Analysis method for precolumn derivatization of hydrogenated impurities of melogabalin besylate intermediate
The derivatization reaction and high-performance liquid chromatography detection of the melogabarin benzenesulfonate intermediate was solved by pre-column derivatization analysis method, which was difficult to detect the hydrogenated impurity MB-2A in the prior art, achieved efficient and accurate detection effects, and ensured product quality control.
Patent Information
- Application Number
- CN202411790318.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-06
- Publication Date
- 2025-03-18
AI Technical Summary
The prior art is difficult to effectively detect the hydrogenated impurity MB-2A of melogabarin benzenesulfonate intermediate, resulting in the inability to accurately separate and quantify it in subsequent steps, affecting the quality of the finished product.
Using pre-column derivatization analysis method, derivatization reactions were performed using derivatization reagents such as 2,4-dinitrofluorobenzene under alkali catalysis to produce stable derivatives, and then used for detection using high performance liquid chromatography-UV detector.
It realizes efficient separation and accurate quantification of MB-2A in MB-2, improves the sensitivity and accuracy of detection, reduces the detection cost, and ensures the control of product quality.
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Figure CN119643737A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of pharmaceutical detection, and in particular relates to an analytical method for pre-column derivatization of hydrogenated impurities of a melogabalin besylate intermediate. Background Art
[0002] Mirogabalin Besilate is a small molecule drug developed by Daiichi Sankyo Co., Ltd. for the treatment of peripheral neuropathic pain (PNP), including diabetic peripheral neuropathy (DPNP) and postherpetic neuralgia (PHN).
[0003] The current mainstream synthetic route of melogabalin besylate is as follows:
[0004]
[0005] MB-4 is used as the raw material, and MB-3 is generated through hydrogenation reaction, followed by chiral separation to obtain the intermediate MB-2, which is then deacidified to obtain MB-1, which is further hydrolyzed into a salt to obtain melogabalin besylate.
[0006] In the above-mentioned production process, MB-2 is a key intermediate of melogabalin besylate, which is easily over-hydrogenated during the reaction process, resulting in the generation of hydrogenated impurities (MB-2A for short). The physical and chemical properties of this impurity are similar to those of the intermediate MB-2, and it is basically impossible to remove it in subsequent steps. The content of MB-2A impurity will directly affect the quality of the finished product of melogabalin besylate, so it is extremely important to control MB-2A in MB-2. The structure of the MB-2A impurity is as follows;
[0007]
[0008] MB-2A impurities have weak UV absorption, and the sensitivity cannot meet the requirements in conventional liquid-UV systems. For such substances with weak UV absorption, GC, LC-CAD, LC-ELSD, LC-MS and other equipment are usually used for analysis, or a derivatization agent is used for pre-column derivatization reaction to generate substances containing UV absorption, and then HPLC is used for detection and analysis. When using GC, MB-2 samples will degrade at the GC injection port due to its thermal instability. As the number of injections increases, the degradation peak will interfere with the test of MB-2A, resulting in inaccurate results; when using LC-CAD and LC-ELSD instruments, because MB-2A is a semi-volatile substance, the sensitivity in this type of detector is also relatively low and cannot meet the control requirements; although LC-MS can achieve higher sensitivity, its instruments are relatively expensive and the detection cost is high, which is not conducive to the reaction monitoring and release of enterprises.
[0009] At present, there is no report on the relevant technology of the detection method of hydrogenation impurities of melogabalin besylate intermediates. Developing a detection method with good separation and high sensitivity is particularly important for the quality of melogabalin besylate products. Summary of the invention
[0010] The purpose of the present invention is to provide a method for detecting hydrogenated impurities of a melogabalin besylate intermediate with good separation and high sensitivity. The analytical method adopts pre-column derivatization, and the derivatized product is stable, and the content of MB-2A in MB-2 can be effectively separated and accurately quantified.
[0011] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:
[0012] A method for pre-column derivatization of hydrogenated impurities in a melogabalin benzenesulfonate intermediate, wherein the melogabalin benzenesulfonate intermediate is MB-2, and the hydrogenated impurities in the melogabalin benzenesulfonate intermediate are MB-2A.
[0013]
[0014] The analysis method comprises:
[0015] (1) using a derivatization reagent to carry out a derivatization reaction on a melogabalin benzenesulfonate intermediate under the catalysis of a base to obtain a derivatization reaction liquid; the derivatization reagent is o-phthalaldehyde, fluorenylmethyl chloroformate or 2,4-dinitrofluorobenzene; taking 2,4-dinitrofluorobenzene as an example, the derivatization reaction equation of MB-2A is as follows:
[0016]
[0017] (2) The derivatization reaction solution is then detected using a high performance liquid chromatography-UV detector to obtain the content of hydrogenated impurities in the melogabalin besylate intermediate.
[0018] Preferably, the base is at least one of sodium hydroxide, triethylamine, sodium carbonate and sodium bicarbonate, more preferably sodium bicarbonate.
[0019] Preferably, the derivatization reagent is 2,4-dinitrofluorobenzene.
[0020] Preferably, step (1) is specifically implemented as follows: a melogabalin besylate intermediate sample, a solvent, a base, and a derivatization reagent are mixed in a container, dissolved, and reacted at 40-80° C. for 10-60 minutes to obtain a derivatization reaction solution; wherein the feed concentrations of the melogabalin besylate intermediate sample, the base, and the derivatization reagent in the reaction solution are 10-50 mg / 15 mL, 10-100 mg / 15 mL, and 10-100 mg / 15 mL, respectively; and the solvent is a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 40-90%:10-60%.
[0021] As a further preference, in the reaction solution of step (1), the feed concentration of the derivatization reagent is 40-60 mg / 15 mL, and more preferably 50 mg / 15 mL.
[0022] As further preferred, in the reaction solution of step (1), the feed concentration of the base is 40-60 mg / 15 mL, preferably 50 mg / 15 mL.
[0023] As a further preference, in the reaction solution of step (1), the feeding concentration of the melogabalin besylate intermediate sample is 20 mg / mL to 30 mg / 15 mL, preferably 25 mg / 15 mL.
[0024] As further preferred, the derivatization reaction temperature is 50-70°C, more preferably 60°C; the reaction time is 20-50 minutes, more preferably 30 minutes.
[0025] Preferably, the chromatographic conditions of the high performance liquid chromatography in step (2) are as follows: the chromatographic column is a C18 column, a phenyl column, a C8 column or a cyano column, the derivatization reaction solution is diluted to acidity with an acidic diluent before detection to obtain a test solution, the mobile phase includes mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is 0.02-0.10 vol% phosphoric acid aqueous solution, 0.02-0.10 vol% formic acid aqueous solution or 0.02-0.10 vol% diethylamine aqueous solution. It is further preferred that the concentration of the phosphoric acid aqueous solution is 0.05 vol%; the concentration of the formic acid aqueous solution is 0.05 vol%; and the concentration of the diethylamine aqueous solution is 0.05 vol%.
[0026] As a further preference, in step (2), the acidic substance in the acidic diluent is potassium dihydrogen phosphate, sodium dihydrogen phosphate, acetic acid or trifluoroacetic acid, and the solvent is a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 40-90%:10-60%. It is further preferred that the feed ratio of potassium dihydrogen phosphate or sodium dihydrogen phosphate to the solvent is 1-3g:1L, and it is further preferred that it is 2g:1L; the feed volume ratio of acetic acid or trifluoroacetic acid to the solvent is 1-3:1000, and it is further preferred that it is 2:1000.
[0027] As a further preference, the chromatographic column is a C18 column. It is further preferred that the C18 column has a filler particle size of 3.5-5 μm, most preferably 5 μm, an inner diameter of 3.0-4.6 mm, most preferably 4.6 mm, and a column length of 100-250 mm, most preferably 250 mm.
[0028] As a further preference, the mobile phase B is an aqueous formic acid solution with a volume percentage concentration of 0.02% to 0.10%, and a further preferred concentration of 0.05%.
[0029] As a further preference, during detection, the column temperature is 25-35°C, and more preferably 30°C; the detection wavelength is 320-370nm, and more preferably 355nm; the injection volume is 5-30μL, and more preferably 10μL; the total flow rate of the mobile phase is 0.8-1.2mL / min, and more preferably 1.0mL / min.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] (1) The analytical method of the present invention can effectively separate and accurately quantify the content of MB-2A in MB-2 with high accuracy, low detection limit and strong method robustness.
[0032] (2) The establishment of the method for detecting hydrogenated impurities in melogabalin besylate described in the present invention can better achieve quality control of the intermediate MB-2 and the finished product melogabalin besylate, provide a simple, accurate and efficient analysis method for product research and development and production, improve the quality of the final product, and ensure its safety and effectiveness in clinical use. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Figure 1 This is the blank solution chromatogram under normal derivatization conditions of Example 1;
[0034] Figure 2 This is the chromatogram of the test sample solution under normal derivatization conditions in Example 1;
[0035] Figure 3 This is the chromatogram of the detection limit solution with a relative concentration of 0.02% in Example 3;
[0036] Figure 4 It is the linear curve graph of MB-2 of Example 3;
[0037] Figure 5 It is the linear curve diagram of Example 3 MB-2A;
[0038] Figure 6This is the chromatogram of the selected sample solution using a Waters Symmetry C18 chromatographic column (250 mm×4.6 mm, 5 μm) in Example 4;
[0039] Figure 7 This is the chromatogram of the control solution of Example 5;
[0040] Figure 8 This is the chromatogram of the sample solution in Example 5;
[0041] Fig. 9 This is a chromatogram of the mixed solution of Example 6;
[0042] Fig.10 This is the chromatogram of the test solution (batch 3) of Example 7. DETAILED DESCRIPTION
[0043] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.
[0044] Example 1: Durability reaction test of the derivatization method
[0045] 1.1 Chromatographic conditions
[0046]
[0047] Gradient Program:
[0048] Time (min) Mobile phase A (%) Mobile phase B (%) 0 87 13 12 87 13 13 100 0 20 100 0 21 87 13 30 87 13
[0049] 1.2 Solution preparation
[0050] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0051] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0052] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0053] ④. Test solution (normal conditions): Weigh 25 mg of MB-2 test sample and place it in a 50 mL volumetric flask. Add 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution respectively. After dissolution, react at 60°C for 30 min, cool to room temperature, and dilute to the scale with a diluent.
[0054] Table 1-1 (Durability conditions for derivatization reactions)
[0055]
[0056]
[0057] 1.3 Test results
[0058] Table 1-2 (Durability reaction results of the derivatization method)
[0059]
[0060] 1.4 Experimental Summary
[0061] The sampling amount of the test sample, the reaction time, the reaction temperature, the amount of the derivatizing agent, and the amount of the alkali are changed. The test results under the changed conditions are basically consistent with those under normal conditions. The derivatization method of the present invention has good durability.
[0062] Example 2: Investigation of Solution Stability of Derivative Products
[0063] 2.1 Chromatographic conditions
[0064]
[0065]
[0066] Gradient Program:
[0067] Time (min) Mobile phase A (%) Mobile phase B (%) 0 87 13 12 87 13 13 100 0 20 100 0 21 87 13 30 87 13
[0068] 2.2 Solution preparation
[0069] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0070] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0071] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0072] ④. Test solution: Weigh 25 mg of MB-2 test sample into a 50 mL volumetric flask, add 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution, respectively. After dissolving, react at 60°C for 30 minutes, cool to room temperature, and dilute to the mark with a diluent. The test solution was tested at 0h and 7h at room temperature.
[0073] 2.3 Test results
[0074] Table 2-1 (Solution stability test results)
[0075] Room temperature storage time MB-2 peak area MB-2A peak area MB-2A peak area relative average deviation 0h 13075176 17314 / 7h 13177008 18006 2.0％
[0076] 2.4 Experimental Summary
[0077] The derivative product is stable within 7h.
[0078] Example 3: Detection limit and linearity
[0079] 3.1 Chromatographic conditions
[0080]
[0081] Gradient Program:
[0082] Time (min) Mobile phase A (%) Mobile phase B (%) 0 87 13 12 87 13 13 100 0 20 100 0 21 87 13 30 87 13
[0083] 3.2 Solution preparation
[0084] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0085] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0086] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0087] ④、Control stock solution①: Weigh 25 mg of MB-2 and MB-2A reference substances respectively and place them in a 50 mL volumetric flask, add 20% acetonitrile aqueous solution to dissolve and make up to volume;
[0088] ⑤. Control stock solution ②: Pipette 1.0 mL of control stock solution ① into a 100 mL volumetric flask and dilute to volume with 50 vol% acetonitrile aqueous solution;
[0089] ⑥. Control solution ①: Pipette 1.0 mL of control stock solution ②, 4.0 mL of acetonitrile, 5.0 mL of sodium bicarbonate solution, and 5.0 mL of 2,4-dinitrofluorobenzene solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0090] ⑦. Control solution ②: Pipette 5.0 mL of control stock solution ②, 5.0 mL of sodium bicarbonate solution, and 5.0 mL of 2,4-dinitrofluorobenzene solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0091] ⑧、Control solution ③: Pipette 1.0mL of control stock solution ①, 4.0mL of acetonitrile, 5.0mL of sodium bicarbonate solution, and 5.0mL of 2,4-dinitrofluorobenzene solution into a 50mL volumetric flask, react at 60℃ for 30min, cool to room temperature, and dilute to the mark with a diluent;
[0092] ⑨. Control solution ④: Pipette 5.0 mL of control stock solution ①, 5.0 mL of sodium bicarbonate solution, and 5.0 mL of 2,4-dinitrofluorobenzene solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0093] 3.3 Test results
[0094] Table 3-1 Detection limit results
[0095] Concentration (ug / mL) S / N of MB S / N of MB-2A 0.1 53 29
[0096] Table 3-2 Linearity results
[0097] name Linear equations Correlation coefficient MB-2 y=30035x-6401 0.9999 MB-2A y=28692x-8686 0.9998
[0098] 3.4 Experimental Summary
[0099] 1. When the relative test sample concentration is 0.02%, the S / N value is greater than 3, and the sensitivity result meets the requirements;
[0100] 2. When the concentration of MB-2A is 0.1ug / mL~10.0ug / mL, the correlation coefficient r>0.999, and the linearity is good.
[0101] Example 4: Chromatographic column durability selection
[0102] 4.1 Chromatographic conditions
[0103]
[0104] Gradient Program:
[0105] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 2 75 25 22 100 0 30 100 0 31 75 25 40 75 25
[0106] 4.2 Solution preparation
[0107] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0108] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0109] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0110] ④, reference stock solution ②: weigh 25 mg of each MB-2 and MB-2A reference substance and place them in a 50 mL volumetric flask, add 20 vol% acetonitrile aqueous solution to dissolve and make up to volume to obtain reference stock solution ①; pipette 1.0 mL of reference stock solution ① into a 100 mL volumetric flask, make up to volume with 50 vol% acetonitrile aqueous solution to obtain reference stock solution ②;
[0111] ⑤、Sampling solution: Take 25 mg of the test sample, add 5.0 mL of control stock solution ②, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, and after dissolution, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0112] 4.3 Test results
[0113] Chromatographic columns Separation between MB-2 and MB-2A AgilentZORBAXExtend-C18250mm×4.6mm, 5μm 6.34 WatersSymmetryC18250mm×4.6mm, 5μm 6.29 ThermoSyncrinisC18250mm×4.6mm, 5μm 2.32 AgilentPoroshellEC-C18250mm×4.6mm, 5μm 4.78 AgilentSB-C18250mm×4.6mm, 5μm 2.84 WatersXBridgeC18250mm×4.6mm, 5μm 4.98
[0114] 4.4 Experimental Summary
[0115] Using the commonly used C18 chromatographic column on the market, good separation can be obtained, indicating that this method has good durability.
[0116] Example 5: Accuracy test
[0117] 5.1 Chromatographic conditions
[0118]
[0119] Gradient Program:
[0120] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 2 75 25 22 100 0 30 100 0 31 75 25 40 75 25
[0121] 5.2 Solution preparation
[0122] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0123] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0124] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0125] ④, reference stock solution ②: weigh 25 mg of each MB-2 and MB-2A reference substance into a 50 mL volumetric flask, add 20 vol% acetonitrile aqueous solution to dissolve and dilute to obtain reference stock solution ①; pipette 1.0 mL of reference stock solution ① into a 100 mL volumetric flask, dilute to volume with 50 vol% acetonitrile aqueous solution to obtain reference stock solution ②;
[0126] ⑤. Control solution: pipette 5.0 mL of control stock solution ②, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0127] ⑥ Test solution: weigh 25 mg of the test sample and place it in a 50 mL volumetric flask. Add 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution. After dissolving, react at 60 °C for 30 min, cool to room temperature, and dilute to the mark with a diluent.
[0128] ⑦. Sample solution: weigh 25 mg of the sample and place it in a 50 mL volumetric flask. Add 5.0 mL of control stock solution ②, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution. After dissolving, react at 60 °C for 30 min, cool to room temperature, and dilute to the mark with a diluent.
[0129] 5.3 Test results
[0130] Control peak area Peak area of test product Peak area Recovery rate 12005 4403 17167 106.3％
[0131] 5.4 Experimental Summary
[0132] The recovery rate is between 80% and 120%, and the recovery rate result meets the requirements.
[0133] Example 7: Specificity test
[0134] 7.1 Chromatographic conditions
[0135]
[0136] Gradient Program:
[0137]
[0138]
[0139] 7.2 Solution preparation
[0140] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0141] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0142] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0143] ④, mixed stock solution ②: weigh 25 mg each of MB-2, MB-2A, MB-2B, MB-2C, MB-2D, MB-2E and MB-4 and place them in a 50 mL volumetric flask, add 90 vol% acetonitrile aqueous solution to dissolve and dilute to obtain mixed stock solution ①; pipette 1.0 mL of mixed stock solution ① into a 100 mL volumetric flask, dilute to volume with 50 vol% acetonitrile aqueous solution to obtain mixed stock solution ②;
[0144] ⑤. Mixed solution: pipette 5.0 mL of mixed stock solution ②, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0145] 6.3 Test results
[0146] MB-2A and MB-2 separation 7.2
[0147] 6.4 Experimental Summary
[0148] According to their chemical structures, impurities MB-2B, MB-2C, MB-2D, MB-2E and MB-4 do not participate in the derivatization reaction according to theoretical analysis. Actual test results show that they do not participate in the derivatization reaction either. The specificity results meet the requirements.
[0149] Example 7: Test sample detection
[0150] 7.1 Chromatographic conditions
[0151]
[0152] Gradient Program:
[0153]
[0154]
[0155] 7.2 Solution preparation
[0156] ①, 2,4-Dinitrofluorobenzene solution: weigh 1.0 g of 2,4-Dinitrofluorobenzene and place it in a 100 mL volumetric flask, add acetonitrile to dissolve and dilute to the scale;
[0157] ②, Sodium bicarbonate solution: weigh 1.0g of sodium bicarbonate and place it in a 100mL volumetric flask, add water to dissolve and dilute to the scale;
[0158] ③. Blank solution: pipette 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0159] ④, reference stock solution ②: weigh 25 mg of each MB-2 and MB-2A reference substance into a 50 mL volumetric flask, add 20 vol% acetonitrile aqueous solution to dissolve and dilute to obtain reference stock solution ①; pipette 1.0 mL of reference stock solution ① into a 100 mL volumetric flask, dilute to volume with 50 vol% acetonitrile aqueous solution to obtain reference stock solution ②;
[0160] ⑤. Control solution (0.10%): pipette 5.0 mL of control stock solution ②, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution into a 50 mL volumetric flask, react at 60°C for 30 min, cool to room temperature, and dilute to the mark with a diluent;
[0161] ⑥ Test solution: weigh 25 mg of the test sample and place it in a 50 mL volumetric flask. Add 5.0 mL of acetonitrile, 5.0 mL of 2,4-dinitrofluorobenzene solution, and 5.0 mL of sodium bicarbonate solution respectively. After dissolution, react at 60 °C for 30 min, cool to room temperature, and dilute to the mark with a diluent.
[0162] 7.3 Test results
[0163] Batch 1 Batch 2 Batch 3 Batch 4 2.0％ 0.43％ 0.03％ 0.04％
[0164] 7.4 Test Summary
[0165] Batch 3 and Batch 4 were used to control the impurity MB-2A after the method was established. The test results in the samples were all less than 0.10%, which met the ICH requirements for impurity quality control.
Claims
1. A method for analyzing the hydrogenated impurities of a melogabalin benzenesulfonate intermediate by pre-column derivatization, wherein the melogabalin benzenesulfonate intermediate is MB-2, and the hydrogenated impurities in the melogabalin benzenesulfonate intermediate are MB-2A. Features The analysis method comprises: (1) using a derivatization reagent to carry out a derivatization reaction on a melogabalin benzenesulfonate intermediate under the catalysis of a base to obtain a derivatization reaction liquid; the derivatization reagent is o-phthalaldehyde, fluorenyl methyl chloroformate or 2,4-dinitrofluorobenzene; (2) The derivatization reaction solution is then detected using a high performance liquid chromatography-UV detector to obtain the content of hydrogenated impurities in the melogabalin besylate intermediate.
2. The analysis method according to claim 1, characterized in that: The base is at least one of sodium hydroxide, triethylamine, sodium carbonate and sodium bicarbonate.
3. The analysis method according to claim 1, characterized in that: Step (1) is specifically implemented as follows: a melogabalin besylate intermediate sample, a solvent, a base, and a derivatization reagent are mixed in a container, dissolved, and reacted at 40-80° C. for 10-60 minutes to obtain a derivatization reaction solution; wherein the feed concentrations of the melogabalin besylate intermediate sample, the base, and the derivatization reagent in the reaction solution are 10-50 mg / 15 mL, 10-100 mg / 15 mL, and 10-100 mg / 15 mL, respectively; and the solvent is a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 40-90%:10-60%.
4. The analysis method according to claim 3, characterized in that: In the reaction solution of step (1), the feed concentration of the derivatization reagent is 40-60 mg / 15 mL, preferably 50 mg / 15 mL; the feed concentration of the base is 40-60 mg / 15 mL, preferably 50 mg / 15 mL; the feed concentration of the melogabalin besylate intermediate sample is 20 mg / mL-30 mg / 15 mL, preferably 25 mg / 15 mL.
5. The analysis method according to claim 1, characterized in that: The derivatization reaction temperature is 50-70°C, preferably 60°C; the reaction time is 20-50 minutes, preferably 30 minutes.
6. The analysis method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography in step (2) are as follows: the chromatographic column is a C18 column, a phenyl column, a C8 column or a cyano column; before detection, the derivatization reaction solution is diluted to acidity with an acidic diluent to obtain a test solution; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is acetonitrile; the mobile phase B is a 0.02-0.10 vol% phosphoric acid aqueous solution (preferably a 0.05 vol% phosphoric acid aqueous solution), a 0.02-0.10 vol% formic acid aqueous solution (preferably a 0.05 vol% formic acid aqueous solution) or a 0.02-0.10 vol% diethylamine aqueous solution (preferably a 0.05 vol% diethylamine aqueous solution).
7. The analysis method according to claim 1, characterized in that: In step (2), the acidic substance in the acidic diluent is potassium dihydrogen phosphate, sodium dihydrogen phosphate, acetic acid or trifluoroacetic acid, and the solvent is a mixed solvent of acetonitrile and water, wherein the volume ratio of acetonitrile to water is 40-90%:10-60%.
8. The analysis method according to claim 7, characterized in that: The feed ratio of potassium dihydrogen phosphate or sodium dihydrogen phosphate to the solvent is 1-3 g:1 L, preferably 2 g:1 L; the feed volume ratio of acetic acid or trifluoroacetic acid to the solvent is 1-3:1000, preferably 2:1000.
9. The analysis method according to claim 1, characterized in that: In step (2), the chromatographic column is a C18 column; preferably, the C18 column packing particle size is 3.5-5 μm (preferably 5 μm), the inner diameter is 3.0-4.6 mm (preferably 4.6 mm), and the column length is 100-250 mm (preferably 250 mm).
10. The analysis method according to claim 9, characterized in that: During the detection of step (2), the column temperature is 25-35°C, preferably 30°C; the detection wavelength is 320-370nm, preferably 355nm; the injection volume is 5-30μL, preferably 10μL; the total flow rate of the mobile phase is 0.8-1.2mL / min, preferably 1.0mL / min.