A method for simultaneously determining a plurality of isomer impurities in meloxicam benzene sulfonic acid

Abstract

This invention relates to the field of pharmaceutical testing technology, and more specifically, to a method for simultaneously determining multiple isomer impurities in meropenem besylate. The method employs reversed-phase high-performance liquid chromatography (RP-HPLC) with a protein-bonded chiral column containing covalently bonded α1-acid glycoprotein on a silica gel surface. The mobile phase is a 5-15 mM phosphate buffer solution. By using a phosphate buffer solution as the mobile phase, baseline noise and drift under low-wavelength UV detection conditions are effectively reduced. Combined with the high stereoselectivity of the protein-bonded chiral column, the method successfully achieves simultaneous separation of meropenem with its enantiomers and diastereomers, with complete baseline separation between all chromatographic peaks. This overcomes the technical challenge of poor separation due to similar structures of meropenem isomers, filling the gap in existing methods that struggle to simultaneously control two types of isomer impurities under the same chromatographic conditions.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing technology, and more specifically, to a method for simultaneously determining multiple isomer impurities in melogabalin besylate. Background Technology

[0002] Mirogabalin, developed by Daiichi Sankyo Co., Ltd. of Japan, is a novel selective calcium channel α2δ ligand. Its mechanism of action involves highly efficient and selective binding to the α2δ-1 subunit of voltage-sensitive calcium channel complexes in the central nervous system (CNS). Compared to traditional drugs, this targeting characteristic endows it with better analgesic efficacy and superior safety potential. On January 8, 2019, miregabalin received formal approval from the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan for marketing in the clinical indication of peripheral neuropathic pain (PNP), specifically covering diabetic peripheral neuropathy (DPNP) and postherpetic neuralgia (PHN), providing a new treatment option for patients suffering from long-term neuropathic pain.

[0003] However, melogabalin's active pharmaceutical ingredient exists in its benzenesulfonate form. While this form offers certain advantages in drug formulation development, its physicochemical properties pose significant challenges to analytical detection and purification processes. Firstly, the relatively small number of double bonds in its molecular structure results in weak UV absorption signals and low sensitivity under conventional UV detectors, placing higher demands on the development of methodologies for content determination and impurity profiling. Secondly, this compound possesses both distinct acidic and basic groups, exhibiting high overall polarity and weak retention in reversed-phase chromatography, increasing the difficulty of optimizing chromatographic separation conditions. Its chemical structural formula is as follows:

[0004] More importantly, meregabalin contains a chiral center and exhibits cis-trans isomerism, with isomers possessing extremely similar physicochemical properties and poor separation. This not only poses a technical bottleneck to the quality control of the active pharmaceutical ingredient (especially the limit testing of isomer impurities) but also imposes stringent requirements on the stereoselectivity of the synthetic route and the crystallization purification process. Therefore, developing an efficient, stable analytical detection method and purification process that can effectively control isomer impurities, based on the aforementioned physicochemical characteristics of meregabalin benzenesulfonate, is a pressing technical problem that needs to be solved in the current industrialization process of this drug. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. Therefore, one aspect of this invention is to provide a method for simultaneously determining multiple isomer impurities in melogabalin benzyl sulfonate. The method employs reversed-phase high-performance liquid chromatography (RP-HPLC), and the specific operational steps of the method are as follows: S1. Test solution: Accurately weigh merogabarine benzylsulfonic acid, dissolve it in solvent and dilute it quantitatively to prepare the test solution; S2. Mixed solution: Take merogabaline benzyl sulfonate, impurity A, impurity B, impurity C and impurity D respectively, weigh them accurately, and dilute them quantitatively with solvent to prepare a mixed solution containing merogabaline benzyl sulfonate and each impurity. S3 Impurity Localization Solution: Accurately weigh impurity A, impurity B, impurity C, and impurity D respectively, and quantitatively dilute them with solvent to prepare a localization solution; S4. Adjust the instrument: The chromatographic column is a protein-bonded chiral column, the mobile phase is A, adjust the column temperature, adjust the mobile phase flow rate, and adjust the detection wavelength; S5. Determination: Accurately measure each solution prepared in S1 to S3, inject them into the liquid chromatograph, and record the chromatogram.

[0006] Preferably, the concentration of the test solution in S1 is 1 mg / ml.

[0007] Preferably, the concentrations of each component in the mixed solution in S2 are 1 mg / ml of melogabalin benzyl sulfonate and 1 μg / ml of each impurity.

[0008] Preferably, the concentration of each positioning solution in S3 is 10 μg / ml.

[0009] Preferably, the solvent is water.

[0010] Preferably, impurity A in S3 is an enantiomer with the following structural formula: Impurity B is diastereomer 1, with the following structural formula: Impurity C is diastereomer 2, with the following structural formula: Impurity D is an isomer with the following structural formula:

[0011] Preferably, the chromatographic column in S4 is a Daicel CHIRALPAK AGP (40mm×150mm, 5μm).

[0012] Preferably, in step S4, the mobile phase A is a 5-15 mM phosphate buffer solution. Preferably, the phosphate buffer solution is adjusted to pH 6.5-7.0 with phosphoric acid.

[0013] Preferably, in step S4, the detection wavelength is 205 nm, the column temperature is 22 °C, the injection volume is 10 μL, and the flow rate is 0.3 mL / min.

[0014] The beneficial effects of this invention are as follows: Simultaneous baseline separation of multiple isomers was achieved: By using phosphate buffer solution as the mobile phase, baseline noise and drift under low-wavelength ultraviolet detection conditions were effectively reduced. At the same time, combined with the high stereoselectivity of protein-bonded chiral chromatographic columns, the simultaneous separation of merogabarine with its enantiomers and diastereomers was successfully achieved, and complete baseline separation (resolution R≥1.5) was achieved between each chromatographic peak. This overcame the technical challenge of poor resolution due to the similar structure of merogabarine isomers and filled the gap in existing methods that could not simultaneously control two types of isomer impurities under the same chromatographic conditions.

[0015] High detection sensitivity meets stringent quality control requirements: The detection sensitivity for various isomers of meregabalin reaches 0.25 μg / mL, effectively detecting trace amounts of isomer impurities in the active pharmaceutical ingredient (API). This high sensitivity ensures accurate and reliable quantitative detection even at extremely low impurity levels, providing strong technical support for the development of quality standards for meregabalin besylate API.

[0016] High specificity and excellent anti-interference ability: Thanks to the synergistic effect of phosphate buffer and chiral stationary phase, this method has excellent specific recognition ability for various isomer impurities. There is no mutual interference between the main component peak and each impurity peak, and the excipient and solvent peaks do not interfere with the determination of the target analyte, ensuring the authenticity and reliability of the detection results.

[0017] The method is stable and reliable, and suitable for quality control in industrial production: the detection method is simple to operate, the chromatographic conditions are mild, and the system has good applicability. It is applicable to the quality control links of the entire production cycle, such as monitoring intermediates in the synthesis of merogabarine besylate API, finished product release inspection, and stability studies. It can effectively ensure the safety and efficacy of drugs and has broad prospects for industrial application.

[0018] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is the chromatogram of the impurity A localization solution in Embodiment 1 of the present invention.

[0020] Figure 2This is the chromatogram of the impurity B localization solution in Embodiment 1 of the present invention.

[0021] Figure 3 This is the chromatogram of the impurity C localization solution in Embodiment 1 of the present invention.

[0022] Figure 4 This is the chromatogram of the impurity D localization solution in Embodiment 1 of the present invention.

[0023] Figure 5 This is a chromatogram of the mixed solution in Embodiment 1 of the present invention.

[0024] Figure 6 This is the chromatogram of the test solution in Example 1 of the present invention.

[0025] Figure 7 This is the blank solvent chromatogram in Example 1 of the present invention. Detailed Implementation

[0026] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0028] This invention provides a method for simultaneously determining enantiomers and diastereomers of merogabarine benzyl sulfonate, using the following conditions: Column: Cairalpak AGP (40mm×150mm, 5μm); Mobile phase: 10 mM diammonium hydrogen phosphate buffer (adjusted to pH 7.0 with phosphoric acid); Detection wavelength: 205 nm, column temperature: 22 ℃, injection volume: 10 μL, flow rate: 0.3 mL / min.

[0029] Test solution: Accurately weigh merogabaline benzyl sulfonic acid, dissolve it in water and dilute quantitatively to prepare a 1 mg / ml test solution; Mixed solution: Take merogabaline benzyl benzyl acid, impurity A, impurity B, impurity C and impurity D respectively, weigh them accurately, and dilute them quantitatively with water to prepare a mixed solution containing 1 mg / ml merogabaline benzyl benzyl acid and 1 μg / ml of each impurity; Impurity localization solution: Weigh impurity A, impurity B, impurity C and impurity D accurately, and dilute with water to prepare a 10 μg / ml localization solution; The specific preparation process of each solution and the experimental verification process and results of the method of the present invention are shown in Experiment Examples 1 to 5.

[0030] Example 1 Specificity test Preparation of the test solution: Weigh approximately 10 mg of melogabalin benzyl sulfonate accurately, place it in a 10 ml volumetric flask, dissolve and dilute with water to the mark, and shake well to obtain the test solution.

[0031] Preparation of the mixed solution: Take appropriate amounts of melogabalin besylate, impurity A, impurity B, impurity C, and impurity D reference standards, dissolve and dilute them with water to prepare a mixed solution containing approximately 1 mg of melogabalin besylate and 1 μg of each impurity per 1 ml.

[0032] Preparation of positioning solutions: Take appropriate amounts of impurity A, impurity B, impurity C, and impurity D reference standards, dissolve them in water, and dilute them quantitatively to prepare solutions containing approximately 10 μg of each impurity per 1 ml. Shake well to obtain positioning solutions for each impurity.

[0033] Accurately measure the above solvent, each positioning solution, test solution, and mixed solution, and inject them separately into the liquid chromatograph, recording the chromatograms. Results are as follows: Figures 1-7 As shown.

[0034] Conclusion: The solvent does not interfere with the detection of the main peak and various isomer impurities, and the components are well separated, indicating that the method has good specificity.

[0035] Example 2 Linearity and Range Test Preparation of linear solutions: Take appropriate amounts of melogabalin benzyl sulfonate, impurity A, impurity B, impurity C, and impurity D reference standards, respectively, and dilute them quantitatively with water to prepare solutions containing 10 μg of each component per ml as linear stock solutions. Accurately measure appropriate amounts and dilute with water to prepare a series of linear solutions.

[0036] Accurately measure each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. The results are shown in Table 2.

[0037] Table 1. Results of Linearity and Range Tests

[0038] Example 3 Limit of Quantification and Limit of Detection Test Limit of Quantification Solution: Based on the signal-to-noise ratio of each component in the linear solution, a solution with a signal-to-noise ratio of not less than 10 is prepared and used as the limit of quantification solution.

[0039] Limit of Detection Solution: Take 3.5 ml of the limit of quantitation solution, place it in a 10 ml volumetric flask, dilute with water to the mark, and shake well to obtain the limit of detection solution.

[0040] Accurately measure each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. The results are shown in Tables 3 and 4.

[0041] Table 2. Results of Limit of Quantitation and Limit of Detection

[0042] Table 3. Repeatability results at the limit of quantitation

[0043] Conclusion: The signal-to-noise ratio (SNR) of the solutions at the limits of quantitation for each component was greater than 10. After six repeated injections, the retention time RSD was less than 1.0% and the peak area RSD was less than 10.0%. The SNR of the solutions at the limits of detection was greater than 3, indicating good method sensitivity.

[0044] Example 4 Recovery rate test Impurity reference standards: Accurately weigh appropriate amounts of impurity A, impurity B, impurity C, and impurity D reference standards, and quantitatively dilute them with water to prepare a mixed solution containing 10 μg per ml, which serves as the reference standard stock solution. Accurately measure 1 ml of the reference standard stock solution, place it in a 10 ml volumetric flask, dilute with water to the mark, and shake well; prepare two parallel aliquots.

[0045] Preparation of accuracy solution: 50% accuracy solution: Take about 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve it, add 0.5 ml of impurity reference stock solution, dilute with water to the mark, and shake well to obtain the solution; prepare 3 parallel portions.

[0046] 100% accuracy solution: Take about 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve it, add 1 ml of impurity reference stock solution, dilute with water to the mark, shake well, and you have the solution; prepare 6 parallel portions.

[0047] 150% accuracy solution: Take about 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve it, add 1.5 ml of impurity reference stock solution, dilute with water to the mark, and shake well to obtain the solution; prepare 3 parallel portions.

[0048] Background solution: Take about 10 mg of this product, place it in a 10 ml volumetric flask, add water to dissolve and quantitatively dilute to the mark, shake well, and the solution is ready.

[0049] 7. Accurately measure each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. The results are shown in Tables 4-7.

[0050] Table 4. Recovery results of impurity A

[0051] Table 5. Recovery results of impurity B

[0052] Table 6. Results of Impurity C Recovery

[0053] Table 7. Results of Impurity D Recovery

[0054] Conclusion: The recovery rate test results show that the recovery rates of each component in the accuracy samples are between 80% and 120%. As can be seen from the table above, the recovery rate meets the determination requirements, indicating that the accuracy of this method is good.

[0055] Example 5 Durability test The solvent, mixed solution, and test solution from Experiment Example 1 were tested at column temperatures of 20℃ and 25℃, and flow rates of 0.2 ml / min and 0.4 ml / min, respectively. The results are shown in Table 8.

[0056] Table 8. Durability Test Results

[0057] Conclusion: Under fine-tuned chromatographic conditions, the solvents did not interfere with the detection of the main peak and various isomer impurities. No isomer impurities were detected in the test solution under all conditions, and the detection results were consistent, indicating that the method has good robustness.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. A method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid, characterized in that: The detection method employs reversed-phase high-performance liquid chromatography (RP-HPLC), and the specific operational steps of the detection method are as follows: S1. Test solution: Accurately weigh merogabarine benzylsulfonic acid, dissolve it in solvent and dilute it quantitatively to prepare the test solution; S2. Mixed solution: Take merogabaline benzyl sulfonate, impurity A, impurity B, impurity C and impurity D respectively, weigh them accurately, and dilute them quantitatively with solvent to prepare a mixed solution containing merogabaline benzyl sulfonate and each impurity. S3 Impurity Localization Solution: Accurately weigh impurity A, impurity B, impurity C, and impurity D respectively, and quantitatively dilute them with solvent to prepare a localization solution; S4. Adjust the instrument: The chromatographic column is a protein-bonded chiral column, the mobile phase is A, adjust the column temperature, adjust the mobile phase flow rate, and adjust the detection wavelength; S5. Determination: Accurately measure each solution prepared in S1 to S3, inject them into the liquid chromatograph, and record the chromatogram.

2. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The concentration of the test solution in S1 is 1 mg / ml.

3. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The concentrations of each component in the mixed solution in S2 are 1 mg / ml of melogabalin benzyl sulfonate and 1 μg / ml of each impurity.

4. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The concentration of each positioning solution in S3 is 10 μg / ml.

5. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The solvent is water.

6. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: In S3, impurity A is an enantiomer with the structural formula: Impurity B is diastereomer 1, with the following structural formula: Impurity C is diastereomer 2, with the following structural formula: Impurity D is an isomer with the following structural formula: .

7. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The chromatographic column in S4 is a Daicel CHIRALPAK AGP.

8. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: In S4, the mobile phase A is a 5-15 mM phosphate buffer solution.

9. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 8, characterized in that: The phosphate buffer solution was adjusted to pH 6.5-7.0 with phosphoric acid.

10. The method for simultaneously determining multiple isomer impurities in melogabalin benzylsulfonic acid according to claim 1, characterized in that: The detection wavelength in S4 is 205 nm, the column temperature is 22 °C, the injection volume is 10 μL, and the flow rate is 0.3 mL / min.

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