CN114705790A - 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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- CN114705790A CN114705790A CN202210214336.2A CN202210214336A CN114705790A CN 114705790 A CN114705790 A CN 114705790A CN 202210214336 A CN202210214336 A CN 202210214336A CN 114705790 A CN114705790 A CN 114705790A
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Abstract
The invention relates to the technical field of pharmaceutical 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 substance solution and a test solution, and detecting the reference substance 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 pharmaceutical analysis, and particularly relates to a detection method of an ethyl (2R,4R) -4-methyl-2-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 FDA in the united states in 2000, and marketed 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-piperidine ethyl formate is an important starting material for synthesizing argatroban, so the quality control of the argatroban has great influence on the synthesis of the argatroban. There are also three other chiral isomers of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate, ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate, ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate and ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate, all the isomers may be impurities in the synthesis of argatroban, and no relevant literature reports a detection method of the isomers at present, therefore, it is urgently needed to develop a detection method capable of effectively controlling the quality of (2R,4R) -4-methyl-2-ethyl piperidinecarboxylate, so as to ensure the effective control of argatroban quality and improve the industrial standard.
Disclosure of Invention
In view of the above, the invention provides a method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer, which has the advantages of strong specificity, high accuracy and excellent linear relationship, 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 detection method of an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer, comprising the following steps:
step one, preparing a reference solution and a test solution:
preparation of a reference solution: preparing a reference substance solution from a (2S,4S) -4-methyl-2-ethyl piperidinecarboxylate reference substance, a (2R,4S) -4-methyl-2-ethyl piperidinecarboxylate reference substance and a (2S,4R) -4-methyl-2-ethyl piperidinecarboxylate reference substance by using a solvent;
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 78-82 ℃, the temperature is increased to 115-125 ℃ at the speed of 9-11 ℃/min, the temperature is maintained for 10-20 min, the temperature is increased to 195-205 ℃ at the speed of 9-11 ℃/min, and 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 detection method of the (2R,4R) -4-methyl-2-ethyl piperidinecarboxylate 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 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 the three chiral isomers thereof, and realizes 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 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 5 min.
The temperature rising program 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 port temperature is 220 ℃.
Optionally, the detector temperature is 250 ℃.
The preferable injection port 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: 1.
Optionally, the split ratio is 10: 1.
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 1 ml/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 are not intended to limit the invention.
Example 1
The embodiment of the invention provides a detection method of a (2R,4R) -4-methyl-2-ethyl piperidinecarboxylate 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 reference substance of ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate, a reference substance of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate and a reference substance of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate, accurately weighing, and preparing a reference solution by using absolute ethyl alcohol, wherein the concentration of ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate in the reference solution is 50 mu g/ml, the concentration of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate is 20 mu g/ml, and the concentration of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate is 20 mu g/ml;
Preparing a test solution: taking a sample of (2R,4R) -4-methyl-2-ethyl piperidinecarboxylate 20mg, precisely weighing, placing in a 20ml volumetric flask, dissolving with absolute ethyl alcohol, diluting to a scale, and shaking up;
preparing a standard sample solution: taking a proper amount of (2R,4R) -4-methyl-2-ethyl piperidine carboxylate, a proper amount of (2S,4S) -4-methyl-2-ethyl piperidine carboxylate reference substances, a proper amount of (2R,4S) -4-methyl-2-ethyl piperidine carboxylate reference substances and a proper amount of (2S,4R) -4-methyl-2-ethyl piperidine carboxylate reference substances, precisely weighing, and preparing a standard sample solution by using absolute ethyl alcohol, wherein the concentration of (2R,4R) -4-methyl-2-ethyl piperidine carboxylate is 1000 mug/ml, the concentration of (2S,4S) -4-methyl-2-ethyl piperidine carboxylate is 50 mug/ml, the concentration of (2R,4S) -4-methyl-2-ethyl piperidine carboxylate is 20 mug/ml, the concentration of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate was 20. mu.g/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:
the 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 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 5 min; 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 10:1, and the sample injection volume is 1 mul.
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 the retention time of 17.363min, a peak of the chromatogram for ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate occurred at the retention time of 19.274min, a peak of the chromatogram for ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate occurred at the retention time of 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 the color spectrum of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate appeared at the retention time of 16.293min, a peak of the color spectrum of ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate appeared at the retention time of 17.362min, a peak of the color spectrum of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate appeared at the retention time of 19.595min, and the degrees of separation of the components were 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-mentioned separation degree detection of each component are shown in table 1 below, and it can be seen from table 1 that the separation degree between the components is good, thereby demonstrating that the detection method of the ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer provided in the present application is good in specificity.
TABLE 1 results of separation detection
| 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 3:1, to obtain the detection limit, the results of which are shown in table 2.
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 spectra were recorded with the signal-to-noise ratio of not less than 10:1 as the limit of quantitation, the results of which are shown in table 2.
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 (A) | 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
Taking appropriate amounts of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate, ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate reference substances, ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate reference substances and ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate reference substances respectively, dissolving with absolute ethyl alcohol, quantitatively diluting to prepare solutions with series concentrations, detecting by adopting gas chromatography, specifically adopting the gas chromatography conditions as shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as an abscissa and taking a peak area as an ordinate, and calculating a regression equation, wherein the results are shown in Table 4. As can be seen from Table 4, ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate had a good 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 had a good 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 had a good 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 had a good linear relationship in the concentration range of 2.01. mu.g/ml to 60.17. mu.g/ml.
TABLE 4 Linear test results
Example 4 recovery
A proper amount of a reference sample of ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate, a proper amount of a reference sample of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate and a proper amount of a reference sample of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate are precisely weighed and quantitatively diluted with absolute ethyl alcohol to prepare a solution containing about 500. mu.g of ethyl (2S,4S) -4-methyl-2-piperidinecarboxylate, 200. mu.g of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate and 200. mu.g of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate per 1ml, which serve as reference stock solutions.
Taking about 10mg of a sample of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate, accurately weighing the sample, placing the sample in a 10ml measuring flask, preparing 9 parts in parallel, respectively and accurately adding appropriate amounts of reference stock solutions to ensure that 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 are respectively in three levels of low, medium and high, fixing the volume to a scale by absolute ethyl alcohol, enabling each concentration to be in parallel for 3 parts, and detecting the sample solution as a recovery rate by adopting gas chromatography, wherein the conditions of the gas chromatography are specifically shown 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 table 6. As can be seen from Table 6, the test results of 6 test solutions are substantially consistent, which indicates 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 content of ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate increased after the sample solution was left at room temperature for 4 hours, and it was found that the sample solution was suggested to be newly prepared.
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 fine-tuning, initial temperatures of 78 ℃ and 82 ℃ 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 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 separation degree was recorded under the same detection conditions as in example 1, 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 comparing examples of the present invention with comparative examples.
Comparative example 1
This 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:
respectively taking a proper amount of a (2R,4R) -4-methyl-2-ethyl piperidine formate reference substance, a proper amount of a (2S,4S) -4-methyl-2-ethyl piperidine reference substance, a proper amount of a (2R,4S) -4-methyl-2-ethyl piperidine reference substance and a proper amount of a (2S,4R) -4-methyl-2-ethyl piperidine reference substance, precisely weighing, respectively dissolving by absolute ethyl alcohol and preparing into a 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 a 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, thus indicating that the detection method provided in comparative example 1 cannot separate ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate and three chiral isomers thereof.
Comparative example 2
This comparative example provides a method for detecting an isomer of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate, comprising the steps of:
step one, preparing a reference substance solution:
preparing a reference substance solution: respectively taking a proper amount of a (2R,4R) -4-methyl-2-ethyl piperidine formate reference substance, a proper amount of a (2S,4S) -4-methyl-2-ethyl piperidine reference substance, a proper amount of a (2R,4S) -4-methyl-2-ethyl piperidine reference substance and a proper amount of 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 mu g/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 30 min; 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 separation degree of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate and ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate is only 0.785, i.e., the two components are not separated, and thus it is understood that the detection method provided by comparative example 2 cannot separate ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate and three chiral isomers thereof.
Comparative example 3
This comparative example provides a method for detecting an isomer of ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate, comprising the steps of:
step one, preparing a reference substance solution:
preparation of a reference solution: respectively taking a proper amount of a (2R,4R) -4-methyl-2-ethyl piperidine formate reference substance, a proper amount of a (2S,4S) -4-methyl-2-ethyl piperidine reference substance, a proper amount of a (2R,4S) -4-methyl-2-ethyl piperidine reference substance and a proper amount of 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 mu g/ml.
Step two, detecting the reference solution by adopting a gas chromatography, and recording a spectrogram, wherein the result is shown in fig. 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 5 min; 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 separation degree of ethyl (2R,4S) -4-methyl-2-piperidinecarboxylate and ethyl (2S,4R) -4-methyl-2-piperidinecarboxylate is only 1.153, i.e., the two components are not separated, and thus it is understood that the detection method provided by comparative example 3 cannot separate ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate and three chiral isomers thereof.
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 (2R,4R) -4-methyl-2-piperidine ethyl formate isomer 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 from a (2S,4S) -4-methyl-2-ethyl piperidinecarboxylate reference substance, a (2R,4S) -4-methyl-2-ethyl piperidinecarboxylate reference substance and a (2S,4R) -4-methyl-2-ethyl piperidinecarboxylate reference substance by using a solvent;
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 78-82 ℃, the temperature is increased to 115-125 ℃ at the speed of 9-11 ℃/min, the temperature is maintained for 10-20 min, the temperature is increased to 195-205 ℃ at the speed of 9-11 ℃/min, and the temperature is maintained for 1-10 min; a hydrogen flame ionization detector is adopted, and the temperature of the detector is 245-255 ℃.
2. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 1, wherein: the temperature programming comprises the following steps: 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 5 min.
3. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 1, wherein: the injection port temperature is 220 ℃.
4. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 1, wherein: the detector temperature was 250 ℃.
5. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 1, wherein: the carrier gas is nitrogen, and the split ratio is 5-20: 1.
6. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 5, wherein: the split ratio was 10: 1.
7. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer 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.
8. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 1, wherein: the capillary column is CYCLOSIL-B.
9. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to claim 8, wherein: the capillary column specification is 30m x 0.32mm x 0.25 μm.
10. The method for detecting an ethyl (2R,4R) -4-methyl-2-piperidinecarboxylate isomer according to any of claims 1 to 9, 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 |
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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 |
| US20140088310A1 (en) * | 2011-04-04 | 2014-03-27 | Lundbeck Pharmaceuticals Italy S.P.A. | Process intermediates and methods for the preparation of process intermediates for the synthesis of argatroban monohydrate |
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