Method for detecting related impurities of piracetam by high performance liquid chromatography

Abstract

The invention discloses a method for detecting related impurities of piracetam by high performance liquid chromatography. The detection method comprises the following steps: preparing a test solution and diluting to obtain a contrast solution; respectively preparing reference substance solutions of impurities A, B, C and D as positioning solutions; under the same chromatographic condition, analyzing each positioning solution, and determining the retention time of an impurity peak; sequentially injecting the test solution and the contrast solution, identifying impurity peaks in the test solution according to retention time, integrating to obtain a peak area (Ai), and integrating a main peak of the contrast solution to obtain a peak area (Astd); the single impurity content (%) is calculated according to the formula: the single impurity content (%) = Ai / (Astd * D) * 100%, and D is the dilution ratio of the contrast solution relative to the test solution. The method has the advantages of strong specificity and high separation degree, and can effectively realize simultaneous separation and quantification of multiple impurities.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection, and in particular relates to a method for detecting piracetam-related impurities by high performance liquid chromatography. Background Technology

[0002] Piracetam, also known as pyrrolidone acetamide or pyrrolidone acetamide, is a derivative of gamma-aminobutyric acid (GABA). This drug activates, protects, and repairs brain cells, improves cerebral hypoxia, increases the ATP / ADP ratio in the brain, promotes the absorption of amino acids and phospholipids, protein synthesis, glucose utilization, and energy storage, thereby enhancing brain metabolism and increasing cerebral blood flow. Furthermore, it accelerates information transmission between the two hemispheres of the brain via the corpus callosum, improving learning, memory, and thinking abilities.

[0003] Clinically, piracetam is commonly used to treat memory and cognitive decline caused by cerebral arteriosclerosis and cerebrovascular accidents, promote the recovery of brain function in patients with carbon monoxide poisoning, and improve the intelligence of children with intellectual developmental delays. It also has some efficacy in treating Alzheimer's disease, senile dementia syndrome, and memory and cognitive impairment caused by traumatic brain injury. The structural formula of piracetam is as follows: .

[0004] In pharmaceutical quality research, the study, analysis, and detection of known drug-related substances are crucial aspects of drug quality control. Regarding the quality control of related substances in piracetam raw material, the United States Pharmacopeia (USP) uses thin-layer chromatography (TLC) for impurity limit testing, which has low sensitivity and cannot identify specific impurities. The 2025 edition of the Chinese Pharmacopoeia uses liquid chromatography (HPLC), controlling only impurities A and D. The 2020 edition of the Chinese Pharmacopoeia also uses HPLC, controlling both as unknown single impurities. The limited number of impurities controlled by the Chinese Pharmacopoeia is detrimental to its quality control. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting piracetam-related impurities using high-performance liquid chromatography (HPLC), thereby solving the problems of limited detectable impurity types and low sensitivity of existing detection technologies.

[0006] The objective of this invention is achieved through the following technical solution: A method for detecting piracetam-related impurities by high performance liquid chromatography includes the following steps: S1. Prepare the test solution and dilute it to obtain the control solution; prepare the control solutions for impurities A, B, C, and D respectively as impurity localization solutions; S2. Perform high performance liquid chromatography analysis on each impurity localization solution to determine the retention time of each impurity peak; S3. Under the same chromatographic conditions as described above, perform high-performance liquid chromatography (HPLC) analysis on the test solution and the control solution sequentially; based on the retention time determined in step S2, identify and integrate each impurity peak in the chromatogram of the test solution to obtain the peak area Ai of each impurity peak; integrate the main peak in the chromatogram of the control solution to obtain its peak area Astd; S4. The content of a single impurity is calculated according to the following formula: The content of a single impurity (%) = Ai / (Astd×D)×100%, where D is the dilution factor of the control solution relative to the test solution.

[0007] Preferably, the concentration of the test solution in step S1 is 0.5 mg / mL.

[0008] Preferably, the concentration of the solution in step S1 is 0.25 μg / mL.

[0009] Preferably, the concentration of the reference solutions for impurities A, B, C, and D in step S1 is 0.9~1.1 mg / mL.

[0010] In this invention, the chemical structures corresponding to impurities A, B, C, and D are as follows: Impurity A: 2-pyrrolidone, with the following chemical structural formula: ; Impurity B: methyl 2-oxo-1-pyrrolidine acetate, with the following chemical structure: ; Impurity C: 1-ethyl acetate-2-pyrrolidone, with the following chemical structure: ; Impurity D: (2-oxo-pyrrolidine-1-yl)-acetic acid, with the following chemical structural formula: .

[0011] Preferably, the concentration of the reference solutions for impurities A, B, C, and D in step S1 is 1.0 mg / mL.

[0012] Preferably, the detection parameters for the high-performance liquid chromatography analysis in step S2 are as follows: mobile phase A is a mixture of phosphoric acid, methanol, and water in a mass ratio of 2:100:900, and mobile phase B is acetonitrile; the gradient elution conditions are as follows: 0~5 min, mobile phase A:mobile phase B = 95:5; 5~20 min, mobile phase A:mobile phase B = 80:20; 20~22 min, mobile phase A:mobile phase B = 95:5; 22~40 min, mobile phase A:mobile phase B = 95:5; the detection wavelength is 205 nm, the mobile phase flow rate is 0.9~1.2 mL / min, and the injection volume is 10 μL.

[0013] Preferably, the phosphoric acid in the mobile phase A is commercially available pure phosphoric acid.

[0014] Preferably, the mobile phase flow rate is 1.0 mL / min.

[0015] Preferably, the high-performance liquid chromatography analysis in step S2 uses a chromatographic column with octadecylsilane-bonded silica gel as the stationary phase, and the column temperature is set to 28~32℃.

[0016] Preferably, the column temperature is set to 30°C.

[0017] Compared with the prior art, the beneficial effects of the present invention include: This invention provides a high-performance liquid chromatography method for detecting multiple related impurities in piracetam. This method has high specificity and high resolution, and can effectively achieve simultaneous separation and quantification of multiple impurities. Attached Figure Description

[0018] Figure 1 The image shows the liquid chromatogram of the test sample described in Example 1.

[0019] Figure 2 This is the liquid chromatogram of the control solution described in Example 1.

[0020] Figure 3 Chromatogram of mobile phase A for phosphoric acid-methanol-water (1:100:900).

[0021] Figure 4 Chromatogram of mobile phase A for phosphoric acid-methanol-water (2:100:900).

[0022] Figure 5 Liquid chromatography was performed for the blank solution and the system suitability solution, where the black curve corresponds to the system suitability solution and the purple curve corresponds to the blank solution. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] Example 1 A high-performance liquid chromatography (HPLC) method for detecting piracetam-related impurities comprises the following steps: (1) Chromatographic conditions Column: Stationary phase was octadecylsilane-bonded silica gel (4.6×250mm×5μm), column temperature was 30℃; Mobile phases: Mobile phase A: phosphoric acid-methanol-water (2:100:900); Mobile phase B: acetonitrile; Elution gradient: as shown in Table 1; Table 1 Elution gradient

[0025] Detection wavelength: 205 nm; Mobile phase flow rate: 1.0 mL / min; Injection volume: 10 μL.

[0026] (2) Sample preparation Solvent (blank solution): methanol-water (10:90).

[0027] Test solution: Take this product, dissolve it in a solvent and dilute it quantitatively to prepare a solution containing 0.5 mg of piracetam per 1 mL.

[0028] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with a solvent to prepare a solution containing 0.25 μg per 1 mL.

[0029] Locational solutions for each impurity: Take appropriate amounts of impurity A, impurity B, impurity C, and impurity D reference standards and prepare solutions with a concentration of 0.1 mg / mL.

[0030] System suitability solution: Accurately weigh 50 mg of piracetam reference standard, place it in a 100 mL volumetric flask, add an appropriate amount of solvent to dissolve it, accurately add 1 mL of each impurity positioning solution, dilute to the mark with solvent, and shake well.

[0031] System suitability requirements: In the system suitability solution, the elution order is piracetam, impurity A, impurity D, impurity B and impurity C, and the resolution between each peak should be greater than 1.5.

[0032] Assay: Accurately measure the test solution and the control solution, and inject them separately into the liquid chromatograph. The corresponding chromatograms are shown in the figure below. Figure 1 and Figure 2 .

[0033] Limits: If there are impurity peaks in the test solution, the peak areas of impurity A, impurity B, impurity C, and impurity D shall not exceed 1 times (0.05%) the area of ​​the main peak of the control solution; the peak area of ​​any other single impurity shall not exceed 1 times (0.05%) the area of ​​the main peak of the control solution; the sum of the peak areas of all impurities shall not exceed 6 times (0.3%) the area of ​​the main peak of the control solution, and those less than 0.6 times (0.03%) the area of ​​the main peak of the control solution can be ignored.

[0034] Analysis method development process: Because piracetam active pharmaceutical ingredient is weakly acidic, an acidic solution was chosen as the aqueous phase, and acetonitrile, with its strong eluting ability, was chosen as the organic phase. Referring to the methods for related substances of piracetam in the 2025 edition of the Chinese Pharmacopoeia, the detection wavelength was set at 205 nm. Referring to the mobile phase for related substances of piracetam in the 2020 edition of the Chinese Pharmacopoeia, the mobile phase is methanol-water (100:900).

[0035] Phosphoric acid was chosen to adjust the acidity of the mobile phase. The following is a system suitability chart for mobile phase A with different proportions of phosphoric acid.

[0036] Chromatogram of mobile phase A: phosphoric acid-methanol-water (1:100:900) is shown below. Figure 3 As shown; b: Chromatogram of mobile phase A: phosphoric acid-methanol-water (2:100:900) is shown below. Figure 4 As shown; according to Figures 3-4 We can see that in the data table, the larger the value under the Resolution (USP) item, the better the resolution. We mainly look at the resolution between impurity A and impurity D. The resolution under condition a is 4.678, which is less than the resolution under condition b (5.075). Therefore, we choose phosphoric acid-methanol-water (2:100:900) as the mobile phase A.

[0037] Example 2 Authenticity Verification The blank solution and system suitability solution were analyzed by liquid chromatography (the preparation of the blank solution and system suitability solution was the same as in Example 1, and the test conditions for liquid chromatography were the same as in Example 1). The corresponding chromatograms are shown below. Figure 5 As shown, the corresponding chromatographic data is shown in Table 2.

[0038] Table 2 Chromatographic data

[0039] See Figure 5 As shown in Table 2, the blank solvent does not interfere with the detection of the main component peak and impurity peak; the resolution between piracetam and adjacent impurities and each known impurity in the system suitability solution is greater than 1.5, indicating that the method has good specificity.

[0040] Example 3 Limit of Quantification Test Take appropriate amounts of impurity A, impurity B, impurity C, and impurity D reference standards, and gradually dilute them with blank solvent to S / N≈10 to obtain their respective limit of quantitation solutions. Detect them according to the chromatographic conditions described in Example 1. The results are shown in Table 3.

[0041] Table 3 Detection results of the limit of quantitation

[0042] As shown in Table 3, the limits of quantification for each impurity are all less than the reporting limit (0.03%), indicating that the method has high sensitivity.

[0043] Example 4 Sample injection precision test Take the system suitability solution and inject it 6 times according to the chromatographic conditions of Example 1. The chromatographic data results are shown in Table 4.

[0044] Table 4. Injection precision results

[0045] Referring to Table 4, we can see that the RSD of the peak area of ​​each impurity and the main component is ≤2.0%, and the RSD of the retention time is ≤2.0%, indicating good precision.

[0046] Example 5 Repeatability test Solution preparation: Solvent (blank solution): methanol-water (10:90).

[0047] Test solution: Take this product, dissolve it in a solvent and dilute it quantitatively to prepare a solution containing 0.5 mg of piracetam per 1 mL.

[0048] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with a solvent to prepare a solution containing 0.25 μg per 1 mL.

[0049] Locational solutions for each impurity: Take appropriate amounts of impurity A, impurity B, impurity C, and impurity D reference standards and prepare solutions with a concentration of 0.1 mg / mL.

[0050] Test steps: (1) Perform high performance liquid chromatography analysis on the solutions of each impurity (see Example 1 for chromatographic test conditions) to determine the retention time of each impurity peak; (2) Under the same chromatographic conditions as described above, the test solution and the control solution were analyzed by high performance liquid chromatography in sequence; according to the retention time determined in step (1), the peaks of each impurity in the chromatogram of the test solution were identified and integrated to obtain the peak area Ai of each impurity peak; the main peak in the chromatogram of the control solution was integrated to obtain its peak area Astd. (3) The content of a single impurity was calculated according to the following formula: The content of a single impurity (%) = Ai / (Astd×D)×100%, where D is the dilution factor of the control solution relative to the test solution.

[0051] The above test steps were repeated 6 times, resulting in 6 sets of test data. The test data are shown in Table 5. Among them, "maximum single impurity" refers to the one with the largest peak area among all other single impurities except impurities A, B, C and D.

[0052] Table 5 Repeatability Test Results

[0053] As shown in Table 5, the range of each impurity content does not exceed 0.05%, proving that the method has good repeatability.

[0054] Example 6 Solution stability test Take the test solution (prepared in the same way as in Example 1), and perform chromatographic testing at 5 time points according to the chromatographic conditions of Example 1. The test results are shown in Table 6.

[0055] Table 6 Solution stability test results

[0056] As shown in Table 6, the range of impurity content detected in the test solution within 23 hours, compared with 0 hours, was no more than 0.006%, the number of impurities was consistent, and the total impurities were basically consistent, indicating that the test solution was stable within 23 hours at room temperature.

[0057] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting piracetam-related impurities by high performance liquid chromatography, characterized in that, Includes the following steps: S1. Prepare the test solution and dilute it to obtain the control solution; prepare the control solutions for impurities A, B, C, and D respectively as impurity localization solutions; S2. Perform high performance liquid chromatography analysis on each impurity localization solution to determine the retention time of each impurity peak; S3. Under the same chromatographic conditions as described above, perform high-performance liquid chromatography (HPLC) analysis on the test solution and the control solution sequentially; based on the retention time determined in step S2, identify and integrate each impurity peak in the chromatogram of the test solution to obtain the peak area Ai of each impurity peak; integrate the main peak in the chromatogram of the control solution to obtain its peak area Astd; S4. The content of a single impurity is calculated according to the following formula: The content of a single impurity (%) = Ai / (Astd×D)×100%, where D is the dilution factor of the control solution relative to the test solution.

2. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 1, characterized in that, The concentration of the test solution in step S1 is 0.5 mg / mL.

3. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 1, characterized in that, The concentration of the solution in step S1 is 0.25 μg / mL.

4. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 1, characterized in that, The concentrations of the reference solutions for impurities A, B, C, and D in step S1 are all 0.9~1.1 mg / mL.

5. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 4, characterized in that, The concentration of the reference solutions for impurities A, B, C, and D mentioned in step S1 is 1.0 mg / mL.

6. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 1, characterized in that, The detection parameters for the high-performance liquid chromatography analysis described in step S2 are as follows: mobile phase A is a mixture of phosphoric acid, methanol, and water in a mass ratio of 2:100:900; mobile phase B is acetonitrile; gradient elution conditions are as follows: 0~5 min, mobile phase A:mobile phase B = 95:5; 5~20 min, mobile phase A:mobile phase B = 80:20; 20~22 min, mobile phase A:mobile phase B = 95:5; 22~40 min, mobile phase A:mobile phase B = 95:5; detection wavelength is 205 nm; mobile phase flow rate is 0.9~1.2 mL / min; injection volume is 10 μL.

7. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 6, characterized in that, The phosphoric acid in the mobile phase A is commercially available pure phosphoric acid.

8. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 6, characterized in that, The mobile phase flow rate was 1.0 mL / min.

9. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 1, characterized in that, The high-performance liquid chromatography analysis in step S2 uses a column with octadecylsilane-bonded silica gel as the stationary phase, and the column temperature is set to 28~32℃.

10. The method for detecting piracetam-related impurities by high performance liquid chromatography according to claim 9, characterized in that, The column temperature is set to 30°C.

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