CN113125577A

CN113125577A - Method for separating and determining bilastine intermediate and isomer thereof by liquid chromatography

Info

Publication number: CN113125577A
Application number: CN201911413510.0A
Authority: CN
Inventors: 贾少华; 刘秋叶; 孙冬雪
Original assignee: Beijing Wanquan Dezhong Medical Biological Technology Co Ltd
Current assignee: Beijing Wanquan Dezhong Medical Biological Technology Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2021-07-16

Abstract

The invention belongs to the field of analytical chemistry, and discloses a method for separating and measuring a bilastine intermediate 2- [4- (chloroacetyl) -phenyl ] -2-methyl-propionic acid and isomers thereof by using a liquid chromatography. The method has the advantages of strong specificity, high accuracy and simple and convenient operation.

Description

Method for separating and determining bilastine intermediate and isomer thereof by liquid chromatography

Technical Field

The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining a bilastine intermediate and isomers thereof by liquid chromatography.

Background

Bilastine is a non-sedating, long-acting antihistamine that is clinically used to treat allergic rhinoconjunctivitis and urticaria. Bilastine selectively antagonizes the peripheral H1 receptor, with no affinity for muscarinic receptors and low affinity for other receptors. Bilastine has the properties of being non-sedating, non-cardiotoxic, non-interacting with cytochrome P450 enzyme substrates and potent antihistamines. Bilastine has a name of Bilastine and a chemical name of 2- [4- (2- (4- [1- (2-ethoxyethyl) -1H-benzimidazole-2-yl)]-piperidin-1-yl) ethyl) phenyl]-2-methylpropionic acid with the molecular formula C₂₈H₃₇N₃O₃. The chemical name of the bilastine intermediate is 2- [4- (chloracetyl) -phenyl]-2-methyl-propionic acid; the molecular formula isC₁₂H₁₃ClO₃. The structural formula of the bilastine intermediate is as follows:

in the process of synthesizing bilastine, the purity of some important intermediates needs to be controlled so as to reduce the occurrence of side reactions and the generation of impurities, thereby improving the yield and purity of the final product. The purity of the bilastine intermediate 2- [4- (chloroacetyl) -phenyl ] -2-methyl-propionic acid is controlled by controlling the content of the isomer 2- [3- (chloroacetyl) -phenyl ] -2-methyl-propionic acid so as to reduce the occurrence of side reactions and the generation of impurities; 2- [3- (chloroacetyl) -phenyl ] -2-methyl-propionic acid of the formula:

incomplete removal of impurities in the bilastine intermediate will cause side reactions and impurity generation, and finally affect the purity and quality of the drug. Therefore, the method for separating and measuring the bilastine intermediate and the isomers thereof has important practical significance in the aspects of bilastine production and quality control.

Disclosure of Invention

The invention aims to provide a method for analyzing a bilastine intermediate and isomers thereof, so that separation and determination of the bilastine intermediate and the isomers thereof are realized, the purity of the bilastine intermediate is ensured, and quality control in a bilastine process is realized.

The method for analyzing the chemical purity of the bilastine intermediate by using the liquid chromatography is a chromatographic column adopting phenyl silane bonded silica gel as a filler, and takes a buffer salt solution-organic phase in a certain proportion as a mobile phase.

The chromatographic column takes phenyl silane bonded silica gel as a filler, and is selected from Agilent, Kromasil, Alltima and other brands.

The organic phase is selected from one or more of the following compounds: methanol, acetonitrile, isopropanol, ethanol, etc., with methanol being preferred.

In the method, the mobile phase buffer salt solution-organic phase adopts isocratic elution.

Said buffer salt may be phosphoric acid, formate, acetate or phosphate, preferably phosphate.

The separation and measurement method of the present invention can be realized by the following method:

1) taking a proper amount of the bilastine intermediate and the isomer thereof, dissolving the sample with acetonitrile, and preparing a sample solution containing 0.1-1.5 mg of the bilastine intermediate and the isomer thereof per 1 mL.

2) Setting the flow rate of the mobile phase to be 0.5-1.5 mL/min, the detection wavelength to be 200-254 nm, and detecting at room temperature.

3) The mobile phase A is 10-50 mmol/L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.0-6.5 by phosphoric acid; the mobile phase B is methanol and is eluted at equal degrees.

4) And (2) injecting 10-50 mu L of the sample solution obtained in the step 1) into a liquid chromatograph to complete the separation and determination of the bilastine intermediate and the isomer thereof. Wherein:

the type of the high performance liquid chromatograph has no special requirements, and the chromatograph adopted by the invention is Shimadzu:

LC-20AT pump, SPD-M20A detector, SIL-20AC autosampler, CBM-20A controller, Lab-solutions workstation

A chromatographic column: phenyl (Alltima, 250X 4.6mm, 5 μm)

Mobile phase: 30mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 2.5 with phosphoric acid) methanol-70: 30;

flow rate: 1.0mL/min

Detection wavelength: 215nm

Sample introduction volume: 20 μ L

The invention adopts Phenyl (Altima, 250 multiplied by 4.6mm, 5 mu m) chromatographic column, and can effectively separate bilastine intermediate and isomer thereof. The invention solves the problems of separation and determination of the bilastine intermediate and the isomers thereof, thereby reducing the occurrence of side reactions, improving the yield and purity of the product and ensuring the quality of bilastine.

Drawings

FIG. 1 is an HPLC plot of the bilastine intermediate and its isomers at the time of example 1;

FIG. 2 is a HPLC chart of the bilastine intermediate for example 1;

FIG. 3 is a blank solvent HPLC plot for example 1;

FIG. 4 is an HPLC plot of the bilastine intermediate and its isomers for example 2;

FIG. 5 is a HPLC chart of the bilastine intermediate for example 2;

FIG. 6 is a blank solvent HPLC plot for example 2;

FIG. 7 is an HPLC plot of the bilastine intermediate and its isomers for example 3;

FIG. 8 is a blank solvent HPLC plot for example 3;

FIG. 9 is an HPLC plot of the bilastine intermediate and its isomers for example 4;

FIG. 10 is a HPLC chart of the bilastine intermediate for example 4;

FIG. 11 is a blank solvent HPLC plot for example 4;

the specific implementation mode is as follows:

the following examples are presented to further understand the present invention, but are not intended to limit the scope of the practice. The following examples are provided to further illustrate the methods for detecting bilastine intermediates and isomers thereof according to the present invention, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following examples, and that any techniques realized based on the above contents of the present invention are within the scope of the present invention.

Example 1

Apparatus and conditions

High performance liquid chromatograph: shimadzu: LC-20AT, CBM-20A, SIL-20AC, SPD-M20A;

a chromatographic column: phenyl (Alltima, 250X 4.6mm, 5 μm);

flow rate: 1.0mL/min

Detection wavelength: 215nm

Sample introduction volume: 20 μ L

Experimental procedure

Taking a proper amount of the bilastine intermediate and the isomer thereof, respectively dissolving the samples with acetonitrile, and preparing sample solutions containing approximately 0.5mg/mL of the bilastine intermediate and the isomer thereof. And preparing a proper amount of bilastine intermediate and isomer solution thereof into system applicability solution. And taking a proper amount of acetonitrile as a blank solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in attached figures 1-3, the chromatographic peak with the retention time of 19.048min in the figure 1 is a bilastine intermediate, and the chromatographic peak with the retention time of 21.472min is a bilastine intermediate isomer; the chromatographic peak with retention time of 19.019min in FIG. 2 is the bilastine intermediate, and the blank solvent chromatogram in FIG. 3.

Example 2

Apparatus and conditions

a chromatographic column: phenyl (Alltima, 250X 4.6mm, 5 μm);

mobile phase: 10mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 2.5 with phosphoric acid) methanol-70: 30;

flow rate: 1.0mL/min

Detection wavelength: 215nm

Sample introduction volume: 20 μ L

Experimental procedure

Taking a proper amount of the bilastine intermediate and the isomer thereof, respectively dissolving the samples with acetonitrile, and preparing sample solutions containing approximately 0.5mg/mL of the bilastine intermediate and the isomer thereof. And preparing a proper amount of bilastine intermediate and isomer solution thereof into system applicability solution. And taking a proper amount of acetonitrile as a blank solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in figures 4-6, the chromatographic peak with the retention time of 19.952min in figure 4 is a bilastine intermediate, and the chromatographic peak with the retention time of 22.512min is a bilastine intermediate isomer; the chromatographic peak with retention time of 19.156min in FIG. 5 is the bilastine intermediate; fig. 6 is a blank solvent chromatogram.

Example 3

Apparatus and conditions

a chromatographic column: phenyl (Alltima, 250X 4.6mm, 5 μm);

mobile phase: 30mmol/L potassium dihydrogen phosphate buffer (pH adjusted to 2.5 with phosphoric acid) acetonitrile-75: 25;

flow rate: 1.0mL/min

Detection wavelength: 215nm

Sample introduction volume: 20 μ L

Experimental procedure

Taking a proper amount of the bilastine intermediate and the isomer thereof, respectively dissolving the samples with acetonitrile, and preparing sample solutions containing approximately 0.5mg/mL of the bilastine intermediate and the isomer thereof. And preparing a proper amount of bilastine intermediate and isomer solution thereof into system applicability solution. And taking a proper amount of acetonitrile as a blank solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in the attached figures 7-8, the chromatographic peak with the retention time of 8.890min in the figure 7 is a bilastine intermediate, and the chromatographic peak with the retention time of 9.666min is a bilastine intermediate isomer; fig. 8 is a blank solvent chromatogram.

Example 4

Apparatus and conditions

a chromatographic column: phenyl (Alltima, 250X 4.6mm, 5 μm);

mobile phase: 50mmol/L sodium dihydrogen phosphate buffer (pH adjusted to 2.5 with phosphoric acid) methanol-70: 30;

flow rate: 1.0mL/min

Detection wavelength: 215nm

Sample introduction volume: 20 μ L

Experimental procedure

Taking a proper amount of the bilastine intermediate and the isomer thereof, respectively dissolving the samples with acetonitrile, and preparing sample solutions containing approximately 0.5mg/mL of the bilastine intermediate and the isomer thereof. And preparing a proper amount of bilastine intermediate and isomer solution thereof into system applicability solution. And taking a proper amount of acetonitrile as a blank solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in figures 9-11, the chromatographic peak with retention time of 22.717min in figure 9 is a bilastine intermediate, and the chromatographic peak with retention time of 25.805min is a bilastine intermediate isomer; the chromatographic peak with retention time of 22.442min in FIG. 10 is the bilastine intermediate; FIG. 11 is a blank solvent chromatogram.

The following items of the bilastine intermediate and isomer analysis method were verified:

system suitability test

The mixed solution of the bilastine intermediate and its isomer was used to analyze whether the chromatographic conditions were satisfactory or not, according to the chromatographic conditions determined in example 1. As can be seen from FIG. 1, under the condition, the separation degree between the bilastine intermediate and the isomer thereof meets the requirement.

Stability of solution

According to the chromatographic conditions determined in example 1, the mixed solution of the bilastine intermediate and the isomer thereof is injected for 0, 2, 4, 6, 8 and 12 hours respectively, and the stability of the solution is checked when the quantitative determination of the sample is carried out, so that the solution is stable within 12 hours.

Durability

To further verify the stability of the method, we fine-tune the conditions of flow rate, wavelength of detection, pH of buffer solution and column brand accordingly to investigate the robustness of the chromatographic conditions.

The results show that the flow rate variation is within the range of +/-0.5 ml/min, no obvious influence is caused on the separation of the sample, and only the retention time has corresponding forward shift and backward shift. The detection wavelength is 200-254 nm, and the separation of the sample is not affected. The pH value of the buffer solution is within the range of 2.0-6.5, and the separation of the sample is not influenced. After the chromatographic columns are changed into Agilent and Kromasil, the retention time of the sample has certain change, and the separation degree of the bilastine intermediate and the isomers thereof meets the requirement. The method has good durability.

Claims

1. A method for separating and measuring a bilastine intermediate 2- [4- (chloroacetyl) -phenyl ] -2-methyl-propionic acid and isomers thereof by liquid chromatography is characterized by comprising the following steps: the chromatographic column with phenyl bonded silica gel as stuffing has certain ratio of buffering salt solution and organic phase as the mobile phase.

2. The method for separating and measuring bilastine intermediate and isomers thereof as claimed in claim 1, wherein the chromatographic column is selected from the group consisting of Agilent, Kromasil and Alltima.

3. The method for separating and measuring bilastine intermediate and isomer thereof according to claim 1, wherein said organic phase is selected from one or more of the following compounds: methanol, acetonitrile, isopropanol, ethanol, and the like.

4. The method for the isolation and determination of bilastine intermediates and isomers thereof as claimed in claim 3, wherein said organic phase is preferably methanol.

5. The method for the isolation and determination of bilastine intermediates and isomers thereof as claimed in claim 1, wherein said buffer salt is selected from the group consisting of: phosphoric acid, formate, acetate or phosphate.

6. The method for the isolation and determination of bilastine intermediates and isomers thereof as claimed in claim 5, wherein said buffer salt is preferably phosphate.

7. The method for separating and measuring bilastine intermediate and isomers thereof as claimed in claim 1, wherein the pH of the buffered saline solution is 2.0-6.5.

8. The method for separating and determining bilastine intermediates and isomers thereof according to claim 1, comprising the following steps:

1) taking a proper amount of the bilastine intermediate and the isomer thereof, dissolving the sample with acetonitrile, and preparing a sample solution containing 0.1-1.5 mg of the bilastine intermediate and the isomer thereof per 1 mL;

2) setting the flow rate of the mobile phase to be 0.5-1.5 mL/min, and the detection wavelength to be 200-254 nm, and detecting at room temperature;

3) the mobile phase A is 10-50 mmol/L potassium dihydrogen phosphate solution, and the pH value is adjusted to 2.0-6.5 by phosphoric acid; the mobile phase B is methanol and is eluted by isocratic elution;

4) and (2) injecting 10-50 mu L of the sample solution obtained in the step 1) into a liquid chromatograph to complete the separation and determination of the bilastine intermediate and the isomer thereof.

9. The method for separating and measuring bilastine intermediate and isomers thereof according to claim 8, wherein: the mobile phase A is 30mmol/L potassium dihydrogen phosphate buffer solution, and the pH value is adjusted to 2.5 by phosphoric acid; the mobile phase B is methanol.

10. The method for separating and measuring bilastine intermediate and isomers thereof according to claim 8, wherein: the chromatographic column is phenyl, the flow rate is 1.0mL/min, and the detection wavelength is 215 nm.