Method for detecting melogabalin besylate and isomer impurities thereof

Abstract

The invention discloses a method for detecting melogabalin besylate and isomer impurities of melogabalin besylate. According to the method, liquid chromatography is adopted, an FXAA reagent and a sample to be tested are subjected to a reaction under the alkaline condition to obtain a solution to be detected, then the solution to be detected is subjected to liquid chromatography separation, the structural formula of the FXAA reagent is # imgabs0 #, R is selected from-CH (CH3) 2,-CH2CH2SCH3 or-C6H5, and the structural formula of the FXAA reagent is as shown in the specification, the structural formula of the FXAA reagent is as shown in the specification, and the structural formula of the FXAA reagent is as shown in the specification, the structural formula of the FXAA reagent is as shown in the specification, the structural formula of the FXAA reagent is as shown in the specification, and the structural formula of the FXAA reagent is as shown in the specification. The test sample comprises melogabalin besylate and isomer impurities thereof, and the liquid chromatography adopts a reversed-phase chromatographic column. According to the method, melogabalin besylate and three isomer impurities thereof can be safely and effectively separated and respectively detected, the detection sensitivity, repeatability and durability are good, and a powerful guarantee is provided for quality control of the medicine.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly to a method for detecting megalamine benzenesulfonate and its isomeric impurities. Background Art

[0002] The raw material of megalamine exists in the form of benzenesulfonate, and there are few conjugated groups in the chemical structure, resulting in weak ultraviolet absorption; the compound has obvious acidic and basic groups and is relatively polar; there is a chiral center in the compound, and there are also cis-trans isomers, with poor separation of isomers. The chemical structural formula of megalamine benzenesulfonate is as follows:

[0003] It is known that in the preparation process, three impurities with known structures will be introduced, including its enantiomers and diastereoisomers, and their structural formulas are as follows: Diastereoisomer 1 (Impurity A) is Enantiomer (Impurity B) is Diastereoisomer 2 (Impurity C) is

[0004] Isomers refer to compounds with the same molecular formula but different structures. Due to their different chemical structures, their physical, chemical properties or toxicity properties vary greatly. In the process of developing the synthesis process of the raw material drug, the control of isomers in the finished product is one of the important contents of quality research. Megalamine benzenesulfonate will introduce a chiral center during the reaction process, and may introduce isomeric impurities with three configurations of (1R,5S,6S), (1S,5R,6R), and (1S,5R,6S) in megalamine (the structural formulas are as above). According to the quality control requirements for megalamine, the control limit of isomeric impurities in the active ingredient shall not exceed 0.5%.

[0005] Patent CN 113740471B discloses a method for detecting milobagrine benzenesulfonate and its enantiomers by high performance liquid chromatography. The applicant tried the method disclosed in Patent CN 113740471B to separate megalamine benzenesulfonate and its enantiomers. The experimental results showed that the peak shape was not sharp and there was tailing. The separation effect between megalamine benzenesulfonate (Formula I) and its enantiomer (Impurity B) was not good, and diastereoisomer 1 (Impurity A) and diastereoisomer 2 (Impurity C) did not show peaks.

[0006] Patent CN 117388402 A discloses a method for detecting milobagrine benzene sulfonate and its isomers by gas chromatography. The detection is carried out using a gas chromatography instrument, with a fused silica capillary column filled with β-cyclodextrin as the chiral chromatographic column, a split ratio of 1:1 - 20:1, and the sample is dissolved completely with a solvent (DMSO, DMF, DMA or sulfolane) before injection. However, this chromatographic column is expensive, and when salts are dissolved and directly injected, it causes great damage to the chromatographic column, resulting in high detection costs. At the same time, the solvents used in gas chromatography are highly toxic, polluting the environment and endangering the health of experimental personnel.

[0007] How to analyze and detect milogabrine benzene sulfonate and its isomeric impurities in a safer way, and then control the impurity content and quality control of this drug is an urgent problem to be solved. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a method for detecting milogabrine benzene sulfonate and its isomeric impurities in view of the shortcomings and deficiencies of the prior art. This method can safely and effectively separate and detect milogabrine benzene sulfonate and its three isomeric impurities respectively, and has good detection sensitivity, repeatability and durability, providing a strong guarantee for the quality control of this drug.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for detecting milogabrine benzene sulfonate and its isomeric impurities, using liquid chromatography. The method first reacts the FXAA reagent with the test sample under alkaline conditions to obtain a test solution, and then separates the test solution by liquid chromatography. The structural formula of the FXAA reagent is wherein, R is selected from -CH(CH3)2, -CH3, -CH2Ph, -CH2CH(CH3)2, -CH2CH2SCH3 or -C6H5; the test sample includes milogabrine benzene sulfonate and its isomeric impurities, and the liquid chromatography uses a reverse-phase chromatographic column.

[0011] In the present invention, Ph refers to a phenyl group.

[0012] Milogabrine benzene sulfonate usually contains three isomeric impurities A, B and C. In the prior art, it is difficult to effectively separate the three isomers when separating them by liquid chromatography. The inventors of the present application found through research that by first derivatizing the test sample with the FXAA reagent having the above specific structural formula, the active C-H bond at the red circle of the FXAA reagent will react with the amino group on milogabrine benzene sulfonate under alkaline conditions The reaction is carried out, and then a chiral carbon atom is introduced at the * of the FXAA reagent. In addition to the chiral center of melogabalin benzenesulfonate itself, the derivatization reaction product also introduces a new chiral center here, and the FXAA reagent also adds a benzene ring structure to the structure of melogabalin benzenesulfonate, so that its ultraviolet absorption is enhanced, the polarity is reduced, and the retention is enhanced, which is also conducive to the separation of the four. The products of melogabalin benzenesulfonate, impurity A, impurity B and impurity C after the derivatization reaction are due to the presence of isomers and because a new chiral center is introduced, and after the applicant explores the conditions of liquid chromatography, after using a reverse phase chromatographic column as a stationary phase, they can be effectively separated. In the prior art, for the separation of isomers, a normal phase chromatographic column is used. However, the inventor of the present application has found through research that in the detection system of the present application, a normal phase chromatographic column is difficult to be effectively separated, while a reverse phase chromatographic column can be effectively separated and can be accurately quantified.

[0013] In some embodiments, R is -CH(CH3)2.

[0014] In some embodiments, the reverse phase chromatography column uses octadecylsilane bonded silica gel as a filler and can be of any size, preferably 4.6×250 mm, 3 μm or 4.6×100 mm, 2.7 μm or a chromatography column with equivalent performance.

[0015] In some embodiments, the mobile phase of the liquid chromatography comprises an aqueous solution of triethylamine and methanol.

[0016] In some embodiments, the volume ratio of the aqueous solution of triethylamine to methanol is 35:50-70.

[0017] In some embodiments, in the aqueous solution of triethylamine, the volume of triethylamine accounts for 0.2%-0.8% of the volume of water.

[0018] In some embodiments, the pH value of the aqueous solution of triethylamine is 2-7.

[0019] In some embodiments, the mobile phase of the liquid chromatography further comprises acetonitrile, and the volume ratio of the aqueous solution of triethylamine, methanol and acetonitrile is 35:40-65:1-25.

[0020] In some embodiments, the alkaline condition is achieved by adding a base, and the base is selected from a combination of one or more of sodium bicarbonate and potassium bicarbonate.

[0021] In some embodiments, the reaction time is 0.5-2 h.

[0022] In some embodiments, the flow rate of the mobile phase of the liquid chromatography is 0.8-1.2 ml / min.

[0023] In some embodiments, the column temperature of the chromatographic column in the liquid chromatography is 30 - 50 °C.

[0024] In some embodiments, the detection wavelength in the liquid chromatography is 339 nm.

[0025] In some embodiments, the elution time of the mobile phase in the liquid chromatography is 50 - 90 min.

[0026] In some embodiments, the injection volume of the test solution is 10 - 50 μL.

[0027] In some embodiments, the isomeric impurities include impurity A impurity B and impurity C

[0028] In some embodiments, the resolution between megalol gabapentin benzenesulfonate and impurity A is 4 - 4.5, the resolution between megalol gabapentin benzenesulfonate and impurity B is 1.6 - 1.8, and the resolution between impurity B and impurity C is 2.6 - 3.1.

[0029] In some embodiments, the concentration of the test solution is 4 mg / ml - 6 mg / ml.

[0030] In some embodiments, the molar ratio of the FXAA reagent to the test sample is 2:1 - 10:1.

[0031] In some embodiments, the FXAA reagent is dissolved in acetone to obtain an acetone solution of FXAA; the test sample is dissolved in an aqueous sodium bicarbonate solution, the acetone solution of FXAA is added to the aqueous sodium bicarbonate solution, and an additional aqueous sodium bicarbonate solution is added for the reaction. The aqueous sodium bicarbonate solution is used for dissolution to neutralize the acid in the drug and facilitate the dissolution of the drug. The subsequent additional addition of the aqueous sodium bicarbonate solution is to provide an alkaline environment for the reaction and facilitate the derivatization reaction.

[0032] In some embodiments, the molar concentration of the aqueous sodium bicarbonate solution is 0.5 - 2 mol / L.

[0033] Due to the implementation of the above technical solutions, the present invention has the following advantages compared with the prior art:

[0034] In this application, a specific FXAA reagent is used to derivatize the test sample. The active C-H bond of the FXAA reagent will react with the amino group on megalol gabapin besylate under alkaline conditions, thereby introducing chiral carbon atoms into the FXAA reagent. In addition to the chiral center of megalol gabapin besylate itself, new chiral centers are introduced here, which is conducive to the separation of megalol gabapin besylate from its isomers.

[0035] In this application, the derivatization reagent FXAA is used to achieve good separation effect of megalol gabapin and its salt forms through a conventional reversed-phase column. Moreover, the method has high sensitivity, uses a conventional chromatographic column, has low detection cost, and is an efficient detection method. This method improves the analysis and detection method of the optical isomers of megalol gabapin or its salts, and is of great significance for the quality analysis, quality control of this drug, and improving the safety and effectiveness of the drug.

[0036] The FXAA reagent can react with megalol gabapin and its isomers under alkaline conditions. Due to the addition of a benzene ring structure to the megalol gabapin structure, its ultraviolet absorption is enhanced, the polarity is reduced, and the retention is enhanced. By introducing new chiral centers of FXAA, megalol gabapin and its three isomers can be effectively separated and accurately quantified. Description of the Drawings

[0037] Figure 1 It is the chromatogram of the blank solution (containing FXAA) in Example 1;

[0038] Figure 2 It is the chromatogram of the mixed solution in Example 1;

[0039] Figure 3 It is the chromatogram of the mixed solution in Comparative Example 1;

[0040] Figure 4 It is the chromatogram of the mixed solution in Comparative Example 3;

[0041] Figure 5 It is the chromatogram of the mixed solution in Comparative Example 8;

[0042] Figure 6 It is the chromatogram of the mixed solution in Comparative Example 3. Detailed Description of the Invention

[0043] The technical solutions of the present invention will be described in detail below in conjunction with specific embodiments, so that those skilled in the art can better understand and implement the technical solutions of the present invention, but the present invention is not limited to the scope of the described examples.

[0044] Unless otherwise specified, the ratio of different solvents in the mobile phase is the volume ratio.

[0045] Example 1

[0046] This embodiment provides a method for detecting megalolabalin besylate and its isomeric impurities, which is as follows:

[0047] The structural formula of the derivatization reagent FXAA is as follows:

[0048] Wherein R is -CH(CH3)2.

[0049] FXAA acetone solution: Take 50 mg of FXAA, place it in a 10 ml volumetric flask, dissolve it with acetone and dilute to the mark, shake well to obtain.

[0050] Preparation of mobile phase:

[0051] 0.5% triethylamine solution (pH 3.0): Take 1 L of water, 5 ml of triethylamine, mix well and adjust the pH to 3.0 with phosphoric acid;

[0052] Mobile phase: Take 700 ml of 0.5% triethylamine solution (pH 3.0) and 1300 ml of methanol, mix well and ultrasonicate to obtain.

[0053] Preparation of solutions:

[0054] Blank solution: Take 0.2 ml of 1 mol / L sodium bicarbonate solution, 1 ml of FXAA acetone solution, 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10 ml volumetric flask, mix well, after reacting in a 40°C water bath for 1 hour, add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute to the mark with the mobile phase.

[0055] Megalolabalin besylate sample: Take 50 mg of megalolabalin besylate sample, place it in a 10 ml volumetric flask, dissolve it ultrasonically with 1 mol / L sodium bicarbonate solution and dilute to the mark; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10 ml volumetric flask, mix well, after reacting in a 40°C water bath for 1 hour, add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute to the mark with the mobile phase.

[0056] Mixed impurity stock solution: Take about 5 mg of each impurity (impurity B, impurity A, impurity C), place them in a 10 ml volumetric flask, dissolve them ultrasonically with 1 mol / L sodium bicarbonate solution and dilute to the mark.

[0057] Mixed solution: Take 50 mg of megalolabalin besylate sample, place it in a 10 ml volumetric flask, measure 0.5 ml of the mixed impurity stock solution, dissolve it ultrasonically with 1 mol / L sodium bicarbonate solution and dilute to the mark; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10 ml volumetric flask, mix well, after reacting in a 40°C water bath for 1 hour, add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute to the mark with the mobile phase.

[0058] Chromatographic conditions:

[0059] Chromatographic column: Octadecylsilyl silica gel as filler (4.6×250mm, 3μm)

[0060] Mobile phase: 0.5% triethylamine solution (pH adjusted to 3.0 with phosphoric acid) - methanol (volume ratio 35 - 65)

[0061] Flow rate: 1.0 ml / min

[0062] Column temperature: 40°C

[0063] Injection volume: 30 μl

[0064] Detection wavelength: 339 nm

[0065] Isocratic elution for 60 minutes.

[0066] Results: The chromatogram of the blank solution (containing FXAA) is as Figure 1 shown, and the chromatogram of the mixed solution is as Figure 2 shown, where Figure 2 the first eluting peak is the derivatizing reagent FXAA, and the megalolabalin besylate sample and the three impurities elute later, with obvious separation.

[0067] Under these chromatographic conditions, the retention times of the diastereomeric impurity (Impurity A) is 19.622 minutes, megalolabalin besylate is 22.089 minutes, the enantiomeric impurity (Impurity B) is 23.143 minutes, and the diastereomeric impurity (Impurity C) is 25.087 minutes. The resolution of Impurity A, Impurity B, and Impurity C are 4.09, 1.63, and 2.82 respectively; indicating that megalolabalin besylate can be effectively separated from its enantiomeric and diastereomeric impurities, and the method has good specificity and selective separation.

[0068] Example 2

[0069] Example 2 provides a method for detecting megalolabalin besylate and its isomeric impurities, which is basically the same as Example 1, except that: the size of the chromatographic column is adjusted to 4.6×100mm, 2.7μm.

[0070] The chromatographic results show that the resolutions of Impurity A, Impurity B, and Impurity C are 4.33, 1.75, and 2.94 respectively. It indicates that megalolabalin besylate can be effectively separated from its enantiomeric and diastereomeric impurities.

[0071] Example 3 (Method sensitivity)

[0072] Stock solution of impurity A: Weigh accurately 20 mg of reference substance of impurity A, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute to the mark, then shake well.

[0073] Stock solution of impurity B: Weigh accurately 20 mg of reference substance of impurity B, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute to the mark, then shake well.

[0074] Stock solution of impurity C: Weigh accurately 20 mg of reference substance of impurity C, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute to the mark, then shake well.

[0075] Stock solution of meloxicam barin benzenesulfonate: Weigh accurately 20 mg of reference substance of meloxicam barin benzenesulfonate, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute to the mark, then shake well.

[0076] Dilute the above stock solutions step by step, derivatize and then inject for analysis. When the ratio of signal to noise is at least 10:1, it is the limit of quantitation; when the ratio of signal to noise is at least 3:1, it is the limit of detection.

[0077] The derivatization method, derivatization reagent, injection analysis method and chromatographic conditions are the same as those in Example 1.

[0078] The results are as follows:

[0079] The limit of quantitation concentration of impurity A is 1.858 μg / ml. Inject continuously for 6 needles, the RSD value (relative standard deviation value) of the peak area is 8.5%, and the average value of S / N (signal-to-noise ratio) is 16.9 (greater than 10:1, meeting the signal requirement for the limit of quantitation); the limit of detection concentration is 0.5574 μg / ml, and the average value of S / N at this time is 6.7 (greater than 3:1, meeting the signal requirement for the limit of detection).

[0080] The limit of quantitation concentration of meloxicam barin benzenesulfonate is 1.799 μg / ml. Inject continuously for 6 needles, the RSD value of the peak area is 2.1%, and the average value of S / N is 19.1; the limit of detection concentration is 0.5396 μg / ml, and the average value of S / N is 7.6.

[0081] The limit of quantitation concentration of impurity B is 1.717 μg / ml. Inject continuously for 6 needles, the RSD value of the peak area is 6.2%, and the average value of S / N is 13.3; the limit of detection concentration is 0.5152 μg / ml, and the average value of S / N is 5.5.

[0082] The limit of quantitation concentration of impurity C is 1.599 μg / ml. Inject continuously for 6 needles, the RSD value of the peak area is 8.6%, and the average value of S / N is 10.3; the limit of detection concentration is 0.4797 μg / ml, and the average value of S / N is 3.9.

[0083] It can be seen that the detection method of the present application has good sensitivity and low detection limit.

[0084] Example 4 (Method Repeatability)

[0085] Solution Preparation:

[0086] Repeatability Solution: Take about 50 mg of melegabalin benzenesulfonate sample and place it in a 10 ml volumetric flask. Add 1 mol / L sodium bicarbonate solution and dissolve it by ultrasonic wave and dilute it to the scale. Take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, and 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10 ml volumetric flask, mix well, react in a 40 °C water bath for 1 hour, then add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute it to the scale with the mobile phase. Prepare 6 portions in parallel.

[0087] Use the detection method of Example 1 to detect the 6 portions respectively. The derivatization method, derivatization reagent, injection analysis method, and chromatographic conditions are the same as those in Example 1. The results are shown in Table 1 below.

[0088] Table 1

[0089]

[0090] From the above results, it can be seen that for the 6 portions of the test solution injected continuously, the relative standard deviations RSDs among the six groups of the detected amounts of melegabalin benzenesulfonate, impurity A, impurity B, and impurity C are 0.01%, 2.67%, 2.66%, and 4.47% respectively. The method has good repeatability, and the deviations of the detection results of different portions of the same sample are small.

[0091] Example 4 (Method Robustness)

[0092] In this example, based on the detection method of Example 1, by changing various detection conditions respectively, such as derivatization reaction process parameters, chromatographic conditions, etc., it is verified that the detection method can effectively separate and detect melegabalin benzenesulfonate and three isomers under various conditions.

[0093] ① Derivatization Reaction Time

[0094] Solution Preparation

[0095] Sample solution: Take about 50 mg of melegabalin benzenesulfonate sample, place it in a 10-ml volumetric flask, add 1 mol / L sodium bicarbonate solution, dissolve it by ultrasonic waves and dilute it to the scale; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, and 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10-ml volumetric flask, mix well, prepare 3 parallel portions, react them in a water bath at 40 °C for 0.5 hour, 1 hour, and 2 hours respectively, then add 0.4 ml of 2 mol / L hydrochloric acid to each, shake well, and dilute it to the scale with the mobile phase to obtain sample solution 1, sample solution 2, and sample solution 3 respectively. The test results are shown in Table 2.

[0096] Table 2

[0097]

[0098] From the above results, it can be seen that the contents of various impurities in the sample can be effectively and accurately detected during the derivatization reaction for 0.5 - 2 hours (the relative standard deviations are relatively small at different reaction times), and the method has good durability.

[0099] ② Flow rate investigation

[0100] Chromatographic conditions:

[0101] Chromatographic column: Packed with octadecylsilane-bonded silica gel (4.6×250 mm, 3 μm); using 0.5% triethylamine solution (pH 3.0)-methanol (35:65) as the mobile phase, isocratic elution for 60 minutes. The flow rate is 0.8 ml - 1.2 ml per minute; the column temperature is 40 °C; the detection wavelength is 339 nm; the injection volume is 30 μl.

[0102] Results:

[0103] When the flow rate is 0.8 ml / min, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.31, 1.72, and 2.94 respectively;

[0104] When the flow rate is 1.0 ml / min, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.33, 1.75, and 2.94 respectively;

[0105] When the flow rate is 1.2 ml / min, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.37, 1.74, and 2.89 respectively.

[0106] Conclusion:

[0107] When the flow rate is 0.8 ml / min - 1.2 ml / min, impurity A, melegabalin benzenesulfonate, impurity B, and impurity C can be effectively separated, and the flow rate has little effect on the determination results, and the method has good durability.

[0108] ③Column temperature investigation

[0109] Chromatographic conditions:

[0110] Chromatographic column: Octadecylsilyl silica gel as the filler (4.6×250 mm, 3 μm); 0.5% triethylamine solution (pH 3.0)-methanol (35:65) as the mobile phase, isocratic elution for 60 minutes. The flow rate is 1.0 ml per minute; the column temperature is 30-50 °C; the detection wavelength is 339 nm; the injection volume is 30 μl.

[0111] Results:

[0112] When the flow rate is at 30 °C, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.42, 1.78, and 3.06 respectively;

[0113] When the flow rate is at 40 °C, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.33, 1.75, and 2.94 respectively;

[0114] When the flow rate is at 50 °C, the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.12, 1.67, and 2.89 respectively.

[0115] Conclusion:

[0116] When the column temperature is between 30 °C and 50 °C, impurity A, melegabalin benzenesulfonate, impurity B, and impurity C can be effectively separated, the column temperature has little effect on the determination results, and the method has good durability.

[0117] ④pH of the aqueous phase in the mobile phase

[0118] Chromatographic conditions:

[0119] Chromatographic column: Octadecylsilyl silica gel as the filler (4.6×250 mm, 3 μm); 0.5% triethylamine solution (pH 2.0-7.0)-methanol (35:65) as the mobile phase, isocratic elution for 60 minutes. The flow rate is 1.0 ml per minute; the column temperature is 40 °C; the detection wavelength is 339 nm; the injection volume is 30 μl.

[0120] Results:

[0121] When the mobile phase is 0.5% triethylamine solution (pH 2.0)-methanol (35:65 volume ratio), the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C are 4.31, 1.73, and 2.96 respectively;

[0122] When the mobile phase is 0.5% triethylamine solution (pH 3.0)-methanol (35:65), the resolution between impurity A, megalamine benzene sulfonate, impurity B, and impurity C are 4.33, 1.75, and 2.94 respectively;

[0123] When the mobile phase is 0.05% triethylamine solution (pH 5.0)-methanol (35:65), the resolution between impurity A, megalamine benzene sulfonate, impurity B, and impurity C are 4.34, 1.73, and 2.96 respectively.

[0124] When the mobile phase is 0.05% triethylamine solution (pH 7.0)-methanol (35:65), the resolution between impurity A, megalamine benzene sulfonate, impurity B, and impurity C are 4.32, 1.73, and 3.00 respectively.

[0125] Conclusion:

[0126] When the pH of the aqueous phase in the mobile phase is in the range of 2.0-7.0, impurity A, megalamine benzene sulfonate, impurity B, and impurity C can be effectively separated, with almost no influence on the measurement results, and the method has good durability.

[0127] ⑤ Proportion of organic phase in the mobile phase

[0128] Chromatographic conditions:

[0129] Chromatographic column: packed with octadecylsilyl silica gel (4.6×250mm, 3μm); change the composition of the mobile phase and perform isocratic elution for 60 minutes. The flow rate is 1.0 ml per minute; the column temperature is 40°C; the detection wavelength is 339 nm; the injection volume is 30 μl.

[0130] Results:

[0131] When the mobile phase is 0.5% triethylamine solution (pH 3.0)-methanol (35:65), the resolution between impurity A, megalamine benzene sulfonate, impurity B, and impurity C are 4.33, 1.75, and 2.94 respectively;

[0132] When the mobile phase is 0.5% triethylamine solution (pH 3.0)-methanol-acetonitrile (35:40:25), the resolution between impurity A, megalamine benzene sulfonate, impurity B, and impurity C are 4.06, 1.71, and 3.09 respectively.

[0133] Conclusion:

[0134] When the proportion of the organic phase in the mobile phase is within the range of 0.5% triethylamine solution (pH 3.0)-methanol (35:65) to 0.5% triethylamine solution (pH 3.0)-methanol-acetonitrile (35:40:25), impurities A, melegabalin benzenesulfonate, impurity B, and impurity C can be effectively separated from each other, with almost no influence on the determination result, and the method has good durability.

[0135] ⑥ The proportion of triethylamine in the aqueous phase of the mobile phase

[0136] Chromatographic conditions:

[0137] Chromatographic column: Packed with octadecylsilane-bonded silica gel (4.6×250 mm, 3 μm); using 0.2% - 0.8% triethylamine solution (the volume percentage of triethylamine in water, adjusted to pH 3.0 with phosphoric acid)-methanol (35:65) as the mobile phase, isocratic elution for 60 minutes. The flow rate is 1.0 ml per minute; the column temperature is 40 °C; the detection wavelength is 339 nm; the injection volume is 30 μl.

[0138] Results:

[0139] When the mobile phase is 0.02% triethylamine solution (pH 3.0)-methanol (35:65), the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C is 4.31, 1.72, and 3.09 respectively;

[0140] When the mobile phase is 0.05% triethylamine solution (pH 3.0)-methanol (35:65), the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C is 4.33, 1.75, and 2.94 respectively;

[0141] When the mobile phase is 0.08% triethylamine solution (pH 3.0)-methanol (35:65), the resolution between impurity A, melegabalin benzenesulfonate, impurity B, and impurity C is 4.47, 1.71, and 2.72 respectively.

[0142] Conclusion:

[0143] When the addition amount of triethylamine in the aqueous phase of the mobile phase is within the range of 0.2% - 0.8%, impurities A, melegabalin benzenesulfonate, impurity B, and impurity C can be effectively separated from each other, with almost no influence on the determination result, and the method has good durability.

[0144] The following provides a comparative experiment in the research and development of the detection method of this application.

[0145] Generally, the separation of isomers is carried out on a normal-phase chromatographic column for detection. In the following Comparative Examples 1 - 6, without derivatization, the samples are directly separated on a normal-phase chromatographic column, but separation cannot be achieved in all cases.

[0146] Comparative Example 1

[0147] Mixed solution: Appropriate amounts of megalolabalin besylate and each isomeric impurity are taken, dissolved in anhydrous ethanol and diluted to prepare a solution containing about 5 mg of megalolabalin besylate and about 25 μg of each isomeric impurity per 1 ml.

[0148] Chromatographic conditions:

[0149] Chromatographic column: Daicel AD-H 250×4.6 mm, 5 μm

[0150] Mobile phase: n-hexane - ethanol (95:5, v / v)

[0151] Flow rate: 1.0 ml / min

[0152] Column temperature: 35 °C

[0153] Injection volume: 20 μl

[0154] Detection wavelength: 210 nm

[0155] Isocratic elution for 60 minutes

[0156] Results: Under these chromatographic conditions, the peak shape is poor, and each impurity overlaps with the main component (megalolabalin besylate), unable to meet the detection requirements. The chromatogram is as Figure 3 shown.

[0157] Comparative Example 2

[0158] The mixed solution is prepared in the same way as in Comparative Example 1.

[0159] Chromatographic conditions:

[0160] Chromatographic column: Daicel AD-H 250×4.6 mm, 5 μm

[0161] Mobile phase: n-hexane - ethanol - trifluoroacetic acid (95:5:0.1, v / v)

[0162] Flow rate: 1.0 ml / min

[0163] Column temperature: 35 °C

[0164] Injection volume: 20 μl

[0165] Detection wavelength: 210 nm

[0166] Isocratic elution for 60 minutes

[0167] Results: Under these chromatographic conditions, the peak shape is not improved, the tailing is serious, and the response cannot meet the detection requirements.

[0168] Comparative Example 3

[0169] The mixed solution is prepared in the same way as in Comparative Example 1.

[0170] Chromatographic conditions:

[0171] Chromatographic column: Daicel AD-H 250×4.6 mm, 5 μm

[0172] Mobile phase: n-hexane - ethanol - diethylamine (95:5:0.1, by volume)

[0173] Flow rate: 1.0 ml / min

[0174] Column temperature: 35 °C

[0175] Injection volume: 20 μl

[0176] Detection wavelength: 210 nm

[0177] Isocratic elution for 60 minutes

[0178] Conclusion: Under these chromatographic conditions, the peak shape is improved, but the peak width is relatively wide, the impurities cannot be effectively separated, and the response cannot meet the detection requirements. The chromatogram is as Figure 4 shown.

[0179] Comparative Example 4

[0180] The mixed solution was prepared in the same manner as in Comparative Example 1.

[0181] Chromatographic conditions:

[0182] Chromatographic column: Daicel IC 250×4.6 mm, 5 μm

[0183] Mobile phase: n-hexane - ethanol - diethylamine (95:5:0.1, by volume)

[0184] Flow rate: 1.0 ml / min

[0185] Column temperature: 35 °C

[0186] Injection volume: 20 μl

[0187] Detection wavelength: 210 nm

[0188] Isocratic elution for 60 minutes

[0189] Conclusion: Under these chromatographic conditions, the peak shape is poor, the impurities cannot be effectively separated, and the response cannot meet the detection requirements.

[0190] Comparative Example 5

[0191] The mixed solution was prepared in the same manner as in Comparative Example 1.

[0192] Chromatographic conditions:

[0193] Chromatographic column: Daicel IC 250×4.6 mm, 5 μm

[0194] Mobile phase: 0.1% triethylamine solution - acetonitrile (90:10)

[0195] Flow rate: 1.0 ml / min

[0196] Column temperature: 35 °C

[0197] Injection volume: 20 μl

[0198] Detection wavelength: 210 nm

[0199] Isocratic elution for 60 minutes

[0200] Conclusion: Under these chromatographic conditions, no peaks of each component appeared, and detection was impossible.

[0201] Comparative Example 6

[0202] The mixed solution was prepared in the same way as in Comparative Example 1.

[0203] Chromatographic conditions:

[0204] Chromatographic column: Daicel ZWIX(+) 250×4.6 mm, 3 μm

[0205] Mobile phase: methanol - ethanol - water (49:49:2), adding 50 mM formic acid and 25 mM diethylamine

[0206] Flow rate: 1.0 ml / min

[0207] Column temperature: 40 °C

[0208] Injection volume: 30 μl

[0209] Detection wavelength: 210 nm

[0210] Isocratic elution for 60 minutes

[0211] Conclusion: Under these chromatographic conditions, no peaks of each component appeared, and detection was impossible.

[0212] In the case of no derivatization and ineffective separation on a normal-phase column, the applicant tried to use the phenylsulfonyl chloride derivatization method for separation on a normal-phase column. The result was still ineffective separation.

[0213] Comparative Example 7 (phenylsulfonyl chloride derivatization method)

[0214] ① Solution preparation:

[0215] Meloxicabalin benzenesulfonate sample solution: Take about 250 mg of meloxicabalin benzenesulfonate sample, place it in a 10-ml volumetric flask, successively add 5 ml of dichloromethane, 0.2 ml of triethylamine, and 4.8 ml of water, and ultrasonicate in an ultrasonic cleaner for 60 minutes; take 1 ml of the above solution, place it in a 10-ml volumetric flask, successively add 0.1 ml of benzenesulfonyl chloride, 0.1 ml of triethylamine, and 1 ml of dichloromethane, seal it, ultrasonicate in an ultrasonic cleaner for 20 minutes, and then dilute to the mark with ethanol and shake well.

[0216] Impurity localization solution: Take about 25 mg of each impurity, place it in a 10-ml volumetric flask, successively add 5 ml of dichloromethane, 0.2 ml of triethylamine, and 4.8 ml of water, and ultrasonicate in an ultrasonic cleaner for 60 minutes; take 1 ml of the above solution, place it in a 10-ml volumetric flask, successively add 0.1 ml of benzenesulfonyl chloride, 0.1 ml of triethylamine, and 1 ml of dichloromethane, seal it, ultrasonicate in an ultrasonic cleaner for 20 minutes, and then dilute to the mark with ethanol and shake well.

[0217] ②Chromatographic conditions:

[0218] Chromatographic column: Daicel AD-H 250×4.6 mm, 5 μm

[0219] Mobile phase: n-hexane - ethanol - diethylamine (95:5:0.1)

[0220] Flow rate: 1.0 ml / min

[0221] Column temperature: 30 °C

[0222] Injection volume: 10 μl

[0223] Detection wavelength: 225 nm

[0224] Isocratic elution for 40 minutes

[0225] Conclusion: Under these chromatographic conditions, the baseline fluctuates greatly, the enantiomeric impurity (impurity B) and the diastereomeric impurity (impurity A) cannot be effectively separated, and the responses of each impurity are small, which cannot meet the detection requirements.

[0226] In the case where the benzenesulfonyl chloride derivatization method and the normal-phase column cannot separate either, the applicant attempts to use the FXAA derivatization method of the present invention in combination with a normal-phase column.

[0227] Comparative Example 8 (FXAA derivatization method, normal-phase column)

[0228] FXAA acetone solution: Take 50 mg of FXAA, place it in a 10-ml volumetric flask, dissolve it with acetone and dilute to the mark, and shake well.

[0229] ①Solution preparation:

[0230] FXAA Acetone Solution: Take about 50 mg of FXAA, place it in a 10-ml volumetric flask, dissolve it with acetone and dilute to the mark, shake well to obtain the solution.

[0231] Mirogabalin Besylate Sample Solution: Take about 50 mg of mirogabalin besylate sample, place it in a 10-ml volumetric flask, add 0.2 ml of triethylamine, dissolve it with water and dilute to the mark, shake well; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution and 0.2 ml of triethylamine, place them in a 10-ml volumetric flask, mix well, react in a 40 °C water bath for 1 hour, add 0.4 ml of hydrochloric acid, shake well and dilute to the mark with the mobile phase.

[0232] Impurity Localization Solution: Take about 25 mg of each impurity respectively, place it in a 10-ml volumetric flask, add 0.2 ml of triethylamine, dissolve it with water and dilute to the mark, shake well; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution and 0.2 ml of triethylamine, place them in a 10-ml volumetric flask, mix well, react in a 40 °C water bath for 1 hour, add 0.4 ml of hydrochloric acid, shake well and dilute to the mark with the mobile phase.

[0233] ② Chromatographic Conditions:

[0234] Chromatographic Column: Daicel AD-H 250×4.6 mm, 5 μm

[0235] Mobile Phase: n-Hexane - Ethanol - Diethylamine (95:5:0.1)

[0236] Flow Rate: 1.0 ml / min

[0237] Column Temperature: 35 °C

[0238] Injection Volume: 30 μl

[0239] Detection Wavelength: 339 nm

[0240] Isocratic Elution for 60 Minutes

[0241] Conclusion: Under these chromatographic conditions, although mirogabalin besylate can be effectively separated from impurities A and C, it cannot be effectively separated from the enantiomeric impurity (impurity B). The chromatogram is as Figure 5 shown.

[0242] Comparative Example 9 (FXAA Derivatization Method, Normal Phase Column)

[0243] On the basis of Comparative Example 8, the applicant tried to change the preparation method of the sample solution.

[0244] ① Solution Preparation:

[0245] FXAA Acetone Solution: Take about 50 mg of FXAA, place it in a 10-ml volumetric flask, dissolve it with acetone and dilute to the mark, shake well to obtain the solution.

[0246] Meloxicabalin benzenesulfonate sample solution: Take about 50 mg of meloxicabalin benzenesulfonate sample, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute it to the mark, shake well; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, and 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10-ml volumetric flask, mix well, react in a 40°C water bath for 1 hour, then add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute to the mark with the mobile phase.

[0247] Impurity localization solution: Take about 25 mg of each impurity respectively, place it in a 10-ml volumetric flask, dissolve it with 1 mol / L sodium bicarbonate solution and dilute it to the mark, shake well; take 0.2 ml of the above solution, 1 ml of FXAA acetone solution, and 0.2 ml of 1 mol / L sodium bicarbonate solution, place them in a 10-ml volumetric flask, mix well, react in a 40°C water bath for 1 hour, then add 0.4 ml of 2 mol / L hydrochloric acid, shake well, and dilute to the mark with the mobile phase.

[0248] ② Chromatographic conditions:

[0249] Chromatographic column: Daicel AD-H 250×4.6 mm, 5 μm

[0250] Mobile phase: n-hexane - ethanol - diethylamine (95:5:0.1)

[0251] Flow rate: 1.0 ml / min

[0252] Column temperature: 35°C

[0253] Injection volume: 30 μl

[0254] Detection wavelength: 339 nm

[0255] Isocratic elution for 60 minutes

[0256] Conclusion: Under these chromatographic conditions, although meloxicabalin benzenesulfonate can be effectively separated from impurities A and C, it cannot be effectively separated from the enantiomeric impurity (impurity B). The chromatogram is as Figure 6 shown.

[0257] It can be seen that it is very difficult to effectively separate and detect meloxicabalin benzenesulfonate from its three impurities. In this application, by derivatizing the sample, then screening a specific reversed-phase chromatographic column, and selecting and adjusting each chromatographic condition, it is possible to effectively separate and detect meloxicabalin benzenesulfonate from its three isomeric impurities, with a low detection limit and high sensitivity.

[0258] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for detecting megalol gabapentin benzenesulfonate and its isomeric impurities, using liquid chromatography, characterized in that: The method first reacts an FXAA reagent with a test sample under alkaline conditions to obtain a test solution, and then separates the test solution by liquid chromatography. The structural formula of the FXAA reagent is wherein, R is selected from -CH(CH3)2, -CH3, -CH2Ph, -CH2CH(CH3)2, -CH2CH2SCH3 or -C6H5; the test sample includes megalolabaline besylate and its isomeric impurities, and the liquid chromatography uses a reverse-phase chromatographic column.

2. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: R is -CH(CH3)2; and / or, the reverse-phase chromatographic column is packed with octadecylsilyl-bonded silica gel.

3. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: The mobile phase of the liquid chromatography comprises an aqueous solution of triethylamine and methanol; preferably, the volume ratio of the aqueous solution of triethylamine to methanol is 35:50 - 70; and / or, in the aqueous solution of triethylamine, the volume of triethylamine accounts for 0.2% - 0.8% of the volume of water; and / or, the pH value of the aqueous solution of triethylamine is 2 - 7.

4. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 3, characterized in that: The mobile phase of the liquid chromatography further comprises acetonitrile, and the volume ratio of the aqueous solution of triethylamine, methanol and acetonitrile is 35:40 - 65:1 - 25.

5. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: The alkaline condition is achieved by adding a base, and the base is selected from one or a combination of more of sodium bicarbonate and potassium bicarbonate; and / or, the reaction time is 0.5 - 2 h.

6. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: The flow rate of the mobile phase of the liquid chromatography is 0.8 - 1.2 ml / min; and / or, the column temperature of the chromatographic column of the liquid chromatography is 30 - 50 °C; and / or, the detection wavelength of the liquid chromatography is 339 nm; and / or, the elution time of the mobile phase of the liquid chromatography is 50 - 90 min; and / or, the injection volume of the test solution is 10 - 50 μL.

7. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: The isomeric impurities include impurity A Impurity B and impurity C 8. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 7, characterized in that: The resolution between megalol gabapentin benzenesulfonate and impurity A is 4 - 4.5, the resolution between megalol gabapentin benzenesulfonate and impurity B is 1.6 - 1.8, and the resolution between impurity B and impurity C is 2.6 - 3.

1.

9. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, characterized in that: The concentration of the test solution is 4 mg / ml - 6 mg / ml; and / or, the molar ratio of the FXAA reagent to the test sample is 2:1 - 10:

1.

10. The method for detecting melegabalin benzenesulfonate and its isomeric impurities according to claim 1, wherein: Dissolve the FXAA reagent in acetone to obtain an acetone solution of FXAA; dissolve the test sample in an aqueous solution of sodium bicarbonate, add the acetone solution of FXAA to the aqueous solution of sodium bicarbonate, and then add an additional aqueous solution of sodium bicarbonate to conduct the reaction.

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