Method for detecting content of lacosamide particles by high performance liquid chromatography

Abstract

The invention discloses a method for detecting the content of lacosamide particles through high performance liquid chromatography, and belongs to the technical field of detection and analysis. The method for detecting the content of lacosamide particles by high performance liquid chromatography comprises the following steps: respectively injecting a reference solution and a test solution into a liquid chromatograph, recording chromatograms, and calculating the content of lacosamide by peak area according to an external standard method. The method solves the problem of poor linearity and repeatability during lacosamide particle content detection, has remarkable advantages, and fills the blank of lacosamide particle content detection in China. Besides, the method is simple and easy to operate, good in specificity, high in precision and sensitivity, stable, reliable and good in durability, various chromatographic conditions are matched with one another, and the method can be used for quality control of the lacosamide particles and is suitable for industrial production.

Description

Technical Field

[0001] The present application relates to a method for detecting the content of lacosamide particles by high performance liquid chromatography, belonging to the technical field of detection and analysis. Background Art

[0002] Lacosamide is a new type of anti-epileptic drug. Its granule preparation has been widely used in clinical practice due to its convenient administration and good stability. Accurate detection of the content of lacosamide granules is very important for quality control and clinical application.

[0003] Chinese patent CN110320291A provides a method for quantitatively detecting the content of lacosamide injection by HPLC, the chromatographic conditions are C18 column, the mobile phase is acetonitrile-methanol-water, and the injection volume is 20μl. However, this method cannot meet the requirements in the content detection of lacosamide granules. In addition, the quality standards of pharmacopoeias of various countries do not include the content detection method of lacosamide granules. Summary of the invention

[0004] According to one aspect of the present application, a method for detecting the content of lacosamide particles by high performance liquid chromatography is provided, wherein the mobile phase is controlled to be aqueous formic acid solution and acetonitrile, the volume content of acetonitrile in the mobile phase is 25-35%, and the length of the chromatographic column is controlled to be 100-250 mm, the inner diameter is 3-4.6 mm, and the particle size is 5 μm, which solves the problem of poor linearity and repeatability in the detection of lacosamide particle content in the prior art, has significant advantages over the prior art, and fills the gap in the detection of lacosamide particle content in China. In addition, the method is simple and easy to operate, the chromatographic conditions cooperate with each other, the specificity is good, the precision and sensitivity are high, stable, reliable, and durable, and can be used for the quality control of lacosamide particles, and is suitable for industrial production.

[0005] A method for detecting the content of lacosamide particles by high performance liquid chromatography comprises: injecting a reference solution and a test solution into a liquid chromatograph respectively, recording a chromatogram, and calculating the content of lacosamide by peak area according to an external standard method.

[0006] Optionally, the filler of the chromatographic column is octylsilane bonded silica gel.

[0007] Optionally, the length of the chromatographic column is 100-250 mm; preferably, the length of the chromatographic column is 250 mm.

[0008] Optionally, the length of the chromatographic column is independently selected from any value of 100 mm, 150 mm, 250 mm, or a range between any two of the above values.

[0009] Optionally, the inner diameter of the chromatography column is 3-4.6 mm.

[0010] Optionally, the particle size of the chromatographic column is 3-5 μm; preferably, the particle size of the chromatographic column is 5 μm.

[0011] Optionally, the detection wavelength is 200-210 nm.

[0012] Optionally, the flow rate of the mobile phase is 0.5-1.5 ml / min; preferably, the flow rate of the mobile phase is 1 ml / min.

[0013] Optionally, the column temperature is 30-40°C; preferably, the column temperature is 35°C.

[0014] Optionally, the injection volume is 3-8 μl; preferably, the injection volume is 5 μl.

[0015] Optionally, the mobile phase is a mixed solution of aqueous formic acid and acetonitrile.

[0016] Optionally, the volume concentration of the formic acid aqueous solution is 0.05-0.15‰; preferably, the volume concentration of the formic acid aqueous solution is 0.1‰.

[0017] Optionally, the volume ratio of the aqueous formic acid solution to acetonitrile is (65-75):(25-35); preferably, the volume ratio of the aqueous formic acid solution to acetonitrile is 70:30.

[0018] Optionally, the concentration of the test solution is 0.04-0.2 mg / mL; preferably, the concentration of the test solution is 0.1 mg / mL.

[0019] Optionally, the concentration of the reference solution is 0.04-0.2 mg / mL; preferably, the concentration of the reference solution is 0.1 mg / mL.

[0020] Optionally, the solvent of the test solution and the reference solution is an aqueous solution of methanol.

[0021] Optionally, in the aqueous solution of methanol, the volume ratio of methanol to water is (10-20):(80-90); preferably, in the aqueous solution of methanol, the volume ratio of methanol to water is 15:85.

[0022] Optionally, the method for preparing the reference solution comprises: mixing lacosamide with a solvent to prepare the reference solution.

[0023] Optionally, the method for preparing the test sample solution includes: grinding the test sample to obtain fine powder; mixing the fine powder with a solvent, and filtering to obtain.

[0024] The beneficial effects of this application include:

[0025] 1. In the prior art, the HPLC method is used to test the content of lacosamide, and during isocratic elution, the content of the organic phase in the mobile phase is generally low, less than 20%. When the inventor used the method to test the content of lacosamide in lacosamide particles, the linearity and repeatability were poor. During the process of adjusting the mobile phase and chromatographic conditions, the inventor found that when the mobile phase was formic acid aqueous solution and acetonitrile, and the volume content of acetonitrile in the mobile phase was controlled at 25-35 Vol%, and the length of the chromatographic column was controlled at 100-250 mm, the inner diameter was 3-4.6 mm, and the particle size was 5 μm, the linearity and repeatability were good in the methodology verification. However, if the content of acetonitrile in the mobile phase is continued to be increased, and the chromatographic column conditions are not within the scope of this application, the retention time of the main component is short and cannot meet the requirements.

[0026] 2. The inventors found in the study that in the process of detecting the content of lacosamide particles, the separation of the main peak and the impurity peak was not obvious. After research, it was found that the present application controls the detection wavelength at 200-210nm, the mobile phase flow rate at 0.5-1.5ml / min, the column temperature at 30-40°C, the injection volume at 3-8μl, and the mobile phase is a mixed solution of formic acid aqueous solution and acetonitrile. The separation effect of the main peak and the impurity peak is obvious, which solves the problem of unclear separation of the main peak and the impurity peak that appeared in the early stage.

[0027] 3. According to the test method for the content of lacosamide injection in the prior art, a C18 chromatographic column was used to test the content of lacosamide particles under the conditions of this application, but the specificity and precision could not meet the requirements. The inventors used the Agilent ZORBAX SB-C8 chromatographic column 4.6×250mm 5μm in this application and controlled the corresponding chromatographic conditions, and finally solved the problems of specificity and precision.

[0028] 4. Under other chromatographic conditions in the present application, the injection volume is controlled at 3-8 μl, and the separation, detection sensitivity and linearity are good. However, the present application uses an injection volume of, for example, 20 μl in the prior art, which results in a broadened peak shape, reduced resolution, and poor linearity. If the injection volume is reduced, it may be necessary to increase the detection limit.

[0029] 5. The HPLC method for detecting the content of lacosamide granules in the present application is simple and easy to operate, the chromatographic conditions cooperate with each other, have good specificity, high precision and sensitivity, stability, reliability and good durability, can be used for quality control of lacosamide granules, and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the liquid chromatogram of the control sample of Example 1;

[0031] Figure 2 It is the liquid chromatogram of the control sample of Comparative Example 1. DETAILED DESCRIPTION

[0032] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0033] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0034] The content calculation formula is as follows:

[0035]

[0036] Where: W 供试品 is the sample size of the test product, g;

[0037] W 平均装量 is the average filling quantity under the filling quantity difference item, g;

[0038] A 供试品 is the main peak area of ​​the test solution;

[0039] D 供试品 is the dilution factor of the test solution;

[0040] The labeled amount is 0.1g.

[0041]

[0042] Where: W 袋+样 is the weight of 10 bags of samples, g;

[0043] W 袋 is the weight of 10 empty bags, g;

[0044]

[0045] Where: W 对照品 The sampling amount of the reference substance, mg;

[0046] P 对照品 is the content of reference substance, %;

[0047] A 对照品 is the main peak area of ​​the reference solution;

[0048] D 对照品 Dilution multiple of the reference solution

[0049] Example 1

[0050] Reference solution: Weigh 10 mg of lacosamide, place in a 100 ml volumetric flask, add solvent to dissolve and dilute to the scale.

[0051] Test solution: Take 10 bags of this product, accurately weigh the weight, pour out the contents completely, wipe the powder in the bag with a cotton swab, accurately weigh the weight of the empty bag, calculate the average filling amount, grind the contents, take 100 mg of fine powder (equivalent to 10 mg of lacosamide), accurately weigh, put it in a 100 ml volumetric flask, add about 60 ml of solvent, ultrasonicate for 5 minutes to dissolve lacosamide, dilute to the scale with solvent, shake well, filter with a filter membrane with a pore size of 0.45 μm, discard 4 ml of the initial filtrate, take the subsequent filtrate, and obtain.

[0052] Inject the reference solution and the test solution into the liquid chromatograph respectively and record the chromatogram.

[0053] The chromatographic conditions were as follows:

[0054] Chromatographic column: Agilent ZORBAX SB-C8 4.6×250mm 5μm

[0055] Detection wavelength: 210nm

[0056] Mobile phase: 0.1 vol‰ formic acid aqueous solution-acetonitrile (volume ratio 70:30)

[0057] Flow rate: 1.0ml / min

[0058] Column temperature: 35°C

[0059] Injection volume: 5 μl

[0060] Solvent: methanol-water (volume ratio 15:85)

[0061] The liquid chromatogram of the control sample is shown in Figure 1 ,from Figure 1 From the test results, it can be seen that the retention time of the main component is about 5.737min, the retention time meets the requirements, the tailing factor is 1.05, the theoretical plate number is 10618, and the peak shape is good.

[0062] The results of the method validation are shown in Table 1.

[0063] Table 1

[0064]

[0065]

[0066] The method validation is as follows:

[0067] 1. Specificity and injection precision Blank solvent: Measure 850 ml of water and 150 ml of methanol, mix well.

[0068] Blank excipient solution: Take 90 mg of blank excipient, accurately weigh, place in a 100 ml volumetric flask, add 60 ml of solvent, ultrasonicate for 5 minutes, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene (pore size 0.45 μm) filter membrane, discard 4 ml of the initial filtrate, and take the subsequent filtrate to obtain.

[0069] Test solution: Take 10 bags of this product, accurately weigh the weight, pour out the contents completely, wipe the powder in the bag with a cotton swab, accurately weigh the empty bag, calculate the average filling amount, grind the contents, take 100 mg of fine powder (equivalent to 10 mg of lacosamide), accurately weigh, put it in a 100 ml volumetric flask, add 60 ml of solvent, ultrasonicate for 5 min to dissolve lacosamide, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of the initial filtrate, take the subsequent filtrate to obtain.

[0070] Reference solution: Take 10 mg of lacosamide reference substance, weigh accurately, put into a 100 ml volumetric flask, add 60 ml of solvent, dissolve the lacosamide by ultrasound, dilute to the scale with solvent, shake well, and obtain.

[0071] Accurately measure the blank solvent, blank excipient solution, reference solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0072] Table 2 Content Methodology-Injection Precision Results

[0073]

[0074] 2. Solution stability

[0075] Take the test solution and the reference solution, place them at room temperature for different periods of time, accurately measure 5 μl and inject it into the liquid chromatograph to record the chromatogram.

[0076] Table 3 Content Methodology - Reference Solution Stability Results

[0077]

[0078]

[0079] Table 4 Content Methodology - Test Solution Stability Results

[0080]

[0081] 3. Linear

[0082] Linear stock solution: Take 25 mg of lacosamide reference substance, accurately weigh it, put it in a 25 ml volumetric flask, add an appropriate amount of solvent, dissolve the lacosamide by ultrasound, dilute to the scale with solvent, shake well, and obtain.

[0083] Prepare each linear solution according to the table below.

[0084] Table 5

[0085]

[0086] Determination method: Take 5 μl of each of the test solution and reference solution mentioned above, inject them into the liquid chromatograph in sequence, record the chromatogram, and the results are shown in the table below.

[0087] Table 6 Assay Methodology - Linearity and Range Test Results

[0088]

[0089] 4. Accuracy

[0090] Reference solution: Take 10 mg of lacosamide reference substance, weigh accurately, place in a 100 ml volumetric flask, add about 60 ml of solvent, dissolve lacosamide by ultrasonication, dilute to scale with solvent, shake well, and prepare 2 portions in parallel.

[0091] 50% recovery solution: Take 180 mg of blank excipient and 10 mg of lacosamide reference substance, weigh accurately, place in the same 200 ml volumetric flask, add 120 ml of solvent, sonicate for 5 min to dissolve lacosamide, bring to room temperature, dilute to scale with solvent, shake well, filter with polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of initial filtrate, take subsequent filtrate as 50% test solution. Prepare 3 portions in parallel.

[0092] 100% recovery solution: Take 90 mg of blank excipient and 10 mg of lacosamide reference substance, weigh accurately, place in the same 100 ml volumetric flask, add 60 ml of solvent, sonicate for 5 min to dissolve lacosamide, bring to room temperature, dilute to scale with solvent, shake well, filter with polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of initial filtrate, take the subsequent filtrate as 100% test solution. Prepare 3 copies in parallel.

[0093] 150% recovery solution: Take 90 mg of blank excipient and 15 mg of lacosamide reference substance, weigh accurately, place in the same 100 ml volumetric flask, add 60 ml of solvent, sonicate for 5 min to dissolve lacosamide, bring to room temperature, dilute to scale with solvent, shake well, filter with polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of initial filtrate, take subsequent filtrate as 150% test solution. Prepare 3 portions in parallel.

[0094] Determination method: Take 5 μl of each of the test solution and reference solution mentioned above, inject them into the liquid chromatograph in sequence, record the chromatogram, and the results are shown in the table below.

[0095] Table 7 Content Methodology - Accuracy Test Results

[0096]

[0097] Calculation formula: measured amount / added amount × 100%

[0098] 5. Precision

[0099] (1) Repeatability

[0100] Test solution: Take 10 bags of this product, accurately weigh them, pour out the contents completely, wipe the powder in the bag with a cotton swab, accurately weigh the empty bag, calculate the average filling amount, grind the contents, take 100 mg of fine powder (equivalent to 10 mg of lacosamide), accurately weigh it, put it in a 100 ml volumetric flask, add about 60 ml of solvent, sonicate for 5 minutes to dissolve lacosamide, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of the initial filtrate, take the subsequent filtrate, and prepare 6 portions in parallel.

[0101] Reference solution: Take 10 mg of lacosamide reference substance, weigh accurately, place in a 100 ml volumetric flask, add 60 ml of solvent, dissolve lacosamide by ultrasonication, dilute to scale with solvent, shake well, and prepare 2 portions in parallel.

[0102] Determination method: Take 5 μl of each of the test solution and reference solution mentioned above, inject them into the liquid chromatograph in sequence, record the chromatogram, and the results are shown in the table below.

[0103] Table 8 Content Methodology - Repeatability Test Results

[0104] Serial number 1 2 3 4 5 6 RSD(%) content(%) 100.0 99.7 100.1 99.9 99.9 100.4 0.25

[0105] (2) Intermediate precision

[0106] In the same laboratory, using different dates, different personnel, and different instruments, the control and test solutions were prepared according to the method under the repeatability item and the determination was carried out using the same method.

[0107] Determination method: Take 5 μl of each of the above solutions, inject them into the liquid chromatograph in sequence, and record the chromatogram. The results are shown in the table below.

[0108] Table 9 Content Methodology - Intermediate Precision Test Results

[0109]

[0110] 6. Durability

[0111] Blank excipient solution: Take 90 mg of blank excipient, accurately weigh, place in a 100 ml volumetric flask, add 60 ml of solvent, ultrasonicate for 5 minutes, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene (pore size 0.45 μm) filter membrane, discard 4 ml of the initial filtrate, and take the subsequent filtrate to obtain.

[0112] Test solution: Take 100 mg of fine powder (equivalent to 10 mg of lacosamide), weigh accurately, place in a 100 ml volumetric flask, add 60 ml of solvent, sonicate for 5 min to dissolve lacosamide, dilute to scale with solvent, shake well, filter with a polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of the initial filtrate, take the subsequent filtrate, and prepare 2 portions in parallel.

[0113] Reference solution: Take 10 mg of lacosamide reference substance, weigh accurately, place in a 100 ml volumetric flask, add 60 ml of solvent, dissolve lacosamide by ultrasonication, dilute to scale with solvent, shake well, and prepare 2 portions in parallel.

[0114] Change the small variable condition:

[0115] (1) Column temperature: 35℃±5℃

[0116] (2) Flow rate: 1.0ml / min±0.1ml / min

[0117] (3) Formic acid ratio: 0.01% ± 0.005%

[0118] (4) Mobile phase: organic phase ratio (±5%)

[0119] (5) Chromatographic columns: Compare different brands of chromatographic columns (Waters Symmetry C85μm, 4.6×250mm or columns of equivalent performance) and columns of the same brand and model with different batches of fillers

[0120] Determination method: Take 5 μl of the above blank solvent, reference solution and test solution respectively, and inject them into the liquid chromatograph in the order described in the table below. The results are shown in the table below.

[0121] Table 10 Content Methodology-Durability Test Results

[0122]

[0123] Note: 1. The tailing factors and theoretical plate numbers of each durability condition are taken from the spectrum of reference solution 1.

[0124] Comparative Example 1

[0125] The chromatographic conditions are as follows, and the others are the same as in Example 1.

[0126] Column: Symmetry C8, 4.6mm×50mm, 3.5μm

[0127] Detection wavelength: 215nm

[0128] Mobile phase: 0.1 vol‰ formic acid aqueous solution-acetonitrile (volume ratio 60:40)

[0129] Flow rate: 1.0ml / min

[0130] Column temperature: 35°C

[0131] Injection volume: 5 μl

[0132] Solvent: methanol-water (10:90)

[0133] The liquid chromatogram of the control sample is shown in Figure 2 ,from Figure 2 From the test results, it can be seen that the retention time of the main component is 1.180min, which is basically unretained.

[0134] Comparative Example 2

[0135] The chromatographic conditions are as follows, and the others are the same as in Example 1:

[0136] Column: Hypersil TM BDS C8 Column 4.6×250mm 5μm

[0137] Detection wavelength: 215nm

[0138] Mobile phase: water-acetonitrile-methanol (volume ratio 60:30:10)

[0139] Flow rate: 1.0ml / min

[0140] Column temperature: 35°C

[0141] Injection volume: 8μl

[0142] Solvent: methanol-water (volume ratio 15:85)

[0143] After testing using this method, the separation effect of the main peak and impurity peak became worse.

[0144] Comparative Example 3

[0145] The chromatographic conditions are as follows, and the others are the same as in Example 1:

[0146] Chromatographic column: Agilent ZORBAX SB-C18 column 4.6×250mm 5μm

[0147] Detection wavelength: 220nm

[0148] Mobile phase: 0.1 vol‰ formic acid aqueous solution-acetonitrile (volume ratio 70:30)

[0149] Flow rate: 1.0ml / min

[0150] Column temperature: 35°C

[0151] Injection volume: 5 μl

[0152] Solvent: methanol-water (volume ratio 15:85)

[0153] Blank excipient solution: Take 90 mg of blank excipient, accurately weigh, place in a 100 ml volumetric flask, add 60 ml of solvent, ultrasonicate for 5 minutes, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene (pore size 0.45 μm) filter membrane, discard 4 ml of the initial filtrate, and take the subsequent filtrate to obtain.

[0154] Test solution: Take 10 bags of this product, accurately weigh the weight, pour out the contents completely, wipe the powder in the bag with a cotton swab, accurately weigh the empty bag, calculate the average filling amount, grind the contents, take 100 mg of fine powder (equivalent to 10 mg of lacosamide), accurately weigh, put it in a 100 ml volumetric flask, add 60 ml of solvent, ultrasonicate for 5 min to dissolve lacosamide, dilute to the scale with solvent, shake well, filter with a polytetrafluoroethylene filter membrane (pore size 0.45 μm), discard 4 ml of the initial filtrate, take the subsequent filtrate to obtain.

[0155] Reference solution: Take 10 mg of lacosamide reference substance, weigh accurately, put into a 100 ml volumetric flask, add 60 ml of solvent, dissolve the lacosamide by ultrasound, dilute to the scale with solvent, shake well, and obtain.

[0156] The blank solvent and blank excipients of this method interfere with the detection of the main component, and the specificity is poor.

[0157] After replacing the chromatographic column, the detection wavelength increased, the peak shape and separation became worse, the reference solution was injected 6 times continuously, the RSD value of the main component peak retention time was 1.57%, which was greater than 1.0%, and the RSD value of the peak area was 2.12%, which was greater than 2.0%, and the injection precision was poor.

[0158] 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 method for detecting the content of lacosamide particles by high performance liquid chromatography, characterized in that: include: The reference solution and the test solution were respectively injected into the liquid chromatograph, the chromatogram was recorded, and the content of lacosamide was calculated by the peak area according to the external standard method.

2. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 1, characterized in that: The mobile phase was a mixture of formic acid aqueous solution and acetonitrile.

3. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 2, characterized in that: The volume concentration of the formic acid aqueous solution is 0.05-0.15‰.

4. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 2, characterized in that: The volume ratio of formic acid aqueous solution to acetonitrile is (65-75):(25-35).

5. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to any one of claims 2 to 4, characterized in that: The filler of the chromatographic column is octylsilane bonded silica gel.

6. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 5, characterized in that: The length of the chromatographic column is 100-250mm.

7. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 5, characterized in that: The inner diameter of the chromatographic column is 3-4.6 mm.

8. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 5, characterized in that: The particle size of the chromatographic column is 3-5 μm.

9. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to any one of claims 6 to 8, characterized in that: The detection wavelength is 200-210nm; Preferably, the column temperature is 30-40°C; Preferably, the injection volume is 3-8 μl.

10. The method for detecting the content of lacosamide particles by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the test solution is 0.04-0.2 mg / mL; Preferably, the concentration of the reference solution is 0.04-0.2 mg / mL.

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