Reversed phase liquid chromatography method for detecting related substances of lacosamide dry syrup

Abstract

The invention discloses a reversed-phase liquid chromatography method for detecting related substances of lacosamide dry syrup, and belongs to the technical field of liquid chromatography analysis. The method comprises the following steps: injecting a test solution and a contrast solution into a liquid chromatograph, recording a chromatogram, and calculating the impurity content by peak area according to a self-contrast method. The method solves the problem that a certain impurity appears late or cannot be detected in the prior art, meanwhile, the response value is high, the separation degree meets the requirement, the blank of detection of the related substances of the lacosamide dry syrup is filled, the specificity is high, the sensitivity is high, and the requirements for purity detection of the substances and quantitative detection of the related substances can be met.

Description

Technical Field

[0001] The present application relates to a reverse phase liquid chromatography method for detecting lacosamide dry syrup-related substances, and belongs to the technical field of liquid chromatography analysis. Background Art

[0002] Impurities present in lacosamide, such as EP impurity A, EP benzylamine, EPLKXA-1, EP impurity E, EPLKXA-ZZ-F, EP impurity B, EPLKXA-ZZ-H, EP impurity C, EP impurity G, EP impurity H, EP impurity I, etc., directly affect the quality and efficacy of the product.

[0003] At present, the detection method of lacosamide related substances can only detect several impurities, the impurity detection is not comprehensive, and the chromatographic peak shape is poor. In addition, EP10.6 currently only provides a method for detecting related substances in lacosamide tablets, and the use of this method for related substance detection in dry syrup dosage forms is not completely applicable. Chinese Patent 202310538220.9 discloses a method for separating and detecting related substances of lacosamide intermediates. The method uses a C18 chromatographic column, which takes a long time to analyze, and long-term use or improper cleaning may cause column contamination, affecting the separation effect. In addition, this patent is a method for detecting lacosamide intermediates. Due to the different types of impurities and molecular polarity of lacosamide intermediates and lacosamide dry syrup, its method cannot be applied to the detection of related substances of lacosamide dry syrup. Summary of the invention

[0004] According to one aspect of the present application, a reversed-phase liquid chromatography method for detecting related substances of lacosamide dry syrup is provided, which solves the problem in the prior art that a certain impurity elutes late or cannot be detected, and at the same time has a high response value and meets the separation requirements, thus filling the gap in the detection of related substances of lacosamide dry syrup, and has strong specificity and high sensitivity, and can meet the requirements for purity detection of the substance and quantitative detection of related substances.

[0005] The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup comprises: injecting a test solution and a control solution into a liquid chromatograph, recording a chromatogram, and calculating the impurity content by peak area according to a self-control method.

[0006] Optionally, the related substances include: LKXA enantiomers, benzylamine, LKXA-0, L KXA-1, LKXA-ZZ-E, LKXA-ZZ-F, LKXA-ZZ-G, LKXA-ZZ-H, LKXA-ZZ-I, LKXA-ZZ-J, LKXA-ZZ-K, LKXA-ZZ-L, LKXA-ZZ-M, LKXA-ZZ-O;

[0007] The chemical name of the LKXA enantiomer is (2S)-2-acetylamino-N-benzyl-3-methoxypropionamide, and the structure is as follows:

[0008]

[0009] The structure of LKXA-0 is as follows:

[0010]

[0011] The chemical name of LKXA-1 is (2R)-2-amino-N-benzyl-3-methoxypropionamide, and its structure is as follows:

[0012]

[0013] The chemical name of LKXA-ZZ-E is (2R)-2-amino-N-benzyl-3-hydroxypropionamide, and its structure is as follows:

[0014]

[0015] The chemical name of LKXA-ZZ-F is (2R)-2-acetylamino-N-benzyl-3-hydroxypropionamide, and its structure is as follows:

[0016]

[0017] The chemical name of LKXA-ZZ-G is (R)-2-acetamido-3-(benzylamino)-3-oxypropyl acetate, and its structure is as follows:

[0018]

[0019] The chemical name of LKXA-ZZ-H is 2-acetylamino-N-benzyl-2-acrylamide, and its structure is as follows:

[0020]

[0021] The structure of LKXA-ZZ-I is as follows:

[0022]

[0023] The chemical name of LKXA-ZZ-J is (R)-3-methoxy-2-(N-methylacetylamino)-N-benzylpropionamide, and its structure is as follows:

[0024]

[0025] The structure of LKXA-ZZ-K is as follows:

[0026]

[0027] The structure of LKXA-ZZ-L is as follows:

[0028]

[0029] The structure of LKXA-ZZ-M is as follows:

[0030]

[0031] The chemical name of LKXA-ZZ-O is benzaldehyde, and its structure is as follows:

[0032]

[0033] Optionally, mobile phase A is a mixed aqueous solution of formic acid-acetonitrile;

[0034] Mobile phase B is a mixed aqueous solution of formic acid and acetonitrile;

[0035] The total volume ratio of formic acid and acetonitrile in the mobile phase A is 10-15%;

[0036] The total volume ratio of formic acid and acetonitrile in the mobile phase B is 85-95%.

[0037] Preferably, the total volume ratio of formic acid and acetonitrile in the mobile phase A is 12%.

[0038] Preferably, the total volume ratio of formic acid and acetonitrile in the mobile phase B is 90%.

[0039] Optionally, the volume ratio of formic acid to acetonitrile in the mobile phase A is 0.1:(95-105); preferably, the volume ratio of formic acid to acetonitrile in the mobile phase A is 0.1:100.

[0040] Optionally, the volume ratio of formic acid to acetonitrile in the mobile phase B is 0.1:(850-950); preferably, the volume ratio of formic acid to acetonitrile in the mobile phase B is 0.1:900.

[0041] Optionally, the solvent of the test solution and the control solution is an aqueous solution of methanol.

[0042] Optionally, in the aqueous solution of methanol, the volume ratio of methanol to water is (10-20):(90-80); preferably, in the aqueous solution of methanol, the volume ratio of methanol to water is 15:85.

[0043] Optionally, gradient elution is employed;

[0044] The elution gradient is as follows:

[0045] 0min, mobile phase A 95-100Vol%, mobile phase B 5-0Vol%;

[0046] 6 min, mobile phase A 95-100 Vol%, mobile phase B 5-0 Vol%;

[0047] 25 min, mobile phase A 65-75 Vol%, mobile phase B 35-25 Vol%;

[0048] 30 min, mobile phase A 5-0 Vol%, mobile phase B 95-100 Vol%;

[0049] 35 min, mobile phase A 95-100 Vol%, mobile phase B 5-0 Vol%;

[0050] 45min, mobile phase A 95-100Vol%, mobile phase B 5-0Vol%.

[0051] Preferably, the elution gradient is as follows:

[0052] 0min, mobile phase A 100Vol%, mobile phase B 0Vol%;

[0053] 6 min, mobile phase A 100 Vol%, mobile phase B 0 Vol%;

[0054] 25 min, mobile phase A 70 Vol%, mobile phase B 30 Vol%;

[0055] 30 min, mobile phase A 0 Vol%, mobile phase B 100 Vol%;

[0056] 35 min, mobile phase A 100 Vol%, mobile phase B 0 Vol%;

[0057] 45 min, mobile phase A 100 Vol%, mobile phase B 0 Vol%.

[0058] Optionally, the filler of the chromatographic column is octylsilane bonded silica gel.

[0059] Optionally, the specifications of the chromatographic column are 150 mm×4.6 mm, 5 μm.

[0060] Optionally, the column temperature is 30-40°C; preferably, the column temperature is 35°C.

[0061] Optionally, the column temperature is independently selected from any one of 30°C, 35°C, 40°C, or a range between any two of the above.

[0062] Optionally, the mobile phase flow rate is 1.2-1.6 ml / min; preferably, the mobile phase flow rate is 1.4 ml / min.

[0063] Optionally, the mobile phase flow rate is independently selected from any value of 1.2 ml / min, 1.4 ml / min, 1.5 ml / min, 1.6 ml / min or a range between any two of the above values.

[0064] Optionally, the detector is a UV detector.

[0065] Optionally, the detection wavelength is 200-230 nm; preferably, the detection wavelength is 215 nm.

[0066] Optionally, the injection volume is 5-10 μl; preferably, the injection volume is 8 μl.

[0067] Impurities are calculated as follows:

[0068]

[0069] Where: A 杂质 is the peak area of ​​a single impurity in the test solution

[0070] A 对照 is the main peak area in the control solution;

[0071]

[0072] Where: ∑A 杂质 is the sum of the peak areas of each impurity in the test solution;

[0073] A 对照 is the main peak area in the control solution.

[0074] The beneficial effects of this application include:

[0075] 1. In the detection process of lacosamide related substances, the prior art generally uses acetonitrile-water as a solvent to dissolve lacosamide in order to dissolve various organic compounds in lacosamide well. However, when the inventors detected the related substances of lacosamide dry syrup, they found that when the conventional acetonitrile-water solvent of the prior art was used for related substance detection, it was found that after the test sample was placed at 2-8°C for a few days, the aniline peak in the chromatogram was easy to bifurcate, which could not meet the requirements for the detection of related substances of lacosamide dry syrup. After various studies by the inventors, it was found that by using methanol-water as a solvent and controlling the volume ratio of the two to (10-20): (90-80), the problem of aniline peak bifurcation was solved on the basis of meeting the separation degree.

[0076] 2. During the experimental process of detecting related substances of lacosamide dry syrup, the inventors found that there was a problem of poor separation. After a lot of research, thinking and improvement, the inventors found that by controlling the mobile phase substance and proportion and adopting the elution gradient of the present application, not only all the related substances of the present application are eluted, but also the separation meets the requirements, and the solvent does not interfere with the detection.

[0077] 3. EP10.6 provides a method for detecting related substances in lacosamide tablets. The related substances in this application include 14 impurities, which are of many types. The method of EP10.6 is not applicable to the dry syrup in this application. When the EP10.6 method is used to detect lacosamide dry syrup, some related substances are not detected and some related substances have late peaks. The inventor has regulated the elution gradient conditions through research, etc., which not only solves the problem of late peaks or inability to detect certain impurities in the prior art, but also has a high response value and meets the requirements for separation. The method of this application fills the gap in the detection of related substances in lacosamide dry syrup, and after methodological verification, the specificity, solution stability, injection precision, linearity and correction factor, quantitative limit and detection limit, accuracy, precision, and durability all meet the requirements, proving that the method of this application can meet the purity detection of the substance and the quantitative detection requirements of related substances. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a liquid chromatogram of the mixed impurity solution of Example 1;

[0079] Figure 2 This is the liquid chromatogram of the mixed impurity solution of Comparative Example 1;

[0080] Figure 3 This is the liquid chromatogram of the mixed impurity solution of Comparative Example 2;

[0081] Figure 4 This is the liquid chromatogram of the mixed impurity solution of Comparative Example 3;

[0082] Figure 5 This is the liquid chromatogram of the mixed impurity solution of Comparative Example 4 after being placed at 2-8°C for 5 days. DETAILED DESCRIPTION

[0083] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0084] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0085] Example 1

[0086] The chromatographic conditions were as follows:

[0087] Chromatographic column: Agilent ZORBAX Eclipse XDB-C8 150mm×4.6mm, 5μm

[0088] Column temperature: 35°C;

[0089] Flow rate: 1.4ml / min;

[0090] Detector: UV detector;

[0091] Detection wavelength: 215nm;

[0092] Injection volume: 8 μl;

[0093] Mobile phase A: formic acid-acetonitrile-water, volume ratio 0.1:120:880;

[0094] Mobile phase B: formic acid-acetonitrile-water, volume ratio of 0.1:900:100.

[0095] Solvent: methanol-water, volume ratio 15:85;

[0096] The elution gradient is as follows:

[0097] Time (minutes) Mobile phase A (Vol%) Mobile phase B (Vol%) 0 100 0 6 100 0 25 70 30 30 0 100 35 100 0 45 100 0

[0098] Take about 2 mg each of impurities LKXA-0, LKXA-1, LKXA-ZZ-E, LKXA-ZZ-F, LKXA-ZZ-G, LKXA-ZZ-H, LKXA-ZZ-I, LKXA-ZZ-J, LKXA-ZZ-K, LKXA-ZZ-L, LKXA-ZZ-M, LKXA-ZZ-O and benzylamine, place them in 10 ml volumetric flasks, add acetonitrile to dissolve and dilute to the scale, shake well, and use them as stock solutions of each impurity.

[0099] Mixed impurity solution: Take 10 mg of lacosamide reference substance (WS-101374-102-00001-1 batch), put it in a 10 ml volumetric flask, add 0.1 ml of each impurity stock solution, add methanol-water (15:85) to dissolve and dilute to the scale, shake well, and prepare a 0.2 wt% impurity mixed solution.

[0100] The liquid chromatogram of the mixed impurity solution is shown in Figure 1 The test results are shown in Table 1 below.

[0101] Table 1

[0102]

[0103]

[0104] from Figure 1From the test results in Table 1, it can be seen that under the chromatographic conditions, the separation between the impurities and between the main component and the impurities is greater than 1.5, and the separation meets the requirements.

[0105] The test was performed again, and the mixed impurity solution was placed at 2-8°C for 32 days and then tested again. The test results are shown in Table 2 below.

[0106] Table 2

[0107]

[0108] Note: The separation of day 0 in Table 1 and Table 2 is within the reasonable range of human error.

[0109] At 2-8°C, after 32 days, the area percentage of each impurity changed by less than 0.1%, the separation degree met the requirements, and the benzylamine chromatographic peak did not change.

[0110] This method was systematically validated according to the Guiding Principles for Validation of Analytical Methods for Pharmaceutical Quality Standards (General Rules 9101 of the 2020 Edition of the Chinese Pharmacopoeia) and High Performance Liquid Chromatography (General Rules 0512 of the 2020 Edition of the Chinese Pharmacopoeia). The validation results all met the requirements. The validation results are summarized in Table 3 below.

[0111] Table 3 Summary of verification results of relevant substance inspection methods

[0112]

[0113]

[0114]

[0115] Comparative Example 1

[0116] The chromatographic conditions were the same as those in Example 1, except that the mobile phase was formic acid-acetonitrile-water, with a volume ratio of 0.75:350:650, the elution was isocratic, and the solvent was acetonitrile-water, with a volume ratio of 15:85. The injection volume was 5 μL.

[0117] The liquid phase spectrum of the impurity mixed solution is shown in Figure 2 .

[0118] Using this comparative chromatographic condition test, Figure 2 The impurity peaks from left to right are as follows: benzylamine, LKXA-ZZ-E, LKXA-1, LKXA-ZZ-F, LKXA-ZZ-K, LKXA-ZZ-H, LKXA-ZZ-G, LKXA-ZZ-I, LKXA-ZZ-L, while LKXA-ZZ-M peaks later (about 115 minutes), and LKXA-0 does not peak.

[0119] Comparative Example 2

[0120] The chromatographic conditions were the same as those in Example 1, except that the solvent was acetonitrile-water, with a volume ratio of 15:85; the mobile phase A was formic acid-acetonitrile-water, with a volume ratio of 0.75:130:870; and the mobile phase B was formic acid-acetonitrile-water, with a volume ratio of 0.75:800:200. The injection volume was 5 μL.

[0121] The elution gradient is as follows:

[0122] Time (min) Mobile phase A (Vol%) Mobile phase B (Vol%) 0 100 0 15 100 0 25 0 100 50 0 100 51 100 0 60 100 0

[0123] The liquid chromatogram of the impurity mixed solution is shown in Figure 3 .

[0124] The chromatographic conditions of this comparative example were used for testing, and the peaks were shown in Figure 3 As shown, chromatographic peaks 1-11 are benzylamine, LKXA-ZZ-E, LKXA-1, LKXA-ZZ-F, LKXA-ZZ-K, LKXA-ZZ-H, LKXA-ZZ-G, LKXA-ZZ-L, LKXA-ZZ-I, LKXA-ZZ-M, and LKXA-0.

[0125] It can be seen from the figure that under the chromatographic conditions, the blank solvent interferes with the LKXA-ZZ-M detection and the separation between adjacent peaks cannot meet the requirements.

[0126] Comparative Example 3

[0127] The chromatographic conditions were the same as those in Example 1, except that the chromatographic column was Diamonsil C8(2) 5 μm 150×4.6 mm; the solvent was acetonitrile-water, with a volume ratio of 15:85; the mobile phase A was formic acid-acetonitrile-water, with a volume ratio of 0.1:100:900; the injection volume was 5 μL; and the elution gradient was as follows:

[0128] Time (in) Mobile phase A (Vol%) Mobile phase B (Vol%) 0 100 0 10 100 0 23 60 40 30 0 100 35 100 0 40 100 0

[0129] The liquid chromatogram of the mixed impurity solution is shown in Figure 4 .

[0130] The test results are shown in Table 4 below.

[0131] Table 4

[0132] Serial number name Retention time (min) RRT S / N Separation 1 Benzylamine 2.262 0.16 904 / 2 LKXA-ZZ-E 3.334 0.23 655 7.15 3 LKXA-1 5.805 0.41 54 13.53 4 LKXA-ZZ-F 8.746 0.61 324 11.02 5 LKXA-ZZ-K 13.235 093 587 16.37 / LqCy 14.23 1.00 212680 4.61 6 LKXA-ZZ-H 15.157 1.07 1018 4.65 7 LKXA-ZZ-G 15.642 1.10 484 2.56 8 LKXA-ZZ-L 16.234 1.14 481 3.53 9 LKXA-ZZ-I 16.676 1.17 590 2.61 10 LKXA-ZZ-J 17.292 1.22 228 3.47 11 LKXA-ZZ-O 18.142 1.27 223 3.60 12 LKXA-ZZ-M 23.16 1.63 840 21.17 13 LKXA-0 24.834 1.75 403 10.02

[0133] From the test results, we can see that the retention time of benzylamine and solvent peaks are close, and the separation degree cannot meet the requirements.

[0134] Comparative Example 4

[0135] The chromatographic conditions were the same as those in Example 1, except that the solvent was acetonitrile-water with a volume ratio of 15:85.

[0136] After the mixed impurity solution was placed at 2-8°C for 5 days, it was tested and the liquid chromatogram was shown in Figure 5 .

[0137] The test results are shown in Table 5 below.

[0138] Table 5

[0139]

[0140] Under the chromatographic conditions, the separation between each impurity and between the main component and the impurity is greater than 1.5, which meets the requirements, but the benzylamine peak is prone to bifurcation.

[0141] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup, characterized in that: include: Inject the test solution and the control solution into the liquid chromatograph, record the chromatogram, and calculate the impurity content by peak area according to the self-control method; The related substances include: LKXA enantiomers, benzylamine, LKXA-0, LKXA-1, LKXA-ZZ-E, LKXA-ZZ-F, LKXA-ZZ-G, LKXA-ZZ-H, LKXA-ZZ-I, LKXA-ZZ-J, LKXA-ZZ-K, LKXA-ZZ-L, LKXA-ZZ-M, LKXA-ZZ-O; The chemical name of the LKXA enantiomer is (2S)-2-acetylamino-N-benzyl-3-methoxypropionamide, and the structure is as follows: The structure of LKXA-0 is as follows: The chemical name of LKXA-1 is (2R)-2-amino-N-benzyl-3-methoxypropionamide, and its structure is as follows: The chemical name of LKXA-ZZ-E is (2R)-2-amino-N-benzyl-3-hydroxypropionamide, and its structure is as follows: The chemical name of LKXA-ZZ-F is (2R)-2-acetylamino-N-benzyl-3-hydroxypropionamide, and its structure is as follows: The chemical name of LKXA-ZZ-G is (R)-2-acetamido-3-(benzylamino)-3-oxypropyl acetate, and its structure is as follows: The chemical name of LKXA-ZZ-H is 2-acetylamino-N-benzyl-2-acrylamide, and its structure is as follows: The structure of LKXA-ZZ-I is as follows: The chemical name of LKXA-ZZ-J is (R)-3-methoxy-2-(N-methylacetylamino)-N-benzylpropionamide, and its structure is as follows: The structure of LKXA-ZZ-K is as follows: The structure of LKXA-ZZ-L is as follows: The structure of LKXA-ZZ-M is as follows: The chemical name of LKXA-ZZ-O is benzaldehyde, and its structure is as follows:

2. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 1, characterized in that: The solvent for the test solution and the control solution was methanol in water.

3. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 2, characterized in that: In the aqueous solution of methanol, the volume ratio of methanol to water is (10-20):(90-80).

4. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to any one of claims 1 to 3, characterized in that: Gradient elution was used; The elution gradient is as follows: 0min, mobile phase A 95-100Vol%, mobile phase B 5-0Vol%; 6 min, mobile phase A 95-100 Vol%, mobile phase B 5-0 Vol%; 25 min, mobile phase A 65-75 Vol%, mobile phase B 35-25 Vol%; 30 min, mobile phase A 5-0 Vol%, mobile phase B 95-100 Vol%; 35 min, mobile phase A 95-100 Vol%, mobile phase B 5-0 Vol%; 45min, mobile phase A 95-100Vol%, mobile phase B 5-0Vol%.

5. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 4, characterized in that: Mobile phase A is a mixed aqueous solution of formic acid and acetonitrile; Mobile phase B is a mixed aqueous solution of formic acid and acetonitrile; The total volume ratio of formic acid and acetonitrile in the mobile phase A is 10-15%; The total volume ratio of formic acid and acetonitrile in the mobile phase B is 85-95%.

6. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 5, characterized in that: The volume ratio of formic acid to acetonitrile in the mobile phase A is 0.1:(95-105).

7. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 5, characterized in that: The volume ratio of formic acid to acetonitrile in the mobile phase B is 0.1:(850-950).

8. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 1, characterized in that: The filler of the chromatographic column is octylsilane bonded silica gel.

9. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 1, characterized in that: The flow rate of the mobile phase is 1.2-1.6 ml / min.

10. The reverse phase liquid chromatography method for detecting related substances of lacosamide dry syrup according to claim 1, characterized in that: The detection wavelength is 200-230nm; Preferably, the injection volume is 5-10 μl.

Images