Method for detecting content of benzyl chloride in lamotrigine key intermediate

Abstract

The invention discloses a method for detecting the content of benzyl chloride in a lamotrigine key intermediate. The detection method based on gas chromatography is good in specificity, precision and repeatability, and the sensitivity meets the control requirements of the impurities. When the method is used for detecting multiple batches of test products, benzyl chloride is not detected.

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 benzyl chloride in a key intermediate of lamotrigine. Background Art

[0002] Lamotrigine is a sodium channel blocker that is clinically used to treat epilepsy. The typical synthesis process of lamotrigine is based on 2,3-dichlorobenzoic acid as the starting compound, 2,3-dichlorobenzoic acid and thionyl chloride (also known as dichlorothionyl, SOCl 2 ) 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 to form a condensation product [chemical name: 2-cyano-(2,3-dichlorophenyl)-2-guanidininylacetonitrile], which then undergoes a cyclization reaction to obtain lamotrigine.

[0003] In the entire synthesis process of lamotrigine and its starting material process, potential trace amounts of mutagenic impurities may be introduced. Among them, the toluene added during the synthesis reaction of the starting material 2,3-dichlorobenzoic acid and the reaction reagent thionyl chloride may produce a small amount of benzyl chloride. The harm of benzyl chloride to the human body includes nervous system damage, skin irritation, respiratory irritation, increased liver and kidney burden, etc., and it is a mutagenic impurity. Therefore, according to ICH "M7: Industry Guidelines for Evaluating and Controlling DNA Reactive (Mutagenic) Impurities in Drugs to Limit Potential Carcinogenic Risks" and "9306 Guidelines for the Control of Genotoxic Impurities" of the 2020 edition (Part IV) of the "Chinese Pharmacopoeia", the Threshold of Toxicological Concern (TTC) of benzyl chloride is 1.5μg / day. According to the instructions for use and dosage of lamotrigine tablets, when this product is used for the treatment of adults or children over 12 years old, the daily dose can reach 200mg, and the control limit of benzyl chloride shall not exceed 7.5ppm. How to accurately detect substances with such low content from samples is a technical problem that needs to be solved urgently by technicians in this field. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for detecting the content of benzyl chloride in the key intermediate of lamotrigine; the present application provides a detection method based on gas chromatography, which has good specificity, precision and repeatability, and the sensitivity meets the control requirements of the impurity. The method was used to detect multiple batches of test products, and no benzyl chloride was detected.

[0005] The technical solution provided by the present invention is as follows: A method for detecting the content of benzyl chloride in a key intermediate of lamotrigine comprises the following steps: Dissolving the sample to be tested and the reference substance in the detection solvent respectively to obtain a test solution and a reference substance solution; The test solution and the reference solution are respectively tested by gas chromatography technology, and the benzyl chloride content is obtained through the gas chromatography test results; The conditions of the gas chromatography are as follows: Chromatographic column: capillary column with (6%) cyanopropylphenyl-(94%) dimethylsiloxane as stationary liquid or a chromatographic column with similar polarity; Chromatographic column heating program: initial temperature is 90-110℃, increase to 170-190℃ at 8-12℃ / min, maintain for 3-8min, then increase to 220-240℃ at 18-22℃ / min, maintain for 1-5min.

[0006] Preferably, the temperature program of the chromatographic column is as follows: the initial temperature is 95-105°C, which is increased to 180-185°C at 9-10°C / min, maintained for 5-6 minutes, and then increased to 230-235°C at 20-21°C / min, maintained for 3-4 minutes.

[0007] Preferably, the detection solvent is N,N-dimethylformamide.

[0008] Preferably, the concentration of the test solution is 120-200 mg / ml; The concentration of the reference solution is 1-1.5 μg / ml.

[0009] Preferably, the sample to be tested is a material sampled during the synthesis of lamotrigine to a condensate.

[0010] Condensation products.

[0011] Preferably, the flow rate is 2.5-3.5 ml / min; The injection port temperature is 220-240℃.

[0012] Preferably, the flow rate is 2.8-3.2 ml / min; The injection port temperature was 225-235°C.

[0013] Preferably, the split ratio is (1.5-2):1; The detector temperature is 240-250°C; The injection volume is 0.8-1.2µl.

[0014] Preferably, the injection method is direct injection of the solution.

[0015] Preferably, the limit of quantification of the detection method is 0.89 µg / ml and the limit of detection is 0.44 µg / ml.

[0016] The present application first provides a method for detecting the content of benzyl chloride in a key intermediate of lamotrigine, using a specific gas chromatography for detection, the method has good specificity, precision and repeatability, and the sensitivity meets the control requirements of the impurity. The method was used to detect multiple batches of test products, and no benzyl chloride was detected.

[0017] 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 reaction in the synthesis process route 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 lamotrigine finished product. Therefore, the present application preferably selects the condensation product as the key intermediate for detection.

[0018] The detection method provided in the present application has a quantitative limit of 0.89 µg / ml and a detection limit of 0.44 µg / ml. The method has good sensitivity, and the detection limit and the quantitative limit are significantly lower than the quality control limit requirements of the trace impurity of benzyl chloride, indicating that the sensitivity of the method meets the control requirements of the impurity. The recovery test proves that the sample processing method and the detection method of the present invention can realize the determination of the content of the trace impurity of benzyl chloride in the condensation product, and can be applied to the quality monitoring of the benzyl chloride impurity in the key intermediate condensation product of lamotrigine. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a chromatogram of the solvent in the solvent selection experiment of the present invention when N,N-dimethylformamide is the solvent; Figure 2 This is a chromatogram of the solvent selected from the experiment of the present invention when dimethyl sulfoxide is the solvent; Figure 3 The chromatogram is the chromatographic condition of method No. 1 in the gradient optimization experiment of the present invention; Figure 4 The chromatogram is the chromatographic condition of method number 2 in the gradient optimization experiment of the present invention; Figure 5 The chromatogram is the chromatographic condition of method number 3 in the gradient optimization experiment of the present invention; Figure 6The chromatogram is the chromatographic condition of method number 4 in the gradient optimization experiment of the present invention; Figure 7 A chromatogram of the system suitability solution in the specificity verification experiment of the present invention; Figure 8 The chromatogram of the test solution in the specificity verification experiment of the present invention; Fig. 9 The chromatogram of the benzyl chloride localization solution in the specificity verification experiment of the present invention; Fig.10 This is a chromatogram of the solvent (N,N-dimethylformamide) in the specificity verification experiment of the present invention; Fig.11 It is the linear correlation diagram obtained in the linear experiment of the present invention. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0022] 1. Exploration of Benzyl Chloride Detection Method (1) Selection of injection method According to the description of the physical properties of benzyl chloride, its boiling point is in the range of 175℃~179℃, and it is a carcinogen. Its limit is calculated with reference to EMA's "Guidelines on Limits of Genotoxic Impurities", which is about 0.00075%. Considering the characteristics of benzyl chloride with a high boiling point and a small limit, direct injection of the solution is temporarily selected as the injection method.

[0023] (2) Solvent selection Description of gas chromatography (GC) conditions: a capillary column with (6%) cyanopropylphenyl-(94%) dimethylsiloxane as the stationary phase (Agilent DB-624 30m×0.530mm×3.0µm); a column flow rate of 2ml / min; a starting temperature of 150°C, which was increased to 230°C at a rate of 10°C per minute and maintained for 2 minutes; an injection port temperature of 230°C; a split ratio of 5:1; a detector temperature of 250°C; and an injection volume of 1µl.

[0024] Take an appropriate amount of benzyl chloride, dissolve it in N,N-dimethylformamide and dimethyl sulfoxide to dilute it into a solution containing about 1 mg per 1 ml, and inject it according to the above chromatographic conditions to examine the interference of the solvent on the detection of benzyl chloride (see Table 1). At the same time, take an appropriate amount of the key intermediate condensate (batch number: 10901-1911001y) and dissolve it in N,N-dimethylformamide and dimethyl sulfoxide to examine the solubility of the key intermediate condensate.

[0025] Table 1 Solvent selection results

[0026] The condensation product has good solubility in both N,N-dimethylformamide and dimethyl sulfoxide. Considering the fact that the solvent peaks have many impurities and may interfere with the smaller limit of benzyl chloride (see Figure 1-2 ), N,N-dimethylformamide is temporarily selected as the solvent for the detection of benzyl chloride of this product.

[0027] (3) Gradient optimization When the low-concentration reference solution was run under the chromatographic conditions tentatively proposed under “(2) Solvent selection”, the baseline changed rapidly before and after the benzyl chloride peak, which was not conducive to the quantification of benzyl chloride. The baseline change near the benzyl chloride peak and the separation of benzyl chloride and adjacent peaks were investigated from the perspectives of initial temperature, heating rate, flow rate, etc. The tentative method is shown in Table 2.

[0028] Table 2

[0029] Take appropriate amounts of the condensate (batch number: 10901-1911001y) and benzyl chloride, dissolve and dilute with N,N-dimethylformamide to a mixed solution containing about 100 mg of the condensate and 1 mg of benzyl chloride per 1 ml, and inject and analyze according to the above-mentioned chromatographic conditions. The results are as follows: Figures 3 to 6 And as shown in Table 3.

[0030] Table 3 Chromatographic conditions optimization results

[0031] Note: "Resolution (front)" and "Resolution (back)" represent the resolution between benzyl chloride and the front peak and the back peak respectively. "-" means that the data does not exist. Considering the change of baseline near the benzyl chloride peak, the separation of benzyl chloride and adjacent peaks, the retention time of benzyl chloride and other factors, method 4 was temporarily selected as the detection method for benzyl chloride in condensation products.

[0032] (4) Confirmation of test solution concentration Take an appropriate amount of benzyl chloride (CFW, batch number: E-501900-NU1, GC purity: 99.79%), dilute it with N,N-dimethylformamide to a solution of appropriate concentration, and analyze it according to the chromatographic conditions of method 4. When the injection concentration is about 0.9µg / ml and 0.4µg / ml, the signal-to-noise ratio of benzyl chloride is about 11 and 6 respectively, which meets the requirements of the limit of quantification and the limit of detection. The limit of benzyl chloride is calculated with reference to the EMA "Guidelines on Limits of Genotoxic Impurities". According to the TCC (Threshold of Toxicological Concern) of 1.5µg / day, the limit = TTC / dose, the daily dose of this product is 200mg, and the limit concentration of benzyl chloride is 0.00075%. Combined with the limit of quantification, the minimum concentration of the test solution that meets the quantitative detection of benzyl chloride is 120mg / ml. In order to ensure that benzyl chloride in the condensate can be accurately quantified, the concentration of the test solution is tentatively set to 200mg / ml.

[0033] 2. Method Description and Validation (1) Method description Test solution: Take an appropriate amount of condensate and dissolve it in N,N-dimethylformamide to make a solution containing 200 mg of condensate per 1 ml.

[0034] Reference solution Take an appropriate amount of benzyl chloride reference substance and dilute it with N,N-dimethylformamide to make a solution containing about 1.5µg of benzyl chloride per 1ml.

[0035] System suitability solution Take appropriate amounts of condensate and benzyl chloride reference substance, dissolve them in N,N-dimethylformamide and dilute to a mixed solution of 200 mg of condensate and 15 µg of benzyl chloride per 1 ml.

[0036] The chromatographic conditions were: a capillary column (Agilent DB-624 30m×0.530mm×3.0µm) with (6%) cyanopropylphenyl-(94%) dimethylsiloxane as the stationary phase; a column flow rate of 3ml / min; a starting temperature of 100°C, which was increased to 180°C at a rate of 10°C per minute and maintained for 5 minutes, then increased to 230°C at a rate of 20°C per minute and maintained for 3 minutes; an injection port temperature of 230°C; a split ratio of 2:1; a detector temperature of 250°C; and an injection volume of 1µl.

[0037] (2) Method validation (2-1) Exclusivity Prepare the system suitability solution and the test solution according to the item "(1) Method Description", and prepare a benzyl chloride fixation solution of appropriate concentration (about 15µg / ml). Take the system suitability solution, the test solution, the benzyl chloride fixation solution and the solvent and test them according to the chromatographic conditions under this item to examine the interference of the benzyl chloride detection. The results are as follows: Figures 7-10 , and as shown in Table 4.

[0038] Table 4 Specificity results

[0039] Note: “-” means the data does not exist or is not detected From the above results, it can be seen that the solvent and condensation product do not interfere with the detection of benzyl chloride, the separation degree of benzyl chloride and adjacent peaks meets the requirements, and the specificity is good.

[0040] (2-2) Linear An appropriate amount of benzyl chloride reference substance (CFW, batch number: E-501900-NU1, GC purity: 99.79%) was diluted with N,N-dimethylformamide to a series of concentrations. The results were tested according to the chromatographic conditions under “(1) Method description” and linear regression was performed with the injection concentration as the horizontal axis and the peak area as the vertical axis. The results are shown in Table 5.

[0041] Table 5 Linearity results

[0042] According to the above results, the linear correlation diagram is as follows Fig.11 The linear range is 0.53-3.33µg / ml, showing a good linear relationship.

[0043] (2-3) Limit of quantification and limit of detection An appropriate amount of benzyl chloride reference substance (CFW, batch number: E-501900-NU1, GC purity: 99.79%) was diluted with N,N-dimethylformamide to a series of concentrations. The concentration with a signal-to-noise ratio of about 10 was the quantification limit concentration, and the concentration with a signal-to-noise ratio of about 3 was the detection limit concentration. The results are shown in Table 6.

[0044] Table 6 Results of limit of quantification and limit of detection

[0045] From the above results, it can be seen that the quantification limit and detection limit concentrations are 0.89µg / ml and 0.44µg / ml, respectively, which are equivalent to 0.0004% and 0.0002% of the test solution concentration, respectively, which are far lower than the quality control requirement of "not exceeding 7.5ppm" (that is, the limit concentration is not exceeding 0.00075%), and the method has good sensitivity.

[0046] (2-4) Injection precision Take 444.86 mg of benzyl chloride reference substance (CFW, batch number: E-501900-NU1, GC purity: 99.79%), put it into a 20 ml volumetric flask, dilute it to the mark with N,N-dimethylformamide, shake well, take 200 µl, put it into a 50 ml volumetric flask, dilute it to the mark with N,N-dimethylformamide, shake well, take another 200 µl, put it into a 10 ml volumetric flask, dilute it to the mark with N,N-dimethylformamide, shake well. Inject the sample 6 times continuously according to the chromatographic conditions under "(1) Method description" to examine the changes in the retention time and peak area of ​​benzyl chloride. The results are shown in Table 7.

[0047] Table 7 Injection precision results

[0048] The benzyl chloride reference substance was injected 6 times continuously, and the RSD of retention time and peak area were 0.04% and 1.40% respectively, indicating that the instrument injection precision was good.

[0049] (5) Solution stability Test solution: Take about 400 mg of the condensate (batch number: 10901-1911001y), accurately weigh it, and add 2 ml of N,N-dimethylformamide to dissolve it.

[0050] Reference solution: Take 444.86 mg of benzyl chloride reference (CFW, batch number: E-501900-NU1, GC purity: 99.79%), place in a 20 ml volumetric flask, dissolve and dilute to the mark with N,N-dimethylformamide, shake well, transfer 200 µl, place in a 50 ml volumetric flask, dilute to the mark with N,N-dimethylformamide, shake well, take another 200 µl, place in a 10 ml volumetric flask, dilute to the mark with N,N-dimethylformamide, shake well.

[0051] According to the chromatographic conditions under “(1) Method description”, the samples were injected at different times to investigate the changes in the peak areas of the condensation product and benzyl chloride over time. The results are shown in Table 8.

[0052] Table 8 Solution stability results

[0053] Note: “ / ” means the data does not exist The peak area RSD of benzyl chloride within 13 hours was 1.70%, and the stability of the test solution after storage for 22 hours was good.

[0054] (2-6) Repeatability and recovery rate Reference solution Take an appropriate amount of benzyl chloride reference (CFW, batch number: E-501900-NU1, GC purity: 99.79%), accurately weigh it, and dilute it with N,N-dimethylformamide to a solution containing about 1.5µg per 1ml.

[0055] Take about 400 mg of the condensate (batch number: 10901-1911001y) of the test solution, accurately weigh it, put it into a 5 ml volumetric flask, quantitatively add 2 ml of the reference solution to dissolve it, and shake well.

[0056] Take an appropriate amount of the condensate (batch number: 10901-1911001y) as the background solution and dissolve it in N,N-dimethylformamide to dilute it into a solution containing about 200 mg per 1 ml.

[0057] The sample was injected according to the chromatographic conditions under “(1) Method Description” and the recovery content of benzyl chloride was investigated. The results are shown in Table 9.

[0058] Table 9 Recovery rate and repeatability results

[0059] The recovery range of benzyl chloride was 94.93%~98.96%, the average recovery was 96.91%, the RSD was 1.54%, and the recovery rate and repeatability were good.

[0060] (2-7) Durability By changing the initial temperature, flow rate (i.e., column flow rate) and injection port temperature, the separation of benzyl chloride and adjacent impurities and the recovery content of benzyl chloride were investigated. The results are shown in Tables 10 and 11.

[0061] Table 10 Durability results - separation

[0062] Table 11 Durability results - content

[0063] By changing the initial temperature, flow rate and injection port temperature, the separation degree of benzyl chloride and adjacent peaks met the requirements and the difference was not large; the content of benzyl chloride under each condition ranged from 93.23% to 101.59%, and the RSD of the content was 2.9%, which had good durability.

[0064] (3) Summary of method validation The summary results of the method validation are shown in Table 12.

[0065] Table 12 Method validation summary

[0066] 3. Sample Testing Several batches of condensation products were prepared and analyzed according to the method described in “(1) Method Description” in Part II to investigate the detection of benzyl chloride in the condensation product of the key intermediate of lamotrigine. The results are shown in Table 13.

[0067] Table 13 Detection results of benzyl chloride

[0068] From the above results, it can be seen that benzyl chloride was not detected in any of the four batches of condensation products.

[0069] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for detecting the content of benzyl chloride in a key intermediate of lamotrigine, characterized in that: The following steps are involved: Dissolving the sample to be tested and the reference substance in the detection solvent respectively to obtain a test solution and a reference substance solution; The test solution and the reference solution are respectively tested by gas chromatography technology, and the benzyl chloride content is obtained through the gas chromatography test results; The conditions of the gas chromatography are as follows: Chromatographic column: capillary column with (6%) cyanopropylphenyl-(94%) dimethylsiloxane as stationary liquid or a chromatographic column with similar polarity; Chromatographic column heating program: initial temperature is 90-110℃, increase to 170-190℃ at 8-12℃ / min, maintain for 3-8min, then increase to 220-240℃ at 18-22℃ / min, maintain for 1-5min.

2. The detection method according to claim 1, characterized in that: Column heating program: initial temperature is 95-105°C, increase to 180-185°C at 9-10°C / min, maintain for 5-6 min, then increase to 230-235°C at 20-21°C / min, maintain for 3-4 min.

3. The detection method according to claim 1, characterized in that: The detection solvent was N,N-dimethylformamide.

4. The detection method according to any one of claims 1 to 3, characterized in that: The concentration of the test solution is 120-200 mg / ml; The concentration of the reference solution is 1-1.5 μg / ml.

5. The detection method according to any one of claims 1 to 3, characterized in that: The sample to be tested is the material sampled when the condensate is synthesized in the lamotrigine synthesis process. Condensation products.

6. The detection method according to claim 1, characterized in that: Flow rate is 2.5-3.5 ml / min; The injection port temperature is 220-240℃.

7. The detection method according to claim 6, characterized in that: Flow rate is 2.8-3.2 ml / min; The injection port temperature was 225-235°C.

8. The detection method according to claim 1, characterized in that: The split ratio is (1.5-2):1; The detector temperature is 240-250°C; The injection volume is 0.8-1.2µl.

9. The detection method according to claim 1, characterized in that: The injection method is direct solution injection.

10. The detection method according to any one of claims 1-3 and 6-9, characterized in that: The limit of quantification and the limit of detection of the assay were 0.89µg / ml and 0.44µg / ml, respectively.

Images