about 10mg of the impurity B reference substances are respectively taken and put into a same 100ml measuring flask, are dissolved and diluted to the scale by acetonitrile, and are uniformly shaken to obtain the product. 2 parts were prepared in parallel.

Impurity control solution B: accurately measuring 1ml of the impurity stock solution B, placing into a 10ml measuring flask, diluting to scale with diluent, and shaking.

50% level test solution B: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 50 μl of impurity stock solution B, diluted to scale with mobile phase A, and shaken well. Triplicate formulations were prepared.

100% level test solution B: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 100 mu l of impurity stock solution B, diluted to scale by mobile phase A, and shaken well. Triplicate formulations were prepared.

150% level test solution B: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 150 μl of impurity stock solution B, diluted to scale with mobile phase A, and shaken well. Triplicate formulations were prepared.

Blank test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, diluted to a scale with mobile phase A, and shaken well.

Control solution: precisely measuring 1ml of each concentration of sample solution, respectively placing into 100ml measuring flask, diluting to scale with diluent, and shaking.

(2) Test results

TABLE 18 impurity J accuracy results

TABLE 19 impurity I accuracy results

TABLE 20 impurity F accuracy results

TABLE 21 impurity E accuracy results

TABLE 22 impurity G accuracy results

TABLE 23 impurity A accuracy results

TABLE 24 impurity H accuracy results

TABLE 25 impurity C accuracy results

TABLE 26 impurity B accuracy results

Conclusion: the recovery rate of each impurity under different concentrations calculated by an external standard method and a correction factor adding main component self-comparison method meets the requirements, the RSD values of 9 recovery rates of each impurity are not more than 10%, the method meets the regulations, and the accuracy of the method is good.

6. Repeatability of

(1) Solution preparation

Impurity stock solution: and (3) examining the storage liquid of each impurity under the item of 1.1 separation degree.

Test solution: about 10mg of the product is taken, put into a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 100 mu l of each of impurity C, impurity E, impurity F, impurity G, impurity H, impurity I and impurity J stock solutions, 50 mu l of each of impurity A and impurity B stock solutions, diluted to scale by using a mobile phase A, and shaken uniformly to obtain the product. 6 parts were prepared in parallel.

(2) Test results

Table 27 repeatability results

Conclusion: in the six samples, the number of impurities is consistent, the RSD of each impurity and the total impurity content is not more than 20%, and the method has good repeatability.

7. Intermediate precision

(1) Sample preparation

Taking the same batch of samples, adopting different testers to test by adopting different instruments at different times according to the preparation method of the repeated test sample solution, carrying out intermediate precision test according to the repeated test operation, preparing six samples of the test sample solution in parallel, measuring relevant substances of the samples, and evaluating the intermediate precision of the method by combining the repeated test results.

(2) Test results

Table 28 intermediate precision results

Conclusion: in 12 samples, the number of impurities is consistent, the RSD of each impurity and the total impurity content is not more than 30%, and the method has good intermediate precision.

8. Solution stability

(1) Solution preparation

Impurity stock solution: and the impurity stock solutions under the same "1.1 selectivity" item.

Test article solution I: about 10mg of the product is taken, put into a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, precisely added with 100 mu l of each of the stock solutions of the impurity A, the impurity C, the impurity E, the impurity F, the impurity G, the impurity H, the impurity I and the impurity J, diluted to the scale by using the mobile phase A, and shaken uniformly to obtain the product.

Test solution II: about 10mg of the product is taken, placed in a 10ml measuring flask, added with 6ml of acetonitrile, shaken to dissolve the sample, 100 mu l of impurity B stock solution is diluted to scale by using a mobile phase A, and shaken uniformly to obtain the product.

(2) Test results

TABLE 29 solution stability test results

Conclusion: the impurity solutions and the control solution have good stability when being placed for 72 hours at room temperature.

9. Durability of

When the chromatographic conditions slightly change, the tolerance of the detection method is examined. Under the conditions of changing flow rate, column temperature, pH value and the like, the durability of the method on detection of related substances is examined.

(1) Solution formulation and parameter set-up

Test solution: repeating the test sample solution.

System applicability solution: and the system applicability solution under the item of 1.1 selectivity.

Table 30-method of analyzing substances verify-durability parameter Range

Under each chromatographic condition, precisely measuring 10 mu l of each of the system applicability solution, the sensitivity solution, the test sample solution and the control solution, respectively injecting into a liquid chromatograph, recording the chromatogram, evaluating the applicability of the chromatographic system, calculating the content of each impurity, and evaluating the tolerance degree of the method to the small variation of the parameters of the measurement conditions.

(2) Test results

Table 31 durability test results

Conclusion: the column temperature (+ -2 ℃) and the flow rate (+ -0.1 ml/min) are changed, the wavelength (+ -2 nm) and the pH value (3.9-4.2) of buffer salt are changed, the chromatographic columns with the same type and different batch numbers are changed, the separation degree among all impurities meets the requirements, the impurity content under all conditions is compared with the original conditions, the content RSD is within 5%, and no obvious influence is caused on the result, so the method has good durability.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The method is characterized in that liquid chromatography is adopted to quantitatively detect the pirenzepine bulk drug, and the detection target is the pirenzepine and the following impurities:

impurity A:3- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridine ] -5' -yl) benzonitrile,

Impurity B:4- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -yl) benzonitrile,

Impurity C:6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -ylbenzene,

Impurity E:2- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [3,3 '-bipyridine ] -5' -yl) benzonitrile,

Impurity F:2- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [4,3 '-bipyridine ] -5' -yl) benzonitrile,

Impurity G:2- (6 '-oxo-1' - (o-tolyl) -1',6' -dihydro- [2,3 '-bipyridine ] -5' -yl) benzonitrile,

Impurity H:2- (6 '-oxo-1' - (p-tolyl) -1',6' -dihydro- [2,3 '-bipyridine ] -5' -yl) benzonitrile,

Impurity I:2- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -yl) benzamide,

Impurity J:2- (6 '-oxo-1' -phenyl-1 ',6' -dihydro- [2,3 '-bipyridyl ] -5' -yl) benzoic acid;

the liquid chromatography adopts octadecylsilane chemically bonded silica as a stationary phase, mobile phase A is ammonium acetate solution with pH of 3-5 and 0.08-0.12%, and mobile phase B is acetonitrile, and the liquid chromatography is separated by gradient elution.

2. The method for detecting related substances in a pirenzepine drug substance according to claim 1, wherein the gradient elution procedure is as follows:

the mobile phase A changes to 53-57% at constant speed within 0-10 min, and the mobile phase B changes to 43-47;

10-35 min, the flow phase ratio is kept unchanged;

50-55 min, the flow phase ratio is kept unchanged;

the flow ratio is kept unchanged for 56-60 min.

3. The method for detecting related substances in a pirenzepine drug substance according to claim 2, wherein,

the gradient elution procedure was as follows:

the initial proportion, mobile phase A is 90% and mobile phase B is 10%;

0-10 min, the mobile phase A changes to 55% at uniform speed, and the mobile phase B is 45;

10-35 min, the flow phase ratio is kept unchanged;

the mobile phase A is changed to 20% at constant speed for 35-50 min, and the mobile phase B is 80%;

50-55 min, the flow phase ratio is kept unchanged;

55-56 min, the mobile phase A is changed to 90% at uniform speed, and the mobile phase B is 10%;

the flow ratio is kept unchanged for 56-60 min.

4. The method for detecting related substances in a pirenzenenapestrap drug substance according to claim 1, wherein the chromatographic column adopts Inertisl ODS-3V 250mm x 4.6mm,5 μm;

alternatively, mobile phase a is a 0.1% ammonium acetate solution, pH 4.0.

5. The method for detecting a substance of interest in a drug substance of pirenzepine as set forth in claim 1, wherein the flow rate of said liquid phase is: 1.0ml/min; column temperature: 25 ℃; wavelength: 290nm.

6. The method for detecting related substances in a pirenzenenape bulk drug according to claim 1, which is characterized by further comprising the steps of preparing a reference substance and a test substance, and adopting acetonitrile-ammonium acetate solution as a solvent to obtain the pirenzenenape bulk drug by constant volume;

the preparation method of the reference substance is as follows: taking a sample to be measured, adding acetonitrile for dissolution, diluting by a mobile phase A, and fixing the volume;

the preparation method of the test sample is as follows: and adding acetonitrile into a sample to be measured for dissolution, and then adding an ethanol-ammonium acetate solution for dilution and volume fixing to obtain the sample.

7. The method for detecting related substances in a crude drug of pirenzepine according to claim 6, further comprising the steps of preparing a system applicability solution and a sensitivity solution, wherein the system applicability solution comprises pirenzepine and one or a part of impurity reference substances, and the sensitivity solution is a dilution solution of the reference substances.

8. The method for detecting related substances in a pirrennet drug substance according to claim 7, wherein the solvent is acetonitrile-0.1% ammonium acetate solution, and glacial acetic acid is used for adjusting the pH value to 4.0;

test solution: taking a proper amount of a sample to be measured, precisely weighing, adding 60% acetonitrile with a fixed volume, dissolving, and quantitatively diluting with a mobile phase A to prepare a solution containing 0.8-1.2 mg in each 1 ml;

system applicability solution: taking a proper amount of each of the pirenzepine reference substance and one reference substance, dissolving and diluting the mixture by adding a solvent to prepare a mixed solution containing 0.8-1.2 mg of the pirenzepine and 0.008-0.012 mg of impurities in each 1 ml;

sensitivity solution: precisely measuring a proper amount of control solution, adding solvent and quantitatively diluting to prepare a solution with the concentration of 0.1-0.3 mug in each 1 ml.

9. The method for detecting related substances in a pirenzeneb drug substance according to claim 1, wherein the correction factor of impurity a is 1.1, the correction factor of impurity B is 1, the correction factor of impurity C is 1.2, the correction factor of impurity E is 1.6, the correction factor of impurity F is 1.2, the correction factor of impurity G is 1.1, the correction factor of impurity H is 1.1, the correction factor of impurity I is 1.2 and the correction factor of impurity J is 1.2.

10. The quality control method of the pirenzepine bulk drug is characterized in that a control product and a test product are prepared according to the method of any one of claims 1-9, liquid phase detection is carried out to obtain impurity content information, and the main component self-control method is adopted to calculate the impurity content.