A method for detecting bromide impurities in semagacestat drug substance

Abstract

The application discloses a method for detecting bromide impurities in semagacestat bulk drug, which comprises the following steps: preparing a test sample, taking semagacestat bulk drug, dissolving the semagacestat bulk drug in methanol, diluting the semagacestat bulk drug with ultrapure water and shaking the semagacestat bulk drug; liquid chromatography separation, adopting a specific specification C18 chromatographic column, using 0.1 mM ammonium fluoride solution and acetonitrile as a mobile phase for gradient elution; mass spectrometry detection, adopting an electrospray negative ion source (ESI-) and a multiple reaction monitoring mode (MRM), and combining isotope ion pair selection and mass spectrometry valve switching technology to complete detection. The application innovatively constructs an integrated detection system of “isotope specific recognition-matrix interference shielding-wide dynamic range quantification”, solves the technical problem of accurately detecting low-limit bromide impurities in a high-concentration bulk drug matrix, and is significantly superior to existing methods in terms of specificity, sensitivity and durability, can realize strict control of the quality of semagacestat bulk drug, and guarantees drug safety.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, specifically to a method for detecting brominated impurities in sambacate active pharmaceutical ingredient. Background Technology

[0002] Sambacote, a widely used antiepileptic drug in clinical practice, relies heavily on the quality of its active pharmaceutical ingredient (API) for its safety and efficacy. Two impurities, 2-bromo-o-chloroacetophenone and 2,2-dibromo-1-(2-chlorophenyl)acetophenone, which are easily generated during the drug's manufacturing process, possess a clear potential mutagenic risk due to their halogenated alkane structures. Based on ICH M7(R2) guidelines and the maximum daily dosage, the control limit for both impurities is set at a stringent standard of 3.7 ppm.

[0003] Current methods for detecting halogenated impurities in pharmaceuticals largely rely on GC-MS or conventional LC-MS techniques. However, there are significant technical bottlenecks for the specific detection of sambacote active pharmaceutical ingredient (API): First, the impurity limit is extremely low (3.7 ppm), while the API matrix concentration is high, easily leading to severe matrix inhibition and insufficient detection sensitivity. Second, the fragmentation pattern of brominated impurities is singular, and conventional ion pair selection is easily interfered with, making it difficult to guarantee specificity. Third, existing methods have a narrow linear range, which cannot simultaneously meet the needs of low-limit screening and high-concentration quantification of abnormal batches. Therefore, developing a detection method with high specificity, high sensitivity, and a wide dynamic range is key to solving the quality control challenges of sambacote API. Summary of the Invention

[0004] This invention aims to provide a method for detecting brominated impurities in sambacote active pharmaceutical ingredient. Through multi-technology collaborative innovation, it achieves accurate quantification of low-limit impurities in complex matrices, providing reliable technical support for drug quality and safety.

[0005] To achieve the above objectives, the technical solution adopted by this invention is as follows: a method for detecting brominated impurities in sambacate raw material, comprising the following steps: Preparation of test samples: Take Sambacote raw material, add methanol to dissolve it, dilute with ultrapure water, shake well to obtain test samples; Liquid chromatography separation: The test sample was separated by liquid chromatography, wherein mobile phase A was 0.1 mM ammonium fluoride solution, mobile phase B was acetonitrile, and the chromatographic column was C18 (3.0 × 150 mm, 2.7 μm); Mass spectrometry detection: The substances separated by liquid chromatography were detected by mass spectrometry to obtain the content of brominated impurities in the Sambacote raw material; the ion source of the mass spectrometer was an electrospray ion source (ESI-), the acquisition mode was multiple reaction monitoring (MRM), and the switching valve was set to 5.5-10 min To MS. At the same time, bromine isotope ion pairs were used for qualitative and quantitative dual verification.

[0006] Preferably, the brominated impurities include 2-bromo-o-chloroacetophenone (1Br, CAS: 5000-66-8) and 2,2-dibromo-1-(2-chlorophenyl)acetophenone (2Br, CAS: 34356-83-7).

[0007] Preferably, the preparation of the test sample includes a precise quantification scheme and a high-sensitivity detection scheme; The precise quantification method includes: accurately weighing 150 mg of Sambacote raw material, placing it in a 10 ml volumetric flask, adding 5 ml of methanol to dissolve it, diluting it to the mark with ultrapure water, shaking it well, filtering it, and taking the filtrate as the final product. The high-sensitivity detection method includes: taking 97 mg of Sambacote raw material, dissolving it in 4.85 ml of methanol, and then adding an equal volume of ultrapure water to dilute it, so as to obtain a test solution with a concentration of about 10 mg / ml.

[0008] Preferably, the column temperature of the liquid chromatography is 30°C, the flow rate is 0.4 ml / min, and the injection volume is 20 μl.

[0009] Preferably, the mass spectrometry detection parameters are as follows: for 2-bromo-o-chloroacetophenone, the quantitative ion pair is 232.9→80.9, the qualitative ion pair is 230.9→78.9, the fragmentor voltage is 100V, and the collision energy is 18V; for 2,2-dibromo-1-(2-chlorophenyl)acetophenone, the quantitative ion pair is 310.8→78.9, the qualitative ion pair is 310.8→80.9, the fragmentor voltage is 110V, and the collision energy is 22V.

[0010] Preferably, during mass spectrometry detection, the diverter valve is switched to the mass spectrometry channel only at 5.5-10 minutes, and switched to the waste liquid channel for the rest of the time.

[0011] Preferably, the method further includes plotting a standard curve, specifically including: Take a series of concentration standard curve solutions and test sample solutions, inject them into the liquid chromatography-mass spectrometry instrument, record the chromatograms, and calculate the regression equation based on the concentration and the corresponding peak area. The concentration range of the standard curve solution covers 0.43-215.0 ppm (2-bromo-o-chloroacetophenone) and 0.49-244.4 ppm (2,2-dibromo-1-(2-chlorophenyl)acetophenone).

[0012] Preferably, the method for obtaining the standard curve includes: Preparation of standard samples: The brominated impurities were diluted with 40% methanol solution to prepare a series of standard curve solutions, with concentration points covering 12%-500% of the limit, suitable for low-limit screening and high-concentration quantification scenarios; Liquid chromatography separation: Standard samples are separated using preset liquid chromatography conditions; Mass spectrometry detection: The substances separated by liquid chromatography are detected using preset mass spectrometry conditions. The limit of quantitation and the limit of detection are calculated based on the signal-to-noise ratio. The regression equation is calculated using the concentration and the corresponding peak area to obtain the standard curve.

[0013] Preferably, the concentration range of the standard samples of different concentrations is: a series of standard curve solutions containing 17ng, 28ng, 56ng, 84ng, and 112ng of 2-bromo-o-chloroacetophenone and 17ng, 28ng, 56ng, 84ng, and 112ng of 2,2-dibromo-1-(2-chlorophenyl)acetophenone per 1ml.

[0014] Preferably, the series of standard curve solutions are prepared by a gradient dilution method, with the highest concentration point being 4.5 μg / ml, which is then diluted 10 times to 4.3 ng / ml, forming 10 concentration gradients.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention involves dissolving sambacate raw material in methanol, diluting it with ultrapure water, and shaking it well; separating it by liquid chromatography using a C18 column of specific specifications, with gradient elution using 0.1 mM ammonium fluoride solution and acetonitrile as the mobile phase; and using an electrospray negative ion source (ESI-) and multiple reaction monitoring mode (MRM), combined with isotope ion pair selection and mass spectrometry valve switching technology to complete the detection. An integrated detection system of "isotope specific recognition - matrix interference shielding - wide dynamic range quantification" has been constructed, which solves the technical problem of accurate detection of low-limit brominated impurities in high-concentration raw material matrix. Attached Figure Description

[0016] Figure 1 The structural formulas of the sambacate active pharmaceutical ingredient and its brominated impurities in this invention are shown below; Figure 2 This is the mass spectrum of the reference solution in this invention; Figure 3 This is the mass spectrum of the blank solution in this invention; Figure 4 This is the mass spectrum of the test sample solution in this invention; Figure 5 This is the mass spectrum of the spiked test solution in this invention; Figure 6This is the standard curve of 2-bromo-o-chloroacetophenone in this invention; Figure 7 This is the standard curve for 2,2-dibromo-1-(2-chlorophenyl)ethyl ketone in this invention; Figure 8 This is a comparison diagram of the ion responses of two substances in different mobile phases in this invention. Detailed Implementation

[0017] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0018] like Figure 1-8 As shown, this invention proposes a method for detecting brominated impurities in sambacate active pharmaceutical ingredient, comprising the following steps: 1. Preparation of test samples Two preparation schemes are provided to suit different scenarios, balancing detection accuracy and flexibility: Precise quantification method: Weigh approximately 150 mg of Sambacote raw material accurately, place it in a 10 ml volumetric flask, add 5 ml of methanol to dissolve it, dilute with ultrapure water to the mark, shake well, filter through a 0.22 μm organic filter membrane, and collect the filtrate. High-sensitivity method: Weigh 97 mg of Sambacote, dissolve it completely in 4.85 ml of methanol, and then add an equal volume of deionized water to dilute it, so as to obtain a test solution with a concentration of about 10 mg / ml. It can be prepared quickly without volumetric flasks, is suitable for rapid on-site screening, and can further improve the detection capability of low-content impurities by increasing the sample concentration.

[0019] 2. Liquid chromatography separation Innovative optimization of the chromatographic separation system to achieve efficient separation of impurities and matrix: Key parameters: C18 column (3.0×150mm, 2.7μm), column temperature 30℃, flow rate 0.4ml / min, injection volume 20μl; Mobile phase: Phase A is 0.1 mM ammonium fluoride solution (significantly improves the ionization efficiency of bromides), and Phase B is acetonitrile; Gradient elution program: 0-2 min maintain phase A at 60% and phase B at 40% to achieve initial matrix separation; Within 2-7 minutes, the content of phase A decreases from 60% to 10% and phase B increases to 90%, completing the elution of impurities. Maintain a high organic phase ratio for 7-10 minutes to ensure complete effluent removal of impurities; 10.1-16 min restores the initial ratio and equilibrates the column; By optimizing the mobile phase ratio and gradient program, the target impurities were baseline separated from the main peak of the active pharmaceutical ingredient and the matrix interference peak, with a separation degree greater than 2.0, effectively eliminating the matrix inhibition effect.

[0020] 3. Mass spectrometry detection Constructing an innovative detection system integrating "isotope identification, valve switching protection, and multiple reaction monitoring": Basic parameters: Ion source is electrospray negative ion source (ESI-), acquisition mode is multiple reaction monitoring (MRM), dry gas temperature is 325℃, dry gas flow rate is 7L / min, sheath gas temperature is 350℃, sheath gas flow rate is 12L / min, nebulizer pressure is 55psi, capillary voltage is 3000V, nozzle voltage is 2000V. Specific ion pair selection: Utilizing the isotopic abundance characteristics of bromine (the abundance ratio of 79Br to 81Br is approximately 1:1), specific qualitative and quantitative ion pairs are designed for each impurity. False positive results are avoided through double verification. Specifically, the quantitative ion pair for 2-bromo-o-chloroacetophenone is 232.9→80.9 (characteristic ion of 81Br), and the qualitative ion pair is 230.9→78.9 (characteristic ion of 79Br). Similarly, the quantitative ion pair for 2,2-dibromo-1-(2-chlorophenyl)acetophenone is 310.8→78.9, and the qualitative ion pair is 310.8→80.9, further enhancing detection specificity. Valve switching protection: Set the impurity detection window to 5.5-10 minutes (from the diverting valve to the MS), and switch to the waste liquid channel after 0-5.5 minutes and 10 minutes to avoid high concentration of raw materials entering the ion source and causing contamination, thereby improving the long-term stability of the instrument by more than 30%. Sensitivity optimization: Through fine adjustment of ion source parameters and optimization of mobile phase additives, the detection limits of the two impurities are as low as 0.12 ppm, and the quantitation limits are 0.43 ppm (2-bromo-o-chloroacetophenone) and 0.49 ppm (2,2-dibromo-1-(2-chlorophenyl)acetophenone), which are far below the control limit of 3.7 ppm, meeting the requirements for accurate detection of low-limit impurities.

[0021] 4. Plotting the Standard Curve and Calculating Results Preparation of standard samples: The brominated impurities were diluted with 40% methanol solution to prepare a series of standard curve solutions, with concentration points covering 12%-500% of the limit, suitable for low-limit screening and high-concentration quantification scenarios; Liquid chromatography separation: The standard sample was separated using preset liquid chromatography conditions (i.e., mobile phase A was 0.1 mM ammonium fluoride solution, mobile phase B was acetonitrile, and the chromatographic column was C18 (3.0 × 150 mm, 2.7 μm)). Mass spectrometry detection: Preset mass spectrometry conditions were used (i.e., the specific parameters for mass spectrometry detection were: quantitative ion pair for 2-bromo-o-chloroacetophenone: 232.9→80.9, qualitative ion pair: 230.9→78.9, Fragmentor voltage: 100V, collision energy: 18V; quantitative ion pair for 2,2-dibromo-1-(2-chlorophenyl)acetophenone: 310.8→78.9, qualitative ion pair: 310.8→80.9, Fragmentor voltage: 100V, collision energy: 18V). Mentor voltage 110V, collision energy 22V;) The substances separated by liquid chromatography were detected. The limit of quantitation and limit of detection were calculated based on the signal-to-noise ratio. Regression equations were calculated using concentration and corresponding peak area: 1Br: y=25.134784×x+4.963489; 2Br: y=476.445291×x−1545.932049, with correlation coefficients R≥0.998 for both, yielding the standard curve.

[0022] The concentration range of the standard samples of different concentrations is as follows: a series of standard curve solutions containing 17ng, 28ng, 56ng, 84ng, and 112ng of 2-bromo-o-chloroacetophenone and 17ng, 28ng, 56ng, 84ng, and 112ng of 2,2-dibromo-1-(2-chlorophenyl)acetophenone per 1ml.

[0023] The series of standard curve solutions were prepared by gradient dilution, with the highest concentration point being 4.5 μg / ml. After being diluted 10 times to 4.3 ng / ml, 10 concentration gradients were formed to ensure linear coverage and detection accuracy.

[0024] The consumables and instruments used in this test method are as follows: 1. Reagents and consumables All reagents used were of analytical grade, and the water was Grade I water.

[0025] 1.1 Methanol: LC-MS grade; 1.2 Acetonitrile: LC-MS grade; 1.3 Ammonium fluoride: Superior grade; 1.4 2-Bromo-o-chloroacetophenone standard: purity not less than 97.6%; 1.5 2,2-Dibromo-1-(2-chlorophenyl)ethyl ketone standard: purity not less than 96.8%; 1.6 1Br stock solution: Weigh an appropriate amount of 2-bromo-o-chloroacetophenone standard, dissolve and dilute it with acetonitrile to prepare a solution containing approximately 250 μg per ml; 1.7 2Br stock solution: Weigh an appropriate amount of 2,2-dibromo-1-(2-chlorophenyl)ethyl ketone standard, dissolve and dilute it with acetonitrile to prepare a solution containing approximately 250 μg per ml; 1.8 Reference stock solution: Accurately measure appropriate amounts of 1Br stock solution and 2Br stock solution respectively, and dilute with 50% methanol solution to prepare a solution containing approximately 560 ng of each of the two impurities per ml; 1.9 Standard Curve Solutions: Accurately measure appropriate amounts of the reference standard stock solution and dilute with 50% methanol solution to prepare a series of standard curve solutions containing 17ng, 28ng, 56ng, 84ng, and 112ng of 1Br and 17ng, 28ng, 56ng, 84ng, and 112ng of 2Br per ml. 1.10 Test solution: Weigh approximately 150 mg of Sambacote raw material accurately, place it in a 10 ml volumetric flask, add 5 ml of methanol to dissolve it, dilute to the mark with ultrapure water, shake well, filter (0.22 μm organic filter membrane), and collect the filtrate. 1.11 Organic filter membrane: 0.22 μm.

[0026] 2. Instruments and equipment 2.1 Liquid Chromatography-Tandem Mass Spectrometry: Equipped with an electrospray ionization (ESI) source (model 1290-6470, Agilent); 2.2 Electronic balance: Model XSR205DU / A (MettlerToledo); 2.3 Oscillator; 2.4 Vortex apparatus; 2.5 Centrifuge: Speed ​​not less than 11000 r / min, equipped with refrigeration function; 2.6 Pipettes; 2.7 Volumetric flasks: 10ml, 100ml, etc.

[0027] 3. Measurement Procedure 3.1 Solution preparation: Prepare blank solution, standard curve solution, test solution and spiked test solution according to the methods in the "Reagents and Consumables" section above; 3.2 Instrument debugging: Set the parameters for liquid chromatography and mass spectrometry. After the instruments stabilize, verify the system suitability. 3.3 Sample determination: Accurately measure the system suitability solution, standard curve solution, blank solution, test solution and spiked test solution in sequence, inject them into the liquid chromatography-mass spectrometry (LC-MS) instrument, and record the chromatogram; 3.4 Result Calculation: Plot the standard curve with the concentration of the standard curve solution as the abscissa and the peak area as the ordinate, and calculate the regression equation; calculate the content of bromine impurities according to the chromatogram of the test solution using the standard curve method.

[0028] 4. System Applicability Requirements 4.1 System Suitability: The peak area RSD of the target impurity peak in the solution chromatogram should not exceed 10%; 4.2 The signal-to-noise ratio of the target impurity peak in the sensitivity solution chromatogram should be no less than 10; 4.3 The correlation coefficient of the standard curve regression equation should not be less than 0.990.

[0029] 5. Limit Requirements The content of 2-bromo-o-chloroacetophenone shall not exceed 3.7 ppm; the content of 2,2-dibromo-1-(2-chlorophenyl)acetophenone shall not exceed 3.7 ppm.

[0030] In summary, this invention utilizes the isotopic characteristics of bromine to design specific ion pairs, enabling both qualitative and quantitative verification. This effectively distinguishes target impurities from interfering substances, demonstrating significantly superior specificity compared to conventional single-ion-pair detection. Through chromatographic gradient optimization and innovative mobile phase additives, it achieves efficient separation of impurities from the active pharmaceutical ingredient matrix and interfering peaks. Furthermore, by combining ESI-ion source mode to reduce matrix inhibition effects, it enables accurate detection of low-concentration impurities even against a high matrix background. By combining mass spectrometry valve switching technology with ion source parameter optimization, this method avoids contamination of the ion source by high concentrations of active pharmaceutical ingredients (APIs) and reduces the detection limit by more than 60% compared to existing methods. The sensitivity at the lowest quantification point can reach 12% of the limit, ensuring sufficient margin for the detection method. The linear range covers 12%-500% of the limit, which can simultaneously meet the needs of routine screening and abnormal batch quantification. The repeatability RSD at low, medium and high concentrations is less than 5%, the sample solution has good stability after being placed at room temperature for more than 13 hours, and small fluctuations in flow rate and column temperature (±10%) do not affect the detection results, making it suitable for industrial batch detection scenarios. This invention provides two sample preparation methods that balance the needs for accurate quantification and rapid screening, without the need for complex derivatization steps, with a detection cycle of only 16 minutes and a 40% reduction in reagent consumption, in line with the concept of green detection.

[0031] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for detecting brominated impurities in sambacate active pharmaceutical ingredient, characterized in that, Includes the following steps: Preparation of test samples: Take Sambacote raw material, add methanol to dissolve it, dilute with ultrapure water, shake well to obtain test samples; Liquid chromatography separation: The test sample was separated by liquid chromatography, wherein mobile phase A was 0.1 mM ammonium fluoride solution, mobile phase B was acetonitrile, and the chromatographic column was C18 (3.0 × 150 mm, 2.7 μm); Mass spectrometry detection: The substances separated by liquid chromatography were detected by mass spectrometry to obtain the content of brominated impurities in the Sambacote raw material; the ion source of the mass spectrometer was an electrospray ion source (ESI-), the acquisition mode was multiple reaction monitoring (MRM), and the switching valve was set to 5.5-10 min To MS. At the same time, bromine isotope ion pairs were used for qualitative and quantitative dual verification.

2. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, The brominated impurities include 2-bromo-o-chloroacetophenone (1Br, CAS: 5000-66-8) and 2,2-dibromo-1-(2-chlorophenyl)acetophenone (2Br, CAS: 34356-83-7).

3. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, The preparation of the test samples includes a precise quantification scheme and a high-sensitivity detection scheme; The precise quantification method includes: accurately weighing 150 mg of Sambacote raw material, placing it in a 10 ml volumetric flask, adding 5 ml of methanol to dissolve it, diluting it to the mark with ultrapure water, shaking it well, filtering it, and taking the filtrate as the final product. The high-sensitivity detection method includes: taking 97 mg of Sambacote raw material, dissolving it in 4.85 ml of methanol, and then adding an equal volume of ultrapure water to dilute it, so as to obtain a test solution with a concentration of about 10 mg / ml.

4. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, The liquid chromatography column temperature was 30℃, the flow rate was 0.4 ml / min, and the injection volume was 20 μl.

5. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, The parameters for mass spectrometry detection are as follows: for 2-bromo-o-chloroacetophenone, the quantitative ion pair is 232.9→80.9, the qualitative ion pair is 230.9→78.9, the fragmentor voltage is 100V, and the collision energy is 18V; for 2,2-dibromo-1-(2-chlorophenyl)acetophenone, the quantitative ion pair is 310.8→78.9, the qualitative ion pair is 310.8→80.9, the fragmentor voltage is 110V, and the collision energy is 22V.

6. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, During mass spectrometry detection, the diverter valve is switched to the mass spectrometry channel only at 5.5-10 minutes, and switched to the waste liquid channel at other times.

7. The method for detecting brominated impurities in sambacate raw material according to claim 1, characterized in that, The method also includes plotting a standard curve, specifically including: Take a series of concentration standard curve solutions and test sample solutions, inject them into the liquid chromatography-mass spectrometry instrument, record the chromatograms, and calculate the regression equation based on the concentration and the corresponding peak area. The concentration range of the standard curve solution covers 0.43-215.0 ppm (2-bromo-o-chloroacetophenone) and 0.49-244.4 ppm (2,2-dibromo-1-(2-chlorophenyl)acetophenone).

8. The method for detecting brominated impurities in sambacate raw material according to claim 7, characterized in that, The method for obtaining the standard curve includes: Preparation of standard samples: The brominated impurities were diluted with 40% methanol solution to prepare a series of standard curve solutions, with concentration points covering 12%-500% of the limit, suitable for low-limit screening and high-concentration quantification scenarios; Liquid chromatography separation: Standard samples are separated using preset liquid chromatography conditions; Mass spectrometry detection: The substances separated by liquid chromatography are detected using preset mass spectrometry conditions. The limit of quantitation and the limit of detection are calculated based on the signal-to-noise ratio. The regression equation is calculated using the concentration and the corresponding peak area to obtain the standard curve.

9. The method for detecting brominated impurities in sambacate raw material according to claim 8, characterized in that, The concentration range of the standard samples of different concentrations is as follows: a series of standard curve solutions containing 17ng, 28ng, 56ng, 84ng, and 112ng of 2-bromo-o-chloroacetophenone and 17ng, 28ng, 56ng, 84ng, and 112ng of 2,2-dibromo-1-(2-chlorophenyl)acetophenone per 1ml.

10. The method for detecting brominated impurities in sambacate raw material according to claim 9, characterized in that, The series of standard curve solutions were prepared by gradient dilution, with the highest concentration point being 4.5 μg / ml. After being diluted 10 times to 4.3 ng / ml, 10 concentration gradients were formed.

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