CN114705790B - Detection method of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate isomer - Google Patents
Detection method of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate isomer Download PDFInfo
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- CN114705790B CN114705790B CN202210214336.2A CN202210214336A CN114705790B CN 114705790 B CN114705790 B CN 114705790B CN 202210214336 A CN202210214336 A CN 202210214336A CN 114705790 B CN114705790 B CN 114705790B
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Abstract
The invention relates to the technical field of drug analysis, and particularly discloses a detection method of a (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate isomer. The detection method comprises the following steps: preparing a reference solution and a test solution, and detecting the reference solution and the test solution by adopting a gas chromatography, wherein the chromatographic conditions of the gas chromatography are as follows: a capillary column which takes 14 percent of cyanopropylphenyl-86 percent of dimethyl polysilane and 30 percent of hepta- (2, 3-di-O-methyl-6-O-tert-butyldimethylsilyl) -beta-cyclodextrin as a stationary liquid is adopted; the temperature of a sample inlet is 215-225 ℃; temperature programming; a hydrogen flame ionization detector was used. The detection method provided by the invention has strong specificity and good linear relation, has excellent accuracy, sensitivity and repeatability, and meets the detection requirements of (2R, 4R) -4-methyl-2-ethyl piperidine formate and three chiral isomers thereof.
Description
Technical Field
The invention relates to the technical field of drug analysis, in particular to a detection method of (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate isomer.
Background
Since the first report of the anticoagulant activity of argatroban by Mitsubishi corporation of japan in 1978, many researchers have conducted intensive studies on its chemical synthesis, biological activity, and clinical application. Argatroban was first marketed in japan in 1990, approved by the us FDA in 2000, and in china in 2002. Argatroban is a piperidine carboxylic acid derivative of L-arginine, has high selectivity, can reversibly and directly inhibit the activity of thrombin, and can be rapidly combined with thrombin in free blood clots in circulation to generate an anticoagulation effect; it can also be used for thrombosis related to thrombocytopenia caused by heparin.
The (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester is an important starting material for synthesizing argatroban, so that the quality control thereof has a great influence on the synthesis of argatroban. Other three chiral isomers (2R, 4R) -4-methyl-2-piperidine ethyl formate, (2R, 4S) -4-methyl-2-piperidine ethyl formate, (2S, 4R) -4-methyl-2-piperidine ethyl formate and (2S, 4S) -4-methyl-2-piperidine ethyl formate exist, the isomers are likely to become impurities during synthesis of argatroban, and no relevant documents report detection methods of the isomers at present, so that a detection method capable of effectively controlling the quality of the (2R, 4R) -4-methyl-2-piperidine ethyl formate is urgently needed to be developed, the effective control of the quality of the argatroban is further ensured, and the industrial standard is perfected.
Disclosure of Invention
In view of the above, the invention provides a method for detecting the ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate isomer, which has the advantages of strong specificity, high accuracy and excellent linear relation, and can meet the detection requirements of other three chiral isomers in the ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
a method for detecting an isomer of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate, comprising the steps of:
step one, preparing a reference solution and a test solution:
preparing a reference substance solution: preparing a reference substance solution from a reference substance of (2S, 4S) -4-methyl-2-piperidine ethyl formate, a reference substance of (2R, 4S) -4-methyl-2-piperidine ethyl formate and a reference substance of (2S, 4R) -4-methyl-2-piperidine ethyl formate by using a solvent;
preparing a test solution: preparing a sample of (2R, 4R) -4-methyl-2-ethyl piperidine formate into a test solution by using a solvent;
step two, detecting the reference substance solution and the test substance solution by adopting a gas chromatography, wherein the chromatographic conditions of the gas chromatography are as follows:
a capillary column which takes 14 percent of cyanopropylphenyl-86 percent of dimethyl polysilane and 30 percent of hepta- (2, 3-di-O-methyl-6-O-tert-butyldimethylsilyl) -beta-cyclodextrin as a stationary liquid is adopted; the temperature of a sample inlet is 215-225 ℃; the temperature programming is as follows: the initial temperature is 78-82 ℃, the temperature is raised to 115-125 ℃ at the speed of 9-11 ℃/min, the temperature is maintained for 10-20 min, the temperature is raised to 195-205 ℃ at the speed of 9-11 ℃/min, the temperature is maintained for 1-10 min; a hydrogen flame ionization detector is adopted, and the temperature of the detector is 245-255 ℃.
Compared with the prior art, the method for detecting the (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester isomer provided by the application has the following advantages:
the detection method of the (2R, 4R) -4-methyl-2-piperidine ethyl formate isomer provided by the application realizes the quantitative and qualitative analysis of the (2R, 4R) -4-methyl-2-piperidine ethyl formate and three chiral isomers thereof, has strong specificity and good linear relation, has excellent accuracy, sensitivity and repeatability, meets the detection requirements of the (2R, 4R) -4-methyl-2-piperidine ethyl formate and three chiral isomers thereof, and realizes the effective control of the quality of the (2R, 4R) -4-methyl-2-piperidine ethyl formate; in addition, the detection method can also be used for rapidly and accurately detecting other three chiral isomers in the (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate bulk drug, the whole operation process is reliable and controllable, and the detection method is suitable for practical application and popularization and has wide application prospect.
Optionally, the programmed temperature rise is: the initial temperature is 80 deg.C, the temperature is increased to 120 deg.C at a rate of 10 deg.C/min, the temperature is maintained for 15min, and then the temperature is increased to 200 deg.C at a rate of 10 deg.C/min, and the temperature is maintained for 5min.
The temperature rising procedure is an important factor influencing the performance of the gas chromatography column, and the initial temperature, the temperature rising rate and the final temperature determine the elution capacity of each component, so the initial temperature, the temperature rising rate and the final temperature are preferably selected in the application, the (2R, 4R) -4-methyl-2-piperidine ethyl formate and three chiral isomers thereof can be rapidly separated, and each component also has excellent separation degree.
Optionally, the injection inlet temperature is 220 ℃.
Optionally, the detector temperature is 250 ℃.
The preferable injection inlet temperature and the detector temperature are favorable for separating isomers existing in the (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate from the main component, and ensure that the peak shape is better and the separation degree is higher.
Optionally, the carrier gas is nitrogen, and the split ratio is 5-20.
Optionally, the split ratio is 10.
The preferable split ratio ensures that the peak shape of the chromatographic peak is better, and avoids the occurrence of situations such as peak deformation and tailing.
Optionally, the flow rate is 0.8 ml/min-1.2 ml/min, and the sample injection volume is 1 μ L.
Further alternatively, the flow rate is 1ml/min.
Optionally, the capillary column is of the type CYCLOSIL-B.
Optionally, the capillary column has a specification of 30m × 0.32mm × 0.25 μm.
Different chromatographic columns have larger difference on the retention performance of the compound, so the capillary column with the model of CYCLOSIL-B and the specification of 30m multiplied by 0.32mm multiplied by 0.25 mu m is adopted in the method, the target can be quickly and effectively separated, and the peak shape is better.
Optionally, the solvent is absolute ethyl alcohol.
The preferable solvent has no interference to the detection of (2R, 4R) -4-methyl-2-piperidine ethyl formate and three chiral isomers thereof, and the accuracy of the detection result is ensured.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a gas chromatogram of a blank solution provided in example 1 of the present invention;
FIG. 2 is a gas chromatogram of a control solution provided in example 1 of the present invention;
FIG. 3 is a gas chromatogram of a test solution provided in example 1 of the present invention;
FIG. 4 is a gas chromatogram of a labeled test solution provided in example 1 of the present invention;
FIG. 5 is a gas chromatogram of a control solution provided in comparative example 1 of the present invention;
FIG. 6 is a gas chromatogram of a control solution provided in comparative example 2 of the present invention;
FIG. 7 is a gas chromatogram of a control solution provided in comparative example 3 of the present invention.
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 do not limit the invention.
Example 1
The embodiment of the invention provides a detection method of a (2R, 4R) -4-methyl-2-piperidine ethyl formate isomer, which comprises the following steps:
step one, preparing a blank solution, a reference solution, a test solution and a standard sample solution:
the above blank solution: absolute ethyl alcohol is used as a blank solution;
preparing a reference substance solution: taking appropriate amounts of a (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, a (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance and a (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, precisely weighing, and preparing a reference solution by using absolute ethyl alcohol, wherein the concentration of the (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester in the reference solution is 50 mug/ml, the concentration of the (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 20 mug/ml, and the concentration of the (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 20 mug/ml;
preparing a test solution: taking 20mg of (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate as a sample, accurately weighing, placing the sample in a 20ml volumetric flask, dissolving the sample with absolute ethyl alcohol, diluting the sample to a scale, and shaking up;
preparing a standard sample solution: taking a proper amount of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester, a proper amount of (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, a proper amount of (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance and a proper amount of (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, precisely weighing, and preparing a standard sample solution by using absolute ethyl alcohol, wherein the concentration of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 1000 mug/ml, the concentration of (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 50 mug/ml, the concentration of (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 20 mug/ml, and the concentration of (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester is 20 mug/ml).
Step two, detecting the blank solution, the reference solution, the test solution and the added standard test solution by adopting a gas chromatography, and recording spectrograms, wherein the results are shown in figures 1-4, wherein the chromatographic conditions of the gas chromatography are as follows:
a capillary column which takes 14 percent of cyanopropyl phenyl-86 percent of dimethyl polysilane and 30 percent of hepta- (2, 3-di-O-methyl-6-O-tert-butyl dimethyl silicon-based) -beta-cyclodextrin as a stationary liquid is adopted, the model is CYCLOSIL-B, and the specification is 30m multiplied by 0.32mm multiplied by 0.25 mu m; the temperature of a sample inlet is 220 ℃; the temperature programming is as follows: the initial temperature is 80 ℃, the temperature is increased to 120 ℃ at the speed of 10 ℃/min, the temperature is maintained for 15min, the temperature is increased to 200 ℃ at the speed of 10 ℃/min, and the temperature is maintained for 5min; a hydrogen flame ionization detector is adopted, the temperature of the detector is 250 ℃, the carrier gas is nitrogen, the flow rate is 1ml/min, the split ratio is 10.
As can be seen from the graphs 1-4, the absolute ethyl alcohol solvent has no interference to the detection of (2R, 4R) -4-methyl-2-piperidine ethyl formate and three chiral isomers thereof, and the accuracy of the detection result is ensured.
As can be seen from FIG. 2, a peak of the chromatogram for ethyl (2S, 4S) -4-methyl-2-piperidinecarboxylate occurred at retention time 17.363min, a peak of the chromatogram for ethyl (2R, 4S) -4-methyl-2-piperidinecarboxylate occurred at retention time 19.274min, a peak of the chromatogram for ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate occurred at retention time 19.593min, and the degrees of separation of the components were good.
As can be seen from FIG. 3, ethyl (2R, 4S) -4-methyl-2-piperidinecarboxylate was not detected, a peak of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate appeared at retention time 16.293min, a peak of ethyl (2S, 4S) -4-methyl-2-piperidinecarboxylate appeared at retention time 17.362min, a peak of ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate appeared at retention time 19.595min, and the separation degree of each component was good.
As can be seen from FIG. 4, a peak of the chromatogram for ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate occurred at retention time 16.289min, a peak of the chromatogram for ethyl (2S, 4S) -4-methyl-2-piperidinecarboxylate occurred at retention time 17.357min, a peak of the chromatogram for ethyl (2R, 4S) -4-methyl-2-piperidinecarboxylate occurred at retention time 19.276min, a peak of the chromatogram for ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate occurred at retention time 19.594min, and the degrees of separation of the components were good.
The results of the above-described measurement of the degree of separation of each component are shown in table 1 below, and it can be seen from table 1 that the degree of separation between the components is good, thereby illustrating that the method for detecting the ethyl (2r,4r) -4-methyl-2-piperidinecarboxylate isomer provided by the present application is good in specificity.
TABLE 1 results of resolution measurements
| Composition (A) | Degree of separation |
| (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester | — |
| (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 4.911 |
| (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 8.824 |
| (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 1.564 |
Example 2 detection and quantitation limits
Detection limit: the reference solution prepared in example 1 was diluted quantitatively with absolute ethanol step by step, and then detected by gas chromatography, the specific conditions of which were as described in example 1, and the spectrum was recorded, with the signal-to-noise ratio not lower than 3.
And (4) quantitative limit: the reference solution prepared in example 1 was diluted quantitatively with absolute ethanol step by step, and then detected by gas chromatography, the specific conditions of which were as described in example 1, and the spectrum was recorded with the signal-to-noise ratio of not less than 10.
TABLE 2
The quantitative limiting solution is repeatedly measured for 6 times, the peak areas are respectively recorded, the results are shown in the following table 3, and the maximum RSD of the peak areas is 6.49% as can be seen from the table 3, so that the detection method provided by the application is proved to be good in quantitative limiting repeatability.
TABLE 3 quantitative limit repeatability test results
| Composition (I) | 1 | 2 | 3 | 4 | 5 | 6 | RSD％ |
| (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 2570 | 2157 | 2328 | 2326 | 2167 | 2303 | 6.49 |
| (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 2486 | 2379 | 2458 | 2454 | 2305 | 2416 | 2.73 |
| (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 2814 | 2867 | 2830 | 2983 | 2708 | 2817 | 3.15 |
| (2S,4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester | 2323 | 2204 | 2227 | 2156 | 2066 | 2182 | 3.86 |
Example 3 Linear relationship
Appropriate amounts of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate, ethyl (2S, 4S) -4-methyl-2-piperidinecarboxylate control, ethyl (2R, 4S) -4-methyl-2-piperidinecarboxylate control and ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate control were taken, dissolved in absolute ethanol and quantitatively diluted to prepare solutions of a series of concentrations, and the detection was carried out by gas chromatography, specifically as shown in example 1, the chromatogram was recorded, the concentration (. Mu.g/ml) was plotted as abscissa and the peak area was plotted as ordinate, and the regression equation was calculated, and the results are shown in Table 4. As can be seen from Table 4, ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate is excellent in linear relationship in the concentration range of 2.38. Mu.g/ml to 28.57. Mu.g/ml, ethyl (2S, 4S) -4-methyl-2-piperidinecarboxylate is excellent in linear relationship in the concentration range of 2.38. Mu.g/ml to 142.92. Mu.g/ml, ethyl (2R, 4S) -4-methyl-2-piperidinecarboxylate is excellent in linear relationship in the concentration range of 2.14. Mu.g/ml to 64.11. Mu.g/ml, and ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate is excellent in linear relationship in the concentration range of 2.01. Mu.g/ml to 60.17. Mu.g/ml.
TABLE 4 results of the Linear test
Example 4 recovery
An appropriate amount of (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester control, of (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester control, and of (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester control was precisely weighed and diluted with absolute ethanol to give a solution containing about 500. Mu.g of (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester, 200. Mu.g of (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester, and 200. Mu.g of (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester per 1ml, and the solution was used as a control stock solution.
About 10mg of a sample of ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate was taken, precisely weighed, placed in a 10ml measuring flask, and 9 parts were prepared in parallel, and then, a proper amount of a control stock solution was precisely added to make the concentrations of ethyl (2s, 4s) -4-methyl-2-piperidinecarboxylate, ethyl (2r, 4s) -4-methyl-2-piperidinecarboxylate and ethyl (2s, 4r) -4-methyl-2-piperidinecarboxylate low, medium and high, respectively, to the scale, and the volume was fixed to 3 parts per concentration in parallel with anhydrous ethanol, and as a recovery rate test sample solution, gas chromatography was used for the test, the conditions of the gas chromatography were specifically as in example 1, and the results are shown in table 5.
TABLE 5 recovery test results
As can be seen from Table 5, the recovery rates of the components are all 85% -120% of quality inspection, and the maximum value of RSD is 6.94%, so that the detection method provided by the application is good in accuracy.
Example 5 reproducibility
Taking the same batch of samples, preparing 6 parts of test solution according to the preparation method of the test solution in the example 1, and detecting by adopting gas chromatography, wherein the specific conditions of the gas chromatography are described in the example 1, and the results are shown in the table 6. As can be seen from Table 6, the test results of 6 test solutions are substantially consistent, indicating that the test method provided by the present application has good repeatability.
TABLE 6 results of the repeatability tests
Example 6 solution stability
The sample solution prepared in example 1 was allowed to stand for 0h, 2h, and 4h, respectively, and then subjected to detection by gas chromatography, the specific conditions of which were as described in example 1, the peak area was recorded, and the RSD (%) was calculated, and the test results are shown in table 7 below. As can be seen from Table 7, the test solution was suggested to be newly prepared for use because the content of ethyl (2S, 4R) -4-methyl-2-piperidinecarboxylate increased after leaving the test solution at room temperature for 4 hours.
TABLE 7 stability test results of solutions
EXAMPLE 7 durability
The preparation method of the labeled test sample solution is as described in example 1, and is not repeated.
And detecting the prepared standard sample solution by adopting gas chromatography, and recording the separation degree.
Initial temperature was adjusted to 78 ℃ and 82 ℃ respectively, and the remaining test conditions were the same as in example 1, and the degree of separation was recorded, and the results are shown in Table 8.
TABLE 8 degrees of separation at different initial temperatures
The temperature rise rate was adjusted to 9 ℃/min and 11 ℃/min in the second step of the programmed temperature rise, respectively, and the remaining test conditions were the same as in example 1, and the degrees of separation were recorded, and the results are shown in table 9.
TABLE 9 degrees of separation at different ramp rates
The inlet temperature was adjusted to 215 ℃ and 225 ℃ respectively, and the remaining test conditions were the same as in example 1, and the degrees of separation were recorded, and the results are shown in Table 10.
TABLE 10 degrees of separation at different injection port temperatures
The detector temperature was adjusted to 245 ℃ and 255 ℃ respectively, and the remaining detection conditions were the same as in example 1, and the degrees of separation were recorded, and the results are shown in table 11.
TABLE 11 degrees of separation of different detector temperatures
As can be seen from tables 8 to 11, the fine-tuning chromatographic conditions have no influence on the detection of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester and three chiral isomers thereof, and the separation degrees of all components meet the requirements, which indicates that the detection method provided by the invention has good durability.
In order to better illustrate the technical solution of the present invention, further comparison is made below by means of a comparative example and an example of the present invention.
Comparative example 1
The present comparative example provides a method for detecting an ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate isomer, comprising the steps of:
step one, preparing a positioning solution:
appropriate amounts of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, (2S, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance, (2R, 4S) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance and (2S, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester reference substance are respectively taken, precisely weighed, dissolved by absolute ethyl alcohol and prepared into single standard solution with the concentration of 0.2 mg/ml.
Step two, detecting the 4 single standard solutions by adopting a gas chromatography, and recording spectrograms, wherein the result is shown in fig. 5, wherein the chromatographic conditions of the gas chromatography are as follows:
adopting 2, 3-di-O-methyl-6-tert-butylsilyl modified beta-cyclodextrin as a capillary column of stationary liquid, wherein the type is B-DA and the specification is 30m multiplied by 0.25mm multiplied by 0.12 mu m; the remaining detection conditions are the same as those in example 1, and are not described again.
As can be seen from fig. 5, the chromatographic peaks of ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate, ethyl (2s, 4s) -4-methyl-2-piperidinecarboxylate, ethyl (2r, 4s) -4-methyl-2-piperidinecarboxylate and ethyl (2s, 4r) -4-methyl-2-piperidinecarboxylate all completely coincide, and thus it is illustrated that the detection method provided in comparative example 1 does not separate ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate and the three chiral isomers thereof.
Comparative example 2
The present comparative example provides a method for detecting an ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate isomer, comprising the steps of:
step one, preparing a reference solution:
preparing a reference substance solution: respectively taking appropriate amounts of a (2R, 4R) -4-methyl-2-ethyl piperidine formate reference substance, a (2S, 4S) -4-methyl-2-ethyl piperidine reference substance, a (2R, 4S) -4-methyl-2-ethyl piperidine reference substance and a (2S, 4R) -4-methyl-2-ethyl piperidine reference substance, precisely weighing, and preparing a reference substance solution by using absolute ethyl alcohol, wherein the concentration of each component in the reference substance solution is 20 mug/ml.
Step two, detecting the reference solution by adopting a gas chromatography, and recording a spectrogram, wherein the result is shown in fig. 6, wherein the chromatographic conditions of the gas chromatography are as follows:
the temperature programming is as follows: the initial temperature is 40 ℃, the temperature is maintained for 5min, the temperature is increased to 180 ℃ at the speed of 10 ℃/min, and the temperature is maintained for 30min; the rest of the detection conditions are the same as those in example 1 and are not described again.
As can be seen from fig. 6, the degree of separation of ethyl (2r, 4s) -4-methyl-2-piperidinecarboxylate and ethyl (2s, 4r) -4-methyl-2-piperidinecarboxylate was only 0.785, i.e., the two components were not separated, and thus it was found that the detection method provided by comparative example 2 did not separate ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate and the three chiral isomers thereof.
Comparative example 3
The present comparative example provides a method for detecting an ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate isomer, comprising the steps of:
step one, preparing a reference substance solution:
preparing a reference substance solution: respectively taking appropriate amounts of a (2R, 4R) -4-methyl-2-ethyl piperidine formate reference substance, a (2S, 4S) -4-methyl-2-ethyl piperidine reference substance, a (2R, 4S) -4-methyl-2-ethyl piperidine reference substance and a (2S, 4R) -4-methyl-2-ethyl piperidine reference substance, precisely weighing, and preparing a reference substance solution by using absolute ethyl alcohol, wherein the concentration of each component in the reference substance solution is 20 mug/ml.
Step two, detecting the reference substance solution by adopting a gas chromatography, and recording a spectrogram, wherein the result is shown in figure 7, wherein the chromatographic conditions of the gas chromatography are as follows:
the temperature programming is as follows: the initial temperature is 50 ℃, the temperature is increased to 120 ℃ at the speed of 5 ℃/min, the temperature is maintained for 15min, the temperature is increased to 200 ℃ at the speed of 5 ℃/min, and the temperature is maintained for 5min; the rest of the detection conditions are the same as those in example 1 and are not described again.
As can be seen from fig. 7, the degree of separation of ethyl (2r, 4s) -4-methyl-2-piperidinecarboxylate and ethyl (2s, 4r) -4-methyl-2-piperidinecarboxylate was only 1.153, i.e., the two components were not separated, and thus it was found that the detection method provided by comparative example 3 could not separate ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate and the three chiral isomers thereof.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (9)
1. A method for detecting an isomer of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester, which is characterized by comprising the following steps: the detection method comprises the following steps:
step one, preparing a reference solution and a test solution:
preparation of a reference solution: preparing a reference substance solution by using a solvent for a reference substance of (2S, 4S) -4-methyl-2-ethyl piperidinecarboxylate, a reference substance of (2R, 4S) -4-methyl-2-ethyl piperidinecarboxylate and a reference substance of (2S, 4R) -4-methyl-2-ethyl piperidinecarboxylate;
preparing a test solution: preparing a sample of (2R, 4R) -4-methyl-2-ethyl piperidinecarboxylate into a test solution by using a solvent;
step two, detecting the reference substance solution and the test substance solution by adopting a gas chromatography, wherein the chromatographic conditions of the gas chromatography are as follows:
a capillary column which takes 14 percent of cyanopropylphenyl-86 percent of dimethyl polysilane and 30 percent of hepta- (2, 3-di-O-methyl-6-O-tert-butyldimethylsilyl) -beta-cyclodextrin as a stationary liquid is adopted; the temperature of a sample inlet is 215-225 ℃; the temperature programming is as follows: the initial temperature is 80 ℃, the temperature is increased to 120 ℃ at the speed of 10 ℃/min, the temperature is maintained for 15min, the temperature is increased to 200 ℃ at the speed of 10 ℃/min, and the temperature is maintained for 5min; a hydrogen flame ionization detector is adopted, and the temperature of the detector is 245-255 ℃.
2. The method for detecting an isomer of ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate according to claim 1, wherein: the injection port temperature is 220 ℃.
3. The method for detecting an isomer of (2r, 4r) -4-methyl-2-piperidinecarboxylic acid ethyl ester according to claim 1, wherein: the detector temperature was 250 ℃.
4. The method for detecting an isomer of ethyl (2r, 4r) -4-methyl-2-piperidinecarboxylate according to claim 1, wherein: the carrier gas is nitrogen, and the flow split ratio is 5 to 20.
5. The method for detecting an isomer of (2R, 4R) -4-methyl-2-piperidinecarboxylic acid ethyl ester according to claim 4, wherein: the flow dividing ratio is 10.
6. The method for detecting an isomer of (2r, 4r) -4-methyl-2-piperidinecarboxylic acid ethyl ester according to claim 1, wherein: the flow rate is 0.8 ml/min-1.2 ml/min, and the sample injection volume is 1 mu L.
7. The method for detecting an isomer of (2r, 4r) -4-methyl-2-piperidinecarboxylic acid ethyl ester according to claim 1, wherein: the type of the capillary column is CYCLOSIL-B.
8. The method for detecting an isomer of ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate according to claim 7, wherein: the capillary column specification is 30m x 0.32mm x 0.25 μm.
9. The method for detecting ethyl (2R, 4R) -4-methyl-2-piperidinecarboxylate isomer according to any of claims 1 to 8, wherein: the solvent is absolute ethyl alcohol.
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| JPH01250061A (en) * | 1988-03-30 | 1989-10-05 | Shimadzu Corp | Gc analysis of compound containing heterocycloamine as molecular structure |
| JPH02212473A (en) * | 1989-02-13 | 1990-08-23 | Mitsubishi Kasei Corp | L-tartaric acid salt of (2r,4r)-4-methyl-2-piperidine-carboxylic acid ethyl ester and its desalted substance |
| CN109734653A (en) * | 2019-02-21 | 2019-05-10 | 北京悦康科创医药科技股份有限公司 | A kind of method for splitting of argatroban starting material isomer impurities |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| ITMI20110545A1 (en) * | 2011-04-04 | 2012-10-05 | Lundbeck Pharmaceuticals Italy S P A | METHOD FOR THE PREPARATION OF PROCESS INTERMEDIATES FOR THE SYNTHESIS OF MONOHYDRATE ARGATROBAN |
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| JPH01250061A (en) * | 1988-03-30 | 1989-10-05 | Shimadzu Corp | Gc analysis of compound containing heterocycloamine as molecular structure |
| JPH02212473A (en) * | 1989-02-13 | 1990-08-23 | Mitsubishi Kasei Corp | L-tartaric acid salt of (2r,4r)-4-methyl-2-piperidine-carboxylic acid ethyl ester and its desalted substance |
| CN109734653A (en) * | 2019-02-21 | 2019-05-10 | 北京悦康科创医药科技股份有限公司 | A kind of method for splitting of argatroban starting material isomer impurities |
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