CN111060629B - Method for detecting related substances of lifusy - Google Patents
Method for detecting related substances of lifusy Download PDFInfo
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- CN111060629B CN111060629B CN201911415386.1A CN201911415386A CN111060629B CN 111060629 B CN111060629 B CN 111060629B CN 201911415386 A CN201911415386 A CN 201911415386A CN 111060629 B CN111060629 B CN 111060629B
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- 239000000126 substance Substances 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 29
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 102
- 239000012535 impurity Substances 0.000 claims abstract description 96
- 239000000243 solution Substances 0.000 claims abstract description 90
- 239000013558 reference substance Substances 0.000 claims abstract description 29
- 239000012085 test solution Substances 0.000 claims abstract description 24
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 14
- 239000007864 aqueous solution Substances 0.000 claims abstract description 13
- 239000012488 sample solution Substances 0.000 claims abstract description 10
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 8
- 238000012360 testing method Methods 0.000 claims abstract description 7
- 238000001514 detection method Methods 0.000 claims description 23
- 238000012937 correction Methods 0.000 claims description 19
- 239000000523 sample Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims description 11
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims description 11
- 230000014759 maintenance of location Effects 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 7
- 239000012088 reference solution Substances 0.000 claims description 7
- XXMFJKNOJSDQBM-UHFFFAOYSA-N 2,2,2-trifluoroacetic acid;hydrate Chemical compound [OH3+].[O-]C(=O)C(F)(F)F XXMFJKNOJSDQBM-UHFFFAOYSA-N 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 230000005526 G1 to G0 transition Effects 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000945 filler Substances 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims 2
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- 238000005259 measurement Methods 0.000 abstract description 2
- 239000003085 diluting agent Substances 0.000 description 10
- 239000011550 stock solution Substances 0.000 description 9
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- 239000003814 drug Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004811 liquid chromatography Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
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- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
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- 208000003556 Dry Eye Syndromes Diseases 0.000 description 2
- 206010013774 Dry eye Diseases 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- JFOZKMSJYSPYLN-QHCPKHFHSA-N lifitegrast Chemical compound CS(=O)(=O)C1=CC=CC(C[C@H](NC(=O)C=2C(=C3CCN(CC3=CC=2Cl)C(=O)C=2C=C3OC=CC3=CC=2)Cl)C(O)=O)=C1 JFOZKMSJYSPYLN-QHCPKHFHSA-N 0.000 description 2
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- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 description 1
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 208000032023 Signs and Symptoms Diseases 0.000 description 1
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- -1 benzofuran-6-carbonyl Chemical group 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 229960005381 lifitegrast Drugs 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
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- OZGNYLLQHRPOBR-DHZHZOJOSA-N naftifine Chemical compound C=1C=CC2=CC=CC=C2C=1CN(C)C\C=C\C1=CC=CC=C1 OZGNYLLQHRPOBR-DHZHZOJOSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention discloses a method for detecting related substances of lifluoshi, which comprises the following specific steps: a. taking lifuste, adding acetonitrile water solution for dissolving to obtain a test sample solution; b. taking a liflstet reference substance and an impurity reference substance, adding acetonitrile aqueous solution to dissolve, and obtaining a separation solution; c. and (3) detecting the separation degree solution, the test solution and the control solution by adopting a high performance liquid chromatography. The method disclosed by the invention can effectively separate the liflstetter from related substances, accurately measure the content of the related substances in the liflstetter, has the advantages of reliable measurement result, strong specificity, high sensitivity, high accuracy and good linearity, and can effectively control the product quality of the liflstetter.
Description
Technical Field
The invention relates to a method for detecting related substances of lifusy, belonging to the field of medicines.
Background
Rifetid (lifitegrast) is developed by Ireland summer development company (fire Dev Llc), is a novel small molecule integrin (integrin) inhibitor, can antagonize lymphocyte function-related antigen-1 (LFA-1), can block the interaction with the homologous ligand intercellular adhesion molecule-1 (ICAM-1), interferes with the ICAM-1 over-expression of cornea and conjunctiva tissues causing dry eye, and is the first medicament for treating dry eye symptoms and signs.
The chemical name of the liffetid is (S) -2- [2- (benzofuran-6-carbonyl) -5, 7-dichloro-1, 2,3, 4-tetrahydroisoquinoline-6-formamido]-3- (3-methanesulfonylphenyl) propionic acid of formula C29H24C12N2O7S, CAS number is 1025967-78-5, and the structural formula is as follows:
the chemical structure and the synthesis process of the liffetid are analyzed, the liffetid at least contains A, B, C, D, E and F6 related substances, the detection method of the 6 related substances is developed, qualitative and quantitative analysis is carried out, the quality of the liffetid can be strictly controlled, and the quality of the product is ensured to meet the requirements. The analysis of the related substances of the lifaste is as follows:
disclosure of Invention
In order to effectively control the quality of the liflstat, the invention provides a detection method for detecting liflstat specific related substances A, B, C, D, E and F by using a high performance liquid chromatography.
The invention provides a method for detecting related substances of lifusy, which adopts a high performance liquid chromatography for detection and comprises the following specific steps:
a. taking lifuste, adding acetonitrile water solution for dissolving to obtain a test sample solution;
b. taking a liflstet reference substance and a related substance reference substance, and dissolving the liflstet reference substance and the related substance reference substance by using acetonitrile aqueous solution to obtain a separation solution;
c. the following chromatographic conditions are adopted to respectively detect the test solution and the separation degree solution:
stationary phase: octadecylsilane chemically bonded silica is used as a filling agent;
mobile phase: using 0.01-1.0% trifluoroacetic acid water solution as a mobile phase A and acetonitrile as a mobile phase B;
the gradient elution procedure was: 0-20 min, 70-80% → 30-40% A, 20-25 min, 30-40% → 5-15% A, 25.01-40 min, 70-80% A;
detection wavelength: 200-250 nm;
the flow rate is 0.5-1.5 ml/min;
the column temperature is 20-40 ℃.
Further, the mass-to-volume ratio of the lifaste and the acetonitrile aqueous solution in the step a) is 0.3-1.0 mg: 1 ml.
Further, the volume ratio of acetonitrile to water in the acetonitrile water solution is 25: 75.
further, the related substances in the step B) are impurity A, impurity B, impurity C, impurity D, impurity E and impurity F; the separation degree solution contains 0.5-1.5 ug of each impurity and 0.3-1.0 mg of the liffetast per 1ml, preferably contains 1ug of each impurity and 0.5mg of the liffetast per 1 ml.
Further, the stationary phase is an Inertsil ODS-3 column; the specification of the chromatographic column is as follows: the inner diameter is 4.6 multiplied by 250mm, 5 um.
Further, in the step c), the detection wavelength is 220nm, the flow rate is 1.0ml/min, the column temperature is 30 ℃, and the sample injection amount is 10 ul.
Further, step c) the gradient elution procedure is: 0-20 min, 75% → 35% A, 20-25 min, 35% → 10% A, 25.01-40 min, 75% A.
Furthermore, a chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of a chromatographic peak of a reference substance in the chromatogram of the resolution solution, namely the test solution contains related substances.
The invention relates to a method for measuring the content of a lyfil specific relevant substance, which comprises the following specific operation steps:
1) adding acetonitrile aqueous solution into the sample solution obtained in the step a to dilute the sample solution by 100-1000 times to obtain a reference solution; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 25: 75;
2) detecting according to the method;
3) the content of the related substances is calculated according to the following formula:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the test solution, A: the area of the peak of the liffetid in the control solution, N is the dilution multiple of the control solution, fiCorrection factors for the substances of interest.
Further, the sample solution of step a is diluted 1000 times with an aqueous solution of acetonitrile.
Further, the relevant substance is impurity a, the correction factor thereof is 1.69, the relevant substance is impurity B, the correction factor thereof is 1.51, the relevant substance is impurity C, the correction factor thereof is 0.33, the relevant substance is impurity D, the correction factor thereof is 0.67, the relevant substance is impurity E, the correction factor thereof is 1.63, the relevant substance is impurity F, the correction factor thereof is 1.75.
The method disclosed by the invention can effectively separate the liflstetter from related substances, accurately measure the content of the related substances in the liflstetter, has the advantages of reliable measurement result, strong specificity, high sensitivity, high accuracy and good linearity, and can effectively control the product quality of the liflstetter.
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 HPLC chart of sample solution in example 1
FIG. 2 example 2 HPLC chart of test solution
FIG. 3 HPLC chart of sample solution in example 3
FIG. 4 is a liquid chromatogram of a blank solution
FIG. 5 is a liquid chromatogram of a resolution solution
FIG. 6 is a liquid chromatogram of a reference solution (b)
Detailed Description
1. Material
Lifeset (Chengdu Vital pharmaceutical Co., Ltd. batch No.: WB104501-180501, WB104501-180502, WB104501-180601)
Reference substance A (Chengdu-bang pharmaceutical Co., Ltd.: WBZ001C)
Reference substance B (Chengdu-bang pharmaceutical Co., Ltd.: WBZ002C)
Reference substance C (Chengdu brand drug industry Co., Ltd.: WBZ003C)
Reference substance D (Chengdu brand drug industry Co., Ltd.: WBZ004C)
Reference substance E (Chengdu-bang pharmaceutical Co., Ltd.: WBZ005C)
Reference substance F (Chengdu-bang pharmaceutical Co., Ltd.: WBZ006C)
2. Main instrument
A high performance liquid chromatograph: shimadzu of Japan
Example 1 determination of the content of impurities involved in Lifilbert
1) Respectively taking 5mg of reference substances of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F, dissolving the reference substances by using 50ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 25:75) and fixing the volume to obtain 100ug/ml solutions of the impurity ABCDEF respectively as impurity stock solutions;
2) weighing 25mg of a liffstet reference substance into a 50ml measuring flask, precisely adding 0.5ml of the impurity stock solution obtained in the step 1), adding a diluent (the volume ratio of acetonitrile to water is 25:75) to dilute to a constant volume to obtain a resolution solution, wherein the liffstet is 0.5mg/ml, and the impurities A, B, C, D, E and F are 1ug/ml respectively.
3) Lifeiste (batch number: WB104501-180501)25mg, adding 50ml acetonitrile water solution (acetonitrile-water volume ratio 25:75) to dissolve, preparing test solution with concentration of 0.5 mg/ml;
4) precisely transferring 1ml of the test solution obtained in the step 3) into a 100ml measuring flask, and adding a volume ratio of 25: the volume of 75 acetonitrile in water is determined, 1ml of the solution is precisely transferred into a 10ml measuring flask, and the volume ratio is increased to 25:75, keeping the volume constant of the acetonitrile water solution to serve as a control solution;
5) the following chromatographic conditions are adopted to respectively detect the test solution, the separation degree solution and the control solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 μm;
mobile phase: using 0.05% trifluoroacetic acid water solution as a mobile phase A and acetonitrile solution as a mobile phase B; detection wavelength: 220 nm; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the sample amount is 10 ul;
the gradient elution procedure was:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.0 | 25 | 75 |
| 20 | 65 | 35 |
| 25 | 90 | 10 |
| 25.01 | 25 | 75 |
| 40 | 25 | 75 |
6) The chromatogram of the test solution (fig. 1) shows chromatographic peaks corresponding to the retention time of chromatographic peaks of impurities C, E and F in the resolution solution, i.e. batch nos. WB104501-180501, liffsite contains related substances: the contents of the impurities C, E and F are calculated by the following formulas:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the control solution, a: the area of the peak of the Lifeiste in the control solution, N is the dilution multiple of the control solution, fiCorrection factor (f) of the substance concernedC＝0.33，fE＝1.63，fF＝1.75)
The specific detection results of related substances of the Lifetid sample batch No. WB104501-180501 are as follows:
example 2 detection of the content of related impurities in Lifetilide
1) Respectively taking 5mg of reference substances of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F, dissolving the reference substances by using 50ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 25:75) and fixing the volume to obtain 100ug/ml solutions of the impurity ABCDEF respectively as impurity stock solutions;
2) weighing 25mg of a liffstet reference substance into a 50ml measuring flask, precisely adding 0.5ml of the impurity stock solution obtained in the step 1), adding a diluent (the volume ratio of acetonitrile to water is 25:75) to dilute to a constant volume to obtain a resolution solution, wherein the liffstet is 0.5mg/ml, and the impurities A, B, C, D, E and F are 1ug/ml respectively.
3) Lifeiste (batch number: WB104501-180502)25mg, adding 50ml acetonitrile water solution (acetonitrile-water volume ratio 25:75) to dissolve, preparing test solution with concentration of 0.5 mg/ml;
4) precisely transferring 1ml of the test solution obtained in the step 3) into a 100ml measuring flask, and adding a volume ratio of 25: the volume of 75 acetonitrile in water is determined, 1ml of the solution is precisely transferred into a 10ml measuring flask, and the volume ratio is increased to 25:75, keeping the volume constant of the acetonitrile water solution to serve as a control solution;
5) the following chromatographic conditions are adopted to respectively detect the test solution, the separation degree solution and the control solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 μm;
mobile phase: using 0.05% trifluoroacetic acid water solution as a mobile phase A and acetonitrile solution as a mobile phase B; detection wavelength: 220 nm; the flow rate is 1.0 ml/min; the column temperature is 30 ℃; the sample amount is 10 ul;
the gradient elution procedure was:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.0 | 23 | 77 |
| 20 | 65 | 35 |
| 25 | 90 | 10 |
| 25.01 | 23 | 77 |
| 40 | 23 | 77 |
6) The chromatogram of the test solution (fig. 2) shows chromatographic peaks corresponding to the retention times of chromatographic peaks of impurities C, E and F in the resolution solution, i.e. batch nos. WB104501-180502, liffsite contains related substances: the contents of the impurities C, E and F are calculated by the following formulas:
wherein XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the control solution, a: the area of the peak of the Lifeiste in the control solution, N is the dilution multiple of the control solution, fiCorrection factor (f) for the substance concernedC＝0.33，fE＝1.63，fF＝1.75)
The specific detection results of related substances of the Lifetid sample batch No. WB104501-180502 are as follows:
example 3 detection of the content of impurities involved in Lifilbert
1) Respectively taking 5mg of reference substances of the impurity A, the impurity B, the impurity C, the impurity D, the impurity E and the impurity F, dissolving the reference substances by using 50ml of acetonitrile aqueous solution (the volume ratio of acetonitrile to water is 25:75) and fixing the volume to obtain 100ug/ml solutions of the impurity ABCDEF respectively as impurity stock solutions;
2) weighing 25mg of a liffstet reference substance into a 50ml measuring flask, precisely adding 0.5ml of the impurity stock solution obtained in the step 1), adding a diluent (the volume ratio of acetonitrile to water is 25:75) to dilute to a constant volume to obtain a resolution solution, wherein the liffstet is 0.5mg/ml, and the impurities A, B, C, D, E and F are 1ug/ml respectively.
3) Lifeiste (batch number: WB104501-180601)25mg, adding 50ml acetonitrile water solution (acetonitrile-water volume ratio 25:75) to dissolve, preparing test solution with concentration of 0.5 mg/ml;
4) precisely transferring 1ml of the test solution obtained in the step 3) into a 100ml measuring flask, and adding a volume ratio of 25: the volume of 75 acetonitrile in water is determined, 1ml of the solution is precisely transferred into a 10ml measuring flask, and the volume ratio is increased to 25:75, keeping the volume constant of the acetonitrile water solution to serve as a control solution;
5) the following chromatographic conditions are adopted to respectively detect the test solution, the separation degree solution and the control solution:
a chromatographic column: inertsil ODS-34.6X 250mm, 5 μm;
mobile phase: using 0.05% trifluoroacetic acid water solution as a mobile phase A and acetonitrile solution as a mobile phase B; detection wavelength: 220 nm; the flow rate is 0.9 ml/min; the column temperature is 30 ℃; the sample amount is 10 ul;
the gradient elution procedure was:
6) the chromatogram of the test solution (fig. 3) shows chromatographic peaks corresponding to the retention times of chromatographic peaks of impurities C, E and F in the resolution solution, i.e. batch nos. WB104501-180601, liffsite contains related substances: the contents of the impurities C, E and F are calculated by the following formulas:
wherein, XiPercent: the content of the relevant substance in the sample, Ai: peak area of the relevant substance in the control solution, a: the area of the peak of the Lifeiste in the control solution, N is the dilution multiple of the control solution, fiCorrection factor (f) of the substance concernedC＝0.33，fE＝1.63，fF＝1.75)
The specific detection results of related substances of the Lifetid sample batch No. WB104501-180601 are as follows:
the beneficial effects of the invention are further illustrated by the following experimental examples:
experimental example 1 System applicability
The instrument comprises the following steps: shimadzu LC-20AT
A chromatographic column: inertsil ODS-34.6X 250mm, 5um, flow rate: 1.0ml/min, column temperature: 30 ℃, sample size 10ul, detector wavelength: 220nm, mobile phase A: 0.05% aqueous trifluoroacetic acid mobile phase B: acetonitrile, gradient elution conditions:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0.0 | 25 | 75 |
| 20 | 65 | 35 |
| 25 | 90 | 10 |
| 25.01 | 25 | 75 |
| 40 | 25 | 75 |
(1) Blank solution: acetonitrile: water 25:75(v/v)
(2) Reference solution (a): precisely weighing 25mg of the liffstet reference substance in a 50ml measuring flask, adding the diluent solution to fix the volume (the concentration of the liffstet is 0.5mg/ml)
(3) And (b) precisely transferring 1ml of the reference solution (a) into a 100ml measuring flask, adding a diluent to the solution to fix the volume, precisely transferring 1ml of the solution into a 10ml measuring flask, and adding the diluent to fix the volume. (Lifesotent concentration: 0.5ug/ml)
(4) Impurity stock solution: precisely weighing 10mg of each of the impurities A, B, C, D, E and F in a 100ml measuring flask, diluting the solution to a constant volume (100 ug/ml of the impurity A, 100ug/ml of the impurity B, 100ug/ml of the impurity C, 100ug/ml of the impurity D, 100ug/ml of the impurity E and 100ug/ml of the impurity F). (5) Resolution solution: precisely weighing 25mg of the liffstet reference substance in a 50ml measuring flask, precisely adding 0.5ml of impurity stock solution, and adding a diluent for diluting to a constant volume. (Lifestat 0.5mg/ml, impurities A, B, C, D, E, F each 1ug/ml)
(6) Impurity localization solution: weighing 5mg of impurities A, B, C, D, E and F respectively, placing in a 50ml measuring flask, adding the diluent solution to a constant volume, weighing 1ml again, placing in a 10ml measuring flask, and adding the diluent to the constant volume. (impurity A: 10ug/ml, impurity B: 10ug/ml, impurity C: 10ug/ml, impurity D: 10ug/ml, impurity E: 10ug/ml, impurity F: 10ug/ml, etc.)
(7) Test solution: precisely weighing 25mg of the liffstet sample into a 50ml measuring flask, and adding the diluent solution to a constant volume.
(8) According to the chromatographic conditions, 1 needle of blank solution, 6 needles of reference solution (b), 1 needle of resolution solution and 1 needle of test solution are respectively injected, and the chromatographic process is recorded, and the results are shown in figures 1-3 and table 1. The relative retention time is calculated as the retention time of the impurity divided by the retention time of the principal component, lifaste.
TABLE 1 System applicability
(9) As a result: the reference solution (b)6 needles were injected repeatedly, the retention time RSD% of lifestet was 0.06%, and the peak area RSD% was 0.59% (standard:. ltoreq.2.0%). The minimum separation degree between the liffstet and each impurity in the separation degree solution is 6.117 (the separation degree is more than or equal to 2.0), and the system applicability requirement is met.
Experimental example 2 specificity
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
The method specifically inspects peak identification and selectivity, samples blank solution and impurity positioning solution respectively, records chromatogram, and the result is shown in Table 2
TABLE 2 specialization
| Name of solution | Name of substance | Concentration of solution | Retention time |
| Specific site solution of liflox | Lifetid | 0.5084mg/ml | 19.338 |
| Positioning solution for impurity A | Impurity A | 10.12ug/ml | 2.645 |
| Positioning solution for impurity B | Impurity B | 10.23ug/ml | 4.441 |
| Positioning solution for impurity C | Impurity C | 10.14ug/ml | 15.371 |
| Impurity D positioning solution | Impurity D | 10.31ug/ml | 22.672 |
| Impurity E localization solution | Impurity E | 10.19ug/ml | 25.368 |
| Impurity F positioning solution | Impurity F | 10.28ug/ml | 26.874 |
As a result: no interference peak with the same retention time as the impurities A, B, C, D, E, F and the liffetid appears in the blank solution, the main peak and the impurities are well separated, and the impurities are well separated.
Experimental example 3 detection and quantitation limits
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
For known impurities, the limit of detection (LOD) and limit of quantitation (LOQ) are determined from the signal-to-noise ratio. Diluting an impurity stock solution with known concentration to low concentration, injecting the sample, determining that the detection signal-to-noise ratio S/N is more than or equal to 10 as a quantification limit, and determining that S/N is 2-4 as a detection limit. For unknown impurities, the detection limit and the quantification limit of a single unknown impurity are examined by using liberty instead. The test results are shown in Table 3.
TABLE 3 quantitation and detection limits
Experimental example 4 linearity and Range
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
For known impurities, 6 points are investigated from the range of LOQ concentration to the index concentration of not less than 150%, the linear relationship is plotted as a function of the measured response signal (peak area) against the analyte concentration, linear regression is carried out by the least squares method, and at least the correlation coefficient R is reported2To confirm a good linear relationship, the linear regression coefficient R is required2Should not be less than 0.990.
For unknown impurities, the linearity and range of the unknown impurities were replaced with liffetid, and the results are shown in Table 4
TABLE 4 Linearity and Range
Correction factors of related substances: respectively measuring the linearity of the Lifstet and the impurity ABCDEF to obtain a linear equation, and then comparing the slope of the Lifstet linear equation with the slope of the linear equation of each impurity to obtain a correction factor of the related substance, wherein the specific results are as follows:
EXAMPLE 5 precision
Experimental conditions, liquid chromatography method and solution preparation As in Experimental example 1
Taking the separation degree solution as a test solution, preparing 6 parts in parallel, sequentially injecting samples, and obtaining the repeatability results shown in Table 5
TABLE 5 repeatability
Intermediate precision was carried out by the same procedure, by different personnel on different instruments, and the results are shown in Table 6.
TABLE 6 intermediate precision
Experimental example 6 accuracy
The accuracy is obtained by adding three impurities with different concentrations of 80%, 100% and 120% of the index into a test sample and measuring the recovery rate of the impurities. The accuracy of the known impurities is determined by adding a known amount of the impurity and measuring the ratio between the amount of the known impurity in the sample to be added and the theoretical value (recovery), expressed as a percentage%. The unknown impurities are replaced by utilizing naftifine. The results are shown in Table 7
TABLE 7 accuracy
The research results of the experimental examples 1-6 show that the method for determining the specific relevant substances of the lifofilt by the high performance liquid chromatography has good specificity, good precision and high accuracy and sensitivity, and is suitable for quality control of the lifofilt synthesis.
Claims (9)
1. A method for detecting a lifusoff substance, which is characterized in that: the detection is carried out by adopting a high performance liquid chromatography, and the method comprises the following specific steps:
a. taking lifuste, adding acetonitrile water solution for dissolving to obtain a test sample solution;
b. taking a liflstet reference substance and a related substance reference substance, and dissolving the liflstet reference substance and the related substance reference substance by using acetonitrile aqueous solution to obtain a separation solution;
c. the following chromatographic conditions are adopted to respectively detect the test solution and the separation degree solution:
stationary phase: octadecylsilane chemically bonded silica is used as a filling agent, and the specification is as follows: inner diameter 4.6 × 250mm, 5 μm;
mobile phase: using 0.01-1.0% trifluoroacetic acid water solution as a mobile phase A and acetonitrile as a mobile phase B;
the gradient elution procedure was: 0-20 min, 75% → 35% A, 20-25 min, 35% → 10% A, 25.01-40 min, 75% A;
detection wavelength: 220 nm;
the flow rate is 1.0 ml/min;
the column temperature is 30 ℃;
the sample amount is 10 mul;
the related substances are impurity A, impurity B, impurity C, impurity D, impurity E and impurity F;
the structural formula of the related substances is as follows:
2. the detection method according to claim 1, characterized in that: the mass-to-volume ratio of the lifastol and the acetonitrile water solution in the step a) is 0.3-1.0 mg: 1 ml.
3. The detection method according to claim 1 or 2, characterized in that: the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 25: 75.
4. the detection method according to claim 1, characterized in that: and c) each 1ml of the resolution solution in the step b) contains 0.5-1.5 ug of each impurity and 0.3-1.0 mg of liffetid.
5. The detection method according to claim 1, characterized in that: the stationary phase is an Inertsil ODS-3 column.
6. The detection method according to claim 1, wherein: and a chromatographic peak corresponding to the retention time of the chromatographic peak of the reference substance in the chromatogram of the resolution solution is presented in the chromatogram of the test solution, namely the test solution contains related substances.
7. A method for measuring the content of a lifluoshi related substance is characterized in that: the specific operation steps are as follows:
1) adding acetonitrile aqueous solution into a sample solution to dilute the sample solution by 100-1000 times to obtain a reference solution; the volume ratio of acetonitrile to water in the acetonitrile aqueous solution is 25: 75;
2) detected according to the method of any one of claims 1 to 6;
3) the content of the related substances is calculated according to the following formula:
wherein the content of the first and second substances,
: the content of the relevant substances in the test sample,
: peak area of the relevant substance in the test sample, A: the area of the peak of the Lifeiste in the control solution, N, the dilution multiple of the control solution, and i, the correction factor of the related substance.
8. The content measurement method according to claim 7, characterized in that: the acetonitrile in water was diluted 1000 times.
9. The content measurement method according to claim 8, characterized in that: the relevant substance is impurity A, the correction factor of which is 1.69, the relevant substance is impurity B, the correction factor of which is 1.51, the relevant substance is impurity C, the correction factor of which is 0.33, the relevant substance is impurity D, the correction factor of which is 0.67, the relevant substance is impurity E, the correction factor of which is 1.63, and the relevant substance is impurity F, the correction factor of which is 1.75.
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| CN107857728A (en) * | 2017-11-20 | 2018-03-30 | 成都惟邦药业有限公司 | A kind of synthetic method of Li Feisite intermediates |
| WO2019053607A1 (en) * | 2017-09-18 | 2019-03-21 | Glenmark Pharmaceuticals Limited | Process for preparation of lifitegrast |
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| WO2019097547A1 (en) * | 2017-11-15 | 2019-05-23 | Cipla Limited | An improved process for the preparation of lifitegrast or salts thereof |
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| WO2019053607A1 (en) * | 2017-09-18 | 2019-03-21 | Glenmark Pharmaceuticals Limited | Process for preparation of lifitegrast |
| WO2019097547A1 (en) * | 2017-11-15 | 2019-05-23 | Cipla Limited | An improved process for the preparation of lifitegrast or salts thereof |
| CN107857728A (en) * | 2017-11-20 | 2018-03-30 | 成都惟邦药业有限公司 | A kind of synthetic method of Li Feisite intermediates |
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Application publication date: 20200424 Assignee: Sichuan meiyugao Biomedical Technology Co.,Ltd. Assignor: CHENGDU WEIBANG PHARMACEUTICAL Co.,Ltd. Contract record no.: X2023980054097 Denomination of invention: A Detection Method for Levofloxacin Related Substances Granted publication date: 20220705 License type: Common License Record date: 20231227 |