Method for detecting hydrazine content in lamotrigine key intermediate

Abstract

The invention discloses a method for detecting the hydrazine content in a lamotrigine key intermediate. The method comprises the following steps: respectively dissolving a test sample and a 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 sample size is 10 microliters; 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 113.3, the quantitative ion pair m / z is 113.3 / 56.4, the quantitative ion collision energy is 14V, the qualitative ion pair m / z is 113.3 / 58.4, and the qualitative ion collision energy is 15V. The methodology verifies that the method is suitable for quantitative detection of the hydrazine content in the lamotrigine key intermediate.

Description

Technical Field

[0001] This application relates to the technical field of drug impurity separation and detection, and particularly to a method for detecting the hydrazine content 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 starts with 2,3-dichlorobenzoic acid. 2,3-dichlorobenzoic acid reacts with thionyl chloride (also called sulfur oxychloride, 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 figure below) 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. Aminoguanidine bicarbonate is synthesized from cyanamide (CH2-NH2) + hydrazine (NH2-NH2) + CO2 + H2O. Among them, hydrazine is the starting material in the synthesis process of aminoguanidine bicarbonate. Since hydrazine has a genotoxic group, quality control research on hydrazine 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" in the fourth part of the Chinese Pharmacopoeia (2020 edition), the Threshold of Toxicological Concern (TTC) for hydrazine 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, then the control limit of hydrazine 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. Hydrazine (N2H4) has high polarity, low relative molecular weight (32.05), no carbon atoms, and no chromophore, making it difficult to directly determine. It is necessary to establish a detection method that can be applied to the hydrazine content 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 hydrazine content in a key intermediate of lamotrigine, including the following steps: Dissolve the test sample and the reference sample in the detection solvent respectively to obtain a test sample solution and a reference sample solution; The test solution and the reference solution were separated by liquid chromatography technology, detected by a mass spectrometry detector, and the content of hydrazine was calculated by the external standard method with the peak area. The liquid chromatography conditions were as follows: C18 chromatographic column, flow rate: 0.5 - 0.7 ml / min; column temperature: 30 - 40 °C; injection volume: 10 μl. The mobile phase was: A: 0.1% formic acid aqueous solution; B: acetonitrile; isocratic elution was carried out with 8% - 12% of A and 92% - 88% of B. The mass spectrometry conditions were: ESI + Positive ion mode, cone voltage was 52 V, precursor ion m / z was 113.3, quantitative ion pair m / z was 113.3 / 56.4, quantitative ion collision energy was 14 V, qualitative ion pair m / z was 113.3 / 58.4, qualitative ion collision energy was 15 V.

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

[0007] The model of the chromatographic column was: Agilent ZORBAX Eclipse Plus C 18 with a specification of 4.6 mm * 150 mm, 3.5 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] Preferably, isocratic elution was carried out with 10% of A and 90% of B.

[0011] The test sample was the material obtained by sampling during the synthesis of lamotrigine to the condensate, , condensate.

[0012] An appropriate amount of the condensate test sample was accurately weighed, dissolved in methanol and quantitatively diluted to prepare a solution containing about 2.6 mg per 1 ml. 200 μl was taken and placed in a centrifuge tube, 1800 μl of acetone - acetic acid solution was added, shaken well, sonicated for 5 - 15 minutes, cooled to room temperature, filtered, and the subsequent filtrate was used as the test solution. An appropriate amount of the hydrazine standard solution was quantitatively diluted with methanol to prepare a hydrazine solution containing about 20 ng per 1 ml. 200 μl was taken and placed in a centrifuge tube, 1800 μl of acetone - acetic acid solution was added, shaken well, sonicated for 5 - 15 minutes, cooled to room temperature, filtered, and the subsequent filtrate was used as the reference solution. The acetone glacial acetic acid solution is prepared by taking 10 ml of acetone and adding 10 μl of glacial acetic acid.

[0013] Preferably, the ultrasonic time is 15 minutes.

[0014] The present application first provides a method for detecting the hydrazine content in a lamotrigine key intermediate. The detection is carried out by using a specific liquid chromatography - mass spectrometry combined method. After methodological verification, this method has good specificity, precision and repeatability, and the sensitivity meets the control requirements of this impurity.

[0015] For the detection method provided by the present application, the limit of quantitation is 0.0010 ng / ml, and the limit of detection is 0.0006 ng / ml. The method has good sensitivity, and the limit of detection and the limit of quantitation are significantly lower than the quality control limit requirements of the hydrazine trace impurity, indicating that the sensitivity of this method meets the control requirements of this impurity. The recovery test proves that the sample treatment method and the detection method of the present invention can realize the determination of the content of the hydrazine trace impurity in the condensate, and can be applied to the quality monitoring of the hydrazine impurity in the lamotrigine key intermediate condensate. Description of the Drawings

[0016] Figure 1 It is the optimized diagram of the impurity hydrazine derivative ion; Figure 2 It is the methanol spectrum of the blank solvent for the methodological verification (specificity) of the impurity hydrazine; Figure 3 It is the negative blank methanol spectrum for the methodological verification (specificity) of the impurity hydrazine; Figure 4 It is the spectrum of the condensate test sample (10901 - 1911001y) for the methodological verification (specificity) of the impurity hydrazine; Figure 5 It is the spectrum of the reference substance for the methodological verification (specificity) of the impurity hydrazine; Figure 6 It is the spectrum of aminoguanidine bicarbonate for the methodological verification (specificity) of the impurity hydrazine; Figure 7 It is the spectrum of the mother liquor diluted 10,000 times for the methodological verification (limit of quantitation and limit of detection) of the impurity hydrazine; Figure 8 It is the detection spectrum of the test sample of batch 10901 - 1911002y; Figure 9 It is the detection spectrum of the test sample of batch 10901 - 1911003y; Figure 10 It is the detection spectrum of the test sample of batch 10901 - 1806005y. Detailed Embodiments

[0017] In the typical synthesis process of lamotrigine, the condensation product formed by the reaction of 2,3-dichlorobenzoyl cyanide with aminoguanidine bicarbonate [chemical name: 2-cyano-(2,3-dichlorophenyl)-2-guanidinoiminoacetonitrile] is the last step in the synthesis process route of lamotrigine. The condensation product is the key intermediate material for synthesizing lamotrigine. Quality control of trace potential genotoxic impurities in the key intermediate condensation product plays a very important role in controlling potential genotoxic impurities in the finished product of lamotrigine. Therefore, this application preferably selects the condensation product as the key intermediate for detection.

[0018] 1 Detection conditions for hydrazine derivatives 1) Principle of hydrazine derivatization reaction The principle of the hydrazine derivatization reaction is as follows:

[0019] 2) Mass spectrometry ion optimization Using the positive ion mode, the ion optimization results of the hydrazine derivative show that the parent ion m / z is 113.3, which is basically consistent with the theoretical molecular weight. The ion optimization results of the hydrazine derivative are shown in Figure 1 .

[0020] 3) Mass spectrometry conditions (see Table 1) Model of mass spectrometry instrument: Thermo TSQ QUANTUM ACCESS MAX (triple quadrupole) Table 1 Mass spectrometry parameters <![CDATA[ESI + (SRM)]]> Compound: Hydrazine Derivative Parent Ion m / z 113.3 Cone Voltage (V) 52 Quantitative Ion Pair m / z 113.3 / 56.4 Quantitative Ion Collision Energy (V) 14 Qualitative Ion Pair m / z 113.3 / 58.4 Qualitative Ion Collision Energy (V) 15 Capillary Temperature (°C) 350 Vaporizer Temperature (°C) 300 Sheath Gas Pressure (Arb) 30 Auxiliary Gas Pressure (Arb) 10 Ion Sweep Gas Pressure (Arb) 2 Spray Voltage (V) 3000 Discharge Current (μA) 4.0 4) Liquid chromatography conditions Model of HPLC instrument: Thermo Fisher ACCELA Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 (4.6mm * 150mm, 3.5μm) Flow rate: 0.6 ml / min; Column temperature: 35°C; Injection volume: 10 μl; Injector temperature: 25°C; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile, isocratic elution with 10%A - 90%B.

[0021] 2 Study on hydrazine derivatization reaction conditions 1) Solution preparation ① Preparation of hydrazine reference stock solution Measure an appropriate amount of hydrazine standard solution (1000 μg / ml), and quantitatively dilute it with methanol to prepare a solution containing 20 ng of hydrazine per 1 ml, that is, obtain.

[0022] ② Preparation of acetone - glacial acetic acid solution: Take 10 ml of acetone, add 10 μl of glacial acetic acid, and mix well to obtain the solution.

[0023] 2) Screening of derivatization reaction ratio Measure 200 μl of hydrazine reference substance stock solution (20 ng / ml) and place it in a 10 - ml centrifuge tube. Take 3 parallel samples respectively, add 1700 μl, 1800 μl, and 1900 μl of acetone - glacial acetic acid solution respectively, ultrasonicate for 15 minutes, conduct reaction ratio investigation, inject samples for analysis according to the above detection conditions, and count the peak areas of different derivatization reaction ratios. The results are shown in Table 2 below.

[0024] Table 2 Results of screening of derivatization reaction ratio Volume of Hydrazine - Acetone - Glacial Acetic Acid Ratio of Hydrazine - Acetone - Glacial Acetic Acid Peak Area 200 μl - 1700 μl 1:8.5 114297 200 μl - 1800 μl 1:9.0 125417 200 μl - 1900 μl 1:9.5 132237 Mean (n = 3) / 123984 RSD% (n = 3) / 7.3 Conclusion: As can be seen from the above table, when the ratio of hydrazine - acetone - glacial acetic acid is 1:8.5, 1:9.0, and 1:9.5 respectively, there is no significant difference in the peak areas of the derivatives (the RSD of peak areas at different ratios is 7.3%). From the derivatization reaction formula, the theoretical reaction molar ratio of hydrazine to acetone is 1:2, which is converted to a mass ratio of 32 g:116 g; the actual reaction mass ratio is 4 ng (20 ng / ml × 200 μl):1404 mg (1800 μl × density 0.78 g / cm 3 ) The amount of reaction reagent acetone - glacial acetic acid used is much higher than that of hydrazine. Therefore, the ratio of hydrazine - acetone - glacial acetic acid is tentatively set at 1:9.0.

[0025] 3) Screening of derivatization reaction time Measure 200 μl of hydrazine reference substance stock solution (20 ng / ml) and place it in a 10 - ml centrifuge tube. Take 3 parallel samples respectively, add 1800 μl of acetone - glacial acetic acid solution respectively, ultrasonicate for 5 minutes, 10 minutes, and 15 minutes respectively, conduct reaction time investigation, inject samples for analysis according to the above detection conditions, and count the peak areas of different derivatization reaction times. The results are shown in Table 3 below.

[0026] Table 3 Results of screening of derivatization reaction time Ultrasonic Reaction Time Peak Area 5 Minutes 130722 10 Minutes 128349 15 Minutes 145286 Mean (n = 3) 134786 RSD (n = 3) 6.8% Conclusion: As can be seen from the above table, when the derivatization reaction times are 5 minutes, 10 minutes, and 15 minutes respectively, there is no significant difference in the peak areas of the derivatives (the RSD of peak areas at different reaction times is 6.8%). The derivatization reaction time is tentatively set at 15 minutes, which is the longest reaction time.

[0027] The accuracy experiment (see the method validation item in Example 2 for details) further verifies the rationality of tentatively setting the ratio of hydrazine - acetone - glacial acetic acid at 1:9.0 and the ultrasonic time at 15 minutes.

[0028] 3 Optimization of liquid chromatography conditions 1) Liquid chromatography conditions Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 (4.6 mm * 150 mm, 3.5 μm) Flow rate: 0.6 ml / min; Column temperature: 35 °C; Injection volume: 10 μl; Injector temperature: 25 °C; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile, isocratic elution with 10%A - 90%B.

[0029] 2) Optimization of chromatographic conditions The flow rate (0.5 ml / min, 0.6 ml / min, 0.7 ml / min), column temperature (30 °C, 35 °C, 40 °C), and mobile phase ratio (0.1% formic acid aqueous solution - acetonitrile, 10:90, 8:92, 12:8) 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 0.6 ml / min, the column temperature was 35 °C, and the ratio of 0.1% formic acid aqueous solution - acetonitrile was 10:90. See the durability experiment for details (see under the methodological verification in Example 2).

[0030] 4 Determined detection method 1) Liquid chromatography conditions Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 (4.6 mm * 150 mm, 3.5 μm) Flow rate: 0.6 ml / min; Column temperature: 35 °C; Injection volume: 10 μl; Injector temperature: 25 °C; Mobile phase: A: 0.1% formic acid aqueous solution; B: acetonitrile, isocratic elution with 10%A - 90%B.

[0031] 2) Mass spectrometry conditions (Table 4) Table 4 Mass spectrometry parameters <![CDATA[ESI + (SRM)]]> Compound: Hydrazine Derivative Parent Ion m / z 113.3 Cone Voltage (V) 52 Quantitative Ion Pair m / z 113.3 / 56.4 Quantitative Ion Collision Energy (V) 14 Qualitative Ion Pair m / z 113.3 / 58.4 Qualitative Ion Collision Energy (V) 15 Capillary Temperature (°C) 350 Vaporizer Temperature (°C) 300 Sheath Gas Pressure (Arb) 30 Auxiliary Gas Pressure (Arb) 10 Ion Sweep Gas Pressure (Arb) 2 Spray Voltage (V) 3000 Discharge Current (μA) 4.0 3) Test solution Weigh an appropriate amount of the condensate test sample accurately, dissolve it with methanol and quantitatively dilute it to make a solution containing about 2.6 mg per 1 ml. Pipette 200 μl into a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, sonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the test solution.

[0032] 4) Reference solution Take an appropriate amount of hydrazine standard solution, quantitatively dilute it with methanol to prepare a hydrazine solution containing about 20 ng per 1 ml. Pipette 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, sonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the reference solution.

[0033] 5) System suitability requirements Take the reference solution and inject it continuously for 5 times. The relative standard deviation of the peak area of the hydrazine derivative shall not exceed 15.0%.

[0034] 6) Assay method Precisely pipette the test solution and the reference solution, and inject them into the liquid chromatograph respectively, and record the chromatogram.

[0035] 7) Limit If there are chromatographic peaks in the chromatogram of the test solution with the same retention time as the hydrazine derivative peak, calculated by the external standard method with the peak area, it shall not exceed 7.5 ppm.

[0036] 1. Specificity 1) Interference of the blank solvent on the hydrazine derivative ① Take an appropriate amount of 1.0 mol / L hydrochloric acid solution to replace the hydrazine standard solution, and prepare it in the same way as the preparation method of the reference solution described in "Item 4" of Example 1, that is, obtain the negative blank solution.

[0037] ② Take an appropriate amount of hydrazine solution and prepare it according to the method of the reference solution described in "Item 4" of Example 1, that is, obtain the reference solution.

[0038] ③ Take an appropriate amount of the condensate test sample (batch number: 10901 - 1911001y) and prepare it according to the method of the test solution described in "Item 4" of Example 1, that is, obtain the test solution.

[0039] Take appropriate amounts of methanol, negative blank solution, reference solution, and test solution, inject and analyze them according to the method described in "Item 4" of Example 1, and examine the interference of methanol and the negative blank on the hydrazine derivative. The results are shown in Table 5 below. Figures 2 - 5 。

[0040] Table 5 Results of blank solvent interference Sample Name Peak Area Retention Time (min) Methanol 13 2.72 Negative Blank Solution 32 2.65 Reference Solution 120954 2.66 Test Solution 13 2.66 Conclusion: The peak areas of methanol and the negative blank at the main peak position are 13 and 32 respectively, which are less than 0.05% of the reference peak area (i.e., 120954×0.05% = 60). Methanol and the negative blank do not interfere with the detection of the hydrazine derivative.

[0041] 2) Interference of other impurities on the hydrazine derivative ①According to the condensate process route, the compounds involved in the condensate synthesis are shown in Table 6 below: Table 6 Compounds Involved in the Condensate Synthesis

[0042] ②Analysis of impurity interference Dicyandiamide, monocyanamide, 2,3-dichlorobenzoic acid, 2,3-dichlorobenzoyl chloride, and 2,3-dichlorobenzoyl cyanide do not have the structural formula for the hydrazine derivative reaction and do not interfere with the detection of hydrazine derivatives. Aminoguanidine bicarbonate has a similar structural formula to hydrazine and may produce hydrazine derivatives, which are verified by the same derivatization method.

[0043] ③Take an appropriate amount of aminoguanidine bicarbonate, dissolve it in methanol and quantitatively dilute it to make an aminoguanidine bicarbonate solution containing about 20 ng per 1 ml. Measure 200 μl and place it in a centrifuge tube. Add 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate.

[0044] Take an appropriate amount of the aminoguanidine bicarbonate derivative solution and inject it for analysis according to the method described in "Item 4" of Example 1 to examine the interference with hydrazine derivatives. See Figure 6 .

[0045] Conclusion: Aminoguanidine bicarbonate does not peak at the main peak position of hydrazine derivatives (peaks with S / N < 3 are ignored) and does not interfere with the detection of hydrazine derivatives.

[0046] 2. Filter membrane adsorption experiment Take an appropriate amount of hydrazine standard solution, quantitatively dilute it with methanol to make a hydrazine solution containing about 20 ng per 1 ml. Measure 400 μl and place it in a centrifuge tube. Add 3600 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, divide it into 3 equal parts, and label them 1#, 2#, and 3# respectively. Centrifuge 1# at 10,000 rpm 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).

[0047] Take the solutions after treatment of 1#, 2#, and 3# respectively, inject them for analysis according to the method described in "Item 4" of Example 1, inject continuously 3 times respectively, record the peak areas, and examine the filter membrane adsorption rate (adsorption rate = 100% - peak area after adsorption / peak area before adsorption × 100%). The results are shown in Table 7 below.

[0048] Table 7 Results of the Hydrazine Filter Membrane Adsorption Experiment Injection Sequence - Sample Number 1 2 3 Mean (n = 3) RSD% (n = 3) Adsorption Rate % 1# 161874 165772 180694 169447 5.86 / 2# 170458 174701 162409 169190 3.69 0.2 3# 163857 158135 201486 174492 13.50 -3.0 Conclusion: The nylon 66 filter membrane (0.22 μm) has no adsorption on hydrazine derivatives.

[0049] 2. Linearity and range Take an appropriate amount of hydrazine standard solution (1000 μg / ml), quantitatively dilute it with methanol to prepare a series of hydrazine reference solutions with concentrations of about 2.0 ng, 6.0 ng, 10.0 ng, 20.0 ng, 30.0 ng, and 40.0 ng per 1 ml. Respectively take 200 μl and place them in centrifuge tubes. Add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid) to each tube, shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the hydrazine derivative solutions of each concentration.

[0050] Inject and analyze according to the method described in "Item 4" of Example 1. Using the peak area of the hydrazine derivative as the ordinate (Y) and the concentration of the hydrazine reference as the abscissa (X), perform linear regression with the peak area Y against the concentration X (ng / ml). The linear equation and correlation coefficient are shown in Table 8 below.

[0051] Table 8 Results of hydrazine linear experiment

[0052] Conclusion: The peak area (Y) of the hydrazine derivative and the concentration (X) show a good linear correlation in the range of 2.0 - 40.0 ng / ml. The linear equation is: y = 10155x - 10998 (r = 0.9983).

[0053] 3. Quantification limit, detection limit Take the 2.0 ng / ml hydrazine derivative solution in the experiment of "2. Linearity and range" above, dilute it with methanol to a suitable concentration, and inject and analyze according to the method described in "Item 4" of Example 1. Calculate according to the signal - to - noise ratio method. The concentration when the signal - to - noise ratio is about 3:1 is the detection limit concentration, and the concentration when the signal - to - noise ratio is about 10:1 is the quantification limit concentration. The results are shown in Table 9 below.

[0054] Table 9 Results of hydrazine quantification limit and detection limit experiment Sample Concentration (ng / ml) Peak Area S / N ppm Equivalent to Test Concentration of Test Sample 2 ng (Stock Solution) 2.0000 19792 919.00 0.7692 Stock Solution Diluted 1000 - fold 0.0020 34 174.30 0.0008 Stock Solution Diluted 2000 - fold (Quantitation Limit) 0.0010 22 120.61 0.0004 Stock Solution Diluted 3333 - fold (Detection Limit) 0.0006 12 46.94 0.0002 Stock Solution Diluted 4000 - fold 0.0005 6 32.46 0.0002 Stock Solution Diluted 5000 - fold 0.0004 5 27.90 0.0002 The mother liquor is diluted 10,000 times (see Figure 7 ). 0.0002 8 90.98 0.0001 Conclusion: Continuously dilute the mother liquor of hydrazine derivative (2 ng / ml). When diluted to 4000 times, 5000 times, and 10000 times, there is no obvious difference in the peak area. Tentatively set the quantification limit concentration of hydrazine to 0.0010 ng / ml, which is equivalent to 0.0004 ppm of the test concentration of the sample. Tentatively set the detection limit concentration of hydrazine to 0.0006 ng / ml, which is equivalent to 0.0002 ppm of the test concentration of the sample. The detection sensitivity of this method meets the requirements for hydrazine control.

[0055] 4. Precision 1) Injection precision Take the 6.0 ng / ml hydrazine derivative solution under the "2. Linearity and Range" experiment above, inject and analyze it according to the method described in "Item 4" of Example 1, inject continuously for 5 times, and count the retention time and peak area. The results are shown in Table 10 below.

[0056] Table 10 Results Table of Injection Precision Experiment of Hydrazine Serial Number 1 2 3 4 5 Mean RSD% Retention Time (min) 2.66 2.66 2.66 2.66 2.66 2.66 0.0 Peak Area (A) 53029 48999 47287 50291 57909 51503 8.1 Conclusion: The hydrazine reference solution was injected continuously for 5 times. The RSDs of the retention time and peak area were 0.0% and 8.1% respectively, and the injection precision was good.

[0057] 2) Repeatability ① Preparation of test solution: Take an appropriate amount of the condensate test sample (batch number: 10901 - 1911001y), weigh it accurately, dissolve it with methanol and quantitatively dilute it to make a solution containing about 2.6 mg per 1 ml. Measure 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the test solution. Prepare 6 portions in parallel.

[0058] ② Preparation of reference solution Take an appropriate amount of the hydrazine standard solution, quantitatively dilute it with methanol to make a hydrazine solution containing about 20 ng per 1 ml. Measure 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the reference solution.

[0059] Take the test solution and the reference solution, inject and analyze them according to the method described in "Item 4" of Example 1. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the hydrazine derivative peak, calculate the amount of hydrazine by the external standard method based on the peak area. The results are shown in Table 11 below.

[0060] Table 11 Results Table of Repeatability Experiment of Hydrazine Sample Number - Hydrazine Content 1 2 3 4 5 6 Mean (n = 6) Content (ppm) 0.0016 0.0025 0.0100 0.0016 0.0035 0.0031 0.0037 Conclusion: As can be seen from the above table, the repeatability of this method is good.

[0061] 5. Solution Stability 1) Stability of reference solution Take an appropriate amount of the hydrazine standard solution, quantitatively dilute it with methanol to make a hydrazine solution containing about 20 ng per 1 ml. Measure 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the reference solution.

[0062] The reference solution was placed at room temperature, and samples were taken at 0 h, 4 h, 5 h, 6 h, 7 h, and 8 h respectively, and injected for analysis according to the method described in "Item 4" of Example 1 to investigate the change in peak area at each time point. The results are shown in Table 12 below.

[0063] Table 12 Results of the stability experiment of the hydrazine reference solution Serial Number 0h 4h 5h 6h 7h 8h Mean RSD% Retention Time (min) 2.66 2.66 2.66 2.66 2.66 2.66 2.66 0.0 Peak Area (A) 193309 215489 184418 168968 175438 169397 184503 9.7 Conclusion: The reference solution of hydrazine was placed at room temperature for 8 hours, and the RSD of the main peak area was 9.7%, indicating good stability.

[0064] 2) Stability of the test solution An appropriate amount of the test sample (batch number: 10901-1911001y) was accurately weighed, dissolved in methanol and quantitatively diluted to a solution containing about 2.6 mg per 1 ml. 200 μl was taken and placed in a centrifuge tube, 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid) was added, shaken well, sonicated for 15 minutes, cooled to room temperature, filtered, and the subsequent filtrate was used as the test solution.

[0065] The test solution was placed at room temperature, and samples were taken at 0 h, 3 h, 4 h, 5 h, 6 h, 7 h, and 8 h respectively, and injected for analysis according to the method described in "Item 4" of Example 1 to investigate the change in peak area at each time point. The results are shown in Table 13 below.

[0066] Table 13 Results of the stability experiment of the test solution Serial Number 0h 3h 4h 5h 6h 7h 8h Mean Peak Area (A) 27 143 29 74 64 66 40 63 Content (ppm) 0.0016 0.0087 0.0018 0.0045 0.0039 0.0040 0.0024 0.0038 Conclusion: The test solution was placed at room temperature for 8 hours, and there was no obvious change in the main peak area, indicating good stability.

[0067] 6. Accuracy 1) Reference solution An appropriate amount of the hydrazine standard solution was taken and quantitatively diluted with methanol to a hydrazine solution containing about 20 ng per 1 ml. 200 μl was taken and placed in a centrifuge tube, 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid) was added, shaken well, sonicated for 15 minutes, cooled to room temperature, filtered, and the subsequent filtrate was used as the reference solution.

[0068] 2) Test solution ① Hydrazine stock solution An appropriate amount of the hydrazine standard solution was taken and quantitatively diluted with methanol to a hydrazine stock solution containing about 200 ng per 1 ml, and that's it.

[0069] ② 50% 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 hydrazine stock solution (200 ng / ml), dissolve it by ultrasonic treatment, cool it to room temperature, take 200 μl and place it in a centrifuge tube, add 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool it to room temperature, filter, take the subsequent filtrate to obtain a 50% accuracy solution, and prepare 3 portions in parallel.

[0070] ③ 100% test sample 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 hydrazine stock solution (200 ng / ml), dissolve it by ultrasonic treatment, cool it to room temperature, take 200 μl and place it in a centrifuge tube, add 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool it to room temperature, filter, take the subsequent filtrate to obtain a 100% accuracy solution, and prepare 3 portions in parallel.

[0071] ④ 150% test sample 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 hydrazine stock solution (200 ng / ml), dissolve it by ultrasonic treatment, cool it to room temperature, take 200 μl and place it in a centrifuge tube, add 1800 μl of acetone glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool it to room temperature, filter, take the subsequent filtrate to obtain a 150% accuracy solution, and prepare 3 portions in parallel.

[0072] ⑤ Take the mean value measured under "Repeatability" as the background quantity of this experiment (this experiment is carried out on the same day as the repeatability experiment).

[0073] Accurately measure the above-mentioned test sample solution and reference substance solution respectively, inject and analyze them according to the method described in "4" under Example 1, calculate the content of hydrazine by the external standard method with the peak area, and examine the recovery rate of this product. The results are shown in Table 14 below.

[0074] Table 14 Results table of hydrazine accuracy experiment

[0075] Conclusion: For 9 test sample solutions, the recovery rate range of hydrazine is 95.6% - 125.2%, the average recovery rate is 107.3%, and the RSD of the recovery rate is 8.2%, meeting the requirements of the limit recovery rate range. The recovery rate of this method is good.

[0076] 7. Robustness 1) Reference solution Take an appropriate amount of hydrazine standard solution, quantitatively dilute it with methanol to prepare a hydrazine solution containing about 20 ng per 1 ml. Pipette 200 μl into a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the reference solution.

[0077] 2) Test solution ① Hydrazine stock solution Take an appropriate amount of hydrazine standard solution, quantitatively dilute it with methanol to prepare a hydrazine stock solution containing about 200 ng per 1 ml, and that's it.

[0078] ② 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 9.7 ml of methanol, add 0.3 ml of hydrazine stock solution (200 ng / ml), ultrasonically dissolve it, cool to room temperature, pipette 200 μl into a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution (take 10 ml of acetone and add 10 μl of glacial acetic acid), shake well, ultrasonicate for 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the test solution.

[0079] To investigate the durability of this method, the chromatographic conditions will be slightly adjusted below, and the system suitability of this product and the content of the test sample will be investigated. The results are shown in Table 15 - 17 below.

[0080] Table 15 Results of changes in chromatographic conditions for durability experiment Standard Conditions Fine - Tuning Factors for Chromatographic Conditions Flow Rate: 0.6 ml / min Flow Rate: 0.5 ml / min, 0.7 ml / min Column Temperature: 35 °C Column temperature: 30 °C, 40 °C 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 16 Results of system suitability investigation for durability experiment

[0081] Table 17 Results of test sample content investigation for durability experiment Chromatographic conditions Hydrazine content (ppm) Unchanged conditions 2.12 Change the flow rate to 0.5 ml / min 2.14 Change the flow rate to 0.7 ml / min 2.03 0.1% formic acid aqueous solution - acetonitrile (12:88) 1.97 0.1% formic acid aqueous solution - acetonitrile (8:92) 1.92 Change the column temperature to 30 °C 2.15 Change the column temperature to 40 °C 2.23 Mean (n = 7) 2.08 RSD% (n = 7) 5.3 Conclusion: Without changing the chromatographic conditions, changing the flow rate (±0.1 ml / min), the proportion of the mobile phase (±2%), and the column temperature (±5°C), under each chromatographic condition, the system suitability meets the requirements, and there is no significant difference in the hydrazine content. This method has good durability.

[0082] Take the condensate test sample and detect it according to the tentative method. The results are shown in Table 18 below, see Figures 8 - 10 。

[0083] Table 18 Test results of the sample Batch number Hydrazine content (ppm) 10901-1911001y 0.0037 10901-1911002 y 0.0078 10901-1911003 y 0.0043 10901-1806005 y 0.0031 Conclusion: The limits of impurity hydrazine in the condensates (batch numbers: 10901-1911001y, 10901-1911002y, 10901-1911003y, 10901-1806005y) all meet the requirements (≤7.5 ppm).

Claims

1. A method for detecting the hydrazine content in a key intermediate of lamotrigine, characterized in that, It includes the following steps: Dissolve the test sample and the reference sample in the detection solvent respectively to obtain a test sample solution and a reference sample solution; Separate the test sample solution and the reference sample solution by liquid chromatography technology respectively, detect them using a mass spectrometry detector, and calculate the content of hydrazine by the external standard method with the peak area; The liquid chromatography conditions are as follows: C18 chromatographic column, flow rate: 0.5 - 0.7 ml / min; column temperature: 30 - 40 °C; injection volume: 10 μl; The mobile phase is: A: 0.1% formic acid aqueous solution; B: acetonitrile; perform isocratic elution with 8% - 12% of A and 92% - 88% of B; The mass spectrometry conditions are as follows: ESI + Positive ion mode, the cone voltage is 52 V, the precursor ion m / z is 113.3, the quantitative ion pair m / z is 113.3 / 56.4, the quantitative ion collision energy is 14 V, the qualitative ion pair m / z is 113.3 / 58.4, and the qualitative ion collision energy is 15 V.

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

3. The detection method according to claim 1, wherein The chromatographic column model: Agilent ZORBAX Eclipse Plus C 18 , with a specification of 4.6 mm * 150 mm, 3.5 μm.

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

5. The detection method according to claim 1, characterized in that The model of the mass spectrometry instrument: Thermo TSQQUANTUM ACCESS MAX, which is a triple quadrupole mass spectrometry detector.

6. The detection method according to claim 1, characterized in that: Perform isocratic elution with 10% of A and 90% of B.

7. The detection method according to any one of claims 1-6, 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.

8. The detection method according to claim 7, wherein: Take an appropriate amount of the condensate test sample, weigh it accurately, dissolve it in methanol and quantitatively dilute it to prepare a solution containing about 2.6 mg per 1 ml. Measure 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution, shake well, ultrasonicate for 5 - 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the test sample solution; Take an appropriate amount of the hydrazine standard solution, quantitatively dilute it with methanol to prepare a hydrazine solution containing about 20 ng per 1 ml. Measure 200 μl and place it in a centrifuge tube, add 1800 μl of acetone - glacial acetic acid solution, shake well, ultrasonicate for 5 - 15 minutes, cool to room temperature, filter, and take the subsequent filtrate as the reference sample solution; The acetone - glacial acetic acid solution is prepared by taking 10 ml of acetone and adding 10 μl of glacial acetic acid.

9. The detection method according to claim 8, wherein: Ultrasonicate for 15 minutes.

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