A chiral chromatographic analysis method for detecting enantiomers

Abstract

The present invention provides a chiral chromatographic analysis method for detecting enantiomers. Specifically, the present invention provides a chiral chromatographic analysis method for detecting enantiomers in (S)-(2-methylaziridine-1-yl) diethyl phosphonate. The detection method adopts capillary column gas chromatography, the chromatographic column uses 2,6-di-O-pentyl-3-trifluoroacetyl-γ-cyclodextrin as the stationary phase, the carrier gas is nitrogen, and the detector is a hydrogen flame ionization detector (FID). In the chiral analysis method provided by the present invention, the separation effect of (S)-(2-methylaziridine-1-yl) diethyl phosphonate and enantiomers is good. This method is simple to operate, accurate and sensitive, has high separation efficiency, and has good specificity and reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical analysis, and relates to a chiral chromatographic analysis method for detecting enantiomers. Specifically, the present invention relates to a chiral chromatographic analysis method for detecting enantiomers in (S)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester. Background Art

[0002] (S)-(2-Methylaziridine-1-yl)phosphonic acid diethyl ester, CAS number is 5890-78-8, molecular formula is C7H 16 NO3P, chemical structure is as follows:

[0003]

[0004] (2-Methylaziridine-1-yl) diethyl phosphonate is an important intermediate for the synthesis of amine compounds. However, due to the presence of chiral molecules in the structure, it mostly exists in the form of a mixture of S and R configurations. As an important intermediate, the control of isomeric impurities of (2-methylaziridine-1-yl) diethyl phosphonate becomes increasingly important. US20150183716A1 discloses a preparation method and purity detection method of (S)-(2-methylaziridine-1-yl) diethyl phosphonate. Technicians have found that the (S)-(2-methylaziridine-1-yl) diethyl phosphonate prepared by the method contains a certain amount of enantiomers, and the detection method provided can only detect impurities, but cannot detect its enantiomer content. Summary of the invention

[0005] Conventional enantiomer analysis methods such as optical rotation method and high performance liquid chromatography are unable to accurately quantify the enantiomers in (S)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester, and thus cannot meet the requirements for content analysis and control of enantiomers in (S)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester. Currently, there is no literature document disclosing an analysis and detection method for enantiomers in (S)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester.

[0006] In view of this, the present invention provides a chiral chromatographic analysis method for accurately and rapidly detecting enantiomers in diethyl (S)-(2-methylaziridine-1-yl)phosphonate, which meets the standards in terms of specificity, repeatability, linear range, quantitative limit, and detection limit and has high sensitivity.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The chiral chromatographic analysis method of enantiomers in (S)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester is a capillary column gas chromatography method, and its chromatographic conditions include: the stationary phase is a γ-cyclodextrin derivative, the carrier gas is nitrogen, and the detector is a hydrogen flame ionization detector (FID). Specifically, the stationary phase is 2,6-di-O-pentyl-3-trifluoroacetyl-γ-cyclodextrin, and most preferably, the stationary phase is 2,6-di-O-pentyl-3-trifluoroacetyl-γ-cyclodextrin. G-TA120 chromatographic column.

[0009] In an embodiment of the present invention, the injection port temperature is 150-300°C. In certain preferred embodiments, the injection port temperature is 250°C.

[0010] In an embodiment of the present invention, the detector temperature is 200-350°C, and in certain preferred embodiments, the detector temperature is 275°C.

[0011] In an embodiment of the present invention, a programmed temperature is adopted, with a starting temperature of 40-60°C, a temperature increased to 150-220°C at a rate of 5-15°C / min, and maintained for 2-10 min. In certain preferred embodiments, the starting temperature is 50°C, a temperature increased to 180°C at a rate of 8°C / min, and maintained for 5 min.

[0012] In an embodiment of the present invention, the carrier gas flow rate is 1.0 ml / min to 3.0 ml / min. In certain preferred embodiments, the carrier gas flow rate is 2.0 ml / min.

[0013] In an embodiment of the present invention, the split ratio is (10:1) to (50:1), and in certain preferred embodiments, the split ratio is 30:1.

[0014] In an embodiment of the invention, the chromatographic method comprises: using G-TA 120 chromatographic column; carrier gas is nitrogen, flow rate: 2.0 ml / min, split ratio: 30:1, injection port temperature: 250°C, detector temperature: 275°C, injection volume: 2.0 μl; the heating program is initial column temperature 50°C, heating to 180°C at a rate of 8°C / min, and maintaining for 5 min; or initial column temperature 50°C, heating to 180°C at a rate of 5°C / min, and maintaining for 5 min; or initial column temperature 50°C, heating to 180°C at a rate of 10°C / min, and maintaining for 5 min.

[0015] The method of the invention adopts a chiral chromatographic column, can quickly and accurately determine the content of the enantiomer in diethyl (S)-(2-methylaziridine-1-yl)phosphonate, and provides reliable analysis data for its quality control.

[0016] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0018] Figure 1 The gas chromatogram was detected for the sample with batch number 6210-20180501 in Example 1.

[0019] Figure 2 This is the gas chromatogram of the sample detection of 6210-20180502 in Example 1.

[0020] Figure 3 This is the gas chromatogram of the sample detection of 6210-20180601 in Example 1.

[0021] Figure 4 The gas chromatogram of the blank solvent methanol.

[0022] Figure 5 Gas chromatogram of enantiomers.

[0023] Figure 6 Gas chromatogram of the sample solution.

[0024] Figure 7 This is the gas chromatogram of the system suitability solution.

[0025] Figure 8 The figure is a standard curve of enantiomers.

[0026] Fig. 9 Gas chromatogram showing the quantification limit of enantiomers.

[0027] Fig.10 Gas chromatogram showing the detection limit of enantiomers.

[0028] Figure 11-1 , Figure 11-2 , Figure 12-1 and Figure 12-2 The chromatograms are obtained in Example 3 at 50 μg / ml and 10 μg / ml of the mixed solution of cyclocompound R isomer in temperature programs 1 and 2, respectively.

[0029] Fig.13 and Fig.14 The chromatograms obtained in temperature programs 1 and 2 of Comparative Example 1 are shown respectively.

[0030] Fig.15 , Fig.16 , Fig.17 and Fig.18 They are the chromatograms obtained in temperature programs 1, 2, 3 and 4 of Comparative Example 2 respectively. DETAILED DESCRIPTION

[0031] The present invention discloses a chiral chromatography analysis method for enantiomers in (S)-(2-methylaziridine-1-yl) diethyl phosphonate. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by way of example, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0032] The raw materials, reagents and instruments used in the specific embodiments of the present invention are all known products and can be purchased from the market.

[0033] Example 1

[0034] Chromatographic conditions:

[0035] 1. Main instruments

[0036] Agilent 7890A gas chromatograph was equipped with an autosampler and a hydrogen flame ionization detector. The control and recording of the chromatographic analysis system was completed by the Dionex chromatography management system.

[0037] 2. Main technical parameters of gas chromatography

[0038] a. Gas chromatograph used: Agilent 7890A gas chromatograph, detector: FID detector, chromatographic column model: G-TA 120, 30m×0.25mm×0.12μm.

[0039] b. The carrier gas is nitrogen, flow rate: 2.0 ml / min, split ratio: 30:1, injection port temperature: 250°C, detector temperature: 275°C, injection volume: 2.0 μl, injection method: direct injection, blank / diluent / needle washing solution: methanol.

[0040] c. The heating program is as follows: initial column temperature 50°C, increase to 180°C at a rate of 8°C / min, and maintain for 5 minutes.

[0041] Sample preparation:

[0042] Test solution: Accurately weigh 25.24 mg, 25.09 mg and 25.40 mg of three batches of 6210-20180501, 6210-20180502 and 6210-20180601 samples respectively, place them in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and obtain.

[0043] The measurement results are as follows Figure 1-3 shown.

[0044] Example 2: Methodological validation

[0045] The chromatographic conditions were the same as in Example 1.

[0046] (I) Determination of enantiomer content in diethyl (S)-(2-methylaziridin-1-yl)phosphonate by gas chromatography

[0047] 1. Solution preparation

[0048] a. Preparation of system suitability solution: Take appropriate amount of diethyl (S)-(2-methylaziridin-1-yl)phosphonate and its enantiomer, add diluent to dissolve and dilute to a mixed solution containing about 1 mg of this product and 50 μg of the enantiomer per 1 ml.

[0049] b. Preparation of enantiomer standard stock solution: Accurately weigh 24.89 mg of the enantiomer, place it in a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and prepare a standard stock solution with a concentration of 0.9956 mg / ml.

[0050] c. Preparation of linear solution: dilute the above-prepared enantiomer standard solution 500 times, 200 times, 100 times, 50 times, and 20 times in sequence.

[0051] d. Preparation of sample solution: Take about 25 mg of sample, weigh accurately, place in a 25 ml volumetric flask, add diluent to dissolve, dilute to the scale, and shake well.

[0052] 2. Gas chromatography detection

[0053] a. Blank solution gas chromatography detection: accurately measure 2.0 μl of methanol for injection and record the spectrum. The result is as follows: Figure 4 shown.

[0054] b. Gas chromatography detection of enantiomer positioning solution: accurately measure 2.0 μl of enantiomer solution and inject it, record the spectrum, and the result is as follows: Figure 5 , the retention time of diethyl (R)-(2-methylaziridin-1-yl)phosphonate is 13.867 min.

[0055] c. Gas chromatography detection of sample solution: accurately measure 2.0 μl of sample solution and inject it, record the spectrum, and the result is as follows Figure 6 According to the peak area normalization method, the enantiomer content of (S)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester in the sample was calculated. After determination, the content of (R)-(2-methylaziridine-1-yl)phosphonic acid diethyl ester in the sample solution was 5.34%.

[0056] (II) System suitability investigation

[0057] Take the system suitability solution prepared by the above method and perform gas chromatography analysis. The results are shown in Figure 7 The retention times of (S)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester and its enantiomer were 13.595 min and 13.901 min, respectively, with a separation degree of 3.88.

[0058] (III) Investigation of linear relationship

[0059] Take 2.0 μl of the linear solution prepared by the above method and inject it, and plot the peak area against the concentration. The results are as follows: Figure 8 As can be seen from the figure, the concentration of the enantiomer standard is linearly related to the peak area, and the linear regression equation is: Y = 0.0047X + 0.0011, where X is the enantiomer concentration, Y is the peak area of ​​the enantiomer, and r = 0.9994.

[0060] (IV) Determination of the quantification limit of enantiomers

[0061] Dilute the enantiomer standard solution 500 times and Fig. 9 , the detection limit concentration was 1.9912μg / ml, equivalent to 0.1991% of the sample solution concentration, and the signal-to-noise ratio was 12.9.

[0062] (V) Determination of enantiomer detection limit

[0063] Take the enantiomer standard solution and dilute it 1000 times. Fig.10 , the detection limit concentration was 0.9956μg / ml, equivalent to 0.0996% of the sample solution concentration, and the signal-to-noise ratio was 3.5.

[0064] (VI) Solution stability study

[0065] The system suitability solution was placed at room temperature for 24 hours, and the enantiomer content was measured at 2, 4, 6, 9, 14, 19, and 22 hours, and the RSD value was calculated. The results are shown in Table 1, where the RSD of the enantiomer content was 2%, indicating that the solution was relatively stable when placed at room temperature for 24 hours.

[0066] Table 1: Changes in enantiomer content in system suitability solutions within 24 hours

[0067]

[0068] Example 3

[0069] Chromatographic conditions:

[0070] The main instruments and main technical parameters of gas chromatography were the same as those in Example 1, and the following heating program was used:

[0071] Temperature program 1: Initial column temperature 50°C, increase to 180°C at a rate of 5°C / min, and maintain for 5 minutes;

[0072] Temperature program 2: Initial column temperature 50°C, increase to 180°C at a rate of 10°C / min, and maintain for 5 minutes.

[0073] Sample preparation:

[0074] Solution of cyclocompound test sample ((R)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester): weigh 25.02 mg of sample 1P13242, place in a 25 ml volumetric flask, add diluent to dissolve, dilute to scale, and shake well.

[0075] Cyclic compound R isomer mixed solution (50 μg / ml): Take 50 μl of cyclocompound impurity DJLY-4 solution and mix it with 950 μl of cyclocompound test solution to obtain;

[0076] Cyclocomplex R isomer mixed solution (10 μg / ml): Take 10 μl of cyclocomplex impurity DJLY-4 solution and mix it with 990 μl of cyclocomplex test sample solution to obtain.

[0077] Experimental results and conclusions:

[0078] Figure 11-1 and Figure 11-2 is the chromatogram obtained under temperature program 1, Figure 12-1 and Figure 12-2 This is the chromatogram obtained under temperature program 2.

[0079] Summary: In choosing When using G-TA 120 30m×0.25mm×0.12μm column, the separation effect can meet the requirements under heating programs 1 and 2.

[0080] Comparative Example 1

[0081] The main instruments and technical parameters of gas chromatography are the same as those in Example 1, using the chromatographic column Beta DEX TM 120 30m×0.25mm×0.25μm (stationary phase is fully methylated cyclodextrin) and the following temperature program:

[0082] Temperature program 1: Initial column temperature 50°C, increase to 220°C at a rate of 10°C / min, and maintain for 15 minutes;

[0083] Temperature program 2: Initial column temperature 50°C, increase to 220°C at a rate of 5°C / min, and maintain for 5 minutes.

[0084] Sample preparation:

[0085] Solution of cyclocompound test sample ((R)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester): weigh 25.02 mg of sample 1P13242, place in a 25 ml volumetric flask, add diluent to dissolve, dilute to scale, and shake well.

[0086] Experimental results and discussion:

[0087] The chromatograms obtained under temperature programs 1 and 2 are shown in Fig.13 and 14 .

[0088] In temperature program 1: (R)-diethyl (2-methylaziridin-1-yl)phosphonate showed no separation trend.

[0089] Under column temperature condition 2: (R)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester has a separation tendency, but complete separation cannot be achieved.

[0090] Comparative Example 2

[0091] The main instruments and technical parameters of gas chromatography are the same as those in Example 1, and the chromatographic column used is Astec B-DM 120 30m×0.25mm×0.12μm (stationary phase is dimethyl derivative of cyclodextrin) and the following temperature program:

[0092] Temperature program 1: initial column temperature 50°C, increase to 200°C at a rate of 10°C / min, and maintain for 15 minutes;

[0093] Temperature program 2: initial column temperature 80°C, increase to 200°C at a rate of 5°C / min, and maintain for 5 minutes;

[0094] Temperature program 3: initial column temperature 80°C, increase the temperature to 140°C at a rate of 2.5°C / min, then increase the temperature to 200°C at a rate of 10°C / min, and maintain for 5 minutes;

[0095] Temperature program 4: Initial column temperature 100°C, increase to 140°C at a rate of 2°C / min, then increase to 200°C at a rate of 10°C / min, and maintain for 5 minutes.

[0096] Sample preparation:

[0097] Take the cyclocompound test sample prepared in Comparative Example 1 and inject it.

[0098] Experimental results and discussion:

[0099] Fig.15 , Fig.16 , Fig.17 and Fig.18 The chromatograms are obtained under temperature programs 1, 2, 3, and 4. It can be seen from the chromatograms that under the conditions of temperature programs 1-4, the separation effect of (R)-(2-methylaziridin-1-yl)phosphonic acid diethyl ester is poor, and the R and S isomers cannot be completely separated.

[0100] The present application describes a plurality of embodiments, but the description is exemplary rather than restrictive, and it is apparent to those skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application. Although many possible feature combinations are shown in the drawings and discussed in the specific embodiments, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

Claims

1. A chiral chromatographic analysis method for detecting enantiomers in diethyl (S)-(2-methylaziridine-1-yl)phosphonate, the method comprising: Using 2,6-di-O-pentyl-3-trifluoroacetyl-γ-cyclodextrin as the stationary phase, the following temperature program was used for separation: starting temperature 40~60℃, heating to 150~220℃ at a rate of 5~15℃ / min, and maintaining for 2~10min.

2. The method according to claim 1, wherein the method uses an Astec® CHIRALDEX® G-TA 120 chromatography column.

3. The method according to claim 1, wherein: The carrier gas was nitrogen and the detector was a hydrogen flame ionization detector (FID).

4. The method according to claim 1, wherein: The injection port temperature is 150~300℃.

5. The method according to claim 4, wherein: The injection port temperature was 250 °C.

6. The method according to claim 1, wherein: The detector temperature is 200~350℃.

7. The method according to claim 6, wherein: The detector temperature was 275°C.

8. The method according to claim 1, wherein: The heating program was as follows: the initial temperature was 50°C, the temperature was increased to 180°C at a rate of 8°C / min, and maintained for 5 min.

9. The method according to claim 1, wherein: The carrier gas flow rate is 1.0 ml / min~3.0 ml / min.

10. The method according to claim 9, wherein: The carrier gas flow rate was 2.0 ml / min.

11. The method according to claim 1, wherein: The split ratio is (10:1)~(50:1).

12. The method according to claim 11, wherein: The split ratio is 30:

1.

13. The method according to claim 1, wherein: The method includes: using an Astec® CHIRALDEX® G-TA 120 chromatographic column; the carrier gas is nitrogen, the flow rate is 2.0 ml / min, the split ratio is 30:1, the injection port temperature is 250°C, the detector temperature is 275°C, the injection volume is 2.0µl, The temperature program is: initial column temperature 50℃, increase to 180℃ at a rate of 8℃ / min, and maintain for 5min; or The temperature program is: initial column temperature 50℃, heating to 180℃ at a rate of 5℃ / min, and maintaining for 5 minutes; or The temperature program was as follows: the initial column temperature was 50°C, the temperature was increased to 180°C at a rate of 10°C / min, and maintained for 5 minutes.

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