阅读说明：本技术 L-谷氨酸二乙酯盐酸盐及其光学异构体的分离检测方法 (Separation and detection method of L-glutamic acid diethyl ester hydrochloride and optical isomer thereof ) 是由 韩林 徐浩宇 董达文 蔡伟 徐瑛 宣景安 吴青青 李博 于向东 夏雨 王莲莲 于 2019-10-15 设计创作，主要内容包括：本发明公开了L-谷氨酸二乙酯盐酸盐及其光学异构体的分离检测方法,所述分离检测方法采用高效液相色谱法,固定相为硅胶类手性柱,流动相为反相溶剂：高氯酸水溶液-乙腈。本发明所述方法具有分离度好、灵敏度高、专属性强等特点,可以快速、准确地对L-谷氨酸二乙酯盐酸盐进行质量控制。(The invention discloses a separation and detection method of L-glutamic acid diethyl ester hydrochloride and optical isomers thereof, which adopts a high performance liquid chromatography, wherein a stationary phase is a silica gel chiral column, and a mobile phase is a reverse phase solvent: aqueous perchloric acid solution-acetonitrile. The method has the characteristics of good separation degree, high sensitivity, strong specificity and the like, and can quickly and accurately control the quality of the L-glutamic acid diethyl ester hydrochloride.)

The separation and detection method of L-glutamic acid diethyl ester hydrochloride and optical isomers thereof comprises the following steps:

1) preparing a test solution: dissolving L-glutamic acid diethyl ester hydrochloride in water and diluting to prepare a test solution of L-glutamic acid diethyl ester hydrochloride;

2) measuring by high performance liquid chromatography, precisely measuring L-glutamic acid diethyl ester hydrochloride test solution, injecting into a liquid chromatograph, and recording chromatogram;

wherein, the chromatographic conditions of the high performance liquid chromatography comprise:

mobile phase: perchloric acid aqueous solution and acetonitrile;

a chromatographic column: chiral crown ether bonded silica gel is used as a stationary phase.

2. The separation detection method according to claim 1, wherein the separation detection method is performed by using a probe

The chromatographic conditions further comprise:

detection wavelength: 200nm to 230nm, preferably 200nm to 210nm, more preferably 205 nm;

column temperature: 20 ℃ to 35 ℃, preferably 25 ℃;

flow rate of mobile phase: 0.4ml/min to 1.2ml/min, preferably 1.0 ml/min;

sample introduction amount: 10. mu.l to 30. mu.l, preferably 20. mu.l.

3. The separation detection method according to claim 1, wherein the volume ratio of the aqueous perchloric acid solution to acetonitrile in the mobile phase is 2:8 to 8:2, preferably (65:35) to (55: 45).

4. The separation detection method according to any one of claims 1 to 3, wherein the pH of the aqueous solution of perchloric acid in the mobile phase is 1.0 to 6.0, preferably 2.0 to 3.0.

5. The separation detection method according to any one of claims 1 to 3, wherein the concentration of L-glutamic acid diethyl ester hydrochloride in the test solution of L-glutamic acid diethyl ester hydrochloride is 3mg/ml to 7mg/ml, preferably 6 mg/ml.

6. The separation detection method according to any one of claims 1 to 3, further comprising formulating a control solution: precisely measuring 1ml of the L-glutamic acid diethyl ester hydrochloride test solution, placing the test solution in a 200ml measuring flask, diluting the test solution to a scale with water, and shaking up to be used as a control solution.

7. The separation detection method according to any one of claims 1 to 3, further comprising preparing an optical isomer stock solution: taking about 30mg of optical isomer of L-glutamic acid diethyl ester hydrochloride, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to scale, shaking up, and taking as optical isomer stock solution.

8. The separation detection method according to claim 7, further comprising preparing a resolution solution: taking about 60mg of L-glutamic acid diethyl ester hydrochloride, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of the optical isomer stock solution, adding water to dilute to scale, and shaking up to obtain the product.

9. The separation detection method according to claim 6, wherein the separation detection method comprises: and precisely measuring the control solution, injecting the control solution into a liquid chromatograph, and recording the chromatogram.

10. The separation detection method according to claim 7, wherein the separation detection method comprises: precisely measuring the optical isomer stock solution, injecting the optical isomer stock solution into a liquid chromatograph, and recording a chromatogram.

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a separation and detection method of L-glutamic acid diethyl ester hydrochloride and an optical isomer thereof.

Background

L-glutamic acid diethyl ester hydrochloride is an important pharmaceutical chemical raw material, can be used for producing raltitrexed, pemetrexed and other medicaments, and has the following chemical structural formula:

a certain amount of optical isomers exist in L-glutamic acid diethyl ester hydrochloride, namely D-glutamic acid diethyl ester hydrochloride, and the chemical structural formula of the D-glutamic acid diethyl ester hydrochloride is as follows:

the optical isomer can be remained in the medicine through subsequent reaction, and the quality of the medicine is influenced. Therefore, quality control of optical isomer impurities in L-glutamic acid diethyl ester hydrochloride is required. The control of the content of the optical isomer in the L-glutamic acid diethyl ester hydrochloride has great significance for improving the medicine quality and ensuring the medication safety of patients.

The separation and detection of amino acid micromolecule optical isomers are always the key and difficult points of quality control in the drug synthesis. A small amount of degradation impurity ethyl pyroglutamate is generated in the storage process of L-glutamic acid diethyl ester hydrochloride, and the structural formula is as follows:

the difficulty of separating and detecting the optical isomers is increased. Document CN104515815A discloses an analysis and detection method of diethyl L-glutamate, which is used for separating diethyl L-glutamate and impurities thereof. However, this method is a method for detecting a substance and is not sufficient for separating and detecting an optical isomer of diethyl L-glutamate. At present, no literature reports that optical isomers are detected by high performance liquid chromatography.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

The invention provides a sensitive, simple, convenient and rapid separation and detection method for L-glutamic acid diethyl ester hydrochloride and optical isomers thereof, which is based on reversed phase liquid chromatography, is mature, universal, simple and rapid, and has the characteristics of good separation degree, high sensitivity, strong specificity and the like.

The invention provides a separation and detection method of L-glutamic acid diethyl ester hydrochloride and an optical isomer thereof, which comprises the following steps:

1) preparing a test solution: dissolving L-glutamic acid diethyl ester hydrochloride in water and diluting to prepare a test solution of L-glutamic acid diethyl ester hydrochloride;

2) measuring by high performance liquid chromatography, precisely measuring L-glutamic acid diethyl ester hydrochloride test solution, injecting into a liquid chromatograph, and recording chromatogram;

wherein, the chromatographic conditions of the high performance liquid chromatography comprise:

mobile phase: perchloric acid aqueous solution and acetonitrile;

a chromatographic column: chiral crown ether bonded silica gel is used as a stationary phase.

In the separation detection method of the present invention, the chromatographic conditions further include:

detection wavelength: 200nm to 230nm, preferably 200nm to 210nm, more preferably 205 nm;

column temperature: 20 ℃ to 35 ℃, preferably 25 ℃;

flow rate of mobile phase: 0.4ml/min to 1.2ml/min, preferably 1.0 ml/min;

sample introduction amount: 10. mu.l to 30. mu.l, preferably 20. mu.l.

In the above separation detection method of the present invention, the volume ratio of the aqueous perchloric acid solution to acetonitrile in the mobile phase is 2:8 to 8:2, preferably (65:35) to (55: 45).

In the above separation detection method of the present invention, the pH of the aqueous solution of perchloric acid in the mobile phase is 1.0 to 6.0, preferably 2.0 to 3.0.

In the above separation and detection method of the present invention, the concentration of L-glutamic acid diethyl ester hydrochloride in the test solution of L-glutamic acid diethyl ester hydrochloride is 3mg/ml to 7mg/ml, preferably 6 mg/ml.

In the above separation detection method of the present invention, the separation detection method further comprises preparing a control solution: precisely measuring 1ml of the L-glutamic acid diethyl ester hydrochloride test solution, placing the test solution in a 200ml measuring flask, diluting the test solution to a scale with water, and shaking up to be used as a control solution.

In the above separation and detection method of the present invention, the separation and detection method further comprises preparing an optical isomer stock solution: taking about 30mg of optical isomer of L-glutamic acid diethyl ester hydrochloride, accurately weighing, placing in a 100ml measuring flask, adding water to dissolve and dilute to scale, shaking up, and taking as optical isomer stock solution.

In the above separation detection method of the present invention, the separation detection method further comprises preparing a separation degree solution: taking about 60mg of L-diethyl glutamate hydrochloride, precisely weighing, placing in a 10ml measuring flask, precisely weighing 1ml of the optical isomer stock solution, adding water to dilute to scale, and shaking up to obtain the final product.

In the above separation detection method of the present invention, the separation detection method includes: precisely measuring the reference solution and/or the optical isomer stock solution and/or the resolution solution, injecting into a liquid chromatograph, and recording the chromatogram.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.

FIG. 1 is a liquid chromatogram of the resolution solution of example 1;

FIG. 2 is a liquid chromatogram of the test solution of example 1;

FIG. 3 is a liquid chromatogram of the resolution solution of example 2;

FIG. 4 is a liquid chromatogram of the resolution solution of example 3;

FIG. 5 is a liquid chromatogram of the resolution solution of example 4;

FIG. 6 is a liquid chromatogram of the resolution solution of example 5;

FIG. 7 is a liquid chromatogram of the mixed solution after replacement of the column in the comparative example;

FIG. 8 is a liquid chromatogram of the mixed solution after the optimization process in the comparative example.

Detailed Description

Hereinafter, embodiments of the present invention will be described in detail in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be noted that the embodiments and features of the embodiments in the present application may be arbitrarily combined with each other without conflict.