CN114527205A - Method for detecting isomer of 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide - Google Patents
Method for detecting isomer of 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- ZEWWJSJMNBJIKR-UHFFFAOYSA-N tert-butyl n-[1-(benzylamino)-3-methoxy-1-oxopropan-2-yl]carbamate Chemical compound CC(C)(C)OC(=O)NC(COC)C(=O)NCC1=CC=CC=C1 ZEWWJSJMNBJIKR-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 238000001514 detection method Methods 0.000 claims abstract description 52
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims abstract description 24
- SBTVLCPCSXMWIQ-UHFFFAOYSA-N (3,5-dimethylphenyl) carbamate Chemical compound CC1=CC(C)=CC(OC(N)=O)=C1 SBTVLCPCSXMWIQ-UHFFFAOYSA-N 0.000 claims abstract description 4
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- 239000012085 test solution Substances 0.000 claims description 16
- -1 (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide Chemical compound 0.000 claims description 10
- ZEWWJSJMNBJIKR-CYBMUJFWSA-N tert-butyl n-[(2r)-1-(benzylamino)-3-methoxy-1-oxopropan-2-yl]carbamate Chemical compound CC(C)(C)OC(=O)N[C@H](COC)C(=O)NCC1=CC=CC=C1 ZEWWJSJMNBJIKR-CYBMUJFWSA-N 0.000 claims description 8
- 238000004587 chromatography analysis Methods 0.000 claims description 2
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- VPPJLAIAVCUEMN-GFCCVEGCSA-N lacosamide Chemical compound COC[C@@H](NC(C)=O)C(=O)NCC1=CC=CC=C1 VPPJLAIAVCUEMN-GFCCVEGCSA-N 0.000 description 12
- 229960002623 lacosamide Drugs 0.000 description 11
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 10
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 10
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- 230000015572 biosynthetic process Effects 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- GCFHZZWXZLABBL-UHFFFAOYSA-N ethanol;hexane Chemical compound CCO.CCCCCC GCFHZZWXZLABBL-UHFFFAOYSA-N 0.000 description 5
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 5
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 5
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- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- HOKKHZGPKSLGJE-GSVOUGTGSA-N N-Methyl-D-aspartic acid Chemical compound CN[C@@H](C(O)=O)CC(O)=O HOKKHZGPKSLGJE-GSVOUGTGSA-N 0.000 description 2
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- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 2
- 238000010200 validation analysis Methods 0.000 description 2
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- LSACYLWPPQLVSM-UHFFFAOYSA-N isobutyric acid anhydride Chemical group CC(C)C(=O)OC(=O)C(C)C LSACYLWPPQLVSM-UHFFFAOYSA-N 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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Abstract
The invention relates to a method for detecting 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer, which comprises the following steps: preparing a solution to be detected; detecting the solution to be detected by adopting a high performance liquid chromatography, wherein the chromatographic conditions comprise: and (3) chromatographic column: chromatographic column with amylose-tris (3, 5-dimethylphenylcarbamate) coated silica gel as filler, mobile phase: the mixed solution of n-hexane and ethanol, wherein the volume ratio of the n-hexane to the ethanol is (78-82) to (18-22). The detection method provided by the invention can effectively reduce and even avoid the interference of other process impurities, realize the effective separation of INT3 and INT3 isomers, and accurately perform quantitative detection on INT3 and INT3 isomer impurities.
Description
Technical Field
The invention relates to the field of analytical chemistry, and particularly relates to a method for detecting 2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide isomers.
Background
Lacosamide (Lacosamide), chemical name: (R) -2-acetamido-N-benzyl-3-methoxypropionamide, a novel glycine site binding antagonist of the N-methyl-D-aspartate (NMDA) receptor, is mainly used for the treatment of epilepsy and neuropathic pain.
At present, lacosamide is mostly synthesized by adopting the following process route:
among them, (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide (INT3) is an important intermediate in the synthesis of lacosamide, and (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide (enantiomer) inevitably occurs during the synthesis process. The (S) -2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide is a process impurity, the effect of inhibiting epilepsy is unclear, and if the effect is not effectively controlled, the INT3 isomer continuously participates in the reaction in the subsequent synthesis process of lacosamide to generate new isomer impurities, so that the quality of the lacosamide is influenced, and therefore, the quality control of the (S) -2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide is the key for ensuring the quality of the lacosamide medicine.
At present, no report about a detection method of 2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide isomer exists in the prior art, so that the development of a method for conveniently, efficiently and accurately quantitatively detecting the 2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide isomer is a technical problem to be solved urgently at present.
Disclosure of Invention
In view of this, the invention provides a method for detecting 2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide isomer, so as to monitor the quality stability of lacosamide and improve the safety of clinical medication.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method for detecting 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer comprises the following steps:
preparing a solution to be detected;
detecting the solution to be detected by adopting a high performance liquid chromatography, wherein the chromatographic conditions comprise:
a chromatographic column: chromatographic column with amylose-tri (3, 5-dimethylphenyl carbamate) coated silica gel as filler,
mobile phase: the mixed solution of n-hexane and ethanol, wherein the volume ratio of the n-hexane to the ethanol is (78-82) to (18-22).
In the process of preparing (R) -2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide (INT3), various process impurities are inevitably generated, so that INT3 and other process impurities can interfere in the process of detecting (S) -2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide (INT3 isomer impurities), and how to avoid the interference of INT3 and other impurities is the key to accurately and quantitatively detecting the INT3 isomer impurities. The inventor of the invention has found that by adopting a specific chiral chromatographic column which takes amylose-tris (3, 5-dimethylphenyl carbamate) coated silica gel as a filler and combining a mixed solution of n-hexane and ethanol in a volume ratio of (78-82) to (18-22) as a mobile phase, the interference of other process impurities can be effectively reduced or even avoided, the effective separation of INT3 and INT3 isomers can be realized, and the quantitative detection of INT3 and INT3 isomer impurities can be accurately carried out.
The detection method of the 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer provided by the invention is verified from the aspects of specificity, detection limit and quantification limit, linearity, recovery rate, repeatability, solution stability, durability and the like, and all the detection method meets the requirements. The detection method provided by the invention has the advantages of good sensitivity, accuracy, reproducibility, recovery rate and stability, accurate and reliable detection result, and provides guarantee for monitoring the quality stability of lacosamide medicines and improving the safety of clinical medication.
Optionally, the concentration of INT3 in the test solution is (1.0-2.0) mg/mL; preferably, the concentration of INT3 in the test solution is 1.5 mg/mL.
The peak shapes of INT3 and INT3 isomers and other impurities are better, the column efficiency is higher, and the integral is more accurate by limiting the concentration of a test solution, so that the effective separation of INT3 and INT3 isomer impurities and other impurities in a test sample is facilitated, and the contents of INT3 and INT3 isomer impurities are more accurately calculated.
Preferably, the volume ratio of the n-hexane to the ethanol is 80: 20.
Optionally, the chromatography column is an AD-H column.
Optionally, the detection wavelength is 200-210 nm.
Preferably, the detection wavelength is 205 nm.
Alternatively, the column temperature is 20-30 ℃.
Preferably, the column temperature is 30 ℃.
Alternatively, the flow rate is 0.9-1.1 mL/min.
Preferably, the flow rate is 1.0 mL/min.
Optionally, the sample size is 8-12 μ L; preferably, the sample size is 10 μ L.
By limiting the detection wavelength, the column temperature, the flow rate, the sample injection amount and the like, the separation degree of INT3 and INT3 isomer impurities and other process impurities can be further improved.
Optionally, the solution to be tested comprises preparation of a test solution and a control solution, wherein the control solution comprises (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide and (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide.
Alternatively, the concentration of the (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide in the control solution is (1.0-2.0) mg/mL, and the concentration of the (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide is (80-120) μ g/mL.
Preferably, the concentration of the (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide in the control solution is 1.5mg/mL, and the concentration of the (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide is 105. mu.g/mL.
By limiting the concentration of each substance in the mixed solution of the reference substance, the INT3 and INT3 isomer impurities in the test solution can be conveniently and accurately located.
Drawings
FIG. 1 is a liquid chromatogram of a mixed solution of a test sample and a control sample of the mobile phase 1) in example 1;
FIG. 2 is a liquid chromatogram of an INT3 isomer mapping solution of mobile phase 1) in example 1;
FIG. 3 is a liquid chromatogram of an INT3-6 localized solution of mobile phase 1) in example 1;
FIG. 4 is a liquid chromatogram of a mixed solution of a test sample and a control sample of the mobile phase 2) in example 1;
FIG. 5 is a liquid chromatogram of a mixed solution of a test sample and a control sample of the mobile phase 3) in example 1;
FIG. 6 is a liquid chromatogram of an INT3-6 localized solution of mobile phase 3) in example 1;
FIG. 7 is a liquid chromatogram of a mixed solution of a test sample and a control sample of the mobile phase 4) in example 1;
FIG. 8 is a liquid chromatogram of an INT3-6 localized solution of mobile phase 4) in example 1;
FIG. 9 is a liquid chromatogram of a blank solvent of the specificity of example 1;
FIG. 10 is a liquid chromatogram of a mixed solution of the test article and the INT3 isomer in the specificity of example 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
According to the existing lacosamide process route, a plurality of impurities are generated during synthesis of INT3, including unreacted INT2 and benzylamine, INT3 isomers ((S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide), INT3-6, dibenzylamine, benzyl alcohol, benzaldehyde, impurity I and the like. Due to the similar polarity of INT3 and its isomers, and the interference of other impurities, the method causes great difficulty in effective separation and quantitative detection of INT3 and its isomers.
Wherein, the structures of INT3-6 and impurity I are as follows:
the inventors finally determined the following detection methods through further research and analysis:
dissolving (R) -2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide with a mobile phase to obtain a test solution with a concentration of (1.0-2.0) mg/mL;
detecting the test solution by adopting a high performance liquid chromatography, wherein the chromatographic conditions are as follows:
a chromatographic column: a xylonite AD-H column;
mobile phase: a mixed solution of n-hexane and ethanol, wherein the volume ratio of the n-hexane to the ethanol is (78-82) to (18-22);
detection wavelength: 200-210 nm;
column temperature: 20-30 ℃;
flow rate: 0.9-1.1 mL/min.
Sample introduction amount: 8-12 μ L.
The method can avoid the interference of various impurities on INT3 and isomers thereof, realize the effective separation and accurate quantification of INT3 and isomers thereof, and provide guarantee for monitoring the stability of the quality of lacosamide medicines and improving the medication safety.
Example 1:
screening of detection method for 2-t-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer:
1.1 solution preparation
Preparing a test solution: accurately weighing an appropriate amount of INT3, adding a solvent to dissolve and diluting to a solution containing 1.5mg of INT3 in every 1mL to obtain the product.
Preparation of each positioning solution: precisely weighing an appropriate amount of INT3 (a test sample), INT2, benzylamine, INT3 isomer, INT3-6, dibenzylamine, benzyl alcohol, benzaldehyde and impurity I reference substances, and respectively dissolving and diluting the appropriate amount of INT2, benzylamine, INT3 isomer, INT3-6, dibenzylamine, benzyl alcohol, benzaldehyde and impurity I reference substances by using a solvent to obtain solutions containing about 80 mu g of INT 31.5 mg, 200 mu g of INT 2500 mu g of benzylamine, 80 mu g of INT3 isomer, INT 3-6200 mu g, 200 mu g of dibenzylamine, 200 mu g of benzyl alcohol, 200 mu g of benzaldehyde and 200 mu g of impurity I per 1mL, thereby obtaining each positioning solution.
Preparation of control solution: respectively and precisely weighing appropriate amounts of INT3 and INT3 isomers as reference substances, dissolving with solvent, diluting, and mixing to obtain a mixed solution containing about 105 μ g of INT31.5 mg and INT3 isomers per 1mL to obtain a reference solution (mixed solution of the test substance and the INT3 isomer).
In the preparation process of each solution, a mixed solution of n-hexane and ethanol (volume ratio of 80:20) is adopted for dissolving and diluting.
The benzylamine, dibenzylamine, benzyl alcohol, benzaldehyde and impurity I reference substances are all commercially available products. INT3 (test article) and INT2 were prepared by the synthesis method described in the background section. The preparation method of the INT3 isomer is similar to that of INT3, and only differs in that L-serine is used as a starting material for preparing the INT3 isomer; INT3-6 was prepared similarly to INT3, except that the BOC anhydride at the time of INT1 synthesis was replaced with isobutyric anhydride only at the time of INT3-6 preparation.
1.2 screening of the detection method
Only the influence of part of impurities and the screening of part of mobile phase in the screening process under the liquid phase condition are listed here.
1.2.1 test methods to be screened:
a chromatographic column: xylonite AD-H column, specification: 4.6mm × 250mm, 5 μm;
detecting the wavelength; 205 nm;
column temperature: 30 ℃, flow rate: 1mL/min, sample size: 10 μ L.
Mobile phase 1) n-hexane-ethanol volume ratio of 10: 90, the elution time is 15 min;
mobile phase 2) n-hexane-ethanol volume ratio of 40: 60, eluting for 15 min;
mobile phase 3) n-hexane-ethanol volume ratio of 65: 35, eluting for 30 min;
mobile phase 4) n-hexane-ethanol volume ratio of 80:20, elution time 40 min.
1.2.2 part of the screening procedure
And taking the mixed solution of the test sample and the reference substance and each positioning solution, and respectively detecting according to the chromatographic conditions. Wherein, the chromatogram of the mixed solution of the sample and the reference substance, the INT3 isomer positioning solution and the INT3-6 positioning solution corresponding to the mobile phase 1) is shown in figure 1-3; the chromatogram of the mixed solution of the test sample and the reference sample corresponding to the mobile phase 2) is shown in FIG. 4; the chromatogram of the test sample and reference sample mixed solution and INT3-6 positioning solution corresponding to the mobile phase 3) is shown in FIG. 5-6; the chromatogram of the test sample and control mixed solution and INT3-6 positioning solution corresponding to the mobile phase 4) is shown in FIGS. 7-8.
As can be seen from the attached figures 1-3, under the condition of the mobile phase 1), the peak emergence time of the INT3 isomer in the INT3 isomer positioning solution is 6.493min, the peak emergence time of the INT3-6 in the INT3-6 positioning solution is 6.363min, the peak emergence time of the INT3-6 and INT3 isomers in the mixed solution of the test product and the reference product is coincided, and the peak emergence time is 6.487 min. Illustrating the inefficient separation of INT3 isomer from INT3-6 under chromatographic conditions for mobile phase 1), INT3-6 interferes with the detection of INT3 isomer.
The INT3 isoform overlaps the peak of INT3-6 under mobile phase 2) conditions, as shown in fig. 4, illustrating that INT3-6 interferes with the detection of INT3 isoform under mobile phase 2) conditions.
As can be seen from the accompanying FIGS. 5-6, under the condition of the mobile phase 3), the peak time of INT3-6 in the INT3-6 positioning solution is 9.793min, the peak time of INT3-6 in the mixed solution of the test sample and the reference sample is 8.853min, and the peak time of INT3 isomer is 9.377min, which indicates that the INT3 isomer can be separated from the impurity INT3-6 under the chromatographic condition of the mobile phase 3), but the separation degree is poor, and the accurate quantification of the INT3 isomer cannot be realized.
Under the condition of the mobile phase 4), as can be seen from the attached figures 7-8, the peak-off time of INT3-6 in the INT3-6 positioning solution is 16.383min, the peak-off time of INT3-6 in the mixed solution of the test sample and the reference sample is 16.903min, and the peak-off time of INT3 isomer is 15.423min, which indicates that the INT3 isomer and the INT3-6 impurity can be effectively separated under the chromatographic condition of the mobile phase 4), and other impurities do not influence the separation effect, so that the accurate quantification of the INT3 isomer can be realized.
1.2.3 Final defined detection method
A chromatographic column: xylonite AD-H column, specification: 4.6mm × 250mm, 5 μm;
detecting the wavelength; 205 nm;
column temperature: 30 ℃, flow rate: 1mL/min, sample size: 10 mu L of the solution;
mobile phase: mixed solution of n-hexane-ethanol (volume ratio of 80: 20).
1.3 methodological validation of the finally defined detection method described above
Methodology validation a batch of INT3 test samples with the best purity was used.
(1) Specialization inspection
Taking a blank solvent (mobile phase), a mixed solution of a test sample and an INT3 isomer and each positioning solution, and respectively detecting according to the detection conditions in 1.2.3, wherein the chromatogram of the blank solvent is shown in FIG. 9, the chromatogram of the mixed solution of the test sample and the INT3 isomer is shown in FIG. 10, and the results are shown in the following Table 1.
TABLE 1 results of the specificity test
The results show that the blank solvent does not interfere with the detection of INT3 and INT3 isomers, the separation degree of each impurity from INT3 is good, the impurities do not interfere with INT3 and INT3 isomers, the requirement of specificity is met, and the detection method of the 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer provided by the invention has good specificity.
(2) Detection limit and quantitative limit investigation
Respectively and precisely weighing appropriate amounts of INT3 and INT3 isomers, respectively dissolving and diluting with mobile phases to obtain INT3 and INT3 isomer solutions with different concentrations, and then detecting according to the detection conditions in 1.2.3; and the signal-to-noise ratio is about 10: the analyte concentration at 1 is the quantitative limit concentration of the analyte, and 6 needles are continuously injected under the concentration condition; a signal-to-noise ratio of about 3 will result: 1 is set as its detection limit concentration, and the specific detection results are shown in the following table.
TABLE 2-1 quantitation limit and detection limit results
Note: corresponding to sample% means the proportion of each substance which accounts for the INT3 content in the test solution.
TABLE 2-2 quantitative limit repeatability results
| Composition (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD％ |
| INT3 | 566297 | 548758 | 566537 | 558070 | 559504 | 544072 | 1.64 |
| INT3 isomer | 553013 | 561368 | 555910 | 554149 | 552581 | 558640 | 0.62 |
As can be seen from the data in the table above, the quantitative limit solution is injected repeatedly for 6 times, the RSD of the peak area of each substance is 1.64% at most, and the quantitative limit and the detection limit of INT3 and INT3 isomers are both microgram grade, which shows that the detection method provided by the invention has high sensitivity and good quantitative limit repeatability, and can accurately and quantitatively detect INT3 and INT3 isomers in a test sample.
(3) Linear survey
Weighing 87.5mg of INT3 and INT3 isomers respectively, precisely weighing, placing in 50mL measuring flasks respectively, ultrasonically dissolving and diluting to scale with a mobile phase, shaking up to obtain stock solutions of INT3 and INT3 isomers, and then diluting to a series of concentrations with the mobile phase according to gradients; the INT3 and INT3 isomer solutions at the respective concentrations and the respective spotting solutions disposed in 1.1 were measured under the measurement conditions of item 1.2.3, and the peak area (A) was plotted as concentration C (. mu.g/mL), and the results are shown in the following tables.
TABLE 3-1 INT3 isomer Linear results
TABLE 3-2 INT3 Linear results
As can be seen from the data in the above table, the INT3 isomer has good linearity in the range of 1.2520-208.6667. mu.g/mL, and the INT3 has good linearity in the range of 1.2582-209.6998. mu.g/mL, which shows that the sample injection amount and the peak area of the detection method provided by the invention have good linear relationship in a certain range.
(4) Investigation of recovery
Precisely weighing about 87.5mg of INT3 isomer, placing the INT3 isomer into a 50mL measuring flask, adding mobile phase for ultrasonic dissolution, diluting to a scale, and shaking up to obtain an INT3 isomer stock solution. 20mL of INT3 isomer stock solution was precisely measured, placed in a 50mL measuring flask, diluted to the scale with a mobile phase, and shaken up to give a stock solution of recovery rate. Precisely measuring 3mL of the recovery stock solution, placing in a 20mL measuring flask, diluting to scale with mobile phase, and shaking to obtain a recovery control solution.
Accurately weighing 75mg of INT3, placing into a 50mL measuring flask, adding a proper amount of mobile phase for ultrasonic dissolution, accurately adding 1mL of recovery rate control solution, adding the mobile phase for dilution to scale, and shaking up to obtain a sample solution with low recovery rate; accurately weighing 30mg of INT3, placing the INT3 in a 20mL measuring flask, adding a proper amount of mobile phase for ultrasonic dissolution, accurately adding 3mL of recovery storage solution, adding the mobile phase for dilution to a scale, and shaking up to obtain a sample solution in the recovery rate; precisely weighing 30mg of INT3, placing the INT3 in a 20mL measuring flask, adding a proper amount of mobile phase for ultrasonic dissolution, precisely adding 4.5mL of recovery stock solution, adding the mobile phase for dilution to a scale, and shaking up to obtain a sample solution with high recovery; 3 parts of each concentration are prepared in parallel, and the detection is carried out according to the detection method under the item 1.2.3, and the detection results are shown in the following table.
Table 4 INT3 isomer recovery results
As can be seen from the data in the table above, the recovery rate of INT3 isomer is between 99% and 110%, and the RSD is 3.15%, which shows that the detection method provided by the invention has good recovery rate.
(5) Repeatability survey
6 parts of the test solution were prepared in parallel according to the method for preparing the test solution in 1.1, and the INT3 isomer content was measured under the detection conditions in 1.2.3, and the results are shown in the following table.
TABLE 5 repeatability results
As can be seen from the data in the above table, the detection method provided by the present invention has good reproducibility.
(6) Stability survey
The test solution was prepared according to the preparation method of the test solution in 1.1, and then the test was carried out for 0h, 2h, 3h, 6h, 8h, 12h, and 15h under the conditions of item 1.2.2, and the test results are shown in the following table.
TABLE 6 solution stability results
| Time of standing | INT3 isomer peak area | INT3 Peak area | INT3 isomer peak area percent (%) |
| 0h | 631744 | 637986049 | 0.10 |
| 1h | 612661 | 634904953 | 0.10 |
| 2h | 623016 | 639318850 | 0.10 |
| 3h | 612631 | 642775236 | 0.10 |
| 6h | 614444 | 649824853 | 0.09 |
| 8h | 639023 | 653760003 | 0.10 |
| 12h | 650791 | 663342538 | 0.10 |
| 15h | 657408 | 673637892 | 0.10 |
| Average peak area | 630215 | 649443797 | 0.10 |
| RSD％ | 2.79 | 2.09 | 3.58 |
As can be seen from the data in the table above, the peak areas of INT3 and INT3 isomers in the test solution are stable within 15h, which indicates that the detection method provided by the invention has good stability.
(7) Durability examination
The sample solution prepared in 1.1 was precisely weighed, the flow rate, column temperature, detection wavelength, and mobile phase ratio under item 1.2.3 were changed, and the detection was performed without changing other detection conditions, and the detection results are shown in the following table.
TABLE 7 results of degree of separation under different test conditions
As can be seen from the data in the table above, the separation degree meets the requirement by changing the conditions of mobile phase proportion, detection wavelength, column temperature, flow rate and the like, and the detection method provided by the invention is good in durability.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A method for detecting 2-tert-butyloxycarbonylamino-N-benzyl-3-methoxypropionamide isomer is characterized by comprising the following steps:
preparing a solution to be detected;
detecting the solution to be detected by adopting a high performance liquid chromatography, wherein the chromatographic conditions comprise:
a chromatographic column: chromatographic column with amylose-tri (3, 5-dimethylphenyl carbamate) coated silica gel as filler,
mobile phase: the mixed solution of n-hexane and ethanol, wherein the volume ratio of the n-hexane to the ethanol is (78-82) to (18-22).
2. The detection method according to claim 1, wherein the volume ratio of the n-hexane to the ethanol is 80: 20.
3. The detection method according to claim 1, wherein the chromatography column is an AD-H column.
4. The detection method according to claim 1, wherein the detection wavelength is 200-210 nm.
5. The detection method according to claim 4, wherein the detection wavelength is 205 nm.
6. The assay of claim 1 wherein the column temperature is from 20 ℃ to 30 ℃.
7. The assay of claim 1, wherein the flow rate is from 0.9 to 1.1 mL/min.
8. The detection method according to claim 1, wherein the amount of sample is 8 to 12 μ L.
9. The detection method according to any one of claims 1 to 8, wherein the test solution comprises a test solution and a control solution, and the control solution comprises (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide and (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide.
10. The assay of claim 9 wherein the control solution comprises (R) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide at a concentration of 1.0 to 2.0mg/mL and (S) -2-tert-butoxycarbonylamino-N-benzyl-3-methoxypropionamide at a concentration of 80 to 120 μ g/mL.
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