A reverse phase liquid chromatography method for detecting lacosamide enantiomers

Abstract

The invention discloses a method for detecting lacosamide enantiomers by using reversed-phase liquid chromatography, which can directly and effectively separate lacosamide from the enantiomers, has good detection result accuracy and high sensitivity, is simpler to operate, easier to balance, has lower cost, is more environmentally friendly, and has a shorter analysis time than the existing normal-phase chromatography, can be directly used for the detection of solution preparations such as lacosamide injection, and is more convenient to operate, thereby providing a better guarantee for the quality control of lacosamide and its preparations.

Description

1. Technical field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for analyzing and determining lacosamide enantiomers by reverse phase liquid chromatography. 2. Background technology

[0002] Lacosamide is a novel N-methyl-D-aspartate (NMDA) receptor glycine site binding antagonist, belonging to a new class of functional amino acids. It is an anti-epileptic drug with a new mechanism of action, and has the characteristics of broad spectrum, high efficiency, good tolerance and ideal pharmacokinetics. Developed by UCB of Belgium, it has been marketed in many countries and regions such as the United States, Japan, Europe, and China for the treatment of partial epileptic seizures. The marketed dosage forms include tablets, injections, and oral solutions.

[0003] The chemical name of lacosamide is (R)-2-acetylamino-N-benzyl-3-methoxypropionamide, and its structural formula is as follows:

[0004]

[0005] Lacosamide has a chiral center, and its enantiomer is in the S configuration. Its chemical name is (S)-2-acetamido-N-benzyl-3-methoxypropionamide, and its structural formula is as follows:

[0006]

[0007] The marketed product of lacosamide is the R configuration, and its S configuration is inactive, so the enantiomer inspection during the production process of lacosamide and its preparations is of great significance to its safety and efficacy.

[0008] By consulting Chinese and foreign literature and patents, it is found that there are relevant literature reports on the use of high performance liquid chromatography to determine the optical isomers of intermediates in the preparation process of lacosamide, which basically use normal phase chromatography systems, and there are no reports on the use of reversed phase high performance liquid chromatography to detect the above-mentioned lacosamide enantiomers. For solution preparations such as lacosamide injection, the use of reversed phase systems can be directly diluted and then injected for detection, which is more conducive to quality control, avoids the situation of immiscibility caused by the use of normal phase solvents, and simplifies the processing steps. Therefore, it is necessary to establish a highly sensitive, simple, and universal reversed phase high performance liquid chromatography method for the determination of lacosamide enantiomers. 3. Summary of the invention

[0009] In order to solve the current technical problems, the present invention provides a method for separating and analyzing the enantiomers of lacosamide using a reverse phase chromatography system after a large number of experimental explorations. The method uses a common ultraviolet detector, is simple and easy to operate, has high general applicability, can achieve high sensitivity, and can better control the product quality of lacosamide.

[0010] The present invention provides a reverse phase liquid chromatography method for detecting lacosamide enantiomers, comprising the following steps:

[0011] (1) Preparation of system suitability solution: Take appropriate amount of lacosamide and its enantiomers, add mobile phase to dissolve and dilute to prepare system suitability solution;

[0012] (2) Preparation of test solution: Take an appropriate amount of lacosamide test sample, dissolve it in the mobile phase and dilute it to prepare the test solution;

[0013] (3) Chromatographic experiment: Take appropriate amounts of system suitability solution and test sample solution, inject them into the liquid chromatograph, and record the chromatogram.

[0014] The specific chromatographic conditions in step (3) are:

[0015] Detector: UV detector;

[0016] Chromatographic column: Silica gel coated with cellulose-tris-(3-chloro-4-methylphenylcarbamate) as filler, polysaccharide derivative reverse phase coated chiral chromatographic column, Daicel Chiralcel OZ-RH 150mm×4.6mm, 5μm;

[0017] Mobile phase: Mobile phase A: ammonium acetate solution, the concentration of which is 0.005-0.1 mol / L, preferably 0.01 mol / L, and the pH value is adjusted to 3.5-6.5, preferably 5.8, with glacial acetic acid; Mobile phase B: organic phase, preferably acetonitrile; Mobile phase A: B = 85-65: 15-35, preferably 75: 25;

[0018] Flow rate: 0.5-1.0 ml / min, preferably 0.5 ml / min;

[0019] Column temperature: 25-35°C, preferably 30°C;

[0020] Detection wavelength: 200-225 nm, preferably 210 nm;

[0021] Injection volume: 10 μl;

[0022] The present invention further provides a reverse phase liquid chromatography method for detecting lacosamide enantiomers, comprising the following steps:

[0023] (1) Preparation of system suitability solution: Take appropriate amount of lacosamide and its enantiomer, dissolve in mobile phase and dilute to make a solution containing approximately 0.01 mg of each in 1 ml.

[0024] (2) Preparation of test solution: Take an appropriate amount of this product, dissolve it in the mobile phase and dilute it to make a solution containing about 0.1 mg of lacosamide per 1 ml, which is used as the test solution.

[0025] (3) Chromatographic experiment: Take appropriate amounts of system suitability solution and test sample solution, inject them into the liquid chromatograph, and record the chromatogram.

[0026] The specific chromatographic conditions in step (3) are:

[0027] Detector: UV detector;

[0028] Chromatographic column: Silica gel coated with cellulose-tris-(3-chloro-4-methylphenylcarbamate) as filler, polysaccharide derivative reverse phase coated chiral chromatographic column, Daicel Chiralcel OZ-RH 150mm×4.6mm, 5μm;

[0029] Mobile phase: Mobile phase A: 0.01 mol / L ammonium acetate solution, pH adjusted to 5.8 with glacial acetic acid; Mobile phase B: acetonitrile; Mobile phase A: B = 75:25;

[0030] Flow rate: 0.5ml / min;

[0031] Column temperature: 30°C;

[0032] Detection wavelength: 210nm;

[0033] Injection volume: 10 μl;

[0034] Compared with the prior art, the present invention has the following technical effects:

[0035] 1. The present invention realizes the effective separation and detection of lacosamide and its enantiomers. The reverse phase chromatography system used has the advantages of stable chromatographic solvent performance, strong separation ability, simple operation process, easier balance, etc., and has stronger universality. It can be directly used for the detection of solution preparations such as lacosamide injection, and the operation is more convenient. It can more conveniently realize the quality control of the product production process, improve the quality of the final product, and ensure the safety and effectiveness of clinical medication.

[0036] 2. The quantitative limit of the present invention is 0.045 μg / ml, and the detection limit is 0.014 μg / ml. It has high sensitivity, high accuracy, good repeatability, and short detection time, providing a simpler, more stable, and more reliable analysis and detection method for product development and production processes. 4. Description of the drawings

[0037] Figure 1 This is the system suitability test spectrum in Example 1.

[0038] Figure 2 This is the test spectrum of the test solution in Example 1.

[0039] Figure 3 This is the accuracy test spectrum of the method in Example 3.

[0040] Figure 4 This is the linear test spectrum in Example 5.

[0041] Figure 5 This is a test diagram of the repeatability of the method in Example 7.

[0042] Figure 6 The test spectra of different chromatographic columns in Example 8 are shown in FIG.

[0043] Figure 7 This is the test spectrum of different column temperatures in Example 8.

[0044] Figure 8 This is the test spectrum of different acetonitrile ratios in Example 8.

[0045] Fig. 9 This is the test spectrum of different mobile phase pH values ​​in Example 8. 5. Specific implementation methods

[0046] To facilitate those skilled in the art to understand the content of the present invention, specific embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0047] Instrument and chromatographic conditions

[0048] Instrument: Waters e2695 high performance liquid chromatograph, UV detector.

[0049] Reagents: Ammonium acetate and glacial acetic acid were of analytical grade, and acetonitrile was of chromatography grade.

[0050] The lacosamide reference substance and enantiomer reference substance were both prepared by the applicant.

[0051] Chromatographic column: Silica gel surface coated with cellulose-tris-(3-chloro-4-methylphenylcarbamate) as the filler.

[0052] Daicel Chiralcel OZ-RH 150mm×4.6mm, 5μm;

[0053] Mobile phase: 0.01 mol / L ammonium acetate solution (pH adjusted to 5.8 with glacial acetic acid)-acetonitrile (75:25);

[0054] Flow rate: 0.5ml / min;

[0055] Column temperature: 30°C;

[0056] Detection wavelength: 210nm;

[0057] Injection volume: 10 μl;

[0058] Solution preparation

[0059] Preparation of system suitability solution: Take appropriate amount of lacosamide and its enantiomer, add mobile phase to dissolve and dilute to make a solution containing approximately 0.01 mg of each in 1 ml.

[0060] Preparation of test solution: Take an appropriate amount of this product, dissolve it in the mobile phase and dilute it to make a solution containing about 0.1 mg of lacosamide per 1 ml, which is used as the test solution.

[0061] Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with the mobile phase to make a solution with about 0.15 μg of lacosamide per 1 ml as the control solution.

[0062] Preparation of sensitivity solution: Take an appropriate amount of the test solution and dilute it with the mobile phase to make a solution containing approximately 0.05 μg of lacosamide per 1 ml.

[0063] Determination method

[0064] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph, record the chromatogram, the peak order is lacosamide and enantiomers, and the separation degree between the two should not be less than 1.5; in the sensitivity solution chromatogram, the signal-to-noise ratio of the peak height of the main component should not be less than 10. Accurately measure 10 μl of the test solution and the control solution, respectively, inject them into the liquid chromatograph, and record the chromatogram. If there is a chromatographic peak in the chromatogram of the test solution with the same retention time as the enantiomer, its peak area shall not be greater than the main peak area of ​​the control solution (0.15%).

[0065] Example 1 Specificity Test

[0066] Take 10 μl of the system suitability solution and inject it into the liquid chromatograph to record the chromatogram. The results are shown in Table 1 and Figure 1In the system suitability solution chromatogram, lacosamide and enantiomers eluted in sequence, and the separation degree between the two was greater than 1.5, which was well separated; the retention time of the main peak of lacosamide was moderate (about 8.5 minutes), the number of theoretical plates was greater than 3000, and the peak shape was good. Take 10μl of the sensitivity solution, inject it into the liquid chromatograph, record the chromatogram, and the results are shown in Table 1. The signal-to-noise ratio of the peak height of the main component is 25, which is greater than 10. Accurately measure 10μl of the test solution and the control solution, inject them into the liquid chromatograph, and record the chromatogram. The results are shown in Table 1 and Figure 2 , enantiomers were not detected in the chromatogram of the test solution, which met the requirements.

[0067] Table 1 Results of suitability test of lacosamide enantiomer test system

[0068]

[0069]

[0070] Example 2 Selection of chromatographic conditions

[0071] The enantiomer system suitability test solution of this product was detected and analyzed by high performance liquid chromatography. Liquid chromatography conditions: Chiralcel OZ-RH (150mm×4.6mm, 5μm) chromatographic column, mobile phase as shown in Table 2, UV detection wavelength of 210nm, flow rate of 0.5ml / min, column temperature of 30℃, injection volume of 10μl, and record the chromatogram.

[0072] Table 2 Selection of chromatographic conditions for the examination of lacosamide enantiomers

[0073]

[0074] From the screening results of the chromatographic conditions in Table 2 and the results in Example Table 1, it can be seen that the mobile phase and chromatographic conditions of Example 1 have good peak shapes for the two peaks, suitable retention times, and a good separation effect is achieved between the lacosamide peak and the lacosamide enantiomer peak.

[0075] Example 3 Accuracy Test

[0076] The accuracy of the method was achieved by examining the accuracy of adding different amounts of enantiomers to the sample. The concentrations of the enantiomers examined included the limit of quantification, 0.075 μg / ml of enantiomer (0.075%), 0.15 μg / ml of enantiomer (0.15%), and 0.225 μg / ml of enantiomer (0.225%). Each concentration was repeated 3 times, and the average recovery rate was consistent with 100±20%, the relative standard deviation was ≤10%, and the method had good accuracy. The results are shown in Table 3 and Figure 3 .

[0077] The accuracy was calculated by measured amount / added amount × 100%.

[0078] Table 3 Lacosamide enantiomer accuracy test results

[0079]

[0080] Example 4 Solution stability test

[0081] Prepare test solution and control solution, place them at room temperature under natural light, and inject samples for detection at 0, 2, 4, 6, 8, and 24 hours, respectively. Compared with 0 hour, calculate the change in peak area or enantiomer amount, which is no more than 10%. The test solution has good stability within 24 hours. The results are shown in Tables 4 and 5.

[0082] Table 4 Lacosamide enantiomer test control solution stability test results

[0083]

[0084] Table 5 Lacosamide enantiomer test solution stability test results

[0085]

[0086] Example 5 Linearity Test

[0087] The linearity of the method was achieved by examining the linearity of at least 6 solutions of different concentrations. The measured peak area was plotted against the concentration, and the regression equation, correlation coefficient and intercept were calculated using the least squares method. The method had good linearity, as shown in Table 6 and Figure 4 .

[0088] Table 6 Lacosamide enantiomer linearity test results

[0089]

[0090]

[0091] Example 6 Quantitative Limit and Detection Limit Test

[0092] Take appropriate amount of lacosamide and lacosamide enantiomers, dissolve and gradually dilute with mobile phase, shake well, accurately measure 10 μl and inject into liquid chromatograph until the response value of each component peak is about 10 times and 3 times higher than the noise signal, which is the quantitative limit and detection limit of the corresponding component. The results are shown in Table 7.

[0093] Table 7 Results of quantification limit and detection limit of lacosamide enantiomers

[0094]

[0095] Example 7 Precision Test

[0096] The precision test was conducted through method repeatability and intermediate precision. The method repeatability was tested by repeatedly preparing the same batch of test samples 6 times (adding enantiomers at 0.15%), calculating the amount of enantiomers detected in each test solution and the relative standard deviation of the detected amount, and the RSD did not exceed 10%; the intermediate precision was tested by repeating the "method repeatability" verification by different analysts, on different dates, and using different instruments, and the RSD did not exceed 10%; and the relative deviation of the results of method repeatability and intermediate precision did not exceed 10%, which met the requirements. The results are shown in Table 8 and Figure 5 .

[0097] Table 8 Lacosamide enantiomer test precision test results

[0098]

[0099] Example 8 Durability Test

[0100] The durability of the test method was investigated by changing parameters such as the chromatographic column batch number, column temperature, mobile phase ratio, and mobile phase pH value. Only one of the parameters was changed in each test, and the results were compared with the test results without changing the parameters. Compared with standard conditions, the separation between the chromatographic peaks of lacosamide and the enantiomers was greater than 1.5 when the chromatographic column batch number was changed, the column temperature changed by ±5°C, the acetonitrile ratio in the mobile phase changed by ±2%, and the mobile phase pH value changed by ±0.2, which met all system suitability test requirements. No enantiomers were detected in the test sample, indicating that the method has good durability. The results are shown in Table 9 and Figure 6 to Figure 9 .

[0101] Table 9 Lacosamide enantiomer inspection durability test results

[0102]

Claims

1. A reverse phase liquid chromatography method for detecting enantiomers of lacosamide, characterized in that: The following steps are involved: (1) Preparation of system suitability solution: Take appropriate amount of lacosamide and its enantiomers, add mobile phase to dissolve and dilute to prepare system suitability solution; (2) Preparation of test solution: Take an appropriate amount of lacosamide test sample, dissolve it in the mobile phase and dilute it to prepare the test solution; (3) Chromatographic test: Take appropriate amounts of system suitability solution and test sample solution, inject them into the liquid chromatograph, and record the chromatogram; Wherein, the specific chromatographic conditions in step (3) are: Detector: UV detector; Chromatographic column: a reverse phase coated chiral chromatographic column with polysaccharide derivatives and cellulose-tris-(3-chloro-4-methylphenylcarbamate) as filler on the surface of silica gel; Mobile phase: Mobile phase A: ammonium acetate solution; Mobile phase B: acetonitrile, the mobile phase A is adjusted to pH 5.8 with glacial acetic acid, and the mobile phase A:B=75:25; Flow rate: 0.5~1.0ml / min; Column temperature: 25-35°C; Injection volume: 10 μl; The detection wavelength is 210 nm.

2. The reverse phase liquid chromatography method for detecting lacosamide enantiomers according to claim 1, characterized in that: The chiral chromatographic column is DaicelChiralcelOZ-RH 150mm×4.6mm, 5μm.

3. The reverse phase liquid chromatography method for detecting lacosamide enantiomers according to claim 1, characterized in that: The concentration of the mobile phase A is 0.005-0.1 mol / L.

4. The reverse phase liquid chromatography method for detecting lacosamide enantiomers according to claim 3, characterized in that: The flow rate is 0.5 ml / min.

5. The reverse phase liquid chromatography method for detecting lacosamide enantiomers according to claim 1, characterized in that: The column temperature was 30°C.

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