Method for detecting genotoxic impurities in lamotrigine

Abstract

The invention relates to the technical field of drug analysis and detection, and particularly discloses a method for detecting genotoxic impurities in lamotrigine. According to the method for detecting the genotoxic impurities in the lamotrigine, a Venusil XBP C18 (L) (150mm * 4.6 mm, 5mu m) chromatographic column is adopted, a mixed solution of a monopotassium phosphate solution-triethylamine and a mixed solution of acetonitrile are used as a mobile phase, and the genotoxic impurities (impurities K) in the lamotrigine are accurately detected in a gradient elution mode through a high performance liquid chromatography method. The detection limit of the impurity K is 0.0013 mu g / mL, the quantification limit is 0.0025 mu g / mL, and the detection requirements of the guiding principle of ICH M7 are met. The method provided by the invention has the advantages of strong specificity, high sensitivity, good linear relationship, good precision and durability, and can be used as a basis for monitoring the quality of lamotrigine drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis and detection, and particularly relates to a method for detecting genotoxic impurities in lamotrigine. Background Art

[0002] Lamotrigine (English name: Lamotrigine), having a triazine skeleton structure, with the structural formula as follows, and the chemical name being 3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine, with the molecular formula: C9H7Cl2N5 and the molecular weight: 256.09, is a novel antiepileptic drug. The results of pharmacological studies show that lamotrigine is a voltage-gated sodium channel blocker with good tolerance, insignificant enzyme induction and enzyme inhibition, and relatively low toxicity during pregnancy compared with classical antiepileptic drugs.

[0003]

[0004] Impurity K is an oxidative degradation impurity of lamotrigine, its structure is a nitrogen oxide, with the structural formula as follows, belonging to potential genotoxic impurities. The daily intake limit of such potential genotoxic impurities is 1.5 μg / day. Based on the maximum daily dose of lamotrigine of 700 mg, the limit index of Impurity K in the finished product of lamotrigine is 2.14 ppm, and its content is much lower than the limit indexes of conventional organic impurities of 0.05% or 0.10%. Therefore, a detection method with better sensitivity is required for analysis. Thus, in order to better monitor the product quality of lamotrigine drugs and further protect the medication safety of patients, a method for detecting potential genotoxic impurity K in lamotrigine drugs needs to be established.

[0005] Summary of the Invention

[0006] In view of the above technical problems, the present invention provides a method for detecting genotoxic impurities in lamotrigine.

[0007] To solve the above technical problems, the technical solution provided by the present invention is:

[0008] A method for detecting genotoxic impurities in lamotrigine, wherein the genotoxic impurity is Impurity K, and high performance liquid chromatography is used for detection, and the chromatographic conditions are:

[0009] Detector: ultraviolet detector;

[0010] Chromatographic column: Venusil XBP C18(L), 150 mm × 4.6 mm, 5 μm;

[0011] Mobile phase A: A mixed solution of potassium dihydrogen phosphate solution with a concentration of 2.6 g / L to 2.8 g / L and triethylamine, wherein the pH of the mobile phase A is 6.95 to 7.05, and the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is (148 to 152):1; Mobile phase B: Acetonitrile;

[0012] Detection wavelength: 219 nm to 221 nm;

[0013] The elution method is gradient elution, and the specific gradient elution sequence is as follows:

[0014] 0 - 15 min, 100% mobile phase A;

[0015] 15.1 - 20 min, 75% mobile phase A, 25% mobile phase B;

[0016] 21 min - 35 min, 100% mobile phase A.

[0017] Compared with the prior art, the method for detecting genotoxic impurities in lamotrigine provided by the present invention uses a Venusil XBP C18(L) (150 mm × 4.6 mm, 5 μm) chromatographic column, and a mixed solution of potassium dihydrogen phosphate solution - triethylamine and acetonitrile as the mobile phase. By high performance liquid chromatography in a gradient elution manner, the accurate detection of genotoxic impurities (impurity K) in lamotrigine is achieved. The detection limit of impurity K is 0.0013 μg / mL, and the quantitation limit is 0.0025 μg / mL, meeting the detection requirements of the ICH M7 guidelines. Moreover, the method provided by the present invention has strong specificity, high sensitivity, and good linear relationship. At the same time, it has good precision and durability, and can achieve trace detection of potential genotoxic impurities (impurity K) in lamotrigine raw materials or their preparations, which can be used as the basis for the quality control of lamotrigine drugs and is beneficial to reducing the medication safety risk of lamotrigine drugs.

[0018] It should be noted that the chemical name of the main genotoxic impurity detected in the present invention is 3,5 - diamino - 6 - (2,3 - dichlorophenyl) - 1,2,4 - triazine - 2 - oxide, and the structural formula is as shown below, hereinafter referred to as impurity K.

[0019]

[0020] Furthermore, the pH of the mobile phase A is adjusted to 6.95 - 7.05 with phosphoric acid.

[0021] Preferably, the pH of the mobile phase A is 7.0.

[0022] The preferred pH value of the mobile phase A can reduce band tailing, improve the peak shape, thereby facilitating the improvement of the separation degree between the raw material and the impurity, and making the accuracy and precision of the detection result higher.

[0023] Preferably, the column temperature is 34°C to 36°C.

[0024] More preferably, the column temperature is 35°C.

[0025] Preferably, the flow rate is 0.95 mL / min to 1.05 mL / min.

[0026] More preferably, the flow rate is 1.0 mL / min.

[0027] Preferably, the injection volume is 100 μL.

[0028] The preferred detection conditions can achieve a higher resolution between lamotrigine and impurity K, ensuring the effective detection of impurity K, thereby achieving the purpose of effectively and accurately controlling the content of impurity K in lamotrigine.

[0029] Further, the preparation of the test solution specifically includes the following steps: Take a lamotrigine sample, dissolve and dilute it with a solvent to obtain a test solution with a concentration of 3 mg / mL.

[0030] Specifically, the solvent is a mixed solution of methanol and hydrochloric acid solution with a volume ratio of 30:70.

[0031] Further, the concentration of the hydrochloric acid solution is 10.3 g / L.

[0032] Further, the preparation of the reference solution specifically includes the following steps: Take a reference substance of impurity K, dissolve and dilute it with methanol to prepare a stock solution with a concentration of 0.1 mg / mL; then dilute it with a solvent to prepare a reference solution with a concentration of 0.006 μg / mL.

[0033] Specifically, the solvent is a mixed solution of methanol and hydrochloric acid solution with a volume ratio of 30:70.

[0034] Further, the concentration of the hydrochloric acid solution is 10.3 g / L.

[0035] The detection method provided by the present invention can effectively separate lamotrigine from impurity K, accurately identify and quantitatively detect trace impurity K in lamotrigine, which is beneficial to effectively control the quality of lamotrigine. Moreover, the operation is simple and fast, with high sensitivity, providing a simple and reliable method for the quality control of lamotrigine products. At the same time, it is also beneficial to improve the quality and safety of pharmaceutical preparations containing lamotrigine, and has high practical value. Description of the Drawings

[0036] Figure 1 It is the chromatogram of the spiked test solution under the specificity item in Example 2. Detailed Embodiments

[0037] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0038] Example 1

[0039] Materials and methods:

[0040] Instruments: High performance liquid chromatograph, ultraviolet detector, volumetric flask, electronic balance.

[0041] Reagents: Acetonitrile, potassium dihydrogen phosphate, triethylamine, phosphoric acid, hydrochloric acid, methanol.

[0042] Blank solvent: Mobile phase.

[0043] 1.1 Preparation of solutions

[0044] Blank solvent: A mixed solution of methanol - hydrochloric acid solution (10.3 g / L) with a volume ratio of 30:70.

[0045] Preparation of test solution: Take lamotrigine sample, dissolve and dilute it with a mixed solution of methanol - hydrochloric acid solution (10.3 g / L) with a volume ratio of 30:70 to obtain a test solution with a concentration of 3 mg / mL.

[0046] Control solution: Take the reference substance of impurity K, dissolve and dilute it with methanol to prepare a stock solution with a concentration of 0.1 mg / mL; then accurately measure an appropriate amount of the stock solution; dilute it with a mixed solution of methanol - hydrochloric acid solution (10.3 g / L) with a volume ratio of 30:70 to prepare a control solution with a concentration of 0.006 μg / mL.

[0047] 1.2 Conditions of high performance liquid chromatography:

[0048] Detector: Ultraviolet detector;

[0049] Chromatographic column: Venusil XBP C18(L), 150 mm × 4.6 mm, 5 μm;

[0050] Mobile phase: A mixed solution of 2.7 g / L potassium dihydrogen phosphate solution and acetonitrile (adjust the pH to 7.0 with phosphoric acid), wherein the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 150:1;

[0051] Flow rate: 1.0 mL / min;

[0052] Column temperature: 35 °C;

[0053] Injection volume: 100 μL;

[0054] Gradient elution; The elution sequence is as shown below.

[0055]

[0056] Example 2

[0057] Methodology Verification:

[0058] 2.1 Specificity

[0059] Stock solution of impurity reference substance: Take 10 mg of impurity K reference substance, weigh accurately, place it in a 100 mL volumetric flask, dissolve it with methanol and dilute to the mark, shake well; then accurately measure 1.2 mL of the above solution, place it in a 20 mL volumetric flask, dilute to the mark with blank solvent, shake well; accurately measure 1 mL of the above solution, place it in a 50 mL volumetric flask, dilute to the mark with blank solvent, shake well, as the stock solution of reference substance.

[0060] Reference solution: Accurately measure 1 mL of the stock solution of reference substance, place it in a 20 mL volumetric flask, dilute to the mark with blank solvent, shake well, as the reference solution.

[0061] Spiked test solution: Take 60 mg of lamotrigine sample, weigh accurately, place it in a 20 mL volumetric flask, then accurately add 1 mL of the stock solution of reference substance, dissolve it with blank solvent and dilute to the mark, shake well, as the spiked test solution.

[0062] Accurately measure the blank solvent and the spiked test solution and inject them according to the above chromatographic conditions, record the chromatogram, as Figure 1 shown, and the results are shown in Table 1.

[0063] Table 1

[0064] Name Retention time (min) Resolution from adjacent peaks Reference solution 7.803 / Spiked test solution 7.782 3.791

[0065] The test results show that the blank solvent does not interfere with the determination of related substances and can be well separated from the adjacent impurity peaks, and can effectively detect impurity K, and the method system suitability is good.

[0066] 2.2 Detection Limit and Quantitation Limit

[0067] Detection limit: Take the reference solution, quantitatively dilute it step by step with blank solvent, perform HPLC detection, and continuously measure 3 times. Calculate the ratio of its peak height to noise (signal-to-noise ratio), and the sample concentration with a signal-to-noise ratio (S / N) above 3 is the detection limit concentration.

[0068] Quantitation limit: Take the reference solution, quantitatively dilute it step by step with blank solvent, perform HPLC detection, and continuously measure 6 times. Calculate the ratio of its peak height to noise (signal-to-noise ratio), and the sample concentration with a signal-to-noise ratio (S / N) above 10 is the quantitation limit concentration.

[0069] Precisely measure the detection limit solution and the quantitation limit solution, inject them continuously for 6 times, record the chromatograms, and the results are shown in Table 2.

[0070] Table 2 Determination Results of Quantitation Limit and Detection Limit

[0071] Name Limit of detection concentration (μg / mL) Limit of quantitation concentration (μg / mL) Impurity K 0.0013 0.0025

[0072] The results show that the detection limit concentration of impurity K by the method of the present invention is 0.0013 μg / mL, the quantitation limit concentration of impurity K is 0.0025 μg / mL, and the repeatability of the 6 - needle quantitation limit and detection limit solutions is good. It proves that this method has high sensitivity and meets the detection requirements for impurities in the finished product.

[0073] 2.3 Linear Range

[0074] Blank solvent: Methanol - 10.3 g / L hydrochloric acid solution with a volume ratio of 30:70.

[0075] Stock solution of impurity reference substance: Take 10 mg of impurity K reference substance, weigh it precisely, place it in a 100 - mL volumetric flask, dissolve it with methanol and dilute to the scale, shake well; then precisely measure 1.2 mL of the above - mentioned solution, place it in a 20 - mL volumetric flask, dilute to the scale with the blank solvent, shake well; precisely measure 1 mL of the above - mentioned solution, place it in a 50 - mL volumetric flask, dilute to the scale with the blank solvent, shake well, and use it as the stock solution of the reference substance.

[0076] Preparation of linear solutions: Precisely measure the stock solution of the reference substance, quantitatively dilute it with the blank solvent to prepare a series of reference substance solutions with various concentrations, carry out the determination according to the above HPLC method, record the chromatograms, draw a standard curve with the concentration as the abscissa (X) and the peak area as the ordinate (Y), and the test results are shown in Table 3.

[0077] Table 3 Standard Curve Equation of Impurity K

[0078] Sample name Standard curve equation R Concentration range (μg / mL) Impurity K y = 481.34x - 0.2297 0.9971 0.0025～0.0212

[0079] The detection results show that impurity K has a good linear relationship in the concentration range of 0.0025 - 0.0212 μg / mL, the linear equation is y = 481.34x - 0.2297, and the linear correlation coefficient R = 0.9971.

[0080] 2.4 Precision

[0081] Stock solution of reference substance: Take 10 mg of impurity K reference substance, weigh it precisely, place it in a 100 - mL volumetric flask, dissolve it with methanol and dilute to the scale, shake well; then precisely measure 1.2 mL of the above - mentioned solution, place it in a 20 - mL volumetric flask, dilute to the scale with the blank solvent, shake well; precisely measure 1 mL of the above - mentioned solution, place it in a 50 - mL volumetric flask, dilute to the scale with the blank solvent, shake well, and use it as the stock solution of the reference substance.

[0082] Reference solution: Accurately measure 1 mL of the reference stock solution, transfer it to a 20-mL volumetric flask, dilute it to the mark with the blank solvent, and mix well to obtain the reference solution.

[0083] Test sample spiked solution: Take 60 mg of this product, accurately weigh it, transfer it to a 20-mL volumetric flask, then accurately add 1 mL of the reference stock solution, dissolve it with the blank solvent and dilute it to the mark, and mix well to obtain the test sample spiked solution. Prepare 6 portions in the same way.

[0084] Accurately measure the blank solvent and the test sample spiked solution and inject them according to the above chromatographic conditions, and record the chromatograms. The intermediate precision is tested by different personnel on different dates using different instruments, and the test results are shown in Table 4.

[0085] Table 4 Detection amounts in the repeatability and intermediate precision tests (equivalent to the test sample solution, %)

[0086]

[0087] From the above results, it can be seen that the intermediate precision of impurity K in the test sample spiked solution is good.

[0088] 2.5 Solution stability

[0089] Place the test sample spiked solution at room temperature and inject it at 0 h, 2 h, 6 h, 11.5 h, 18 h, and 24 h respectively according to the above chromatographic conditions, and record the chromatograms. The test results are shown in Table 5.

[0090] Table 5 Solution stability

[0091]

[0092]

[0093] From the above results, it can be seen that at room temperature, the test sample spiked solution can remain stable within 24 h.

[0094] 2.6 Accuracy

[0095] Reference stock solution: Take 10 mg of the reference substance of impurity K, accurately weigh it, transfer it to a 100-mL volumetric flask, dissolve it with methanol and dilute it to the mark, and mix well; then accurately measure 1.2 mL of the above solution, transfer it to a 20-mL volumetric flask, dilute it to the mark with the blank solvent, and mix well; accurately measure 1 mL of the above solution, transfer it to a 50-mL volumetric flask, dilute it to the mark with the blank solvent, and mix well to obtain the reference stock solution.

[0096] Reference solution: Accurately measure 1 mL of the reference stock solution, transfer it to a 20-mL volumetric flask, dilute it to the mark with the blank solvent, and mix well to obtain the reference solution.

[0097] Test solution: Weigh accurately about 60 mg of this product, place it in a 20 mL volumetric flask, dissolve and dilute to the mark with the blank solvent, shake well, and use it as the test solution.

[0098] Spiked test solution: Weigh accurately about 60 mg of this product, place it in a 20 mL volumetric flask, weigh 9 portions in parallel, with every three portions as a group, accurately add 0.5 mL, 1.0 mL, and 1.5 mL of the reference stock solution respectively, dissolve and dilute to the mark with the blank solvent, shake well, and you will get it.

[0099] Precisely measure the above solutions, inject samples according to the above chromatographic conditions, record the chromatogram, and calculate the detection results of impurity K. The results are shown in Table 6.

[0100] Table 6 Results of accuracy test

[0101]

[0102] From the above results, it can be seen that the recoveries of impurity K are all between 80% and 115%, the RSD of the recoveries are all less than 10%, and the accuracy is good.

[0103] 2.7 Robustness

[0104] Make slight adjustments to the important parameters in the proposed chromatographic conditions (hereinafter referred to as the original conditions) to form a series of chromatographic conditions for robustness investigation. Determine the chromatographic conditions to measure the spiked test solution. The chromatographic conditions are shown in Table 8. Compare the detected amounts measured under each robustness condition with the original conditions, and the results are shown in Table 9.

[0105] Table 8 Parameters of chromatographic conditions for robustness investigation

[0106] Robustness Normal chromatographic conditions Robustness - 1 Robustness - 2 Column temperature 35℃ 34℃ 36℃ Flow rate 1.0 mL / min 0.95 mL / min 1.05 mL / min Mobile phase buffer pH pH 7.0 pH 6.95 pH 7.05 Detector wavelength 220 nm 219 nm 221 nm

[0107] Table 9 Results of robustness investigation - detected amount (equivalent to % of the test solution)

[0108] Name Detected amount of Impurity K (%) Original conditions 0.000216 Flow rate 0.95 mL / min 0.000226 Flow rate 1.05 mL / min 0.000226 Mobile phase buffer pH 6.95 0.000256 Mobile phase buffer pH 7.05 0.000228 Detector wavelength 219 nm 0.000220 Detector wavelength 221 nm 0.000212

[0109] The results of the robustness test show that on the basis of the original chromatographic conditions, when the column temperature changes by ±1 °C, the flow rate changes by ±0.05 mL / min, the buffer pH value of the mobile phase changes by ±0.05 °C, and the detector wavelength changes by ±1 nm, it has no effect on the detection results, and the robustness of this method is good.

[0110] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting genotoxic impurities in lamotrigine, characterized in that: The genotoxic impurity is impurity K, which is detected by high performance liquid chromatography under the following chromatographic conditions: Detector: UV detector; Chromatographic column: Venusil XBP C18 (L), 150 mm × 4.6 mm, 5 μm; Mobile phase A: a mixed solution of potassium dihydrogen phosphate solution-triethylamine with a concentration of 2.6 g / L to 2.8 g / L, wherein the pH of the mobile phase A is 6.95 to 7.05, and the volume ratio of potassium dihydrogen phosphate solution to acetonitrile is (148 to 152):1; Mobile phase B: acetonitrile; Detection wavelength: 219nm~221nm; The elution method is gradient elution, and the specific gradient elution sequence is: 0-15 min, 100% mobile phase A; 15.1-20 min, 75% mobile phase A, 25% mobile phase B; 21min~35min, 100% mobile phase A.

2. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: Phosphoric acid was used to adjust the pH of mobile phase A to 6.95-7.

05.

3. The method for detecting genotoxic impurities in lamotrigine according to claim 2, characterized in that: The pH of the mobile phase A is 7.

0.

4. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: The column temperature is 34℃~36℃.

5. The method for detecting genotoxic impurities in lamotrigine according to claim 4, characterized in that: The column temperature was 35°C.

6. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: The flow rate is 0.95mL / min~1.05mL / min.

7. The method for detecting genotoxic impurities in lamotrigine according to claim 6, characterized in that: The flow rate was 1.0 mL / min.

8. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: The injection volume was 100 μL.

9. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: The concentration of the test solution was 3 mg / mL.

10. The method for detecting genotoxic impurities in lamotrigine according to claim 1, characterized in that: The concentration of the control solution was 0.006 μg / mL.

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