CN109781894B

CN109781894B - Method for detecting lifuster R isomer

Info

Publication number: CN109781894B
Application number: CN201910138630.8A
Authority: CN
Inventors: 随裕敏; 李连明; 敬方梨
Original assignee: Chengdu Weibang Pharmaceutical Co ltd
Current assignee: Chengdu Weibang Pharmaceutical Co ltd
Priority date: 2019-02-25
Filing date: 2019-02-25
Publication date: 2021-12-14
Anticipated expiration: 2039-02-25
Also published as: CN109781894A

Abstract

The invention provides a method for detecting a lifaste R isomer, which adopts normal phase chromatography for detection and comprises the following operation steps: (1) preparing a solution; (2) detecting; (3) and (6) analyzing the chromatogram. According to the method for detecting the R isomer of the liffetilite, provided by the invention, the impurities of the liffetilite and the R isomer can be simply, quickly and accurately separated and detected by selecting a specific chromatographic condition, so that the quality control of the liffetilite and the preparation thereof is ensured. Plays a role in guiding the development of the synthesis process and is beneficial to the formulation of the quality standard of the bulk drugs.

Description

Method for detecting lifuster R isomer

Technical Field

The invention relates to a method for detecting a lifaste R isomer, and belongs to the technical field of medicines.

Background

Rifisett (lifitegrast) was developed by Ireland summer development company (Shire Dev Llc), is a novel small molecule integrin (integrin) inhibitor, antagonizes lymphocyte function-associated antigen-1 (LFA-1), blocks interaction with its cognate ligand intercellular adhesion molecule-1 (ICAM-1). Interferon causes ICAM-1 overexpression in corneal and conjunctival tissues of dry eye.2015.4.9 days provides a new drug application to the United states Food and Drug Administration (FDA) and qualifies for prior review.after supplementing the validity and safety data of clinical trials, FDA approval was obtained at 2016.7.11 days to market, which is the first drug to treat dry eye symptoms and signs.

The chemical structural formula of lifaste is shown as follows:

the liffetilide is a chiral chemical drug, has the drug effect of S type, and the R type isomer is strictly controlled in pharmaceutical research as an impurity. Because the content of the R isomer in the medicine has strict limit regulation in the quality of the medicine, the regulation of the item is more strict particularly for the raw medicine. Therefore, the quantitative detection of the R isomer of the liffetid plays a guiding role in the development of the synthesis process and is essential for the formulation of the quality standard of the raw material medicaments.

The chemical structural formula of the lifaste R isomer is shown as follows:

an easy-to-operate test method for the lifuster R isomer needs to be established so as to better promote the development of the drug and ensure the product quality.

Disclosure of Invention

In order to better guide and promote the research and development of the liffetiliast bulk drug and the establishment of quality standard, the invention aims to: provides an analysis method for detecting the R isomer of the liffetid, which is effectively used for research and development and quality analysis of the liffetid synthesis process.

The invention provides a method for detecting a lifaste R isomer, which adopts normal phase chromatography for detection and comprises the following operation steps:

(1) preparing solution

a. Weighing a Lifexist sample, and dissolving the Lifexist sample by adding a mobile phase to obtain a solution A;

b. weighing a reference substance of the R isomer of the Lifetid, adding a mobile phase for dissolving, and taking the reference substance as a solution B;

c. mixing A, B solutions, adding mobile phase for dilution, and shaking to obtain system applicability solution;

d. taking the solution A, and diluting the solution A by using a mobile phase to obtain a test solution;

e. taking the solution B, and diluting with a mobile phase to serve as a reference substance solution;

(2) detection of

Respectively injecting the reference substance solution, the system applicability solution and the test solution into a liquid chromatograph under the following chromatographic conditions:

a chromatographic column: amylose-tri (3, 5-xylyl carbamate) is covalently bonded on the surface of silica gel as a stationary phase; mobile phase: n-hexane (a) -isopropanol (B) -ethanol (C) -trifluoroacetic acid (D), mobile phase ratio: b, C, D is 60-90: 5-30: 5-30: 0 to 0.5;

(3) analytical chromatograms

Further, the solution A in the step a contains 1-3mg, preferably 2.5mg, of the liflstet-containing sample per 1 ml.

Further, the solution B in the step B contains 1-3mg, preferably 2.5mg of the R isomer of lifilter per 1 ml.

Further, the ratio of the solution A to the solution B to the mobile phase in the system applicability solution in the step c is 1: 1: 10.

further, the concentration of the liffetid in the test solution in the step d is 0.2-1.0 mg/mL, preferably 0.5 mg/mL.

Further, the concentration of the Lifetid R isomer in the control solution in the step e is 0.5 mg/mL.

Further, the volume of the solution injected into the liquid chromatograph in the step 2) is 10 μ l; and/or the mobile phase ratio of A to B to C to D is 75: 15: 15: 0.1; and/or the wavelength in the chromatographic condition is 200-260 nm, the column temperature is 20-40 ℃, and the flow velocity of the mobile phase is 0.5-1.5 ml/min.

Further, the wavelength was 220nm, the column temperature was 30 ℃ and the mobile phase flow rate was 0.8 ml/min.

Further, in the chromatogram in the step 3), determining the retention time of a chromatographic peak of the libertine R isomer according to a chromatogram of a reference solution, and determining the separation degree of a principal peak of the libertine and the chromatographic peak of the libertine R isomer according to a chromatogram of a system applicability solution; and calculating the content of the Lifetid R isomer in the Lifetid sample according to the chromatogram of the test solution.

Further, the separation degree of the main sharp-edged peak and the sharp-edged R isomer chromatographic peak in the system applicability solution chromatogram is more than 2.

The "liffetilit" in the invention refers to S-type liffetilit.

The R isomer of the Lifetilide and the R isomer of the Lifetilide are R-type Lifetilide.

According to the method for detecting the R isomer of the liffetilite, provided by the invention, the impurities of the liffetilite and the R isomer can be simply, quickly and accurately separated and detected by selecting a specific chromatographic condition, so that the quality control of the liffetilite and the preparation thereof is ensured. Plays a role in guiding the development of the synthesis process and is beneficial to the formulation of the quality standard of the bulk drugs.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is an HPLC chromatogram of a solution suitable for use in the system of example 1

FIG. 2 is an HPLC chromatogram of the test solution of example 1

FIG. 3 is an HPLC chromatogram of a system suitability solution of example 2

FIG. 4 is an HPLC chromatogram of the test solution of example 2

Detailed Description

The starting materials used in the embodiments of the present invention are obtained by purchasing commercially available products.

1) Material

Lifetid R isomer impurity control (provided by Chengdu-bang pharmaceutical Co., Ltd., lot number 20160819)

Lifetid raw material (provided by Chengdu-bang pharmaceutical Co., Ltd., lot number 20170601,20170602)

2) Main instrument

High performance liquid chromatograph LC-20AT Japan Shimadzu

CHIRALPAK IA chromatographic column 4.6X 250mm Japanese xylonite

Example 1 determination of the R isomer content of Lifetilide according to the invention

1. Main technical parameters of high performance liquid chromatography

a. A high performance liquid chromatograph;

b. a chiral chromatographic column taking amylose-tri (3, 5-xylyl carbamate) covalently bonded on the surface of silica gel as a stationary phase;

c. mobile phase n-hexane-isopropanol-ethanol-trifluoroacetic acid 75: 15: 15: 0.1, the flow rate is 0.8 ml/min;

d. the detection wavelength is 220nm, and the column temperature is 30 ℃.

2. Assay method

(1) Preparing solution

a. Rifilast (batch No. 20170601, available from David pharmaceutical Co., Ltd.) 24.98mg was weighed precisely into a 10ml volumetric flask and diluted to the scale with mobile phase as solution A.

b. Precisely weigh 25.36mg of the lixisenatide R isomer into a 10ml volumetric flask and dilute to the mark with the mobile phase as solution B.

c. System applicability solution: putting 1ml of the solution A and 1ml of the solution B into a volume of 10ml, diluting the solution A and the solution B to scale marks by using a mobile phase, and shaking up to obtain the system applicability solution.

d. Test solution: taking 2ml of the A solution, putting the A solution into a 10ml measuring flask, and diluting the A solution to a scale mark by using a mobile phase to obtain a test solution.

e. And taking the solution B, and diluting the solution B by using a mobile phase to obtain a reference substance solution, wherein the concentration of the R isomer in the reference substance solution is 0.5 mg/mL.

(2) High performance liquid chromatography

a. And respectively taking 10 mu l of the test solution and the reference solution, injecting into a liquid chromatograph, and recording the chromatogram to obtain the peak positions of the Lifeistel peak and the R isomer peak thereof.

b. The system applicability solution 10 μ l is taken, injected into liquid chromatograph, and chromatogram is recorded, as shown in figure 1 and table 1.

TABLE 1 HPLC SEPARATION DATA FOR SYSTEM-APPLICABILITY SOLUTIONS

Peak number	Retention time	Area of	Area%	Degree of separation
					1	13.33	2096410	49.663	0.00
2	16.233	2124821	50.337	3.07

And (4) conclusion: the separation degree of the Lifetid peak and the R isomer peak is 3.07, which meets the requirement.

c. A sample solution (10. mu.l) was taken and injected into a liquid chromatograph, and a chromatogram was recorded as shown in FIG. 2 and Table 2. If there is R isomer peak in the chromatogram of the test solution, the content is calculated according to the area normalization method.

TABLE 2 HPLC chromatogram data of test solutions

Peak number	Retention time	Area of	Area%
				1	16.343	4096212	100.000

(3) Calculation of the content of the R isomer of Lifetid

The measurement results are as follows: the Lifetid R isomer content in the Lifetid starting material (batch No. 20170601) was not detected.

Example 2 determination of the content of the R isomer of Lifetilide by high performance liquid chromatography

1. Main technical parameters of high performance liquid chromatography

a. A high performance liquid chromatograph;

d. the detection wavelength is 220nm, and the column temperature is 20 ℃.

2. Assay method

(1) Preparing solution

a. Rifilast (batch No. 20170602, available from David pharmaceutical Co., Ltd.) 24.18mg was precisely weighed into a 10ml volumetric flask and diluted to the mark with mobile phase as solution A.

b. Precisely weigh 25.41mg of the R isomer of Lifetid in a 10ml volumetric flask and dilute to the mark with the mobile phase as solution B.

(2) High performance liquid chromatography

a. And respectively taking 10 mu l of the solution C and the solution D, injecting the solution C and the solution D into a liquid chromatograph, and recording a chromatogram to obtain the peak positions of the Riofilter peak and the R isomer peak thereof.

b. The system applicability solution 10 μ l is taken and injected into liquid chromatograph, and chromatogram is recorded, as shown in figure 3 and table 3.

TABLE 3 HPLC SEPARATION DATA FOR SYSTEM-APPLICABILITY SOLUTIONS

Peak number	Retention time	Area of	Area%	Degree of separation
					1	13.322	2083610	49.275	0.00
2	16.289	2144923	50.725	3.22

And (4) conclusion: the separation degree of the Lifetid peak and the R isomer peak is 3.22, which meets the requirement.

c. The control solution and the sample solution were measured 10. mu.l each, injected into a liquid chromatograph, and chromatograms were recorded as shown in FIG. 4 and Table 4. If there is R isomer peak in the chromatogram of the test solution, the content is calculated according to the area normalization method.

TABLE 4 HPLC chromatogram data of test solutions

Peak number	Retention time	Area of	Area%
				1	16.323	4078306	100.000

(3) Calculation of the content of the R isomer of Lifetid

The Lifetid R isomer content in the Lifetid starting material (batch No. 20170602) was not detected.

The following methodological validation results of the invention are illustrated by way of experimental examples:

experimental example 1 System suitability examination

Solution preparation: a proper amount of a liflstet raw material (batch No. 20170602) and a liflstet R isomer reference substance are precisely weighed, and a systemic applicable solution containing 0.2520mg of the liflstet raw material and 0.2429mg of the liflstet R isomer per ml is prepared by using a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid: 75: 15: 15: 0.1) to serve as a test solution.

A sample solution of 10. mu.l was taken and injected into a liquid chromatograph for 6 times, and the retention time and the relative standard deviation of the peak area were examined, and the results are shown in Table 5.

TABLE 5 summary of System suitability solution test results

And (4) conclusion: the sample solution is continuously injected for 6 times, the RSD of the peak area is not more than 2%, the RSD of the retention time is not more than 1.0%, and the method accords with the methodology verification standard specified in Chinese pharmacopoeia 2015 edition, which shows that the system has good applicability.

Experimental example 2 Special Property experiment

Precisely weighing 3 parts of 5.82mg, 5.46mg and 5.29mg of liffetid raw material (batch number 20170601), respectively adding 2ml of 1M hydrochloric acid, 1M sodium hydroxide and 2% hydrogen peroxide, standing for 6 hours, neutralizing an acid-base sample, diluting the neutralized acid-base sample and an oxidized sample to 0.5mg/ml by using a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid: 75: 15: 15: 0.1) to obtain a A, B, C test solution; then, 10.64mg of the Lifept raw material (lot No. 20170601) was precisely weighed, and the sample was placed in a 100 ℃ high-temperature test chamber for 6 hours to dilute the sample to 0.5mg/ml with a mobile phase to obtain a D sample solution.

10. mu.l of each sample solution A, B, C, D was taken, injected into a liquid chromatograph, and the chromatogram was recorded and the degree of separation was calculated. The results were:

the degrees of separation between the main peak and each adjacent peak are all more than 2.0 (pharmacopeia standard is not less than 1.5), and the degree of separation is good.

Experimental example 3 detection and quantitation limits

The results of the detection limit and the quantification limit are shown in Table 6.

The lixisenatide R isomer (lot 20160819) was precisely weighed, dissolved in a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid 75: 15: 15: 0.1) to prepare a mother liquor of 10 μ g/ml, diluted step by step, and subjected to detection limit and quantitative limit determination tests.

TABLE 6 determination of detection limit and quantitation limit

And (4) conclusion: the absolute value is converted, the detection limit is 0.3ng, the quantification limit is 1ng, and the measurement requirement can be met.

Experimental example 4 linearity and Range

Preparing a solution: 6 parts of the Lifexite R isomer (batch No. 20160819) are precisely weighed, diluted by a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid 75: 15: 15: 0.1) and prepared into No. 1-6 test solution with the concentration of 0.9960 mu g/ml, 1.494 mu g/ml, 1.992 mu g/ml, 2.490 mu g/ml, 2.998 mu g/ml and 3.984 mu g/ml respectively.

Precisely measuring 10 μ l of each of No. 1-6 sample solutions, injecting into a liquid chromatograph, injecting each sample into each sample solution for 3 times, and examining the relative standard deviation of peak area, wherein the results are shown in Table 7.

TABLE 7 results of linearity and range measurements

And (4) conclusion: the linear range is 0.1028-5.1400 mug/ml, the requirement of Chinese pharmacopoeia verification scheme is met (LOQ is not lower than 150% index concentration), the linear equation is Y7427X-108 in the range, the linear regression coefficient R2 is 0.9994, and the requirement is met (R2 is more than 0.9990);

y-axis intercept is within 25% of 100% concentration response (108<3999 × 0.25 ═ 999.75); the RSD value of the response factor is 5.40 percent, which meets the requirement (the RSD is less than or equal to 10 percent); a good linear relationship was confirmed.

Experimental example 5 precision experiment

Preparing a solution: 6 parts of the Lifeiste R isomer (batch No. 20160819) were precisely weighed, diluted with a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid 75: 15: 15: 0.1) to prepare No. 1-6 test solutions having concentrations of 0.5012. mu.g/ml, 0.5007. mu.g/ml, 0.4993. mu.g/ml, 0.5021. mu.g/ml, 0.5019. mu.g/ml and 0.4996. mu.g/ml, respectively.

The sample solutions 1 to 6, 10. mu.l each, were precisely measured and injected into a liquid chromatograph, and the relative standard deviations of the peak areas were examined, and the results are shown in Table 8.

TABLE 8 results of measurement of precision

And (4) conclusion: the RSD value of 6 test solutions is 2.72 percent, which meets the requirement (RSD is less than 10 percent).

Experimental example 6 recovery rate

Preparing a solution: precisely weighing 3 parts of the Lifstetr isomer (batch No. 20160819), diluting the Lifstetr isomer with a mobile phase (n-hexane-isopropanol-ethanol-trifluoroacetic acid (75: 15: 15: 0.1), preparing No. 1 to No. 3 test sample solutions with the concentrations of 1.225 mu g/ml, 2.4499 mu g/ml and 3.6749 mu g/ml respectively, taking 10 mu l of each test sample solution, injecting the solution into a liquid chromatograph for 3 times, and calculating the recovery rate, wherein the results are shown in Table 9.

TABLE 9 results of recovery measurement

And (4) conclusion: the recovery rate is between 97.3 and 100.1 percent, and meets the verification requirement (80 to 120 percent); the RSD value of the recovery rate is 1.6 percent, and the RSD value meets the verification requirement (less than or equal to 10 percent); the process was confirmed to have good recovery.

Experimental example 7 durability

The influence of different column temperatures, mobile phase ratios and different batches of chromatographic columns on the measurement of the specific structure of the liffss is examined, and specific variable parameters are shown in a table 10.

Preparing a test solution: precisely weighing 5.18mg of the Lifetid R isomer (batch number 20160819), placing the Lifetid R isomer into a 100ml volumetric flask, and diluting the Lifetid R isomer to a scale mark by using a mobile phase; precisely measuring 1ml of the solution, placing the solution in a 100ml volumetric flask, and diluting the solution to a scale by using a mobile phase to obtain a test solution.

Precisely measuring 10 μ l of the test solution, injecting into a liquid chromatograph, recording peak area, and calculating recovery rate.

TABLE 10 chromatographic Change parameters

TABLE 11 Effect of different conditions on the assay

And (4) conclusion: through tests, different column temperatures are adopted for inspection, and the peak area and the recovery rate are basically consistent. The results show that the durability is good when the column temperature is changed within the range of 30-40 ℃.

Through tests, different flow phase ratios are adopted for inspection, and the peak area and the recovery rate are basically consistent. The results show that the durability is good when the mobile phase ratio varies within. + -. 5%.

The amount of impurities is almost unchanged under the condition of fine adjustment of chromatographic conditions, the recovery rate is 98.2-105.6%, and the method meets the requirement (80.0-120.0%), and the method is proved to have better durability.

In conclusion, the invention provides a method for determining the content of R isomer of liffetid by high performance liquid chromatography, the result meets the requirement through methodological verification, and the impurities of the liffetid and the R isomer thereof can be simply, rapidly and accurately separated and detected by selecting specific chromatographic conditions, thereby ensuring the quality controllability of the liffetid bulk drug and the preparation thereof. Plays a guiding role in the development of the synthesis process and is beneficial to the formulation of the quality standard of the bulk drugs.

Claims

1. A method for detecting a lifaste R isomer is characterized in that: the detection is carried out by adopting normal phase chromatography, and the operation steps are as follows:

(1) preparing solution

(2) detection of

a chromatographic column: amylose-tri (3, 5-xylyl carbamate) is covalently bonded on the surface of silica gel as a stationary phase; mobile phase: n-hexane (a) -isopropanol (B) -ethanol (C) -trifluoroacetic acid (D), mobile phase ratio: a, B, C, D75: 15: 15: 0.1;

(3) analyzing the chromatogram;

the volume of the solution injected into the liquid chromatograph in the step 2) is 10 mul; and/or the wavelength in the chromatographic condition is 200-260 nm, the column temperature is 20-40 ℃, and the flow velocity of the mobile phase is 0.5-1.5 ml/min;

and d, the concentration of the Lifexist sample in the test solution is 0.2-1.0 mg/ml.

2. The detection method according to claim 1, characterized in that: step a, 1-3mg of the solution A containing the liflstet sample per 1 ml.

3. The detection method according to claim 1, characterized in that: step a, 2.5mg of the solution A is added into each 1ml of the sample containing the liflstet.

4. The detection method according to claim 1, characterized in that: and B, 1-3mg of the Lifeiste R isomer is contained in every 1ml of the solution B.

5. The detection method according to claim 1, characterized in that: step B the solution B contains 2.5mg of the lixisenatide R isomer per 1 ml.

6. The detection method according to claim 1, characterized in that: c, in the system applicability solution, the proportion of the solution A to the solution B to the mobile phase is 1: 1: 10.

7. the detection method according to claim 1, characterized in that: and d, the concentration of the Lifexist sample in the test solution is 0.5 mg/mL.

8. The detection method according to claim 1, characterized in that: and e, the concentration of the R isomer in the reference substance solution in the step e is 0.5 mg/mL.

9. The detection method according to claim 1, characterized in that: the wavelength is 220nm, the column temperature is 30 ℃, and the flow rate of the mobile phase is 0.8 ml/min.

10. The detection method according to claim 1, characterized in that: in the chromatogram in the step 3), determining the retention time of a chromatographic peak of a lixisent R isomer according to a chromatogram of a reference solution, and determining the separation degree of a lixisent main peak and the lixisent R isomer chromatographic peak according to a system applicability solution chromatogram; and calculating the content of the Lifetid R isomer in the Lifetid according to the chromatogram of the test solution.

11. The detection method according to claim 10, characterized in that: the separation degree of a main sharp-pointed peak and a chromatographic peak of a sharp-pointed R isomer in a system applicability solution chromatogram is more than 2.