CN115097035B - LLTS intermediate and detection method and application of related impurities thereof - Google Patents
LLTS intermediate and detection method and application of related impurities thereof Download PDFInfo
- Publication number
- CN115097035B CN115097035B CN202210706463.4A CN202210706463A CN115097035B CN 115097035 B CN115097035 B CN 115097035B CN 202210706463 A CN202210706463 A CN 202210706463A CN 115097035 B CN115097035 B CN 115097035B
- Authority
- CN
- China
- Prior art keywords
- llts
- solution
- solvent
- intermediates
- performance liquid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000012535 impurity Substances 0.000 title claims abstract description 139
- 238000001514 detection method Methods 0.000 title claims abstract description 121
- 239000000543 intermediate Substances 0.000 claims abstract description 91
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 64
- 238000000034 method Methods 0.000 claims abstract description 46
- 239000012488 sample solution Substances 0.000 claims abstract description 44
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 30
- 238000010828 elution Methods 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000003908 quality control method Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 215
- 239000002904 solvent Substances 0.000 claims description 130
- YWLXLRUDGLRYDR-UHFFFAOYSA-N 10-deacetylbaccatin Chemical compound CC(=O)OC12COC1CC(O)C(C(C(O)C1=C(C)C(O)CC3(O)C1(C)C)=O)(C)C2C3OC(=O)C1=CC=CC=C1 YWLXLRUDGLRYDR-UHFFFAOYSA-N 0.000 claims description 35
- 239000012085 test solution Substances 0.000 claims description 27
- 239000013558 reference substance Substances 0.000 claims description 26
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 12
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 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
- 238000004458 analytical method Methods 0.000 abstract description 16
- 239000003814 drug Substances 0.000 abstract description 15
- 229940079593 drug Drugs 0.000 abstract description 14
- 239000000126 substance Substances 0.000 abstract description 14
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 11
- 239000004480 active ingredient Substances 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 97
- 238000012360 testing method Methods 0.000 description 85
- 238000005303 weighing Methods 0.000 description 48
- 238000007865 diluting Methods 0.000 description 40
- 239000011550 stock solution Substances 0.000 description 40
- 239000012490 blank solution Substances 0.000 description 34
- 238000002347 injection Methods 0.000 description 33
- 239000007924 injection Substances 0.000 description 33
- 238000002360 preparation method Methods 0.000 description 16
- 238000011084 recovery Methods 0.000 description 14
- 239000000047 product Substances 0.000 description 13
- 230000014759 maintenance of location Effects 0.000 description 12
- 239000012088 reference solution Substances 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 6
- 238000013112 stability test Methods 0.000 description 6
- 238000004364 calculation method Methods 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000012795 verification Methods 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 3
- 241001290564 Fimbristylis Species 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000011003 system suitability test Methods 0.000 description 2
- 238000012404 In vitro experiment Methods 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 229930012538 Paclitaxel Natural products 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012084 conversion product Substances 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229960001592 paclitaxel Drugs 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Abstract
The invention discloses a LLTS intermediate and a detection method and application of relevant impurities thereof, which comprise the following steps: detecting the sample solution by adopting high performance liquid chromatography; wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-232nm; mobile phase a: water, mobile phase B: acetonitrile; the elution mode is gradient elution of a specific program. According to the synthetic process route and impurity properties of LLTS raw medicines, LLTS intermediates and related impurities thereof are determined, and related substance analysis and detection methods are formulated, so that the one-time high-efficiency separation is realized on the basis of ensuring the high-efficiency separation of each impurity and active ingredients, and the analysis and detection have good specificity, durability and sensitivity, thereby better realizing the quality control of LLTS intermediates.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis, in particular to LLTS intermediates, and a detection method and application of related impurities thereof.
Background
LLTS the raw material medicine is Lalotataxel, and the structural formula isLLTS as a new generation of taxane compounds, in vitro experiments, the ralostazol shows obvious proliferation inhibition effect on various human tumor cell lines, and has stronger antitumor activity than paclitaxel and docetaxel and stronger resistance to multiple drugs.
In the production process of LLTS raw medicines, two intermediates are mainly used: LLTS-M1 and LLTS-M2, wherein the structural formula of LLTS-M1 isLLTS-M2 has the structural formula/>LLTS-M1 and LLTS-M2 are taken as intermediates of LLTS bulk drugs, if the intermediates contain impurities, the impurities or the conversion products of the impurities can enter subsequent reactions in the process of producing the bulk drugs, so that the production quality of the bulk drugs is affected. Thus, control of impurities in LLTS-M2 is critical. When LLTS-M1 and LLTS-M2 are used as intermediates, the impurities contained in the intermediates have certain similarity in structure and are not easy to separate and detect, further, the impurities are difficult to control, and the LLTS intermediates and related impurities are not loaded in the current pharmacopoeia of various countries. Therefore, in order to further improve the quality of LLTS raw materials, a method capable of detecting LLTS intermediates and impurities existing therein is needed.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide LLTS intermediates, and a detection method and application of related impurities thereof, so as to solve the technical problems.
The invention is realized in the following way:
In a first aspect, the present invention provides a method for detecting LLTS intermediates and related impurities thereof, comprising the steps of: detecting the sample solution by adopting high performance liquid chromatography; wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-232nm; mobile phase a: water, mobile phase B: acetonitrile; the elution mode is gradient elution, and the elution program is as follows:
Alternatively, LLTS intermediates are LLTS-M1 or LLTS-M2; LLTS-M1 has the structural formula: LLTS-M2 has the structural formula: /(I)
Optionally, the volume ratio of mobile phase a to mobile phase B is maintained constant during the elution procedure for a period of 0-5min and 25-30 min.
Alternatively, when LLTS intermediate is LLTS-M1, the elution procedure for high performance liquid chromatography is:
alternatively, when LLTS intermediate is LLTS-M1, the elution procedure for high performance liquid chromatography is:
when LLTS intermediate is LLTS-M2, the elution procedure for high performance liquid chromatography is:
Alternatively, when LLTS intermediate is LLTS-M2, the elution procedure for high performance liquid chromatography is:
in a second aspect, the invention also provides the use of the above LLTS intermediates and their related impurities detection methods in quality control of LLTS intermediates.
The invention has the following beneficial effects: according to the synthetic process route and impurity properties of LLTS raw medicines, LLTS intermediates and related impurities thereof are determined, and related substance analysis and detection methods are formulated, so that the one-time high-efficiency separation is realized on the basis of ensuring the high-efficiency separation of each impurity and active ingredients, and the analysis and detection have good specificity, durability and sensitivity, thereby better realizing the quality control of LLTS intermediates.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram of a blank solution of example 1 with LLTS intermediate LLTS-M1;
FIG. 2 is a chromatogram of the system applicability solution of example 1 with LLTS intermediate LLTS-M1;
FIG. 3 is a chromatogram of the 10-DAB solution of example 1 with LLTS intermediate LLTS-M1;
FIG. 4 is a chromatogram of a LLTS-M1I1 solution of example 1 with LLTS intermediate LLTS-M1 intermediate;
FIG. 5 is a chromatogram of the test solution of example 1 with LLTS intermediate LLTS-M1;
FIG. 6 is a chromatogram of a test solution of comparative example 1 with LLTS intermediate LLTS-M1;
FIG. 7 is a graph showing the linear relationship of test examples to 10-DAB provided LLTS intermediate LLTS-M1;
FIG. 8 is a graph showing the linear relationship of test examples providing LLTS-M1 when LLTS intermediate is LLTS-M1 intermediate;
FIG. 9 is a graph showing the linear relationship of test example provided LLTS-M1I1 when LLTS intermediate is LLTS-M1 intermediate;
FIG. 10 is a chromatogram of a blank solution of example 12 with LLTS intermediate LLTS-M2;
FIG. 11 is a chromatogram of the system applicability solution of example 12 with LLTS intermediate LLTS-M2;
FIG. 12 is a chromatogram of the LLTS-M1 solution of example 12 with LLTS intermediate LLTS-M2;
FIG. 13 is a chromatogram of a LLTS-M2I1 solution of example 12 with LLTS intermediate LLTS-M2 intermediate;
FIG. 14 is a chromatogram of the test solution of example 12 with LLTS intermediate LLTS-M2;
FIG. 15 is a chromatogram of a test solution of comparative example 2 with LLTS intermediate LLTS-M2;
FIG. 16 is a graph showing the linear relationship of test example provided LLTS-M1 for LLTS intermediate LLTS-M2;
FIG. 17 is a graph showing the linear relationship of test examples providing LLTS-M2 for LLTS intermediate LLTS-M2;
FIG. 18 is a graph showing the linear relationship of test example provided LLTS-M2I1 when LLTS intermediate was LLTS-M2 intermediate.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the specific implementation mode, the verification of the items such as specificity, detection limit and quantitative limit, precision, linearity and range, accuracy, solution stability and the like is carried out according to the method verification technical guidelines of the quality control analysis method of chemical drugs, the standardized process technical guidelines established by the quality standards of chemical drugs, the chemical drug impurity research technical guidelines, the chemical drug residual solvent research technical guidelines and the guidelines related to the appendix of the current edition of the pharmacopoeia of the people's republic of China.
The following specifically describes a LLTS intermediate and a detection method and application of related impurities thereof.
At present, no detection method of related substances of LLTS intermediates and impurities thereof is related to the pharmacopoeia of each country and related documents, based on the detection method, the inventor determines LLTS intermediates and impurities thereof according to the relevant requirements of the standardized process technical guidelines established by the chemical drug quality standard and the chemical drug impurity research technical guidelines, and the synthetic process route and impurity properties of the product, and the following detection method is proposed through a great deal of research and practice.
Some embodiments of the present invention provide a LLTS intermediate and a method for detecting related impurities thereof, comprising the steps of: and detecting the sample solution by adopting high performance liquid chromatography. Wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-232nm; mobile phase a: water, mobile phase B: acetonitrile; the elution mode is gradient elution, and the elution program is as follows:
By selecting specific detection wavelength, mobile phase and gradient elution program, the method realizes the one-time high-efficiency separation of related substances of controlled impurities in LLTS intermediates, has strong specificity, good durability and high sensitivity, and can be used for quality control of LLTS intermediates. In order to achieve a better separation and detection effect, in some embodiments, the volume ratio of mobile phase a to mobile phase B is maintained unchanged during the elution procedure for a period of 0-5min and 25-30 min.
It should be noted that LLTS intermediates are mainly LLTS-M1 or LLTS-M2; LLTS-M1 has the structural formula: LLTS-M2 has the structural formula: /(I)
On the other hand, when LLTS intermediates are selected differently, the corresponding impurities in the intermediates are also different, and the corresponding detection methods are slightly different in parameters of the elution procedure, namely when LLTS intermediate is LLTS-M1, LLTS intermediate is LLTS-M1, the related impurities are 10-DAB and LLTS-M1I1, and the structural formulas of 10-DAB and LLTS-M1I1 are as follows: in the detection method, the elution program of the high performance liquid chromatography is as follows:
Preferably, when LLTS intermediate is LLTS-M1, the elution procedure of high performance liquid chromatography is:
When LLTS intermediate is LLTS-M2, the structural formulas of the related impurities are LLTS-M1 and LLTS-M2I1, LLTS-M1 and LLTS-M2I1 in sequence are as follows: in the detection method, the elution program of the high performance liquid chromatography is as follows:
Preferably, when LLTS is LLTS-M2, the elution procedure of the high performance liquid chromatography is:
further, in some embodiments, the flow rate of the gradient elution is from 0.8mL/min to 1.2mL/min, preferably 1.0mL/min.
In some embodiments, the chromatographic column used in the high performance liquid chromatography is octadecylsilane chemically bonded silica chromatographic column, and the column temperature of the chromatographic column is 25-35 ℃ during detection;
specifically, the chromatographic column used for high performance liquid chromatography can be Agilent SB-C18.6mm×250mm,5 μm, and the column temperature of the chromatographic column is 30deg.C when detecting.
In some embodiments, the content of the relevant impurities in the sample solution is calculated by a self-contrast method; specifically, the method for calculating the content of the related impurities comprises the following steps: and respectively injecting the sample solution and the reference substance solution into a high performance liquid chromatograph, measuring the corresponding chromatographic peak area, and calculating by a self-comparison method to obtain the content of related impurities in the sample solution.
Further, in some embodiments, the test solution and the control solution of the related impurities used in the detection process are acetonitrile-water, ethanol-water solution, and LLTS is LLTS-M1, where the volume ratio of acetonitrile to water is 45-55:45-55, preferably 50:50; LLTS the volume ratio of ethanol to water is 88-92 when LLTS-M2 is used as an intermediate: 12-8, preferably 90:10.
For example, when LLTS intermediate is LLTS-M1, the sample formulation procedure is:
10-DAB control solution: about 2mg of 10-DAB was weighed precisely, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (200. Mu.g/ml). 10-DAB positioning solution: 2ml of 10-DAB reference solution is precisely measured, placed in a20 ml measuring flask, diluted to the scale with solvent and shaken well (20. Mu.g/ml). LLTS-M1I1 control solution: about 2mg LLTS-M1I1 was weighed precisely, placed in a20 ml measuring flask, dissolved in a solvent and diluted to the scale, shaken well, (100. Mu.g/ml). LLTS-M1I1 positioning solution: accurately weighing LLTS-M1I1 control solution 0.3ml, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking (10 μg/ml). Test solution: about 10mg LLTS-M1 was weighed precisely, placed in a 10ml measuring flask, dissolved in a solvent and diluted to the scale, and shaken well (1 mg/ml). Control solution: 1ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale with a solvent, and shaken well (100. Mu.g/ml). System applicability solution: taking LLTS-M1, 10-DAB reference substance and LLTS-M1I1 reference substance, dissolving with appropriate amount of solvent, and diluting to obtain mixed solution containing LLTS-M1.1 mg, 10-DAB 20 μg and LLTS-M1I 13 μg per 1 ml.
When LLTS intermediate is LLTS-M2, the sample preparation process is:
LLTS-M1 control solution: about 5mg LLTS-M1 was weighed precisely, placed in a 100ml measuring flask, dissolved in a solvent and diluted to the scale, and shaken well (50. Mu.g/ml). LLTS-M1 positioning solution: accurately weighing LLTS-M1 control solution 2ml, placing into a20 ml measuring flask, diluting to scale with solvent, and shaking (5 μg/ml). LLTS-M2I1 control solution: about 4mg of LLTS-M2I1 control was taken, precisely weighed, placed in a20 ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (200. Mu.g/ml). LLTS-M2I1 positioning solution: accurately weighing LLTS-M2I1 control solution 2ml, placing into a20 ml measuring flask, diluting to scale with solvent, and shaking (20 μg/ml). LLTS-M3 control solution: about 10mg LLTS-M3 was weighed precisely, placed in a10 ml measuring flask, dissolved in a solvent and diluted to the scale, and shaken well (1 mg/ml). LLTS-M3 positioning solution: 1ml of LLTS-M3 control solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken well (10. Mu.g/ml). Test solution: about 10mg of the product is taken, precisely weighed, placed in a10 ml measuring flask, dissolved by a solvent and diluted to a scale, and shaken well (1 mg/ml). Control solution: 1ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale with a solvent, and shaken well (10. Mu.g/ml). System applicability solution: about 10mg of the product is taken, put into a10 ml measuring flask, added with a proper amount of solvent to dissolve, precisely added with LLTS-M1 reference substance solution and LLTS-M2I1 reference substance solution respectively in 1ml, diluted to scale by the solvent, and shaken uniformly (LLTS-M2 1mg/ml, LLTS-M1 5 mug/ml and LLTS-M2I120 mug/ml).
Some embodiments of the present invention specifically further provide a method for detecting LLTS-M1 and related impurities thereof, comprising: detecting the sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography include: chromatographic column: agilent SB-C18.6mm.times.250 mm,5 μm; mobile phase a: water, mobile phase B: acetonitrile; gradient elution was performed as follows:
Wherein, the velocity of flow: 1.0ml/min, detection wavelength: 230nm, column temperature: 30 ℃, sample injection amount: 10 μl, solvent: acetonitrile-water (50:50).
Sample solution preparation: about 10mg of LLTS-M1 intermediate was taken, precisely weighed, placed in a 10ml measuring flask, dissolved in a solvent and diluted to the scale, and shaken well (1 mg/ml).
Some embodiments of the present invention specifically further provide a method for detecting LLTS-M2 and related impurities thereof, comprising: detecting the sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography include: chromatographic column: agilent SB-C18.6mm.times.250 mm,5 μm; mobile phase a: water, mobile phase B: acetonitrile; gradient elution was performed as follows:
wherein, the velocity of flow: 1.0ml/min, detection wavelength: 230nm, column temperature: 30 ℃, sample injection amount: 10 μl, solvent: ethanol-water (90:10).
Sample solution preparation: about 10mg of LLTS-M2 intermediate was taken, precisely weighed, placed in a 10ml measuring flask, dissolved in a solvent and diluted to the scale, and shaken well (1 mg/ml).
Some embodiments of the invention also provide applications of LLTS intermediates and detection methods of related impurities thereof in the above embodiments in quality control of LLTS intermediates.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
The sample solution in the following examples is mainly prepared from LLTS-M1 or LLTS-M2 and the respective corresponding impurities, and in actual detection, LLTS-M1 or LLTS-M2 to be detected is dissolved and diluted to prepare the sample solution.
Example 1
The present embodiment provides a method for detecting LLTS intermediate (LLTS-M1), comprising:
preparing a test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a solvent and diluted to a scale, and shaken uniformly to serve as a sample solution (1 mg/ml).
Detecting the sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography include: chromatographic column: agilent SB-C18.6mm.times.250 mm,5 μm; mobile phase a: water, mobile phase B: acetonitrile; gradient elution was performed as follows:
Wherein, the velocity of flow: 1.0ml/min, detection wavelength: 230nm, column temperature: 30 ℃, sample injection amount: 10 μl, solvent: acetonitrile-water (50:50).
Example 2
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the column temperature was 25 ℃.
Example 3
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the column temperature is 35 ℃.
Example 4
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection condition, the wavelength is 228nm.
Example 5
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection condition, the column temperature is 232nm.
Example 6
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the flow rate was 0.9ml/min.
Example 7
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the flow rate was 1.1ml/min.
Example 8
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection condition, the volume ratio of mobile phases A and B is maintained at 62:38 for 0-5 min; the volume ratio of mobile phases A and B was maintained at 62:38 for 25-30 min.
Example 9
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection condition, the volume ratio of mobile phases A and B of 0-5 min is maintained at 58:42; the volume ratio of mobile phases A and B was maintained at 58:42 for 25-30 min.
Example 10
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the chromatographic column is Agilent ZORBAX XDB-C18.6mm×250mm,5 μm (different batch numbers from the manufacturer).
Example 11
This embodiment differs from embodiment 1 only in that: in the high performance liquid chromatography detection conditions, the chromatographic column is Fimbristylis Titank C, 4.6mm×250mm,5 μm (different batch numbers of different manufacturers).
Comparative example 1
Detecting LLTS intermediate (LLTS-M1) test sample solution by adopting high performance liquid chromatography; the only difference from the examples is that the elution procedure is as follows:
test example 1
System applicability test (LLTS-M1 detection)
Sample preparation:
10-DAB control solution: about 2mg of 10-DAB was weighed precisely, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (200. Mu.g/ml) (weighing 2.065 mg). 10-DAB positioning solution: 2ml of 10-DAB reference solution is precisely measured, placed in a 20ml measuring flask, diluted to the scale with solvent and shaken well (20. Mu.g/ml). LLTS-M1I1 control solution: about 2mg of LLTS-M1I1 was weighed precisely, placed in a 20ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (100. Mu.g/ml) (weighing 2.022 mg). LLTS-M1I1 positioning solution: accurately weighing LLTS-M1I1 control solution 0.3ml, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking (3 μg/ml). Test solution: about 10mg of LLTS-M1 was weighed precisely, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (1 mg/ml) (weighing 10.12 mg). Control solution: 1ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale with a solvent, and shaken well (100. Mu.g/ml). System applicability solution: taking LLTS-M1, 10-DAB reference substance and LLTS-M1I1 reference substance, dissolving with appropriate amount of solvent, and diluting to obtain mixed solution containing LLTS-M11.0mg, 10-DAB 20 μg and LLTS-M1I 13 μg per 1 ml. Blank solution: solvent (acetonitrile-water (50:50)).
The detection step comprises: taking 10 mu l of each of blank solution, control solution, system applicability solution, test sample solution and impurity positioning solution, performing sample injection detection, recording a chromatogram, performing sample injection according to the sequence of table 1, and performing detection according to the detection conditions in examples 1-11. The chromatograms of the solutions obtained under the conditions of example 1 are shown in FIGS. 1 to 5 in this order. The chromatogram obtained under the condition of comparative example 1 is shown in FIG. 6, and the separation degree of LLTS-M1 from adjacent unknown impurities in the chromatogram of LLTS-M1 sample is less than 1.5.
TABLE 1 sample injection order and requirements
The results were analyzed as follows:
table 2 results of system suitability test-relative retention time
TABLE 3 System applicability test results-theoretical plate count
TABLE 4 System applicability test results-tailing factor
Table 5 results of system suitability test-degree of separation
TABLE 6 impurity detection results of test samples
From the above results, it can be seen that: under each condition, the blank solution does not interfere with the determination of the main component and impurities; in the system applicability solution, the separation degree between a main peak and known impurities and adjacent impurity peaks is more than 1.5, the number of theoretical plates of the main peak and each known impurity is not less than 5000, and the tailing factors are less than 2.0; the number of each impurity, total impurities and impurities in the sample solution is detected to have no obvious change; under each condition, the control solution is continuously injected for 5 needles, and the peak area RSD is smaller than 5.0%; therefore, the detection method of the embodiment can stably and effectively detect the related substances LLTS-M1.
Test example 2
Sensitivity test (LLTS-M1), comprising the steps of:
(1) Sample preparation
Solvent: acetonitrile-water (50:50); blank solution: a solvent; LLTS-M1 stock solution: about 10mg of LLTS-M1 control is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale with a solvent, and shaken well to serve as LLTS-M1 stock solution (1 mg/ml) (weighing 9.98 mg). Control solution: 1ml of LLTS-M1 control stock solution was precisely measured, placed in a 100ml measuring flask, diluted to scale with solvent, and shaken well (10. Mu.g/ml). 10-DAB control stock solution: about 2mg of 10-DAB reference substance is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by a solvent, and shaken well. (200. Mu.g/ml) (2.052 mg in a sample); 10-DAB solution: precisely weighing 1ml of 10-DAB control stock solution, placing into a 10ml measuring flask, diluting to scale with solvent, shaking, precisely weighing 1ml, placing into a20 ml measuring flask, diluting to scale with solvent, and shaking (1 μg/ml). LLTS-M1I1 control stock: about 2mg of LLTS-M1I1 control was taken, precisely weighed, placed in a20 ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (100. Mu.g/ml) (weighing 2.001 mg). LLTS-M1I1 solution: accurately measuring LLTS-M1I1 reference stock solution 0.3ml, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking (3 μg/ml). Quantitative limiting solution: precisely measuring 0.4ml of reference substance solution, 1.5ml of 10-DAB solution and 0.5ml of LLTS-M1I1 solution respectively, placing into the same 10ml measuring flask, diluting to scale with solvent, shaking, precisely measuring 3ml, placing into 10ml measuring flask, diluting to scale with solvent, shaking, and S/N is more than or equal to 10. Preparing a detection limit solution: precisely measuring 3ml of quantitative limiting solution, placing in a 10ml measuring flask, diluting to scale with solvent, shaking uniformly, and S/N is more than or equal to 3.
(2) High performance liquid chromatography detection conditions
Same as in example 1
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, reference solution, quantitative limit solution and detection limit solution, carrying out sample injection detection, recording a chromatogram, and carrying out sample injection according to the sequence of the table.
TABLE 7 sample injection order and frequency requirement
| Sequential order | Sample name | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | System applicability solution | 1 Needle |
| 3 | Reference substance solution | 5 Needles |
| 4 | Quantitative limiting solution | 6 Needle |
| 5 | Detection limiting solution | 2 Needle |
| 6 | Control solution | 1 Needle |
(4) Analysis results
The sensitivity test results are shown in tables 8 to 11.
Table 8 LLTS-M1 quantitative limit test results
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 1.48 | 1.47 | 1.45 | 1.46 | 1.51 | 1.49 | 1.48 | 1.41 |
| Retention time (min) | 8.365 | 8.355 | 8.350 | 8.349 | 8.340 | 8.340 | 8.350 | 0.11 |
| S/N | 26.21 | 26.46 | 17.00 | 14.66 | 18.25 | 19.64 | / | / |
TABLE 9 quantitative limit test results for 10-DAB
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 0.70 | 0.68 | 0.70 | 0.70 | 0.70 | 0.66 | 0.69 | 2.78 |
| Retention time (min) | 4.281 | 4.277 | 4.276 | 4.275 | 4.270 | 4.269 | 4.275 | 0.10 |
| S/N | 18.79 | 18.65 | 12.50 | 10.83 | 13.47 | 14.33 | / | / |
Table 10LLTS-M1I1 quantitative limit test results
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 0.58 | 0.59 | 0.60 | 0.56 | 0.56 | 0.58 | 0.58 | 2.44 |
| Retention time | 12.948 | 12.943 | 12.941 | 12.938 | 12.936 | 12.937 | 12.941 | 0.03 |
| S/N | 17.78 | 18.16 | 11.89 | 10.12 | 12.36 | 13.16 | / | / |
TABLE 11 sensitivity test results
The calculation formula is as follows:
Quantitative limit/detection limit concentration (μg/ml) =sample size×impurity content/dilution factor
Quantitative limit/detection limit (ng) =concentration×sample size
Quantitative limit accounts for sample ratio (%) = quantitative limit concentration/sample concentration×100%
From the above sensitivity test results, it can be seen that:
the quantitative limiting solution is continuously tested for 6 times, the peak areas RSD of LLTS-M1 and various impurities are not more than 10.0%, and the retention time RSD is not more than 2.0%; the quantification limits were all less than the reported limit (0.05% test concentration).
Test example 3
A linearity test comprising the steps of:
(1) Sample preparation
Solvent: acetonitrile-water (50:50); blank solution: a solvent; 10-DAB control solution: about 4mg of 10-DAB reference substance is taken, precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to a scale by a solvent, and shaken well. (200. Mu.g/ml) (weighing 4.052 mg); LLTS-M1I1 control solution: about 2mg of LLTS-M1I1 reference substance is taken, precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to a scale with a solvent, and shaken well. (100. Mu.g/ml) (weighing 2.051 mg); LLTS-M1 control stock solution: about 10mg of LLTS-M1 reference substance is taken, precisely weighed, placed in a10 ml measuring flask, dissolved and diluted to a scale with a solvent, and shaken well. (1 mg/ml) (9.96 mg in sample); LLTS-M1 control solution: accurately measuring LLTS-M1 reference stock solution 1ml, placing into a 100ml measuring flask, diluting to scale with solvent, and shaking. (10. Mu.g/ml); impurity mixing stock solution: precisely weighing 5ml of 10-DAB control solution, 1.5ml of LLTS-M1I1 control solution and 1.5ml of LLTS-M1 control stock solution, placing into the same 25ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 1: the solution was limited quantitatively. Linear solution 2 (10%): precisely measuring 0.5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 3 (20%): precisely measuring 1ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 4 (50%): accurately measuring 2.5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 5 (100%): precisely measuring 5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 6 (200%): mixing the impurities into the stock solution.
(2) High performance liquid chromatography detection conditions
Same as in example 1
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, LLTS-M1 control solution and each of the linear solutions, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
TABLE 12 sample injection order and requirements
(4) Analysis results
The results of the linear tests are shown in tables 13-15.
TABLE 13 10-DAB Linear test results
TABLE 14LLTS-M1 Linear test results
TABLE 15LLTS-M1I1 Linear test results
The linear correlation coefficient r is more than or equal to 0.990; the y-axis intercept is less than 25% of the peak area of the 100% limit concentration, and the RSD of the response factors is not more than 10.0%. The concentration C (μg/mL) is taken as an abscissa, and the corresponding peak area is taken as an ordinate, so that a linear regression equation is obtained, and the linear relation diagrams are shown in FIG. 7, FIG. 8 and FIG. 9. The linear relation between LLTS-M1, 10-DAB and LLTS-M1I1 concentration and peak area is good, and the verification requirement is met.
Test example 4
Accuracy test (LLTS-M1), comprising the steps of:
(1) Sample preparation
Solvent: acetonitrile-water (50:50); blank solution: a solvent; test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by a solvent and diluted to a scale, and shaken well (1 mg/ml). 10-DAB control solution: about 10mg of 10-DAB reference substance is taken, precisely weighed, placed in a 25ml measuring flask, dissolved by a solvent and diluted to a scale, and shaken well (2 parts are prepared in parallel, 400 mug/ml) (weighing amounts of 10.10mg and 10.25 mg). LLTS-M1I1 control stock: about 2mg of LLTS-M1I1 control is taken, precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to a scale with a solvent, and shaken well (2 parts are prepared in parallel, 100 mug/ml) (weighing amounts of 2.069mg, 2.074 mg). Impurity mixing stock solution: precisely measuring 10ml of 10-DAB control solution and 6ml of LLTS-M1I1 control stock solution, placing into the same 100ml measuring flask, diluting to scale with solvent, and shaking. 2 parts of the liquid are prepared in parallel in one-to-one correspondence with the stock solution. Control solution: precisely measuring 5ml of impurity mixed stock solution, placing into a 10ml measuring flask, diluting to scale with solvent, and shaking. 2 parts of mixed stock solution are prepared in parallel in one-to-one correspondence with the impurity. Recovery solution (preparing recovery solution from impurity mixed stock solution-1); 50% solution: taking about 10mg of a sample to be tested, precisely weighing, placing the sample into a 10ml measuring flask, adding a proper amount of solvent for dissolution, precisely adding 2.5ml of impurity mixed stock solution, diluting to a scale with the solvent, and shaking uniformly. 3 portions (weighing 10.08mg, 10.10 mg) were prepared in parallel. 100% solution: taking about 10mg of a sample, precisely weighing, placing into a 10ml measuring flask, adding a proper amount of solvent for dissolution, precisely adding 5ml of impurity mixed stock solution, diluting to a scale with the solvent, and shaking uniformly. 3 portions (weighing 10.37mg, 10.24mg, 10.10 mg) were prepared in parallel. 150% solution: taking about 10mg of a sample to be tested, precisely weighing, placing the sample into a 10ml measuring flask, adding a proper amount of solvent for dissolution, precisely adding 7.5ml of impurity mixed stock solution, diluting to a scale with the solvent, and shaking uniformly. 3 parts (weighing 10.04mg, 10.20 mg) were prepared in parallel.
(2) High performance liquid chromatography detection conditions
Same as in example 1
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, reference solution and quantitative limit and detection limit solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 16 sample injection order and requirements
| Sequential order | Sample of | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | System applicability solution | 1 Needle |
| 3 | Reference substance solution-1 | 5 Needles |
| 4 | Reference substance solution-2 | 2 Needle |
| 5 | Test solution | 1 Needle |
| 6 | 50% -1 Solution | 1 Needle |
| 7 | 50% -2% Solution | 1 Needle |
| 8 | 50% -3% Solution | 1 Needle |
| 9 | Reference substance solution-1 | 1 Needle |
| 10 | 100% -1 Solution | 1 Needle |
| 11 | 100% -2 Solution | 1 Needle |
| 12 | 100% -3% Solution | 1 Needle |
| 13 | Reference substance solution-1 | 1 Needle |
| 14 | 150% -2 Solution | 1 Needle |
| 15 | 150% -3% Solution | 1 Needle |
| 16 | Reference substance solution-1 | 1 Needle |
Note that: the calculation formula is as follows: percent recovery = (measured-content in sample)/addition x 100%
(4) Analysis results
The accuracy test results are shown in tables 17-18.
TABLE 17 10-DAB recovery test results
TABLE 18LLTS-M1I1 recovery test results
The impurity limit concentration is taken as 100%, the impurities are quantitatively added into the sample according to three concentrations of 50%, 100% and 150%, the impurity recovery rate is in the range of 90.0% -110.0% at each concentration level, and the 9-part recovery rate RSD is less than 5.0%, so that the accuracy is good.
Test example 5
Repeatability test (LLTS-M1), comprising the steps of:
(1) Sample preparation
Solvent: acetonitrile-water (50:50); blank solution: a solvent; test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by a solvent, diluted to a scale and shaken well. (6 parts, 1 mg/ml) in parallel (weighing 10.11mg, 10.26mg, 10.08mg, 10.03mg, 10.00mg, 10.35 mg); control solution: 1ml of the sample solution was precisely measured, placed in a 100ml measuring flask, diluted to the scale with the solvent, and shaken well (6 parts, 1. Mu.g/ml were prepared in parallel).
(2) High performance liquid chromatography detection conditions
Same as in example 1
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, control solution and test sample solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
TABLE 19 sample injection order and requirements
(4) Analysis results
The results of the repeatability test are shown in Table 20.
Table 20 repeatability test results
| Test article | 10-DAB(％) | LLTS-M1I1 | Maximum unknown single impurity | Total impurity (%) | Number of impurities |
| 1 | 1.44 | 0.19 | 0.07 | 1.98 | 14 |
| 2 | 1.45 | 0.20 | 0.07 | 1.97 | 13 |
| 3 | 1.47 | 0.20 | 0.07 | 2.00 | 13 |
| 4 | 1.44 | 0.20 | 0.07 | 1.96 | 13 |
| 5 | 1.46 | 0.20 | 0.07 | 1.99 | 13 |
| 6 | 1.46 | 0.20 | 0.07 | 1.99 | 13 |
| Mean value of | 1.45 | 0.20 | 0.07 | 1.98 | / |
| RSD(％) | 0.83 | 2.06 | 0.00 | 0.74 | / |
6 Parts of test samples, basically consistent detection of impurities, no obvious change of total impurities and impurity number and good repeatability.
Test example 6
(1) Solution stability test (LLTS-M1), comprising the steps of:
system applicability solution uses system applicability example 1; the test solution and the control solution were prepared as in example 1.
Sample preparation
Solvent: acetonitrile-water (50:50); blank solution: a solvent; test article solution 1: taking 10mg of the product, precisely weighing, placing into a 10ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking uniformly. (1 mg/ml) (weighing 10.15 mg); control solution 1: 1ml of the sample solution was precisely measured, placed in a 100ml measuring flask, diluted to a scale with a solvent, and shaken well (1. Mu.g/ml). Test article solution 2: taking 10mg of the product, precisely weighing, placing into a 10ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking uniformly. (1 mg/ml) (weighing 10.33 mg); control solution 2: 2 ml of the sample solution was precisely measured, placed in a 100ml measuring flask, diluted to the scale with the solvent, and shaken well (1. Mu.g/ml).
(2) High performance liquid chromatography detection conditions
Same as in example 1
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, control solution and test sample solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 21 sample injection order and requirements
/>
/>
(4) Analysis results
The results of the solution stability test are shown in tables 22 and 23.
TABLE 22 stability results of test solutions
| Time of | 10-DAB(％) | LLTS-M1I1 | Maximum unknown sheet | Total impurity (%) | Number of impurities |
| 0 Hours | 1.43 | 0.20 | 0.07 | 1.94 | 13 |
| 1 Hour | 1.43 | 0.20 | 0.07 | 1.95 | 13 |
| For 2 hours | 1.43 | 0.20 | 0.07 | 1.95 | 13 |
| 4 Hours | 1.43 | 0.20 | 0.07 | 1.96 | 13 |
| For 6 hours | 1.43 | 0.20 | 0.07 | 1.96 | 13 |
| 8 Hours | 1.43 | 0.20 | 0.07 | 1.96 | 13 |
| For 10 hours | 1.43 | 0.20 | 0.07 | 1.96 | 13 |
| For 12 hours | 1.43 | 0.20 | 0.07 | 1.97 | 13 |
| 16 Hours | 1.43 | 0.20 | 0.07 | 1.97 | 13 |
| 20 Hours | 1.42 | 0.20 | 0.07 | 1.97 | 14 |
| 24 Hours | 1.42 | 0.20 | 0.07 | 1.98 | 14 |
| For 28 hours | 1.42 | 0.20 | 0.07 | 1.99 | 15 |
| 32 Hours | 1.43 | 0.20 | 0.07 | 2.00 | 15 |
| 36 Hours | 1.43 | 0.20 | 0.07 | 2.01 | 15 |
| 40 Hours | 1.44 | 0.20 | 0.07 | 2.02 | 15 |
| 44 Hours | 1.44 | 0.20 | 0.07 | 2.03 | 15 |
| 48 Hours | 1.44 | 0.20 | 0.07 | 2.04 | 15 |
| Mean value of | 1.43 | 0.20 | 0.07 | 1.98 | / |
| RSD | 0.43 | 0.00 | 0.00 | 1.52 | / |
Table 23 control solution stability test results
The sample solution is placed for 48 hours at room temperature, the number of single impurities, total impurities and impurities are not obviously changed, and the content RSD at each time point is less than 5.0 percent; the total impurities and the number of impurities are not obviously changed, which indicates that the sample solution is stable after being placed for 48 hours at room temperature; the control solution had a main peak area RSD of not more than 5.0% for 48.5 hours, indicating that the control solution was stable for 48.5 hours at room temperature.
Example 12
The present embodiment provides a method for detecting LLTS intermediate (LLTS-M2), comprising:
preparing a test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, dissolved by adding a solvent and diluted to a scale, and shaken uniformly to serve as a sample solution (1 mg/ml).
Detecting the sample solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography include: chromatographic column: agilent SB-C18.6mm.times.250 mm,5 μm; mobile phase a: water, mobile phase B: acetonitrile;
Gradient elution was performed as follows:
Wherein, the velocity of flow: 1.0ml/min, detection wavelength: 230nm, column temperature: 30 ℃, sample injection amount: 10 μl, solvent: acetonitrile-water (90:10).
Example 13
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the column temperature was 25 ℃.
Example 14
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the column temperature is 35 ℃.
Example 15
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection condition, the wavelength is 228nm.
Example 16
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection condition, the column temperature is 232nm.
Example 17
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the flow rate was 0.9ml/min.
Example 18
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the flow rate was 1.1ml/min.
Example 19
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the initial ratio is 60:40.
Example 20
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, starting ratio 56:44.
Example 21
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the chromatographic column is Agilent ZORBAX XDB-C18.6mm×250mm,5 μm (different batch numbers from the manufacturer).
Example 22
This embodiment differs from embodiment 12 only in that: in the high performance liquid chromatography detection conditions, the chromatographic column is Fimbristylis Titank C, 4.6mm×250mm,5 μm (different batch numbers of different manufacturers).
Comparative example 2
Detecting LLTS intermediate (LLTS-M2) test sample solution by adopting high performance liquid chromatography; the only difference from the examples is that the elution procedure is as follows:
Test example 7
System applicability test (LLTS-M2 detection)
Sample preparation:
LLTS-M1 control solution: about 5mg of LLTS-M1 control was taken, precisely weighed, placed in a 100ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (500. Mu.g/ml) (weighing 5.010 mg). LLTS-M1 positioning solution: accurately weighing LLTS-M1 control solution 2ml, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking (5 μg/ml). LLTS-M2I1 control solution: about 4mg of LLTS-M2I1 was weighed precisely, placed in a 20ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (200. Mu.g/ml) (weighing 4.005 mg). LLTS-M2I1 positioning solution: accurately weighing LLTS-M2I1 control solution 2ml, placing into a 20ml measuring flask, diluting to scale with solvent, and shaking (20 μg/ml). LLTS-M3 control solution: about 10mg LLTS-M3 was weighed precisely, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (1 mg/ml) (weighing 0.01002 g). LLTS-M3 positioning solution: 1ml of LLTS-M3 control solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken well (10. Mu.g/ml). Test solution: about 10mg of LLTS-M2 intermediate was taken, precisely weighed, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well to obtain a sample solution (1 mg/ml) (weighing 0.01012 g). Control solution: 1ml of the sample solution is precisely measured, placed in a 100ml measuring flask, diluted to a scale with a solvent, and shaken well (100. Mu.g/ml). System applicability solution: taking LLTS-M2 intermediate 10mg, placing into a 10ml measuring flask, adding appropriate amount of solvent to dissolve, precisely adding LLTS-M1 reference substance solution and LLTS-M2I1 reference substance solution 1ml each, diluting to scale with solvent, and shaking. (LLTS-M2 1mg/ml, LLTS-M1 5. Mu.g/ml, LLTS-M2I 1. Mu.g/ml). Blank solution: solvent (acetonitrile-water (90:10)) (sample size 0.01002 g).
The detection step comprises: taking 10 mu l of each of blank solution, control solution, system applicability solution, test sample solution and impurity positioning solution, performing sample injection detection, recording a chromatogram, performing sample injection according to the sequence of table 1, and performing detection according to the detection conditions in examples 1-11. The chromatograms of the solutions obtained under the conditions of example 1 are shown in FIGS. 10 to 14 in order. The chromatogram of LLTS-M2 sample obtained under the condition of comparative example 2 is shown in FIG. 15, and LLTS-M2 and the peak time of each impurity are later.
Table 24 sample injection order and requirements
| Sequential order | Sample name | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | Control solution | Continuous 5-needle |
| 3 | System applicability solution | 1 Needle |
| 4 | Test solution | 1 Needle |
| 5 | LLTS-M1 positioning solution | 1 Needle |
| 6 | LLTS-M2I1 positioning solution | 1 Needle |
| 7 | LLTS-M3 positioning solution | 1 Needle |
The results were analyzed as follows:
Table 25 sample injection precision test results
| Name of the name | Retention time (min) | Peak area |
| Control solution 1 | 14.922 | 118.12 |
| Control solution 2 | 14.920 | 118.31 |
| Control solution 3 | 14.915 | 117.69 |
| Control solution 4 | 14.915 | 117.60 |
| Control solution 5 | 14.915 | 117.85 |
| Mean value of | 14.917 | 117.91 |
| RSD(％) | 0.03 | 0.25 |
Table 26 results of system suitability test-relative retention time
Table 27 System applicability test results-theoretical plate count
Table 28 System suitability test results-tailing factor
Table 29 System suitability test results-degree of separation
TABLE 30 impurity detection results for samples
/>
From the above results, it can be seen that: under each condition, the blank solution does not interfere with the determination of the main component and impurities; in the system applicability solution, the separation degree between a main peak and known impurities and adjacent impurity peaks is more than 1.5, the number of theoretical plates of the main peak and each known impurity is not less than 5000, and the tailing factors are less than 2.0; the number of each impurity, total impurities and impurities in the sample solution is detected to have no obvious change; under each condition, the control solution is continuously injected for 5 needles, and the peak area RSD is less than 5.0 percent. Therefore, the detection method of the invention can stably and effectively detect the related substances LLTS-M2.
Test example 8
Sensitivity test (LLTS-M2), comprising the steps of:
(1) Sample preparation
Solvent: ethanol-water (90:10); blank solution: a solvent; LLTS-M1 control solution ①: accurately weighing LLTS-M1 control solution 1ml, placing into a 10ml measuring flask, diluting with solvent to scale, shaking, accurately weighing 5ml, placing into a 10ml measuring flask, diluting with solvent to scale, and shaking (50 μg/ml). LLTS-M2 control stock solution: about 10mg of LLTS-M2 control was taken, precisely weighed, placed in a 10ml measuring flask, dissolved in a solvent and diluted to a scale, and shaken well (1 mg/ml) (weighing 0.01007 g). LLTS-M2 control solution: accurately measuring LLTS-M2 control stock solution 1ml, placing into a 25ml measuring flask, diluting with solvent to scale, shaking, accurately measuring 1ml, placing into a 20ml measuring flask, diluting with solvent to scale, and shaking (1 μg/ml). LLTS-M2I1 control solution ①: 1ml of LLTS-M2I1 control solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken well (2. Mu.g/ml). Quantitative limiting solution: accurately weighing LLTS-M1 reference solution ① 1.5.5 ml, LLTS-M2 reference solution 1ml and LLTS-M2I1 reference solution ① ml, placing into the same 10ml measuring flask, diluting with solvent to scale, and shaking. Preparing a detection limit solution: 3ml of quantitative limiting solution is precisely measured, placed in a 10ml measuring flask, diluted to a scale by a solvent and shaken well.
(2) High performance liquid chromatography detection conditions
Same as in example 12
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, reference solution, quantitative limit solution and detection limit solution, carrying out sample injection detection, recording a chromatogram, and carrying out sample injection according to the sequence of the table.
Table 31 sample injection order and frequency requirement
| Sequential order | Sample name | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | System applicability solution | 1 Needle |
| 4 | Quantitative limiting solution | 6 Needle |
| 5 | Detection limiting solution | 2 Needle |
(4) Analysis results
The sensitivity test results are shown in tables 32 to 35.
Table 32LLTS-M1 quantitative limit test results
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 6.75 | 7.22 | 6.87 | 6.79 | 7.18 | 7.39 | 7.03 | 3.77 |
| Retention time (min) | 7.881 | 7.881 | 7.878 | 7.877 | 7.877 | 7.874 | 7.878 | 0.04 |
Table 33LLTS-M2 quantitative limit test results
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 2.08 | 2.12 | 2.02 | 2.12 | 2.08 | 2.19 | 2.10 | 2.71 |
| Retention time | 14.915 | 14.914 | 14.914 | 14.912 | 14.91 | 14.91 | 14.913 | 0.02 |
Table 34LLTS-M2I1 quantitative limit test results
| Name of the name | 1 | 2 | 3 | 4 | 5 | 6 | Average of | RSD(％) |
| Peak area | 2.41 | 2.49 | 2.45 | 2.34 | 2.45 | 2.41 | 2.43 | 2.11 |
| Retention time | 15.915 | 15.913 | 15.914 | 15.911 | 15.909 | 15.909 | 15.912 | 0.02 |
Table 35 sensitivity test results
The calculation formula is as follows:
Quantitative limit/detection limit concentration (μg/ml) =sample size×impurity content/dilution factor
Quantitative limit/detection limit (ng) =concentration×sample size
Quantitative limit accounts for sample ratio (%) = quantitative limit concentration/sample concentration×100%
From the above sensitivity test results, it can be seen that:
The quantitative limiting solution is continuously tested for 6 times, the peak areas RSD of LLTS-M2 and various impurities are not more than 10.0%, and the retention time RSD is not more than 2.0%; the quantification limits were all less than the reported limit (0.05% test concentration).
Test example 9
A linearity test (LLTS-M2) comprising the steps of:
(1) Sample preparation
The product LLTS-M2 contains hydroxyl, can be oxidized into LLTS-M2I1, and linearly expands the main component and each impurity to the limit of 3 times in order to cope with the increase of LLTS-M2I1 content in storage (LLTS-M2 is in the range of quantitative limit-15.0% of sample concentration, LLTS-M1 is in the range of quantitative limit-1.5% of sample concentration, and LLTS-M2I1 is in the range of quantitative limit-6.0% of sample concentration); solvent: ethanol-water (90:10); blank solution: a solvent; LLTS-M2 control stock solution: about 20mg of LLTS-M2 reference substance is taken, precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to a scale with a solvent, and shaken well. (1 mg/ml) (sample amount 0.02003 g); LLTS-M2 control solution: 1ml of LLTS-M2 control stock solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken well (10. Mu.g/ml). ; impurity mixing stock solution: accurately weighing LLTS-M1 control solution 4ml, LLTS-M2I1 control solution 4ml, LLTS-M2 control stock solution 2ml, placing into the same 20ml measuring flask, diluting with solvent to scale, and shaking.
Linear solution 1: the solution was limited quantitatively. Linear solution 2 (10%): precisely measuring 0.5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 3 (20%): precisely measuring 1ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 4 (50%): accurately measuring 2.5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 5 (100%): precisely measuring 5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. Linear solution 6 (200%): mixing the impurities into the stock solution. Linear solution 7 (300%): accurately weighing LLTS-M1 control solution 3ml, LLTS-M2I1 control solution 3ml, LLTS-M2 control stock solution 1.5ml, placing into a10 ml measuring flask, diluting with solvent to scale, and shaking.
(2) High performance liquid chromatography detection conditions
Same as in example 12
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, LLTS-M2 control solution and each of the linear solutions, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 36 sample injection order and requirements
(4) Analysis results
The results of the linear tests are shown in tables 37-39.
Table 37LLTS-M1 Linear test results
TABLE 38LLTS-M2 Linear test results
TABLE 39LLTS-M2I1 Linear test results
The linear correlation coefficient r is more than or equal to 0.990; the y-axis intercept is less than 25% of the peak area of the 100% limit concentration, and the RSD of the response factors is not more than 10.0%. The concentration C (μg/mL) is taken as an abscissa, and the corresponding peak area is taken as an ordinate, so that a linear regression equation is obtained, and the linear relation diagrams are shown in FIG. 16, FIG. 17 and FIG. 18. The linear relation between LLTS-M1, LLTS-M2 and LLTS-M2I1 concentration and peak area is good, and the verification requirement is met.
Test example 10
Accuracy test (LLTS-M2), comprising the steps of:
(1) Sample preparation
Solvent: ethanol-water (90:10); blank solution: a solvent; test solution: about 10mg of the product is taken, precisely weighed, placed in a10 ml measuring flask, dissolved by a solvent and diluted to a scale, and shaken well (1 mg/ml, sample weighing 0.01017 g). LLTS-M1 control solution: about 10mg of LLTS-M1 reference substance is taken, precisely weighed, placed in a20 ml measuring flask, dissolved by a solvent and diluted to a scale, and shaken uniformly (2 parts are prepared in parallel, 500 mug/ml, and the weighing amounts are 0.01001g and 0.01000g respectively). LLTS-M2I1 control solution: about 10mg of LLTS-M2I1 reference substance is taken, precisely weighed, placed in a10 ml measuring flask, dissolved and diluted to a scale by a solvent, and shaken uniformly (2 parts are prepared in parallel, 1mg/ml, and the weighing amounts are 0.01005g and 0.01000g respectively). Impurity mixing stock solution: accurately weighing LLTS-M1 control solution 2ml and LLTS-M2I1 control solution 4ml, placing into the same 100ml measuring flask, diluting with solvent to scale, shaking, and preparing 2 parts (LLTS-M110 μg/ml and LLTS-M2I 140 μg/ml) in parallel. Impurity control solution: precisely measuring 5ml of impurity mixed stock solution, placing into a10 ml measuring flask, diluting to scale with solvent, and shaking. 2 portions (LLTS-M1 5. Mu.g/ml, LLTS-M2I 120. Mu.g/ml) were prepared in parallel. Recovery solution (preparing recovery solution from impurity mixed stock solution-1); 50% solution: about 10mg of the sample is taken, precisely weighed, placed in a10 ml measuring flask, added with a proper amount of solvent to dissolve, precisely added with 2.5ml of impurity mixed stock solution, diluted to scale by the solvent, and uniformly shaken (3 parts of sample are prepared in parallel, and the sample weighing amounts are 0.01009g, 0.00988g and 0.01006g respectively). 100% solution: about 10mg of the sample is taken, precisely weighed, placed in a10 ml measuring flask, added with a proper amount of solvent to dissolve, precisely added with 5ml of impurity mixed stock solution, diluted to scale by the solvent, and uniformly shaken (3 parts of sample are prepared in parallel, and the weighing amounts are 0.01011g, 0.01010g and 0.01013g respectively). 150% solution: about 10mg of the sample is taken, precisely weighed, placed in a10 ml measuring flask, added with a proper amount of solvent to dissolve, precisely added with 7.5ml of impurity mixed stock solution, diluted to scale by the solvent, and uniformly shaken (3 parts of sample are prepared in parallel, and the sample weighing amounts are 0.01012g, 0.01009g and 0.01005g respectively). ()
(2) High performance liquid chromatography detection conditions
Same as in example 12
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, reference solution and quantitative limit and detection limit solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 40 sample injection order and requirements
| Sequential order | Sample of | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | Reference substance solution | 2 Needle |
| 3 | Test solution | 1 Needle |
| 4 | 50% -1 Solution | 1 Needle |
| 5 | 50% -2% Solution | 1 Needle |
| 6 | 50% -3% Solution | 1 Needle |
| 7 | 100% -1 Solution | 1 Needle |
| 8 | 100% -2 Solution | 1 Needle |
| 9 | 100% -3% Solution | 1 Needle |
| 10 | 150% -1 Solution | 1 Needle |
| 11 | 150% -2 Solution | 1 Needle |
| 12 | 150% -3% Solution | 1 Needle |
Note that: the calculation formula is as follows: percent recovery = (measured-content in sample)/addition x 100%
(4) Analysis results
The accuracy test results are shown in tables 41-42.
TABLE 41LLTS-M1 recovery test results
Table 42LLTS-M2I1 recovery test results
The impurity limit concentration is taken as 100%, the impurities are quantitatively added into the sample according to three concentrations of 50%, 100% and 150%, the impurity recovery rate is in the range of 90.0% -110.0% at each concentration level, and the 9-part recovery rate RSD is less than 5.0%, so that the accuracy is good.
Test example 11
Repeatability test (LLTS-M2), comprising the steps of:
(1) Sample preparation
Solvent: ethanol-Water (90:10)
Blank solution: solvent(s)
Test solution: about 10mg of the product is taken, precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to a scale by a solvent, and shaken uniformly (6 parts, 1mg/ml are prepared in parallel, and the weighing amounts are 0.01015g, 0.01011g, 0.01014g, 0.01002g, 0.01004g and 0.01006g respectively).
Control solution: precisely measuring 1ml of the sample solution, placing in a 100ml measuring flask, diluting to scale with solvent, and shaking. (6 parts, 10. Mu.g/ml in parallel)
(2) High performance liquid chromatography detection conditions
Same as in example 12
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, control solution and test sample solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 43 sample injection order and requirements
| Sequential order | Sample of | Needle count |
| 1 | Blank solution | 1 Needle |
| 2 | System applicability solution | 1 Needle |
| 3 | Test article-1-6 | 1 Needle |
| 4 | Control solutions-1 to 6 | 1 Needle |
(4) Analysis results
The results of the repeatability test are shown in table 44.
Table 44 repeatability test results
| Test article | LLTS-M1 | LLTS-M2I1 | Maximum unknown single impurity | Total impurity (%) | Number of impurities |
| 1 | 0.284 | 1.889 | 0.637 | 4.038 | 9 |
| 2 | 0.269 | 2.098 | 0.613 | 4.174 | 9 |
| 3 | 0.274 | 2.016 | 0.627 | 4.091 | 9 |
| 4 | 0.280 | 1.989 | 0.610 | 4.137 | 9 |
| 5 | 0.279 | 1.997 | 0.623 | 4.121 | 9 |
| 6 | 0.269 | 1.854 | 0.612 | 3.995 | 9 |
| Mean value of | 0.28 | 1.97 | 0.62 | 4.09 | / |
| RSD(％) | 2.24 | 4.51 | 1.71 | 1.62 | / |
6 Parts of test samples, basically consistent detection of impurities, no obvious change of total impurities and impurity number and good repeatability.
Test example 12
(1) Solution stability test (LLTS-M2), comprising the steps of:
System applicability solution uses system applicability example 12; test solution and control solution were prepared as in example 12.
Sample preparation
Solvent: ethanol-Water (90:10)
Blank solution: solvent(s)
Preparing two parts of test solution and control solution in parallel, taking control solution-1 to examine the stability of the system, taking control solution-2 to examine the stability of the control solution, and taking test solution-2 to examine the stability of the test solution (the sample weighing amounts of test 1 and test 2 are 0.01011g and 0.01013g respectively).
(2) High performance liquid chromatography detection conditions
Same as in example 12
(3) Detection step
Taking 10 mu l of each of blank solution, system applicability solution, control solution and test sample solution, carrying out sample injection detection, and recording a chromatogram. Sample introduction was performed in the order of the following table.
Table 45 sample injection order and requirements
(4) Analysis results
The results of the solution stability test are shown in tables 46 and 47.
TABLE 46 stability results of test solutions
Test results of stability of test sample solution
Table 47 control solution stability test results
| Time (h) | Retention time (min) | Peak area |
| 0.5 Hour | 14.909 | 107.46 |
| 1.5 Hours | 14.911 | 107.31 |
| 2.5 Hours | 14.909 | 106.85 |
| 4.5 Hours | 14.902 | 106.70 |
| 6.5 Hours | 14.896 | 106.56 |
| 8.5 Hours | 14.896 | 106.15 |
| 10.5 Hours | 14.894 | 105.85 |
| 12.5 Hours | 14.891 | 105.69 |
| Mean value of | 14.901 | 106.57 |
| RSD(％) | 0.06 | 0.61 |
The sample solution is placed at room temperature for 12 hours, the average content of the impurity LLTS-M1 at each time point is 0.27%, and the RSD is 7.40% and less than 10.0%; the average content of the impurity LLTS-M2I1 at each time point is 2.02%, and the RSD is 1.90% and less than 5.0%; the average content of the maximum unknown single impurity at each time point is 0.62%, and the RSD is 1.80% and less than 5.0%; the average content of the total impurities at each time point is 4.14%, the RSD is 7.40% and is more than 5.0%; indicating that the test solution was unstable when left at room temperature for 12 hours.
The sample solution is placed for 4 hours at room temperature, the average content of the impurity LLTS-M1 at each time point is 0.26%, the RSD is 8.45%, and the content is less than 10.0%; the average content of the impurity LLTS-M2I1 at each time point is 2.04%, and the RSD is 1.62% and less than 5.0%; the average content of the maximum unknown single impurity at each time point is 0.62%, and the RSD is 0.98% and less than 5.0%; the average content of the total impurities at each time point is 3.90%, the RSD is 4.65% and less than 5.0%; indicating that the test solution is stable after being left at room temperature for 4 hours, and should be used for new preparation.
The control solution had a main peak area RSD of 0.61% or less over 12.5 hours, indicating that the control solution was stable for 12.5 hours at room temperature.
In summary, the detection method of the embodiment of the invention ensures that each related substance is separated from the effective component with good specificity, repeatability and accuracy on the basis of ensuring the high efficiency, thereby better realizing the quality control of LLTS-M1 or LLTS-M2.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. A method for detecting LLTS intermediates and related impurities thereof, comprising the steps of: detecting the sample solution by adopting high performance liquid chromatography;
wherein, the detection conditions of the high performance liquid chromatography comprise:
detection wavelength: 228-232nm;
Mobile phase a: water, mobile phase B: acetonitrile;
The elution mode is gradient elution, and the elution program is as follows:
The LLTS intermediate is LLTS-M1 or LLTS-M2; the structural formula of LLTS-M1 is as follows: the structural formula of LLTS-M2 is as follows: /(I)
When the LLTS intermediate is LLTS-M1, the related impurities are 10-DAB and LLTS-M1I1, and the structural formulas of the 10-DAB and the LLTS-M1I1 are as follows in sequence:
when the LLTS intermediate is LLTS-M2, the related impurities are LLTS-M1 and LLTS-M2I1, and the structural formulas of LLTS-M1 and LLTS-M2I1 are as follows:
The chromatographic column adopted by the high performance liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column, and the column temperature of the chromatographic column is 25-35 ℃ during detection;
The solvent adopted by the test solution and the control solution of related impurities used in the detection process is acetonitrile-water solution or ethanol-water solution, and when the LLTS intermediate is LLTS-M1, the volume ratio of acetonitrile to water is 45-55:45-55, wherein when the LLTS intermediate is LLTS-M2, the volume ratio of ethanol to water is 88-92:12-8.
2. The method for detecting LLTS intermediates and related impurities according to claim 1, wherein when the LLTS intermediates are LLTS-M1, the elution procedure of the high performance liquid chromatography is:
Or, the LLTS intermediate is LLTS-M2, and the elution program of the high performance liquid chromatography is as follows:
3. the method for detecting LLTS intermediates and related impurities according to claim 1, wherein when the LLTS intermediates are LLTS-M1, the elution procedure of the high performance liquid chromatography is:
Or, when the LLTS intermediate is LLTS-M2, the elution procedure of the high performance liquid chromatography is as follows:
4. The method for detecting LLTS intermediates and related impurities according to claim 1, wherein the flow rate of the gradient elution is 0.8mL/min to 1.2mL/min.
5. The method for detecting LLTS intermediates and related impurities according to claim 1, wherein the flow rate of the gradient elution is 1.0mL/min.
6. The method for detecting LLTS intermediates and related impurities according to claim 1, wherein the chromatographic column used in the high performance liquid chromatography is Agilent SB-C18.6 mm x 250mm,5 μm, and the column temperature of the chromatographic column is 30 ℃ during detection.
7. The method for detecting LLTS intermediates and related impurities according to claim 1, wherein the content of related impurities in the sample solution is calculated by self-contrast method.
8. The method for detecting LLTS intermediates and related impurities thereof according to claim 7, wherein the method for calculating the content of related impurities comprises: and respectively injecting the sample solution and the reference substance solution into a high performance liquid chromatograph, measuring the corresponding chromatographic peak area, and calculating the content of related impurities in the sample solution by a self-contrast method.
9. Use of LLTS intermediates as defined in any one of claims 1 to 8 and their related impurities in quality control of LLTS intermediates.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210706463.4A CN115097035B (en) | 2022-06-21 | LLTS intermediate and detection method and application of related impurities thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210706463.4A CN115097035B (en) | 2022-06-21 | LLTS intermediate and detection method and application of related impurities thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115097035A CN115097035A (en) | 2022-09-23 |
| CN115097035B true CN115097035B (en) | 2024-06-04 |
Family
ID=
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001007040A1 (en) * | 1999-07-28 | 2001-02-01 | Kobe Natural Products & Chemicals Co., Ltd. | Multidrug-resistant cancer overcoming agents and process for producing the same |
| CN104710389A (en) * | 2013-12-14 | 2015-06-17 | 苏州雷纳药物研发有限公司 | Taxane derivative purifying method |
| CN109206386A (en) * | 2017-06-30 | 2019-01-15 | 江苏汉邦科技有限公司 | A kind of preparation method of La Luotasai |
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001007040A1 (en) * | 1999-07-28 | 2001-02-01 | Kobe Natural Products & Chemicals Co., Ltd. | Multidrug-resistant cancer overcoming agents and process for producing the same |
| CN104710389A (en) * | 2013-12-14 | 2015-06-17 | 苏州雷纳药物研发有限公司 | Taxane derivative purifying method |
| CN109206386A (en) * | 2017-06-30 | 2019-01-15 | 江苏汉邦科技有限公司 | A kind of preparation method of La Luotasai |
Non-Patent Citations (3)
| Title |
|---|
| Degradation kinetics of larotaxel and identification of its degradation products in alkaline condition;Xiaoming Liang et al.;Journal of Pharmaceutical Analysis;全文 * |
| Isolation and characterization of process-related impurities and degradation products in larotaxel;Xin Che et al.;Journal of Pharmaceutical and Biomedical Analysis;全文 * |
| 拉洛他赛的波谱解析及高效液相色谱法对其脂质体含量的测定;李雪琦;李建伟;李秋红;阎妍;段嘉伦;崔一诺;苏展博;罗倩;许佳瑞;杜亚菲;王桂玲;谢英;吕万良;;北京大学学报(医学版)(第03期);全文 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110320293B (en) | Method for determining residual solvent in phthalide compound | |
| CN104655751B (en) | A kind of detect the method for organic solvent residual in dapoxetine | |
| CN112394127B (en) | Method for determining content of 3-amino-1-adamantanol and L-prolinamide in vildagliptin | |
| CN111751459A (en) | Method for simultaneously detecting multiple residual solvents in sitafloxacin | |
| CN104749269A (en) | Method for determining enantiomer impurity in alogliptin crude drug and preparation by virtue of HPLC | |
| CN111521698B (en) | Method for detecting residual solvent in setastine hydrochloride raw material medicine | |
| CN110672734B (en) | Analysis method of related substances in amiodarone hydrochloride injection | |
| CN115097035B (en) | LLTS intermediate and detection method and application of related impurities thereof | |
| CN110261508B (en) | Analysis method of related substances in amiodarone hydrochloride raw material medicine | |
| CN114280191B (en) | Method for detecting related substances in double-cysteine and preparation thereof | |
| CN113820409B (en) | Method for detecting related substances in mother nucleus of moxifloxacin | |
| CN109765317B (en) | Composition and detection method of related substances in tofacitinib citrate | |
| CN114755346A (en) | Method for measuring substances related to cyclosporine soft capsules | |
| CN115436525B (en) | LLTS-M3 and detection method and application of related substances thereof | |
| CN112881538B (en) | Method for detecting impurities and enantiomers in fudosteine and fudosteine tablets | |
| CN115097035A (en) | LLTS intermediate and detection method and application of related impurities thereof | |
| CN107976489B (en) | Method for determining residual pyridine in pregabalin | |
| CN115326999B (en) | Detection method of oseltamivir epoxy intermediate and isomer thereof | |
| CN112034058B (en) | Method for detecting isomer impurities in vincamine | |
| CN114280190B (en) | Kit for detecting related substances of double cysteines | |
| CN111380991B (en) | Method for detecting content of degradation impurities in vitamin C medicament | |
| CN116183759A (en) | Detection method and application of related substances of pezopanib hydrochloride intermediate PZP-M1 | |
| CN116124912A (en) | Detection method and application of 17-iodo-androstane-5, 16-diene-3 beta-alcohol related substances | |
| CN116735730A (en) | Method for detecting antiviral component and application thereof | |
| CN117825552A (en) | Evaporation light scattering detection method for lysine hydrochloride and calcium hydrophosphate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |