An impurity detection method for piracetam injection

Abstract

The present invention relates to the field of drug impurities, and particularly to a method for detecting impurities in piracetam injection, comprising the following steps: S1, preparing a test solution, wherein the test solution includes: a test sample solution, a control solution, a system suitability solution, and a sensitivity solution; S2, detecting the contents of various impurities in the test solution of step S1 by using a liquid chromatography system; S3, obtaining the contents of various impurities in piracetam according to the chromatogram of the test solution, and comparing the contents of various impurities with preset values to determine the quality of piracetam drugs. The method of the present invention can improve the detection ability of various process impurities and degradation impurities, especially the detection of new impurity E, solves the technical problems of impurity detection and quantification in piracetam injection preparations, improves the drug quality, and reduces its potential harm to the human body.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical impurities, and particularly to a method for detecting impurities in piracetam injection. Background Art

[0002] Piracetam is a cyclic derivative of γ-aminobutyric acid (GABA), and is the earliest marketed nootropic drug. It has a protective effect on brain hypoxia injury, promotes the synthesis of acetylcholine and enhances the conduction of nerve excitation, has a promoting effect on brain metabolism, and can improve the cognitive impairment of patients. Its chemical name is 2-oxo-1-pyrrolidineacetamide, the molecular formula is C6H 10 N2O2, and the structural formula is:

[0003] Piracetam injection was developed by UCB Pharma and has been successively launched in many countries in Europe, the Middle East, Latin America and Asia for improving memory disorders, dizziness, decreased attention, mood instability, etc. At present, this original research product has not been imported into China. Domestic piracetam injection was first launched in China in the 1980s and is clinically used for memory decline and mild to moderate brain dysfunction caused by various reasons such as acute and chronic cerebrovascular diseases, brain trauma, and various toxic encephalopathies, and is also used for children's mental retardation. It is used in single dose, and the administration routes are intramuscular injection, intravenous injection and drip infusion.

[0004] The 2020 edition of the Chinese Pharmacopoeia includes the piracetam injection standard not included in other countries. This standard describes the detection method of the impurities of this drug and the limit control of the total impurities in related substances (1.0%), but does not control the limit of individual impurities, and the related substances detection method included also cannot detect the degradation impurity D (2-oxo-1-pyrrolidineacetic acid) and the new impurity E (2,5-dioxo-1-pyrrolidineacetic acid); the EP / BP Pharmacopoeia includes the piracetam bulk drug standard, but this standard also cannot detect the new impurity E, which may affect the stability and safety of the product. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting impurities in piracetam injection, which can improve the detection ability of process impurities and degradation impurities, especially the detection of the new impurity E, solve the technical problems of impurity detection and quantification in piracetam injection, improve the drug quality, and reduce its potential harm to the human body.

[0006] The present invention provides a method for detecting impurities in piracetam injection, comprising the following steps:

[0007] S1. Prepare a test solution, and the test solution includes: a test article solution, a control solution, a system suitability solution and a sensitivity solution;

[0008] S2. Use a liquid chromatography system to detect the content of each impurity in the test solution in step S1;

[0009] S3. Obtain the content of each impurity in piracetam based on the chromatogram of the test solution, and compare the content of each impurity with the preset value to determine the quality of the piracetam drug.

[0010] Preferably, the impurities include one or more of: 2-pyrrolidone, methyl 2-oxo-1-pyrrolidineacetate, ethyl 2-oxo-1-pyrrolidineacetate, 2-oxo-1-pyrrolidineacetic acid, 2,5-dioxo-1-pyrrolidineacetic acid.

[0011] Preferably, the preparation step of the test solution in step S1 is specifically: take an appropriate amount of piracetam injection, dilute it with water to prepare a solution containing 0.5 mg of piracetam per 1 ml.

[0012] Preferably, the preparation step of the control solution in step S1 is specifically: take an appropriate amount of the prepared test solution, dilute it with water to prepare a solution containing 2.5 μg of piracetam per 1 ml.

[0013] Preferably, the preparation step of the sensitivity solution in step S1 is specifically: take an appropriate amount of the prepared control solution, dilute it with water to prepare a solution containing 0.25 μg of piracetam per 1 ml.

[0014] Preferably, the preparation step of the system suitability solution in step S1 is specifically: take an appropriate amount of piracetam injection, 2-pyrrolidone, and 2-oxo-1-pyrrolidineacetic acid, dissolve and dilute them with water to prepare a mixed solution containing 0.5 mg of piracetam, 0.5 μg of 2-pyrrolidone, and 5 μg of 2-oxo-1-pyrrolidineacetic acid per 1 ml.

[0015] Preferably, the chromatographic column used in the liquid chromatography system in step S2 is ODS C18, and gradient elution is performed with mobile phase A and mobile phase B.

[0016] Preferably, mobile phase A is a phosphate buffer solution, and mobile phase B is methanol.

[0017] Preferably, the detection conditions of the liquid chromatography system in step S2 are: detection wavelength 210 nm, column temperature 30 - 33 °C, flow rate 1.0 - 0.1 mL / min, injection volume 5 - 20 μL.

[0018] Preferably, in the chromatogram of the system suitability solution in step S3, the resolution between the piracetam peak and the adjacent peak should not be less than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main peak should be not less than 10.

[0019] Beneficial effects:

[0020] The technical solution of the present invention uses a liquid chromatography system to detect impurities in the test solution (test sample solution, control solution, system suitability solution, and sensitivity solution), which can effectively detect the related impurities in piracetam injection, especially the detection of new impurity E, improving the detection ability of process impurities and degradation impurities in piracetam injection, solving the technical problems of impurity detection and quantification in piracetam injection, and thus effectively controlling the quality of piracetam injection and reducing its potential harm to the human body. Brief Description of the Drawings

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the chromatogram of the piracetam injection sample in Example 1 of the present invention;

[0023] Figure 2 It is the chromatogram of the system suitability solution in Example 2 of the present invention;

[0024] Figure 3 It is the chromatogram of the sensitivity solution in Example 2 of the present invention;

[0025] Figure 4 It is the chromatogram of the impurity mixed solution in Example 2 of the present invention;

[0026] Figure 5 It is the chromatogram of the impurity E quantitation limit solution in Example 2 of the present invention;

[0027] Figure 6 It is the chromatogram of the impurity mixed solution in Comparative Example 1 of the present invention;

[0028] Figure 7 It is the chromatogram of the impurity mixed solution in Comparative Example 2 of the present invention. Detailed Description of the Invention

[0029] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application pertains. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular forms also include the plural forms. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0030] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0031] I. Impurity Detection Method for Piracetam Injection in Example 1

[0032] This example provides an impurity detection method for piracetam injection, and the experimental steps are as follows:

[0033] Step 1: Prepare the test solutions, which include: the test sample solution, the control solution, the system suitability solution, and the sensitivity solution. The specific preparation methods for each solution are as follows:

[0034] (1) Test sample solution: Take an appropriate amount of piracetam and dilute it with water to prepare a solution containing about 0.5 mg of piracetam per 1 ml.

[0035] (2) Control solution: Accurately measure an appropriate amount of the test sample solution and dilute it with water to prepare a solution containing about 2.5 μg of piracetam per 1 ml.

[0036] (3) Sensitivity solution: Accurately measure an appropriate amount of the control solution and quantitatively dilute it with water to prepare a solution containing about 0.25 μg of piracetam per 1 ml.

[0037] (4) System suitability solution: Take appropriate amounts of piracetam, impurity A reference substance, and impurity D reference substance, dissolve and dilute them with water to prepare a mixed solution containing about 0.5 mg of piracetam, 0.5 μg of impurity A, and 5 μg of impurity D per 1 ml.

[0038] Among them, impurity A is 2-pyrrolidone, and impurity D is 2-oxo-1-pyrrolidineacetic acid.

[0039] Step 2: Use a liquid chromatography system to detect the contents of each impurity in the test solutions prepared in Step 1.

[0040] The detection method is as follows: A sampling needle sucks 10 μL of the sample solution and injects it into the injection port of the liquid chromatograph to detect the impurities in the sample solution. The parameters are as follows:

[0041] The detection wavelength is 210 nm; the column temperature is 30 - 33 °C; the mobile phase flow rate is 1.0 - 0.1 mL / min;

[0042] The chromatographic column is ODS C18, and the mobile phases A and B are phosphate buffer solution (pH = 6.0) and methanol respectively, and elute according to the concentration gradient in Table 1.

[0043] Table 1 Elution conditions

[0044] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 7.0 90 10 35.0 75 25 35.1 90 10 45.0 90 10

[0045] Step 3: Obtain the contents of various impurities of piracetam from the chromatogram of the solution to be tested, and compare the contents of various impurities with the preset values (refer to the requirements for the limits of related substances in piracetam injection or bulk drug substances included in the Chinese Pharmacopoeia and EP / BP Pharmacopoeias) to determine the quality of the piracetam drug.

[0046] Using the impurity detection method of the piracetam injection in this example, a piracetam injection sample (specification 5 mL: 1 g) was detected. The detection results are shown in the chromatogram of the piracetam injection sample ( Figure 1 ) and Table 2.

[0047] Table 2 Detection results of the piracetam injection sample

[0048]

[0049] Chromatogram of the piracetam injection sample ( Figure 1 ) and Table 2 results show that: Impurities A, B, and C were not detected in the piracetam injection sample, the detected amount of impurity D was 0.08%, and the piracetam injection sample met the requirements for the limits of related substances in piracetam injection or bulk drug substances included in the Chinese Pharmacopoeia and EP / BP Pharmacopoeias; in addition, a new impurity E was detected by the impurity detection method of the piracetam injection in this example, with a detected amount of 0.03%, and the new impurity E could not be detected by the existing piracetam detection methods.

[0050] II. Related verification tests for the impurity detection method of piracetam injection in Example 2

[0051] (1) System suitability test

[0052] Inject 10 μL each of the system suitability solution and the sensitivity solution into the liquid chromatograph. The detection results are shown in the chromatogram of the system suitability solution ( Figure 2 ) and the chromatogram of the sensitivity solution (Figure 3 ) and Table 3.

[0053] Table 3 Results of System Suitability Test

[0054]

[0055] Chromatogram of System Suitability Solution ( Figure 2 ) and the results in Table 3 show that the resolution (≥1.5) between the piracetam peak and impurity A is 3.969, and the resolution (≥1.5) between the piracetam peak and impurity D is 2.459; the chromatogram of the sensitivity solution ( Figure 3 ) and the results in Table 3 show that the signal-to-noise ratio (≥10) of the piracetam peak is 42.79, meeting the requirements of system suitability.

[0056] (2) Specificity Test

[0057] Inject 10 μl each of the blank solution, test solution, control solution, impurity E location solution, and impurity mixed solution into the liquid chromatograph. The specific preparation methods for each solution are as follows:

[0058] Blank solution: Take water to obtain it.

[0059] Preparation of stock solution of impurity A reference substance: Take 10 mg of impurity A reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with water and dilute to the mark. Take 1 ml and place it in a 100 ml volumetric flask, dilute it with water to the mark, and shake well to obtain it.

[0060] Preparation of stock solution of impurity B reference substance: Take 10 mg of impurity B reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with water and dilute to the mark. Take 1 ml and place it in a 100 ml volumetric flask, dilute it with water to the mark, and shake well to obtain it.

[0061] Preparation of stock solution of impurity C reference substance: Take 10 mg of impurity C reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with water and dilute to the mark. Take 1 ml and place it in a 100 ml volumetric flask, dilute it with water to the mark, and shake well to obtain it.

[0062] Preparation of stock solution of impurity D reference substance: Take 15 mg of impurity D reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with water and dilute to the mark. Take 1 ml and place it in a 10 ml volumetric flask, shake well to obtain it.

[0063] Preparation of stock solution of impurity E reference substance: Take 15 mg of impurity E reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it with a small amount of methanol, dilute it with water to the mark, and shake well to obtain it.

[0064] Impurity E location solution: Take 1 ml of the stock solution of impurity E reference substance, place it in a 100 ml volumetric flask, dilute it with water to the mark, and shake well to obtain it.

[0065] Impurity mixed solution: Take 1 ml of piracetam injection and place it in a 100-ml volumetric flask. Dilute it to the mark with water; then take 5 ml from the 100-ml volumetric flask and place it in a 20-ml volumetric flask. Add 2 ml of the reference substance stock solution of impurity A, 2 ml of the reference substance stock solution of impurity B, 2 ml of the reference substance stock solution of impurity C, 2 ml of the reference substance stock solution of impurity D, and 2 ml of the reference substance stock solution of impurity E. Dilute it to the mark with water and shake well to obtain the solution.

[0066] Among them, impurity A is 2-pyrrolidone, impurity B is methyl 2-oxo-1-pyrrolidineacetate, impurity C is ethyl 2-oxo-1-pyrrolidineacetate, impurity D is 2-oxo-1-pyrrolidineacetic acid, and impurity E is 2,5-dioxo-1-pyrrolidineacetic acid.

[0067] Table 4 Results of specificity test

[0068]

[0069] Chromatogram of impurity mixed solution ( Figure 4 ) and the results in Table 4 show that: the blank solution does not interfere with the elution of each known impurity in the test solution and the impurity mixed solution; the resolution between piracetam and the adjacent impurity and each known impurity in the impurity mixed solution meets the requirements, indicating that the method has good specificity.

[0070] (3) Sensitivity test

[0071] Take the impurity E localization solution (prepared with reference to the preparation method of the impurity E localization solution in the specificity test), and dilute it step by step to make the signal-to-noise ratio approximately equal to 10, which is the limit of quantitation concentration. Inject 10 μl into the liquid chromatograph.

[0072] Table 5 Results of sensitivity test

[0073]

[0074] Chromatogram of impurity E limit of quantitation solution ( Figure 5 ) and the results in Table 5 show that: the limit of quantitation concentration of impurity E is 0.0151 μg / ml, which is lower than the reporting limit concentration (0.25 μg / ml), proving that the method has sufficient sensitivity for the detection of impurity E.

[0075] (4) Accuracy test

[0076] After the system suitability meets the requirements, accurately measure the blank solution, test solution, control solution, reference substance solution, and each spiked recovery solution, and inject them into the liquid chromatograph respectively. The specific preparation methods of the reference substance solution and each spiked recovery solution are as follows:

[0077] Reference solution: Take 1 ml of the reference stock solution of piracetam, place it in a 50-ml volumetric flask, dilute it to the mark with water, take 1 ml and place it in a 20-ml volumetric flask, dilute it to the mark with water, and shake well to obtain the solution.

[0078] Test sample stock solution: Take 5 ml of piracetam injection, place it in a 20-ml volumetric flask, dilute it to the mark with water, and shake well.

[0079] Reference solution for impurity E: Take 1 ml of the reference stock solution of impurity E, place it in a 20-ml volumetric flask, dilute it to the mark with water, and shake well. Spiked recovery solution (1): Accurately measure 1 ml of the test sample stock solution and 1 ml of the reference solution for impurity E, place them in the same 100-ml volumetric flask, dilute it to the mark with water, and shake well.

[0080] Spiked recovery solution (2): Accurately measure 1 ml of the test sample stock solution and 2 ml of the reference solution for impurity E, place them in the same 100-ml volumetric flask, dilute it to the mark with water, and shake well.

[0081] Spiked recovery solution (3): Accurately measure 1 ml of the test sample stock solution and 3 ml of the reference solution for impurity E, place them in the same 100-ml volumetric flask, dilute it to the mark with water, and shake well.

[0082] Calculate the recovery rate of impurity E by the external standard method based on the peak area. The test results are shown in Table 5. The test results show that: the average recovery rates of impurity E in 9 test samples at the concentration levels of 50%, 100%, and 150% are between 98.1% and 100.3%, and the RSD (coefficient of variation) is 1.30%, indicating that the accuracy of this method is good.

[0083] Table 6 Test results of the accuracy of impurity E

[0084]

[0085] III. Comparative Example 1 is detected by the detection method of piracetam injection in Pharmacopoeia ChP2020

[0086] The piracetam impurity mixed solution in Example 2 is detected according to the detection method of piracetam raw material / injection standard in Pharmacopoeia ChP2020. The test results are shown in the chromatogram of the piracetam impurity mixed solution ( Figure 6 ) and Table 7.

[0087] Table 7 Test results of the piracetam impurity mixed solution

[0088]

[0089] Chromatogram of the impurity mixed solution ( Figure 6) and the results in Table 7 show that: the standard detection methods for piracetam raw material / injection in Pharmacopoeia ChP2020 can only detect Impurity A and Impurity B, and cannot detect Impurity C, Impurity D, and Impurity E.

[0090] IV. Comparative Example 2 is detected by the detection method for piracetam raw material in Pharmacopoeia EP / BP

[0091] The piracetam impurity mixed solution in Example 2 is detected according to the standard detection method for piracetam raw material in Pharmacopoeia EP / BP. The detection results are shown in the chromatogram of the piracetam impurity mixed solution ( Figure 7 ) and Table 8.

[0092] Table 8 Detection Results of Piracetam Impurity Mixed Solution

[0093]

[0094] Chromatogram of the impurity mixed solution ( Figure 7 ) and the detection results in Table 8 show that: the standard detection method for piracetam raw material in Pharmacopoeia EP / BP also cannot detect Impurity E.

[0095] In summary, in the method of the present invention, an ODS C18 chromatographic column is used, a mixture of phosphate buffer solution and methanol is used as the mobile phase, and gradient elution is carried out through the mobile phase, so as to effectively detect piracetam and related impurities (Impurity A, Impurity B, Impurity C, Impurity D, and Impurity E). In particular, the detection of the new impurity E has not been detected in the existing piracetam detection methods at present, thereby effectively controlling the quality of piracetam injection and reducing its potential harm to the human body.

[0096] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An impurity detection method for piracetam injection, characterized in that, It includes the following steps: S1. Prepare the test solution, which includes: the test sample solution, the control solution, the system suitability solution, and the sensitivity solution; S2. Use a liquid chromatography system to detect the content of each impurity in the test solution in step S1; S3. Obtain the content of each impurity of piracetam according to the chromatogram of the test solution, and judge the quality of the piracetam drug by comparing the content of each impurity with the preset value; The impurities include: 2-pyrrolidone, methyl 2-oxo-1-pyrrolidineacetate, ethyl 2-oxo-1-pyrrolidineacetate, 2-oxo-1-pyrrolidineacetic acid, and 2,5-dioxo-1-pyrrolidineacetic acid; In step S2, the chromatographic column used in the liquid chromatography system is ODS C18, and gradient elution is performed with mobile phase A and mobile phase B; The mobile phase A is a phosphate buffer solution, and the mobile phase B is methanol; Specifically, elute according to the concentration gradient in the following table: 。 2. The impurity detection method for piracetam injection according to claim 1, characterized in that, The specific preparation steps of the test sample solution in step S1 are as follows: Take an appropriate amount of piracetam injection, and dilute it with water to prepare a solution containing 0.5 mg of piracetam per 1 ml.

3. The impurity detection method for piracetam injection according to claim 2, characterized in that, The specific preparation steps of the control solution in step S1 are as follows: Take an appropriate amount of the prepared test sample solution, and dilute it with water to prepare a solution containing 2.5 μg of piracetam per 1 ml.

4. The impurity detection method for piracetam injection according to claim 3, characterized in that, The specific preparation steps of the sensitivity solution in step S1 are as follows: Take an appropriate amount of the prepared control solution, and dilute it with water to prepare a solution containing 0.25 μg of piracetam per 1 ml.

5. The impurity detection method for piracetam injection according to claim 2, characterized in that, The specific preparation steps of the system suitability solution in step S1 are as follows: Take an appropriate amount of piracetam injection, 2-pyrrolidone, and 2-oxo-1-pyrrolidineacetic acid, dissolve and dilute them with water to prepare a mixed solution containing 0.5 mg of piracetam, 0.5 μg of 2-pyrrolidone, and 5 μg of 2-oxo-1-pyrrolidineacetic acid per 1 ml.

6. The impurity detection method for piracetam injection according to claim 1, characterized in that, The detection conditions of the liquid chromatography system in step S2 are: detection wavelength 210 nm, column temperature 30 ± 3 °C, flow rate 1.0 ± 0.1 mL / min, injection volume 5 - 20 μL.

7. The impurity detection method for piracetam injection according to claim 1, characterized in that, In the chromatogram of the system suitability solution in step S3, the resolution between the piracetam peak and the adjacent peak is not less than 1.5; in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main peak is not less than 10.

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