Determination method of L-alaninamide hydrochloride enantiomer

Abstract

The invention provides a determination method of an L-alaninamide hydrochloride enantiomer, which adopts an HPLC (High Performance Liquid Chromatography) method, takes a chromatographic column taking (S)-18-crown-6 ether coated silica gel as a filler as a stationary phase, and takes a perchloric acid aqueous solution as a mobile phase; the mobile phase is used for dissolving a test sample to prepare a test sample solution with the concentration not lower than 4 mg / ml. The detection method provided by the invention can be suitable for accurate quantitative analysis of D-alaninamide hydrochloride, and has the advantages of good specificity, high sensitivity, good accuracy, simple operation, no need of sample pretreatment, and accurate determination of the enantiomer D-alaninamide hydrochloride of L-alaninamide hydrochloride.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to quantitative analysis of the enantiomer of L-alaninamide hydrochloride, D-alaninamide hydrochloride. Background Art

[0002] As an important starting material, L-alaninamide hydrochloride has a chiral center and is widely used in the synthesis of drugs, such as safinamide mesylate. The enantiomeric impurity D-alaninamide of L-alaninamide hydrochloride is one of the main sources of chiral impurities in safinamide mesylate, so the content of its enantiomer needs to be strictly controlled.

[0003] In recent years, the requirements for isomers in drugs have become increasingly stringent, not just the limit requirements, but the requirements for accurate determination of their content. At present, there is no pharmacopoeia standard for L-alaninamide hydrochloride and its enantiomers. The detection method reported in the literature is to chirally derivatize L-alanine isopropyl ester hydrochloride and its isomers with o-phthalaldehyde / N-acetyl-L-cysteine ​​(OPA / NAC) to generate a pair of diastereomeric derivatives with ultraviolet absorption (wavelength of 333nm), separated by 5-fluorophenyl chromatographic column, and gradient eluted with a mobile phase system of 0.1% trifluoroacetic acid aqueous solution-methanol. In this method, the sample needs to be pre-treated, the operation is complicated, and the quality of the derivatization reagent and the derivatization time affect the detection results of the isomers. Therefore, it is very necessary to establish an accurate, reliable, sensitive, simple to operate, and sample pre-treatment-free liquid chromatography method for the determination of L-alaninamide hydrochloride isomers. Summary of the invention

[0004] The first object of the present invention is to provide a method for determining the enantiomer of L-alaninamide hydrochloride, which is suitable for the quantitative analysis of the enantiomer D-alaninamide hydrochloride of L-alaninamide hydrochloride.

[0005] The invention provides a method for determining the enantiomers of L-alaninamide hydrochloride, which adopts HPLC method, uses a chromatographic column with (S)-18-crown-6 ether coated with silica gel as a filler as a stationary phase, and uses a perchloric acid aqueous solution as a mobile phase; and uses the mobile phase to dissolve a test sample to prepare a test sample solution with a concentration of not less than 4 mg / ml.

[0006] The determination method provided by the present invention adopts a high performance liquid chromatograph as the equipment, adopts the above-mentioned stationary phase and mobile phase, and dissolves the test sample in the mobile phase to prepare a test sample solution with a concentration of not less than 5 mg / ml, which can ensure that its enantiomer sensitivity meets the requirements.

[0007] Preferably, the pH value of the perchloric acid aqueous solution is 1.0 to 1.4, preferably 1.1 to 1.2. The present invention can effectively improve the separation degree of enantiomers from other single impurities and L-alaninamide by optimizing the pH of the perchloric acid aqueous solution.

[0008] More preferably, the pH value of the perchloric acid aqueous solution is 1.2. Using the perchloric acid aqueous solution of the above concentration as the mobile phase and dissolving the test solution can help improve the chromatographic peak shape.

[0009] In the determination method provided by the present invention, the concentration of the test solution is not less than 4 mg / ml, preferably not less than 5 mg / ml, in particular 5 mg / ml. The test solution at this concentration can better ensure the sensitivity and quantitative accuracy of the detection, thereby effectively ensuring the reliability of the overall analysis results.

[0010] In the determination method provided by the present invention, the chromatographic column is preferably a CROWNPAK CR (+) column with a specification of 150 mm×4.0 mm and 5 μm. The stationary phase has higher separation efficiency and selectivity for L-alaninamide hydrochloride and its enantiomers.

[0011] The determination method provided by the present invention preferably has a detection wavelength not exceeding 205 nm, preferably 200 nm, at which wavelength the sensitivity of L-alaninamide hydrochloride to its enantiomers is better.

[0012] In the determination method provided by the present invention, the column temperature during detection is preferably 25±5°C, more preferably 25°C. The above column temperature has a higher match with the mobile phase, which is beneficial to the stability of the retention time, as well as the separation efficiency and selectivity.

[0013] The determination method provided by the present invention preferably has a flow rate of no more than 0.5 ml / min during detection, more preferably 0.2±0.02 ml / min, so as to better separate D-alaninamide hydrochloride, other single impurities and L-alaninamide hydrochloride, and effectively ensure the separation degree, analysis time and column efficiency.

[0014] In the determination method provided by the present invention, the injection volume of the test solution is preferably 5 μl to 20 μl, such as 5 μl, 8 μl, 10 μl, 20 μl, etc. The above injection volume is conducive to the quantitative detection of D-alaninamide hydrochloride.

[0015] As a preferred embodiment of the present invention, the determination method comprises the following steps: (1) Take the sample to be tested, accurately weigh it and place it in a volumetric flask, add the mobile phase to dissolve and dilute to the scale to make a sample solution with a concentration of not less than 4 mg / ml; (2) Accurately measure the test sample solution and inject it into the liquid chromatograph, and record the chromatogram.

[0016] The specific equipment selection and operation are within the skill of those skilled in the art and are not particularly limited in the present invention.

[0017] The detection method provided by the present invention can be applied to the accurate quantitative analysis of D-alaninamide hydrochloride. The detection method has good specificity, high sensitivity, good accuracy, simple operation, and no sample pretreatment is required. The enantiomer of L-alaninamide hydrochloride, D-alaninamide hydrochloride, can be accurately determined. L-alaninamide hydrochloride is used as a sample for method development, and HPLC method is attempted to quantitatively detect D-alaninamide hydrochloride. The results show that D-alaninamide at 0.0359 μg / ml and above can be quantitatively analyzed. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is an overlapping diagram of the interference test for verifying the determination method of the present invention, wherein a: blank solvent b: system suitability solution c: L-alanine methyl ester d: D-alaninamide e: hydrochloric acid f: test solution.

[0020] Figure 2 It is a linear relationship diagram of L-alaninamide verified by the determination method of the present invention.

[0021] Figure 3 It is a linear relationship diagram of D-alaninamide verified by the determination method of the present invention.

[0022] Figure 4 This is a superimposed diagram of the determination method described in the present invention - Method 1.

[0023] Figure 5 It is a superimposed diagram of the determination method-method 2 described in the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Combine the following Figure 1-Figure 5Specific embodiments of the present invention are described.

[0026] Example 1

[0027] This embodiment provides a method for determining the enantiomers of L-alaninamide hydrochloride, comprising: (1) Solution preparation

[0028] Blank solution: mobile phase

[0029] Test solution: Take about 50 mg of the product, accurately weigh it, place it in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale (5 mg / ml), and shake well.

[0030] System suitability solution: Take appropriate amount of L-alaninamide hydrochloride and D-alaninamide reference substance, add mobile phase to dissolve and dilute to make a mixed solution containing about 5 mg of L-alaninamide hydrochloride and about 5 μg of D-alaninamide per 1 ml.

[0031] Impurity localization solution: Take appropriate amount of D-alaninamide and L-alanine methyl ester reference substances, add them to the mobile phase to dissolve and dilute to make a solution containing about 50 μg of D-alaninamide and L-alanine methyl ester per 1 ml.

[0032] (2) Determination

[0033] Take 20 μl of the test solution and the control solution respectively and inject them into the high performance liquid chromatograph to record the chromatogram. Calculate the content of optical isomers according to the self-control method. The specific detection conditions are shown in Table 1.

[0034] Table 1

[0035] Test Example 1

[0036] This test example verifies the effect of different pH perchloric acid aqueous solutions on the test results, wherein method 2 is the method described in Example 1, and the only difference between method 1 and method 1 is that the pH of the perchloric acid aqueous solution is 1.0. The results are shown in Tables 2 and Figure 4 , Figure 5 .

[0037] Table 2

[0038] When the pH of the perchloric acid aqueous solution is 1.2, other impurities do not interfere with the detection of D-alaninamide, indicating that the mobile phase of the perchloric acid aqueous solution with a pH of 1.2 can meet the detection of the L-alaninamide enantiomer.

[0039] Test Example 2

[0040] (1) Exclusivity

[0041] Solution preparation: Blank solution: mobile phase

[0042] Test solution: Take about 50 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add mobile phase to dissolve and dilute to the scale, and shake well.

[0043] System suitability solution: Take appropriate amount of L-alaninamide hydrochloride and D-alaninamide reference substance, add mobile phase to dissolve and dilute to make a mixed solution containing about 5 mg of L-alaninamide hydrochloride and about 5 μg of D-alaninamide per 1 ml.

[0044] Impurity localization solution: Take appropriate amount of D-alaninamide and L-alanine methyl ester reference substances, add them to the mobile phase to dissolve and dilute to make a solution containing about 50 μg of D-alaninamide and L-alanine methyl ester per 1 ml.

[0045] Take the above solutions and inject them respectively, examine the specificity, record the chromatogram, and the results are shown in Figure 1 and Table 3.

[0046] Table 3

[0047] Result analysis: Blank solvent and hydrochloric acid do not interfere with the detection of known impurities in the test solution; the impurity L-alanine methyl ester does not show a peak under this chromatographic condition; in the system suitability solution, the separation degree between the impurity D-alaninamide and the main peak is 2.950, which is greater than 1.5, and the specificity is good.

[0048] (2) Detection limit

[0049] Take appropriate amount of L-alaninamide hydrochloride and D-alaninamide reference substance, dissolve them in mobile phase and dilute them step by step to make solutions of appropriate concentration. The detection limit is when the signal-to-noise ratio is about 3:1. Calculate the percentage (TC%) of the test solution concentration and the limit concentration percentage (LC%). The results are shown in Table 4.

[0050] Table 4

[0051] Result analysis: The detection limit concentration of L-alaninamide is 0.0193μg / ml, which is equivalent to 0.0004% of the test solution concentration; the detection limit concentration of impurity D-alaninamide is 0.0179μg / ml, which is equivalent to 0.0004% of the test solution concentration; this shows that more than 0.0004% of D-alaninamide can be qualitatively detected with high detection sensitivity.

[0052] (3) Limit of quantification

[0053] Take appropriate amount of L-alaninamide hydrochloride and D-alaninamide reference substance, dissolve them in mobile phase and dilute them step by step to make solutions of appropriate concentration. The quantitative limit is when the signal-to-noise ratio is about 10:1. The sample is injected 6 times continuously to examine the repeatability of the quantitative limit, and calculate the percentage equivalent to the concentration of the test solution (TC%) and the limit concentration percentage (LC%). The results are shown in Table 5.

[0054] Table 5

[0055] Analysis of results: The quantitative limit concentration of the impurity D-alaninamide was 0.0359μg / ml, which was equivalent to 0.0007% of the test solution concentration and was less than the reporting limit (0.05%). The detection limit concentration of L-alaninamide was 0.0387μg / ml, which was equivalent to 0.0008% of the test solution concentration, indicating that D-alaninamide above 0.0007% could be quantitatively detected with high detection sensitivity. The quantitative limit solution was injected 6 times continuously, and the retention time RSD values ​​of D-alaninamide and L-alaninamide were 0.2% and 0.1%, respectively, which were less than 1%, and the peak area RSD values ​​were 6.8% and 3.6%, respectively, which were both less than 10%, indicating good injection precision.

[0056] (4) Linearity and range

[0057] Take appropriate amount of L-alaninamide hydrochloride and D-alaninamide reference substance, weigh accurately, add mobile phase to dissolve and dilute step by step to make mixed solutions equivalent to 20%, 50%, 100%, 200%, 400% of the impurity limit concentration, that is, mixed solutions with concentrations of 1, 2.5, 5, 10, 20µg / ml (corresponding to linear solution-1 to linear solution-5, respectively), as linear solutions.

[0058] Accurately measure 20µl of each of the above linear solutions and inject them into the liquid chromatograph to record the chromatogram. Perform linear regression with concentration as the horizontal axis and peak area as the vertical axis. The results are shown in Table 6, Table 7, Figure 2, and Figure 3.

[0059] Table 6

[0060] Table 7

[0061] Analysis of results: Within the range of limit of quantification to 400%, linear fitting was performed with concentration and peak area. The linear regression equations of the main component and isomer were y=8.56×104x+5.879×103 and y=8.91×104x+8.41×102, respectively. The linear correlation coefficients r were all greater than 0.999, the Y-axis intercepts were all less than 25% at 100% response value, and the residual sums of squares were 102166674 and 156666082, respectively. The proposed method had a good linear relationship.

[0062] (5) Correction factor

[0063] Correction factor: The correction factor for each known impurity is obtained by comparing the ratio of the slope in the linear regression equation of the principal component to the slope of the known impurity. The correction factor results are shown in Table 8.

[0064] Table 8

[0065] Result analysis: The correction factor of the impurity D-alaninamide is 1.0. In the range of 0.9 to 1.1, the principal component self-control method without adding the correction factor is used to calculate the detection amount of D-alaninamide.

[0066] (6) Accuracy

[0067] In order to confirm that the proposed method can accurately determine the concentration range of D-alaninamide, the accuracy of this method will be specifically investigated. The experimental process is as follows: D-Alaninamide stock solution: Take an appropriate amount of D-Alaninamide reference substance, weigh it accurately, add mobile phase to dissolve it and quantitatively dilute it to make a solution containing about 0.05 mg of impurity D-Alaninamide per 1 ml.

[0068] D-Alaninamide reference solution: Take 1 ml of D-Alaninamide stock solution and place it in a 10 ml volumetric flask, dilute to scale with mobile phase, shake well, and the solution is ready. Prepare 2 portions in the same way.

[0069] Recovery solution: Take about 50 mg of L-alaninamide, accurately weigh it, put it in a 10 ml volumetric flask, weigh 9 portions in parallel, and make 1 group of 3 portions. Accurately add 0.5 ml, 1 ml, and 1.5 ml of D-alaninamide stock solution respectively, dissolve it with an appropriate amount of mobile phase and dilute it to the scale, shake well, and use them as recovery solutions of 50%, 100%, and 150%.

[0070] Test solution: Take about 50 mg of L-alaninamide, accurately weigh it, place it in a 10 ml volumetric flask, dissolve it in an appropriate amount of mobile phase and dilute it to the scale, and shake well.

[0071] Accurately measure 20 µl of the reference solution and test solution prepared above, inject them into the liquid chromatograph, and record the chromatogram. The recovery rate is used as the evaluation index, and the specific results are shown in Table 9.

[0072] Table 9

[0073] Result analysis: The recovery rates of D-alaninamide solutions with limit concentrations of 50%, 100% and 150% were between 93.7% and 103.7%, within the range of 90% to 108%; the RSD of 9 samples was 3.9%, less than 10%, indicating that this method has high accuracy.

[0074] (7) Durability

[0075] The influence of slight changes in column temperature, flow rate and pH value on the measurement results was investigated. The durability range of the chromatographic conditions and the results are shown in Tables 10 to 12.

[0076] Table 10

[0077] Table 11

[0078] Table 12

[0079] Analysis of results: Under all conditions, the separation degree of isomers and main components in the system suitability solution met the requirements; the column temperature was 25±5℃, the flow rate was 0.2±0.02ml / min, the mobile phase pH was in the range of 1.1-1.2, the detection amount of enantiomers in the test solution was between 0.032% and 0.039%, and the proposed method had good durability; the pH was fine-tuned to 1.3, the detection amount of enantiomers in the test solution was 0.013%, and the absolute change compared with the proposed conditions was 0.021%, which was greater than 0.02%. In subsequent experiments, attention should be paid to the mobile phase pH value.

[0080] (7) Sample measurement

[0081] Table 13 Summary of L-alaninamide test results

[0082] Result analysis: All inspection items of multiple batches of samples meet the limit requirements.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for determining enantiomers of L-alaninamide hydrochloride, characterized in that: The HPLC method is adopted, with a chromatographic column filled with (S)-18-crown-6 ether coated with silica gel as the stationary phase and a perchloric acid aqueous solution as the mobile phase; the test sample is dissolved in the mobile phase to prepare a test sample solution with a concentration of not less than 4 mg / ml.

2. The measuring method according to claim 1, characterized in that The pH value of the perchloric acid aqueous solution is 1-1.4, preferably 1.1-1.

2.

3. The measuring method according to claim 2, characterized in that The pH value of the perchloric acid aqueous solution is 1.

2.

4. The assay method according to any one of claims 1 to 3, characterized in that The concentration of the test solution is 5 mg / ml.

5. The assay method according to any one of claims 1 to 4, characterized in that The chromatographic column is a CROWNPAK CR (+) column, and preferably the specification of the chromatographic column is 150 mm×4.0 mm, 5 μm.

6. The assay method according to any one of claims 1 to 5, characterized in that The detection wavelength does not exceed 205 nm, preferably 200~205 nm.

7. The assay method according to any one of claims 1 to 6, characterized in that The column temperature during detection is 25±5°C, preferably 25°C.

8. The assay method according to any one of claims 1 to 7, characterized in that The flow rate during detection is not higher than 0.5 ml / min, preferably 0.2±0.02 ml / min.

9. The determination method according to any one of claims 1 to 8, characterized in that The injection volume of the test solution is 5μl~20μl.

10. The determination method according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Take the sample to be tested, accurately weigh it and place it in a volumetric flask, add the mobile phase to dissolve and dilute to the scale to make a sample solution with a concentration of not less than 4 mg / ml; (2) Accurately measure the test sample solution and inject it into the liquid chromatograph, and record the chromatogram.

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