Method for detecting cyanoacetate in tofacitinib citrate
Through gas chromatography combined with external standard method, the problem of difficulty in accurately detecting the impurity content of ethyl cyanoacetate and methyl cyanoacetate in torosybutybrate citrate in the prior art was solved, and the torosybrate citrate citrate was realized. The accurate determination of cyanoacetate impurities in the cloth ensures the control of drug quality.
Patent Information
- Application Number
- CN202411591771.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2025-02-18
AI Technical Summary
It is difficult to accurately detect the impurity content of ethyl cyanoacetate and methyl cyanoacetate in Tofatibu citrate, and the residue of methyl cyanoacetate will affect the safety and effectiveness of the drug.
The gas chromatography combined with the external standard method was used to prepare the test and reference solution, and the samples were analyzed using preset chromatography conditions, and the contents of methyl cyanoacetate and ethyl cyanoacetate were calculated.
The simultaneous determination of the content of methyl cyanoacetate and ethyl cyanoacetate in Tofatibu citrate was achieved, ensuring the control of drug quality and improving the accuracy and reliability of detection.
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Figure CN119470696A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting cyanoacetates in tofacitinib citrate, and belongs to the technical field of quality detection methods for high-temperature easily decomposable raw materials. Background Art
[0002] Tofacitinib citrate is a JAK3 (Janus kinase 3) inhibitor developed for the prevention of acute rejection, treatment of rheumatoid arthritis (RA), Crohn's disease, dry eye disease and psoriasis. Tofacitinib citrate is a potent and selective inhibitor of the JAK kinase family, with selectivity for JAK3 (1nM) approximately 1000 times stronger than the other 82 kinases tested.
[0003] Ethyl cyanoacetate is one of the starting materials for the synthesis of tofacitinib citrate, and methyl cyanoacetate is the main impurity of ethyl cyanoacetate, both of which may remain in tofacitinib citrate. In order to ensure the safety and effectiveness of the use of tofacitinib citrate, it is very necessary to control and detect cyanoacetate impurities. At present, the gas chromatography method for detecting ethyl cyanoacetate in tofacitinib citrate has been retrieved, but the sample processing method may cause pre-column thermal decomposition of tofacitinib citrate, and the decomposition products remain at the column head to interfere with the detection. Moreover, with the increase in the number of injections, the recovery rate decreases, the interference is significant, and the impurity amount of ethyl cyanoacetate cannot be accurately detected.
[0004] In addition, there may be two cyanoacetate impurities in tofacitinib citrate, methyl cyanoacetate and ethyl cyanoacetate. Measuring only ethyl cyanoacetate to evaluate the quality of the drug is inaccurate and not conducive to a comprehensive evaluation of its quality.
[0005] The LD50 of methyl cyanoacetate in guinea pigs by oral and intraperitoneal injection is 400-800 mg / kg, which is moderately toxic. Therefore, the residual methyl cyanoacetate in tofacitinib citrate will affect its safety and efficacy, so it is particularly necessary to control the content of methyl cyanoacetate in tofacitinib citrate. Currently, there is no method to detect methyl cyanoacetate in tofacitinib citrate. Summary of the invention
[0006] The purpose of the present application is to provide a method for detecting cyanoacetates in tofacitinib citrate, which can simultaneously determine methyl cyanoacetate and ethyl cyanoacetate impurities in tofacitinib citrate, thereby ensuring the quality control of the tofacitinib citrate product.
[0007] The present application provides a method for detecting cyanoacetates in tofacitinib citrate, using gas chromatography to determine impurities in tofacitinib citrate, the impurities include methyl cyanoacetate and ethyl cyanoacetate, and the detection method includes:
[0008] S1. Prepare a test solution; dissolve and dilute the test sample tofacitinib citrate to obtain a test solution;
[0009] S2, preparing reference substance solutions; including ethyl cyanoacetate reference substance solution and methyl cyanoacetate reference substance solution;
[0010] S3, performing gas chromatography analysis on the test solution and the reference solution using preset chromatographic conditions;
[0011] The preset chromatographic conditions are as follows: the chromatographic column is a capillary column with an inner diameter of 0.32 mm, a length of 30 m, and a film thickness of 1.80 μm; the stationary liquid is 6% cyanopropylphenyl-94% dimethylpolysiloxane; the column temperature program is a starting temperature of 50°C, maintained for 5 minutes, then heated to 200°C at a rate of 20°C per minute, and maintained for 5 minutes; the carrier gas is high-purity helium; the injection volume is 1 μl; the injection port temperature is 220°C; the detector temperature is 250°C;
[0012] S4. The contents of methyl cyanoacetate and ethyl cyanoacetate in tofacitinib citrate were obtained according to the chromatographic analysis by the external standard method.
[0013] In one feasible scheme, the preparation process of the test solution is as follows:
[0014] Take a predetermined amount of the test sample tofacitinib citrate, add a predetermined amount of 0.2 mol / L hydrochloric acid solution to dissolve it, shake and extract with dichloromethane for several times, combine the dichloromethane liquids and place them in a volumetric flask, dilute to the scale with dichloromethane, shake well, and obtain the test sample solution.
[0015] In one feasible scheme, the preparation process of the test solution is as follows:
[0016] Take 1.0 g of the test sample Tofacitinib Citrate, accurately weigh it, place it in a separatory funnel, add 50 ml of 0.2 mol / L hydrochloric acid solution to dissolve it, shake and extract it with dichloromethane three times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane liquids and place them in a 20 ml volumetric flask, dilute to the scale with dichloromethane, and shake well.
[0017] In one feasible scheme, the purity of the reference substance ethyl cyanoacetate is ≥99%.
[0018] In an operative scheme, the preparation process of the ethyl cyanoacetate reference substance solution is as follows: take the reference substance ethyl cyanoacetate, and quantitatively dilute it with dichloromethane to prepare the ethyl cyanoacetate reference substance solution containing 0.1 mg of ethyl cyanoacetate per 1 ml.
[0019] In one feasible scheme, the purity of the reference substance methyl cyanoacetate is ≥99%.
[0020] In an practicable scheme, the preparation process of the methyl cyanoacetate reference solution is as follows: take the reference substance methyl cyanoacetate, and quantitatively dilute it with dichloromethane to prepare a methyl cyanoacetate reference solution containing 0.1 mg of methyl cyanoacetate per 1 ml.
[0021] Compared with the prior art, the beneficial effects of the present application include at least the following:
[0022] The present application provides a detection method for determining cyanoacetate impurities in tofacitinib citrate, using respective reference substances of methyl cyanoacetate and ethyl cyanoacetate as reference substances, and calculating the contents of methyl cyanoacetate and ethyl cyanoacetate in tofacitinib citrate by an external standard method, thereby being able to simultaneously determine the methyl cyanoacetate and ethyl cyanoacetate impurities in tofacitinib citrate, thereby being able to ensure the quality control of the tofacitinib citrate product. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0024] Figure 1 A flow chart of a detection method shown in an embodiment of the present invention;
[0025] Figure 2 It is the solvent dichloromethane chromatogram under the specificity test item;
[0026] Figure 3 It is the chromatogram of the ethyl cyanoacetate localization solution under the specificity test item;
[0027] Figure 4 It is the chromatogram of the methyl cyanoacetate localization solution under the specificity test item;
[0028] Figure 5 It is the chromatogram of the test sample solution under the specificity test item;
[0029] Figure 6 It is the chromatogram of the positive test solution of ethyl cyanoacetate under the specificity test item;
[0030] Figure 7 It is the chromatogram of the positive test solution of methyl cyanoacetate under the specificity test item;
[0031] Figure 8 It is a linear relationship-ethyl cyanoacetate content determination linear graph; wherein, the ordinate is the peak area and the abscissa is the concentration (μg / ml);
[0032] Fig. 9It is a linear relationship - linear graph of methyl cyanoacetate content determination; wherein, the ordinate is the peak area and the abscissa is the concentration (μg / ml). DETAILED DESCRIPTION
[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.
[0034] Therefore, the following detailed description of the embodiments of the present application provided in the accompanying drawings is not intended to limit the scope of the present application for which protection is sought, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.
[0035] like Figure 1 As shown, the embodiment of the present application provides a method for detecting cyanoacetate impurities in tofacitinib citrate, the method being characterized in that gas chromatography is used to determine the impurities, the impurities include methyl cyanoacetate and ethyl cyanoacetate, and the detection method includes:
[0036] S1. Prepare a test solution; dissolve and dilute the test sample tofacitinib citrate to obtain a test solution;
[0037] S2, preparing reference substance solutions; including ethyl cyanoacetate reference substance solution and methyl cyanoacetate reference substance solution;
[0038] S3, performing gas chromatography analysis on the test solution and the reference solution using preset chromatographic conditions;
[0039] The preset chromatographic conditions are as follows: the chromatographic column is a capillary column with an inner diameter of 0.32 mm, a length of 30 m, and a film thickness of 1.80 μm; the stationary liquid is 6% cyanopropylphenyl-94% dimethylpolysiloxane; the column temperature program is a starting temperature of 50°C, maintained for 5 minutes, then heated to 200°C at a rate of 20°C per minute, and maintained for 5 minutes; the carrier gas is high-purity helium; the injection volume is 1 μl; the injection port temperature is 220°C; the detector temperature is 250°C;
[0040] S4. The contents of methyl cyanoacetate and ethyl cyanoacetate in tofacitinib citrate were obtained according to the chromatographic analysis by the external standard method.
[0041] The embodiment of the present application provides a detection method for determining cyanoacetate impurities in tofacitinib citrate, using respective reference substances of methyl cyanoacetate and ethyl cyanoacetate as reference substances, and calculating the contents of methyl cyanoacetate and ethyl cyanoacetate in tofacitinib citrate by an external standard method, thereby being able to simultaneously determine the methyl cyanoacetate and ethyl cyanoacetate impurities in tofacitinib citrate, thereby ensuring the quality control of the tofacitinib citrate product.
[0042] In this embodiment, the preparation process of the test solution can be:
[0043] Take a predetermined amount of the test sample tofacitinib citrate, add a predetermined amount of 0.2 mol / L hydrochloric acid solution to dissolve it, shake and extract with dichloromethane for several times, combine the dichloromethane liquids and place them in a volumetric flask, dilute to the scale with dichloromethane, shake well, and obtain the test sample solution.
[0044] In this embodiment, the detailed preparation process of the test solution can be:
[0045] Take 1.0g of the test sample Tofacitinib Citrate, accurately weigh it, place it in a separatory funnel or conical flask, add 50ml of 0.2mol / L hydrochloric acid solution to dissolve it, shake and extract it with dichloromethane three times, 10ml, 5ml, and 5ml respectively, combine the dichloromethane liquids and place them in a 20ml volumetric flask, dilute to the scale with dichloromethane, and shake well.
[0046] In this embodiment, the purity of the reference substance ethyl cyanoacetate is ≥99%. For example, the purity of the ethyl cyanoacetate used in this embodiment reaches 99.539%.
[0047] In this embodiment, the purity of the reference substance methyl cyanoacetate is ≥99%. For example, the purity of the methyl cyanoacetate used in this embodiment reaches 99.169%.
[0048] In this embodiment, the preparation process of the ethyl cyanoacetate reference substance solution can be: take the reference substance ethyl cyanoacetate, and quantitatively dilute it with dichloromethane to prepare the ethyl cyanoacetate reference substance solution containing 0.1 mg of ethyl cyanoacetate per 1 ml.
[0049] In this embodiment, the preparation process of the methyl cyanoacetate reference solution can be: take the reference substance methyl cyanoacetate, and quantitatively dilute it with dichloromethane to prepare a methyl cyanoacetate reference solution containing 0.1 mg of methyl cyanoacetate per 1 ml.
[0050] In the present embodiment, in order to verify the results of this detection method, a stock solution is provided, and the stock solution includes methyl cyanoacetate stock solution and ethyl cyanoacetate stock solution. The preparation process is as follows: 49.14 mg of ethyl cyanoacetate and 52.63 mg of methyl cyanoacetate are weighed respectively, placed in 50 ml measuring bottles respectively, diluted to the mark with dichloromethane, and shaken up, as methyl cyanoacetate stock solution and ethyl cyanoacetate stock solution.
[0051] The present application also conducts applicability tests and interference tests on the detection method of this embodiment, and the steps are as follows:
[0052] S011. Prepare a positioning solution; the positioning solution includes an ethyl cyanoacetate positioning solution and a methyl cyanoacetate positioning solution;
[0053] Accurately measure 1 ml of ethyl cyanoacetate stock solution and methyl cyanoacetate stock solution, respectively, and place them in 10 ml volumetric flasks, dilute to the mark with dichloromethane, and shake well to serve as ethyl cyanoacetate positioning solution and methyl cyanoacetate positioning solution;
[0054] S012. Prepare the reference solution for the test: weigh about 1.00816 g of tofacitinib citrate, place it in a conical flask, add 18 ml of 0.2 mol / L hydrochloric acid solution to 1000 ml (59 ml is used in this method), shake to dissolve, extract with dichloromethane by shaking for 3 times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane solution, place it in a 20 ml volumetric flask, dilute to the scale with dichloromethane, shake well, and use it as the test solution for the test;
[0055] S013. Prepare a positive test solution. Accurately weigh 1.00629 g of tofacitinib citrate, place in a conical flask, add 50 ml of 0.2 mol / L hydrochloric acid solution to dissolve, add 2 ml of ethyl cyanoacetate stock solution, shake to dissolve, extract with dichloromethane by shaking for 3 times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane solutions, place in a 20 ml volumetric flask, dilute to the mark with dichloromethane, shake well, and use as the positive test solution for ethyl cyanoacetate. Test solution: Weigh 1.00387 g of tofacitinib citrate, place in a conical flask, add 0.2 ml of methyl cyanoacetate stock solution (1 mg / ml), add 50 ml of 0.2 mol / L hydrochloric acid solution, shake to dissolve, extract with dichloromethane by shaking for 3 times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane solutions and place in a 20 ml volumetric flask, dilute to the scale with dichloromethane, shake well, and use as the positive test solution for methyl cyanoacetate.
[0056] S014. Accurately measure 1 μl of each solvent, each positioning solution, test sample solution and positive test sample solution, and inject them into the gas chromatograph respectively. Record the chromatogram. Figure 2-Figure 7The results showed that the solvent and tofacitinib citrate did not interfere with the detection of ethyl cyanoacetate and methyl cyanoacetate, and the method had good specificity. The results are shown in Table 1.
[0057] Table 1 Ethyl cyanoacetate inspection specificity test results
[0058]
[0059] like Figure 2-Figure 7 The chromatogram shown and the analysis based on the data in Table 1 are as follows.
[0060] According to the results of the chromatogram: the solvent eluted within 3 minutes, which means that the peak of the solvent is separated from the peaks of ethyl cyanoacetate and methyl cyanoacetate, so the solvent will not interfere with the detection of these two compounds.
[0061] The retention time of ethyl cyanoacetate in the fixation solution was 10.819 minutes, and in the positive test solution was 10.822 minutes, but it was not detected in the experimental test solution, which indicates that the presence of tofacitinib citrate did not lead to a false positive in the detection of ethyl cyanoacetate.
[0062] The retention time of methyl cyanoacetate in the fixation solution was 10.208 minutes, in the positive test solution was 10.822 minutes, and was not detected in the experimental test solution, which indicated that the presence of tofacitinib citrate did not lead to a false positive in the detection of ethyl cyanoacetate.
[0063] In summary, since the peak of the solvent is separated from the peak of the target compound, and the presence of tofacitinib citrate does not lead to false positive detection of the target compound, we can conclude that neither the solvent nor tofacitinib citrate interferes with the detection of ethyl cyanoacetate and methyl cyanoacetate, and this method has good specificity.
[0064] The present application also conducted sensitivity tests, including tests on the quantitative limit and detection limit of ethyl cyanoacetate and methyl cyanoacetate, and tests on the precision of the quantitative limit of ethyl cyanoacetate.
[0065] ① Test the quantitative limit and detection limit of ethyl cyanoacetate and methyl cyanoacetate
[0066] According to the above content, ethyl cyanoacetate and methyl cyanoacetate reference solutions were accurately prepared, gradually diluted to a certain concentration and injected. The sample concentration with a signal-to-noise ratio (S / N) of about 3-5 was the detection limit concentration, and the detection limit was injected with 1 injection; the sample concentration with a signal-to-noise ratio (S / N) of about 9-15 was the quantification limit concentration, and the quantification limit was injected with 6 consecutive injections, and the signal-to-noise ratio RSD value corresponding to the quantification limit was calculated.
[0067] Accurately measure an appropriate amount of the reference solution and dilute it step by step into solutions with concentrations of 3μg / ml, 5μg / ml, and 10μg / ml as the test solutions for the detection limit and quantification limit.
[0068] Take 1ul of the solvent and the above solution, respectively, and inject them into the gas chromatograph, record the ion chromatogram, and record the detection limit experimental results, see Table 2.
[0069] Table 2 Detection limit test results
[0070]
[0071] ② Conduct quantitative limit precision test of ethyl cyanoacetate
[0072] Accurately measure 1 μl of ethyl cyanoacetate limit of quantification solution and methyl cyanoacetate limit of quantification solution, inject 6 times in succession, record the chromatogram, and calculate the RSD of the peak area of each component. The results show that the precision of the limit of quantification is good, and the results are shown in Table 3.
[0073] Table 3 Results of the precision test of ethyl cyanoacetate quantitative limit
[0074]
[0075] The examples of the present application also examine the preparation of the standard curve and the linear relationship.
[0076] Accurately measure 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml of ethyl cyanoacetate and methyl cyanoacetate stock solutions (about 1 mg / ml each) and place them in 5 10 ml volumetric flasks respectively. Dilute to the mark with dichloromethane and shake well to prepare linear series solution 2. # ~6 # .
[0077] Accurately measure 1 μl of each linear series solution, inject it into the gas chromatograph, and record the chromatogram.
[0078] Take the quantitation limit solution spectrum as the linear series solution 1 # Participate in the calculation, use concentration C as the horizontal coordinate and peak area A as the vertical coordinate, perform linear regression, and calculate the linear regression equation and correlation coefficient r.
[0079] The results are shown in Tables 4 and 5 below. The linear correlation coefficient r of ethyl cyanoacetate and methyl cyanoacetate is greater than 0.998 and the intercept / 100% response value is less than 25% within the range of the quantitative limit to 200% of the limit concentration, indicating a good linear relationship. Figure 8 , the linear relationship of methyl cyanoacetate content determination can be found in Fig. 9 .
[0080] Table 4 Ethyl cyanoacetate standard curve results
[0081]
[0082] Table 5 Methyl cyanoacetate standard curve results
[0083]
[0084] The examples of the present application also verify the injection precision.
[0085] Weigh 10.53 mg of ethyl cyanoacetate and 10.21 mg of methyl cyanoacetate, place in the same 100 ml volumetric flask, dilute to scale with dichloromethane, shake well, and use as reference solution. Accurately measure 1 μl, inject 6 times continuously, and record the chromatogram. The results show that the injection precision is good. The results are shown in Table 6.
[0086] Table 6 Precision data table
[0087]
[0088] The present invention also conducts stability tests, including stability tests of reference solution and stability tests of test solution.
[0089] ①Stability test of reference solution
[0090] Take the reference solution under the above precision test item, place it at room temperature, and accurately measure 1μl at 0, 1, 2, 4, 6 and 8 hours, inject it into the gas chromatograph, and record the chromatogram. The results show that the reference solution is stable within 8 hours at room temperature. The results are shown in Table 7.
[0091] Table 7 Stability data of reference solution
[0092] Time (h) 0 1 2 4 6 8 RSD Ethyl cyanoacetate A 18.55 18.67 18.68 18.48 18.87 18.82 0.80％ Methyl cyanoacetate A 17.21 17.33 17.32 17.19 17.32 17.29 0.35％
[0093] ② Stability of test solution
[0094] Weigh 1.00699g of tofacitinib citrate, put it in a conical flask, add 50ml of 0.2mol / L hydrochloric acid solution, shake to dissolve, extract 3 times with dichloromethane shaking, respectively 10ml, 5ml, 5ml, combine dichloromethane liquid, put it in a 20ml measuring bottle, dilute to the scale with dichloromethane, shake well, as the test solution, place at room temperature, at 0, 2, 4, 6 and 8 hours, accurately measure 1μl, inject into gas chromatograph, and record chromatogram. The results show that the test solution is stable within 8 hours at room temperature. The results are shown in Table 8.
[0095] Table 8 Stability data of test solution
[0096] Time (h) 0 2 4 6 8 RSD(%) Ethyl cyanoacetate (%) 0 0 0 0 0 - Methyl cyanoacetate(%) 0 0 0 0 0 -
[0097] The present application example also carried out a repeatability test.
[0098] Weigh 10.35 mg of ethyl cyanoacetate and 10.21 mg of methyl cyanoacetate, place in a 100 ml volumetric flask, dilute to scale with dichloromethane, shake well, and obtain a reference solution. Weigh about 1 g of tofacitinib citrate, accurately weigh, place in a conical flask, add 50 ml of 0.2 mol / L hydrochloric acid solution, shake to dissolve, and extract with dichloromethane by shaking for 3 times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane solution and place in a 20 ml volumetric flask, dilute to scale with dichloromethane, shake well, and use as the test solution. Prepare 6 portions (1 # ~6 # ). Accurately measure 1 μl of each of the above solutions, inject into the gas chromatograph, and record the chromatogram. The results show that ethyl cyanoacetate was not detected, and the method has good repeatability. The results are shown in Table 9.
[0099] Table 9 Determination repeatability data table
[0100] Serial number Weighing(g) Methyl cyanoacetate(%) Ethyl cyanoacetate (%) 1 1.00699 0 0 2 1.00835 0 0 3 1.00253 0 0 4 0.99981 0 0 5 1.00489 0 0 6 1.00368 0 0
[0101] The present application example also conducts an accuracy test.
[0102] Weigh 49.28 mg of ethyl cyanoacetate, place in a 50 ml volumetric flask, dilute to scale with water, shake well, and use as ethyl cyanoacetate stock solution. Weigh about 1.0 g of tofacitinib citrate, weigh accurately, and place 1 in 9 portions. # ~9 # In a conical flask, accurately measure 0.2 ml of ethyl cyanoacetate stock solution, a total of 3 portions, and place 1 # ~3 # In a conical flask, add 50ml of 0.2mol / L hydrochloric acid solution to dissolve, shake and extract with dichloromethane three times, 10ml, 5ml, and 5ml respectively, combine the dichloromethane liquid and put it in a 20ml volumetric flask, dilute to the mark with dichloromethane, shake well, and use it as 10% test solution 1 # ~3 # ; Accurately measure 2.0 ml of ethyl cyanoacetate stock solution, a total of 3 portions, and place them in 4 # ~6 # In a conical flask, operate in the same way as 100% test solution 4 # ~6 # ; Accurately measure 3 ml of ethyl cyanoacetate stock solution, a total of 3 portions, and place them at 7 # ~9 # In a conical flask, operate in the same way as 150% test solution 7 # ~9 # .
[0103] Accurately measure 1 μl of the above test solution and the reference solution under the repeatability test, inject them into the gas chromatograph, record the chromatogram, calculate the recovery rate of ethyl cyanoacetate by peak area according to the external standard method, and compare them. The results show that the recovery rate of ethyl cyanoacetate is in the range of 93.87% to 100.58%, with an RSD of 1.90%, and the accuracy of this method is good. The test results are shown in Table 10.
[0104] Table 10 Ethyl cyanoacetate recovery data table
[0105]
[0106] Note: The original percentage content of tofacitinib ethyl cyanoacetate in this example was tested using the test sample under the repeatability test item: not detected.
[0107] The embodiments of the present application also undergo a durability test.
[0108] According to the established inspection method, the reference solution and the test solution were taken for durability test. By changing the column temperature, flow rate, different batches of chromatographic columns and injection port temperature, the precision of the reference solution and the detection amount of ethyl cyanoacetate in the test sample were examined; the tolerance of the ethyl cyanoacetate inspection under the established chromatographic conditions was evaluated.
[0109] By changing the test conditions, the durable range of the chromatographic conditions was confirmed. The results are shown in Tables 11 and 12.
[0110] Table 11 Inspection method durability data table
[0111] Inspection conditions Test method conditions Confirmed durability range Column temperature 200℃ 190℃～210℃ Flow rate 3.0ml / min 2.9ml / min~3.1ml / min Inlet temperature 220℃ 210℃～230℃ Chromatographic columns Agilent DB-624 30m×0.32mm Different batch numbers
[0112] Table 12 Tofacitinib citrate residual solvent determination durability test results
[0113]
[0114] It should be noted that the melting point of tofacitinib citrate (204°C-208°C, decomposes simultaneously when melted) is similar to the boiling points of ethyl cyanoacetate (about 210°C) and methyl cyanoacetate (about 207°C). When it is dissolved in a solvent and directly injected into the gas chromatography method for determination, thermal decomposition occurs before the column. The decomposition products remain at the column head and interfere with the detection. Moreover, as the number of injections increases, the recovery rate decreases, and the interference is significant, making it impossible to accurately detect the amount of cyanoacetate impurities. When high-temperature easily decomposable raw materials are dissolved in a solvent and directly injected into the gas chromatography method for determination, thermal decomposition occurs. The decomposition products remain in the chromatographic column, affecting the quality and life of the chromatographic column, and the detection reproducibility is poor and the detection cost is high.
[0115] Therefore, in order to solve the aforementioned problems, the present application describes the determination of the preparation method of the test solution.
[0116] First, N,N-dimethylacetamide is used as a solvent for direct dissolution.
[0117] Weigh 100.27 mg of methyl cyanoacetate and 101.82 mg of ethyl cyanoacetate, place in a 50 ml volumetric flask, dilute to scale with N, N-dimethylacetamide, shake well, and use as reference substance stock solution; accurately measure 1 ml of reference substance solution, place in a 10 ml volumetric flask, dilute to scale with N, N-dimethylacetamide, shake well, and use as reference substance solution. Weigh about 1 g of tofacitinib citrate, accurately weigh, a total of 9 portions, place in 10 ml volumetric flasks (1 # ~9 # ) in the well; accurately measure 0.5 ml of the reference stock solution, a total of 3 portions, and place 1 # ~3 # In a volumetric flask, dilute to the mark with N,N-dimethylacetamide and shake well to prepare the 50% test solution. Accurately measure 1.0 ml of the reference stock solution, a total of 3 portions, and place them in 4 # ~6 # In a volumetric flask, dilute to the mark with N,N-dimethylacetamide and shake well to prepare the 100% test solution. Accurately measure 1.5 ml of the reference stock solution, a total of 3 portions, and place them in 7 # ~9 # In a volumetric flask, dilute to the mark with N,N-dimethylacetamide and shake well to prepare the 150% test solution.
[0118] Accurately measure 1 μl of each reference solution and test solution, inject into the gas chromatograph, record the chromatogram, and calculate the recovery of methyl cyanoacetate and ethyl cyanoacetate by peak area according to the external standard method. The results show that tofacitinib citrate undergoes pre-column thermal decomposition, and the decomposition products remain at the column head to interfere with the detection; as the number of injections increases, the recovery rate decreases, the interference is significant, and the impurity amount of methyl cyanoacetate and ethyl cyanoacetate cannot be accurately detected. The results are shown in Table 13 below.
[0119] Table 13 Accuracy results of methyl cyanoacetate and ethyl cyanoacetate
[0120]
[0121] Secondly, the liquid-liquid extraction steps are specifically determined, including the selection of extraction solvent, the selection of extraction solvent volume, and the determination of the number of extractions.
[0122] a. Selection of extraction solvent
[0123] Weigh 103.22 mg of ethyl cyanoacetate, place in a 50 ml volumetric flask, dilute to scale with water, shake well, and use as ethyl cyanoacetate stock solution. Weigh about 1 g of tofacitinib citrate, a total of 5 portions, accurately weigh, place in conical flasks, add 1.0 ml of ethyl cyanoacetate stock solution, add 100 ml of 0.1 mol / L hydrochloric acid solution, shake to dissolve, transfer to a 125 ml separatory funnel, shake and extract with 10 ml of toluene, benzene, cyclohexane, n-heptane, and dichloromethane, respectively, and take the organic solvent layer as the test solution.
[0124] Accurately measure 1 μl of the extraction solvent and the test solution, inject them into the gas chromatograph, and record the chromatogram. The extraction capacity of each solvent for ethyl cyanoacetate is determined by the size of the ethyl cyanoacetate peak area in the test solution spectrum. The results show that dichloromethane is not dry for determination; and dichloromethane has the strongest extraction capacity, so dichloromethane is determined to be the extraction solvent for preparing the test solution. The results are shown in Table 14 below.
[0125] Table 14 Selection results of extraction solvents
[0126] Extract name Weighing amount (g) Peak area Toluene 1.00153 7.62 benzene 0.97758 11.34 Cyclohexane 0.98018 0 n-Heptane 0.98274 0 Dichloromethane 0.97581 39.45
[0127] b. Selection of extraction solvent volume
[0128] Weigh 47.29 mg of ethyl cyanoacetate, place in a 50 ml volumetric flask, dilute to scale with water, shake well, and use as ethyl cyanoacetate stock solution; weigh 20.77 mg of ethyl cyanoacetate, place in a 100 ml volumetric flask, dilute to scale with dichloromethane, shake well, and use as reference solution. Measure 50 ml of 0.2 mol / L hydrochloric acid solution, place in a 125 ml separatory funnel, and add 4 portions (1 # ~4 # ), and add 1g of tofacitinib citrate respectively, take 1 # , 2 # Add 2 ml of ethyl cyanoacetate stock solution respectively, shake well, extract with dichloromethane three times, 5 ml, 3 ml, 2 ml respectively, combine the dichloromethane solution and put it in a 10 ml volumetric flask, dilute to the scale with dichloromethane, shake well, filter, and use it as the test solution 1 # , 2 # ; Take 3 # , 4 # Add 2 ml of ethyl cyanoacetate stock solution respectively, shake well, extract with dichloromethane three times, 10 ml, 5 ml, 5 ml respectively, combine the dichloromethane solution and put it in a 20 ml volumetric flask, dilute to the scale with dichloromethane, shake well, filter, and use it as the test solution 3 # , 4 # .
[0129] Accurately measure 1 μl of the test solution and reference solution, inject them into the gas chromatograph, record the chromatogram, and calculate the recovery rate of ethyl cyanoacetate by peak area according to the external standard method. The results show that the extraction effect is better when the total volume of dichloromethane is 20 ml. The results are shown in Table 15 below.
[0130] Table 15 Selection results of extraction solvent volume
[0131]
[0132] c. Determination of extraction times
[0133] Weigh 52.15 mg of ethyl cyanoacetate, place in a 50 ml volumetric flask, dilute to scale with water, shake well, as ethyl cyanoacetate stock solution; weigh 10.86 mg of ethyl cyanoacetate, place in a 100 ml volumetric flask, dilute to scale with dichloromethane, shake well, as reference solution. Measure 50 ml of 0.2 mol / L hydrochloric acid solution, place in a 125 ml separatory funnel, add 1 g of tofacitinib citrate, add 2 ml of ethyl cyanoacetate stock solution, shake well, a total of 8 portions (1 # ~8 # ); take 1 # , 2 # Extract once with 20 ml of dichloromethane, take the dichloromethane solution, put it in a 20 ml volumetric flask, dilute to the mark with dichloromethane, shake well, and use it as the test solution①; take 3 # , 4 # Extract twice with dichloromethane, 10 ml each time, combine the dichloromethane solutions, place in a 20 ml volumetric flask, dilute to scale with dichloromethane, shake well, and use as the test solution②; take 5 # , 6 # Extract with dichloromethane by shaking for 3 times, 10ml, 5ml, and 5ml respectively, combine the dichloromethane solutions, place in a 20ml volumetric flask, dilute to the mark with dichloromethane, shake well, and use as the test solution ③; take 7 # , 8 # Extract with dichloromethane by shaking for 4 times, 10ml, 5ml, 3ml, 2ml respectively, combine the dichloromethane solutions, place in a 20ml volumetric flask, dilute to the scale with dichloromethane, shake well, and use as the test solution④.
[0134] Accurately measure 1 μl of each of the test solution and reference solution, inject into the gas chromatograph, record the chromatogram, and calculate the recovery rate of ethyl cyanoacetate by peak area according to the external standard method. The results show that the effect of extracting with dichloromethane three times is better, so the number of extractions is determined to be 3 times, and the volumes are 10ml, 5ml, and 5ml respectively. The results are shown in Table 16.
[0135] Table 16 Selection results of extraction times
[0136]
[0137] In summary, the method for preparing the test solution is preferably as follows: take 1.0 g of the test sample tofacitinib citrate, accurately weigh it, place it in a separatory funnel or a conical flask, add 50 ml of 0.2 mol / L hydrochloric acid solution, shake it to dissolve it, and extract it with dichloromethane by shaking for 3 times, 10 ml, 5 ml, and 5 ml respectively, combine the dichloromethane liquids, place them in a 20 ml volumetric flask, dilute to the scale with dichloromethane, shake well, and use it as the test solution.
[0138] This method of treating the test solution can extract cyanoacetates from tofacitinib citrate to prevent tofacitinib citrate from thermally decomposing when entering a gas chromatograph. The decomposition products remain in the chromatographic column to interfere with the detection results and affect the quality and life of the chromatographic column, thereby improving the feasibility and reproducibility of the detection method and reducing the high detection cost.
[0139] The above description is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for detecting cyanoacetates in tofacitinib citrate, characterized in that: Gas chromatography is used to determine the impurities in tofacitinib citrate, wherein the impurities include methyl cyanoacetate and ethyl cyanoacetate, and the detection method includes: S1. Prepare a test solution; dissolve and dilute the test sample tofacitinib citrate to obtain a test solution; S2, preparing reference substance solutions; including ethyl cyanoacetate reference substance solution and methyl cyanoacetate reference substance solution; S3, performing gas chromatography analysis on the test solution and the reference solution using preset chromatographic conditions; The preset chromatographic conditions are as follows: the chromatographic column is a capillary column with an inner diameter of 0.32 mm, a length of 30 m, and a film thickness of 1.80 μm; the stationary liquid is 6% cyanopropylphenyl-94% dimethylpolysiloxane; the column temperature program is a starting temperature of 50°C, maintained for 5 minutes, then heated to 200°C at a rate of 20°C per minute, and maintained for 5 minutes; the carrier gas is high-purity helium; the injection volume is 1 μl; the injection port temperature is 220°C; the detector temperature is 250°C; S4. The contents of methyl cyanoacetate and ethyl cyanoacetate in tofacitinib citrate were obtained according to the chromatographic analysis by the external standard method.
2. The method for detecting cyanoacetates in tofacitinib citrate according to claim 1, characterized in that: The preparation process of the test solution is as follows: Take a predetermined amount of the test sample tofacitinib citrate, add a predetermined amount of 0.2 mol / L hydrochloric acid solution to dissolve it, shake and extract with dichloromethane for several times, combine the dichloromethane liquids and place them in a volumetric flask, dilute to the scale with dichloromethane, shake well, and obtain the test sample solution.
3. The method for detecting cyanoacetates in tofacitinib citrate according to claim 1, characterized in that: The preparation process of the test solution is as follows: Take 1.0g of the test sample Tofacitinib Citrate, accurately weigh it, place it in a separatory funnel or conical flask, add 50ml of 0.2mol / L hydrochloric acid solution to dissolve it, shake and extract it with dichloromethane three times, 10ml, 5ml, and 5ml respectively, combine the dichloromethane liquids and place them in a 20ml volumetric flask, dilute to the scale with dichloromethane, and shake well.
4. The method for detecting cyanoacetates in tofacitinib citrate according to claim 1, characterized in that: The purity of the reference substance ethyl cyanoacetate is ≥99%.
5. The method for detecting cyanoacetates in tofacitinib citrate according to claim 4, characterized in that: The preparation process of the ethyl cyanoacetate reference substance solution is as follows: taking the reference substance ethyl cyanoacetate, quantitatively diluting it with dichloromethane to prepare the ethyl cyanoacetate reference substance solution containing 0.1 mg of ethyl cyanoacetate per 1 ml.
6. The method for detecting cyanoacetates in tofacitinib citrate according to claim 1, characterized in that: The purity of the reference substance methyl cyanoacetate is ≥99%.
7. The method for detecting cyanoacetates in tofacitinib citrate according to claim 6, characterized in that: The preparation process of the methyl cyanoacetate reference substance solution is as follows: taking the reference substance methyl cyanoacetate, quantitatively diluting it with dichloromethane to prepare a methyl cyanoacetate reference substance solution containing 0.1 mg of methyl cyanoacetate per 1 ml.