Method for detecting enantiomer and diastereomer of lumepirone tosylate

Abstract

The invention relates to a method for detecting enantiomers and diastereoisomers of lumepirone tosylate, which is used for detecting enantiomers and diastereoisomers of lumepirone tosylate by adopting a high performance liquid chromatography. In the high performance liquid chromatography detection process, a filling agent of a chromatographic column is amylose-tri (4-chloro-3-methyl phenyl carbamate), and a mobile phase comprises alkane, alcohol and amine. The detection method has the advantages of good separation degree, high sensitivity and simple method, and is suitable for inspection and quality control of the lumepirone tosylate isomer in the medical industry, so that the safety of clinical medication is improved.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, and specifically relates to a method for detecting the enantiomers and diastereomers of lumepirobenone tosylate. Background Technology

[0002] Lumateperone tosylate (trade name: Lumateperone tosylate) Developed by Intra-Cellular Therapies Inc., this drug is used to treat schizophrenia and bipolar disorder in adults. It was first approved for marketing in the United States on December 20, 2019, in capsule form. This drug is a novel antipsychotic with a unique mechanism of action, exerting its potential antipsychotic effects by modulating neurotransmission of 5-HT, DA, and glutamate. It has a high affinity for 5-HT2A receptors and is a dopamine phosphoprotein modulator, acting simultaneously as a partial agonist of presynaptic D2 receptors and an antagonist of postsynaptic D2 receptors. It also indirectly modulates glutamate neurotransmission by acting on D1 receptors.

[0003] The structural formula of rumeperone tosylate is as follows:

[0004]

[0005] The structure of lumepirobenzyl tosylate shows that it has two chiral centers. Therefore, in addition to the above compound, there are three isomers: one enantiomer and a pair of diastereomer impurities.

[0006] Chiral drugs are enantiomers that are mirror images of each other, formed by introducing a chiral center into the drug molecule. Chiral drugs exhibit stereoselectivity in absorption, distribution, metabolism, and excretion within the body. Different isomers of chiral drugs often display different pharmacological and toxicological properties, as well as different pharmacokinetic properties. Different configurational isomers may have opposite pharmacodynamic effects; therefore, controlling isomer impurities (including enantiomers and diastereomers) in products is crucial for the safe use of pharmaceuticals. However, due to their highly similar structures, isomers are difficult to separate using conventional detection methods, necessitating the development of targeted isomer detection methods.

[0007] Currently, there are no reports in patents or literature regarding quality research on the isomers of rumepiride tosylate. Therefore, in order to better control the quality of rumepiride tosylate, especially to conduct quality research on isomers and develop suitable isomer detection methods, is a pressing technical problem that needs to be solved to ensure the quality and safety of the drug on the market. Summary of the Invention

[0008] To accurately reflect the content of isomers in lumepirox sylate raw materials or preparations, and to provide a reasonable basis for the formulation of quality standards, so as to better control and manage product quality and improve the safety of clinical medication, the present invention aims to provide a method for detecting enantiomers and diastereomers of lumepirox sylate. High-performance liquid chromatography (HPLC) is used to detect the enantiomers and diastereomers of lumepirox sylate. The chromatographic column packing material in the HPLC detection process is amylose-tris(4-chloro-3-methylphenylcarbamate), and the mobile phase includes alkanes, alcohols, and amines.

[0009] In a further preferred embodiment of the present invention, isocratic elution is used to pass the mobile phase through the chromatographic column during the high performance liquid chromatography detection process.

[0010] In a further preferred embodiment of the present invention, the alkane is n-hexane; the alcohol is one or more of methanol, ethanol, propanol, and butanol, preferably anhydrous ethanol; and the amine is one or more of diethylamine, triethylamine, butylamine, and ethanolamine, preferably diethylamine.

[0011] In a further preferred embodiment of the present invention, the particle size of the chromatographic column is 4-6 μm.

[0012] In a further preferred embodiment of the present invention, the chromatographic column is a Phenomen Chiral NX(2) column (4.6×250mm, 5μm).

[0013] In a further preferred embodiment of the present invention, the volume ratio of the alkane, alcohol and amine is (85-95):(5-15):(0.05-0.2), preferably (85-90):(10-15):(0.08-0.15).

[0014] In a further preferred embodiment of the present invention, the volume ratio of n-hexane, anhydrous ethanol and diethylamine is (85-95):(5-15):(0.05-0.2), preferably (85-90):(10-15):(0.08-0.15), preferably 87:13:0.1, preferably 88:12:0.1, and preferably 89:11:0.1.

[0015] In a further preferred embodiment of the present invention, the column temperature of the chromatographic column is 25-40℃, preferably 25-35℃, preferably 25-30℃, and preferably 25-27℃.

[0016] In a further preferred embodiment of the present invention, the flow rate of the mobile phase is 0.5-1.2 ml / min, preferably 0.6-1.1 ml / min, more preferably 0.6-1.0 ml / min, more preferably 0.6-0.9 ml / min, and even more preferably 0.7 ml / min.

[0017] In a further preferred embodiment of the present invention, the detection wavelength is 225-235nm, preferably 228-232nm, and more preferably 230nm.

[0018] In a further preferred embodiment of the present invention, the injection volume is 8-12 μl, preferably 9-11 μl, and more preferably 10 μl.

[0019] In a further preferred embodiment of the present invention, the content of the isomers of rumeperone tosylate is calculated by the external standard method.

[0020] In a further preferred embodiment of the present invention, the enantiomers and diastereomers of lumepirobentosulfonate have the following structures:

[0021]

[0022] In a further preferred embodiment of the present invention, the detection method is used for the detection and analysis of isomer impurities in lumepirobentosulfonate raw materials or preparations.

[0023] In a further preferred embodiment of the present invention, a method for detecting the enantiomers and diastereomers of lumepirobenone tosylate is provided. High-performance liquid chromatography (HPLC) is used to detect the enantiomers and diastereomers of lumepirobenone tosylate. The HPLC detection process uses linear starch-tris(4-chloro-3-methylphenylcarbamate) as the column packing material, and the mobile phase consists of n-hexane, anhydrous ethanol, and diethylamine in a volume ratio of (85-95):(5-15):(0.05-0.2). The flow rate is 0.6-1.2 ml / min, the detection wavelength is 225-235 nm, the column temperature is 30-40 °C, and the injection volume is 8-12 μl.

[0024] In a further preferred embodiment of the present invention, during the high performance liquid chromatography detection process, each solution is diluted with a diluent before detection. The diluent is a mixed solution of alkanes and alcohols, preferably n-hexane and anhydrous ethanol.

[0025] In a further preferred embodiment of the present invention, the diluent is n-hexane and anhydrous ethanol, wherein the volume ratio of n-hexane to anhydrous ethanol is 40-90:10-60, preferably 60-80:20-40.

[0026] In a further preferred embodiment of the present invention, the diluent is n-hexane and anhydrous ethanol, wherein the volume ratio of n-hexane to anhydrous ethanol is 90:10.

[0027] In a further preferred embodiment of the present invention, the diluent is n-hexane and anhydrous ethanol, wherein the volume ratio of n-hexane to anhydrous ethanol is 70:30.

[0028] In a further preferred embodiment of the present invention, the diluent is n-hexane and anhydrous ethanol, wherein the volume ratio of n-hexane to anhydrous ethanol is 60:40.

[0029] In a further preferred embodiment of the present invention, each solution must be stored away from light. The solutions refer to mixed solutions, mixed impurity solutions, separation degree solutions, reference solutions, test solutions, etc.

[0030] In a further preferred embodiment of the present invention, the separation degree between the isomer and the adjacent impurity peak is greater than 1.5.

[0031] In a further preferred embodiment of the present invention, the detection method includes the following detection steps: preparing a mixed solution or a mixed impurity solution, a separation solution, and a test sample solution respectively, and then injecting each solution into a liquid chromatograph and recording the chromatogram.

[0032] In a further preferred embodiment of the present invention, the method for preparing the mixed impurity solution is as follows: measure the stock solution of each impurity reference standard and put it into a bottle, and dilute it with a diluent to prepare a solution containing 0.8-1.2 μg of each impurity per 1 ml.

[0033] In a further preferred embodiment of the present invention, the mixed solution is prepared by measuring lumepirozol tosylate and stock solutions of various impurity reference standards into a bottle, and diluting them with a diluent to prepare a solution containing 400-600 μg of lumepirozol tosylate and 0.8-1.2 μg of each impurity per 1 ml.

[0034] In a further preferred embodiment of the present invention, the separation solution is prepared by measuring lumepirozoline toluenesulfonate and stock solutions of various isomer impurities into a bottle, and diluting them with a diluent to prepare a solution containing 400-600 μg of lumepirozoline toluenesulfonate and 0.8-1.2 μg of various isomer impurities per 1 ml.

[0035] In a further preferred embodiment of the present invention, the preparation method of the reference solution is as follows: measure the stock solution of lumepirozol tosylate reference standard into a bottle, and dilute it with a diluent to prepare a solution containing 0.8-1.2 μg of lumepirozol tosylate per 1 ml.

[0036] In a further preferred embodiment of the present invention, the test solution is prepared by measuring lumepirozoline toluenesulfonate into a bottle and diluting it with a diluent to prepare a solution containing 400-600 μg of lumepirozoline toluenesulfonate per 1 ml.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] 1. This invention provides a method for detecting isomers in rumetrol tosylate. This method has good separation, high sensitivity, and is simple, making it suitable for the inspection and quality control of rumetrol tosylate isomers in the pharmaceutical industry, thereby improving the safety of clinical medication.

[0039] 2. This invention scientifically screened the chromatographic column, mobile phase, and the combined effects of flow rate and column temperature on separation and detection, achieving optimal detection results with a resolution greater than 1.5 for both isomers and adjacent impurity peaks. This method can not only effectively separate enantiomers and diastereomers in rumeperone tosylate, but also rapidly and efficiently separate process or degradation impurities detected in related substances under the same chromatographic conditions without interfering with the detection of isomer impurities. The analytical method was subsequently validated, demonstrating high sensitivity, strong specificity, rapid and simple operation, and ease of use. It can effectively control drug quality and is suitable for separating and detecting three isomers. Attached Figure Description

[0040] Figure 1 Resolution solution chromatogram in Example 1;

[0041] Figure 2 Spectrum of mixed impurity solution in Example 1

[0042] Figure 3 Resolution solution chromatogram in Example 2;

[0043] Figure 4 The spectrum of the mixed impurity solution in Example 2;

[0044] Figure 5 Example 3 shows the resolution solution chromatogram obtained using a Chiral NX(2) column in hexane-anhydrous ethanol-diethylamine (88:12:0.1) at a column temperature of 35°C.

[0045] Figure 6 Example 3: Chromatography of a mixed solution of hexane-anhydrous ethanol-diethylamine (88:12:0.1) at 35°C using a Chiral NX(2) column;

[0046] Figure 7 The resolution solution chromatogram of the reversed-phase system in Comparative Example 1;

[0047] Figure 8 The resolution solution chromatogram of the column in Comparative Example 2 in a normal phase system;

[0048] Figure 9 The chromatogram of the column in Comparative Example 2 in a normal-phase system with mixed impurity solution;

[0049] Figure 10 The resolution solution chromatogram of the column in Comparative Example 3 in a normal phase system;

[0050] Figure 11 The chromatogram of the column in Comparative Example 3 in a normal-phase system with mixed impurity solution;

[0051] Figure 12 The chromatographic column in Comparative Example 4 showed the resolution solution chromatogram in a normal phase system;

[0052] Figure 13 The chromatogram of the column in Comparative Example 4 in a normal-phase system with mixed impurity solution;

[0053] Figure 14 The resolution solution chromatogram of the Chiral NX(2) column in the hexane-anhydrous ethanol system in Comparative Example 5;

[0054] Figure 15 The resolution solution chromatogram of the Chiral NX(2) column in the hexane-isopropanol-diethylamine system in Comparative Example 6;

[0055] Figure 16 The chromatogram of the Chiral NX(2) column in Comparative Example 6 in a mixed impurity solution of hexane-isopropanol-diethylamine system. Detailed Implementation

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0057] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] Example 1

[0059] (1) Instruments and testing conditions:

[0060] Instrument: Shimadzu LC-20AD high performance liquid chromatograph;

[0061] Chromatographic column: Phenomenology Chiral NX(2) (4.6×250mm, 5μm);

[0062] Mobile phase: n-hexane-anhydrous ethanol-diethylamine (89:11:0.1)

[0063] Flow rate: 0.6 ml / min;

[0064] Column temperature: 30℃;

[0065] Wavelength: 230nm;

[0066] Injection volume: 10 μl;

[0067] Elution procedure: isocratic elution.

[0068] (2) Test Procedure

[0069] Operate in the dark.

[0070] Diluent: n-hexane-anhydrous ethanol (90:10), the same applies below.

[0071] Impurity reference standard stock solution: Accurately weigh appropriate amounts of isomer impurities XV (enantiomer), XVI (diastereomer), XVIII (diastereomer) and related substances impurities (XXI*, XXVI*, XXXVIII*, XXXXII) reference standards, place them in different volumetric flasks, dissolve and dilute with isopropanol to prepare a solution containing approximately 200 μg per ml.

[0072] Note: Impurities marked with an asterisk (*) in the relevant substances contain isomers and may appear as two peaks in chiral column separation.

[0073] Mixed impurity solution: Measure 0.1 ml of each impurity reference standard stock solution, place it in a 20 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately 1 μg of each impurity per 1 ml.

[0074] Separation solution: Weigh an appropriate amount of lumepirozoline tosylate, accurately weigh it, place it in a 20 ml volumetric flask, add 0.1 ml of stock solution of the three isomer impurity reference standards, dissolve and dilute with diluent to prepare a solution containing approximately 500 μg of the main component and approximately 1 μg of each impurity per ml.

[0075] Test solution: Weigh an appropriate amount of lumepirozol toluenesulfonic acid, accurately weigh it, place it in a 20ml volumetric flask, dissolve and dilute it with diluent to prepare a solution containing approximately 500μg of the main component per ml.

[0076] Accurately measure 10 μl each of the diluent, mixed impurity solution, resolution solution, and test solution, inject them into the liquid chromatograph, and record the chromatograms. See the chromatograms for the resolution solution and mixed impurity solution. Figures 1-2 .

[0077] Conclusion: In the mixed impurity solution, the resolution between isomer impurities XV, XVI, and XVIII and adjacent impurities as well as the main peak is greater than 1.5. Process impurities / degradation impurities detected by related substances do not interfere with the detection of isomer impurities, indicating good resolution. In the resolution solution, the resolution between isomer impurities XV, XVI, and XVIII and adjacent chromatographic peaks is greater than 2.0.

[0078] Example 2

[0079] (1) Instruments and testing conditions:

[0080] Instrument: Shimadzu LC-20AD high performance liquid chromatograph;

[0081] Chromatographic column: Phenomenology Chiral NX(2) (4.6×250mm, 5μm);

[0082] Mobile phase: n-hexane-anhydrous ethanol-diethylamine (89:11:0.1)

[0083] Flow rate: 0.7 ml / min;

[0084] Column temperature: 30℃;

[0085] Wavelength: 230nm;

[0086] Injection volume: 10 μl;

[0087] Elution procedure: isocratic elution.

[0088] (2) Test Procedure

[0089] Operate in the dark.

[0090] Diluent: n-hexane-anhydrous ethanol (90:10), the same applies below.

[0091] Impurity reference standard stock solution: Accurately weigh appropriate amounts of isomer impurities XV (enantiomer), XVI (diastereomer), XVIII (diastereomer) and related substances impurities (XXI*, XXVI*, XXXVIII*, XXXXII) reference standards, place them in different volumetric flasks, dissolve and dilute with isopropanol to prepare a solution containing approximately 200 μg per ml.

[0092] Note: Impurities marked with an asterisk (*) in the relevant substances contain isomers and may appear as two peaks in chiral column separation.

[0093] Toluene-4-sulfonic acid (toluenesulfonic acid) stock solution: Weigh about 11 mg of toluene-4-sulfonic acid into a 50 ml volumetric flask, dilute with isopropanol and bring to volume.

[0094] Mixed impurity solution: Measure 0.1 ml of each impurity reference standard stock solution, place it in a 20 ml volumetric flask, and dilute with diluent to prepare a solution containing approximately 1 μg of each impurity per 1 ml.

[0095] Separation solution: Weigh an appropriate amount of lumepirozoline tosylate, accurately weigh it, place it in a 20 ml volumetric flask, add 0.1 ml of stock solution of 3 isomer impurity reference standards, dissolve and dilute with diluent to prepare a solution containing approximately 500 μg of the main component and approximately 1 μg of each impurity per ml.

[0096] Test solution: Weigh an appropriate amount of lumepirozol toluenesulfonic acid, accurately weigh it, place it in a 20ml volumetric flask, dissolve and dilute it with diluent to prepare a solution containing approximately 500μg of the main component per ml.

[0097] Test sample + toluene-4-sulfonic acid (toluenesulfonic acid) solution: Measure 1 ml of the test sample solution and add 0.1 ml of toluene-4-sulfonic acid (toluenesulfonic acid) stock solution, then mix well.

[0098] Accurately measure 10 μl each of the diluent, mixed impurity solution, resolution solution, test solution, and test solution + toluene-4-sulfonic acid (toluenesulfonic acid) solution, inject them into the liquid chromatograph, and record the chromatograms. See the chromatograms for the resolution solution and mixed impurity solution. Figures 3-4 .

[0099] Conclusion: In the mixed impurity solution, the resolution between isomer impurities XV, XVI, and XVIII and adjacent impurities as well as the main peak was greater than 1.5. Process impurities / degradation impurities detected by related substances did not interfere with the detection of isomer impurities, indicating good resolution. The resolution between isomer impurities XV, XVI, and XVIII and adjacent chromatographic peaks in the solution was greater than 2.0. The peak position of toluenesulfonic acid under these chromatographic conditions was confirmed by localization.

[0100] Example 3

[0101] This application provides methodological validation for a high-performance liquid chromatography (HPLC) method for separating and detecting lumepirobenone isomers of tosylate (impurities XV, XVI, and XVIII), including:

[0102] (1) Instruments and testing conditions:

[0103] Instrument: Shimadzu LC-20AD high performance liquid chromatograph;

[0104] Chromatographic column: Phenomenology Chiral NX(2) (4.6×250mm, 5μm);

[0105] Mobile phase: n-hexane-anhydrous ethanol-diethylamine (88:12:0.1)

[0106] Flow rate: 0.7 ml / min;

[0107] Column temperature: 25℃;

[0108] Wavelength: 230nm;

[0109] Injection volume: 10 μl;

[0110] Elution procedure: isocratic elution.

[0111] (2) Test Procedure

[0112] Operate in the dark.

[0113] Diluent: n-hexane-anhydrous ethanol (60:40), the same applies below.

[0114] Impurity reference stock solution: Accurately weigh appropriate amounts of isomer impurities XV (enantiomer), XVI (diastereomer), XVIII (diastereomer) and process impurities and degradation impurities (XXI, XXVI, XXXVIII, XXXXII) reference standards detected by related substances, place them in different volumetric flasks, dissolve and dilute with isopropanol to prepare a solution containing approximately 200 μg per ml.

[0115] For each impurity reference solution: accurately measure 50 μl of each impurity reference standard stock solution and place it in a 10 ml volumetric flask. Dilute with diluent and bring to volume to prepare a solution containing approximately 1 μg per ml.

[0116] Reference stock solution: Accurately weigh an appropriate amount of rumeperone tosylate reference standard, dissolve and dilute it with isopropanol to prepare a solution containing approximately 200 μg of rumeperone per 1 ml.

[0117] Toluene-4-sulfonic acid (toluenesulfonic acid) positioning: Weigh about 11 mg of toluene-4-sulfonic acid into a 50 ml volumetric flask, dilute with diluent and bring to volume.

[0118] Reference solution: Accurately transfer 0.5 ml of the reference stock solution into a 100 ml volumetric flask, dilute with diluent and bring to volume to prepare a solution containing approximately 1 μg per ml.

[0119] Mixed solution: Weigh an appropriate amount of lumepirozoline tosylate, accurately weigh it, and place it in a 20 ml volumetric flask; then add 0.1 ml of stock solution of each impurity reference standard, dissolve and dilute with diluent to prepare a solution containing approximately 500 μg of the main component and approximately 1 μg of each impurity per 1 ml.

[0120] Separation solution: Weigh an appropriate amount of lumepirozoline tosylate, accurately weigh it, and place it in a 20 ml volumetric flask; then add 0.1 ml of stock solution of the three isomer impurity reference standards, dissolve and dilute with diluent to prepare a solution containing approximately 500 μg of the main component and approximately 1 μg of each impurity per ml.

[0121] Test solution: Weigh an appropriate amount of lumepirozol toluenesulfonic acid, accurately weigh it, place it in a 20ml volumetric flask, dissolve and dilute it with diluent to prepare a solution containing approximately 500μg of the main component per ml.

[0122] (3) Exclusivity

[0123] Accurately measure 10 μl each of the above-mentioned diluent, impurity localization solution, mixed solution, resolution solution, reference solution, and test solution, inject them into the liquid chromatograph, record the chromatograms, and the results are shown in Table 1. The chromatograms of the resolution solution and mixed solution are shown in Table 1. Figures 5-6 .

[0124] Table 1 Specificity Results

[0125]

[0126] Conclusions: ① The diluent did not interfere with the determination of lumepirocin and impurities XV, XVI, and XVIII. ② The resolution between isomer impurities XV, XVI, and XVIII and adjacent impurities and the main peak in the mixed solution was greater than 1.5. Process impurities / degradation impurities detected in the related substances did not interfere with the detection of the three isomer impurities, indicating good resolution. The resolution between isomer impurities XV, XVI, and XVIII and adjacent chromatographic peaks in the separation solution was greater than 2.0.

[0127] (4) Limit of detection, limit of quantitation

[0128] Take appropriate amounts of the stock solutions of the three isomer impurities under test step (2), dilute them with diluent to prepare solutions of a certain concentration, and inject the prepared solutions into the liquid chromatograph in sequence according to the planned chromatographic conditions, and record the chromatograms. The results are shown in Table 2.

[0129] Table 2 Sensitivity Test Results

[0130]

[0131] Conclusion: All impurities exhibit high sensitivity.

[0132] (5) Linearity and range

[0133] Accurately weigh appropriate amounts of the three isomer impurities and lumepirozol tosylate, and dilute with diluent to prepare linear solutions of 0.1, 0.4, 0.8, 1.0, 1.5, and 2.5 μg / mL (equivalent to 0.02%, 0.08%, 0.16%, 0.2%, 0.3%, and 0.5% of the test sample concentration). Inject the linear solutions into the liquid chromatograph under the planned chromatographic conditions and record the chromatograms. Perform linear regression with the injection concentration as the x-axis and the peak area as the y-axis; the results are shown in Table 3.

[0134] Table 3. Results of Linearity and Range Tests

[0135] name Linear equations r Lumepiride Y = 67496X - 87.59 1.0000 Impurities XV Y = 67863X - 269.26 1.0000 Impurities XVI Y = 68904X - 882.27 1.0000 Impurity XVIII Y = 67363X - 667.98 1.0000

[0136] Conclusions: Lumepiride showed good linearity in the concentration range of 0.1023 μg / ml to 2.5581 μg / ml; impurity XV showed good linearity in the concentration range of 0.1048 μg / ml to 2.6207 μg / ml; impurity XVI showed good linearity in the concentration range of 0.1137 μg / ml to 2.8419 μg / ml; and impurity XVIII showed good linearity in the concentration range of 0.1144 μg / ml to 2.8596 μg / ml.

[0137] (6) Stability test

[0138] For the preparation of stock solutions of impurity XV, XVI, and XVIII reference standards, see “(2) Test Procedure”.

[0139] Sample preparation solution: Weigh an appropriate amount of lumepirozol tosylate, accurately weigh it, and place it in a 20ml brown volumetric flask. Accurately transfer 0.1ml of the stock solution of impurity XV, XVI, and XVIII reference standards, dissolve and dilute it with diluent to prepare a solution containing approximately 500μg of the main component and 1μg of each impurity per 1ml.

[0140] The chromatogram was injected into the liquid chromatograph under the prescribed chromatographic conditions, and the results were recorded. The results are shown in Table 4.

[0141] Table 4. Stability Test Results

[0142]

[0143] Conclusion: The test solution showed good stability after being left to stand for 26 hours.

[0144] (7) Precision test

[0145] For the preparation of stock solutions of impurity XV, XVI, and XVIII reference standards, see “(2) Test Procedure”.

[0146] Impurity reference solution: Accurately transfer 0.1 ml of the stock solution of impurity XV, XVI, and XVIII reference standards into the same 20 ml brown volumetric flask, dissolve and dilute with diluent to prepare a solution of about 1 μg per ml.

[0147] Sample preparation solution: Weigh an appropriate amount of lumepirozol tosylate, accurately weigh it, and place it in a 20ml brown volumetric flask. Accurately transfer 0.1ml of the stock solution of impurity XV, XVI, and XVIII reference standards, dissolve and dilute it with diluent to prepare a solution containing approximately 500μg of the main component and 1μg of each impurity per 1ml.

[0148] Inject the chromatogram into the liquid chromatograph under the planned chromatographic conditions and record the chromatogram.

[0149] Conclusion: ① Repeatability: The number of impurities in the 6 sample solutions was consistent. The average contents of impurities XV, XVI and XVIII were 0.20%, 0.22% and 0.22% respectively, and the RSDs were 1.3%, 2.2% and 1.5% respectively, indicating good repeatability.

[0150] ② Intermediate precision: The number of impurities in the 6 sample test solutions was consistent, and the average contents of impurities XV, XVI, and XVIII were 0.21%, 0.18%, and 0.22%, respectively, with RSDs of 3.0%, 2.9%, and 2.8%, respectively. The number of impurities in the 12 sample test solutions was consistent, and the average contents of impurities XV, XVI, and XVIII were 0.20%, 0.20%, and 0.22%, respectively, with RSDs of 3.0%, 12.5%, and 2.3%, respectively, indicating good intermediate precision.

[0151] (8) Recovery rate test

[0152] For the preparation of stock solutions of impurity XV, XVI, and XVIII reference standards, see “(2) Test Procedure”.

[0153] Concentrated solution: Accurately measure 2 ml each of the stock solutions of impurity XV, XVI, and XVIII into the same 20 ml brown volumetric flask, and dilute with diluent to prepare a solution containing approximately 20 μg of the substance per ml.

[0154] Test solution: Weigh an appropriate amount of lumepirozol toluenesulfonic acid, accurately weigh it, place it in a 20ml brown volumetric flask, dissolve and dilute it with diluent to prepare a solution containing about 500μg per ml.

[0155] Impurity reference solution: Accurately transfer 0.1 ml of the stock solution of impurity XV, XVI, and XVIII reference standards into the same 20 ml brown volumetric flask, dissolve and dilute with diluent to prepare a solution of about 1 μg per ml.

[0156] LOQ solution: Accurately weigh approximately 10 mg of lumepirozol tosylate and place it in a 20 ml brown volumetric flask. Add 0.1 ml of concentrated solution, then dilute and bring to volume. Prepare 3 portions.

[0157] Low concentration solution: Accurately weigh approximately 10 mg of lumepirozol tosylate and place it in a 20 ml brown volumetric flask. Add 0.5 ml of concentrated solution, then dilute and bring to volume. Prepare 3 portions.

[0158] Medium concentration solution: Accurately weigh approximately 10 mg of lumepirozol tosylate and place it in a 20 ml brown volumetric flask. Add 1 ml of concentrated solution, then dilute and bring to volume. Prepare 3 portions.

[0159] High-concentration solution: Accurately weigh approximately 10 mg of lumepirozol tosylate and place it in a 20 ml brown volumetric flask. Add 2.5 ml of concentrated solution, then dilute and bring to volume. Prepare 3 portions.

[0160] Take each solution, inject it into the liquid chromatograph under the planned chromatographic conditions, and record the chromatogram. Calculate the recovery rate of each impurity using the external standard method. The results are shown in Tables 5-7.

[0161] Table 5. Recovery rate of impurity XV

[0162] Recovery rate name Average recovery rate RSD (%) Quantitative limit solution 86.1 3.7 low concentration solution 98.0 0.4 medium concentration solution 101.2 1.5 high concentration solution 104.8 0.3

[0163] Table 6 Recovery of Impurity XVI

[0164] Recovery rate name Average recovery rate RSD (%) Quantitative limit solution 85.4 8.0 low concentration solution 95.0 1.5 medium concentration solution 98.1 1.4 high concentration solution 103.5 0.4

[0165] Table 7 Recovery of Impurity XVIII

[0166] Recovery rate name Average recovery rate RSD (%) Quantitative limit solution 91.0 7.6 low concentration solution 96.5 2.2 medium concentration solution 98.9 1.4 high concentration solution 103.9 0.2

[0167] Conclusion: The above experimental results show that the recovery rate of each impurity in the 9 samples is between 80% and 120%, and the RSD is within 10%, which meets the relevant technical requirements and indicates that the proposed chromatographic conditions are accurate.

[0168] (9) Durability

[0169] The robustness of the experimental analytical method was evaluated by changing the column temperature (25±2℃), flow rate (0.7±0.1ml / min), wavelength (230±2nm), mobile phase ratio (87:13:0.1, 89:11:0.1), and different batches of chromatographic columns.

[0170] For the preparation of stock solutions of impurity XV, XVI, and XVIII reference standards, see “(2) Test Procedure”.

[0171] Impurity reference solution: Accurately transfer 0.1 ml of the stock solution of impurity XV, XVI, and XVIII reference standards into the same 20 ml brown volumetric flask, dissolve and dilute with diluent to prepare a solution of about 1 μg per ml.

[0172] System suitability solution / sample solution: Weigh approximately 10 mg of lumepirozol tosylate, accurately, and place it in a 20 ml brown volumetric flask. Accurately transfer 0.1 ml of the stock solution of impurity XV, XVI, and XVIII reference standards, dissolve and dilute with diluent to prepare a solution containing approximately 500 μg of the main component and 1 μg of each impurity per ml.

[0173] Inject the sample into the liquid chromatograph under the chromatographic conditions described above and record the chromatogram.

[0174] Under fine-tuned chromatographic conditions, the resolution between the three isomer impurities and adjacent peaks in the system suitability solution is greater than 1.5, the content of each impurity in the sample solution is basically consistent, and the system exhibits good robustness.

[0175] Comparative example:

[0176] Comparative examples provide high-performance liquid chromatography (HPLC) detection of samples using different chromatographic columns and mobile phase compositions. Specific methods include:

[0177] (1) High performance liquid chromatography was used to detect lumepirozoline toluenesulfonic acid and its isomers (impurity XV, impurity XVI, impurity XVIII). The specific conditions were: the chromatographic column and different mobile phase compositions in Table 8, and the wavelength of the ultraviolet detector was 230 nm.

[0178] (2) Solution preparation:

[0179] Mixed impurity solution: Take appropriate amounts of the process impurities / degradation impurities (impurity XXI, impurity XXVI, impurity XXXVIII and impurity XXXXII) and isomer impurities XV, XVI, and XVI detected by related substances of lumepirobenone toluenesulfonate, add them to a volumetric flask, dissolve and dilute to the mark with isopropanol, and prepare an impurity stock solution containing 0.2 mg of each impurity per ml. Accurately measure an appropriate amount and dilute with n-hexane-anhydrous ethanol (90:10) to prepare a mixed solution containing 1 μg per ml.

[0180] Separation solution: Weigh an appropriate amount of lumepirozoline toluenesulfonate and place it in a 20 ml volumetric flask. Add an appropriate amount of stock solution of the three isomer impurities and dilute with n-hexane-anhydrous ethanol (90:10) to prepare a solution containing 0.5 mg of the main component and 1 μg of the isomer impurities per ml.

[0181] The mixed impurity solution and the separation solution were tested using a high-performance liquid chromatograph according to the mobile phase and elution method in Table 8. The experimental results are shown in Table 8.

[0182] Table 8 Comparison of different chromatographic columns and mobile phase compositions.

[0183]

[0184]

[0185] Conclusion: The reversed-phase chromatography system could not effectively separate the three isomer impurities; the chiral columns with different packing materials showed different separation degrees for isomer impurities. The ChiralNX(2) column not only met the separation degree requirements for the three isomer impurities, but also effectively separated the process impurities / degradation impurities detected in the related substances from the isomer impurities, thus meeting the quantitative requirements; the addition of diethylamine to the mobile phase improved the peak shape of the main component; different alcohols showed selectivity in the separation degree of isomer impurities. Compared with the comparative examples, Examples 1, 2 and 3 met the separation degree requirements for isomer impurities and adjacent peaks, and also effectively separated the process impurities / degradation impurities detected in the related substances from the isomer impurities, demonstrating high method specificity.

Claims

1. A method for detecting the enantiomers and diastereomers of lumepirobentosulfonate, characterized in that, High performance liquid chromatography (HPLC) was used to detect the enantiomers and diastereomers of lumepirobenone tosylate. The column packing material in the HPLC detection process was amylose-tris(4-chloro-3-methylphenylcarbamate), and the mobile phase included alkanes, alcohols, and amines.

2. The detection method as described in claim 1, wherein isocratic elution is used to pass the mobile phase through the chromatographic column during the high performance liquid chromatography detection process.

3. The detection method according to claim 1 or 2, wherein the alkane is n-hexane; the alcohol is one or more of methanol, ethanol, propanol, and butanol; and the amine is one or more of diethylamine, triethylamine, butylamine, and ethanolamine.

4. The detection method according to any one of claims 1-3, wherein the volume ratio of the alkane, alcohol and amine is (85-95):(5-15):(0.05-0.2), preferably (85-90):(10-15):(0.08-0.15).

5. The detection method according to any one of claims 1-4, wherein the column temperature of the chromatographic column is 25-40℃, preferably 25-35℃, and more preferably 25-30℃.

6. The detection method according to any one of claims 1-5, wherein the flow rate of the mobile phase is 0.5-1.2 ml / min, preferably 0.6-1.1 ml / min, more preferably 0.6-1.0 ml / min, and even more preferably 0.6-0.9 ml / min.

7. The detection method according to any one of claims 1-6, wherein the detection wavelength is 225-235nm, preferably 228-232nm.

8. The detection method according to any one of claims 1-7, wherein the injection volume is 8-12 μl, preferably 9-11 μl.

9. The detection method according to any one of claims 1-8, wherein the detection method is used for the detection and analysis of isomer impurities in lumepirobentosylate raw materials or preparations.

10. The detection method according to any one of claims 1-9, wherein during the high performance liquid chromatography detection process, each solution is diluted with a diluent before detection, wherein the diluent is a mixed solution of alkane and alcohol, preferably n-hexane and anhydrous ethanol, and the volume ratio of n-hexane to anhydrous ethanol is 40-90:10-60.

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