阅读说明：本技术 一种l-丙氨酸异丙酯盐酸盐与其对映异构体的分离方法 (Separation method of L-alanine isopropyl ester hydrochloride and enantiomer thereof ) 是由 张洪飞 郭赛 尚金凤 廖正华 于 2020-11-06 设计创作，主要内容包括：本发明公开了一种L-丙氨酸异丙酯盐酸盐与其对映异构体的分离方法,所述分离方法包括在碱性条件下,使用GITC溶液对L-丙氨酸异丙酯盐酸盐及其异构体进行衍生化,衍生化后的L-丙氨酸异丙酯盐酸盐及其异构体采用HPLC色谱法进行分离和检测。该分离方法能够有效的分离L丙氨酸异丙酯盐酸盐和其对映异构体D-丙氨酸异丙酯盐酸盐,并能够准确的测定其对映异构体的含量。本方法采用特定的反相方法和等度洗脱方式,简单且易于操作,峰型较好,分离度合格,方法学验证显示本方法专属性强,灵敏度高,准确度以及线性均比较良好。(The invention discloses a separation method of L-alanine isopropyl ester hydrochloride and an enantiomer thereof, which comprises the steps of using GITC solution to perform derivatization on the L-alanine isopropyl ester hydrochloride and the enantiomer thereof under an alkaline condition, and separating and detecting the derivatized L-alanine isopropyl ester hydrochloride and the enantiomer thereof by HPLC chromatography. The separation method can effectively separate L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof, and can accurately determine the content of the enantiomer. The method adopts a specific reverse phase method and an isocratic elution mode, is simple and easy to operate, has good peak pattern and qualified separation degree, and is proved by methodology to have strong specificity, high sensitivity, and good accuracy and linearity.)

1. The method for separating the L-alanine isopropyl ester hydrochloride and the enantiomer thereof is characterized by comprising the steps of derivatizing the L-alanine isopropyl ester hydrochloride and the enantiomer thereof by using a GITC solution in an alkaline solution, and separating and detecting the derivatized L-alanine isopropyl ester hydrochloride and the enantiomer thereof by using HPLC chromatography.

2. The separation method of claim 1, wherein the basic solution is a triethylamine solution.

3. The separation method of claim 2, wherein the triethylamine solution comprises triethylamine and acetonitrile solution.

4. The separation method of claim 3, wherein the triethylamine solution is prepared by: and (3) taking 1-2 ml of triethylamine solution, placing the triethylamine solution in a 100ml measuring flask, and diluting the triethylamine solution to a scale with acetonitrile to obtain the triethylamine-based measuring flask.

5. The isolation process of claim 1, wherein the GITC solution is prepared by: and taking about 78mg of GITC, putting the GITC into a 20ml volumetric flask, and adding acetonitrile to dilute the GITC to the scale.

6. The separation method according to any one of claims 1 to 5, wherein the chromatographic column in the HPLC chromatography is a bonded silica gel chromatographic column, and the mobile phase is a mixture of a mobile phase and a solvent in a volume ratio of 50: 50 parts of water (A) and methanol (B).

7. The separation method of claim 6, wherein the HPLC chromatography further comprises the following detection conditions: the bonded silica gel chromatographic column is YMC-Pack ODS-AQ, the specification of the chromatographic column is 150 x 4.6mm x 3 μm, the column temperature is 25 ℃, the detection wavelength is 246nm, the flow rate is 1.0ml/min, and the sample volume is 10 μ l.

8. The separation method according to claim 7, characterized in that it comprises the steps of:

(1) preparing a solution: respectively preparing a blank solution, a test solution, a D-alanine isopropyl ester hydrochloride reference solution and a system applicability solution; the test solution comprises an L-alanine isopropyl ester hydrochloride sample and an acetonitrile solution; the system suitability solution comprises D-alanine isopropyl ester hydrochloride and L-alanine isopropyl ester hydrochloride;

(2) and (3) HPLC determination: the prepared solutions were injected into a chromatograph, and chromatograms were recorded.

9. The method of claim 8, wherein the sample solution is prepared by: taking about 150mg of L-alanine isopropyl ester hydrochloride, precisely weighing, placing in a 50ml measuring flask, adding an appropriate amount of acetonitrile, ultrasonically dissolving, cooling to room temperature, adding acetonitrile to dilute to a scale, and shaking up; precisely measuring 0.5ml, placing in a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution and 0.5ml of GITC solution respectively, vortex mixing uniformly, and standing at room temperature for 1h to obtain a sample solution.

10. The method of claim 8, wherein the system suitability solution is prepared by: respectively weighing appropriate amount of L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof, adding acetonitrile to dissolve and dilute into mixed solution containing 0.3mg of D-alanine isopropyl ester hydrochloride and L-alanine isopropyl ester hydrochloride in each 1 ml; precisely measuring 0.5ml, placing into a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution and 0.5ml of GITC solution respectively, vortex mixing uniformly, and standing at room temperature for 1h to obtain the system applicability solution.

Technical Field

The invention relates to the field of drug detection and analysis, and more particularly relates to a separation method of L-alanine isopropyl ester hydrochloride and an enantiomer thereof.

Background

The L-alanine isopropyl ester hydrochloride is an important medical intermediate and an important starting material for synthesizing raw medicines such as tenofovir alafenamide and the like, and the structure of the L-alanine isopropyl ester hydrochloride has a chiral center. When raw material medicines such as tenofovir alafenamide and the like are synthesized by taking L-alanine isopropyl ester hydrochloride as a starting material, enantiomer D-alanine isopropyl ester hydrochloride thereof may also participate in the reaction to generate corresponding isomer impurities of the raw material medicines, for example, D-alanine isopropyl ester hydrochloride is one of the sources of tenofovir alafenamide chiral impurities. Therefore, the research on material impurities is helpful for optimizing the synthesis process and controlling the quality of the medicine, the research on the separation detection method of the L-alanine isopropyl ester hydrochloride and the enantiomer D-alanine isopropyl ester hydrochloride thereof can provide better reference for evaluating the quality of the L-alanine isopropyl ester hydrochloride, also provide technical basis for optimizing the synthesis process of the L-alanine isopropyl ester hydrochloride, and have great significance for determining adverse reactions caused by the enantiomer of the D-alanine isopropyl ester hydrochloride in the L-alanine isopropyl ester hydrochloride.

As can be seen from the literature, very few studies have been reported on the separation method of L-alanine isopropyl ester hydrochloride and its enantiomer D-alanine isopropyl ester hydrochloride. For example, Chennan et al (Chennan et al, high performance liquid chromatography for separation and detection of enantiomer impurity [ J ] of L-alanine isopropyl ester hydrochloride, Zhongnan pharmacy 2020, (06): 997-. However, the method has strict requirements on the chromatographic column, also has strict limitation on the derivatization reaction time before pre-column derivatization, and has higher detection cost; the stability time of the derivatization reaction product is shorter, and is only 2.5h, so that the accuracy of the experimental result is possibly influenced; these are clearly disadvantageous for the resolution of the L-alanine isopropyl ester hydrochloride enantiomer.

Patent CN110849980A discloses a method for detecting L-alanine isopropyl ester hydrochloride and its enantiomer, wherein the chromatographic column filler is crown ether chiral column (silica gel surface is coated with chiral crown ether), and strong acid mobile phase, such as perchloric acid aqueous solution with pH value of 1.0-2.0, is required, which has great damage to chromatographic column and strict requirement for temperature in the detection process.

The invention provides a convenient, efficient and accurate separation method for solving the problem that an L-alanine isopropyl ester hydrochloride isomer and an enantiomer thereof are difficult to separate.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings in the prior art and provide a method for separating L-alanine isopropyl ester hydrochloride and an enantiomer thereof, which adopts GITC solution to carry out pre-column derivatization on the L-alanine isopropyl ester hydrochloride under alkaline conditions and combines specific HPLC detection conditions, thereby realizing the separation and determination of the L-alanine isopropyl ester hydrochloride and the enantiomer D-alanine isopropyl ester hydrochloride thereof, ensuring that the method completely accords with the guiding principle of Chinese pharmacopoeia method verification in the aspects of system applicability, specificity, linearity, range and the like and can be used for quality control of raw medicines or medicines prepared by taking the L-alanine isopropyl ester hydrochloride as a starting material or an intermediate.

The above purpose of the invention is realized by the following technical scheme:

a separation method of L-alanine isopropyl ester hydrochloride and enantiomer thereof comprises the steps of using acetyl Glucose Isothiocyanate (GITC) solution to conduct derivatization on L-alanine isopropyl ester hydrochloride and enantiomer thereof in the presence of alkaline solution, and separating and detecting the derivatized L-alanine isopropyl ester hydrochloride and enantiomer thereof by HPLC chromatography.

Preferably, the basic solution is a triethylamine solution.

Preferably, the triethylamine solution comprises triethylamine and acetonitrile solution.

Preferably, the GITC solution is prepared by: and taking about 78mg of GITC, putting the GITC into a 20ml volumetric flask, and adding acetonitrile to dilute the GITC to the scale.

Preferably, the chromatographic column in the HPLC chromatography is a bonded silica gel chromatographic column, and the mobile phase is a mixture of a volume ratio of 50: 50 parts of water (A) and methanol (B).

Preferably, the HPLC chromatography further comprises the following detection conditions: the bonded silica gel chromatographic column is YMC-Pack ODS-AQ, the specification of the chromatographic column is 150 x 4.6mm x 3 μm, the column temperature is 25 ℃, the detection wavelength is 246nm, the flow rate is 1.0ml/min, and the sample volume is 10 μ l.

Preferably, the separation method comprises the steps of:

(1) preparing a solution: respectively preparing a blank solution, a test solution, a D-alanine isopropyl ester hydrochloride reference solution and a system applicability solution; the test solution comprises an L-alanine isopropyl ester hydrochloride sample and an acetonitrile solution; the system suitability solution comprises D-alanine isopropyl ester hydrochloride and L-alanine isopropyl ester hydrochloride;

(2) and (3) HPLC determination: the prepared solutions were injected into a chromatograph, and chromatograms were recorded.

Preferably, the preparation method of the test solution comprises the following steps: taking about 150mg of L-alanine isopropyl ester hydrochloride, precisely weighing, placing in a 50ml measuring flask, adding an appropriate amount of acetonitrile, ultrasonically dissolving, cooling to room temperature, adding acetonitrile to dilute to a scale, and shaking up; precisely measuring 0.5ml, placing in a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution and 0.5ml of GITC solution respectively, vortex mixing uniformly, and standing at room temperature for 1h to obtain a sample solution.

Preferably, the preparation method of the system suitability solution is as follows: respectively weighing appropriate amount of L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof, adding acetonitrile to dissolve and dilute into mixed solution containing 0.3mg of D-alanine isopropyl ester hydrochloride and L-alanine isopropyl ester hydrochloride in each 1 ml; precisely measuring 0.5ml, placing into a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution and 0.5ml of GITC solution respectively, vortex mixing uniformly, and standing at room temperature for 1h to obtain the system applicability solution.

Compared with the prior art, the invention has the following beneficial effects:

(1) the separation method can effectively separate L-alanine isopropyl ester hydrochloride and enantiomer thereof, and the separation degree can reach more than 3.0.

(2) Under alkaline conditions, GITC is used as a derivatization agent to perform derivatization reaction with L-alanine isopropyl ester hydrochloride, and the obtained derivatization product has good stability within 24 hours.

(3) The method adopts a reversed-phase high performance liquid chromatography method to separate L-alanine isopropyl ester hydrochloride and D-alanine isopropyl ester hydrochloride, adopts a common bonded silica gel chromatographic column and isocratic elution, has simple operation, low requirements on the chromatographic column and low cost, and obtains a good chromatographic peak type with the separation degree being as high as more than 3.0. The methodology verification shows that the method has strong specificity and good linearity, and is suitable for accurately and quickly separating and detecting the enantiomer impurity of the L-alanine isopropyl ester hydrochloride.

Drawings

FIG. 1 is a graph showing the results of HPLC detection of the white solution in example 1.

FIG. 2 is a diagram showing the results of HPLC analysis of a solution suitable for use in the system of example 1.

FIG. 3 is a graph showing the results of HPLC analysis of the sample solution in example 1.

FIG. 4 is a graph showing the results of HPLC analysis of the control solution in example 1.

FIG. 5 is a graph of the linear relationship of the D-alanine isopropyl ester hydrochloride control of example 1.

Detailed Description

In order to more clearly and completely describe the technical scheme of the invention, the invention is further described in detail by the specific embodiments, and it should be understood that the specific embodiments described herein are only used for explaining the invention, and are not used for limiting the invention, and various changes can be made within the scope defined by the claims of the invention.

Example 1

A method for separating L-alanine isopropyl ester hydrochloride from its enantiomer, comprising the steps of:

(1) preparation of the solution

GITC solution: weighing about 78mg of GITC, putting the GITC in a 20ml volumetric flask, adding acetonitrile to dissolve the GITC, quantitatively diluting the GITC to a scale, and uniformly shaking the GITC to obtain the product;

triethylamine solution: measuring 1ml of triethylamine solution, placing the triethylamine solution in a 100ml measuring flask, quantitatively diluting the triethylamine solution to a scale with acetonitrile to obtain triethylamine solution A, precisely measuring 2ml of triethylamine solution A, placing the triethylamine solution A in the 100ml measuring flask, carrying out constant volume on the acetonitrile to the scale, and shaking up to obtain triethylamine solution B;

blank solution: taking 0.5ml of acetonitrile, placing the acetonitrile into a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution A and 0.5ml of GITC solution respectively, uniformly mixing by vortex, and standing at room temperature for 1h to serve as a blank solution;

control solution of isopropyl D-alaninate: taking 7.5mg of D-isopropyl alanine reference substance, precisely weighing, placing in a 100ml measuring flask, adding acetonitrile to dissolve and dilute to a scale, shaking uniformly, taking 2ml to place in a 50ml measuring flask, adding acetonitrile to dilute to a scale, shaking uniformly, precisely measuring 0.5ml, placing in a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution B and 0.5ml of GITC solution respectively, vortex mixing uniformly, and placing at room temperature for 1h to serve as a reference substance solution;

test solution: taking about 150mg of L-alanine isopropyl ester hydrochloride, precisely weighing, placing in a 50ml measuring flask, adding an appropriate amount of acetonitrile, ultrasonically dissolving, cooling to room temperature, adding acetonitrile to dilute to a scale, and shaking up; precisely measuring 0.5ml, placing in a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution A and 0.5ml of GITC solution respectively, vortex mixing uniformly, and standing at room temperature for 1h to serve as a sample solution;

system applicability solution: respectively weighing appropriate amount of L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof, adding acetonitrile to dissolve and dilute into mixed solution containing 0.3mg of D-alanine isopropyl ester hydrochloride and L-alanine isopropyl ester hydrochloride in each 1 ml; precisely measuring 0.5ml, placing in a 2ml automatic sample feeding bottle, sequentially and precisely adding 0.5ml of triethylamine solution A and 0.5ml of GITC solution respectively, uniformly mixing by vortex, and standing at room temperature for 1h to serve as a system applicability solution;

(2) and (4) HPLC detection:

the blank solution, the system suitability solution and the test solution were injected into a liquid chromatograph, respectively, and chromatograms were recorded under chromatographic conditions as shown in table 1.

TABLE 1

Chromatographic column	Bonded silica gel chromatographic column YMC-Pack ODS-AQ
		Specification of chromatographic column	150*4.6mm*3μm
Column temperature	25℃
		Wavelength of light	246nm
Flow rate of flow	1.0ml/min
		Sample volume	10μl
Mobile phase	Water (a) -methanol (B); a: b is 50: 50
		Run time	45min

Detection results and analysis:

(1) the specificity and detection result analysis of the separation method of the invention

The chromatographic detection results of the blank solution, the system applicability solution, the test solution and the reference solution are shown in figures 1-4 respectively, and it can be seen from the figures that the blank solvent does not interfere with the detection of L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof, which indicates that the separation method of the invention has excellent specificity.

In the attached figure 2, L-alanine isopropyl ester hydrochloride and enantiomer D-alanine isopropyl ester hydrochloride thereof are respectively shown from left to right, the separation degree is more than 3.0 and much more than 1.5, baseline separation can be achieved, and the separation method can completely separate the L-alanine isopropyl ester hydrochloride from the enantiomer D-alanine isopropyl ester hydrochloride.

(2) Linearity and range of the separation method of the invention

Blank solvent: acetonitrile

Linear solution: the enantiomeric isomer D-alanine isopropyl ester hydrochloride control was used in appropriate amounts to prepare solutions of a series of concentrations, and the results of the linearity and range measurements of the D-alanine isopropyl ester hydrochloride control were shown in Table 2, according to the chromatographic method of example 1.

TABLE 2

The linear relationship of D-alanine isopropyl ester hydrochloride control is shown in figure 5.

And (4) conclusion: the D-alanine isopropyl ester hydrochloride reference substance has good linear relation in the concentration range of 0.09 mu g/ml to 0.6 mu g/ml. Coefficient of linear regression R²0.9982, meets the requirements of the scheme (R)²≥0.99)。

(3) Recovery test of the separation method of the present invention

The experimental steps are as follows: accurately weighing an appropriate amount of L-alanine isopropyl ester hydrochloride, placing the L-alanine isopropyl ester hydrochloride into a measuring flask, respectively adding an appropriate amount of enantiomer D-alanine isopropyl ester hydrochloride, dissolving acetonitrile, diluting to scale, preparing solutions with relative concentrations of 50%, 100% and 150%, and shaking uniformly to obtain a test solution. And precisely weighing a proper amount of L-alanine isopropyl ester hydrochloride, dissolving and diluting the L-alanine isopropyl ester hydrochloride to scales by using acetonitrile, shaking up the scales to obtain a blank sample solution, precisely weighing a proper amount of L-alanine isopropyl ester hydrochloride enantiomer D-alanine isopropyl ester hydrochloride, dissolving and diluting the enantiomer D-alanine isopropyl ester hydrochloride into a solution with a proper concentration by using the acetonitrile to obtain a reference solution. Each 10 μ l of the above solutions was precisely measured, and each solution was injected into a liquid chromatograph, followed by detection by the chromatography method in example 1, and a color development spectrum and a peak area were recorded to calculate a recovery rate. The results are shown in Table 3.

TABLE 3

And (4) conclusion: the recovery rate of L-alanine isopropyl ester hydrochloride enantiomer D-alanine isopropyl ester hydrochloride under each concentration accords with the regulation of pharmacopoeia, and the method has good accuracy.

(4) Stability analysis of derivatized products

Under the condition of room temperature, the test solution and the reference solution are placed for 24 hours, and detection is carried out at the time interval point of every 1 hour, the result shows that the RSD of the main peak of each solution is less than 2.0%, and impurities which increase and interfere the detection of the solutions are added in the test solution and the reference solution, which indicates that the obtained derivatization product has good stability within 24 hours.

The present invention has been described in detail with reference to the above examples using specific embodiments and experiments, but it will be apparent to those skilled in the art that modifications or improvements can be made thereto without departing from the spirit of the present invention. Accordingly, such modifications and improvements do not depart from the spirit of the invention and are intended to be included within the scope of the invention.