Method for detecting content of aminoguanidine in lamotrigine key intermediate
Through the combined use of liquid chromatography-mass spectrometry, the problem of aminoguanidine detection in key lamotrigine intermediates was solved, and high-sensitivity quantitative detection of aminoguanidine was achieved to meet the requirements of drug impurity control.
Patent Information
- Application Number
- CN202510563838.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-18
AI Technical Summary
The prior art is difficult to accurately detect aminoguanidine with extremely low content in key lamotrigine intermediates, and its structure has no characteristic absorption groups, resulting in difficulty in detection.
The quantitative detection of aminoguanidine was performed by using liquid chromatography-mass spectrometry technology through C8 columns, specific mobile phases and gradient elution conditions, combined with the ESI+ mode of mass spectrometry and specific ion collision energy.
It realizes high sensitivity detection for aminoguanidine, with a detection limit of 0.0019ng/ml, which meets the quality control requirements of ICH and Chinese Pharmacopoeia, and is suitable for the quality control of aminoguanidine in key lamotrigine intermediates.
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Figure CN120334411A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of drug impurity detection, and specifically relates to a method for detecting the content of aminoguanidine in a key intermediate of lamotrigine. Background Art
[0002] Lamotrigine is a sodium channel blocker and is clinically used for the treatment of epilepsy. The typical synthesis process of lamotrigine uses 2,3-dichlorobenzoic acid as the starting compound. 2,3-dichlorobenzoic acid reacts with thionyl chloride (also called dichlorosulfoxide, SOCl₂) to obtain 2,3-dichlorobenzoyl chloride, which then reacts with cuprous cyanide (CuCN) to obtain 2,3-dichlorobenzoyl cyanide; 2,3-dichlorobenzoyl cyanide reacts with aminoguanidine bicarbonate (see the following formula) to form a condensate [chemical name: 2-cyano-(2,3-dichlorophenyl)-2-guanidinoiminoacetonitrile], and then undergoes a cyclization reaction to obtain lamotrigine. Aminoguanidine bicarbonate is a reaction reagent in the synthesis process of lamotrigine. The aminoguanidine bicarbonate reaction reagent will decompose into aminoguanidine during the reaction. Since both the aminoguanidine bicarbonate reaction reagent and its aminoguanidine have genotoxic groups, quality control research on aminoguanidine should be carried out.
[0003]
[0004] According to the ICH "M7: Industry Guide for the Assessment and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk" and the "Guideline for the Control of Genotoxic Impurities" (Part 4) of the Chinese Pharmacopoeia 2020 Edition, the Threshold of Toxicological Concern (TTC) for aminoguanidine is 1.5 μg / day. According to the usage and dosage instructions of the lamotrigine tablets, when this product is used for the treatment of adults or children over 12 years old, the daily dose can reach 200 mg, so the control limit of aminoguanidine should not exceed 7.5 ppm. How to accurately detect a substance with such a low content from the sample is a technical problem that needs to be solved urgently by those skilled in the art. The relative molecular weight of aminoguanidine (CH₆N₄) is 74.09, and the relative molecular weight is relatively small. There is no characteristic absorption group in the structural formula, and there is currently no literature report on the detection of aminoguanidine. It is urgent to establish a set of detection methods that can be applied to the determination of the content of aminoguanidine in the key intermediate of lamotrigine. Summary of the Invention
[0005] To solve the above technical problems, the purpose of the present invention is to provide a method for detecting the content of aminoguanidine in a key intermediate of lamotrigine, including the following steps: Dissolve the test sample and the reference substance in the detection solvent respectively to obtain a test sample solution and a reference substance solution; The test solution and the reference solution were separated by liquid chromatography technology, detected by a mass spectrometry detector, and the content of aminoguanidine was calculated by the external standard method with the peak area. The liquid chromatography conditions were as follows: C8 chromatographic column, flow rate: 0.95 - 1.05 ml / min; column temperature: 30 - 40 °C; The mobile phase was: A: 0.1% formic acid aqueous solution; B: acetonitrile; Gradient elution: 0.0 - 0.8 min, 90% of A; 0.8 - 2.0 min, 90% - 10% of A; 2.0 - 4.0 min, 10% A; The mass spectrometry conditions were: ESI + Positive ion mode, cone voltage was 38 V, precursor ion m / z was 75.2, quantitative ion pair m / z was 75.2 / 58.4, quantitative ion collision energy was 11 V, qualitative ion pair m / z was 75.2 / 43.5, qualitative ion collision energy was 36 V.
[0006] Preferably, the flow rate was 1.0 ml / min; the column temperature was 35 °C.
[0007] The model of the chromatographic column was: Thermo BDS Hypersil C8, with a specification of 4.6 mm * 150 mm, 5 μm.
[0008] The model of the liquid chromatography instrument was: Thermo Fisher ACCELA.
[0009] The model of the mass spectrometry instrument was: Thermo TSQ QUANTUM ACCESS MAX, which was a triple quadrupole mass spectrometry detector.
[0010] The test sample was the material obtained by sampling during the synthesis of the condensate in the lamotrigine synthesis process, , condensate.
[0011] Test solution: Take an appropriate amount of the condensate test sample, weigh it precisely, dissolve it with 50% acetonitrile solution and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the subsequent filtrate to obtain it; Reference solution: Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it with water and quantitatively dilute it to make a solution containing about 1 mg of aminoguanidine per 1 ml, precisely measure an appropriate amount, and quantitatively dilute it with 50% acetonitrile solution to make a solution containing about 7.5 ng of aminoguanidine per 1 ml This application provides a method for detecting the content of aminoguanidine in a key intermediate of lamotrigine. By using a specific liquid chromatography - mass spectrometry coupling method for detection, through methodological verification, this method has good specificity, precision and repeatability, and the sensitivity meets the control requirements of this impurity.
[0012] The detection method provided by this application has a quantitative limit of 0.0038 ng / ml and a detection limit of 0.0019 ng / ml. The method has good sensitivity, and the detection limit and quantitative limit are significantly lower than the quality control limit requirements for aminoguanidine trace impurities, indicating that the sensitivity of this method meets the control requirements for this impurity. The recovery test proves that the sample treatment method and detection method of the present invention can achieve the determination of the content of aminoguanidine trace impurities in the condensate, and can be applied to the quality control of aminoguanidine impurities in the condensate of the key intermediate of lamotrigine. Description of the Drawings
[0013] Figure 1 It is the optimized graph of the impurity aminoguanidine derivative ion; Figure 2 It is the graph of the blank solvent 50% acetonitrile for the method validation (specificity) of the impurity aminoguanidine; Figure 3 It is the graph of the condensate test sample for the method validation (specificity) of the impurity aminoguanidine; Figure 4 It is the graph of the reference substance for the method validation (specificity) of the impurity aminoguanidine; Figure 5 It is the graph of dicyandiamide for the method validation (specificity) of the impurity aminoguanidine. Detailed Description of the Invention
[0014] In the typical synthesis process of lamotrigine, the condensate [chemical name: 2-cyano-(2,3-dichlorophenyl)-2-guanidinoiminoacetonitrile] formed by the reaction of 2,3-dichlorobenzoyl cyanide and aminoguanidine bicarbonate is the last step in the synthesis process route of lamotrigine. The condensate is a key intermediate material for the synthesis of lamotrigine. The quality control of trace potential genotoxic impurities in the key intermediate condensate plays a very important role in controlling the potential genotoxic impurities in the finished product of lamotrigine. Therefore, this application preferably selects the condensate as the key intermediate for detection.
[0015] Example 1: Method Exploration and Description Mass Spectrometry Ion Optimization Using the positive ion mode, the aminoguanidine ion optimization results show that the parent ion m / z is 75.2, which is basically consistent with the theoretical molecular weight. The aminoguanidine ion optimization results are shown in Figure 1 .
[0016] Liquid Chromatography Condition Optimization 1) Liquid Chromatography Conditions HPLC instrument model: Thermo Fisher ACCELA Chromatographic column: Thermo BDS Hypersil C8 (4.6 mm * 150 mm, 5 μm) Flow rate: 1.0 ml / min; Column temperature: 35 °C; Injection volume: 5 μl; Injector temperature: 25 °C; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile, gradient elution, the elution program is shown in the following table.
[0017] Time (min) A（%） B（%） 0.00 90.0 10.0 0.80 90.0 10.0 2.00 10.0 90.0 4.00 10.0 90.0 4.01 90.0 10.0 5.50 90.0 10.0 2) Optimization of chromatographic conditions The flow rate (0.95 ml / min, 1.0 ml / min, 1.05 ml / min), column temperature (30 °C, 35 °C, 40 °C), and mobile phase ratio (initial ratio of 0.1% formic acid aqueous solution - acetonitrile, 90:10, 92:8, 88:12) were investigated respectively. The results showed that different flow rates, column temperatures, and mobile phase ratios had no significant effect on the detection results. The tentative flow rate was 0.5 ml / min, the column temperature was 35 °C, and the ratio of 0.1% formic acid aqueous solution - acetonitrile was 10:90. See the robustness experiment in Example 2 for details.
[0018] Detection conditions for aminoguanidine 1) Liquid chromatography conditions Chromatographic column: Thermo BDS Hypersil C8 (4.6 mm * 150 mm, 5 μm) Flow rate: 1.0 ml / min; Column temperature: 35 °C; Injection volume: 5 μl; Injector temperature: 25 °C; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile, gradient elution, the elution program is shown in the following table.
[0019] Time (min) A（%） B（%） 0.00 90.0 10.0 0.80 90.0 10.0 2.00 10.0 90.0 4.00 10.0 90.0 4.01 90.0 10.0 5.50 90.0 10.0 2) Mass spectrometry conditions (Table 1) Model of mass spectrometry instrument: Thermo TSQ QUANTUM ACCESS MAX (triple quadrupole) Table 1 Mass spectrometry parameters <![CDATA[ESI + (SRM)]]> Compound: Aminoguanidine Parent Ion m / z 75.2 Cone Voltage (V) 38 Quantitative Ion Pair m / z 75.2 / 58.4 Quantitative Ion Collision Energy (V) 11 Qualitative Ion Pair m / z 75.2 / 43.5 Qualitative Ion Collision Energy (V) 36 Capillary Temperature (°C) 350 Vaporizer Temperature (°C) 300 Sheath Gas Pressure (Arb) 30 Aux Gas Pressure (Arb) 10 Ion Sweep Gas Pressure (Arb) 2 Spray Voltage (V) 3000 Discharge Current (μA) 4.0 3) Test solution Take an appropriate amount of the condensate test sample, weigh it accurately, dissolve it with 50% acetonitrile solution and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter, and take the subsequent filtrate to obtain.
[0020] 4) Reference solution Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it with water and quantitatively dilute it to make a solution containing about 1 mg of aminoguanidine per 1 ml. Accurately measure an appropriate amount and quantitatively dilute it with 50% acetonitrile solution to make a solution containing about 7.5 ng of aminoguanidine per 1 ml.
[0021] 5) System suitability requirements Take the reference solution and inject it continuously for 5 times. The relative standard deviation of the peak area of aminoguanidine shall not exceed 15.0%.
[0022] 6) Determination method Precisely measure appropriate volumes of the test solution and the reference solution, and inject them into the liquid chromatograph respectively. Record the chromatograms.
[0023] 7) Limit If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the aminoguanidine peak, calculate by the external standard method based on the peak area, and it shall not exceed 7.5 ppm.
[0024] Example 2: Methodology verification (method under item "3" described in Example 1) 1. Specificity 1) Interference of the blank solvent on aminoguanidine ① Take an appropriate amount of the aminoguanidine bicarbonate reference solution and prepare it according to the method of the reference solution under item "3" described in Example 1 to obtain the reference solution.
[0025] ② Take an appropriate amount of the condensate test sample (batch number: 10901 - 1911001y) and prepare it according to the method of the test solution under item "3" described in Example 1 to obtain the test solution.
[0026] Take appropriate amounts of the 50% acetonitrile solution, the reference solution, and the test solution, and inject them for analysis according to the method under item "3" described in Example 1 to examine the interference of 50% acetonitrile, the blank solvent, on aminoguanidine. The results are shown in Table 2 below. Figures 2 - 4 .
[0027] Table 2 Results of blank solvent interference Conclusion: 50% acetonitrile does not show a peak at the main peak position (peaks with S / N < 3 are ignored), and does not interfere with the detection of aminoguanidine.
[0028] 2) Interference of other impurities on aminoguanidine ① According to the condensate process route, the compounds involved in the condensate synthesis process are: dicyandiamide, aminoguanidine carbonate, monocyanamide, hydrazine, 2,3 - dichlorobenzoic acid, 2,3 - dichlorobenzoyl chloride, 2,3 - dichlorobenzoyl cyanide. The molecular structures and molecular weights are as follows:
[0029] ② Impurity interference analysis The quantitative ion of aminoguanidine has an m / z of 58.4. The molecular weights of cyanamide and hydrazine are both less than 58.4, and after mass spectrometry collision, they will not produce daughter ions with an m / z of 58.4. From the structural formula analysis, 2,3-dichlorobenzoic acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide are not likely to produce daughter ions with an m / z of 58.4 after mass spectrometry collision. Some groups of dicyandiamide are similar to those of aminoguanidine, which may affect the detection of aminoguanidine. By preparing a dicyandiamide reference solution for injection, it is investigated whether it interferes with the detection of aminoguanidine.
[0030] Take an appropriate amount of dicyandiamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 7.5 ng per 1 ml, which is used as the dicyandiamide reference solution.
[0031] Take an appropriate amount of the dicyandiamide reference solution, inject and analyze it according to the method described in "Item 3" of Example 1, and investigate the interference of dicyandiamide on aminoguanidine. The results are shown in Table 3 below, see Appendix Figure 5 。
[0032] Table 3 Results of dicyandiamide interference Sample Name Peak Area Retention Time (min) Dicyandiamide Reference Solution 8 2.17 Reference Solution 58366 2.18 Conclusion: The peak area of dicyandiamide at the main peak position is 8, which is less than 0.05% of the peak area of the reference substance (58366×0.05% = 29). Dicyandiamide does not interfere with the detection of aminoguanidine. Through comprehensive analysis, each impurity does not interfere with the detection of aminoguanidine in the ion channel with an m / z of 68.4.
[0033] 2. Filter membrane adsorption Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it in 50% acetonitrile and quantitatively dilute it to make a solution containing about 7.5 ng per 1 ml. Divide it into 3 equal parts and number them 1#, 2#, and 3# respectively. Centrifuge 1# at 10,000 revolutions per minute for 10 minutes; discard the initial filtrate of 0.1 ml for 2# (nylon 66 material, 0.22 μm); discard the initial filtrate of 0.3 ml for 3# (nylon 66 material, 0.22 μm).
[0034] Take the solutions after treatment of 1#, 2#, and 3# respectively, inject and analyze them according to the method described in "Item 3" of Example 1, inject continuously 3 times respectively, record the peak area, and investigate the filter membrane adsorption rate (adsorption rate = 100% - peak area after adsorption / peak area before adsorption × 100%). The results are shown in Table 4 below.
[0035] Table 4 Experimental results of aminoguanidine filter membrane adsorption Table 4 Experimental results of aminoguanidine filter membrane adsorption Injection Sequence Sample Number 1 2 3 Mean (n = 3) RSD% (n = 3) Adsorption Rate % 1# 44287 44360 41933 43527 3.2 / 2# 44683 42842 42883 43469 2.4 0.1 3# 42879 42139 42493 42503 0.9 2.4 Conclusion: The nylon 66 filter membrane (0.22 μm) has no adsorption of aminoguanidine.
[0036] 3. Linearity and range Take an appropriate amount of aminoguanidine bicarbonate reference substance, quantitatively dilute it with 50% acetonitrile to prepare a series of aminoguanidine reference substance solutions with a concentration of about 0.75 ng, 1.50 ng, 3.75 ng, 7.50 ng, 15.00 ng, and 22.50 ng of aminoguanidine per 1 ml.
[0037] Take the aminoguanidine reference substance solutions with different concentrations, inject and analyze them according to the method described in "Item 3" of Example 1. Use the peak area of aminoguanidine as the ordinate (Y) and the concentration of aminoguanidine reference substance as the abscissa (X), and perform linear regression of the peak area Y against the concentration X (ng / ml). The linear equation and correlation coefficient are shown in Table 5 below.
[0038] Table 5 Results of the linear experiment of aminoguanidine
[0039] Conclusion: There is a good linear correlation between the peak area (Y) of aminoguanidine and the concentration (X) in the range of 0.75 - 22.50 ng / ml. The linear equation is: y = 5140.1x - 1108.6 (r = 0.9920).
[0040] 4. Quantification limit, detection limit Take an appropriate amount of aminoguanidine bicarbonate reference substance, quantitatively dilute it with 50% acetonitrile to prepare a solution containing about 0.75 ng of aminoguanidine per 1 ml, and then dilute it with 50% acetonitrile to a suitable concentration. Inject and analyze it according to the method described in "Item 3" of Example 1. Calculate according to the signal-to-noise ratio method. When the signal-to-noise ratio is about 3:1, the concentration is the detection limit concentration, and when the signal-to-noise ratio is about 10:1, the concentration is the quantification limit concentration. The results are shown in Table 6 below.
[0041] Table 6 Results of the experiment on the quantification limit and detection limit of aminoguanidine Sample Concentration (ng / ml) Peak Area S / N ppm Equivalent to Test Concentration of Test Sample 0.75 ng (Stock Solution) 0.7500 5168 680.79 0.7500 Stock Solution Diluted 200 - fold (Quantitation Limit) 0.0038 38 21.44 0.0038 Stock Solution Diluted 400 - fold (Detection Limit) 0.0019 94 48.00 0.0019 50% Acetonitrile NA NA NA NA Conclusion: Continuously dilute the aminoguanidine stock solution (0.75 ng / ml). When diluted 200 times, the S / N is 21.44. The tentative quantification limit concentration of aminoguanidine is 0.0038 ng / ml, which is equivalent to 0.0038 ppm of the test concentration of the sample; when diluted 400 times, the S / N is 48.00. The tentative detection limit concentration of aminoguanidine is 0.0019 ng / ml, which is equivalent to 0.0019 ppm of the test concentration of the sample. The detection sensitivity of this method meets the control requirements for aminoguanidine.
[0042] 5. Precision 1) Injection precision Take the 7.50 ng / ml aminoguanidine solution in the experiment of "2. Linearity and range" above, inject and analyze it according to the method described in "Item 3" of Example 1, and inject continuously 5 times. Statistically analyze the retention time and peak area. The results are shown in Table 7 below.
[0043] Table 7 Results of the injection precision experiment of aminoguanidine Serial Number 1 2 3 4 5 Mean RSD% Retention Time (min) 2.27 2.27 2.27 2.27 2.27 2.27 0.0 Peak Area (A) 31204 36157 33165 36776 39140 35288 8.9 Conclusion: The aminoguanidine reference solution was injected continuously for 5 times, and the RSDs of retention time and peak area were 0.0% and 8.9% respectively, indicating good injection precision.
[0044] 2) Repeatability ① Preparation of test solution: An appropriate amount of the condensate test sample (batch number: 10901-1911001y) was accurately weighed, dissolved in 50% acetonitrile and quantitatively diluted to a solution containing about 1 mg per 1 ml, filtered, and the subsequent filtrate was taken as the test solution. Six parallel portions were prepared.
[0045] ② Preparation of reference solution An appropriate amount of aminoguanidine bicarbonate reference was taken, dissolved in 50% acetonitrile and quantitatively diluted to a solution containing about 7.5 ng per 1 ml as the reference solution.
[0046] The test solution and the reference solution were taken and injected for analysis according to the method described in "3 below" in Example 1. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the aminoguanidine peak, the content of aminoguanidine was calculated by the external standard method based on the peak area. The results are shown in Table 8 below.
[0047] Table 8 Results of the repeatability experiment of aminoguanidine Sample Number Aminoguanidine Content 1 2 3 4 5 6 Mean (n = 6) RSD% (n = 6) Content (ppm) 1.18 1.65 1.89 1.69 1.64 1.62 1.61 14.5 Conclusion: As can be seen from the above table, among the six test solution portions, the average content of aminoguanidine was 1.61 ppm and the RSD was 14.5%, indicating good repeatability of this method.
[0048] 6. Solution stability 1) Stability of reference solution An appropriate amount of aminoguanidine bicarbonate reference was taken, dissolved in 50% acetonitrile and quantitatively diluted to a solution containing about 7.5 ng per 1 ml as the reference solution.
[0049] The reference solution was placed at room temperature, and samples were taken at 0 h, 3 h, 8 h, and 11 h respectively and injected for analysis according to the method described in "3 below" in Example 1 to investigate the change of peak area at each time point. The results are shown in Table 9 below.
[0050] Table 9 Results of the stability experiment of aminoguanidine reference solution Serial Number 0h 3h 8h 11h Mean RSD% Retention Time (min) 2.30 2.30 2.30 2.31 2.30 0.2 Peak Area (A) 44807 37280 34549 32433 37267 14.5 Conclusion: The aminoguanidine reference solution was placed at room temperature for 11 hours, and the RSD of the main peak area was 14.5%, indicating good stability.
[0051] 2) Stability of test solution Take an appropriate amount of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it with 50% acetonitrile and quantitatively dilute it to prepare a solution containing about 1 mg per 1 ml, filter, and take the subsequent filtrate as the test sample solution.
[0052] Let the test sample solution stand at room temperature, sample at 0 h, 2 h, 4 h, and 6 h respectively, and inject and analyze according to the method described in "Item 3" of Example 1, and examine the changes in the peak areas at each time point. The results are shown in Table 10 below.
[0053] Table 10 Results of the stability experiment of the aminoguanidine test sample solution Serial Number 0h 2h 4h 6h 8h Mean RSD% Peak Area (A) 5819 7333 5311 5141 5791 5879 13.2 Content (ppm) 1.18 1.48 1.07 1.04 1.17 1.19 13.2 Conclusion: When the test sample solution stands at room temperature for 8 hours, the RSD of the main peak area is 13.2%, and the stability is good.
[0054] 7. Accuracy 1) Reference solution Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it with 50% acetonitrile and quantitatively dilute it to prepare a solution containing about 7.5 ng per 1 ml as the reference solution.
[0055] 2) Test sample solution ① Aminoguanidine stock solution Take an appropriate amount of aminoguanidine bicarbonate reference substance, quantitatively dilute it with 50% acetonitrile to prepare an aminoguanidine reference stock solution containing about 75 ng per 1 ml, and you will get it.
[0056] ② 50% test sample solution Take about 10 mg of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 9.5 ml of 50% acetonitrile, add 0.5 ml of the aminoguanidine reference stock solution (75 ng / ml), dissolve it by ultrasonic treatment, cool it to room temperature, filter, and take the subsequent filtrate as the 50% accuracy solution, and prepare 3 portions in parallel.
[0057] ③ 100% test sample solution: Take about 10 mg of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 9 ml of 50% acetonitrile, add 1 ml of the aminoguanidine reference stock solution (75 ng / ml), dissolve it by ultrasonic treatment, cool it to room temperature, filter, and take the subsequent filtrate as the 100% accuracy solution, and prepare 3 portions in parallel.
[0058] ④ 150% test sample solution Take about 10 mg of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10-ml centrifuge tube, add 8.5 ml of 50% acetonitrile, add 0.5 ml of the aminoguanidine reference substance stock solution (75 ng / ml), dissolve it by ultrasound, cool it to room temperature, filter it, and take the consecutive filtrate as the 150% accuracy solution. Prepare 3 portions in parallel.
[0059] ⑤Take an appropriate amount of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it with 50% acetonitrile and quantitatively dilute it to prepare a solution containing about 1 mg per 1 ml, filter it, and take the consecutive filtrate as the test sample solution. Prepare 2 portions in parallel, and the test results serve as the background quantity of this experiment.
[0060] Accurately measure the above-mentioned test sample solution and reference substance solution respectively, inject and analyze them according to the method described in "3 under" in Example 1, calculate the content of aminoguanidine by the external standard method based on the peak area, and investigate the recovery rate of this product. The results are shown in Table 11 below.
[0061] Table 11 Results of the aminoguanidine accuracy experiment
[0062] Conclusion: For 9 test sample solutions, the recovery rate range of aminoguanidine is 101.1% - 129.6%, the average recovery rate is 116.9%, and the RSD of the recovery rate is 8.9%, meeting the requirements of the limit recovery rate range. The recovery rate of this method is good.
[0063] 8. Robustness 1) Reference substance solution Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it with 50 acetonitrile and quantitatively dilute it to prepare a solution containing about 7.5 ng per 1 ml as the reference substance solution.
[0064] 2) Test sample solution Take an appropriate amount of the condensate test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it with 50 acetonitrile and quantitatively dilute it to prepare a solution containing about 1 mg per 1 ml, filter it, and take the consecutive filtrate as the test sample solution.
[0065] To investigate the robustness of this method, the chromatographic conditions will be slightly adjusted below, and the system suitability solution and the content of the test sample of this product will be investigated. The results are shown in Tables 12 - 14 below.
[0066] Table 12 Results of the changes in the chromatographic conditions of the robustness experiment Standard Conditions Fine - Tuning Factors for Chromatographic Conditions Flow Rate: 0.5 ml / min Flow Rates: 0.95 ml / min, 1.05 ml / min Column Temperature: 35 °C Column Temperatures: 30 °C, 40 °C 0.1% Formic Acid Aqueous Solution - Acetonitrile (10:90) 0.1% Formic Acid Aqueous Solution - Acetonitrile (88:12), 0.1% Formic Acid Aqueous Solution - Acetonitrile (92:8) Table 13 Results of the investigation of the system suitability of the robustness experiment
[0067] Table 14 Results of the content investigation of the test articles in the durability experiment Chromatographic Conditions Aminoguanidine Content (ppm) Unchanged Conditions 0.73 Change Flow Rate to 0.95 ml / min 0.53 Change Flow Rate to 1.05 ml / min 0.65 0.1% Formic Acid Aqueous Solution - Acetonitrile (89:12) 0.65 0.1% Formic Acid Aqueous Solution - Acetonitrile (92:8) 0.74 Change Column Temperature to 30 °C 0.75 Change Column Temperature to 40 °C 0.68 Mean (n = 7) 0.67 RSD% (n = 7) 11.4 Conclusion: Without changing the chromatographic conditions, changing the flow rate (±10%), the proportion of the mobile phase (±2%), and the column temperature (±5°C), under each chromatographic condition, the system suitability meets the requirements (all less than 15.0%), and there is no significant difference in the content of aminoguanidine (RSD < 15.0%). This method has good durability.
[0068] Example 3: Sample detection
[0069] Take the condensate test article and detect it according to the method of the present invention. The results are shown in Table 15 below.
[0070] Table 15 Sample detection results Batch Number Aminoguanidine Content (ppm) 10901-1911001y 1.61 10901-1911002 y 1.52 10901-1911003 y 1.73 10901-1806005 y 1.64 Conclusion: For the condensates (batch numbers: 10901-1911001y, 10901-1911002y, 10901-1911003y, 10901-1806005y), the limits of the impurity aminoguanidine all meet the requirements (≤7.5 ppm).
Claims
1. A method for detecting the content of aminoguanidine in a key intermediate of lamotrigine, characterized in that, It includes the following steps: Dissolve the test sample and the reference substance in the detection solvent respectively to obtain a test sample solution and a reference substance solution; Separate the test sample solution and the reference substance solution by liquid chromatography technology respectively, detect them with a mass spectrometry detector, and calculate the content of aminoguanidine by the external standard method based on the peak area; The liquid chromatography conditions are as follows: C8 chromatographic column, flow rate: 0.95 - 1.05 ml / min; column temperature: 30 - 40 °C; The mobile phase is: A: 0.1% formic acid aqueous solution; B: acetonitrile; Gradient elution: 0.0 - 0.8 min, 90% of A; 0.8 - 2.0 min, 90% - 10% of A; 2.0 - 4.0 min, 10% A; The mass spectrometry conditions are as follows: ESI + Positive ion mode, cone voltage is 38 V, precursor ion m / z is 75.2, quantitative ion pair m / z is 75.2 / 58.4, quantitative ion collision energy is 11 V, qualitative ion pair m / z is 75.2 / 43.5, qualitative ion collision energy is 36 V.
2. The detection method according to claim 1, wherein Flow rate: 1.0 ml / min; column temperature: 35 °C.
3. The detection method according to claim 1, characterized in that The model of the chromatographic column: Thermo BDS Hypersil C8, the specification is 4.6 mm * 150 mm, 5 μm.
4. The detection method according to claim 1, characterized in that, The model of the liquid chromatography instrument: Thermo Fisher ACCELA.
5. The detection method according to claim 1, characterized in that The model of the mass spectrometry instrument: Thermo TSQ QUANTUM ACCESS MAX, which is a triple quadrupole mass spectrometry detector.
6. The detection method according to any one of claims 1-5, characterized in that The test sample is the material obtained by sampling during the synthesis of lamotrigine to the condensate in the synthesis process, ， Condensate.
7. According to the detection method described in claim 6, it is characterized in that: Test sample solution: Take an appropriate amount of the condensate test sample, accurately weigh it, dissolve it with 50% acetonitrile solution and quantitatively dilute it to prepare a solution containing about 1 mg per 1 ml, filter it, and take the subsequent filtrate to obtain; Reference substance solution: Take an appropriate amount of aminoguanidine bicarbonate reference substance, dissolve it with water and quantitatively dilute it to prepare a solution containing about 1 mg of aminoguanidine per 1 ml, accurately measure an appropriate amount, and quantitatively dilute it with 50% acetonitrile solution to prepare a solution containing about 7.5 ng of aminoguanidine per 1 ml.