Method for detecting isomer content in brivaracetam oral solution
The tandem method solves the problem of difficult isomer separation in briracetam oral solution in the prior art, achieving high efficiency, specificity, system applicability and robustness of chromatographic conditions, and providing an effective detection method.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANDONG QIDU PHARMA
- Filing Date
- 2026-03-12
- Publication Date
- 2026-07-10
AI Technical Summary
Existing technologies are difficult to achieve high-resolution separation and accurate quantitative detection of bricetramine and its isomers in oral bricetramine solutions, and the operation is cumbersome, easily affected by excipients, and has poor applicability.
High-performance liquid chromatography (HPLC) was used to separate the isomers by using a series of octadecylsilane-bonded silica gel and linear starch-tris-(4-chloro-3-methylphenylcarbamate) chiral columns, combined with gradient elution and valve switching pathways, and using 0.1% phosphoric acid solution and acetonitrile as the mobile phase.
It achieves effective separation of isomers in briracetam oral solution, avoids interference from excipients, and achieves the desired detection effect with high accuracy, strong applicability, and good detection results.
Smart Images
Figure CN121831003B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of pharmaceutical quality testing technology, specifically relating to a method for detecting the content of isomers in bricetrastran oral solution. Background Technology
[0002] Brivaceratam is a new generation of pyrrolidone antiepileptic drug, with the structural formula... It has advantages such as rapid onset of action, good tolerability, and low risk of drug-drug interactions. This molecule contains two chiral centers, and the (2S, 4R) configuration is used clinically; its enantiomers and other stereoisomers may have different pharmacological activities or toxicities, therefore, pharmacopoeias of various countries and ICH guidelines all require strict control of isomer impurities in formulations.
[0003] Biriracetam oral solution is a liquid formulation containing purified water as the solvent and various water-soluble excipients such as sweeteners, flavoring agents, and buffer salts. Because the matrix of this formulation is aqueous, traditional normal-phase chromatographic systems (such as the n-hexane-isopropanol system) are not suitable due to the polarity mismatch with the sample, easily causing peak tailing, decreased sensitivity, or even failure to inject the sample. While conventional reversed-phase high-performance liquid chromatography (RP-HPLC) is compatible with aqueous matrices, the diverse types and wide polarity range of excipients in oral solutions easily lead to co-elution or matrix interference near the target peak, severely affecting the specific separation and accurate quantification of isomers.
[0004] Patent CN112834674A discloses an HPLC method using a normal-phase system (n-hexane-isopropanol-organic base) combined with a surface-coated amylose-tris(3,5-dimethylcarbamate) silica chiral column for the separation and determination of bricetam and its enantiomers and diastereomers. However, this method uses an organic solvent system, which is incompatible with aqueous oral solution matrices, requires complex sample pretreatment (such as extraction, concentration, and solvent displacement), is cumbersome, prone to introducing errors, and is not conducive to high-throughput detection.
[0005] Patent CN114428134A proposes a gas chromatography method based on a cyclodextrin-modified chiral column for the detection of briracetam intermediate isomers. It employs a temperature-programmed mode, making it suitable for the analysis of volatile intermediates. However, briracetam itself is a low-volatility compound, and oral solutions contain a large amount of non-volatile excipients; direct injection can easily contaminate the chromatographic system. Therefore, this method is not suitable for the detection of finished pharmaceutical products.
[0006] Patent CN114076799A discloses a reversed-phase HPLC method for the detection of isomers in bricetam injection, employing a phosphate buffer-acetonitrile system and a linear starch-tris(3-chloro-5-methylphenylcarbamate) bonded silica chiral column. While this method is suitable for aqueous matrices, injection solutions use simple excipients, whereas oral solutions contain large amounts of highly polar excipients (such as sugar alcohols and buffer salts), which tend to accumulate at the column head, leading to a rapid decrease in column efficiency and reduced isomer resolution.
[0007] Therefore, there is an urgent need to develop an efficient, specific, and durable detection method that can achieve high-resolution separation and accurate quantification of briracetam and its isomers in complex oral solution matrices, filling the gap in existing technologies. Summary of the Invention
[0008] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for detecting the content of isomers in briracetam oral solution. The method is easy to operate and control, has high sensitivity, and has good specificity, system applicability, solution stability and chromatographic condition robustness, thus providing an effective prescription analysis method for drug development.
[0009] The method for detecting the content of isomers in the bricetam oral solution involves preparing the solution and using high performance liquid chromatography (HPLC) with chromatographic columns 1 and 2 connected in series and a valve switching pathway for detection.
[0010] The structural formula of the enantiomer is: The structural formula of diastereomer I is: The structural formula of diastereomer II is: .
[0011] The chromatographic conditions for high performance liquid chromatography include:
[0012] Column 1: octadecylsilane-bonded silica gel as the packing material;
[0013] Column 2: Packed with amylose-tris-(4-chloro-3-methylphenylcarbamate);
[0014] Mobile phase A: Organic acid solution;
[0015] Mobile phase B: Acetonitrile;
[0016] Elution method: gradient elution;
[0017] The gradient elution conditions are as follows:
[0018] 0→9min, the volume percentage of mobile phase A is 65±2%, and the volume percentage of mobile phase B is 35±2%;
[0019] 9→14min, mobile phase A volume percentage 50±2%, mobile phase B volume percentage 50±2%;
[0020] 14→55min, mobile phase A volume percentage 65±2%, mobile phase B volume percentage 35±2%;
[0021] Valve switching settings: Column 1 is connected to the main channel, and Column 2 and the two-way valve are connected to different parallel channels. Column 2 channel is used at 6 min and 20 min, and the two-way valve channel is used at 9 min and 50 min.
[0022] Chromatographic conditions also include: flow rate: 0.5±0.02 mL / min; column temperature: 30~40℃; detection wavelength: 200~300 nm; injection volume: 5~20 μL.
[0023] The organic acid solution is a 0.1% phosphoric acid solution.
[0024] Column 1 is a YMC-Triart C18 with dimensions of 4.6 mm × 150 mm and a packing particle size of 5 μm; Column 2 is a linear starch-tris(4-chloro-3-methylphenylcarbamate) column with dimensions of 4.6 mm × 250 mm and a packing particle size of 5 μm.
[0025] The volumetric flow rates of mobile phase A and mobile phase B are 0.5 ± 0.02 mL / min.
[0026] The prepared solutions are: test solution, mixed spiked test solution, and reference solution.
[0027] Preparation of the test solution: Take briceracetam oral solution, dilute with solvent, shake well, and prepare a solution containing 2 mg / mL of briceracetam as the test solution.
[0028] Preparation of mixed spiking test solution: Take briracetam reference standard, enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute them with solvent to prepare a mixed solution containing 2 mg / mL briracetam and 4 μg / mL each of enantiomer, diastereomer I, and diastereomer II, as the mixed spiking test solution.
[0029] Preparation of reference solutions: Accurately weigh the enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute them with solvent to prepare solutions of enantiomer 1.2~8.0 μg / mL, diastereomer I 3.0~20.2 μg / mL, and diastereomer II 1.2~8.0 μg / mL.
[0030] The solvent is water.
[0031] Specifically, the method for detecting the content of isomers in the briceracetam oral solution includes the following steps:
[0032] (1) Solution preparation:
[0033] The specific preparation of the test solution is as follows: Take 2 mL of bricetrastane oral solution, place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the test solution.
[0034] Preparation of mixed spiking test solution: Take briracetam reference standard, enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute with water to prepare a mixed solution containing 2 mg / mL briracetam and 4 μg / mL each of enantiomer, diastereomer I, and diastereomer II, as the mixed spiking test solution.
[0035] Preparation of reference solutions: Accurately weigh the enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute them with solvent to prepare a 4 μg / mL solution, which shall be used as the reference solution.
[0036] (2) Detection: High performance liquid chromatography was used for separation using a gradient elution program, and the content of isomers was controlled by external standard method.
[0037] Compared with the prior art, the beneficial effects of the present invention are:
[0038] The present invention provides a method for detecting the content of isomers in briceceran oral solution, which achieves effective separation of briceceran, enantiomers, diastereomer I, and diastereomer II; at the same time, it avoids interference from other related substances of briceceran and excipients on the detection of isomers, and accurately detects the content of each isomer in briceceran oral solution. Attached Figure Description
[0039] Figure 1 This is the chromatogram of the solvent.
[0040] Figure 2 This is a chromatogram of the mixed spiked test solution.
[0041] Figure 3 This is the chromatogram of the test solution.
[0042] Figure 4 This is the chromatogram of the reference solution.
[0043] Figure 5 This is the chromatogram of the sample detection in Example 7. Detailed Implementation
[0044] The present invention will be further described below with reference to specific embodiments.
[0045] Unless otherwise specified, all raw materials and additives used in the following examples and comparative examples are commercially available.
[0046] Briracetam oral solution: UCB, a Belgian pharmaceutical company, specification BRIVIACT (brivaracetam) oral solution, 10 mg / mL.
[0047] Bricetan reference standard: Heyuan Guangpu Biotechnology Co., Ltd.
[0048] Enantiomer reference standard: Heyuan Broad Spectrum Biotechnology Co., Ltd.
[0049] Diastereomer I reference standard: Heyuan Broad Spectrum Biotechnology Co., Ltd.
[0050] Diastereomer II reference standard: Heyuan Broad Spectrum Biotechnology Co., Ltd.
[0051] Example 1
[0052] The method for detecting the content of isomers in the bricetam oral solution includes the following steps:
[0053] (1) Solution preparation:
[0054] Solvent: Water.
[0055] Preparation of test solution: Take an appropriate amount of bricetrast oral solution and dilute it with water to prepare a solution containing 2 mg of bricetrast per 1 mL.
[0056] Preparation of enantiomer stock solution: Accurately weigh 4.103 mg of enantiomer reference standard, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and record as enantiomer stock solution.
[0057] Preparation of diastereomer I stock solution: Accurately weigh 4.094 mg of diastereomer I reference standard, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and record it as diastereomer I stock solution.
[0058] Preparation of diastereomer II stock solution: Accurately weigh 3.982 mg of diastereomer II reference standard, place it in a 10 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and record it as diastereomer II stock solution.
[0059] Mixed spiked test solution: Accurately measure 2.0 mL of bricetam oral solution and place it in a 10 mL volumetric flask. Then add 1 mL each of the enantiomer stock solution, diastereomer I stock solution, and diastereomer II stock solution. Dilute with water to the mark and shake well.
[0060] (2) Detection: High performance liquid chromatography was used. The mixed spiked test solution, solvent and test solution were injected into the high performance liquid chromatograph and separated by gradient elution program. The content of isomers was controlled by external standard method.
[0061] The chromatographic conditions are as follows:
[0062] Chromatographic columns: linear starch-tris-(4-chloro-3-methylphenylcarbamate) was used as the packing material (Chiral NX(2)-RH reversed-phase chiral column, 4.6 mm × 250 mm, 5 μm); octadecylsilane-bonded silica gel was used as the packing material (YMC-TriartC18 reversed-phase chiral column, 4.6 mm × 150 mm, 5 μm); detection was performed by connecting chromatographic column 1 and chromatographic column 2 in series and using a valve switching method; valve switching settings: chromatographic column 1 was connected to the main channel, and chromatographic column 2 and the two-way valve were connected to different parallel channels respectively. The chromatographic column 2 channel was used at 6 min and 20 min, and the two-way valve channel was used at 9 min and 50 min.
[0063] At 0 min, the valve is set to run through two ports, and the flow path is column 1 → two ports → detector;
[0064] At 6 minutes, the valve switches to column 2, and the flow path is column 1 → column 2 → detector;
[0065] At 9 minutes, the valve is switched back, and the flow path is column 1 → two-way port → detector.
[0066] At 20 minutes, the valve is switched off again and the flow path is column 2, which is column 1 → column 2 → detector.
[0067] At 50 minutes, the valve is switched back, and the flow path is column 1 → two-way valve → detector.
[0068] UV detector: Detection wavelength is 210nm;
[0069] Flow rate: 0.5 mL / min;
[0070] Injection volume: 10 μL;
[0071] Mobile phase A: 0.1% aqueous solution of phosphoric acid (prepared by adding water to phosphoric acid);
[0072] Mobile phase B: Acetonitrile;
[0073] The gradient elution was performed using the following procedure, as shown in Table 1, where % represents the volume percentage.
[0074] Table 1 Elution Procedure Gradient Table
[0075]
[0076] After testing, the chromatogram of the solvent water is as follows: Figure 1 As shown, the chromatogram of the mixed spiked test solution is as follows: Figure 2 As shown, the chromatogram of the test solution is as follows: Figure 3 As shown in the figure, the blank solvent has no interference, and the resolution between each isomer and between the mixed spiked test solution and the main peak is greater than 1.5, thus exhibiting good specificity. The specific results of the specificity detection are shown in Table 2.
[0077] Table 2 Specificity Detection Results
[0078]
[0079] Example 2
[0080] System applicability verification
[0081] Preparation of reference solutions: Accurately weigh appropriate amounts of bricetam reference standard, enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute with water to a mixed solution containing 10 μg / mL bricetam and 4 μg / mL each of enantiomer I and diastereomer II.
[0082] The detection was performed using the chromatographic conditions of Example 1, and the chromatogram of the reference solution is shown below. Figure 4 As shown in the results, the peak retention time RSDs for bricetam, its enantiomer, diastereomer I, and diastereomer II are 0.1%, 0.1%, 0.1%, and 0.1%, respectively, all less than 1.0%, and the peak area RSDs are 0.7%, 1.0%, 0.6%, and 0.8%, respectively, all less than 2.0%. The results indicate that the system has good applicability.
[0083] Example 3
[0084] Linear verification
[0085] Preparation of mixed stock solution of isomers: Accurately weigh appropriate amounts of enantiomer reference standard, diastereomer I reference standard and diastereomer II reference standard, dissolve and dilute with water to prepare a mixed solution of 0.4 mg / mL enantiomer, 1.0 mg / mL diastereomer I and 0.4 mg / mL diastereomer II.
[0086] Take an appropriate amount of the mixed stock solution of isomers and gradually dilute it with water to prepare solutions of different concentrations: enantiomers 1.2 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 6.0 μg / mL, 8.0 μg / mL; diastereomer II 1.2 μg / mL, 2.0 μg / mL, 4.0 μg / mL, 6.0 μg / mL, 8.0 μg / mL; and diastereomer I 3.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 15.0 μg / mL, 20.0 μg / mL, thus preparing a series of linear solutions.
[0087] The linearity results obtained by detecting the sample under the chromatographic conditions of Example 1 are shown in Table 3.
[0088] Table 3 Linearity Results
[0089]
[0090] Example 4
[0091] Accuracy verification
[0092] Preparation of isomer reference solution: Accurately weigh appropriate amounts of enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute with water to a mixed solution containing 4 μg / mL of enantiomer and diastereomer II, and 10 μg / mL of diastereomer I, as the isomer reference solution.
[0093] Preparation of test solution: Take 2 mL of brucetam oral solution, place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the test solution.
[0094] Prepare three samples of each isomer at different concentrations using briracetam oral solution, enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard (the test solution prepared with briracetam solution is used as a solvent; 30%, 100%, and 150% refer to the concentration of the isomer). Prepare three test samples for each concentration, and evaluate the recovery rate using nine samples. The chromatographic conditions of Example 1 were used for detection, and the test results are shown in Tables 4, 5, and 6.
[0095] Table 4. Results of Enantiomer Accuracy Test
[0096]
[0097] Table 5. Accuracy test results for diastereomer I
[0098]
[0099] Table 6. Accuracy Test Results of Diastereomer II
[0100]
[0101] Example 5
[0102] Limit of detection and limit of quantitation
[0103] Solvent: Water
[0104] Isomer Mixed Stock Solution 1: Accurately weigh 4.138 mg of enantiomer reference standard, 10.101 mg of diastereomer I reference standard and 4.011 mg of diastereomer II reference standard and place them in a 10 mL volumetric flask. Dissolve and dilute to the mark with solvent and shake well.
[0105] Isomer Mixed Stock Solution 2: Accurately measure 1 mL of isomer mixed stock solution 1, place it in a 20 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.
[0106] Isomer reference solution: Accurately measure 2 mL of isomer mixed stock solution 2, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.
[0107] Limit of Quantification Solution: Accurately measure 3.0 mL of the isomer reference solution, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.
[0108] Detection limit solution: Accurately measure 3.3 mL of the quantitation limit solution and place it in a 10 mL volumetric flask. Dilute to the mark with solvent and shake well.
[0109] Accurately pipette 10 μL of the limit of detection (LOD) and limit of quantitation (LOQ) solutions and inject them into the liquid chromatograph. Record the signal-to-noise ratio (S / N) and calculate the RSD of the peak areas of the LOD solution over six injections. The LOD solution S / N should be ≥3; the LOD solution S / N should be ≥10; and the RSD of the peak areas over six injections should be ≤10%. The experimental results are shown in Tables 7-9.
[0110] Table 7 Results of determination of enantiomer detection limit and quantitation limit
[0111]
[0112] Table 8 Results of determination of detection limit and quantitation limit for diastereomer I
[0113]
[0114] Table 9 Results of determination of the detection limit and quantitation limit of diastereomer II
[0115]
[0116] Example 6
[0117] Durability test
[0118] Solvent: Water
[0119] Isomer Mixed Stock Solution 1: Accurately weigh 4.138 mg of enantiomer reference standard, 10.101 mg of diastereomer I reference standard and 4.011 mg of diastereomer II reference standard and place them in a 10 mL volumetric flask. Dissolve and dilute to the mark with solvent and shake well.
[0120] Isomer Mixed Stock Solution 2: Accurately measure 1 mL of isomer mixed stock solution 1, place it in a 20 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.
[0121] Isomer reference solution: Accurately measure 2 mL of isomer mixed stock solution 2, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.
[0122] Spiked test solution: Accurately measure 1.0 mL of bricetam oral solution and place it in a 5 mL volumetric flask. Then accurately add 1 mL of isomer mixed stock solution 2, dilute to the mark with solvent, and shake well.
[0123] The experimental parameters were varied in terms of flow rate, column temperature, and mobile phase ratio. For each change in parameter, 10 μL each of the solvent, reference solution, and spiked test solution were injected sequentially into the liquid chromatograph, and the peak areas were recorded. The contents of enantiomers, diastereomer I, and diastereomer II in the spiked test solution were calculated using the external standard method.
[0124] Table 10 Durability Test Results
[0125]
[0126] Based on the test results of the above embodiments, it can be seen that the method for detecting the content of briracetam oral solution isomers described in this invention is simple to operate, easy to control, highly sensitive, and has good specificity, system applicability, linearity, and accuracy, providing an effective detection method.
[0127] Example 7
[0128] Sample testing
[0129] Solvent: Water
[0130] Isomer Mixed Stock Solution 1: Accurately weigh 4.138 mg of enantiomer reference standard, 10.101 mg of diastereomer I reference standard and 4.011 mg of diastereomer II reference standard and place them in a 10 mL volumetric flask. Dissolve and dilute to the mark with solvent and shake well.
[0131] Isomer Mixed Stock Solution 2: Accurately measure 1 mL of isomer mixed stock solution 1, place it in a 20 mL volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.
[0132] Isomer reference solution: Accurately measure 2 mL of isomer mixed stock solution 2, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well.
[0133] Test solution: Accurately measure 1.0 mL of bricetam oral solution (UCB, a Belgian pharmaceutical company) into a 5 mL volumetric flask, dissolve and dilute to the mark with solvent, and shake well.
[0134] Accurately pipette 10 μL of each of the above solutions and inject them sequentially into the high-performance liquid chromatograph (chromatographic conditions are exactly the same as in Example 1), and record the chromatograms, as shown below. Figure 5 As shown in the figure, the test results show that the content of diastereomer I in the sample is 0.56%, while neither enantiomer nor diastereomer II was detected.
Claims
1. A method for detecting the content of isomers in briracetam oral solution, characterized in that: The solution was prepared and detected by high performance liquid chromatography, using chromatographic columns 1 and 2 connected in series and a valve switching method. The chromatographic conditions for high performance liquid chromatography include: Column 1: octadecylsilane-bonded silica gel as the packing material; Column 2: Packed with amylose-tris-(4-chloro-3-methylphenylcarbamate); Mobile phase A: 0.1% phosphoric acid solution; Mobile phase B: Acetonitrile; Elution method: gradient elution; The gradient elution conditions are as follows: 0→9min, the volume percentage of mobile phase A is 65±2%, and the volume percentage of mobile phase B is 35±2%; 9→14min, mobile phase A volume percentage 50±2%, mobile phase B volume percentage 50±2%; 14→55min, mobile phase A volume percentage 65±2%, mobile phase B volume percentage 35±2%; Valve switching settings: At 0 min, the valve is set to use the two-way valve, with the flow path being column 1 → two-way valve → detector; at 6 min, the valve switches to use column 2, with the flow path being column 1 → column 2 → detector; at 9 min, the valve switches back to the two-way valve, with the flow path being column 1 → two-way valve → detector; at 20 min, the valve switches back to use column 2, with the flow path being column 1 → column 2 → detector; at 50 min, the valve switches back to the two-way valve, with the flow path being column 1 → two-way valve → detector. The structural formula of the enantiomer is: The structural formula of diastereomer I is: The structural formula of diastereomer II is: ; The prepared solutions are: test solution, mixed spiked test solution, and reference solution; Preparation of test solution: Take briceracetam oral solution, dilute with solvent, shake well, and prepare a solution containing 2 mg / mL of briceracetam as the test solution; Preparation of mixed spiking test solution: Take briracetam reference standard, enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute them with solvent to prepare a mixed solution containing 2 mg / mL briracetam and 4 μg / mL each of enantiomer, diastereomer I, and diastereomer II, as the mixed spiking test solution; Preparation of reference solutions: Accurately weigh the enantiomer reference standard, diastereomer I reference standard, and diastereomer II reference standard, dissolve and dilute them with solvent to prepare solutions of enantiomer 1.2~8.0 μg / mL, diastereomer I 3.0~20.2 μg / mL, and diastereomer II 1.2~8.0 μg / mL.
2. The method for detecting the content of isomers in briracetam oral solution according to claim 1, characterized in that: Chromatographic conditions also include: flow rate: 0.5±0.02 mL / min; column temperature: 30~40℃; detection wavelength: 200~300 nm; injection volume: 5~20 μL.
3. The method for detecting the content of isomers in briracetam oral solution according to claim 2, characterized in that: Column 1 is a YMC-Triart C18 with dimensions of 4.6 mm × 150 mm and a packing particle size of 5 μm; Column 2 is a linear starch-tris(4-chloro-3-methylphenylcarbamate) column with dimensions of 4.6 mm × 250 mm and a packing particle size of 5 μm.
4. The method for detecting the content of isomers in briracetam oral solution according to claim 1, characterized in that: The solvent is water.