A method for detecting meloxicam and its isomers by high performance liquid chromatography

Abstract

The present application relates to a kind of methods for detecting meloxicam and its isomers by high performance liquid chromatography, which uses high performance liquid chromatography for detection, and the chromatographic column uses covalent bond and cellulose-tris (3, 5-dichlorophenyl carbamate) as filler;With n-hexane-ethanol-trifluoroacetic acid-triethylamine as mobile phase;Isocratic elution;Column temperature is 20~30 ℃;Flow rate is 0.8~1.1ml / min;Detection wavelength is 215nm.The high performance liquid chromatography detection method of meloxicam and its isomers provided by the present application can realize the complete separation of meloxicam main component and its three isomer impurities, and this method has strong specificity, high sensitivity and good durability, and can be used for the quality control of meloxicam bulk drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to a method for detecting melogabalin besylate and its isomers by high performance liquid chromatography. Background Technology

[0002] Merogabalin besylate is a drug developed by Daiichi Sankyo Co., Ltd., similar to drugs such as gabapentin and pregabalin. Merogabalin is a new generation of selective calcium channel modulator that selectively binds to the α2δ-1 subunit of voltage-gated calcium channels. It shows sustained analgesic effects on various chronic peripheral neuropathic pain conditions, including diabetic peripheral neuropathic pain and postherpetic neuralgia, and its efficacy is significantly higher than that of the second-generation pregabalin. Its structural formula is as follows:

[0003]

[0004] The content of impurities in pharmaceutical products directly reflects the stereoselectivity of the synthesis process and the stability of the product, and is a key indicator for evaluating drug purity and ensuring efficacy. Isomeric impurities not only lack therapeutic activity but may also alter target binding selectivity, increasing the risk of adverse reactions. Merogabalin besylate contains multiple chiral centers, including enantiomers (Y-1) and diastereomers (Y-2, Y-3). Developing methods for detecting merogabalin besylate and its isomeric impurities is a necessary prerequisite for ensuring drug safety, efficacy, and compliance with registration regulations.

[0005]

[0006] Patent CN117388402 A discloses a gas chromatography method for detecting melobalin besylate and its isomers. This method uses a fused silica capillary column filled with β-cyclodextrin as a chiral column, and employs DMSO, DMF, DMA, or sulfolane to completely dissolve the sample before separation and detection in a gas chromatograph. However, this method involves expensive columns and high detection costs. Patent CN120334387 A discloses a method for detecting melobalin besylate and its isomers. This method first reacts the sample with FXAA reagent under alkaline conditions to obtain a test solution, which is then separated by liquid chromatography. This method has a cumbersome pretreatment process and poor practicality. Patent CN113740471 A discloses a high-performance liquid chromatography method for detecting melobalin and its enantiomers, but it does not cover the separation and detection of diastereomers, thus limiting its application scope.

[0007] Therefore, establishing accurate, reliable, simple, and rapid analytical methods for the detection of impurities in melogabalin besylate and its isomers is a crucial issue that urgently needs to be addressed in the quality control of this drug. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a high-performance liquid chromatography (HPLC) method for the detection of merogabarine benzyl sulfonate and its isomers. This method is highly specific, sensitive, and robust, and can accurately quantify both the main component and isomer impurities, making it suitable for the quality control of merogabarine benzyl sulfonate.

[0009] The merogabaline benzyl sulfonic acid structure in this invention is as follows:

[0010]

[0011] The enantiomer (Y-1) and diastereomer impurities (Y-2, Y-3) in this invention have the following structural formulas:

[0012]

[0013] The technical solution of the present invention is as follows:

[0014] Specifically, this invention provides a method for detecting melogabalin besylate and its isomers, using high-performance liquid chromatography (HPLC) to detect the isomers in melogabalin besylate.

[0015] The high-performance liquid chromatography (HPLC) detection conditions are as follows: a CHIRALPAK IC-3 column with covalent bonds and cellulose-tris(3,5-dichlorophenylcarbamate) as the packing material is used; the column temperature is 20~30℃; the column flow rate is 0.8~1.1 ml / min; the detection wavelength is 215 nm; the mobile phase is n-hexane-ethanol-trifluoroacetic acid-triethylamine in a ratio of 900~907:100~93:1:1; isocratic elution is performed for 0~26 min; and the injection volume is 1 mL.

[0016] Preferably, the column temperature is 25°C and the column flow rate is 1.0 ml / min.

[0017] Furthermore, the test solution was prepared using hexane-ethanol as a diluent in a ratio of 50:50.

[0018] Furthermore, the chromatographic column has dimensions of 250 mm × 4.6 mm and a diameter of 3 μm.

[0019] Furthermore, the impurity content (%) was calculated using a self-comparison method.

[0020] The specific detection and calculation methods provided by this invention are as follows: using n-hexane-ethanol (50:50) as a blank solution; dissolving the reference standards of melogabalin besylate, its enantiomer (Y-1), and diastereomer impurities (Y-2, Y-3) in 5 ml of ethanol by sonication, and then diluting them with a diluent to prepare a positioning solution with a concentration of 1 mg / ml; dissolving the reference standards of melogabalin besylate in 5 ml of ethanol by sonication, and then diluting them with a diluent to prepare a test solution with a concentration of 1 mg / ml; taking an appropriate amount of the test solution and diluting it with a diluent to prepare a solution with a concentration of 15 μg / ml; injecting each solution sequentially into the chromatograph and collecting chromatograms; calculating the impurity content (%) using the self-comparison method.

[0021] formula:

[0022]

[0023] In the formula:

[0024] Ai: Peak area of ​​each impurity in the test solution;

[0025] Ar: Peak area of ​​the control solution;

[0026] X: Control solution concentration level, 0.15%.

[0027] The beneficial effects of this invention are:

[0028] 1. The high-performance liquid chromatography (HPLC) method for the detection of melogabalin besylate and its isomers provided by this invention can achieve complete separation (R≥2.0) of the main component and three isomer impurities (enantiomer Y-1, diastereomers Y-2 and Y-3). It has high specificity and can effectively avoid the interference of isomer impurities on the detection of the main component. It can be used for the quality control of melogabalin besylate raw material.

[0029] 2. Compared with the prior art, the present invention does not require derivatization pretreatment, is easy to operate, has mild detection conditions, good durability, and the method has high sensitivity and can accurately quantify, which can meet the needs of detecting impurity content in melogabalin besylate raw materials. Attached Figure Description

[0030] Figure 1 This is the high-performance liquid chromatogram of a blank solution (n-hexane-ethanol).

[0031] Figure 2 The high-performance liquid chromatogram of melogabalin benzyl sulfonic acid localization solution.

[0032] Figure 3 High performance liquid chromatogram of the solution for isomer (Y-1).

[0033] Figure 4High performance liquid chromatogram of the solution for isomer (Y-2).

[0034] Figure 5 High performance liquid chromatogram of the solution for isomer (Y-3).

[0035] Figure 6 High-performance liquid chromatography (HPLC) chromatogram for localization of melogabalin benzyl sulfonic acid and its isomer mixture.

[0036] Figure 7 The graph shows the regression equation curve for melogabalin benzyl sulfonate.

[0037] Figure 8 The graph shows the regression equation curve for the isomer (Y-1).

[0038] Figure 9 The graph shows the regression equation curve for the isomer (Y-2).

[0039] Figure 10 The graph shows the regression equation curve for the isomer (Y-3).

[0040] Figure 11 The degree of separation between melogabalin and its isomers under different conditions. Detailed Implementation

[0041] The following embodiments are only used to further illustrate the present invention and do not limit the scope of the invention to these embodiments.

[0042] The high-performance liquid chromatograph used in this invention is manufactured by Shimadzu, and its model is LC-2010C HT.

[0043] The chromatographic column used in this invention is manufactured by DACEL and its model is CHIRALPAK IC-3.

[0044] The merogabalin besylate raw material used in this invention was manufactured in-house by North China Pharmaceutical New Drug Group Co., Ltd. The reference standards for merogabalin besylate (purity 99.5%), isomer (Y-1, purity 93.7%), isomer (Y-2, purity 96.7%), and isomer (Y-3, purity 97.0%) were all manufactured by Nanjing Weikaier Company.

[0045] The manufacturers of n-hexane, ethanol, and trifluoroacetic acid are all Merck.

[0046] Triethylamine is an Aladdin reagent.

[0047] Example 1: Specificity of the present invention of mesologabarin and its three isomers

[0048] 1. Sample preparation:

[0049] Diluent (blank solution): n-hexane-ethanol (50:50)

[0050] Merogabalin positioning solution: Accurately measure about 35.2 mg of merogabalin besylate reference standard, place it in a 20 ml volumetric flask, add 5 ml of ethanol and sonicate to dissolve, then dilute to the mark with diluent and shake well.

[0051] Isomer (Y-1~Y-3) positioning solution: Accurately weigh 20mg each of isomers Y-1, Y-2 and Y-3, place them in 20ml volumetric flasks, add 5ml of ethanol and sonicate to dissolve, then dilute to the mark with diluent and shake well.

[0052] Merogabalin and isomer mixed solution: Accurately weigh 5 ml each of the merogabalin positioning solution and the isomer (Y-1~Y-3) positioning solution and place them in the same 20 ml volumetric flask, and shake well.

[0053] 2. High-performance liquid chromatography (HPLC) detection:

[0054] Inject 10 μL of each of the blank solution, positioning solution, and mixed solution into the high-performance liquid chromatograph (HPLC) and record the chromatograms. The chromatographic conditions are as follows:

[0055] Column: DAICEL CHIRALPAK IC-3 250mm × 4.6mm × 3μm

[0056] Flow rate: 1.0 ml / min

[0057] Column temperature: 25℃

[0058] Detection wavelength: 215nm

[0059] Mobile phase: n-hexane-ethanol-trifluoroacetic acid-triethylamine (905:95:1:1)

[0060] Isocratic elution for 0-26 minutes.

[0061] 3. Results:

[0062] The blank solution did not interfere with the detection of the meropenem peak. The retention times of each component in the mixed positioning solution were consistent with their respective positioning solutions, and the elution order was Y-1, meropenem, Y-3, and Y-2. The minimum resolution (USP) between the meropenem peak and all impurity peaks reached 2.844, indicating complete separation. Data are shown in Table 1. Chromatograms are shown below. Figure 1-6 .

[0063] Table 1. Specificity of melogabalone and its three isomers

[0064]

[0065] Example 2: Sensitivity test of the present invention for mesologabarin and its three isomers

[0066] The chromatographic conditions were the same as in Example 1.

[0067] 1. Sample preparation:

[0068] Diluent (blank solution): n-hexane-ethanol (50:50)

[0069] Limit of Quantification (LOQ) Solution: Take the melogabalin and its isomer positioning solutions from Example 1, and quantitatively dilute them stepwise with diluent to a low concentration solution. Prepare 6 parallel portions of each component.

[0070] Limit of Detection (LOD) solution: Take 5 ml of the LOQ solution under this test and place it in a 10 ml volumetric volumetric flask. Dilute to the mark with diluent and mix well. Prepare two parallel portions of each component.

[0071] 2. High-performance liquid chromatography (HPLC) detection:

[0072] 10 μl each of the blank solution, LOQ solution, and LOD solution of each component were injected into the high-performance liquid chromatograph, the chromatograms were recorded, and the signal-to-noise ratio (S / N) was calculated. The concentration with a S / N ≥ 10 was taken as the LOQ, and the concentration with a S / N ≥ 3 was taken as the LOD.

[0073] 3. Results:

[0074] The LOQ of isomer (Y-1) was 2.86 μg / ml, and the LOD was 1.43 μg / ml; the LOQ of melogabalin was 3.01 μg / ml, and the LOD was 1.50 μg / ml; the LOQ of isomer (Y-3) was 2.89 μg / ml, and the LOD was 1.44 μg / ml; the LOQ of isomer (Y-2) was 2.95 μg / ml, and the LOD was 1.47 μg / ml. See Tables 2-3. The signal-to-noise ratio (S / N) of the six LOQ solutions for each component was greater than 10, the RSD (%) of the retention time was less than 1, and the RSD (%) of the peak area was less than 10; the signal-to-noise ratio (S / N) of the peak area in the LOD solutions was greater than 3. This method has high sensitivity for the determination of melogabalin besylate and its three isomers.

[0075] Table 2. LOQ solutions of merogabaline and its three isomers

[0076]

[0077] Table 3 LOD solutions of melogabalin and its three isomers

[0078]

[0079] Example 3: Linearity and Range Experiments of the Invention on Mid-Logabalin and its Three Isomers

[0080] The chromatographic conditions were the same as in Example 1.

[0081] 1. Sample preparation:

[0082] Diluent (blank solution): n-hexane-ethanol (50:50)

[0083] Merogabalin stock solution: Accurately weigh approximately 52.7 mg of melogabalin besylate reference standard into a 20 ml volumetric flask containing 5 ml of ethanol, dissolve, and dilute to the mark with diluent. Shake well.

[0084] Stock solutions of isomers (Y-1, Y-3): Accurately weigh approximately 30 mg each of Y-1 and Y-3 reference standards into the same 20 ml volumetric flask containing 5 ml of ethanol, dissolve, dilute to the mark with diluent, and shake well.

[0085] Isomer (Y-2) stock solution: Accurately weigh about 40 mg of Y-2 reference standard into a 20 ml volumetric flask containing 5 ml of ethanol, dissolve, dilute to the mark with diluent, and shake well.

[0086] Linear stock solution: Accurately weigh 2 ml each of the isomer (Y-1, Y-3) stock solution and the melogabalin stock solution into the same 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0087] Linear solutions: Accurately measure the linear stock solution and the isomer (Y-2) stock solution respectively to prepare linear solutions of merogabarine and the three isomer impurities according to the schemes listed in Table 4, and dilute to the mark with diluent.

[0088] The theoretical concentrations of the linear solutions of the obtained melogabalone and its three isomer impurities are shown in Table 4.

[0089] 2. High-performance liquid chromatography (HPLC) detection:

[0090] Inject the blank solution and each linear solution into the high-performance liquid chromatograph, 10 μl of each, and record the chromatogram.

[0091] Table 4 Linear Solution Preparation Scheme

[0092]

[0093] 3. Results:

[0094] Linear curves were plotted based on the concentration (X) and measured peak area (Y) of each component, as shown in Table 5. The linear equations for the obtained isomers were: Y = 3884.7288X - 2493.7624 (r = 0.9999; n = 6), for melogabalone Y = 4116.4463X - 1626.6861 (r = 0.9999; n = 6), for isomer Y = 4352.4991X - 362.0872 (r = 0.9997; n = 6), and for isomer Y = 3935.3355X - 5550.4528 (r = 0.9989; n = 6). All components showed good linearity within the LOQ to 200% limit concentration range. See [link to table]. Figures 7-10 .

[0095] Table 5. Linear solutions of meropenem and its three isomers

[0096]

[0097] Example 4: Recovery rate experiment of the three isomers in this invention

[0098] The chromatographic conditions were the same as in Example 1.

[0099] 1. Sample preparation:

[0100] Diluent (blank solution): n-hexane-ethanol (50:50)

[0101] Recovery stock solution: Weigh 18.8 mg of each of the isomers (Y-1~Y-3) reference standards and place them in the same 25 ml volumetric flask containing 5 ml of ethanol. Dissolve and dilute to volume with diluent. Take 5 ml of the above solution, place it in a 25 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0102] Test solution: Take 352 mg of merogabaline besylate raw material, place it in a 20 ml volumetric flask, add 5 ml of ethanol and sonicate to dissolve, then dilute to the mark with diluent and shake well to obtain the background solution.

[0103] Recovery solutions: Take 352 mg of merogabarine besylate raw material, place it in a 20 ml volumetric flask, add 5 ml of ethanol to dissolve it, add 1.0 ml, 2.0 ml and 3.0 ml of recovery stock solution respectively, and dilute to the mark with diluent to prepare recovery solutions with limits of 50%, 100% and 150% respectively. Prepare 3 replicates for each limit.

[0104] Control solution: Take 1 ml of the test solution and the recovery solution of each concentration, put them into a 100 ml volumetric flask, dilute to volume with diluent, shake well, then accurately measure 1.5 ml, put it into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0105] 2. High-performance liquid chromatography (HPLC) detection:

[0106] Inject 10 μl each of blank solution, test solution, accuracy solutions at each limit concentration, and control solution into the high-performance liquid chromatograph and record the chromatograms.

[0107] 3. Results:

[0108] None of the three isomers were detected in the test solution. The recoveries of each impurity in the solution were calculated using the self-comparison method, as shown in Tables 6-8. At the three concentration limits of 50%, 100%, and 150%, the recoveries of the three isomers were all between 90% and 110%, with RSD% less than 10.0% (n=9). This method is effective for the determination of merogabarine and its three isomers.

[0109] Table 6 Recovery of isomer (Y-1)

[0110]

[0111] Table 7 Recovery of isomer (Y-3)

[0112]

[0113] Table 8 Recovery of isomer (Y-2)

[0114]

[0115] Example 5: Durability test of the three isomers in this invention

[0116] The durability test items are examined according to the scheme in Table 9.

[0117] Table 9 Durability Test Items

[0118]

[0119] During the durability test, the remaining chromatographic conditions were the same as in Example 1.

[0120] 1. Sample preparation:

[0121] Diluent (blank solution): n-hexane-ethanol (50:50)

[0122] Impurity stock solution: Weigh 18.8 mg of each of the isomers (Y-1~Y-3) reference standards and place them in different 25 ml volumetric flasks containing 5 ml of ethanol. Dissolve and dilute to volume with diluent. Take 4 ml of each of the above solutions, place them in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0123] Mixed impurity solution: Dissolve 352 mg of melogabalin benzyl sulfonate reference standard in a 20 ml volumetric flask containing 5 ml of ethanol, add 2 ml of mixed impurity stock solution, dilute to the mark with diluent, and shake well.

[0124] Test solution: Take 352 mg of melogabalin besylate raw material, place it in a 20 ml volumetric flask, add 5 ml of ethanol to dissolve it, dilute to the mark with diluent, and shake well.

[0125] Spiked test solution: Take 352 mg of melogabalin besylate raw material, place it in a 20 ml volumetric flask, add 5 ml of ethanol to dissolve it, add 2 ml of impurity stock solution, dilute to the mark with diluent, and shake well.

[0126] Control solution: Measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to volume with diluent and shake well. Then accurately measure 1.5 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0127] 2. High-performance liquid chromatography (HPLC) detection:

[0128] Inject 10 μl each of the blank solution, mixed impurity solution, spiked test solution, and control solution into the high-performance liquid chromatograph (HPLC) and record the chromatograms. Examine the changes in resolution. Calculate the content of the three isomers in the spiked test solution using the self-comparison method.

[0129] 3. Results:

[0130] Under the analytical conditions for isomer impurities of this product, when there were minor changes in flow rate, column temperature, and mobile phase ratio, the peak elution order remained consistent with the original conditions. The maximum difference in the content of the three isomer impurities in the labeled test solution compared to the original conditions was 0.02% (see Table 10). The minimum resolution between each peak was 2.10, indicating complete separation. Figure 11 The high-performance liquid chromatography (HPLC) method for the detection of melogabalin and its isomers established in this invention has good robustness.

[0131] Table 10 Comparison of isomer impurity content under different conditions with data under original conditions

[0132]

[0133] Comparative Example 1: Determination of isomers in melogabalin benzyl sulfonate by high performance liquid chromatography

[0134] 1. Sample preparation:

[0135] Diluent (blank solution): Water

[0136] Merogabalin raw material and isomer mixed solution: Take appropriate amounts of merogabalin besylate and isomer (Y-1~Y-3) reference standards, add ethanol and sonicate to dissolve, and dilute quantitatively with diluent to prepare a solution containing about 2 mg of merogabalin besylate and 5 μg of other impurities per ml.

[0137] 2. High-performance liquid chromatography (HPLC) detection:

[0138] Inject 10 μL of each of the blank solution and mixed solution into the high-performance liquid chromatograph (HPLC) and record the chromatograms. The chromatographic conditions are as follows:

[0139] Chromatographic column: DAICEL CHIRALCEL OJ-RH (4.6mm × 150mm, 5μm)

[0140] Flow rate: 1.0 ml / min

[0141] Column temperature: 25℃

[0142] Detection wavelength: 215nm

[0143] Mobile phase: 0.05% phosphoric acid solution: acetonitrile (95:5)

[0144] Isocratic elution for 0-30 minutes.

[0145] 3. Results: Under these conditions, the retention time of melogabalone was 4.637 min, and the retention times of the isomer impurities (Y-1~Y-3) were all the same (2.868 min), which not only resulted in excessively rapid elution but also made it impossible to separate the impurities.

Claims

1. A method for detecting melogabalin besylate and its isomers by high performance liquid chromatography, characterized in that: 1) The high-performance liquid chromatography (HPLC) detection conditions are as follows: a chromatographic column with covalent bonds and cellulose-tris(3,5-dichlorophenylcarbamate) as the packing material is used; the column temperature is 20~30℃; the column flow rate is 0.8~1.1ml / min; the detection wavelength is 215nm; the mobile phase is n-hexane-ethanol-trifluoroacetic acid-triethylamine in a ratio of 900~907:100~93:1:1, isocratic elution is performed, and the injection volume is 1mL. 2) Diluent: n-hexane-ethanol; 3) Determination: Accurately measure the test solution and inject it into the high performance liquid chromatograph, record the chromatogram, and calculate the impurity content using the self-comparison method.

2. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The chromatographic column was a DAICL CHIRALPAK IC-3 with dimensions of 250 mm × 4.6 mm × 3 μm.

3. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The column temperature was 25°C.

4. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The flow rate is 1.0 ml / min.

5. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The ratio of n-hexane-ethanol-trifluoroacetic acid-triethylamine in the mobile phase is 905:95:1:

1.

6. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The ratio of n-hexane-ethanol-trifluoroacetic acid-triethylamine in the mobile phase is 900:100:1:

1.

7. The method for detecting meloxicam and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The ratio of n-hexane-ethanol-trifluoroacetic acid-triethylamine in the mobile phase is 907:93:1:

1.

8. The method for detecting melogabalin besylate and its isomers by high performance liquid chromatography according to claim 1, characterized in that, The diluent is hexane-ethanol in a ratio of 50:50.

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