Method for detecting content of cyanamide 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 cyanamide 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 substance solution by adopting a liquid chromatography technology, detecting by adopting a mass spectrometry detector, and calculating the content of hydrazine by adopting an external standard method according to a peak area; the liquid chromatography conditions are as follows: a C18 chromatographic column is adopted, and the flow rate is 0.5-0.7 ml / min; the column temperature is 30-40 DEG C; the mobile phase is A: 0.1% formic acid aqueous solution; b: acetonitrile; 3%-7% of A and 97%-93% of B are used for isocratic elution; the mass spectrometry conditions are as follows: in an ESI-anion mode, the taper hole voltage is 52V, the parent ion m / z is 274.2, the quantitative ion pair m / z is 274.2 / 257.9, the quantitative ion collision energy is 31V, the qualitative ion pair m / z is 274.2 / 194.0, and the qualitative ion collision energy is 39V. The methodology verifies that the method can be used for quantitatively detecting the content of cyanamide in the lamotrigine key intermediate.

Description

Technical Field

[0001] The present application relates to the technical field of drug impurity separation and detection, and specifically to a method for detecting the content of monocyanamide 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 figure 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 cyanamide (CH2-NH2) + hydrazine (NH2-NH2) + CO2 + H2O, where cyanamide (also known as aminocyanamide) is the starting material in the aminoguanidine bicarbonate synthesis process. Since cyanamide contains genotoxic groups, quality control studies should be conducted on cyanamide.

[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 cyanamide is 1.5 μg / day. According to the instructions for use and dosage of lamotrigine tablets, the daily dose of this product for adults or children over 12 years of age can reach 200 mg. Therefore, the control limit for cyanamide should not exceed 7.5 ppm. Accurately detecting such low levels of this substance in samples is a technical challenge urgently needed by those skilled in the art. Cyanamide (CH2N2) is highly polar, has a low relative molecular weight (42.04), and lacks a chromophore. Ultraviolet (UV) detection has low sensitivity, necessitating the development of a method applicable to the detection of cyanamide 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 cyanamide 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 hydrazine content was calculated by peak area using the external standard method; The liquid chromatography conditions are: C18 chromatographic column, flow rate: 0.5-0.7 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 3%-7% A and 97%-93% B; The mass spectrometry conditions are: ESI - In negative ion mode, the cone voltage was 52 V, the parent ion m / z was 274.2, the quantitative ion pair m / z was 274.2 / 257.9, the quantitative ion collision energy was 31 V, the qualitative ion pair m / z was 274.2 / 194.0, and the qualitative ion collision energy was 39 V.

[0006] Preferably, the flow rate is 0.6 ml / min and the column temperature is 35°C.

[0007] The chromatographic column model: Agilent ZORBAX Eclipse Plus C 18 , specifications are 4.6mm*150mm, 3.5μm.

[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 mobile phase is 5% A and 95% B for isocratic elution.

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

[0012] Furthermore, an appropriate amount of the condensate test sample was taken, accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution containing approximately 2.6 mg per 1 ml. 1 ml was placed in a centrifuge tube, 1 ml of a sodium carbonate-sodium bicarbonate mixed solution of pH 8.00-pH 10.50 was added, 1 ml of a dansyl chloride acetone solution of 8 mg / ml-14 mg / ml was added, mixed, and derivatized in a water bath at 40°C-60°C for 40-80 minutes. The product was cooled to room temperature, filtered, and 1 ml of the filtrate was taken, 1 ml of methanol was added, and mixed to obtain the test solution.

[0013] Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.00-pH 10.50, add 1 ml of dansyl chloride acetone solution with a pH of 8.00-10.50, mix well, and derivatize in a water bath at 40-60°C for 40-80 minutes. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and the reference substance solution is obtained.

[0014] Preferably, Take an appropriate amount of the condensation product test sample, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 2.6 mg per 1 ml, take 1 ml and place it in a centrifuge tube, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.96, add 1 ml of 10 mg / ml dansyl chloride acetone solution, mix well, derivatize it in a 60°C water bath for 1 hour, cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and the test solution is obtained.

[0015] Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.96 and 1 ml of 10 mg / ml dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, and mix well to obtain the reference substance solution.

[0016] 0.2 mol / L sodium carbonate solution: Take 0.53 g of sodium carbonate and dissolve it in 25 ml of water; 0.2 mol / L sodium bicarbonate solution: Take 1.68 g of sodium bicarbonate and dissolve it in 100 ml of water; Sodium carbonate-sodium bicarbonate mixed solution with pH 8.96: Mix 4 ml of 0.2 mol / L sodium carbonate solution and 46 ml of 0.2 mol / L sodium bicarbonate solution to obtain the solution.

[0017] The present application first provides a method for detecting the content of monocyanamide in the key intermediate of lamotrigine, which uses a specific liquid chromatography-mass spectrometry method for detection. After methodological verification, the method has good specificity, precision and repeatability, and the sensitivity meets the control requirements of the impurity.

[0018] The detection method provided in the present application has a limit of quantification of 0.0010 ng / ml and a limit of detection of 0.0005 ng / ml. The method has good sensitivity, and the limit of detection and the limit of quantification are significantly lower than the quality control limit requirements of the trace impurity of monocyanamide, 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 content determination of the trace impurity of monocyanamide in the condensation product, and can be applied to the quality monitoring of the monocyanamide impurity in the key intermediate condensation product of lamotrigine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Optimization diagram for the impurity cyanamide derivative ion; Figure 2 Methanol spectrum of blank solvent for method validation (specificity) of impurity cyanamide; Figure 3 This is the negative blank methanol spectrum for the methodological validation (specificity) of the impurity cyanamide; Figure 4 This is the sample spectrum of the condensation product of the impurity cyanamide for method validation (specificity); Figure 5 This is the reference substance spectrum for the methodology validation (specificity) of the impurity cyanamide; Figure 6 Dicyandiamide spectrum for methodological validation (specificity) of impurity cyanamide; Figure 7 This is the test spectrum of the test product batch 10901-1911002; Figure 8 This is the test spectrum of the test product batch 10901-1911003; Figure 9 This is the test spectrum of the test product batch 10901-1806005. DETAILED DESCRIPTION

[0020] 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.

[0021] 1. Detection conditions of cyanamide derivatives 1) The principle of cyanamide derivatization reaction is as follows:

[0022] 2) Mass spectrometry ion optimization Using negative ion mode, the optimization results of cyanamide derivative ions showed that the parent ion m / z was 274.2. The optimization results of cyanamide derivative ions are shown in Figure 1 .

[0023] 3) Liquid chromatography conditions HPLC instrument model: Thermo Fisher ACCELA Column: Agilent ZORBAX Eclipse Plus C 18 (4.6mm*150mm, 3.5μm) Flow rate: 0.6 ml / min; column temperature: 35°C; injection volume: 5 μl; injector temperature: 25°C; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile, eluted isocratically from 5% A to 95% B.

[0024] 4) Mass spectrometry conditions (Table 1) Mass spectrometer model: Thermo TSQ QUANTUM ACCESS MAX (triple quadrupole) Table 1 Mass spectrometry parameters <![CDATA[ESI - (SRM)]]> Compound: Cyanamide derivative Precursor ion m / z 274.2 Cone voltage (V) 52 Quantitative ion pair m / z 274.2 / 257.9 Quantitative ion collision energy (V) 31 Qualifier ion pair m / z 274.2 / 194.0 Qualifying ion collision energy (V) 39 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 2 Study on the reaction conditions of cyanamide derivatization 1) Solution preparation ① Preparation of cyanamide reference stock solution Take an appropriate amount of cyanamide reference substance, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 20 ng of cyanamide per 1 ml.

[0025] ② Preparation of dansyl chloride acetone solution Take an appropriate amount of dansyl chloride, accurately weigh it, dissolve it in acetone and quantitatively dilute it to make a solution containing approximately 10 mg of dansyl chloride per 1 ml.

[0026] ③ Preparation of sodium carbonate-sodium bicarbonate mixed solution: Sodium carbonate solution (0.2 mol / L): Dissolve 0.53 g of sodium carbonate in 25 ml of water; Sodium bicarbonate solution (0.2 mol / L): Dissolve 1.68 g of sodium bicarbonate in 100 ml of water. Sodium carbonate-sodium bicarbonate mixed solution: Mix 4 ml of 0.2 mol / L sodium carbonate solution and 46 ml of 0.2 mol / L sodium bicarbonate solution.

[0027] 2) pH screening of sodium carbonate-sodium bicarbonate mixed solution for derivatization reaction Take an appropriate amount of a sodium carbonate-sodium bicarbonate mixed solution (pH 8.96) and adjust the pH to 8.00 and 10.50 with 0.1 mol / L hydrochloric acid and 0.1 mol / L sodium hydroxide solution, respectively. Place 1 ml of the cyanamide reference stock solution (20 ng / ml) in 10 ml centrifuge tubes. Take three aliquots and add 1 ml of each sodium carbonate-sodium bicarbonate mixed solution at pH 8.00, pH 8.96, and pH 10.50, respectively. Add 1 ml of dansyl chloride acetone solution to each aliquot and mix thoroughly. Derivatize in a 60°C waterbath for 1 hour. Cool to room temperature, filter, and take 1 ml of each derivatization filtrate. Add 1 ml of methanol to each aliquot and mix thoroughly. Inject and analyze according to the above detection conditions. Calculate the peak area at different derivatization pH values. The results are shown in Table 2 below.

[0028] Table 2 Derivatization reaction pH screening results Sample number Peak area pH8.00 70477 pH8.96 86102 pH10.50 48492 Conclusion: From the above table, it can be seen that when the pH of the sodium carbonate-sodium bicarbonate mixed solution of the derivatization reaction is 8.96, the reaction is complete, and the derivative peak area is significantly larger than that of pH 8.00 and pH 10.50. The pH of the sodium carbonate-sodium bicarbonate mixed solution of the derivatization reaction is tentatively determined to be 8.96 (that is, the sodium carbonate-sodium bicarbonate mixed solution can be used directly without adjusting the pH after preparation).

[0029] 3) Screening of dosage of derivatization reagent (dansyl chloride) Accurately weigh an appropriate amount of dansyl chloride, dissolve in acetone, and quantitatively dilute to produce solutions containing approximately 8 mg, 10 mg, 12 mg, and 14 mg of dansyl chloride per 1 ml. Place 1 ml of the cyanamide reference stock solution (20 ng / ml) in 10 ml centrifuge tubes. Take four aliquots, add 1 ml of a sodium carbonate-sodium bicarbonate mixture to each aliquot, and then add 1 ml of 8 mg / ml, 10 mg / ml, 12 mg / ml, and 14 mg / ml dansyl chloride acetone solutions, respectively. Mix thoroughly. Derivatize in a 60°C waterbath for 1 hour. Cool to room temperature, filter, and take 1 ml of each filtrate. Add 1 ml of methanol to each aliquot, mix thoroughly, and analyze according to the above detection conditions. Calculate the peak area for each dansyl chloride dosage as the derivatization reagent. The results are shown in Table 3 below.

[0030] Table 3 Screening results of different dosages of dansyl chloride Dansyl chloride concentration mg / ml Peak area 8 57095 10 57688 12 58453 14 57198 Mean (n=4) 57608 RSD% (n=4) 1.1 Conclusion: From the above table, it can be seen that when the concentrations of dansyl chloride are 8 mg / ml, 10 mg / ml, 12 mg / ml and 14 mg / ml, there is no significant difference in the peak area of the derivatives (RSD of the peak area of different concentrations of dansyl chloride is 1.1%). The concentration of dansyl chloride is tentatively set to 10 mg / ml.

[0031] 4) Screening of derivatization reaction time Take 1 ml of the monocyanamide reference stock solution (20 ng / ml) and place it in a 10 ml centrifuge tube. Take three parallel aliquots, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution to each aliquot, add 1 ml of dansyl chloride acetone solution to each aliquot, mix well, and derivatize in a 60°C water bath for 40 min, 60 min, and 80 min, respectively. Cool to room temperature, filter separately, take 1 ml of each filtrate, add 1 ml of methanol to each aliquot, mix well, and analyze according to the above detection conditions. The peak areas at different derivatization reaction times are counted. The results are shown in Table 4 below.

[0032] Table 4 Screening results of different derivatization reaction times Derivatization reaction time (min) Peak area 40 84102 60 87719 80 83798 Mean (n=3) 85206 RSD% (n=3) 2.6 Conclusion: From the above table, it can be seen that there is no significant difference in the peak area of the derivatives when the derivatization reaction is 40min, 60min, and 80min (the RSD of the peak area at different reaction times is 2.6%), and the tentative reaction time is 60min.

[0033] 5) Screening of derivatization reaction temperature Take 1 ml of the monocyanamide reference stock solution (20 ng / ml) and place it in a 10 ml centrifuge tube. Take three parallel aliquots, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution to each aliquot, add 1 ml of dansyl chloride acetone solution to each aliquot, mix well, and derivatize in a water bath at 40°C, 50°C, and 60°C for 1 hour. Cool to room temperature, filter separately, take 1 ml of each filtrate, add 1 ml of methanol to each aliquot, mix well, and analyze according to the above detection conditions. The peak areas at different derivatization reaction temperatures are counted. The results are shown in Table 5 below.

[0034] Table 5 Screening results of different derivatization reaction temperatures Derivatization reaction temperature (℃) Peak area 40 60958 50 57838 60 57262 Mean (n=3) 58686 RSD% (n=3) 3.4 Conclusion: From the above table, it can be seen that when the derivatization reaction temperatures are 40℃, 50℃n and 60℃, there is no significant difference in the peak area of the derivatives (RSD of the peak area at different reaction temperatures is 3.4%). The tentative reaction temperature is 60℃.

[0035] In summary, the derivatization reaction conditions for cyanamide are as follows: 1 ml of a 20 ng / ml cyanamide reference stock solution is placed in a 10 ml centrifuge tube, 1 ml of a sodium carbonate-sodium bicarbonate solution is added, and 1 ml of a dansyl chloride acetone solution is added. Mix thoroughly, and derivatize in a 60°C water bath for 1 hour to obtain the product. Accuracy experiments (see Example 2, Methodology Validation) further validated the rationality of the tentative cyanamide derivatization reaction conditions.

[0036] 3 Optimization of liquid chromatography conditions 1) Liquid chromatography conditions Column: Agilent ZORBAX Eclipse Plus C 18(4.6mm*150mm, 3.5μm) Flow rate: 0.6 ml / min; column temperature: 35°C; injection volume: 5 μl; injector temperature: 25°C; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile, eluted isocratically from 5% A to 95% B.

[0037] 2) Optimization of chromatographic conditions The flow rates (0.5 ml / min, 0.6 ml / min, 0.7 ml / min), column temperatures (30°C, 35°C, 40°C), and mobile phase ratios (0.1% formic acid aqueous solution-acetonitrile, 5:95, 7:93, 3:97) 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 0.6 ml / min, the column temperature at 35°C, and the ratio of 0.1% formic acid aqueous solution-acetonitrile at 5:95. For details, see the durability experiment (see Example 2 under Methodology Verification).

[0038] 4 Determined detection methods 1) Liquid chromatography conditions Column: Agilent ZORBAX Eclipse Plus C 18 (4.6mm*150mm, 3.5μm) Flow rate: 0.6 ml / min; column temperature: 35°C; injection volume: 5 μl; injector temperature: 25°C; Mobile phase: A: 0.1% formic acid in water; B: acetonitrile, eluted isocratically from 5% A to 95% B.

[0039] 2) Mass spectrometry conditions (Table 6) Table 6 Mass spectrometry parameters <![CDATA[ESI - (SRM)]]> Compound: Cyanamide derivative Precursor ion m / z 274.2 Cone voltage (V) 52 Quantitative ion pair m / z 274.2 / 257.9 Quantitative ion collision energy (V) 31 Qualifier ion pair m / z 274.2 / 194.0 Qualifying ion collision energy (V) 39 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 condensate sample, accurately weigh it, dissolve it in methanol and quantitatively dilute it to make a solution containing about 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution [注1] , add 1ml dansyl chloride acetone solution [注2] , mix well, derivatize in a 60℃ water bath for 1 hour, cool to room temperature, filter, take 1ml of the filtrate, add 1ml of methanol and mix well.

[0040] 4) Reference solution Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, and mix well.

[0041] 5) System suitability requirements Take the reference solution and inject it 5 times continuously. The relative standard deviation of the peak area of cyanamide derivatives shall not exceed 15.0%.

[0042] 6) Determination method Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0043] 7) Limits If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the peak of the cyanamide derivative, the peak area calculated by the external standard method shall not exceed 7.5ppm.

[0044] [Note 1]: Preparation of sodium carbonate-sodium bicarbonate mixed solution: Sodium carbonate solution (0.2 mol / L): Take 0.53 g of anhydrous sodium carbonate and dissolve it in 25 ml of water; Sodium bicarbonate solution (0.2 mol / L): Dissolve 1.68 g of sodium bicarbonate in 100 ml of water. Sodium carbonate-sodium bicarbonate mixed solution: Mix 4 ml of 0.2 mol / L sodium carbonate solution and 46 ml of 0.2 mol / L sodium bicarbonate solution, shake well, and the solution is obtained.

[0045] [Note 2]: Preparation of dansyl chloride acetone solution: Take an appropriate amount of dansyl chloride, accurately weigh it, dissolve it in acetone and quantitatively dilute it to make a solution containing approximately 10 mg of dansyl chloride per 1 ml.

[0046] Example 2: Methodological Verification (Method under Item 4 of Example 1) 1. Exclusivity 1) Interference of blank solvent on cyanamide derivatives ① Take an appropriate amount of methanol to replace the monocyanamide reference solution and prepare the negative blank solution in the same way as the reference solution preparation method under item "4" in Example 1.

[0047] ② Take an appropriate amount of cyanamide reference solution and prepare it according to the method for the reference solution under item "4" of Example 1 to obtain the reference solution.

[0048] ③ Take an appropriate amount of the condensation product test sample (batch number: 10901-1911001y) and prepare it according to the test sample solution method under item "4" of Example 1 to obtain the test sample solution.

[0049] Take appropriate amounts of methanol, negative blank solution, reference solution, and test solution, and analyze them according to the method under "4" described in Example 1 to investigate the interference of methanol and negative blank on the monocyanamide derivatives. The results are shown in Table 7 below. Figure 2-5 .

[0050] Table 7 Blank solvent interference results Sample name Peak area Retention time (min) Methanol NA NA Negative blank solution 4 2.16 Reference solution 66642 2.13 Test solution 29912 2.16 Conclusion: Methanol does not appear at the main peak position (peaks with S / N < 3 are negligible), and the peak area of the negative blank at the main peak position is 4, which is less than 0.05% of the peak area of the reference substance (i.e., 66642×0.05%=33). Methanol and the negative blank do not interfere with the detection of cyanamide derivatives.

[0051] 2) Interference of other impurities on cyanamide derivatives ① According to the condensation process route, the compounds involved in the condensation synthesis process include dicyandiamide, aminoguanidine carbonate, monocyanamide, hydrazine, 2,3-dichlorobenzoyl acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide.

[0052] ②Impurity interference analysis The compounds aminoguanidine carbonate, hydrazine, 2,3-dichlorobenzoyl acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide do not have the structural formula of cyanamide derivatization reaction and do not interfere with the detection of cyanamide derivatives. Dicyandiamide has a structural formula similar to cyanamide and may produce cyanamide derivatives, which are verified by the same derivatization method.

[0053] ③ Take an appropriate amount of dicyandiamide reference substance, dissolve it in methanol and quantitatively dilute it to make a dicyandiamide reference substance solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, and mix well to obtain a dicyandiamide derivative solution.

[0054] Take an appropriate amount of dicyandiamide derivative solution and analyze it according to the method under "4" of Example 1 to examine the interference with monocyanamide derivatives. Figure 6 .

[0055] Conclusion: The peak area of the main peak of the dicyandiamide derivative solution is 7, which is less than 0.05% of the peak area of the reference substance (i.e., 66642×0.05%=33). Dicyandiamide does not interfere with the detection of monocyanamide derivatives.

[0056] 2. Filter membrane adsorption Take an appropriate amount of monocyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml, take 2 ml and place it in a centrifuge tube, add 2 ml of sodium carbonate-sodium bicarbonate mixed solution, add 2 ml of dansyl chloride acetone solution, mix well, and react in a 60℃ water bath for 1 hour. Cool to room temperature and divide it into 3 equal parts, numbered 1#, 2#, and 3# respectively. Centrifuge 1# at 10,000 rpm for 10 minutes; discard 0.1 ml of the initial filtrate of 2# (nylon 66 material, 0.22μm); discard 0.3 ml of the initial filtrate of 3# (nylon 66 material, 0.22μm), take 1 ml of the treated solutions of 1#, 2#, and 3# respectively, add 1 ml of methanol to each, and mix well.

[0057] The diluted solutions of 1#, 2#, and 3# were taken respectively, and sampled and analyzed according to the method under "4" described in Example 1. The samples were injected three times continuously, and the peak areas were recorded. The adsorption rate of the filter membrane was examined (adsorption rate = 100% - peak area after adsorption / peak area without adsorption × 100%). The results are shown in Table 8 below.

[0058] Table 8 Cyanamide filter membrane adsorption test results Injection number Sample number 1 2 3 Mean (n=3) RSD% (n=3) Adsorption rate% 1# 40491 53931 51214 48545 14.6 / 2# 46454 48190 49266 47970 3.0 1.2 3# 51034 50095 51226 50785 1.2 -4.6 Conclusion: Nylon 66 filter membrane (0.22μm) has no adsorption on cyanamide derivatives.

[0059] 3. Linearity and range Take an appropriate amount of cyanamide reference substance and add methanol for quantitative dilution to prepare a series of cyanamide reference substance solutions containing approximately 2.0 ng, 6.0 ng, 10.0 ng, 20.0 ng, 30.0 ng, and 40.0 ng per 1 ml. Take 1 ml of each solution and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution respectively, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, and take 1 ml of the filtrate respectively, add 1 ml of methanol, and mix well to prepare the cyanamide derivative solutions of various concentrations.

[0060] The samples were analyzed according to the method under "4" of Example 1, with the peak area of the cyanamide derivative as the ordinate (Y) and the concentration of the cyanamide reference substance as the abscissa (X). A linear regression was performed using the peak area Y against the concentration X (ng / ml). The linear equation and correlation coefficient are shown in Table 9 below.

[0061] Table 9 cyanamide linearity test results

[0062] Conclusion: The peak area (Y) of cyanamide derivatives was well linearly correlated with the concentration (X) in the range of 2.0-40.0 ng / ml, and the linear equation was: y=2848.9x+1255.9 (r=0.9912).

[0063] 4. Limit of quantification and detection limit A 2.0 ng / mL solution of a cyanamide derivative from the experiment described in "2. Linearity and Range" was diluted with methanol to an appropriate concentration. Samples were then injected and analyzed according to the method described in "4" of Example 1. Using the signal-to-noise ratio method, the detection limit (LOD) was determined when the signal-to-noise ratio was approximately 3:1, and the quantification limit (LOQ) was determined when the signal-to-noise ratio was approximately 10:1. The results are shown in Table 10 below.

[0064] Table 10 Quantitative limit and detection limit experimental results of monocyanamide sample Concentration (ng / ml) Peak area S / N Equivalent to the test concentration in ppm 2ng (mother solution) 2.0000 4311 792.71 0.7692 The stock solution was diluted 1000 times 0.0020 33 372.44 0.0008 Dilute the stock solution 2000 times (limit of quantification) 0.0010 13 11.10 0.0004 The mother liquor was diluted 3333 times 0.0006 19 311.87 0.0002 The mother solution was diluted 4000 times (detection limit) 0.0005 5 75.11 0.0002 Methanol / 3 2.85 / Conclusion: The peak area of the cyanamide derivative mother solution (2 ng / ml) was 5 when diluted 4000 times, close to the peak area of the blank solvent methanol. The provisional limit of quantification for cyanamide is 0.0010 ng / ml, equivalent to 0.0004 ppm of the test sample concentration. The provisional limit of detection for cyanamide is 0.0005 ng / ml, equivalent to 0.0002 ppm of the test sample concentration. The sensitivity of this method meets the control requirements for cyanamide.

[0065] 5. Precision 1) Injection precision A 10.0 ng / ml solution of a cyanamide derivative under the experiment "3. Linearity and Range" was injected and analyzed according to the method under "4" described in Example 1. Five injections were made continuously, and the retention times and peak areas were calculated. The results are shown in Table 11 below.

[0066] Table 11 Cyanamide injection precision test results Serial number 1 2 3 4 5 mean RSD% Retention time (min) 2.12 2.12 2.13 2.13 2.13 2.13 0.2 Peak area (A) 26435 30052 32345 30145 31586 30112 7.6 Conclusion: The cyanamide reference solution was injected five times continuously, and the RSDs of retention time and peak area were 0.2% and 7.6% respectively, indicating good injection precision.

[0067] 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 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize it in a 60°C water bath for 1 hour. Cool it to room temperature, filter it, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and prepare 6 parallel portions.

[0068] ② Preparation of reference solution Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, and mix well.

[0069] Take the test solution and the reference solution, and analyze them according to the method under "4" of Example 1. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the peak of the cyanamide derivative, the amount of cyanamide is calculated by the peak area according to the external standard method. The results are shown in Table 12 below.

[0070] Table 12: Results of cyanamide repeatability test Sample No. Cyanamide Content 1 2 3 4 5 6 Mean (n=6) RSD% (n=6) Content (ppm) 1.94 1.76 1.98 2.00 2.12 1.77 1.93 7.3 Conclusion: As can be seen from the above table, the RSD of cyanamide content in 6 parallel samples is 7.3%, and this method has good repeatability.

[0071] 6. Solution stability 1) Stability of reference solution Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and use as the reference substance solution.

[0072] 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 method under "4" of Example 1 to examine the changes in peak area at each time point. The results are shown in Table 13 below.

[0073] Table 13 Cyanamide reference solution stability test results Serial number 0h 2h 4h 6h 8h mean RSD% Retention time (min) 2.12 2.13 2.13 2.14 2.14 2.13 0.4 Peak area (A) 62320 50973 45321 54025 48541 52236 12.4 Conclusion: The control solution was placed at room temperature for 8 hours, and the RSD of the main peak area was 12.4%, indicating good stability.

[0074] 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 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize it in a 60°C water bath for 1 hour. Cool it to room temperature, filter it, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and use it as the test solution.

[0075] The test solution was placed at room temperature, and samples were taken at 0 h, 2 h, 4 h, 6 h, and 8 h, respectively. Samples were injected and analyzed according to the method under "4" described in Example 1 to examine the changes in peak area at each time point. The results are shown in Table 14 below.

[0076] Table 14 Cyanamide test solution stability test results Serial number 0h 2h 4h 6h 8h mean RSD% Peak area (A) 12010 11276 12025 12466 12488 12053 3.6 Content (ppm) 1.94 1.82 1.94 2.01 2.02 1.95 3.6 Conclusion: When the test solution was placed at room temperature for 8 hours, the RSD of the main peak area was 3.6%, indicating good stability.

[0077] 7. Accuracy 1) Reference solution Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and use as the reference substance solution.

[0078] 2) Test solution ①Cyanamide stock solution Take an appropriate amount of cyanamide reference substance and add methanol to quantitatively dilute it to make a stock solution containing about 200 ng of cyanamide per 1 ml.

[0079] ②50% test solution Take about 26 mg of the 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 monocyanamide stock solution (200 ng / ml), ultrasonically dissolve it, cool to room temperature, take 1 ml and place it in a centrifuge tube, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution, add 1 ml of dansyl chloride acetone solution, mix well, derivatize it in a 60°C water bath for 1 hour, cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well to obtain a 50% accuracy solution, and prepare 3 parallel copies.

[0080] ③100% test solution: Take about 26 mg of the 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 monocyanamide stock solution (200 ng / ml), dissolve it by ultrasonication, cool to room temperature, take 1 ml and place it in a centrifuge tube, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution, add 1 ml of dansyl chloride acetone solution, mix well, derivatize it in a 60°C water bath for 1 hour, cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well to obtain a 100% accuracy solution, and prepare 3 parallel copies.

[0081] ④150% test solution Take about 26 mg of the test sample (batch number: 10901-1911001y), accurately weigh it, place it in a 10 ml centrifuge tube, add 8.5 ml of methanol, add 1.5 ml of monocyanamide stock solution (200 ng / ml), dissolve it by ultrasonication, cool to room temperature, take 1 ml and place it in a centrifuge tube, add 1 ml of sodium carbonate-sodium bicarbonate mixed solution, add 1 ml of dansyl chloride acetone solution, mix well, derivatize it in a 60°C water bath for 1 hour, cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well to obtain a 100% accuracy solution, and prepare 3 parallel copies.

[0082] 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).

[0083] Accurately measure the above test solution and reference solution respectively, and analyze the samples according to the method under "4" of Example 1. Calculate the content of monocyanamide by peak area according to the external standard method. The recovery of this product is examined. The results are shown in Table 15 below.

[0084] Table 15 Cyanamide accuracy test results

[0085] Conclusion: The recovery of cyanamide in 9 test solutions ranged from 90.3% to 129.7%, with an average recovery of 110.9% and an RSD of 11.8%, which met the recovery limit requirements. The recovery of this method was good.

[0086] 8. Durability 1) Reference solution Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and use as the reference substance solution.

[0087] 2) Test solution ①Cyanamide stock solution Take an appropriate amount of hydrazine standard solution and add methanol to quantitatively dilute it to make a stock solution containing about 200ng of monocyanamide per 1ml.

[0088] ②Test 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 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution and 1 ml of dansyl chloride acetone solution, mix well, and derivatize it in a 60°C water bath for 1 hour. Cool it to room temperature, filter it, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and use it as the test solution.

[0089] 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 16-18 below.

[0090] Table 16 Results of chromatographic condition changes in durability experiment Standard conditions Factors for fine-tuning chromatographic conditions Flow rate: 0.6ml / min Flow rate: 0.5ml / min, 0.7ml / min Column temperature: 35°C Column temperature: 30℃, 40℃ 0.1% formic acid aqueous solution-acetonitrile (5:95) 0.1% formic acid aqueous solution-acetonitrile (3:97) 0.1% formic acid aqueous solution-acetonitrile (7:93) Table 17 Results of the suitability investigation of the durability test system

[0091] Table 18 Durability test results of sample content Chromatographic conditions Cyanamide content (ppm) Unchanged conditions 2.00 Change the flow rate to 0.5ml / min 2.06 Change the flow rate to 0.7 ml / min 1.86 0.1% formic acid aqueous solution-acetonitrile (3:97) 1.80 0.1% formic acid aqueous solution-acetonitrile (7:93) 1.78 Change the column temperature to 30℃ 1.77 Change the column temperature to 40℃ 1.69 Mean (n=7) 1.85 RSD% (n=7) 7.2 Conclusion: Without changing the chromatographic conditions, the flow rate (±0.1ml / min), mobile phase ratio (±2%), and column temperature (±5℃) 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 monocyanamide content (RSD < 15.0%). This method has good durability.

[0092] Take the condensation product sample and test it according to the method under "4" of Example 1. The results are shown in Table 19. Figures 7-9 .

[0093] Table 19 Sample test results batch number Cyanamide content (ppm) 10901-1911001y 1.93 10901-1911002 y 2.29 10901-1911003 y 1.96 10901-1806005 y 1.71 Conclusion: The limits of impurity cyanamide in condensation products (batch numbers: 10901-1911001y, 10901-1911002y, 10901-1911003y, 10901-1806005y) all meet the requirements (≤7.5ppm).

Claims

1. A method for detecting the content of cyanamide 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 hydrazine content was calculated by peak area using the external standard method; The liquid chromatography conditions are: C18 chromatographic column, flow rate: 0.5-0.7 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 3%-7% A and 97%-93% B; The mass spectrometry conditions are: ESI - In negative ion mode, the cone voltage was 52 V, the parent ion m / z was 274.2, the quantitative ion pair m / z was 274.2 / 257.9, the quantitative ion collision energy was 31 V, the qualitative ion pair m / z was 274.2 / 194.0, and the qualitative ion collision energy was 39 V.

2. The detection method according to claim 1, characterized in that Flow rate: 0.6 ml / min; column temperature: 35°C.

3. The detection method according to claim 1, wherein The chromatographic column model: Agilent ZORBAXEclipse Plus C 18 , specifications are 4.6mm*150mm, 3.5μm.

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 5% A and 95% 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: 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 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of a sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.00-pH 10.50, add 1 ml of a dansyl chloride acetone solution with a pH of 8.00-10.50, mix well, and derivatize it in a water bath at 40-60°C for 40-80 minutes. Cool it to room temperature, filter it, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and the test solution is obtained. Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.00-pH 10.50, add 1 ml of dansyl chloride acetone solution with a pH of 8.00-10.50, mix well, and derivatize in a water bath at 40-60°C for 40-80 minutes. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, mix well, and the reference substance solution is obtained.

9. The detection method according to claim 8, wherein: 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 2.6 mg per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.96 and 1 ml of 10 mg / ml dansyl chloride acetone solution. Mix well. Derivatize it in a 60°C water bath for 1 hour. Cool it to room temperature, filter it, take 1 ml of the filtrate, add 1 ml of methanol, and mix well to obtain the test solution. Take an appropriate amount of cyanamide reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 20 ng per 1 ml. Take 1 ml and place it in a centrifuge tube. Add 1 ml of sodium carbonate-sodium bicarbonate mixed solution with a pH of 8.96 and 1 ml of 10 mg / ml dansyl chloride acetone solution, mix well, and derivatize in a 60°C water bath for 1 hour. Cool to room temperature, filter, take 1 ml of the filtrate, add 1 ml of methanol, and mix well to obtain the reference substance solution.

10. The detection method according to claim 9, characterized in that: 0.2 mol / L sodium carbonate solution: Take 0.53 g of sodium carbonate and dissolve it in 25 ml of water; 0.2 mol / L sodium bicarbonate solution: Dissolve 1.68 g of sodium bicarbonate in 100 ml of water; Sodium carbonate-sodium bicarbonate mixed solution with pH 8.96: Mix 4 ml of 0.2 mol / L sodium carbonate solution and 46 ml of 0.2 mol / L sodium bicarbonate solution to obtain the solution.

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