High performance liquid chromatography determination method for polymer impurities in lipoic acid injection

Abstract

The invention provides a high performance liquid chromatography determination method for polymer impurities in a lipoic acid injection, and particularly relates to the field of medicines. The solvent is acetonitrile and water, and the volume ratio of the acetonitrile to the water is (29-31): (69-71); precisely measuring a proper amount of a to-be-detected product, and quantitatively diluting the to-be-detected product with a solvent to prepare a solution containing about 4mg of lipoic acid per 1ml; and precisely measuring a proper amount of the test solution, and quantitatively diluting the test solution with a solvent to prepare a solution containing about 8 [mu] g of lipoic acid per 1ml. Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent; a 0.02% phosphoric acid solution is used as a mobile phase A, acetonitrile and the 0.02% phosphoric acid solution are used as mobile phases B, the volume ratio of the acetonitrile to the phosphoric acid solution is (59-61): (39-41), and gradient elution is carried out; the flow rate is 0.9 to 1.1 ml per minute; the detection wavelength is 214 to 216 nm; the column temperature is 24-26 DEG C; the sample injection volume is 20 microliters; precisely measuring the test solution and the contrast solution, respectively injecting the test solution and the contrast solution into a liquid chromatograph, and recording chromatograms; the method is high in specificity, high in sensitivity and simple and convenient to operate.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a high performance liquid chromatography determination method for polymer impurities in lipoic acid injection. Background Art

[0002] Lipoic acid is optically active, and its racemic form can undergo polymerization at relatively low temperatures. The polymerization is intensified under acidic catalysis and light conditions. Therefore, it is necessary to control the polymer in lipoic acid injection.

[0003] The European Pharmacopoeia (EP11.3), the British Pharmacopoeia (BP2023), and the United States Pharmacopoeia (USP2023) contain standards for polymers in lipoic acid APIs, all of which are controlled by thin-layer chromatography, but this method has poor specificity when determining polymers in lipoic acid injection. The National Drug Administration standard for lipoic acid injection uses high-performance liquid chromatography to determine polymers, and uses the molecular exclusion method of a gel chromatography column for detection, but this method has poor reproducibility. Summary of the invention

[0005] The invention aims to provide a high performance liquid chromatography method for determining polymer impurities in lipoic acid injection, which has strong specificity, high sensitivity and is easy to operate.

[0006] The present invention provides the following technical solutions:

[0007] A high performance liquid chromatography method for determining polymer impurities in lipoic acid injection, the steps are as follows:

[0008] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 29-31:69-71;

[0009] Test solution: Accurately measure an appropriate amount of the test product and quantitatively dilute it with a solvent to make a solution containing about 4 mg of lipoic acid per 1 ml;

[0010] Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with a solvent to make a solution containing approximately 8 μg of lipoic acid per 1 ml.

[0011] Chromatographic conditions: octadecylsilane bonded silica gel was used as filler; 0.02% phosphoric acid solution was used as mobile phase A, acetonitrile and 0.02% phosphoric acid solution were used as mobile phase B, the volume ratio of acetonitrile to phosphoric acid solution was 59-61:39-41, and gradient elution was performed;

[0012] The flow rate is 0.9-1.1 ml per minute; the detection wavelength is 214-216 nm; the column temperature is 24-26°C; the injection volume is 20 μl; accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0013] Preferably, the volume ratio of acetonitrile to water is 30:70.

[0014] Preferably, the volume ratio of acetonitrile to 0.02% phosphoric acid solution is 60:40.

[0015] Preferably, the flow rate is 1.0 ml per minute.

[0016] Preferably, the detection wavelength is 215 nm.

[0017] Preferably, the column temperature is 25°C.

[0018] Preferably, gradient elution is performed according to the following table:

[0019]

[0020] Beneficial effects:

[0021] The prior art has poor specificity and reproducibility in detecting polymers in lipoic acid injection. The present invention makes up for the shortcomings of the prior art and has the advantages of strong specificity, high sensitivity and simple operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0023] Figure 1 is the blank solvent chromatogram;

[0024] Figure 2 is the chromatogram of the control solution;

[0025] Figure 3 The chromatogram of the test solution. DETAILED DESCRIPTION

[0026] Example 1

[0027] Specificity test:

[0028] Blank solvent interference

[0029] The blank solvent was directly injected for testing to investigate whether it interfered with the determination of the polymer of this product.

[0030] Interference of blank excipients

[0031] The blank auxiliary material solution was sampled and tested to examine whether it interfered with the determination of the polymer of this product.

[0032] Location of impurities A and C

[0033] The reference solutions of impurity A (concentration of about 200ug / ml) and impurity C (concentration of about 500ug / ml) were injected and tested respectively to investigate whether they interfered with the determination of the polymer of this product.

[0034] Forced degradation test

[0035] The blank solvent, blank excipients and lipoic acid injection were subjected to forced degradation tests in acid, alkali, oxidation, high temperature and light, and their changes under various conditions were investigated, as follows:

[0036]

[0037] The test results are as follows:

[0038]

[0039]

[0040] Embodiment 2:

[0041] Injection precision test

[0042] Test standard: The relative standard deviation of the main peak area should be ≤2.0%; the relative standard deviation of the main peak retention time should be ≤1.0%.

[0043] Take 20 μl of each reference solution and number them as injection precision-1 to injection precision-6. The test conditions are as follows:

[0044] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 30:70; Test solution: accurately measure an appropriate amount of the test product, and dilute it with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the following table:

[0045]

[0046] The flow rate was 1.0 ml per minute; the detection wavelength was 215 nm; the column temperature was 25 °C; the injection volume was 20 μl; the test results are as follows:

[0047] Sample name Retention time (min) Peak area Injection precision -1 41.100 1.4022 Injection precision -2 41.100 1.4057 Injection precision -3 41.100 1.4096 Injection precision -4 41.103 1.4095 Injection precision -5 41.103 1.4050 Injection precision -6 41.097 1.4084 RSD 0.1％ 0.3％

[0048] From the above results, it can be seen that the retention time RSD of lipoic acid is less than 10%, and the peak area RSD is less than 2.0%. The method has good injection precision.

[0049] Example 3

[0050] Repeatability test: The test standard is that the RSD of the total amount of polymer in 6 samples should be ≤5.0%.

[0051] Take 20 μl of each test solution and number them as repeatability-1 to repeatability-6. The test conditions are as follows: solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 30:70; test solution: accurately measure an appropriate amount of the test product, and dilute it quantitatively with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; control solution: accurately measure an appropriate amount of the test solution, and dilute it quantitatively with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the following table;

[0052]

[0053] The flow rate was 1.0 ml per minute; the detection wavelength was 215 nm; the column temperature was 25 °C; the injection volume was 20 μl; the test results are as follows:

[0054] Sample name Total polymer Repeatability -1 0.18％ Repeatability -2 0.19％ Repeatability - 3 0.19％ Repeatability - 4 0.19％ Repeatability -5 0.19％ Repeatability - 6 0.18％ At-the-money 0.19％ RSD 2.4％

[0055] From the above experimental data, it can be seen that the RSD of the total amount of polymer in lipoic acid injection is less than 5.0%, which meets the limit requirement, indicating that the repeatability of this method is good.

[0056] Example 4

[0057] Intermediate precision: The test standard is 12 samples of total polymer RSD ≤ 10.0%.

[0058] Take 20 μl of the test solution and number them as repeatability-1A to repeatability-6A, intermediate precision-1 to intermediate precision-6. The test conditions are as follows:

[0059] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 30:70; Test solution: accurately measure an appropriate amount of the test product, and dilute it with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the following table:

[0060]

[0061] The flow rate was 1.0 ml per minute; the detection wavelength was 215 nm; the column temperature was 25 °C; the injection volume was 20 μl; the test results are as follows:

[0062]

[0063] The average value of the total amount of polymer in 12 test solutions of lipoic acid injection was 0.18%, and the RSD value was 7.8%, which was less than 10.0%, meeting the limit requirement, indicating that the intermediate precision of this method was good.

[0064] Example 5

[0065] Solution stability: The test standard is that the ratio of the peak area of ​​the last point of the control solution to 0h is between 98.0% and 102.0%, and the main peak area RSD ≤ 2.0%, which can be judged as stable. The absolute value change of the total polymer content of the last point of the test solution is ≤ 0.05%, and RSD% ≤ 5.0%, which can be judged as stable.

[0066] 20 μl of the control solution and the test solution were added and placed at room temperature and protected from light. The results showed that both the control solution and the test solution were stable within 25 hours.

[0067] Example 6

[0068] Durability test

[0069] Test condition 1 is as follows:

[0070] Take 20 μl of the test solution and number them a1 to a6 respectively. The test conditions are as follows:

[0071] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 29:69; Test solution: accurately measure an appropriate amount of the test product, and dilute it with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the table below;

[0072]

[0073] The flow rate was 0.9 ml per minute; the detection wavelength was 214 nm; the column temperature was 24 °C; the injection volume was 20 μl; the test results are as follows:

[0074] Sample name Total polymer a1 0.18％ a2 0.19％ a3 0.19％ a4 0.19％ a5 0.19％ a6 0.18％ Average 1 0.185％

[0075] The optimal parameters for test condition 2 are as follows:

[0076] Take 20 μl of the test solution and number them b1 to b6 respectively. The test conditions are as follows:

[0077] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 30:70; Test solution: accurately measure an appropriate amount of the test product, and dilute it with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the following table:

[0078]

[0079] The flow rate was 1.0 ml per minute; the detection wavelength was 215 nm; the column temperature was 25 °C; the injection volume was 20 μl; the test results are as follows:

[0080] Sample name Total polymer b1 0.18％ b2 0.19％ b3 0.19％ b4 0.19％ b5 0.19％ b6 0.18％ Average 2 0.187％

[0081] The blank solvent, control solution and test solution were injected according to the above detection conditions, and the chromatograms were as follows: Figure 1 , Figure 2 and Figure 3 shown.

[0082] Test condition 3 is as follows:

[0083] Take 20 μl of the test solution and number them c1 to c6 respectively. The test conditions are as follows:

[0084] Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 31:71; Test solution: accurately measure an appropriate amount of the test product, and dilute it with the solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: accurately measure an appropriate amount of the test solution, and dilute it with the solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: octadecylsilane bonded silica gel is used as the filler; 0.02% phosphoric acid solution is used as the mobile phase A, acetonitrile-0.02% phosphoric acid solution (60:40) is used as the mobile phase B, and gradient elution is performed according to the following table;

[0085]

[0086]

[0087] The flow rate was 1.1 ml per minute; the detection wavelength was 216 nm; the column temperature was 26°C; the injection volume was 20 μl; the test results are as follows:

[0088] Sample name Total polymer c1 0.18％ c2 0.19％ c3 0.19％ c4 0.19％ c5 0.19％ c6 0.18％ Average 3 0.185％

[0089] The proportion of mobile phase, detection wavelength, chromatographic column, column temperature, flow rate, etc. were moderately changed. The absolute difference of the total amount of polymer in test conditions 1 and 3 was ≤0.05% and the RSD was ≤10.0% compared with the test condition 2 with the best parameters. This method has good durability.

[0090] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for determining polymer impurities in lipoic acid injection, characterized in that: Here are the steps: Solvent: acetonitrile and water, the volume ratio of acetonitrile to water is 29-31:69-71; Test solution: Accurately measure an appropriate amount of the test product and quantitatively dilute it with a solvent to make a solution containing about 4 mg of lipoic acid per 1 ml; Control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with a solvent to make a solution containing about 8 μg of lipoic acid per 1 ml; Chromatographic conditions: Octadecylsilane bonded silica gel was used as filler; Using 0.02% phosphoric acid solution as mobile phase A, acetonitrile and 0.02% phosphoric acid solution as mobile phase B, the volume ratio of acetonitrile to phosphoric acid solution is 59-61:39-41, and gradient elution is performed; The flow rate is 0.9-1.1 ml per minute; the detection wavelength is 214-216 nm; the column temperature is 24-26°C; the injection volume is 20 μl; accurately measure the test solution and the control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

2. A lipoic acid injection polymer impurity high performance liquid chromatography determination method according to claim 1, characterized in that: The volume ratio of acetonitrile to water is 30:

70.

3. A lipoic acid injection polymer impurity high performance liquid chromatography determination method according to claim 1, characterized in that, The volume ratio of acetonitrile and 0.02% phosphoric acid solution is 60:

40.

4. A lipoic acid injection polymer impurity high performance liquid chromatography determination method according to claim 3, characterized in that: The flow rate was 1.0 ml per minute.

5. The method for determining polymer impurities in lipoic acid injection by high performance liquid chromatography according to claim 1, characterized in that: The detection wavelength is 215 nm.

6. The method for determining polymer impurities in lipoic acid injection by high performance liquid chromatography according to claim 1, characterized in that: The column temperature was 25°C.

7. The method for determining polymer impurities in lipoic acid injection by high performance liquid chromatography according to claim 1, characterized in that: Perform gradient elution according to the table above.

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