CN115436525A - LLTS-M3 and detection method and application of related substances thereof - Google Patents
LLTS-M3 and detection method and application of related substances thereof Download PDFInfo
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- 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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- 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
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a detection method and application of LLTS-M3 and related substances thereof, wherein the detection method of the LLTS-M3 and the related substances thereof comprises the following steps: detecting the test solution by high performance liquid chromatography; the detection conditions of the chromatogram comprise: the detection wavelength is 230nm; a mobile phase A: water; mobile phase B: acetonitrile; gradient elution was performed by a specific elution procedure using mobile phase a and mobile phase B. Under the high performance liquid chromatography detection condition, the invention can simultaneously realize the one-time high-efficiency separation of the related substances of five impurities controlled in the LLTS-M3 on the basis of ensuring the high-efficiency separation of the related substances and the intermediate, so that the analysis and detection has good specificity, durability and sensitivity, thereby better realizing the quality control of the LLTS-M3 intermediate.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis, in particular to a detection method and application of LLTS-M3 and related substances thereof.
Background
Related substances (related substances) refer to starting materials, reagents, intermediates, byproducts and the like brought in the production of the raw material medicaments, and may also be degradation products, polymers or special impurities such as crystal transformation and the like generated in the production, storage and transportation processes of the preparation. The types of related substances are closely related to the synthetic route and the production process of the medicine, and the impurity spectrum of the medicine can also be changed by different synthetic routes and production processes.
LLTS bulk drug is larotaxel, and the structural formula is
LLTS is taken as a new generation of taxane compound, in vitro tests, larotaxel shows obvious proliferation inhibition effect on various human tumor cell lines, the antitumor activity of the larotaxel is stronger than that of paclitaxel and docetaxel, and the larotaxel has stronger multidrug resistance.
In the production process of the LLTS bulk drug, the structural formula of the intermediate LLTS-M3 is shown as
LLTS-M3 is used as an intermediate of LLTS bulk drug, if related substances contained in the LLTS-M3 or converted substances thereof can enter subsequent reactions, the production quality of the bulk drug is affected. Thus, it is important for the control of the substances involved in LLTS-M3. At present, the intermediate LLTS-M3 and a detection method of related impurities thereof are not collected in the pharmacopoeias of various countries. Therefore, in order to further improve the production quality of the LLTS bulk drug, a method capable of detecting the LLTS-M3 and the impurities existing in the LLTS bulk drug is needed.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide a method for detecting LLTS-M3 and related substances thereof, which can efficiently detect the related substances of LLTS-M3 at a time, and which is highly specific and durable.
The second purpose of the invention is to provide the application of the detection method of LLTS-M3 and related substances thereof in the quality control of LLTS-M3 intermediates.
The invention is realized by the following steps:
in a first aspect, the present invention provides a method for detecting LLTS intermediates and related impurities thereof, comprising the steps of: detecting the test solution by adopting high performance liquid chromatography; wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-232nm; a mobile phase A: water, mobile phase B: acetonitrile; the elution mode is gradient elution, and the elution procedure is as follows:
optionally, the volume ratio of mobile phase a and mobile phase B is maintained constant for time periods of 0-5min and 40-45min during the elution procedure.
Optionally, the elution procedure of high performance liquid chromatography is:
optionally, the elution procedure of the high performance liquid chromatography is:
optionally, the chromatographic column used in the high performance liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column, and during detection, the column temperature of the chromatographic column is 35-45 ℃, and the flow rate of the gradient elution is 0.9-1.1 mL/min.
In a second aspect, the invention also provides the application of the detection method of the LLTS-M3 and related substances thereof in the quality control of the LLTS intermediate.
The invention has the following beneficial effects: according to the synthesis process route and the impurity properties of LLTS bulk drugs, the LLTS-M3 intermediate and related substances thereof are determined, and a related substance analysis and detection method is drawn up, so that on the basis of ensuring the efficient separation of the related substances from the intermediate, the one-time efficient separation of the related substances of five impurities controlled in LLTS-M3 is realized, the analysis and detection have good specificity, durability and sensitivity, and the quality control of the LLTS-M3 intermediate is better realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and those skilled in the art can also obtain other related drawings based on the drawings without inventive efforts.
FIG. 1 is a chromatogram of a blank solution of chromatographic conditions of example 1;
FIG. 2 is a chromatogram of a solution of LLTS-M3I1 (1. Mu.g/ml) under the chromatographic conditions of example 1;
FIG. 3 is a chromatogram of a solution of LLTS-M3I2 (8. Mu.g/ml) under the chromatographic conditions of example 1;
FIG. 4 is a chromatogram of a LLTS-M3I3 solution (1. Mu.g/ml) under the chromatographic conditions of example 1;
FIG. 5 is a chromatogram of a solution of LLTS-M3I4 (3. Mu.g/ml) under the chromatographic conditions of example 1;
FIG. 6 is a chromatogram of an LTS-M2 solution (1. Mu.g/ml) under chromatographic conditions of example 1;
FIG. 7 is a chromatogram of the test solution of example 1;
FIGS. 8-12 are graphs showing the linear relationship between the concentrations of LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, and LLTS-M3I4 and the peak areas.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specific implementation mode, the verification of the items such as specificity, detection limit and quantification limit, precision, linearity and range, accuracy, solution stability and the like is performed according to the technical guide principles of verification of chemical drug quality control analysis method, the technical guide principles of standardized process established by chemical drug quality standards, the technical guide principles of chemical drug impurity research, the technical guide principles of chemical drug residual solvent research and the related guide principles in the appendix of the current edition of pharmacopoeia of the people's republic of China.
The following is a detailed description of the method for detecting LLTS-M3 and related substances and the application thereof.
At present, the pharmacopoeia of various countries and related documents do not relate to a detection method of LLTS-M3 intermediate and related substances of impurities thereof, based on the detection method, the inventor determines LLTS-M3 and related substances thereof according to the relevant requirements of normalized process technical guide principles established by chemical drug quality standards and chemical drug impurity research technical guide principles, and according to the synthesis process route and the impurity properties of the product, and provides the following detection method through a large amount of research and practice.
Some embodiments of the present invention provide a detection method of LLTS-M3 and related substances, comprising the following steps: and detecting the test 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 procedure is as follows:
by selecting specific detection wavelength, mobile phase and gradient elution program, the related substances of the controlled impurities in the LLT-M3 intermediate are efficiently separated at one time, and the detection method has strong specificity, good durability and high sensitivity, and can be used for quality control of the LLTS-M3 intermediate.
In some embodiments, for better separation detection, the volume ratio of mobile phase a to mobile phase B is maintained constant during the elution procedure for time periods of 0-5min and 40-45 min.
It is noted that the intermediate of LLTS-M3 has the structural formula
Related substances in the LLTS-M3 intermediate comprise LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, LLTS-M3I4 and LLTS-M2, wherein the structural formulas of LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, LLTS-M3I4 and LLTS-M2 are sequentially represented by formula I-formula V:
Optionally, the volume ratio of mobile phase a and mobile phase B is maintained constant for time periods of 0-5min and 40-45min during the elution procedure.
In order to further improve the detection effect, in some embodiments, the elution procedure of the high performance liquid chromatography is optimized again, specifically, the elution procedure is as follows:
more preferably, the elution procedure of the high performance liquid chromatography is:
further, in some embodiments, the flow rate of the gradient elution is 0.9mL/min to 1.1mL/min, for example, 0.9mL/min, 1.0mL/min, or 1.1mL/min, preferably 1.0mL/min, can be selected.
In some embodiments, the HPLC uses an octadecylsilane chemically bonded silica column, and the column temperature during detection is 35-45 ℃.
Specifically, the high performance liquid chromatography can use Agilent5TC-C18 (2), 4.6mm × 250mm,5 μm, and the column temperature is 40 deg.C.
In some embodiments, the content of the substance in the test solution is calculated by a self-comparison method; specifically, the method for calculating the content of the related substances comprises the following steps: respectively injecting the test solution and the reference solution into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas, and calculating by using a self-contrast method to obtain the content of related substances in the test solution.
Further, in some embodiments, the solvent used for the test solution and the control solution of the substance used in the assay process is acetonitrile.
For example, when the LLTS intermediate is LLTS-M1, the sample is formulated as follows:
LLTS-M3I1 control stock solution: about 2mg of the LLTS-M3I1 control sample is precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (20 mug/ml).
LLTS-M3I2 control stock solution: about 2mg of the LLTS-M3I2 control sample is precisely weighed, placed in a 25ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (80 mug/ml).
LLTS-M3I3 control stock solution: about 2mg of the LLTS-M3I3 control sample is precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (20 mug/ml).
LLTS-M3I4 control stock solution: about 2mg of the LLTS-M3I4 control sample is precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (100 mu g/ml).
LLTS-M2 control stock solution: about 2mg of LLTS-M2 control was weighed precisely, placed in a 100ml measuring flask, dissolved and diluted to the mark with solvent, shaken up (20. Mu.g/ml).
Test solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up (1 mg/ml).
Control solution: 1ml of the test solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with the solvent and shaken up (10. Mu.g/ml).
System applicability solution: taking about 10mg of the product, placing into a 10ml measuring flask, adding appropriate amount of solvent to dissolve, adding accurately 0.5ml of LLTS-M3I1 control stock solution, 1ml of LLTS-M3I2 control stock solution, 0.5ml of LLTS-M3I3 control stock solution, 0.3ml of LLTS-M3I4 control stock solution, and 0.5ml of LLTS-M2 control stock solution, diluting with solvent to scale, and shaking uniformly (about 1mg/ml of LLTS-M3, about 1 μ g/ml of LLTS-M3I1, about 8 μ g/ml of LLTS-M3I2, about 1 μ g/ml of LLTS-M3I3, about 3 μ g/ml of LLTS-M3I4, and about 1 μ g/ml of LLTS-M2).
Some embodiments of the present invention specifically provide a method for detecting LLTS-M3 and related substances, comprising: detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
and (3) chromatographic column: agilent5TC-C18 (2) 4.6mm × 250mm,5 μm;
mobile phase A: water;
mobile phase B: acetonitrile;
gradient elution was performed as follows:
wherein, the flow rate: 1.0ml/min, detection wavelength: 230nm, column temperature: 40 ℃, sample introduction: 10. Mu.l, solvent: and (3) acetonitrile.
Test solution: taking 10mg of LLTS-M3 intermediate sample, precisely weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking up (1 mg/ml).
The features and properties of the present invention are described in further detail below with reference to examples.
The system applicability solution in the following embodiment is mainly prepared from LLTS-M3, impurities I, II, III, IV and V, and during actual detection, the LLTS-M3 to be detected is dissolved and diluted to prepare a test solution.
Example 1
The embodiment provides a method for detecting LLTS-M3 and related substances thereof, which comprises the following steps:
preparing a test solution: taking 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking uniformly to obtain a test solution (1 mg/ml). The detection conditions of the high performance liquid chromatography comprise: a chromatographic column: agilent5TC-C18 (2) 4.6 mm. Times.250mm, 5 μm; mobile phase A: water; and (3) mobile phase B: acetonitrile; gradient elution was performed as follows:
wherein, the flow rate: 1.0ml/min, detection wavelength: 230nm, column temperature: 40 ℃, sample introduction: 10. Mu.l, solvent: and (3) acetonitrile.
Example 2
This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 35 ℃.
Example 3
This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 45 ℃.
Example 4
This example differs from example 1 only in that: the wavelength is 228nm under the detection condition of high performance liquid chromatography.
Example 5
This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 232nm.
Example 6
This example differs from example 1 only in that: the flow rate is 0.9ml/min under the detection condition of high performance liquid chromatography.
Example 7
This example only differs from example 1 in that: the flow rate is 1.1ml/min under the detection condition of high performance liquid chromatography.
Example 8
This example only differs from example 1 in that: in the detection conditions of the high performance liquid chromatography, the volume ratio of the mobile phases A and B is maintained at 62; the volume ratio of mobile phases A and B was maintained at 62-30 min.
Example 9
This example differs from example 1 only in that: in the detection conditions of the high performance liquid chromatography, the volume ratio of the mobile phases A and B is maintained at 58; the volume ratio of mobile phases A and B was maintained at 58.
Example 10
This example only differs from example 1 in that: in the detection condition of the high performance liquid chromatography, the chromatographic column is Agilent5TC-C18 (2) 4.6mm multiplied by 250mm and 5 mu m (different batches of the same manufacturer).
Example 11
This example differs from example 1 only in that: in the detection conditions of high performance liquid chromatography, the chromatographic column is Waters Symmetry C18.6 mm × 250mm,5 μm (different manufacturers).
Test example 1
Test of system suitability
Sample preparation:
LLTS-M3I1 control stock solution: about 2mg of the LLTS-M3I1 control was weighed precisely, placed in a 100ml measuring flask, dissolved in a solvent and diluted to the mark, and shaken up (20. Mu.g/ml). Wherein, the sample weight is 2.010mg. LLTS-M3I1 localization solution: accurately weighing 5ml of LLTS-M3I1 reference stock solution, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking up (10 μ g/ml). LLTS-M3I2 control stock solution: about 2mg of the LLTS-M3I2 control sample is precisely weighed, placed in a 25ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (80 mug/ml). Wherein, the sample amount is 2.002mg. LLTS-M3I2 localization solution: the LLTS-M3I2 reference stock solution is precisely measured by 5ml, placed in a 10ml measuring flask, diluted to the scale with the solvent and shaken up (40 mug/ml). LLTS-M3I3 control stock solution: about 2mg of the LLTS-M3I3 control sample is precisely weighed, placed in a 100ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (20 mug/ml). Wherein, the sample weight is 2.004mg. LLTS-M3I3 localization solution: the LLTS-M3I3 reference stock solution is precisely measured by 5ml, placed in a 100ml measuring flask, diluted to the scale with the solvent and shaken up (10 mug/ml). LLTS-M3I4 control stock solution: about 2mg of the LLTS-M3I4 control sample is precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (100 mu g/ml). Wherein, the sample amount is 2.006mg. LLTS-M3I4 localization solution: precisely measure 3ml of LLTS-M3I4 control stock solution, place in a 10ml measuring flask, dilute to the mark with solvent, and shake up (30. Mu.g/ml). Test solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up (1 mg/ml). Wherein the sample weight is 0.01006g. Control solution: 1ml of the test solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with the solvent and shaken up (10. Mu.g/ml). System applicability solution: taking about 10mg of the product, placing into a 10ml measuring flask, adding appropriate amount of solvent to dissolve, adding 0.5ml of LLTS-M3I1 control stock solution, 1ml of LLTS-M3I2 control stock solution, 0.5ml of LLTS-M3I3 control stock solution, and 0.3ml of LLTS-M3I4 control stock solution precisely, diluting with solvent to scale, and shaking uniformly (about 1mg/ml of LLTS-M3, about 1 μ g/ml of LLTS-M3I1, about 8 μ g/ml of LLTS-M3I2, about 1 μ g/ml of LLTS-M3I3, and about 3 μ g/ml of LLTS-M3I 4). Blank solution: and (3) acetonitrile.
A detection step: taking 10 mu l of each of blank solution, control solution, system applicability solution, test sample solution and impurity positioning solution, injecting samples, detecting, recording chromatogram, injecting samples according to the sequence of table 1, and detecting according to the detection conditions in examples 1-11. Chromatograms of each solution obtained under the conditions of example 1 are shown in fig. 1 to 7 in this order.
TABLE 1 sample introduction sequence and requirements
| Sequence of | Sample name | Number of |
| 1 | |
1 |
| 2 | |
1 |
| 4 | Control solution | Continuous 5 |
| 5 | |
1 |
| 6 | LLTS- |
1 |
| 7 | LLTS- |
1 |
| 8 | LLTS- |
1 needle |
| 9 | LLTS- |
1 needle |
The results were analyzed as follows:
TABLE 2 System suitability test results-relative retention time
TABLE 3 System applicability test results-number of theoretical plates
TABLE 4 System applicability test results-tailing factor
TABLE 5 System suitability test results-degree of separation
From the above results, it can be seen that: under each condition, the blank solution does not interfere with the determination of main components and impurities; in the system applicability solution, the separation degrees between the main peak and the known impurities and between each known impurity and the adjacent impurities are not less than 1.5, and the number of the main peak and each impurity theoretical plate is not less than 5000; the tailing factors are not more than 2.0; therefore, the detection method of the invention can stably and effectively detect the related substances of the LLTS-M3.
Test example 2
A sensitivity assay comprising the steps of:
(1) Sample preparation
Solvent: acetonitrile; blank solution: a solvent; system applicability solution: the "system suitability solution" prepared under the section of "test example 1" was used. LLTS-M3 control stock solution: taking about 10mg of LLTS-M3 reference substance, precisely weighing, placing in a 10ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking up (1 mg/ml). Wherein the sample weight is 0.01027g. LLTS-M3 control solution: 1ml of LLTS-M3 reference stock solution is precisely weighed, placed in a 100ml measuring flask, diluted to the scale with a solvent and shaken up (10 mug/ml). LLTS-M3 control dilutions: 4ml of the LLTS-M3 control solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale with the solvent and shaken up (4 mug/ml). LLTS-M3I1 control stock solution: about 2mg of the LLTS-M3I1 control sample is precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to the scale by adding a solvent, and shaken up (100 mu g/ml). Wherein, the sample weight is 2.095mg. LLTS-M3I1 solution: precisely measuring 1ml of LLTS-M3I1 reference substance stock solution, placing the LLTS-M3I1 reference substance stock solution into a 10ml measuring flask, diluting the LLTS-M3I1 reference substance stock solution to a scale with a solvent, and shaking up; precisely measure 5ml, put into a 10ml measuring flask, dilute to the mark with solvent, shake up (5 mug/ml). LLTS-M3I2 control stock solution: taking about 2mg of LLTS-M3I2 reference substance, precisely weighing, placing in a 20ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking up (100 μ g/ml). Wherein, the sample weight is 2.009mg. LLTS-M3I2 solution: precisely measuring 1ml of LLTS-M3I2 reference substance stock solution, placing the LLTS-M3I2 reference substance stock solution in a 10ml measuring flask, diluting the LLTS-M3I2 reference substance stock solution to a scale with a solvent, and shaking up; 3ml of the solution is precisely measured, placed in a 10ml measuring flask, diluted to the mark with the solvent and shaken up (3. Mu.g/ml). LLTS-M3I3 control stock solution: taking about 2mg of LLTS-M3I3 reference substance, precisely weighing, placing in a 20ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking up (100 μ g/ml). Wherein, the sample weight is 2.024mg. LLTS-M3I3 control solution: precisely measuring 1ml of LLTS-M3I3 reference substance stock solution, placing the LLTS-M3I3 reference substance stock solution in a 10ml measuring flask, diluting the LLTS-M3I3 reference substance stock solution to a scale with a solvent, and shaking up; precisely measure 5ml, put into a 10ml measuring flask, dilute to the mark with solvent, shake up (5 mug/ml). LLTS-M3I4 control stock solution: about 2mg of LLTS-M3I4 control was weighed precisely, placed in a 20ml measuring flask, dissolved in solvent and diluted to the mark, shaken up (100. Mu.g/ml). Wherein, the sample weight is 2.037mg. LLTS-M3I4 control solution: precisely measuring 1ml of LLTS-M3I4 reference stock solution, placing in a 10ml measuring flask, diluting with solvent to scale, shaking up, precisely measuring 2ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking up (2 μ g/ml). Quantitative limiting solution: precisely measuring 1ml of LLTS-M3 reference substance diluent, 1ml of LLTS-M3I1 reference substance solution, 1ml of LLTS-M3I2 reference substance solution, 0.8ml of LLTS-M3I3 reference substance solution and 2ml of LLTS-M3I4 reference substance solution respectively, putting the solution in the same 10ml measuring flask, diluting the solution to a scale with a solvent, and shaking up (S/N is approximately equal to 10). Preparing a detection limiting solution: precisely measuring 3ml of the quantitative limiting solution, placing the quantitative limiting solution into a 10ml measuring flask, diluting the quantitative limiting solution to a scale mark with a solvent, and shaking up (S/N is approximately equal to 3).
(2) High performance liquid chromatography detection conditions
Same as example 1
(3) Detection step
Taking 10 μ l of each of blank solution, system applicability solution, reference solution, quantitative limit solution and detection limit solution, performing sample injection detection, recording chromatogram, and performing sample injection in the sequence shown in Table 6.
TABLE 6 sample introduction sequence and frequency requirements
| Sequence of | Sample name | Number of |
| 1 | |
1 |
| 2 | |
1 |
| 3 | |
6 |
| 4 | |
2 needles |
(4) Analysis results
The results of the sensitivity tests are shown in tables 7 to 10.
TABLE 7 System suitability solution test results
TABLE 8 LLTS-M3I1 quantitation limit test results
TABLE 9 LLTS-M3I2 quantitation limit test results
TABLE 10 LLTS-M3I3 quantitation Limit test results
TABLE 11 LLTS-M3I4 quantitation limit test results
TABLE 12 LLTS-M3 quantitation limit test results
TABLE 13 sensitivity test results
Calculating the formula:
limit of quantitation/limit of detection concentration (μ g/ml) = sample amount × impurity content/dilution multiple
Quantitative limit/detection limit (ng) = concentration x sample injection amount
The quantitative limit accounts for the ratio of the sample (%) = the quantitative limit concentration/the sample concentration x 100%
From the sensitivity test results, it is found that: the quantitative limiting solution is continuously tested for 6 times, the LLTS-M3 and the RSD of each impurity peak area are not more than 10.0 percent, and the retention time RSD is not more than 2.0 percent; the quantification limits were all less than the reporting limit (0.05% of the test article concentration).
Test example 3
A linearity test comprising the steps of:
(1) Sample preparation
Reference is made to test example 1 for LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, and LLTS-M3I4 reference stock solutions; LLTS-M3I1 solution, LLTS-M3I2 solution, LLTS-M3I3 solution, LLTS-M3I4 solution, and LLTS-M3 reference dilution see test example 2; solvent: acetonitrile; blank solution: a solvent; system applicability solution: the "system suitability solution" prepared under "example 1" was used. LLTS-M3 control stock solution: taking about 10mg of LLTS-M3 reference substance, precisely weighing, placing in a 10ml measuring flask, adding solvent to dissolve and dilute to scale, and shaking up (1 mg/ml). Wherein the sample weight is 0.01009g. LLTS-M3 control solution: 1ml of LLTS-M3 reference stock solution is precisely weighed, placed in a 100ml measuring flask, diluted to the scale with a solvent and shaken up (10 mug/ml). Quantitative limiting solution: precisely measuring 1ml of LLTS-M3 reference substance diluent, 1ml of LLTS-M3I1 solution, 1ml of LLTS-M3I2 solution, 0.8ml of LLTS-M3I3 solution and 2ml of LLTS-M3I4 solution, placing the solution in the same 10ml measuring flask, diluting the solution to the scale with a solvent, and shaking up. Impurity mixed mother liquor: precisely measuring 5ml of LLTS-M3I1 stock solution, 1.5ml of LLTS-M3 reference stock solution, 10ml of LLTS-M3I2 stock solution, 5ml of LLTS-M3I3 stock solution and 3ml of LLTS-M3I4 stock solution, putting the solutions in a same 50ml measuring flask, diluting the solutions to scales by using a solvent, and shaking the solutions uniformly. Linear solution 1: and (4) quantifying the limiting solution. Linear solution 2 (80%): precisely measuring 4ml of impurity mixed mother liquor, placing the impurity mixed mother liquor into a 10ml measuring flask, diluting the impurity mixed mother liquor to a scale with a solvent, and shaking up. Linear solution 3 (100%): precisely measuring 5ml of impurity mixed mother liquor, placing the impurity mixed mother liquor into a 10ml measuring flask, diluting the impurity mixed mother liquor to a scale with a solvent, and shaking up. Linear solution 4 (120%): precisely measuring 6ml of impurity mixed mother liquor, placing the impurity mixed mother liquor into a 10ml measuring flask, diluting the impurity mixed mother liquor to a scale with a solvent, and shaking up. Linear solution 5 (150%): precisely measuring 7.5ml of impurity mixed mother liquor, placing the impurity mixed mother liquor into a 10ml measuring flask, diluting the impurity mixed mother liquor to a scale with a solvent, and shaking up. Linear solution 6 (200%): mixing the impurities with the mother liquor.
(2) High performance liquid chromatography detection conditions
Same as example 1
(3) Detection step
And taking 10 mu l of blank solution, system applicability solution, LLTS-M1 control solution and each linear solution respectively, carrying out sample injection detection, and recording a chromatogram. Samples were injected in the order of Table 14.
TABLE 14 sample introduction sequence and requirements
| Sequence of | Sample name | Number of |
| 1 | |
1 |
| 2 | |
1 |
| 3 | |
Each 1 needle |
(4) Analysis results
The results of the linearity test are shown in tables 15-21.
TABLE 15 System applicability solution test results
TABLE 16 LLTS-M3I1 Linear test results
TABLE 17 LLTS-M3I2 Linear test results
TABLE 18 LLTS-M3I3 Linear test results
TABLE 19 LLTS-M3I4 Linear test results
TABLE 20 LLTS-M3 Linear test results
TABLE 21 correction factors for impurities of the substances involved
| Name(s) | LLTS-M3I1 | LLTS-M3I2 | LLTS-M3I3 | LLTS-M3I4 | LLTS-M3 |
| Slope of | 13.33335 | 18.63144 | 16.58886 | 18.71513 | 16.49472 |
| Correction factor | 1.24 | 0.89 | 0.99 | 0.88 | - |
The linear correlation coefficients r are all more than or equal to 0.990; the y-axis intercept is less than 25% of the peak area of the limit concentration of 100%, and the RSD of the response factor is not more than 10.0%. The linear regression equation was obtained by using the concentration C (. Mu.g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIGS. 8, 9, 10, 11, and 12. The linear relation between the concentrations of LLTS-M3I1, LLTS-M3I2, LLTS-M3I3 and LLTS-M3I4 and the peak area is good, and the method meets the verification requirement.
Test example 4
And (3) an accuracy test, which comprises the following steps:
(1) Sample preparation
Solvent: acetonitrile; blank solution: solvent system applicability solution: the "system suitability solution" prepared under the section of "test example 1" was used. Test solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up (1 mg/ml). Wherein the sample weight is 0.01013g. LLTS-M3I1 control stock solution: taking about 2mg of LLTS-M3I1 reference substance, precisely weighing, placing in a 20ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up. 2 parts of the mixture are prepared in parallel, 100 mu g/ml, and the sample weight is 2.037mg and 2.038mg. LLTS-M3I2 control stock solution: taking about 2mg of LLTS-M3I2 reference substance, precisely weighing, placing in a 25ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up. 2 parts of the mixture are prepared in parallel, 80 mu g/ml, and the sample weight is 2.023mg and 2.023mg. LLTS-M3I3 control stock solution: taking about 2mg of LLTS-M3I3 reference substance, accurately weighing, placing in a 20ml measuring flask, adding a solvent to dissolve and dilute to a scale mark, and shaking up. 2 portions of the mixture are prepared in parallel, 100 mu g/ml, and the sample weights are 2.012mg and 2.013mg. LLTS-M3I4 control stock solution: taking about 2mg of LLTS-M3I4 reference substance, accurately weighing, placing in a 20ml measuring flask, adding a solvent to dissolve and dilute to a scale mark, and shaking up. 2 parts of the mixture are prepared in parallel, 100 mu g/ml, and the sample weights are 2.036mg and 2.036mg. Impurity mixed mother liquor: precisely measuring 2ml of LLTS-M3I1 reference substance storage solution, 2ml of LLTS-M3I3 reference substance storage solution and 6ml of LLTS-M3I4 reference substance storage solution, placing the solutions in the same 100ml measuring flask, diluting with solvent to scale, and shaking up. The two solutions are prepared in parallel in one-to-one correspondence with the stock solutions. Control solution: precisely measuring 5ml of impurity mixed mother liquor and 1ml of LLTS-M3I2 reference stock solution, placing the mixture in the same 10ml measuring flask, diluting the mixture to the scale with a solvent, and shaking up. And 2 parts of the mixed mother liquor is prepared in parallel in a one-to-one correspondence with the impurity mixed mother liquor. And (4) preparing a recovery rate solution (taking the impurity mixed mother liquor-1). 50% solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding appropriate amount of solvent for dissolving, accurately adding 2.5ml of impurity mixed mother liquor and 0.5ml of LLTS-M3I2 stock solution, diluting with solvent to scale, and shaking. 3 parts of the mixture are prepared in parallel, and the sample weights are 0.01020g, 0.01020g and 0.01020g respectively. 100% solution: precisely weighing about 10mg of the product, placing the product in a 10ml measuring flask, adding a proper amount of solvent for dissolving, precisely adding 5ml of impurity mixed mother liquor and 1ml of LLTS-M3I2 stock solution, diluting with the solvent to scale, and shaking uniformly. 3 parts of the mixture were prepared in parallel, and the sample weights were 0.01011g, 0.01011g and 0.01011g, respectively. 150% solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding appropriate amount of solvent for dissolving, accurately adding 7.5ml of impurity mixed mother liquor and 1.5ml of LLTS-M3I2 stock solution, diluting with solvent to scale, and shaking. 3 parts of the mixture are prepared in parallel, and the sample weights are 0.01035g, 0.01014g and 0.01014g respectively.
(2) High performance liquid chromatography detection conditions
Same as example 1
(3) Detection step
Taking 10 mul of blank solution, system applicability solution, reference solution, quantitative limit solution and detection limit solution respectively, carrying out sample introduction and detection, and recording a chromatogram map. Samples were injected in the order of Table 22.
TABLE 22 sample introduction sequence and requirements
| Sequence of | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
1 |
| 3 | Control solution-1 | 5 |
| 4 | Control solution-2 | 2 |
| 5 | |
1 |
| 6 | 50% -1 |
1 |
| 7 | 50% -2 |
1 |
| 8 | 50% -3 |
1 needle |
| 9 | 100% -1 |
1 |
| 10 | 100% -2 |
1 needle |
| 11 | 100% -3 |
1 |
| 12 | 150% -1 |
1 needle |
| 13 | 150% -2 |
1 |
| 14 | 150% -3 |
1 needle |
Note: calculating the formula: percent recovery = (measured amount-content in sample)/addition × 100%
(4) Analysis results
The results of the accuracy tests are shown in tables 23-27.
TABLE 23 System applicability solution test results
TABLE 24 LLTS-M3I1 recovery test results
TABLE 25 LLTS-M3I2 recovery test results
TABLE 26 LLTS-M3I3 recovery test results
TABLE 27 LLTS-M3I4 recovery test results
The limit concentration of the impurities is taken as 100%, the impurities are quantitatively added into the sample according to three concentrations of 50%, 100% and 150%, under each concentration level, the recovery rate of the impurities is within the range of 90.0-110.0%, the recovery rate RSD of 9 parts is less than 5.0%, and the accuracy is good.
Test example 5
The repeatability test comprises the following steps:
(1) Sample preparation
Solvent: acetonitrile; blank solution: a solvent; system applicability solution: the "system suitability solution" prepared under "example 1" was used. Test solution: weighing about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to the scale, and shaking up. 6 parts of the mixture are prepared in parallel, and the sample weights are respectively 0.01024g, 0.01016g, 0.01019g, 0.01001g, 0.01011g and 0.01014g. Control solution: precisely measuring 1ml of the test solution, placing the test solution into a 100ml measuring flask, diluting the test solution to a scale with a solvent, and shaking up. 6 parts are prepared in parallel.
(2) High performance liquid chromatography detection conditions
Same as example 1
(3) Detection step
And taking 10 mu l of each of the blank solution, the system applicability solution, the control solution and the test solution, carrying out sample introduction detection, and recording a chromatogram. Samples were injected in the order of Table 28.
TABLE 28 sample introduction sequence and requirements
| Sequence of events | Sample (I) | Number of |
| 1 | |
1 |
| 2 | |
1 |
| 4 | 1 to 6 portions of test solution | Each 1 |
| 5 | Control solutions-1 to 6 | Each 1 needle |
(4) Analysis results
The results of the reproducibility tests are shown in tables 29 to 30.
TABLE 29 System applicability solution test results
TABLE 30 repeatability test results
The results show that 6 samples have basically consistent impurity detection, no obvious change in total impurities and impurity number, and good repeatability.
Test example 6
(1) The solution stability test comprises the following steps:
system applicability solution: the "system suitability solution" prepared under the section of "test example 1" was used.
Sample preparation
Solvent: acetonitrile; blank solution: a solvent; the preparation of the test solution and the control solution was the same as that of the solution in test example 1. Preparing the test solution and the control solution in two parts in parallel, taking the control solution-1 to investigate the stability of the system, taking the control solution-2 to investigate the stability of the control solution, and taking the test solution-2 to investigate the stability of the test solution.
(2) High performance liquid chromatography detection conditions
Same as example 1
(3) Detection step
Taking 10 mul of blank solution, system applicability solution, control solution and sample solution respectively, sampling and detecting, and recording chromatogram. Samples were injected in the order of Table 31.
TABLE 31 sample introduction sequence and requirements
(4) Analytical results the results of the solution stability test are shown in tables 32 to 34.
TABLE 32 System applicability solution test results
TABLE 33 test article solution stability results
TABLE 34 control solution stability test results
Placing the test solution at room temperature for 40 hours until no impurities LLTS-M3I1 and LLTS-M3I3 are detected; the content average value of the impurities LLTS-M3I2 at each time point is 0.43 percent, the RSD is 0.87 percent, and is less than 10.0 percent; the content average value of the impurities LLTS-M3I4 at each time point is 0.40 percent, the RSD is 0.58 percent and is less than 20.0 percent; the content of the maximum unknown single impurity at each time point is 0.08 percent, the RSD is 2.39 percent and is less than 20.0 percent; the content average value of all impurities at each time point is 1.05 percent, the RSD is 1.86 percent and is less than 5.0 percent; the number of impurities has no obvious change, which indicates that the sample solution is stable after being placed at room temperature for 40 hours. When the control solution is placed at room temperature for 41 hours, the RSD of the main peak area is 0.72 percent and is not more than 5.0 percent, which shows that the control solution is stable when placed at room temperature for 41 hours.
In conclusion, the detection method provided by the embodiment of the invention has good specificity, repeatability and accuracy on the basis of ensuring high-efficiency separation of various related substances and effective components, thereby better realizing quality control of LLTS-M3.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting LLTS-M3 and related substances thereof is characterized by comprising the following steps: detecting the test 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 procedure is as follows:
2. the method of claim 1, wherein the LLTS-M3 has the formula:
preferably, the volume ratio of mobile phase a and mobile phase B is maintained constant during the elution procedure for time periods of 0-5min and 40-45 min.
3. The method for detecting LLTS-M3 and related substances according to claim 2, wherein the elution procedure of said HPLC is as follows:
preferably, the elution procedure of the high performance liquid chromatography is:
4. the method of claim 2, wherein the related substances include LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, LLTS-M3I4 and LLTS-M2, and the structures of LLTS-M3I1, LLTS-M3I2, LLTS-M3I3, LLTS-M3I4 and LLTS-M2 are sequentially represented by formula I-formula V:
5. the method of claim 2, wherein the test solution and the control solution of the substances involved in the assay are acetonitrile.
6. The method for detecting LLTS-M3 and related substances according to any one of claims 1 to 5, wherein said gradient elution has a flow rate of 0.9-1.1 mL/min, preferably 1.0mL/min.
7. The method for detecting LLTS-M3 and related substances according to any one of claims 1 to 5, wherein the HPLC uses octadecylsilane chemically bonded silica, and the column temperature is 35-45 ℃ during detection;
preferably, the high performance liquid chromatography adopts Agilent5TC-C18 (2), 4.6mm x 250mm and 5 μm of chromatographic column, and the temperature of the chromatographic column is 40 ℃ when detecting.
8. The method for detecting LLTS-M3 and related substances according to any one of claims 1 to 5, wherein the content of related substances in said test solution is calculated by a self-control method;
preferably, the method for calculating the content of the related substance includes: respectively injecting the test solution and the reference solution into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas, and calculating the content of related substances in the test solution by using a self-contrast method.
9. The method for detecting LLTS-M3 and related substances according to any one of claims 1 to 3, wherein the HPLC column is an octadecylsilane chemically bonded silica gel column, and the column temperature of said column is 35-45 ℃ and the flow rate of said gradient elution is 0.9-1.1 mL/min during detection.
10. Use of the method of LLTS-M3 and related substances according to any of claims 1 to 9 for the quality control of LLTS-M3 intermediates.
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