Method for detecting content of dicyandiamide in lamotrigine key intermediate

Abstract

The invention relates to the technical field of drug impurity detection, in particular to a method for detecting the content of dicyandiamide in a lamotrigine key intermediate. Respectively dissolving the test sample and the reference substance in a detection solvent to obtain a test sample solution and a reference substance solution; respectively separating the test solution and the reference solution by adopting a liquid chromatography technology, detecting by adopting a mass spectrometry detector, and calculating the content of dicyandiamide by adopting an external standard method according to a peak area; the liquid chromatography conditions are as follows: a C8 chromatographic column is adopted, and the flow rate is 0.45-0.55 ml / min; the column temperature is 30-40 DEG C; the mobile phase is A: 0.1% formic acid aqueous solution; b: acetonitrile; 8%-12% of A and 92%-88% of B are used for isocratic elution; the mass spectrometry conditions are as follows: in an ESI + positive ion mode, the taper hole voltage is 52V, the parent ion m / z is 85.2, the quantitative ion pair m / z is 85.2 / 68.4, the quantitative ion collision energy is 16V, the qualitative ion pair m / z is 85.2 / 43.5, and the qualitative ion collision energy is 14V. The methodology verifies that the method can be used for quantitatively detecting the content of dicyandiamide in the lamotrigine key intermediate.

Description

Technical Field

[0001] The present application relates to the technical field of drug impurity detection, and in particular to a method for detecting the content of dicyandiamide in a key intermediate of lamotrigine. Background Art

[0002] Lamotrigine is a sodium channel blocker clinically used to treat epilepsy. The typical synthesis of lamotrigine uses 2,3-dichlorobenzoic acid as the starting compound. 2,3-Dichlorobenzoic acid reacts with thionyl chloride (also known as dichlorothionyl, SOCl2) to produce 2,3-dichlorobenzoyl chloride, which then reacts with cuprous cyanide (CuCN) to produce 2,3-dichlorobenzoyl cyanide. 2,3-Dichlorobenzoyl cyanide then reacts with aminoguanidine bicarbonate (see the formula below) to form a condensation product (chemically named: 2-cyano-(2,3-dichlorophenyl)-2-guanidininylacetonitrile), which then undergoes a cyclization reaction to produce lamotrigine. Aminoguanidine bicarbonate is a reaction reagent in the lamotrigine synthesis process. Aminoguanidine bicarbonate is synthesized from monocyanamide (CH2-NH2) + hydrazine (NH2-NH2) + CO2 + H2O. Dicyandiamide is a by-product of the aminoguanidine bicarbonate synthesis process. Since dicyandiamide contains genotoxic groups, quality control research on dicyandiamide should be carried out.

[0003]

[0004] According to the ICH "M7: Guidance for Industry on the Evaluation and Control of DNA-Reactive (Mutagenic) Impurities in Pharmaceuticals to Limit Potential Carcinogenic Risk" and the "9306 Guidelines for the Control of Genotoxic Impurities" of the 2020 edition (Part IV) of the Chinese Pharmacopoeia, the Threshold of Toxicological Concern (TTC) for dicyandiamide is 1.5 μg / day. According to the instructions for use and dosage of lamotrigine tablets, when used in adults or children over 12 years of age, the daily dose can reach 200 mg. Therefore, the control limit for dicyandiamide should not exceed 7.5 ppm. Accurately detecting such low levels of this substance in samples is a pressing technical challenge for those skilled in the art. Dicyandiamide (also known as dicyandiamide, C2H4N4) has a relative molecular weight of 84.08. Direct determination is difficult and subject to significant interference. Therefore, it is necessary to develop a method that can be applied to the detection of dicyandiamide in key lamotrigine intermediates. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the content of dicyandiamide in a key intermediate of lamotrigine, comprising the following steps: Dissolve the test sample and reference sample in the detection solvent to obtain a test sample solution and a reference sample solution respectively; The test solution and the reference solution were separated by liquid chromatography, detected by mass spectrometry, and the content of dicyandiamide was calculated by peak area using the external standard method; The liquid chromatography conditions are: C8 chromatographic column, flow rate: 0.45-0.55 ml / min; column temperature: 30-40°C; The mobile phases were: A: 0.1% formic acid in water; B: acetonitrile; isocratic elution was performed with 8%-12% A and 92%-88% B; The mass spectrometry conditions are: ESI + In positive ion mode, the cone voltage was 52 V, the parent ion m / z was 85.2, the quantitative ion pair m / z was 85.2 / 68.4, the quantitative ion collision energy was 16 V, the qualitative ion pair m / z was 85.2 / 43.5, and the qualitative ion collision energy was 14 V.

[0006] Preferably, the flow rate is 0.5 ml / min and the column temperature is 35°C.

[0007] The chromatographic column model is: Thermo BDS Hypersil C8, with specifications of 4.6 mm*150 mm and 5 μm.

[0008] The liquid chromatography instrument model is: Thermo Fisher ACCELA.

[0009] The mass spectrometer model is: Thermo TSQ QUANTUM ACCESS MAX, which is a triple quadrupole mass spectrometer detector.

[0010] Preferably, the elution is performed isocratically with 10% A and 90% B.

[0011] The test sample is a material sampled from the lamotrigine synthesis process when the condensate is synthesized. condensation products.

[0012] Test solution: Take an appropriate amount of the condensate test sample, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate; Reference solution: Take an appropriate amount of dicyandiamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 7.5 ng per 1 ml.

[0013] The present application provides a method for detecting the content of dicyandiamide in a key intermediate of lamotrigine, which uses a specific liquid chromatography-mass spectrometry method for detection. According to methodological verification, the method has good specificity, precision and repeatability, and the sensitivity meets the control requirements of the impurity.

[0014] The detection method provided in the present application has a limit of quantification of 0.0150 ng / ml and a limit of detection of 0.0075 ng / ml. The method has good sensitivity, and the detection limit and the limit of quantification are significantly lower than the quality control limit requirements of the dicyandiamide trace impurity, indicating that the sensitivity of the present method meets the control requirements of the impurity. The recovery rate test proves that the sample processing method and the detection method of the present invention can realize the determination of the content of the dicyandiamide trace impurity in the condensation product, and can be applied to the quality monitoring of the dicyandiamide impurity in the condensation product of the key intermediate of lamotrigine. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Optimization diagram for the impurity dicyandiamide ion; Figure 2 Methanol spectrum of blank solvent for method validation (specificity) of impurity dicyandiamide; Figure 3 This is the sample spectrum of the condensation product of the impurity dicyandiamide for methodological validation (specificity); Figure 4 This is the reference sample spectrum for the methodology validation (specificity) of the impurity dicyandiamide. DETAILED DESCRIPTION

[0016] In the typical synthesis process of lamotrigine, the condensation product [chemical name: 2-cyano-(2,3-dichlorophenyl)-2-guanidine iminoacetonitrile] generated by the reaction of 2,3-dichlorobenzoyl cyanide and aminoguanidine bicarbonate is the last step in the synthesis process of lamotrigine. The condensation product is a key intermediate material for the synthesis of lamotrigine. The quality control of trace amounts of potential genotoxic impurities in the key intermediate condensation product plays a very important role in controlling the potential genotoxic impurities in the finished lamotrigine product. Therefore, this application preferably selects the condensation product as the key intermediate for detection.

[0017] 1 Optimization of liquid chromatography conditions 1) Liquid chromatography conditions HPLC instrument model: Thermo Fisher ACCELA Chromatographic column: Thermo BDS Hypersil C8 (4.6mm*150mm, 5μm) Flow rate: 0.5 ml / min; column temperature: 35°C; injection volume: 10 μl; injector temperature: 25°C; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile, isocratic elution with 10% A and 90% B.

[0018] 2) Optimization of chromatographic conditions The flow rates (0.45 ml / min, 0.5 ml / min, 0.55 ml / min), column temperatures (30°C, 35°C, 40°C), and mobile phase ratios (0.1% formic acid aqueous solution-acetonitrile, 10:90, 8:92, 12:88) 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 flow rate was tentatively set at 0.5 ml / min, the column temperature was 35°C, and the 0.1% formic acid aqueous solution-acetonitrile ratio was 10:90. For details, see Example 2 Durability Experiment.

[0019] 2 Mass spectrometry ion optimization Using the positive ion mode, the dicyandiamide ion optimization results showed that the parent ion m / z was 85.2, which is consistent with the theoretical molecular weight. The dicyandiamide ion optimization results are shown in Figure 1 .

[0020] 3 Dicyandiamide detection conditions 1) Liquid chromatography conditions Chromatographic column: Thermo BDS Hypersil C8 (4.6mm*150mm, 5μm) Flow rate: 0.5 ml / min; column temperature: 35°C; injection volume: 10 μl; injector temperature: 25°C; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile, isocratic elution with 10% A and 90% B.

[0021] 2) Mass spectrometry conditions (Table 1) Mass spectrometer model: Thermo TSQ QUANTUM ACCESS MAX (triple quadrupole) Table 1 Mass spectrometry parameters <![CDATA[ESI + (SRM)]]> Compound: Dicyandiamide Precursor ion m / z 85.2 Cone voltage (V) 52 Quantitative ion pair m / z 85.2 / 68.4 Quantitative ion collision energy (V) 16 Qualifier ion pair m / z 85.2 / 43.5 Qualifying ion collision energy (V) 14 Capillary Temperature (℃) 350 Vaporizer Temperature (℃) 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 condensation product sample, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate.

[0022] 4) Reference solution Take an appropriate amount of dicyandiamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 7.5 ng per 1 ml.

[0023] 5) System suitability requirements Take the reference solution and inject it continuously for 5 times. The relative standard deviation of the dicyandiamide peak area shall not exceed 15.0%.

[0024] 6) Determination method Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0025] 7) Limits If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the dicyandiamide peak, the peak area calculated by the external standard method shall not exceed 7.5ppm.

[0026] Example 2: Methodological Verification (Method under "3" in Example 1) 1. Exclusivity 1) Interference of blank solvent on dicyandiamide ① Take an appropriate amount of dicyandiamide reference solution and prepare it according to the method for the reference solution under "3" described in Example 1 to obtain a reference solution.

[0027] ② Take an appropriate amount of the condensation product test sample (batch number: 10901-1911001y) and prepare the test sample solution according to the method for the test sample solution under "Item 3" described in Example 1 to obtain the test sample solution.

[0028] Take appropriate amounts of methanol, reference solution, and test solution, and analyze the sample according to the method "under 3" described in Example 1 to investigate the interference of blank solvent methanol on dicyandiamide. The results are shown in Table 2 below. Figures 2-4 .

[0029] Table 2 Blank solvent interference results Sample name Peak area Retention time (min) Methanol 4 3.24 Reference solution 25602 3.29 Test solution 2212 3.29 Conclusion: The peak area of methanol at the main peak position is 4, which is less than 0.05% of the peak area of the reference substance (i.e.: 25602×0.05%=13). The blank solvent methanol does not interfere with the detection of dicyandiamide.

[0030] 2) Interference of other impurities on dicyandiamide ① According to the condensation process route, the compounds involved in the condensation synthesis process are aminoguanidine carbonate, cyanamide, hydrazine, 2,3-dichlorobenzoyl acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide.

[0031] ②Impurity interference analysis The m / z of the quantitative ion of dicyandiamide is 68.4. The molecular weights of monocyanamide and hydrazine are both less than 68.4. After mass spectrometry collision, no daughter ion with m / z of 68.4 is generated. According to the structural analysis, aminoguanidine carbonate, compounds 2,3-dichlorobenzoyl acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide are unlikely to generate daughter ions with m / z of 68.4 after mass spectrometry collision. Comprehensive analysis shows that the above compounds do not interfere with the detection of dicyandiamide at the ion channel with m / z of 68.4.

[0032] 2. Filter membrane adsorption 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. Divide it into 3 equal parts and number them 1#, 2# and 3#. Centrifuge 1# at 10,000 rpm for 10 minutes; discard 0.1 ml of the initial filtrate from 2# (made of nylon 66, 0.22 μm); discard 0.3 ml of the initial filtrate from 3# (made of nylon 66, 0.22 μm).

[0033] The solutions treated with 1#, 2#, and 3# were respectively sampled and analyzed according to the method under "item 3" described in Example 1. The samples were injected three times continuously, and the peak areas were recorded to examine the adsorption rate of the filter membrane (adsorption rate = 100% - peak area after adsorption / peak area without adsorption × 100%). The results are shown in Table 3 below.

[0034] Table 3 Dicyandiamide filter membrane adsorption test results Injection number 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: Nylon 66 filter membrane (0.22μm) has no adsorption to dicyandiamide.

[0035] 3. Linearity and range Take an appropriate amount of dicyandiamide reference substance and add methanol to quantitatively dilute it to prepare a series of dicyandiamide reference substance solutions containing approximately 0.75ng, 1.50ng, 3.75ng, 7.50ng, 15.00ng, and 22.50ng per 1ml.

[0036] The reference solution of dicyandiamide at each concentration was sampled and analyzed according to the method under "item 3" described in Example 1. The peak area of dicyandiamide was used as the ordinate (Y), and the concentration of the dicyandiamide reference substance was used as the abscissa (X). A linear regression was performed on the peak area Y versus the concentration X (ng / ml). The linear equation and correlation coefficient are shown in Table 4 below.

[0037] Table 4 Dicyandiamide linearity test results

[0038] Conclusion: There was a good linear correlation between the peak area (Y) and concentration (X) of dicyandiamide in the range of 0.75-22.50 ng / ml, and the linear equation was: y=2887.6x+473.13 (r=0.9992).

[0039] 4. Limit of quantification and detection limit An appropriate amount of dicyandiamide reference substance was quantitatively diluted with methanol to produce a solution containing approximately 1.50 ng per 1 ml. This solution was then diluted with methanol to an appropriate concentration and analyzed using the method described in "Item 3" of Example 1. Using the signal-to-noise ratio method, the concentration was the detection limit when the signal-to-noise ratio was approximately 3:1, and the quantification limit when the signal-to-noise ratio was approximately 10:1. The results are shown in Table 5 below.

[0040] Table 5 Dicyandiamide quantitative limit detection limit experimental results sample Concentration (ng / ml) Peak area S / N Equivalent to the test concentration in ppm 1.5ng (mother solution) 1.5000 4113 717.79 1.5000 Dilute the stock solution 100 times (limit of quantification) 0.0150 60 53.06 0.0150 Dilute the stock solution 200 times (detection limit) 0.0075 27 1.23 0.0075 Methanol / 12 0.88 / Conclusion: The S / N ratio of the dicyandiamide mother solution (1.5 ng / ml) was continuously diluted to 100-fold, reaching a value of 53.06. The provisional limit of quantification (LOQ) for dicyandiamide was 0.0150 ng / ml, equivalent to a test sample concentration of 0.0150 ppm. The S / N ratio was 1.23 at a 200-fold dilution, resulting in a provisional limit of detection (LOD) of 0.0075 ng / ml, equivalent to a test sample concentration of 0.0075 ppm. The sensitivity of this method meets the control requirements for dicyandiamide.

[0041] 5. Precision 1) Injection precision A 7.50 ng / ml dicyandiamide solution under the experimental item "2. Linearity and Range" was injected and analyzed according to the method described in "3" of Example 1. The injections were repeated five times, and the retention times and peak areas were calculated. The results are shown in Table 6 below.

[0042] Table 6 Dicyandiamide injection precision test results Serial number 1 2 3 4 5 mean RSD% Retention time (min) 3.35 3.35 3.34 3.35 3.35 3.35 0.1 Peak area (A) 19264 20258 20571 21081 22628 20760 6.0 Conclusion: The dicyandiamide reference solution was injected 5 times continuously, and the RSDs of retention time and peak area were 0.1% and 6.0%, respectively, indicating good injection precision.

[0043] 2) Repeatability ① Preparation of test solution: Take an appropriate amount of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate as the test sample solution. Prepare 6 parallel portions.

[0044] ② Preparation of reference solution 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 as the reference substance solution.

[0045] Take the test solution and the reference solution, and analyze the sample according to the method under "3" described in Example 1. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the dicyandiamide peak, the content of dicyandiamide is calculated by the peak area according to the external standard method. The results are shown in Table 7 below.

[0046] Table 7 Dicyandiamide repeatability test results Sample No. Dicyandiamide Content 1 2 3 4 5 6 Mean (n=6) RSD% (n=6) Content (ppm) 0.78 0.90 0.75 0.84 0.75 0.78 0.80 7.3 Conclusion: As can be seen from the above table, the RSD of dicyandiamide content in 6 parallel samples was 7.3%, and the repeatability of this method was good.

[0047] 6. Solution stability 1) Stability of reference solution 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 as the reference substance solution.

[0048] The reference solution was placed at room temperature, and samples were taken at 0 h, 2 h, 4 h, 6 h, and 8 h, respectively, and analyzed according to the "3 items" method described in Example 1 to examine the changes in peak area at each time point. The results are shown in Table 8 below.

[0049] Table 8 Dicyandiamide reference solution stability test results Serial number 0h 2h 4h 6h 8h mean RSD% Retention time (min) 3.34 3.35 3.35 3.35 3.35 3.35 0.1 Peak area (A) 19264 19632 19140 20109 19711 19571 2.0 Conclusion: The dicyandiamide reference solution was placed at room temperature for 8 hours, and the RSD of the main peak area was 2.0%, indicating good stability.

[0050] 2) Stability of test solution Take an appropriate amount of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate as the test sample solution.

[0051] The test solution was placed at room temperature, and samples were taken at 0 h, 2 h, 4 h, and 6 h, respectively, and the sample was analyzed according to the "3 items" method described in Example 1 to examine the changes in peak area at each time point. The results are shown in Table 9 below.

[0052] Table 9 Dicyandiamide test solution stability test results Serial number 0h 2h 4h 6h mean RSD% Peak area (A) 2277 2745 2102 2101 2306 13.2 Content (ppm) 0.78 0.94 0.72 0.72 0.79 13.2 Conclusion: After the test solution was placed at room temperature for 6 hours, the RSD of the main peak area was 13.2%, indicating good stability.

[0053] 7. Accuracy 1) Reference solution 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 as the reference substance solution.

[0054] 2) Test solution ①Dicyandiamide stock solution Take an appropriate amount of dicyandiamide reference substance, add methanol to quantitatively dilute it to make a dicyandiamide reference substance stock solution containing approximately 75ng per 1ml.

[0055] ②50% test solution Take about 10 mg of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 9.5 ml of methanol, add 0.5 ml of dicyandiamide reference stock solution (75 ng / ml), dissolve it by ultrasonication, cool to room temperature, filter, and take the filtrate as the 50% accuracy solution, and prepare 3 parallel portions.

[0056] ③100% test solution: Take about 10 mg of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 9 ml of methanol, add 1 ml of dicyandiamide reference stock solution (75 ng / ml), ultrasonically dissolve it, cool to room temperature, filter, and take the filtrate as the 100% accuracy solution. Prepare three parallel aliquots.

[0057] ④150% test solution Take about 10 mg of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 8.5 ml of methanol, add 0.5 ml of dicyandiamide reference substance stock solution (75 ng / ml), dissolve it by ultrasonication, cool to room temperature, filter, and take the filtrate as the 150% accuracy solution, and prepare three parallel portions.

[0058] ⑤ Take the mean value measured under the "Repeatability" item as the background amount of this experiment (this experiment and the repeatability experiment are carried out on the same day).

[0059] The above test solution and reference solution were accurately measured respectively, and the samples were analyzed according to the method under "3" described in Example 1. The content of dicyandiamide was calculated by peak area according to the external standard method. The recovery of this product was investigated. The results are shown in Table 10 below.

[0060] Table 10 Dicyandiamide accuracy test results

[0061] Conclusion: The recovery of dicyandiamide in 9 test solutions ranged from 72.2% to 89.0%, with an average recovery of 81.3% and an RSD of 8.2%, which met the recovery limit requirements. The recovery of this method was good.

[0062] 8. Durability 1) Reference solution 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 as the reference substance solution.

[0063] 2) Test solution Take an appropriate amount of the condensation product test sample (batch number: 10901-1911001y), accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate as the test sample solution.

[0064] In order to examine the robustness of this method, the chromatographic conditions will be slightly adjusted to examine the system suitability of the solution and the content of the test sample. The results are shown in Tables 11-13 below.

[0065] Table 11 Results of chromatographic condition changes in durability experiment Standard conditions Factors for fine-tuning chromatographic conditions Flow rate: 0.5ml / min Flow rate: 0.45ml / min, 0.55ml / min Column temperature: 35°C Column temperature: 30℃, 40℃ 0.1% formic acid aqueous solution-acetonitrile (10:90) 0.1% formic acid aqueous solution-acetonitrile (8:92) 0.1% formic acid aqueous solution-acetonitrile (12:88) Table 12 Results of the suitability investigation of the durability test system

[0066] Table 13 Durability test results of sample content Chromatographic conditions Dicyandiamide content (ppm) Unchanged conditions 0.57 Change the flow rate to 0.45ml / min 0.60 Change the flow rate to 0.55ml / min 0.56 0.1% formic acid aqueous solution-acetonitrile (8:92) 0.58 0.1% formic acid aqueous solution-acetonitrile (12:88) 0.54 Change the column temperature to 30℃ 0.55 Change the column temperature to 40℃ 0.37 Mean (n=7) 0.54 RSD% (n=7) 14.1 Conclusion: Without changing the chromatographic conditions, the flow rate (±10%), mobile phase ratio (±2%), and column temperature (±5°C) were changed. Under each chromatographic condition, the system suitability met the requirements (all less than 15.0%), and there was no significant difference in the dicyandiamide content (RSD < 15.0%). This method has good durability.

[0067] The condensation product sample was tested according to the method under "3" described in Example 1. The results are shown in Table 14 below.

[0068] Table 14 Sample test results batch number Dicyandiamide content (ppm) 10901-1911001y 0.80 10901-1911002 y 0.76 10901-1911003 y 0.68 10901-1806005 y 0.83 Conclusion: The condensation products (batch numbers: 10901-1911001y, 10901-1911002y, 10901-1911003y, 10901-1806005y), the limit of impurity dicyandiamide all met the requirements (≤7.5ppm).

Claims

1. A method for detecting the content of dicyandiamide in a key intermediate of lamotrigine, characterized in that: The following steps are involved: Dissolve the test sample and reference sample in the detection solvent to obtain a test sample solution and a reference sample solution respectively; The test solution and the reference solution were separated by liquid chromatography, detected by mass spectrometry, and the content of dicyandiamide was calculated by peak area using the external standard method; The liquid chromatography conditions are: C8 chromatographic column, flow rate: 0.45-0.55 ml / min; column temperature: 30-40°C; The mobile phases were: A: 0.1% formic acid in water; B: acetonitrile; isocratic elution was performed with 8%-12% A and 92%-88% B; The mass spectrometry conditions are: ESI + In positive ion mode, the cone voltage was 52 V, the parent ion m / z was 85.2, the quantitative ion pair m / z was 85.2 / 68.4, the quantitative ion collision energy was 16 V, the qualitative ion pair m / z was 85.2 / 43.5, and the qualitative ion collision energy was 14 V.

2. The detection method according to claim 1, wherein Flow rate: 0.5 ml / min; column temperature: 35°C.

3. The detection method according to claim 1, wherein The chromatographic column model is: Thermo BDS Hypersil C8, with specifications of 4.6 mm*150 mm and 5 μm.

4. The detection method according to claim 1, wherein The liquid chromatography instrument model is: ThermoFisher ACCELA.

5. The detection method according to claim 1, wherein The mass spectrometer model is Thermo TSQQUANTUM ACCESS MAX, which is a triple quadrupole mass spectrometer detector.

6. The detection method according to claim 1, wherein: Elution was performed isocratically with 10% A and 90% B.

7. The detection method according to any one of claims 1 to 6, characterized in that The test sample is the material obtained by sampling during the synthesis of lamotrigine to the condensation product. condensation products.

8. The detection method according to claim 7, wherein: Test solution: Take an appropriate amount of the condensate test sample, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 1 mg per 1 ml, filter it, and take the filtrate; Reference solution: Take an appropriate amount of dicyandiamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 7.5 ng per 1 ml.

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