A detection method for isobutyl chloroformate in lacosamide

Abstract

The present invention relates to the field of chemical analysis technology, and in particular to a method for detecting isobutyl chloroformate in lacosamide. The detection method uses gas chromatography-mass spectrometry technology to accurately determine the residual impurity isobutyl chloroformate in lacosamide. The detection method has strong specificity, good linear relationship, high sensitivity, accurate and reliable quantitative analysis, and good precision, repeatability, durability and solution stability. It can be used for trace detection of isobutyl chloroformate in lacosamide as a basis for quality monitoring of lacosamide.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and in particular to a method for detecting isobutyl chloroformate in lacosamide. Background Art

[0002] Lacosamide is a novel glycine-binding antagonist of the N-methyl-D-aspartate (NMDA) receptor. Its chemical name is (R)-2-acetamido-N-benzyl-3-methoxypropionamide lacosamide. It is an anticonvulsant drug with a new dual mechanism of action, used to treat epilepsy and neuropathic pain. It has few side effects, a unique mechanism of action, does not require liver metabolism, and does not produce clinically significant drug interactions with other anti-epileptic drugs. It can be used as a new treatment option for some patients with epileptic seizures. Its chemical structure is:

[0003]

[0004] Isobutyl chloroformate (IBCF), with the molecular formula of C5H9ClO2 and the structural formula shown below, is an important chemical raw material, commonly used in organic synthesis and drug synthesis. It is one of the important condensation agents for synthesizing peptide compounds, and its quality has a great influence on the reaction of peptide compounds. Isobutyl chloroformate is a genotoxic impurity of halogenated alkanes. As one of the raw materials for producing lacosamide drugs, it may produce trace levels of residues in lacosamide drugs in the existing lacosamide production process. Therefore, it is necessary to detect the residual amount of isobutyl chloroformate in lacosamide. However, since the residual amount of isobutyl chloroformate is generally at the trace level, the sensitivity of conventional gas chromatography or liquid chromatography detection methods cannot meet the detection requirements of isobutyl chloroformate, a genotoxic impurity of halogenated alkanes in lacosamide raw materials (ICH Guideline M7 requires control at 1.5μg / d / maximum daily dose). Therefore, it is urgent to establish a detection method with high sensitivity and capable of accurately and reliably detecting isobutyl chloroformate in lacosamide raw materials.

[0005] Summary of the invention

[0006] In view of this, the present invention provides a method for detecting isobutyl chloroformate in lacosamide, which realizes accurate determination of the residual impurity isobutyl chloroformate in lacosamide by gas chromatography-mass spectrometry.

[0007] In order to solve the above technical problems, the present invention provides a method for detecting isobutyl chloroformate in lacosamide, wherein the content of isobutyl chloroformate in lacosamide is detected by gas chromatography-mass spectrometry, and the gas chromatography conditions in the gas chromatography-mass spectrometry method include:

[0008] Chromatographic column: capillary column;

[0009] Carrier gas: helium;

[0010] Column temperature: first maintain at 40℃ for 1 min, then increase the temperature to 240℃ at a rate of 10℃ / min and maintain for 5 min;

[0011] Transmission line temperature: 240°C;

[0012] Injector oven temperature: 90°C;

[0013] Inlet temperature: 220°C;

[0014] Column flow rate: 1 mL / min;

[0015] Split ratio: 10:1;

[0016] Injection volume: 1mL.

[0017] The detection method of isobutyl chloroformate in lacosamide provided by the present invention realizes the detection of trace levels of isobutyl chloroformate remaining in lacosamide by setting specific gas chromatography conditions. The detection method has high sensitivity (detection limit concentration as low as 76.41 ng / mL, quantitative limit concentration as low as 152.83 ng / mL), good linear relationship (linear correlation coefficient r=0.9956), good accuracy (recovery rate between 96.55% and 106.79%), good precision and durability, meets the detection requirements of the guiding principles of ICH M7, is suitable for the detection of residual impurity isobutyl chloroformate in lacosamide raw materials, and can be used as the basis for its quality monitoring.

[0018] In combination with the first aspect, the capillary chromatographic column is a highly polar chromatographic column, which can ensure good separation effect and peak shape.

[0019] Preferably, the stationary liquid of the capillary chromatographic column is polyethylene glycol.

[0020] In combination with the first aspect, the equilibration time of the chromatographic column is 5 to 10 minutes.

[0021] In combination with the first aspect, the purity of the helium is at least 99.999%.

[0022] In combination with the first aspect, the mass spectrometry conditions in the gas chromatography-mass spectrometry method include:

[0023] Mass spectrometer: triple quadrupole tandem mass spectrometer;

[0024] Quadrupole temperature: 150℃;

[0025] Ion mode: EI ion source;

[0026] Ion source temperature: 250°C;

[0027] Ionization energy: 70 eV;

[0028] Ion detection mode: MRM multiple reaction monitoring mode;

[0029] name Quantitative ion pair Collision Energy Qualifying ion pairs Collision Energy Isobutyl chloroformate 77 / 33 15V 90 / 45 10V .

[0030] In combination with the first aspect, the method for detecting isobutyl chloroformate in lacosamide comprises the following steps: preparing a standard series of reference solution and a test solution of the lacosamide sample to be tested, and using the gas chromatography-mass spectrometry method to detect the residual impurity isobutyl chloroformate in the test solution of the lacosamide sample to be tested by a standard curve method.

[0031] The detection method provided by the present invention can realize accurate detection of isobutyl chloroformate in lacosamide without using an internal standard method, and the detection method is simple.

[0032] In combination with the first aspect, the method for detecting isobutyl chloroformate in lacosamide specifically comprises the following steps:

[0033] Step a, using a mixture of acetonitrile and methanol as solvent to prepare a test solution and a standard series reference solution of the residual impurity isobutyl chloroformate;

[0034] Step b, measuring the standard series reference substance solution by gas chromatography-mass spectrometry, and drawing a standard curve according to the peak area and the concentration of the standard series reference substance solution;

[0035] Step c, measuring the test solution by gas chromatography-mass spectrometry, and calculating the concentration of the residual impurity isobutyl chloroformate in the test solution using the standard curve obtained in step b.

[0036] In combination with the first aspect, the concentration range of the standard series reference solution is 0.15-1.5 μg / mL. When the solution is measured within this concentration range, there is an optimal linear relationship between the concentration of isobutyl chloroformate and the mass spectrometry peak area.

[0037] The present invention adopts a detection method of gas chromatography and mass spectrometry to achieve accurate detection of isobutyl chloroformate in lacosamide. The detection method has strong specificity, good linear relationship, high sensitivity, accurate and reliable quantification, and good precision, repeatability and durability. The method can be used for trace detection of isobutyl chloroformate in lacosamide and serve as a basis for quality monitoring of lacosamide. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the mass spectrum of the blank solution of Example 1;

[0039] Figure 2 is the mass spectrum of the isobutyl chloroformate limit concentration reference solution;

[0040] Figure 3 is the mass spectrum of the lacosamide test solution;

[0041] Figure 4 It is the mass spectrum of the mixed solution of lacosamide test substance and isobutyl chloroformate limit concentration reference substance;

[0042] Figure 5 This is the standard curve of isobutyl chloroformate reference solution. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] In the following examples of the present invention, the isobutyl chloroformate used was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with a purity of 98.8%; the chromatographic column used was Agilent VF-WAXms with a specification of 30m*0.25mm*1.0μm.

[0045] Example 1

[0046] The embodiment of the present invention provides a method for detecting isobutyl chloroformate in lacosamide, which is detected by gas chromatography-mass spectrometry; wherein:

[0047] (1) The chromatographic conditions of gas chromatography are:

[0048] Chromatographic column: capillary column;

[0049] Carrier gas: helium, purity 99.999%;

[0050] Column temperature: first maintain at 40℃ for 1 min, then increase the temperature to 240℃ at a rate of 10℃ / min and maintain for 5 min;

[0051] Transmission line temperature: 240°C;

[0052] Injector oven temperature: 90°C;

[0053] Inlet temperature: 220°C;

[0054] Column flow rate: 1 mL / min;

[0055] Split ratio: 10:1;

[0056] Injection volume: 1mL;

[0057] Equilibration time: 10 min.

[0058] (2) Mass spectrometry conditions are:

[0059] Mass spectrometer: triple quadrupole tandem mass spectrometer;

[0060] Quadrupole temperature: 150℃;

[0061] Ion mode: EI ion source;

[0062] Ion source temperature: 250°C;

[0063] Ionization energy: 70 eV;

[0064] Ion detection mode: MRM multiple reaction monitoring mode;

[0065] name Quantitative ion pair Collision Energy Qualifying ion pairs Collision Energy Isobutyl chloroformate 77 / 33 15V 90 / 45 10V .

[0066] (3) Preparation of reference solution and test solution:

[0067] Blank solution: Mix acetonitrile and methanol in a volume ratio of 3:7 as a blank solution, accurately measure 1 mL and place it in a headspace bottle, and seal it with a cap;

[0068] Isobutyl chloroformate mother liquor: weigh about 10 mg of isobutyl chloroformate, accurately weigh it, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing 1000 μg of isobutyl chloroformate per 1 ml as the impurity mother liquor;

[0069] Impurity stock solution: Take an appropriate amount of impurity mother solution and add acetonitrile to prepare a stock solution containing 50 μg of isobutyl chloroformate per 1 ml;

[0070] Reference substance stock solution: accurately measure 0.5 ml of impurity stock solution, place in a 10 ml volumetric flask, add acetonitrile to the mark, shake well, and make a solution containing 2500 ng of isobutyl chloroformate per 1 ml;

[0071] Limit concentration reference solution: Accurately measure 0.3 ml of reference stock solution, place in a headspace bottle, add 0.7 ml of methanol, and seal the bottle as the limit concentration reference solution;

[0072] Biaoqu series reference solution: accurately measure 0.1ml, 0.15ml, 0.25ml, 0.4ml, 0.5ml, 0.6ml, 1ml of impurity stock solution and place them in 10ml volumetric flasks 1-7 respectively, add acetonitrile to the scale line, and make solutions containing 0.50, 0.75, 1.25, 2.00, 2.50, 3.00, 5.00ug of isobutyl chloroformate per 1ml, which are used as impurity reference solutions 1-7 respectively. Accurately measure 0.3ml of each impurity reference solution 1-7 and place them in 7 headspace bottles numbered 1-7 respectively, then add 0.7ml of methanol respectively, cover and seal, and use them as Biaoqu series reference solution;

[0073] Low recovery solution: Take about 200 mg of the test sample, weigh accurately, place in an empty bottle, add 0.3 ml of impurity reference solution 4, then add 0.7 ml of methanol, cover and seal, prepare three times in parallel;

[0074] Medium recovery solution: take about 200 mg of the test sample, accurately weigh it, place it in an empty bottle, add 0.3 ml of impurity reference solution 5, then add 0.7 ml of methanol, cover and seal, and prepare three copies in parallel;

[0075] High recovery solution: Take about 200 mg of the test sample, weigh it accurately, place it in an empty bottle, add 0.3 ml of impurity reference solution 6, then add 0.7 ml of methanol, cover and seal, and prepare three parallel batches.

[0076] Test solution: Take about 200 mg of the test sample, accurately weigh it, and place it in a headspace bottle. Add 0.3 ml of acetonitrile and 0.7 ml of methanol to the headspace bottle, cover and seal it as the test solution;

[0077] Mixed solution of test sample and limit concentration reference substance: Take about 200 mg of the test sample, weigh accurately, place in an empty bottle, add 0.3 ml of the reference substance stock solution, then add 0.7 ml of methanol, and seal with a cap.

[0078] Test example

[0079] According to the detection conditions of Example 1 and using the solution prepared in Example 1, the detection method was respectively investigated and verified in terms of specificity, linearity, detection limit, accuracy, precision, repeatability, intermediate precision and durability, and the detection method was used to detect actual samples. The specific detection process is as follows:

[0080] 1. Specificity test:

[0081] The blank solution, the limit concentration reference solution, the test solution, and the mixed solution of the test sample and the limit concentration reference were respectively tested by HS-GCMS under the above-mentioned detection conditions, and the spectra were recorded. The retention time of isobutyl chloroformate in each solution is shown in Table 1, and the spectra are shown in Figure 1 to Figure 4 shown.

[0082] Table 1 Retention time of isobutyl chloroformate in each solution

[0083]

[0084] From Table 1 and Figures 1 to 4 It can be seen that under the detection conditions, the blank solution and other components or impurities in the test sample have no interference with the detection of isobutyl chloroformate, indicating that the detection method has good specificity.

[0085] 2. Linear relationship verification:

[0086] The standard series of reference solutions prepared according to the above method were tested by HS-GCMS, and the test results are shown in Table 2. The standard curve (as shown in Table 2) was drawn with the concentration of isobutyl chloroformate (ng / mL) as the horizontal axis and the mass spectrum peak area as the vertical axis. Figure 5 The results show that isobutyl chloroformate has a good linear relationship within this concentration range, and the linear regression equation is: y = 9.9544x + 37.721, and the linear correlation coefficient R 2 =0.9913.

[0087] Table 2 Linearity test results

[0088]

[0089] 3. Detection limit and quantification limit test:

[0090] Detection limit: Take an appropriate amount of the reference stock solution, dilute it quantitatively step by step with the blank solution, and perform the test according to the above test conditions. The detection limit is obtained when the signal-to-noise ratio is not less than 3:1;

[0091] Limit of quantitation: Take an appropriate amount of the reference stock solution, dilute it step by step with the blank solution, and perform the test according to the above test conditions. The limit of quantitation is obtained when the signal-to-noise ratio is not less than 10:1;

[0092] The results of the detection limit and quantification limit are shown in Table 3, respectively.

[0093] Table 3 Detection limit and quantification limit test results

[0094]

[0095] Repeatability test of quantitation limit: Six quantitation limit solutions (referred as solution 1 to solution 6) were prepared in parallel and tested by HS-GCMS to observe the precision of quantitation limit. The results are shown in Table 4.

[0096] Table 4 Quantitation limit repeatability test results

[0097]

[0098] It can be seen from Tables 3 and 4 that the detection limit and quantification limit obtained by this method can meet the detection requirements.

[0099] 4. Repeatability test:

[0100] According to the preparation method of the test solution, 6 parallel samples (referred to as solution 1 to solution 6) were prepared in parallel. The 6 parallel samples were tested according to the above-mentioned test conditions. The test results are shown in Table 5.

[0101] Table 5 Repeatability test results

[0102]

[0103] From the results in Table 5, it can be seen that the results of the 6 parallel test solutions were all undetectable, indicating that the detection method has good repeatability.

[0104] 5. Accuracy test:

[0105] According to the above solution preparation method, three portions of low recovery solution (80% limit recovery solution), medium recovery solution (100% limit recovery solution) and high recovery solution (120% limit recovery solution) were prepared respectively, and the above detection method was used for detection. The detection results are shown in Table 6, and the spiked recovery rates were calculated according to the formula: Spiked recovery rate (%) = (measured amount - original amount) / added amount × 100%.

[0106] Table 6 Accuracy test results

[0107]

[0108] From the results in Table 6, it can be seen that within the limit concentration range of 80% to 120% for isobutyl chloroformate, the recovery rate is between 96.55% and 106.79%, and the RSD between the recoveries is less than 10%, indicating that the detection method has good accuracy.

[0109] 6. Precision test:

[0110] According to the preparation method of the above-mentioned limit concentration reference solution, 6 limit concentration reference solutions (referred to as solution 1 to solution 6) were prepared in parallel, and the above-mentioned detection method was used for detection. The detection results are shown in Table 7.

[0111] Table 7 Precision test results

[0112]

[0113] From the test results in Table 7, it can be seen that the relative standard deviation of the peak areas corresponding to the six parallel solutions is 3.47%, which meets the requirement of the relative standard deviation of precision of ≤10% in the art, indicating that the precision of the detection method of the present invention is good.

[0114] 7. Durability test:

[0115] The injection port temperature, column flow rate, starting temperature and furnace temperature in the gas chromatography conditions were fine-tuned respectively, and then the limit concentration reference solution was tested using the fine-tuned test conditions. The test results are shown in Table 8.

[0116] Table 8 Durability test results

[0117]

[0118] It can be seen from Table 8 that fine-tuning the chromatographic conditions of the gas chromatography has no effect on the detection of isobutyl chloroformate, indicating that the detection method has good durability.

[0119] 8. Intermediate precision test:

[0120] According to the above preparation method, 6 test solutions (referred to as solution 1 to solution 6) were prepared in parallel, and tested using the above detection method to investigate the intermediate precision of the detection method, that is, the detection date and detection personnel of the repeatability test of this embodiment and embodiment 4 were different, but the detection conditions were the same as those of embodiment 4. The specific test results are shown in Table 9.

[0121] Table 9 Durability test results

[0122]

[0123] As shown in Table 9, the test results of the 6 parallel samples prepared from the test solution were all undetectable, which is consistent with the repeatability test results of Example 4, indicating that the intermediate precision of the detection method is good.

[0124] 9. Stability test:

[0125] The test solution and the limit concentration reference solution were prepared according to the above solution preparation method, left to stand at room temperature, and tested according to the above test conditions every 0 h, 2 h, 4 h, 6 h, and 8 h. The test results are shown in Tables 10 and 11, respectively.

[0126] Table 10 Test solution stability test results

[0127]

[0128] Table 11 Stability test results of limit concentration reference solution

[0129]

[0130] It can be seen from the test results in Table 10 that the test solution was left standing for 0-8h at room temperature, and the test results of isobutyl chloroformate in the test solution after standing for different time periods were all undetectable; it can be seen from the test results in Table 11 that the limit concentration reference solution was left standing for 0-8h at room temperature, and the relative standard deviation of the mass spectrum peak area of ​​isobutyl chloroformate after standing for different time periods was 7.65%, which met the requirement of ≤10%; the test results in Tables 10 and 11 indicate that the solution used in the detection method has good stability.

[0131] 10. Actual sample testing:

[0132] According to the above detection method, isobutyl chloroformate in three batches of lacosamide was detected respectively. The test results are shown in Table 12.

[0133] Table 12 Sample test results

[0134] Content of isobutyl chloroformate (ng / mg) Batch 1 Not detected Batch 2 Not detected Batch 3 Not detected

[0135] From the results in Table 12, it can be seen that isobutyl chloroformate was not detected in the three batches of lacosamide raw material samples, which is in compliance with relevant regulations.

[0136] What is described above is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for detecting isobutyl chloroformate in lacosamide, characterized in that: The content of isobutyl chloroformate in lacosamide is detected by gas chromatography-mass spectrometry, and the gas chromatography conditions in the gas chromatography-mass spectrometry method include: Chromatographic column: capillary column; Carrier gas: helium; Column temperature: first maintain at 40℃ for 1 min, then increase the temperature to 240℃ at a rate of 10℃ / min and maintain for 5 min; Transmission line temperature: 240°C; Injector oven temperature: 90°C; Inlet temperature: 220°C; Column flow rate: 1 mL / min; Split ratio: 10:1; Injection volume: 1mL; The capillary chromatographic column is a highly polar chromatographic column; The mass spectrometry conditions in the gas chromatography-mass spectrometry method include: Mass spectrometer: triple quadrupole tandem mass spectrometer; Quadrupole temperature: 150°C; Ion mode: EI ion source; Ion source temperature: 250°C; Ionization energy: 70 eV; Ion detection mode: MRM multiple reaction monitoring mode; 。 2. The method for detecting isobutyl chloroformate in lacosamide according to claim 1, characterized in that: The stationary liquid of the capillary chromatographic column is polyethylene glycol.

3. The method for detecting isobutyl chloroformate in lacosamide according to claim 1, characterized in that: The equilibration time of the chromatographic column is 5 to 10 minutes.

4. The method for detecting isobutyl chloroformate in lacosamide according to claim 1, characterized in that: The purity of the helium is at least 99.999%.

5. The method for detecting isobutyl chloroformate in lacosamide according to any one of claims 1 to 4, characterized in that, The following steps are involved: A standard series of reference substance solutions and a test solution of the lacosamide sample to be tested were prepared, and the residual impurity isobutyl chloroformate in the test solution of the lacosamide sample to be tested was detected by the gas chromatography-mass spectrometry method through a standard curve method.

6. The method for detecting isobutyl chloroformate in lacosamide according to claim 5, characterized in that: The specific steps include: Step a, using a mixture of acetonitrile and methanol as solvent to prepare a test solution and a standard series reference solution of the residual impurity isobutyl chloroformate; Step b, measuring the standard series reference substance solution by gas chromatography-mass spectrometry, and drawing a standard curve according to the peak area and the concentration of the standard series reference substance solution; Step c, measuring the test solution by gas chromatography-mass spectrometry, and calculating the concentration of the residual impurity isobutyl chloroformate in the test solution using the standard curve obtained in step b.

7. The method for detecting isobutyl chloroformate in lacosamide according to claim 6, characterized in that: The concentration range of the standard series reference solution is 0.15~1.5μg / mL.

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