CN109781894A - A kind of detection method of Li Feisite R isomers - Google Patents
A kind of detection method of Li Feisite R isomers Download PDFInfo
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- 238000004587 chromatography analysis Methods 0.000 claims abstract description 5
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- 239000012085 test solution Substances 0.000 claims description 32
- 239000007788 liquid Substances 0.000 claims description 23
- 239000013558 reference substance Substances 0.000 claims description 13
- 238000010790 dilution Methods 0.000 claims description 11
- 239000012895 dilution Substances 0.000 claims description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 230000014759 maintenance of location Effects 0.000 claims description 8
- SBTVLCPCSXMWIQ-UHFFFAOYSA-N (3,5-dimethylphenyl) carbamate Chemical compound CC1=CC(C)=CC(OC(N)=O)=C1 SBTVLCPCSXMWIQ-UHFFFAOYSA-N 0.000 claims description 4
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Abstract
The present invention provides a kind of detection methods of Li Feisite R isomers, it is detected using normal phase chromatography, and operating procedure is as follows: (1) configuring solution；(2) it detects；(3) chromatogram is analyzed.A kind of detection method of Li Feisite R isomers provided by the invention simply, can be separated quickly and accurately, detect Li Feisite and R isomer impurities by selecting specific chromatographic condition, to ensure that the quality controllable of Li Feisite and its preparation.Play the role of directing to the exploitation of synthesis technology, helps to work out the quality standard of bulk pharmaceutical chemicals.
Description
Technical field
The present invention relates to a kind of detection methods of Li Feisite R isomers, belong to pharmaceutical technology field.
Background technique
Li Feisite (lifitegrast) is researched and developed by Irish Xia Er development company (Shire Dev Llc), is novel
Small molecule integrin (integrin) depressant, energy antagonism lymphocyte function-associated antigen-1 (LFA-1) block homologous with it
The interaction interference of ligand intercellular adhesion molecule-1 (ICAM-1) causes the cornea of scheroma and the ICAM-1 of conjunctival tissue
.2015 April 9 is over-expressed to Food and Drug Adminstration of the US (FDA) proposition New Drug Application, and obtains preferential examination money
Lattice ratifies to list after the validity and safety data of supplement clinical test, in acquisition FDA on July 11st, 2016, is first
The drug for treating dry eye symptoms and sign.
The chemical structural formula of Li Feisite is as follows:
Li Feisite is chiral chemistry drug, and playing drug action is S type, and R type isomers is ground as impurity in pharmacy
Study carefully middle strict control.Since the R content of isomer in drug has stringent limit regulation in drug quality, especially to original
Expect for medicine, this purpose regulation is more stringent.So to the quantitative detection of Li Feisite R isomers not only to synthesis technology
Exploitation play directive function, the quality standards of bulk pharmaceutical chemicals is worked out even more essential.
Li Feisite R isomers chemical structural formula is as follows:
A kind of easy-operating method of inspection about Li Feisite R isomers need to be established, to advantageously promote the drug
Research and development, it is ensured that product quality.
Summary of the invention
In order to preferably instruct and promote the research and development to Li Feisite bulk pharmaceutical chemicals and the formulation of quality standard, this hair
Bright purpose is: a kind of analysis method for detecting Li Feisite R isomers is provided, effective for Li Feisite synthesis technology
Research and development and quality analysis.
The present invention provides a kind of detection methods of Li Feisite R isomers, it is detected using normal phase chromatography, behaviour
Steps are as follows for work:
(1) solution is configured
A, Li Feisite sample is weighed, adds flowing phased soln, as solution A；
B, Li Feisite R isomer control product are weighed, add flowing phased soln, as B solution；
C, A, B solution are mixed, adds flowing phase dilution, shake up, as system suitability solution；
D, solution A is taken, with phase dilution is flowed, as test solution；
E, B solution is taken, with phase dilution is flowed, as reference substance solution；
(2) it detects
Reference substance solution, system suitability solution, test solution injection liquid chromatograph are taken respectively, and chromatographic condition is such as
Under:
Chromatographic column: it is solid for being covalently bonded with amylose-three (3,5- xylyl carbamate) with Silica Surface
Determine phase；Mobile phase: n-hexane (A)-isopropanol (B)-ethyl alcohol (C)-trifluoroacetic acid (D), mobile phase ratio: A:B:C:D=60-
90:5-30:5-30:0-0.5；
(3) chromatogram is analyzed
Further, every 1ml sample containing the Li Feisite 1-3mg, preferably 2.5mg of solution A described in step a.
Further, every 1ml R containing Li Feisite the isomers 1-3mg, preferably 2.5mg of B solution described in step b.
Further, solution A, B solution and mobile phase ratio are 1:1:10 in system suitability solution described in step c.
Further, Li Feisite concentration is 0.2~1.0mg/ml in test solution described in step d, preferably
0.5mg/mL。
Further, Li Feisite R isomer concentration is 0.5mg/mL in reference substance solution described in step e.
Into one, the liquor capacity of step 2) the injection liquid chromatograph is 10 μ l；And/or mobile phase ratio A:B:
C:D=75:15:15:0.1；And/or a length of 200~260nm of chromatographic condition medium wave, column temperature are 20~40 DEG C, mobile phase
Flow velocity is 0.5~1.5ml/min.
Further, the wavelength is 220nm, and column temperature is 30 DEG C, flow rate of mobile phase 0.8ml/min.
Further, in the step 3) chromatogram, Li Feisite R isomery body colour is determined according to reference substance solution chromatogram
Spectral peak retention time determines Li Feisite main peak and Li Feisite R isomers chromatographic peak according to system suitability solution chromatogram
Separating degree；The content of Li Feisite R isomers in Li Feisite sample is calculated according to test solution chromatogram.
Further, Li Feisite main peak and Li Feisite R isomers chromatography in the system suitability solution chromatogram
The separating degree at peak is greater than 2.
Heretofore described " Li Feisite " refers to S type Li Feisite.
Heretofore described " Li Feisite R isomers " and " R isomers " all refer to R type Li Feisite.
A kind of detection method of Li Feisite R isomers provided by the invention can letter by selecting specific chromatographic condition
List quickly and accurately separates, detects Li Feisite and R isomer impurities, to ensure that the matter of Li Feisite and its preparation
Amount is controllable.Play the role of directing to the exploitation of synthesis technology, helps to work out the quality standard of bulk pharmaceutical chemicals.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Specific embodiment by the following examples is described in further detail above content of the invention again.
But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all real based on above content institute of the present invention
Existing technology all belongs to the scope of the present invention.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
The specific embodiment of form by the following examples remakes further specifically above content of the invention
It is bright.But the range that this should not be interpreted as to the above-mentioned theme of the present invention is only limitted to example below.It is all to be based on above content of the present invention
The technology realized all belongs to the scope of the present invention.
Detailed description of the invention
Fig. 1 is the HPLC map of 1 system suitability solution of embodiment
Fig. 2 is the HPLC map of 1 test solution of embodiment
Fig. 3 is the HPLC map of 2 system suitability solution of embodiment
Fig. 4 is the HPLC map of 2 test solution of embodiment
Specific embodiment
Raw material used in the specific embodiment of the invention is obtained by purchase commercial product.
1) material
Li Feisite R isomer impurities reference substance (Chengdu Wei Bang pharmaceutcal corporation, Ltd provides, lot number 20160819)
Li Feisite raw material (Chengdu Wei Bang pharmaceutcal corporation, Ltd provides, lot number 20170601,20170602)
2) key instrument
High performance liquid chromatograph LC-20AT Japan Shimadzu
CHIRALPAK IA 4.6 × 250mm of chromatographic column Japan Daicel
1 present invention measurement Li Feisite R content of isomer of embodiment
1, high performance liquid chromatography important technological parameters
A, high performance liquid chromatograph；
B, being covalently bonded with amylose-three (3,5- xylyl carbamate) with Silica Surface is stationary phase
Chiral chromatographic column；
C, mobile phase: n-hexane-isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1, flow velocity 0.8ml/min；
D, Detection wavelength be 220nm, 30 DEG C of column temperature.
2, measuring method
(1) solution is configured
A, precision weighs Li Feisite (lot number 20170601, Chengdu Wei Bang pharmaceutcal corporation, Ltd provide) 24.98mg and is placed in
In 10ml volumetric flask, scale is diluted to mobile phase, as solution A.
B, precision weighs Li Feisite R isomers 25.36mg and is placed in 10ml volumetric flask, is diluted to scale with mobile phase and makees
For B solution.
C, system suitability solution: respectively taking above-mentioned solution A 1ml and B solution 1ml to be placed in 10ml capacity, dilute with mobile phase
It releases to graduation mark, shakes up to get system suitability solution.
D, test solution: taking solution A 2ml in 10ml measuring bottle, is diluted to scale with mobile phase, molten as test sample
Liquid.
E, B solution is taken, to flow phase dilution, obtains reference substance solution, wherein the concentration of R isomers is in reference substance solution
0.5mg/mL。
(2) high performance liquid chromatography
A, 10 μ l of test solution and control solution is taken respectively, injects liquid chromatograph, records chromatogram, get profit non-department
The peak position out at special peak and its R isomers peak.
B, 10 μ l of this product system suitability solution is taken, liquid chromatograph is injected, records chromatogram, as shown in figure 1 and table 1.
The HPLC of 1 system suitability solution of table separates situation data
| Peak number | Retention time | Area | Area % | Separating degree |
| 1 | 13.33 | 2096410 | 49.663 | 0.00 |
| 2 | 16.233 | 2124821 | 50.337 | 3.07 |
Conclusion: the separating degree at the peak Li Feisite and its R isomers peak is 3.07, is met the requirements.
C, 10 μ l of test solution is taken, liquid chromatograph is injected, chromatogram is recorded, as shown in Fig. 2 and table 2.Test sample is molten
In the chromatogram of liquid, if any R isomers peak, content is calculated by area normalization method.
The HPLC spectrum data of 2 test solution of table
| Peak number | Retention time | Area | Area % |
| 1 | 16.343 | 4096212 | 100.000 |
(3) calculating of Li Feisite R content of isomer
Measurement result are as follows: in Li Feisite raw material (lot number 20170601), Li Feisite R content of isomer is to be not detected.
2 high effective liquid chromatography for measuring Li Feisite R content of isomer of embodiment
1, high performance liquid chromatography important technological parameters
A, high performance liquid chromatograph；
B, being covalently bonded with amylose-three (3,5- xylyl carbamate) with Silica Surface is stationary phase
Chiral chromatographic column；
C, mobile phase: n-hexane-isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1, flow velocity 0.8ml/min；
D, Detection wavelength be 220nm, 20 DEG C of column temperature.
2, measuring method
(1) solution is prepared
A, precision weighs Li Feisite (lot number 20170602, Chengdu Wei Bang pharmaceutcal corporation, Ltd provide) 24.18mg and is placed in
In 10ml volumetric flask, scale is diluted to mobile phase, as solution A.
B, precision weighs Li Feisite R isomers 25.41mg and is placed in 10ml volumetric flask, is diluted to scale with mobile phase and makees
For B solution.
C, system suitability solution: respectively taking above-mentioned solution A 1ml and B solution 1ml to be placed in 10ml capacity, dilute with mobile phase
It releases to graduation mark, shakes up to get system suitability solution.
D, test solution: taking solution A 2ml in 10ml measuring bottle, is diluted to scale with mobile phase, molten as test sample
Liquid.
E, B solution is taken, to flow phase dilution, obtains reference substance solution, wherein the concentration of R isomers is in reference substance solution
0.5mg/mL。
(2) high performance liquid chromatography
A, 10 μ l of C solution and solution D is taken respectively, injects liquid chromatograph, records chromatogram, and get profit the peak Fei Site and its R
Isomers peak goes out peak position.
B, 10 μ l of this product system suitability solution is taken, liquid chromatograph is injected, chromatogram is recorded, as shown in Fig. 3 and table 3.
The HPLC of 3 system suitability solution of table separates situation data
| Peak number | Retention time | Area | Area % | Separating degree |
| 1 | 13.322 | 2083610 | 49.275 | 0.00 |
| 2 | 16.289 | 2144923 | 50.725 | 3.22 |
Conclusion: the separating degree at the peak Li Feisite and R isomers peak is 3.22, is met the requirements.
C, contrast solution and 10 μ l of test solution are measured respectively, injects liquid chromatograph, records chromatogram, such as Fig. 4 and
Shown in table 4.In the chromatogram of test solution, if any R isomers peak, content is calculated by area normalization method.
The HPLC spectrum data of 4 test solution of table
| Peak number | Retention time | Area | Area % |
| 1 | 16.323 | 4078306 | 100.000 |
(3) calculating of Li Feisite R content of isomer
In Li Feisite raw material (lot number 20170602), Li Feisite R content of isomer is to be not detected.
Illustrate methodology validation result of the invention with the mode of experimental example below:
1 system suitability of experimental example is investigated
Solution is prepared: precision weighs Li Feisite raw material (lot number 20170602) and Li Feisite R isomer control product are suitable
It is former containing Li Feisite to be configured to every ml with mobile phase (n-hexane-isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1) for amount
Expect the system suitability solution of 0.2520mg, Li Feisite R isomers 0.2429mg, is test solution.
10 μ l of test solution is taken, liquid chromatograph is injected, sample introduction 6 times, investigates the opposite mark of retention time and peak area
Quasi- deviation, the results are shown in Table 5.
5 system suitability solution testing result of table summarizes
Conclusion: test solution continuous sample introduction 6 times, the RSD of peak area is not more than 2%, and the RSD of retention time is not more than
1.0%, meet methodology validation standard as defined in Chinese Pharmacopoeia version in 2015, shows that system suitability is good.
The experiment of 2 specificity of experimental example
Precision weighs 3 parts of the Li Feisite raw material (lot number 20170601) of 5.82mg, 5.46mg, 5.29mg, is separately added into
1M hydrochloric acid, 1M sodium hydroxide, each 2ml of 2% hydrogen peroxide are placed 6 hours, with stream together with oxidation sample after soda acid sample is neutralized
Dynamic phase (n-hexane-isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1) diluted concentration is supplied to 0.5mg/ml as A, B, C
Test sample solution；Another precision weighs Li Feisite raw material (lot number 20170601) 10.64mg, and it is small to be placed in 100 DEG C of high-temperature test chambers 6
When, with mobile phase diluted concentration to 0.5mg/ml, as D test solution.
Each 10 μ l of test solution A, B, C, D is taken, liquid chromatograph is injected, records chromatogram, calculates separating degree.As a result
Are as follows:
Separating degree between main peak and each adjacent peak is equal > 2.0 (standards of pharmacopoeia is not less than 1.5), separating degree is good.
The detection of experimental example 3 limit and quantitative limit
Detection limit and quantitative limit measurement result are shown in Table 6.
Precision weighs Li Feisite R isomers (lot number 20160819), with mobile phase (n-hexane-isopropanol-ethyl alcohol-three
Fluoroacetic acid=75:15:15:0.1) dissolution, it is configured to the mother liquor of 10 μ g/ml, is diluted step by step, detection limit is carried out and quantitative limit determines
Test.
The detection of table 6 limit and quantitative limit measurement result
Conclusion: being converted into absolute value, and detection is limited to 0.3ng, is quantitatively limited to 1ng, can satisfy measurement and requires.
4 linearity and range of experimental example
The preparation of solution: precision weighs 6 parts of Li Feisite R isomers (lot number 20160819), with mobile phase (n-hexane-
Isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1) dilution, being configured to concentration is respectively 0.9960 μ g/ml, 1.494 μ g/
1~No. 6 test solution of ml, 1.992 μ g/ml, 2.490 μ g/ml, 2.998 μ g/ml, 3.984 μ g/ml.
Precision measures each 10 μ l of 1-6 test solution, injects liquid chromatograph, each number solution sample introduction 3 times, investigates
The relative standard deviation of peak area, the results are shown in Table 7.
7 linearity and range measurement result of table
Conclusion: the range of linearity is 0.1028-5.1400 μ g/ml, meets Chinese Pharmacopoeia proof scheme and requires that (LOQ is not to low
In 150% index concentration), linear equation is Y=7427X-108 within this range, and linear regression coeffficient R2 is 0.9994, is met
It is required that (R2 > 0.9990)；
Y intercept is within the 25% of 100% concentration-response value (108 < 3999*0.25=999.75)；Response factor
RSD value is 5.40%, and meet the requirements (RSD≤10%)；Confirm good linear relationship.
5 Precision Experiment of experimental example
The preparation of solution: precision weighs 6 parts of Li Feisite R isomers (lot number 20160819), with mobile phase (n-hexane-
Isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1) dilution, being configured to concentration is respectively 0.5012 μ g/ml, 0.5007 μ g/
1~No. 6 test solution of ml, 0.4993 μ g/ml, 0.5021 μ g/ml, 0.5019 μ g/ml, 0.4996 μ g/ml.
Precision measures each 10 μ l of 1-6 test solution, injects liquid chromatograph, and the relative standard for investigating peak area is inclined
Difference the results are shown in Table 8.
8 precision measurement result of table
Conclusion: the RSD value of 6 parts of test liquid contents is 2.72%, and meet the requirements (RSD < 10%).
6 rate of recovery of experimental example
The preparation of solution: precision weighs 3 parts of Li Feisite R isomers (lot number 20160819), with mobile phase (n-hexane-
Isopropanol-ethyl alcohol-trifluoroacetic acid=75:15:15:0.1) dilution, being configured to concentration is respectively 1.225 μ g/ml, 2.4499 μ g/
1~No. 3 test solution of ml, 3.6749 μ g/ml, each test solution take 10 μ l, inject liquid chromatograph, every kind of solution
Sample introduction 3 times, the rate of recovery is calculated, the results are shown in Table 9.
9 determination of recovery rates result of table
Conclusion: the rate of recovery meets verifying and requires (80%-120%) between 97.3%-100.1%；The RSD of the rate of recovery
Value is 1.6%, meets verifying and requires (≤10%)；Confirm that this method has the good rate of recovery.
7 durability of experimental example
The influence that different column temperatures, mobile phase ratio, different batches chromatographic column measure Li Feisite isomers is investigated, specifically
Variable parameter is shown in Table 10.
Test solution is prepared: precision weighs Li Feisite R isomers (lot number 20160819) 5.18mg, is placed in 100ml
In volumetric flask, scale is diluted to mobile phase；Precision measures the 1ml solution and is placed in 100ml volumetric flask, is diluted to mobile phase
Scale is test solution.
Precision measures 10 μ l of test solution, injects liquid chromatograph, records peak area, calculates the rate of recovery.
10 chromatography variable parameter of table
Influence of 13 different condition of table to measurement
Conclusion: it through testing, is investigated using different column temperatures, peak area and the rate of recovery are almost the same.Column temperature as the result is shown
When changing within the scope of 30~40 DEG C, good tolerance.
It through testing, is investigated using different mobile phase ratios, peak area and the rate of recovery are almost the same.It flows as the result is shown
Phase Proportion is when ± 5% encloses interior variation, good tolerance.
The amount of impurity is almost unchanged in the case where chromatographic condition fine tuning, and rate of recovery 98.2%-105.6% meets
It is required that (80.0%-120.0%), it was demonstrated that this method durability is preferable.
To sum up, the present invention provides a kind of method of high effective liquid chromatography for measuring Li Feisite R content of isomer, classical prescriptions
Science of law verifying, as a result meets the requirements, and by selecting specific chromatographic condition, can simply, quickly and accurately separate, detect benefit
Fei Site and its R isomer impurities, to ensure that the quality controllability of Li Feisite bulk pharmaceutical chemicals and its preparation.To synthesis technology
Exploitation play directive function, help to work out the quality standards of bulk pharmaceutical chemicals.
Claims (10)
1. a kind of detection method of Li Feisite R isomers, it is characterised in that: it is detected using normal phase chromatography, operation
Steps are as follows:
(1) solution is configured
A, Li Feisite sample is weighed, adds flowing phased soln, as solution A；
B, Li Feisite R isomer control product are weighed, add flowing phased soln, as B solution；
C, A, B solution are mixed, adds flowing phase dilution, shake up, as system suitability solution；
D, solution A is taken, with phase dilution is flowed, as test solution；
E, B solution is taken, with phase dilution is flowed, as reference substance solution；
(2) it detects
Reference substance solution, system suitability solution, test solution injection liquid chromatograph are taken respectively, and chromatographic condition is as follows:
Chromatographic column: amylose-three (3,5- xylyl carbamate) is covalently bonded with as stationary phase with Silica Surface；
Mobile phase: n-hexane (A)-isopropanol (B)-ethyl alcohol (C)-trifluoroacetic acid (D), mobile phase ratio: A:B:C:D=60-90:5-
30:5-30:0-0.5；
(3) chromatogram is analyzed.
2. detection method according to claim 1, it is characterised in that: the every 1ml sample containing Li Feisite of solution A described in step a
Product 1-3mg, preferably 2.5mg.
3. detection method according to claim 1, it is characterised in that: the every 1ml R containing Li Feisite of B solution described in step b is different
Structure body 1-3mg, preferably 2.5mg.
4. detection method according to claim 1, it is characterised in that: solution A, B in system suitability solution described in step c
Solution and mobile phase ratio are 1:1:10.
5. detection method according to claim 1, it is characterised in that: Li Feisite sample in test solution described in step d
Product concentration is 0.2~1.0mg/ml, preferably 0.5mg/mL.
6. detection method according to claim 1, it is characterised in that: R isomer concentration in reference substance solution described in step e
For 0.5mg/mL.
7. detection method according to claim 1, it is characterised in that: the solution body of step 2) the injection liquid chromatograph
Product is 10 μ l；And/or mobile phase ratio A:B:C:D=75:15:15:0.1；And/or the chromatographic condition medium wave a length of 200
~260nm, column temperature are 20~40 DEG C, and flow rate of mobile phase is 0.5~1.5ml/min.
8. detection method according to claim 7, it is characterised in that: the wavelength is 220nm, and column temperature is 30 DEG C, flowing
Phase flow velocity is 0.8ml/min.
9. detection method according to claim 1, it is characterised in that: molten according to reference substance in the step 3) chromatogram
Liquid chromatography figure determines Li Feisite R isomers chromatographic peak retention time, determines Li Feisi according to system suitability solution chromatogram
The separating degree of special main peak and Li Feisite R isomers chromatographic peak；It is non-that benefit in Li Feisite is calculated according to test solution chromatogram
Take charge of the content of spy's R isomers.
10. detection method according to claim 9, it is characterised in that: benefit is non-in the system suitability solution chromatogram
The separating degree for taking charge of special main peak and Li Feisite R isomers chromatographic peak is greater than 2.
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Application publication date: 20190521 Assignee: Sichuan meiyugao Biomedical Technology Co.,Ltd. Assignor: CHENGDU WEIBANG PHARMACEUTICAL Co.,Ltd. Contract record no.: X2023980054097 Denomination of invention: A detection method for the isomer of Levofloxacin R Granted publication date: 20211214 License type: Common License Record date: 20231227 |