Detection method of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester related substances
The quality detection of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester was solved by high performance liquid chromatography and the self-control method of adding the correction factor. The problem of this problem achieves high sensitivity, accuracy and low cost quality control to ensure the safety of drug use.
Patent Information
- Application Number
- CN202411289493.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-14
- Publication Date
- 2024-12-27
AI Technical Summary
The prior art lacks effective detection methods to ensure the quality of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, especially in its long synthetic route. In the case of this, it is difficult to achieve simple, reliable and low-cost analysis methods, which affects the safety of drug use.
High performance liquid chromatography was used for detection, using octadecylsilane bonded silica gel chromatography column, gradient elution program and specific chromatography conditions, combined with the self-control method of adding correction factors, the impurity content was calculated, and multiple impurities were achieved. separation and quality control.
It realizes high sensitivity and accuracy detection of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, providing a simple and reliable quality control method. Meet relevant regulations and reduce testing costs.
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Figure CN119198991A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester. Background Art
[0002] (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester is an important starting material for chemical drugs, and its structural formula is shown in formula (I):
[0003]
[0004] The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guideline "Q11 API Development and Manufacturing (Chemical Entities and Biotechnology / Biological Entities)" points out that comprehensive impurity research and effective quality control should be carried out on starting materials to ensure the quality of APIs and the safety of patients. The "Notice on the Release of the Requirements for the Application Materials for the New Registration Classification of Chemical Drugs (Trial) (No. 80 of 2016)" issued by the former State Food and Drug Administration clearly requires that the impurities of starting materials should be comprehensively analyzed and controlled in accordance with relevant technical guidelines and technical requirements, and the impurities that may affect subsequent reactions or may be introduced into the final product should be identified, and on this basis, appropriate analytical methods should be used for control.
[0005] The synthetic route of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester is relatively long, and there is no literature report on the analytical method of this compound. In order to ensure the quality of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester and ensure the safety of patients' medication, it is necessary to provide a simple, effective, stable, reliable and low-cost analytical method to achieve the separation and detection of related substances of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester. Summary of the invention
[0006] The invention provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester. The detection method has good specificity, high sensitivity and good accuracy, can meet the requirements of relevant laws and regulations and guiding principles, and can provide a simple and reliable detection method for the large-scale production of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester.
[0007] In order to achieve the above technical objectives, the technical solution of the present invention is:
[0008] A method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, comprising the following steps: using high performance liquid chromatography for detection, the chromatographic conditions are: using an octadecylsilane bonded silica gel chromatographic column as the stationary phase, mobile phase A being a phosphoric acid aqueous solution, mobile phase B being acetonitrile, and performing gradient elution;
[0009] The gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 60%-70%; within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 15%-25%; within 35-50 minutes, the volume fraction of mobile phase A is 15%-25%; within 50-60 minutes, the volume fraction of mobile phase A is 60%-70%.
[0010] The octadecylsilane bonded silica gel chromatographic column adopts Waters Xselect HSS T3, with a specification of 4.6 mm×250 mm, and a particle size of the chromatographic column filler is 3.5 μm.
[0011] Wherein, the column temperature of the chromatographic column is 25°C-40°C, preferably, the column temperature of the chromatographic column is 30°C; the flow rate of the chromatographic column is 0.8ml / min-1.2ml / min, preferably, the flow rate of the chromatographic column is 1.0ml / min.
[0012] The mass percentage concentration of phosphoric acid in the mobile phase A is 0.05-0.2%. Preferably, the mass percentage concentration of phosphoric acid in the mobile phase A is 0.1%.
[0013] The elution is performed in a gradient elution mode, and the gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 65%; within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 20%; within 35-50 minutes, the volume fraction of mobile phase A is 20%; within 50-60 minutes, the volume fraction of mobile phase A is 65%.
[0014] The detection wavelength is 205nm-220nm, preferably, the detection wavelength is 210nm.
[0015] The injection volume is 5 μL-20 μL, preferably, the injection volume is 10 μL.
[0016] Among them, the relevant substances include: one or more of impurities B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q (impurities B and impurity C are controlled together, and impurities E and impurity D are controlled together). The structural formula of each impurity is as follows:
[0017]
[0018] Among them, the main component self-control method with correction factor is used to calculate the content of known impurities and unknown impurities of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester. In this field, impurity calculation methods include: internal standard method, external standard method, main component self-control method with correction factor, main component self-control method without correction factor and area normalization method. The main component self-control method with correction factor has the advantage of low detection cost compared with the external standard method. Its steps are generally as follows: prepare blank solution, test solution and main component self-control solution respectively, use the above-mentioned chromatographic conditions, inject samples in sequence for detection, record chromatograms, and calculate the content of each impurity according to the peak area of each impurity, the peak area of the main component self-control and the correction factor of each impurity.
[0019] Among them, the test solution is prepared using pure organic phase as a diluent with a concentration of 1-5 mg / ml. Preferably, acetonitrile is used as a diluent to prepare the test solution with a concentration of 3 mg / ml; the test solution is used and diluted 100-1000 times with a diluent to prepare a main component self-control solution. Preferably, the test solution is used and diluted 1000 times with acetonitrile to prepare a main component self-control solution.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention selects different chromatographic conditions to detect multiple impurities of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, and finally screens out the chromatographic conditions of the present invention. Except for impurities B and impurity C (impurities B and impurity C are enantiomers and cannot be separated in a non-chiral environment), the separation between each impurity peak and between the main component and the adjacent impurity peak can be greater than 1.5; at the same time, each impurity correction factor is obtained according to the linearity and range experiment between each impurity and the main component, and the main component self-comparison method with the correction factor is used to calculate the content of each impurity. The method has good specificity, high sensitivity, and low detection cost, and provides a simple and reliable detection method for the quality control of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1Overlay of HPLC chromatograms of blank solution, system suitability solution and test solution of Example 1;
[0023] Figure 2 The HPLC chromatograms of the blank solution, the main component and the mixed solution of each impurity in Example 3 are superimposed;
[0024] Figure 3 is the HPLC chromatogram of the system suitability solution of Example 4;
[0025] Figure 4 HPLC chromatogram of the system suitability solution of Comparative Example 1;
[0026] Figure 5 This is the HPLC chromatogram of the system suitability solution of Comparative Example 2. DETAILED DESCRIPTION
[0027] The technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0028] Example 1
[0029] The present embodiment provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, comprising the following steps: using a high performance liquid chromatograph (WatersArc HPLC) for detection, the chromatographic conditions are: using Waters Xselect HSS T3 as the stationary phase; mobile phase A is 0.1% phosphoric acid aqueous solution; mobile phase B is acetonitrile; gradient elution is performed, and the gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 65%, within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 20%, within 35-50 minutes, the volume fraction of mobile phase A is 20%, and within 50-60 minutes, the volume fraction of mobile phase A is 65%; the chromatographic column temperature is 30°C; the detection wavelength is 210nm; the flow rate is 1.0ml / min; and the injection volume is 10μL.
[0030] Solution preparation:
[0031] Blank solution: Use acetonitrile as blank solution.
[0032] Test solution: Take an appropriate amount of (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, accurately weigh it, add acetonitrile to dissolve it and dilute it to make a solution containing about 3 mg per 1 ml.
[0033] Main component self-control solution: Accurately measure 0.1 ml of the test solution and quantitatively dilute it to 100 ml with acetonitrile.
[0034] System suitability solution: Accurately weigh appropriate amounts of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester reference substance, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q concentration of 9 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0035] operate:
[0036] Take 10 μL of blank solution, system suitability solution, test solution and main component self-control solution, respectively, and record the chromatogram. The results are as follows: Figure 1 And as shown in Table 1.
[0037] Figure 1 HPLC chromatograms of blank solution, system suitability solution and test solution of Example 1 are overlaid
[0038] Table 2 is the results of the system suitability solution test of Example 1.
[0039] Table 2 System suitability solution test results
[0040] Peak name Retention time min Relative retention time Separation Theoretical plates Impurity G 16.2935 0.62 / 73295 Impurity H 17.8450 0.67 6.24 83465 Impurity F 21.2179 0.80 13.73 129276 Impurity E 21.7272 0.82 2.12 132379 Impurity Q 25.1120 0.95 14.29 188795 <![CDATA[Principal component * > 26.4888 1.00 5.87 208028 Impurity B 27.5492 1.04 4.52 233231 Impurity D 45.4228 1.71 57.36 225155
[0041] Note: The main component refers to (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester.
[0042] It can be seen from Table 1 and Table 2 that this method has good specificity.
[0043] Example 2
[0044] According to the "9101 Validation Guidelines for Analytical Methods" of the "Chinese Pharmacopoeia", pre-validation work was carried out on the key items of the analytical method of this method.
[0045] The detection method is the same as in Example 1.
[0046] Solution preparation:
[0047] Linear solution - 20%: Accurately weigh an appropriate amount of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester reference substance, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester with a concentration of 1.8 μg / ml
[0048] Linear solution - 40%: Accurately weigh an appropriate amount of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester reference substance, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester with a concentration of 3.6 μg / ml
[0049] Linear solution - 100%: Accurately weigh an appropriate amount of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester reference substance, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester with a concentration of 9 μg / ml
[0050] Linear solution - 200%: Accurately weigh appropriate amounts of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester reference substance, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester with a concentration of 18 μg / ml.
[0051] 20% recovery solution: Accurately weigh an appropriate amount of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q reference substances and (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester test sample, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q concentration of about 1.8 μg / ml and a (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml
[0052] 50% recovery solution: Accurately weigh appropriate amounts of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q reference substances and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester test sample, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q concentration of approximately 4.5 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0053] 100% recovery solution: Accurately weigh an appropriate amount of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q reference substances and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester test sample, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q concentration of approximately 9 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0054] 150% recovery solution: Accurately weigh appropriate amounts of impurity B, impurity D, impurity E, impurity F, impurity G, impurity H, impurity Q reference substances and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester test sample, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity B, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q concentration of approximately 13.5 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0055] The measured recovery rates and correction factors of each impurity are shown in Table 3.
[0056] Table 3 Recovery rate and correction factor of each impurity
[0057] Peak name 20% recovery rate 50% recovery rate 100% recovery rate 150% recovery rate Correction Factor Impurity G 109.75 106.24 105.30 100.80 0.86 Impurity H 99.82 103.36 104.60 100.63 0.46 Impurity F 106.94 106.41 105.87 101.67 0.55 Impurity E 114.68 113.72 104.80 112.76 0.70 Impurity Q 108.77 105.22 105.15 101.02 0.92 Impurity B 109.17 106.85 107.00 102.63 1.00 Impurity D 92.08 94.62 109.47 84.41 1.06
[0058] Except for impurities D and E, the recovery rates of other impurities meet the requirements. The solution stability was examined using a 100% recovery solution, and it was found that the content of impurity D gradually decreased, while the content of impurity E gradually increased. Based on the structural analysis of impurities D and E, it was believed that impurity D could be decomposed into impurity E, so impurities D and impurity E were controlled together.
[0059] The recovery rate of impurity E was examined again, and each solution was prepared as follows:
[0060] 10% recovery solution: Accurately weigh an appropriate amount of impurity E and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity E concentration of 0.9 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0061] 50% recovery solution: Accurately weigh an appropriate amount of impurity E and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity E concentration of 4.5 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0062] 100% recovery solution: Accurately weigh an appropriate amount of impurity E and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity E concentration of 9 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0063] 150% recovery solution: Accurately weigh an appropriate amount of impurity E and (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, dissolve them in acetonitrile and quantitatively dilute them to prepare a mixed solution with an impurity E concentration of 13.5 μg / ml and a (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester concentration of 3 mg / ml.
[0064] The recovery rate of the 10% recovery solution was 101.49%, the recovery rate of the 50% recovery solution was 99.89%, the recovery rate of the 100% recovery solution was 100.29%, and the recovery rate of the 150% recovery solution was 100.85%.
[0065] Example 3
[0066] This embodiment provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylate benzyl ester, which is different from Embodiment 1 in that:
[0067] The gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 70%; within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 25%; within 35-50 minutes, the volume fraction of mobile phase A is 25%; within 50-60 minutes, the volume fraction of mobile phase A is 70%.
[0068] The mass percentage concentration of phosphoric acid in mobile phase A is 0.05%.
[0069] The column temperature was 25°C and the column flow rate was 1.2 ml / min.
[0070] The detection wavelength is 205 nm.
[0071] The injection volume was 5 μL.
[0072] Use the test solution and dilute it 100-1000 times with diluent to prepare the main component self-control solution.
[0073] Other testing conditions are the same as those in Example 1.
[0074] The results of the test are as follows Figure 2 The retention time and separation degree of each impurity and main component are shown in Table 4:
[0075] Table 4 Example 3 measurement results
[0076] Peak name Retention time min Relative retention time Separation Theoretical plates Impurity G 11.1216 0.55 / 44490 Impurity H 12.5382 0.62 6.40 52395 Impurity F 15.5866 0.77 13.8 83104 Impurity E 15.8574 0.78 1.5 76976 Impurity Q 19.0631 0.94 13.3 116783 principal component 20.3468 1.00 4.6 133779 Impurity B 21.3362 1.05 4.4 146305 Impurity D 36.6415 1.80 62.8 365463
[0077] Example 4
[0078] This embodiment provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylate benzyl ester, which is different from Embodiment 1 in that:
[0079] The gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 60%; within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 15%; within 35-50 minutes, the volume fraction of mobile phase A is 15%; within 50-60 minutes, the volume fraction of mobile phase A is 60%.
[0080] The mass percentage concentration of phosphoric acid in mobile phase A is 0.2%.
[0081] The column temperature was 40°C and the column flow rate was 0.8 ml / min.
[0082] The detection wavelength is 220 nm.
[0083] The injection volume was 20 μL.
[0084] Use the test solution and dilute it 100-1000 times with diluent to prepare the main component self-control solution.
[0085] Other testing conditions are the same as those in Example 1.
[0086] The results of the test are as follows Figure 3 The retention time and separation degree of each impurity and main component are shown in Table 5:
[0087] Table 5 Example 4 Measurement results
[0088] Peak name Retention time min Relative retention time Separation Theoretical plates Impurity G 16.2935 0.62 / 73295 Impurity H 17.8450 0.67 6.24 83465 Impurity F 21.2179 0.80 13.73 129276 Impurity E 21.7272 0.82 2.12 132379 Impurity Q 25.1120 0.95 14.29 188795 principal component 26.4888 1.00 5.87 208028 Impurity B 27.5492 1.04 4.52 233231 Impurity D 45.4228 1.71 57.36 225155
[0089] Comparative Example 1
[0090] The present embodiment provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, comprising the following steps: using a high performance liquid chromatograph (WatersArc HPLC) for detection, the chromatographic conditions are: using Waters Xselect HSS T3 as the stationary phase; mobile phase A is 0.1% phosphoric acid aqueous solution; mobile phase B is acetonitrile; gradient elution is performed, and the gradient elution program is: within 0-25 minutes, the volume fraction of mobile phase A is 65%, within 25-30 minutes, the volume fraction of mobile phase A gradually changes to 20%, within 30-40 minutes, the volume fraction of mobile phase A is 20%, and within 40.01-45 minutes, the volume fraction of mobile phase A is 65%; the temperature of the chromatographic column is 35°C; the detection wavelength is 210nm; the flow rate is 1.0ml / min; and the injection volume is 10μL. Under this specific implementation method, the peak shape of impurities G and impurity H is not good, and impurities F and impurity E are not successfully separated. The HPLC chromatogram of the system suitability solution of Comparative Example 1 is as follows Figure 4 shown.
[0091] Comparative Example 2
[0092] The present embodiment provides a method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester, comprising the following steps: using a high performance liquid chromatograph (WatersArc HPLC) for detection, the chromatographic conditions are: using Agilent InfinityLab Poroshell 120Phenyl Hexyl as the stationary phase; mobile phase A is 0.1% phosphoric acid aqueous solution; mobile phase B is acetonitrile; gradient elution is performed, and the gradient elution program is: within 0-70 minutes, the volume fraction of mobile phase A gradually changes from 90% to 20%, within 70-80 minutes, the volume fraction of mobile phase A is 20%, and within 80.01-90 minutes, the volume fraction of mobile phase A is 90%; the chromatographic column temperature is 30°C; the detection wavelength is 210nm; the flow rate is 1.0ml / min; and the injection volume is 10μL. Under this specific implementation method, the peak shape and separation degree of each component are not good. The HPLC chromatogram of the system suitability solution of Comparative Example 2 is shown in Figure 5 shown.
[0093] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.
Claims
1. A method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylate, characterized in that The method comprises the following steps: using high performance liquid chromatography to detect, the chromatographic conditions are: using an octadecylsilane bonded silica gel chromatographic column as the stationary phase, mobile phase A being a phosphoric acid aqueous solution, mobile phase B being acetonitrile, and performing gradient elution; The gradient elution program is: within 0-5 minutes, the volume fraction of mobile phase A is 60%-70%; within 5-35 minutes, the volume fraction of mobile phase A gradually changes to 15%-25%; within 35-50 minutes, the volume fraction of mobile phase A is 15%-25%; within 50-60 minutes, the volume fraction of mobile phase A is 60%-70%.
2. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The octadecylsilane bonded silica gel chromatographic column adopts Waters Xselect HSST3, with a specification of 4.6 mm×250 mm, and a particle size of the chromatographic column filler is 3.5 μm.
3. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 2, characterized in that: The column temperature of the chromatographic column is 25° C.-40° C.; the flow rate of the chromatographic column is 0.8 ml / min-1.2 ml / min.
4. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The mass percentage concentration of phosphoric acid in mobile phase A is 0.05-0.2%.
5. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The elution was performed in a gradient elution mode, and the gradient elution program was as follows: within 0-5 minutes, the volume fraction of mobile phase A was 65%; within 5-35 minutes, the volume fraction of mobile phase A gradually changed to 20%; within 35-50 minutes, the volume fraction of mobile phase A was 20%; within 50-60 minutes, the volume fraction of mobile phase A was 65%.
6. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The detection wavelength is 205nm-220nm.
7. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The injection volume is 5μL-20μL.
8. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: The relevant substances include: one or more of impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H and impurity Q. The structural formula of each impurity is as follows: Among them, impurities B and impurity C are controlled together, and impurities E and impurity D are controlled together.
9. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 8, characterized in that: The contents of known and unknown impurities in (3R,4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester were calculated by principal component self-reference method with correction factor.
10. The method for detecting related substances of (3R, 4S)-3-(2-bromoacetyl)-4-ethylpyrrolidine-1-carboxylic acid benzyl ester according to claim 1, characterized in that: Use pure organic phase as diluent to prepare the test solution with a concentration of 1-5 mg / ml; use the test solution and dilute it 100-1000 times with the diluent to prepare the main component self-control solution.