CN115097035A

CN115097035A - LLTS intermediate and detection method and application of related impurities thereof

Info

Publication number: CN115097035A
Application number: CN202210706463.4A
Authority: CN
Inventors: 王潞娜; 王红芳; 张辉; 宋荧荧; 关茹群
Original assignee: Shanxi Zhendong Pharmaceutical Co ltd
Current assignee: Shanxi Zhendong Pharmaceutical Co ltd
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2022-09-23
Anticipated expiration: 2042-06-21
Also published as: CN115097035B

Abstract

The invention discloses a detection method and application of LLTS intermediate and related impurities thereof, which comprises the following steps: detecting the test solution by high performance liquid chromatography; wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-232 nm; a mobile phase A: water, mobile phase B: acetonitrile; the elution mode is a gradient elution of a specific procedure. According to the synthesis process route and the impurity properties of the LLTS bulk drug, the LLTS intermediate and the related impurities thereof are determined, and a related substance analysis and detection method is drawn up, so that one-time high-efficiency separation is realized on the basis of ensuring high-efficiency separation of each impurity and effective components, and the analysis and detection have good specificity, durability and sensitivity, thereby better realizing the quality control of the LLTS intermediate.

Description

LLTS intermediate and detection method and application of related impurities thereof

Technical Field

The invention relates to the technical field of chemical drug analysis, in particular to a LLTS intermediate and a detection method and application of related impurities thereof.

Background

LLTS bulk drug is larotaxel, and the structural formula is

LLTS is taken as a new generation of taxane compound, in vitro tests, larotaxel has 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.

There are two main intermediates in the production process of LLTS bulk drug: LLTS-M1 and LLTS-M2, wherein the LLTS-M1 has a structural formula

The structural formula of LLTS-M2 is

LLTS-M1 and LLTS-M2 are used as LLTS bulk drug intermediates, and if the LLTS bulk drug intermediates contain impurities, the impurities or converted substances of the impurities can enter subsequent reactions along with the LLTS bulk drug intermediates in the bulk drug production process, so that the production quality of the bulk drugs is influenced. Thus, it is important for impurity control in LLTS-M2. When LLTS-M1 and LLTS-M2 are used as intermediates, the impurities contained in the intermediates have certain similarity with the intermediates in structure, separation and detection are not easy, and further, the impurity control is difficult, and the detection methods of the LLTS intermediates and related impurities are not loaded in the current 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 intermediate and the impurities existing in the intermediate is urgently needed.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a detection method and application of LLTS intermediate and related impurities thereof so as to improve the technical problem.

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 high performance liquid chromatography; wherein, the detection conditions of the high performance liquid chromatography comprise: detection wavelength: 228-; mobile phase A: water, mobile phase B: acetonitrile; the elution mode is gradient elution, and the elution procedure is as follows:

alternatively, the LLTS intermediate is LLTS-M1 or LLTS-M2; the structural formula of LLTS-M1 is:

the structural formula of LLTS-M2 is:

alternatively, the volume ratio of mobile phase a and mobile phase B is maintained constant for time periods of 0-5min and 25-30min during the elution procedure.

Alternatively, when the LLTS intermediate is LLTS-M1, the elution procedure for high performance liquid chromatography is:

when the LLTS intermediate is LLTS-M2, the elution procedure of high performance liquid chromatography is as follows:

alternatively, when the LLTS intermediate is LLTS-M2, the elution procedure for high performance liquid chromatography is:

in a second aspect, the invention also provides the application of the detection method of the LLTS intermediate and related impurities 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 the LLTS bulk drug, the LLTS intermediate and the related impurities thereof are determined, and a related substance analysis and detection method is drawn up, so that one-time high-efficiency separation is realized on the basis of ensuring high-efficiency separation of the impurities and the effective components, and the analysis and detection has good specificity, durability and sensitivity, thereby better realizing the quality control of the LLTS intermediate.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used 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 for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a chromatogram of a blank solution of example 1 when the LLTS intermediate is a LLTS-M1 intermediate;

FIG. 2 is a chromatogram of the system suitability solution of example 1 when the LLTS intermediate is a LLTS-M1 intermediate;

FIG. 3 is a chromatogram of the 10-DAB solution of example 1 when the LLTS intermediate is the LLTS-M1 intermediate;

FIG. 4 is a chromatogram of the LLTS-M1I1 solution of example 1 when the LLTS intermediate is the LLTS-M1 intermediate;

FIG. 5 is a chromatogram of the test solution of example 1 when the LLTS intermediate is the LLTS-M1 intermediate;

FIG. 6 is a chromatogram of the test solution of comparative example 1 when the LLTS intermediate is LLTS-M1 intermediate;

FIG. 7 is a graph showing the linear relationship of 10-DAB provided by the experimental examples when the LLTS intermediate is LLTS-M1 intermediate;

FIG. 8 is a graph showing the linear relationship of LLTS-M1 provided in the experimental examples when the LLTS intermediate is LLTS-M1 intermediate;

FIG. 9 is a graph showing the linear relationship of LLTS-M1I1 provided in the experimental examples when the LLTS intermediate is LLTS-M1 intermediate;

FIG. 10 is a chromatogram of a blank solution of example 12 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 11 is a chromatogram of the system suitability solution of example 12 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 12 is a chromatogram of the LLTS-M1 solution of example 12 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 13 is a chromatogram of the LLTS-M2I1 solution of example 12 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 14 is a chromatogram of the test solution of example 12 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 15 is a chromatogram of the test solution of comparative example 2 when the LLTS intermediate is the LLTS-M2 intermediate;

FIG. 16 is a graph showing the linear relationship of LLTS-M1 provided in the experimental examples when the LLTS intermediate is LLTS-M2 intermediate;

FIG. 17 is a graph showing the linear relationship of LLTS-M2 provided in the experimental examples when the LLTS intermediate is LLTS-M2 intermediate;

FIG. 18 is a graph showing the linear relationship of LLTS-M2I1 provided in the test example when the LLTS intermediate is LLTS-M2 intermediate.

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 principle of verification of chemical drug quality control analysis method, the technical guide principle of standardized process established by chemical drug quality standard, the technical guide principle of chemical drug impurity research, the technical guide principle of chemical drug residual solvent research and the related guide principle in the appendix of the current edition of the pharmacopoeia of the people's republic of China.

The following is a detailed description of the LLTS intermediate and the detection method and application of related impurities thereof proposed by the present invention.

At present, the pharmacopoeia of various countries and related documents do not relate to a detection method of related substances of LLTS intermediates and impurities thereof, and based on the detection method, the inventor determines the LLTS intermediates and the impurities thereof according to the related requirements of normalized process technical guidelines established by chemical drug quality standards and chemical drug impurity research technical guidelines and according to the synthesis process route and the impurity properties of the product, and provides the following detection method through a large number of researches and practices.

Some embodiments of the present invention provide a method for detecting LLTS intermediates and related impurities thereof, comprising the steps of: 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-; 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 method realizes one-time high-efficiency separation of related substances of controlled impurities in the LLTS intermediate, has strong specificity, good durability and high sensitivity, and can be used for quality control of the LLTS intermediate. In order to achieve better separation detection, in some embodiments, the volume ratio of mobile phase a to mobile phase B is maintained constant during the elution process for 0-5min and 25-30 min.

It is noted that the LLTS intermediate is mainly LLTS-M1 or LLTS-M2; the structural formula of LLTS-M1 is:

the structural formula of LLTS-M2 is:

in summary, when the selection of the LLTS intermediate is different, the corresponding impurities in the intermediate are also different, and the corresponding detection methods are slightly different in the parameters of the elution procedure, that is, when the LLTS intermediate is LLTS-M1 and the LLTS intermediate is LLTS-M1, the related impurities are 10-DAB and LLTS-M1I1, 10-DAB and the structural formula of LLTS-M1I1 are as follows:

in the detection method, the elution procedure of the high performance liquid chromatography is as follows:

preferably, when the LLTS intermediate is LLTS-M1, the elution procedure of high performance liquid chromatography is as follows:

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:

preferably, when the LLTS intermediate is LLTS-M2, the elution procedure of high performance liquid chromatography is as follows:

further, in some embodiments, the flow rate of the gradient elution is from 0.8mL/min to 1.2mL/min, preferably 1.0 mL/min.

In some embodiments, 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 25-35 ℃;

specifically, the high performance liquid chromatography can adopt Agilent SB-C184.6mm × 250mm, 5 μm, and the column temperature of the chromatographic column is 30 ℃ during detection.

In some embodiments, the content of the relevant impurities in the test solution is calculated by a self-comparison method; specifically, the method for calculating the content of the impurities includes: 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 impurities in the test solution.

Further, in some embodiments, the test solution and the control solution of the impurities used in the assay process are acetonitrile-water, ethanol-water solutions, and when the LLTS intermediate is LLTS-M1, the volume ratio of acetonitrile to water is 45-55: 45-55, preferably 50: 50; when the LLTS intermediate is LLTS-M2, the volume ratio of ethanol to water is 88-92: 12-8, preferably 90: 10.

for example, when the LLTS intermediate is LLTS-M1, the sample was formulated as follows:

10-DAB control solution: about 2mg of 10-DAB is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale mark by a solvent, and shaken up (200 mug/ml). 10-DAB localization solution: 2ml of 10-DAB reference solution is precisely measured, placed in a 20ml measuring flask, diluted to the scale with solvent and shaken up (20 mug/ml). LLTS-M1I1 control solution: about 2mg of LLTS-M1I1 was weighed out precisely, placed in a 20ml measuring flask, dissolved and diluted to the mark with solvent, shaken well (100. mu.g/ml). LLTS-M1I1 localization solution: 0.3ml of LLTS-M1I1 reference solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale with solvent and shaken up (10 mug/ml). Test solution: about 10mg of LLTS-M1 was weighed out precisely, placed in a 10ml measuring flask, dissolved and diluted to the mark with solvent, and shaken 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 (100. mu.g/ml). System applicability solution: taking appropriate amount of 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 μ g, and LLTS-M1I 13 μ g per 1 ml.

When the LLTS intermediate is LLTS-M2, the sample was prepared by the following steps:

LLTS-M1 control solution: about 5mg of LLTS-M1 was weighed out precisely, placed in a 100ml measuring flask, dissolved and diluted to the mark by adding solvent, and shaken up (50. mu.g/ml). LLTS-M1 localization solution: 2ml of the LLTS-M1 control solution is precisely measured, placed in a 20ml measuring flask, diluted to the mark with the solvent and shaken up (5. mu.g/ml). LLTS-M2I1 control solution: about 4mg of the LLTS-M2I1 control was weighed precisely, placed in a 20ml measuring flask, dissolved and diluted to the mark with solvent, and shaken well (200. mu.g/ml). LLTS-M2I1 localization solution: 2ml of the reference LLTS-M2I1 solution was measured accurately, placed in a 20ml measuring flask, diluted to the mark with solvent and shaken up (20. mu.g/ml). LLTS-M3 control solution: about 10mg of LLTS-M3 was weighed out precisely, placed in a 10ml measuring flask, dissolved and diluted to the mark with solvent, shaken up (1 mg/ml). LLTS-M3 localization solution: 1ml of LLTS-M3 control solution is precisely measured, placed in a 100ml measuring flask, diluted to the mark with solvent and shaken up (10. mu.g/ml). Test solution: about 10mg of the product is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale by a solvent, and shaken 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, precisely adding LLTS-M1 reference solution and LLTS-M2I1 reference solution each 1ml, diluting with solvent to scale, and shaking up (LLTS-M21 mg/ml, LLTS-M15 μ g/ml, LLTS-M2I120 μ g/ml).

Some embodiments of the present invention specifically provide a method for detecting LLTS-M1 and related impurities, comprising: detecting the test solution by high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: a chromatographic column: agilent SB-C184.6mm × 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: 30 ℃, sample introduction: 10. mu.l, solvent: acetonitrile-water (50: 50).

Preparing a test solution: about 10mg of the LLTS-M1 intermediate is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale with solvent, and shaken up (1 mg/ml).

Some embodiments of the present invention specifically provide a method for detecting LLTS-M2 and related impurities, comprising: detecting the test solution by adopting high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: a chromatographic column: agilent SB-C184.6mm × 250mm, 5 μm; a 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: 30 ℃, sample introduction: 10. mu.l, solvent: ethanol-water (90: 10).

Preparing a test solution: about 10mg of the LLTS-M2 intermediate is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale with solvent, and shaken up (1 mg/ml).

Some embodiments of the present invention also provide applications of the detection methods of the LLTS intermediates and their related impurities in the above embodiments in quality control of the LLTS intermediates.

The features and properties of the present invention are described in further detail below with reference to examples.

The test solution in the following embodiments is mainly prepared from LLTS-M1 or LLTS-M2 and corresponding impurities, and during actual detection, the LLTS-M1 or LLTS-M2 to be detected is dissolved and diluted to prepare the test solution.

Example 1

The present embodiment provides a method for detecting an LLTS intermediate (LLTS-M1), which includes:

preparing a test solution: taking about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, adding a solvent to dissolve and dilute to a scale, and shaking up to obtain a test solution (1 mg/ml).

Detecting the test solution by high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: a chromatographic column: agilent SB-C184.6mm × 250mm, 5 μm; mobile phase A: water, mobile phase B: acetonitrile; gradient elution was performed as follows:

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 25 ℃.

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 35 ℃.

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 232 nm.

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 differs from example 1 only in that: the flow rate is 1.1ml/min under the detection condition of high performance liquid chromatography.

Example 8

This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, the volume ratio of the mobile phase A to the mobile phase B is maintained at 62:38 within 0-5 min; the volume ratio of mobile phase A and B is maintained at 62:38 for 25-30 min.

Example 9

This example only differs from example 1 in that: in the detection condition of the high performance liquid chromatography, the volume ratio of the mobile phase A to the mobile phase B is maintained at 58:42 within 0-5 min; the volume ratio of mobile phase A and B is maintained at 58:42 for 25-30 min.

Example 10

This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, a chromatographic column is Agilent ZORBAX XDB-C184.6mm multiplied by 250mm, and is 5 mu m (different batches of the same manufacturer).

Example 11

This example differs from example 1 only in that: in the detection condition of the high performance liquid chromatography, a chromatographic column is Feilomen Titank C184.6mm multiplied by 250mm and 5 mu m (different batches of different manufacturers).

Comparative example 1

Detecting a LLTS intermediate (LLTS-M1) test 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 suitability test (LLTS-M1 test)

Sample preparation:

10-DAB control solution: about 2mg of 10-DAB is precisely weighed, placed in a 10ml measuring flask, dissolved by a solvent and diluted to the scale mark, and shaken up (200 mug/ml) (the sample weight is 2.065 mg). 10-DAB localization solution: 2ml of 10-DAB reference solution is precisely measured, placed in a 20ml measuring flask, diluted to the scale with solvent and shaken up (20 mug/ml). LLTS-M1I1 control solutions: about 2mg of LLTS-M1I1 was weighed out precisely, placed in a 20ml measuring flask, dissolved and diluted to the mark with solvent, shaken up (100. mu.g/ml) (sample weight 2.022 mg). LLTS-M1I1 localization solution: 0.3ml of LLTS-M1I1 reference solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale with solvent and shaken up (3 mug/ml). Test solution: about 10mg of LLTS-M1 was weighed out precisely, placed in a 10ml measuring flask, dissolved and diluted to the mark with solvent, and shaken up (1mg/ml) (weighing 10.12 mg). 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 (100. mu.g/ml). System applicability solution: taking appropriate amount of 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 μ g, and LLTS-M1I 13 μ g per 1 ml. Blank solution: solvent (acetonitrile-water (50: 50)).

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 sequence in fig. 1-5. The chromatogram obtained under the conditions of comparative example 1 is shown in FIG. 6, and the separation degree of LLTS-M1 from adjacent unknown impurities in the chromatogram of the LLTS-M1 test sample is less than 1.5.

TABLE 1 sample introduction sequence and requirements

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

TABLE 6 test article impurity detection results

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 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 tailing factors are less than 2.0; the number of each impurity, total impurities and impurities in the test solution is detected to have no obvious change; under each condition, the contrast solution is continuously injected with 5 needles, and the peak area RSD is less than 5.0 percent; therefore, the detection method of the embodiment can stably and effectively detect the related substances of the LLTS-M1.

Test example 2

Sensitivity test (LLTS-M1), comprising the following steps:

(1) sample preparation

Solvent: acetonitrile-water (50: 50); blank solution: a solvent; LLTS-M1 stock solution: about 10mg of LLTS-M1 control sample was weighed precisely, placed in a 10ml measuring flask, dissolved and diluted to the mark with solvent, shaken up to obtain LLTS-M1 stock solution (1mg/ml) (weighing 9.98 mg). Control solution: 1ml of LLTS-M1 control stock solution is precisely measured, placed in a 100ml measuring flask, diluted to the mark with solvent and shaken up (10. mu.g/ml). 10-DAB reference stock solution: taking about 2mg of 10-DAB reference substance, accurately weighing, placing in a 10ml measuring flask, dissolving with solvent, diluting to scale, and shaking up. (200. mu.g/ml) (sample weight 2.052 mg); 10-DAB solution: precisely measuring 1ml of 10-DAB reference substance stock solution, placing in a 10ml measuring flask, diluting with solvent to scale, shaking, precisely measuring 1ml, placing in a 20ml measuring flask, diluting with solvent to scale, and shaking (1 μ g/ml). LLTS-M1I1 control stock solution: about 2mg of the LLTS-M1I1 control sample was weighed precisely, placed in a 20ml measuring flask, dissolved and diluted to the mark with solvent, and shaken well (100. mu.g/ml) (sample weighing 2.001 mg). LLTS-M1I1 solution: 0.3ml of LLTS-M1I1 reference stock solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale with solvent and shaken up (3 mug/ml). Quantitative limiting solution: respectively precisely measuring 0.4ml of reference solution, 1.5ml of 10-DAB solution and 0.5ml of LLTS-M1I1 solution, putting the solutions into the same 10ml measuring flask, diluting the solutions to the scale by using a solvent, shaking up, precisely measuring 3ml of the solutions, putting the solutions into the 10ml measuring flask, diluting the solutions to the scale by using the solvent, shaking up, wherein S/N is more than or equal to 10. Preparing a detection limiting solution: precisely measuring 3ml of quantitative limiting solution, placing the quantitative limiting solution into a 10ml measuring flask, diluting the quantitative limiting solution to a scale with a solvent, and shaking up until S/N is more than or equal to 3.

(2) High performance liquid chromatography detection conditions

Same as example 1

(3) Detection step

Taking blank solution, system applicability solution, reference solution, quantitative limit solution and detection limit solution, respectively 10 μ l, sampling, detecting, recording chromatogram, and sampling according to the sequence of the following table.

TABLE 7 sample introduction sequence and frequency requirements

Sequence of	Sample name	Number of needles
			1	Blank solution	1 needle
2	System applicability solution	1 needle
			3	Control solution	5 needles
4	Quantitative limiting solution	6 needles
			5	Detection limiting solution	2 needles
6	Control solution	1 needle

(4) Analysis results

The results of the sensitivity tests are shown in tables 8 to 11.

TABLE 8LLTS-M1 quantitation limit test results

Name (R)	1	2	3	4	5	6	Average	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 910 DAB quantitation limit test results

Name(s)	1	2	3	4	5	6	Average	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 (R)	1	2	3	4	5	6	Average out	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

Calculating the formula:

limit of quantitation/limit of detection (μ g/ml) is the weight of sample x impurity content/dilution multiple

Quantitative limit/detection limit (ng) ═ concentration x sample volume

The quantitative limit accounts for the ratio (%) of the sample to the quantitative limit concentration/sample concentration × 100%

From the sensitivity test results, it is found that:

the quantitative limiting solution is tested continuously for 6 times, the LLTS-M1 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 quantitative limits are all less than the reported limit (0.05% of the test article 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: taking about 4mg of 10-DAB reference substance, accurately weighing, placing in a 20ml measuring flask, dissolving and diluting to the scale mark by using a solvent, and shaking up. (200. mu.g/ml) (weighing 4.052 mg); LLTS-M1I1 control solution: taking about 2mg of LLTS-M1I1 reference substance, accurately weighing, placing in a 20ml measuring flask, dissolving with solvent and diluting to scale, and shaking up. (100. mu.g/ml) (sample weight 2.051 mg); LLTS-M1 control stock solution: taking about 10mg of LLTS-M1 reference substance, precisely weighing, placing in a 10ml measuring flask, dissolving with solvent and diluting to scale, and shaking up. (1mg/ml) (sample weight 9.96 mg); LLTS-M1 control solution: precisely measuring 1ml of LLTS-M1 reference stock solution, placing in a 100ml measuring flask, diluting with solvent to scale, and shaking. (10. mu.g/ml); impurity mixed stock solution: precisely measuring 5ml of 10-DAB reference substance solution, 1.5ml of LLTS-M1I1 reference substance solution and 1.5ml of LLTS-M1 reference substance stock solution, placing in the same 25ml measuring flask, diluting with solvent to scale, and shaking. Linear solution 1: and (4) quantifying the limiting solution. Linear solution 2 (10%): precisely measuring 0.5ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 3 (20%): precisely measuring 1ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 4 (50%): 2.5ml of impurity mixed stock solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale by using a solvent and shaken up. Linear solution 5 (100%): precisely measuring 5ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 6 (200%): mixing the impurities with the stock solution.

(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, injecting a sample, detecting, and recording a chromatogram. Samples were injected in the order of the following table.

TABLE 12 sample introduction sequence and requirements

(4) Analysis results

The results of the linear tests are shown in tables 13-15.

TABLE 1310-DAB Linear test results

TABLE 14LLTS-M1 Linear test results

TABLE 15LLTS-M1I1 Linear test results

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 concentration C (μ g/mL) is used as the abscissa, and the corresponding peak area is used as the ordinate, so as to obtain a linear regression equation, and the linear relationship diagrams are shown in FIG. 7, FIG. 8 and FIG. 9. The linear relation between the concentrations of LLTS-M1, 10-DAB and LLTS-M1I1 and the peak area is good, and the method meets the verification requirement.

Test example 4

Accuracy test (LLTS-M1), comprising the following steps:

(1) sample preparation

Solvent: acetonitrile-water (50: 50); blank solution: a solvent; test solution: about 10mg of the product is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale by a solvent, and shaken up (1 mg/ml). 10-DAB control solution: about 10mg of 10-DAB reference substance is precisely weighed, placed in a 25ml measuring flask, dissolved by a solvent and diluted to the scale, and shaken up (2 parts are prepared in parallel, 400 mu g/ml) (the sample amount is 10.10mg and 10.25 mg). LLTS-M1I1 control stock solution: about 2mg of LLTS-M1I1 control sample is precisely weighed, placed in a 20ml measuring flask, dissolved and diluted to the scale with solvent, and shaken up (2 parts prepared in parallel, 100. mu.g/ml) (sample weights 2.069mg, 2.074 mg). Impurity mixed stock solution: precisely measuring 10ml of 10-DAB reference substance solution and 6ml of LLTS-M1I1 reference substance stock solution, placing in a 100ml measuring flask, diluting with solvent to scale, and shaking. And 2 parts of the solution is prepared in parallel in one-to-one correspondence with the stock solution. Control solution: precisely measuring 5ml of impurity mixed stock solution, placing the impurity mixed stock solution into the same 10ml measuring flask, diluting the impurity mixed stock solution to the scale with a solvent, and shaking up. And 2 parts of the mixed stock solution is prepared in parallel in one-to-one correspondence with the impurity mixed stock solution. Recovering solution (mixing impurity with stock solution-1 to prepare recovering solution); 50% solution: taking about 10mg of a test sample, accurately weighing, placing in a 10ml measuring flask, adding a proper amount of solvent for dissolving, accurately adding 2.5ml of impurity mixed stock solution, diluting to scale with the solvent, and shaking uniformly. 3 parts (sample weights 10.08mg, 10.10mg) were prepared in parallel. 100% solution: taking about 10mg of a sample, precisely weighing, placing in a 10ml measuring flask, adding a proper amount of solvent for dissolving, precisely adding 5ml of impurity mixed stock solution, diluting to scale with the solvent, and shaking up. 3 parts (sample amounts 10.37mg, 10.24mg, 10.10mg) were prepared in parallel. 150% solution: taking about 10mg of a sample, precisely weighing, placing in a 10ml measuring flask, adding a proper amount of solvent for dissolving, precisely adding 7.5ml of impurity mixed stock solution, diluting to scale with the solvent, and shaking up. 3 parts (sample amounts 10.04mg, 10.20mg) were prepared in parallel.

(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 the following table.

TABLE 16 sample introduction sequence and requirements

Sequence of events	Sample (I)	Number of needles
			1	Blank solution	1 needle
2	System applicability solution	1 needle
			3	Control solution-1	5 needles
4	Control solution-2	2 needles
			5	Test solution	1 needle
6	50% -1 solution	1 needle
			7	50% -2 solution	1 needle
8	50% -3 solution	1 needle
			9	Control solution-1	1 needle
10	100% -1 solution	1 needle
			11	100% -2 solution	1 needle
12	100% -3 solution	1 needle
			13	Control solution-1	1 needle
14	150% -2 solution	1 needle
			15	150% -3 solution	1 needle
16	Control solution-1	1 needle

Note: calculating the formula: percent recovery [% ] (measured amount-content in sample)/amount added × 100%

(4) Analysis results

The results of the accuracy tests are shown in tables 17-18.

TABLE 1710-DAB recovery test results

TABLE 18LLTS-M1I1 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%, the recovery rate of the impurities is within a range of 90.0-110.0% in each concentration level, the RSD (recovery yield) of 9 parts is less than 5.0%, and the accuracy is good.

Test example 5

Repeatability tests (LLTS-M1) comprising the following steps:

(1) sample preparation

Solvent: acetonitrile-water (50: 50); blank solution: a solvent; test solution: taking about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, dissolving and diluting to the scale mark by using a solvent, and shaking up. (6 parts, 1mg/ml) were prepared in parallel (sample weights 10.11mg, 10.26mg, 10.08mg, 10.03mg, 10.00mg, 10.35 mg); 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 (6 parts in parallel, 1. mu.g/ml).

(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 the following table.

TABLE 19 sample introduction sequence and requirements

(4) Analysis results

The results of the reproducibility test are shown in Table 20.

TABLE 20 repeatability test results

Test article	10-DAB(％)	LLTS-M1I1	Maximum unknown simple impurity	Total impurities (%)	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	1.45	0.20	0.07	1.98	/
RSD(％)	0.83	2.06	0.00	0.74	/

6 parts of test sample has basically consistent impurity detection, no obvious change of total impurities and impurity number and good repeatability.

Test example 6

(1) The solution stability test (LLTS-M1) comprising the steps of:

system applicability the solution is used under the item of system applicability example 1; the test solution and the control solution were prepared in the same manner as in example 1.

Sample preparation

Solvent: acetonitrile-water (50: 50); blank solution: a solvent; test solution 1: taking 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. (1mg/ml) (sample weight 10.15 mg); control solution 1: 11 ml of the test solution is precisely measured, placed in a 100ml measuring flask, diluted to the mark with the solvent and shaken up (1 mu g/ml). Sample solution 2: weighing 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. (1mg/ml) (sample weight 10.33 mg); control solution 2: 21 ml of the test solution is precisely measured, placed in a 100ml measuring flask, diluted to the scale with the solvent and shaken up (1. mu.g/ml).

(2) High performance liquid chromatography detection conditions

Same as example 1

(3) Detection step

TABLE 21 sample introduction sequence and requirements

(4) Analysis results

The results of the solution stability tests are shown in tables 22 and 23.

TABLE 22 test article solution stability results

Time	10-DAB(％)	LLTS-M1I1	Maximum unknown sheet	Total impurities (%)	Number of impurities
						0 hour	1.43	0.20	0.07	1.94	13
1 hour (h)	1.43	0.20	0.07	1.95	13
						2 hours	1.43	0.20	0.07	1.95	13
4 hours	1.43	0.20	0.07	1.96	13
						6 hours	1.43	0.20	0.07	1.96	13
8 hours	1.43	0.20	0.07	1.96	13
						10 hours	1.43	0.20	0.07	1.96	13
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
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	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 at room temperature for 48 hours, the number of single impurities, total impurities and impurities has no obvious change, and the content of RSD at each time point is not more than 5.0 percent; the total impurities and the number of impurities are not obviously changed, which shows that the sample solution is stable after being placed at room temperature for 48 hours; the peak area RSD of the main peak of the control solution at 48.5 hours is not more than 5.0 percent, which shows that the control solution is stable when placed at room temperature for 48.5 hours.

Example 12

The present embodiment provides a method for detecting an LLTS intermediate (LLTS-M2), which includes:

Detecting the test solution by high performance liquid chromatography; the detection conditions of the high performance liquid chromatography comprise: a chromatographic column: agilent SB-C184.6mm × 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: 30 ℃, sample size: 10. mu.l, solvent: acetonitrile-water (90: 10).

Example 13

This example differs from example 12 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 25 ℃.

Example 14

This example differs from example 12 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 35 ℃.

Example 15

This example differs from example 12 only in that: the wavelength is 228nm under the detection condition of high performance liquid chromatography.

Example 16

This example differs from example 12 only in that: in the detection condition of the high performance liquid chromatography, the column temperature is 232 nm.

Example 17

This example differs from example 12 only in that: the flow rate is 0.9ml/min under the detection condition of high performance liquid chromatography.

Example 18

This example differs from example 12 only in that: the flow rate is 1.1ml/min under the detection condition of high performance liquid chromatography.

Example 19

This example differs from example 12 only in that: in the detection conditions of the high performance liquid chromatography, the initial ratio is 60: 40.

example 20

This example differs from example 12 only in that: in the detection conditions of the high performance liquid chromatography, the initial ratio is 56: 44.

example 21

This example differs from example 12 only in that: in the detection condition of the high performance liquid chromatography, a chromatographic column is Agilent ZORBAX XDB-C184.6mm multiplied by 250mm, 5 mu m (different batches of the same manufacturer).

Example 22

This example differs from example 12 only in that: in the detection condition of the high performance liquid chromatography, a chromatographic column is Feilomen titanium C184.6mm multiplied by 250mm, 5 mu m (different batches of different manufacturers).

Comparative example 2

Detecting a LLTS intermediate (LLTS-M2) test 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 suitability test (LLTS-M2 test)

Sample preparation:

LLTS-M1 control solution: about 5mg of LLTS-M1 control was weighed precisely, placed in a 100ml measuring flask, dissolved and diluted to the mark with solvent, shaken up (500. mu.g/ml) (sample weight 5.010 mg). LLTS-M1 localization solution: 2ml of the reference LLTS-M1 solution was measured accurately, placed in a 20ml measuring flask, diluted to the mark with solvent and shaken well (5. mu.g/ml). LLTS-M2I1 control solution: about 4mg of LLTS-M2I1 was weighed out precisely, placed in a 20ml measuring flask, dissolved and diluted to the mark with solvent, shaken well (200. mu.g/ml) (weighing 4.005 mg). LLTS-M2I1 localization solution: 2ml of the reference LLTS-M2I1 solution was measured accurately, placed in a 20ml measuring flask, diluted to the mark with solvent and shaken up (20. mu.g/ml). LLTS-M3 control solution: about 10mg of LLTS-M3 was weighed out precisely, placed in a 10ml measuring flask, dissolved and diluted to the mark with solvent, and shaken up (1mg/ml) (weighing 0.01002 g). LLTS-M3 localization solution: 1ml of LLTS-M3 control solution is precisely measured, placed in a 100ml measuring flask, diluted to the mark with solvent and shaken up (10. mu.g/ml). Test solution: about 10mg of the LLTS-M2 intermediate is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale mark by a solvent, and shaken up to be used as a test solution (1mg/ml) (the sample amount is 0.01012 g). 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 (100. mu.g/ml). System applicability solution: taking 10mg of LLTS-M2 intermediate, placing in a 10ml measuring flask, adding appropriate amount of solvent to dissolve, precisely adding 1ml of each of LLTS-M1 control solution and LLTS-M2I1 control solution, diluting with solvent to scale, and shaking. (LLTS-M21 mg/ml, LLTS-M15. mu.g/ml, LLTS-M2I 120. mu.g/ml). Blank solution: solvent (acetonitrile-water (90:10)) (sample weight 0.01002 g).

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 sequence in fig. 10-14. FIG. 15 shows the chromatogram of the LLTS-M2 sample obtained under the conditions of comparative example 2, in which the LLTS-M2 and the respective impurities showed relatively late peaks.

TABLE 24 sample introduction sequence and requirements

Sequence of	Sample name	Number of needles
			1	Blank solution	1 needle
2	Control solution	Continuous 5 needles
			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 results of sample introduction precision measurement

Name (R)	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	14.917	117.91
RSD(％)	0.03	0.25

TABLE 26 measurement of System suitability-relative Retention time

TABLE 27 number of theoretical plates for System suitability test results

TABLE 28 System suitability test results-tailing factor

TABLE 29 System suitability test results-degree of separation

TABLE 30 test article impurity detection results

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 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 tailing factors are less than 2.0; the number of each impurity, total impurities and impurities in the test solution is not obviously changed; under each condition, the control solution is continuously injected with 5 needles, and the peak area RSD is less than 5.0%. Therefore, the detection method of the present invention can stably and effectively detect the related substances of LLTS-M2.

Test example 8

Sensitivity test (LLTS-M2), comprising the following steps:

(1) sample preparation

Solvent: ethanol-water (90: 10); blank solution: a solvent; LLTS-M1 control solution (r): precisely measuring 1ml of LLTS-M1 reference solution, placing in a 10ml measuring flask, diluting with solvent to scale, shaking, precisely measuring 5ml, placing in a 10ml measuring flask, diluting with solvent to scale, and shaking (50 μ g/ml). LLTS-M2 control stock solution: about 10mg of LLTS-M2 control sample is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the mark by solvent, and shaken up (1mg/ml) (the sample weight is 0.01007 g). LLTS-M2 control solutions: precisely measuring 1ml of LLTS-M2 reference stock solution, placing in a 25ml measuring flask, diluting to scale with solvent, shaking, precisely measuring 1ml, placing in a 20ml measuring flask, diluting to scale with solvent, and shaking (1 μ g/ml). LLTS-M2I1 control solution (r): 1ml of LLTS-M2I1 control solution is precisely weighed, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken up (2 mug/ml). Quantitative limiting solution: precisely measuring 1.5ml of LLTS-M1 reference substance solution, 1ml of LLTS-M2 reference substance solution and 1ml of LLTS-M2I1 reference substance solution in a same 10ml measuring flask, diluting the solution to scale with a solvent, and shaking up. Preparing a detection limiting solution: precisely measuring 3ml of the limiting solution, placing the limiting solution into a 10ml measuring flask, diluting the limiting solution to a scale with a solvent, and shaking up.

(2) High performance liquid chromatography detection conditions

Same as example 12

(3) Detection step

TABLE 31 sample introduction sequence and times requirements

Sequence of events	Sample name	Number of needles
			1	Blank solution	1 needle
2	System applicability solution	1 needle
			4	Quantitative limiting solution	6 needles
5	Detection limiting solution	2 needles

(4) Analysis results

The results of the sensitivity tests are shown in tables 32 to 35.

TABLE 32LLTS-M1 quantitative Limit test results

Name (R)	1	2	3	4	5	6	Average out	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(s)	1	2	3	4	5	6	Average	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 (R)	1	2	3	4	5	6	Average	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

Calculating the formula:

Quantitative limit/detection limit (ng) × concentration × sample size

From the sensitivity test results, it is found that:

the quantitative limiting solution is tested continuously for 6 times, the LLTS-M2 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 9

Linear assay (LLTS-M2) comprising the following steps:

(1) sample preparation

The product has a LLTS-M2 structure containing hydroxyl, can be oxidized into LLTS-M2I1, and linearly expands main components and impurities to 3 times of limit in order to cope with the increase of the content of LLTS-M2I1 in storage (LLTS-M2 is in the range of quantitative limit to 15.0% of the concentration of a test sample, LLTS-M1 is in the range of quantitative limit to 1.5% of the concentration of the test sample, and LLTS-M2I1 is in the range of quantitative limit to 6.0% of the concentration of the test sample); solvent: ethanol-water (90: 10); blank solution: a solvent; LLTS-M2 control stock solution: taking about 20mg of LLTS-M2 reference substance, precisely weighing, placing in a 20ml measuring flask, dissolving with solvent and diluting to scale, and shaking up. (1mg/ml) (sample weight 0.02003 g); LLTS-M2 control solution: 1ml of LLTS-M2 reference stock solution is precisely weighed, placed in a 100ml measuring flask, diluted to the scale with solvent and shaken up (10 mug/ml). (ii) a Impurity mixed stock solution: precisely measuring 4ml of LLTS-M1 reference substance solution, 4ml of LLTS-M2I1 reference substance solution and 2ml of LLTS-M2 reference substance stock solution, placing the solutions in the same 20ml measuring flask, diluting the solutions to the scale with a solvent, and shaking up.

Linear solution 1: and (4) quantifying the limiting solution. Linear solution 2 (10%): precisely measuring 0.5ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 3 (20%): precisely measuring 1ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 4 (50%): 2.5ml of impurity mixed stock solution is precisely measured, placed in a 10ml measuring flask, diluted to scale by a solvent and shaken up. Linear solution 5 (100%): precisely measuring 5ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. Linear solution 6 (200%): mixing the impurities with the stock solution. Linear solution 7 (300%): precisely measuring 3ml of LLTS-M1 reference solution, 3ml of LLTS-M2I1 reference solution and 1.5ml of LLTS-M2 reference stock solution, placing the solutions in a 10ml measuring flask, diluting the solutions to the scale with a solvent, and shaking up.

(2) High performance liquid chromatography detection conditions

Same as example 12

(3) Detection step

And taking 10 mu l of each blank solution, system applicability solution, LLTS-M2 control solution and each linear solution, injecting samples, detecting, and recording chromatograms. Samples were introduced in the order of the table below.

TABLE 36 sample introduction sequence 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 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 concentration C (μ g/mL) is taken as the abscissa and the corresponding peak area is taken as the ordinate, so as to obtain a linear regression equation, and the linear relationship graphs are shown in FIG. 16, FIG. 17 and FIG. 18. The linear relation between the concentrations of the LLTS-M1, the LLTS-M2 and the LLTS-M2I1 and the peak area is good, and the verification requirements are met.

Test example 10

Accuracy test (LLTS-M2), comprising the following steps:

(1) sample preparation

Solvent: ethanol-water (90: 10); blank solution: a solvent; test solution: about 10mg of the product is precisely weighed, placed in a 10ml measuring flask, dissolved and diluted to the scale mark by a solvent, and shaken up (1mg/ml, the weighing amount is 0.01017 g). LLTS-M1 control solution: taking about 10mg of LLTS-M1 reference substance, precisely weighing, placing in a 20ml measuring flask, dissolving with solvent and diluting to scale, shaking up (preparing 2 parts in parallel, 500 μ g/ml, weighing 0.01001g, 0.01000g respectively). LLTS-M2I1 control solution: taking about 10mg of LLTS-M2I1 reference substance, precisely weighing, placing in a 10ml measuring flask, dissolving with solvent and diluting to scale, shaking up (preparing 2 parts in parallel, 1mg/ml, weighing sample amounts of 0.01005g and 0.01000g respectively). Impurity mixed stock solution: 2ml of LLTS-M1 reference substance solution and 4ml of LLTS-M2I1 reference substance solution are precisely measured, placed in the same 100ml measuring flask, diluted to scale by solvent, shaken up and prepared into 2 parts (LLTS-M110 mug/ml, LLTS-M2I 140 mug/ml) in parallel. Impurity control solution: precisely measuring 5ml of impurity mixed stock solution, placing the impurity mixed stock solution into a 10ml measuring flask, diluting the impurity mixed stock solution to a scale with a solvent, and shaking up. 2 aliquots were prepared in parallel (LLTS-M15. mu.g/ml, LLTS-M2I 120. mu.g/ml). Recovering rate solution (preparing recovering rate solution by mixing impurity with stock solution-1); 50% solution: taking about 10mg of a sample, accurately weighing, placing in a 10ml measuring flask, adding a proper amount of solvent to dissolve, accurately adding 2.5ml of impurity mixed stock solution, diluting to scale with the solvent, and shaking up (preparing 3 parts in parallel, weighing 0.01009g, 0.00988g and 0.01006g respectively). 100% solution: precisely weighing about 10mg of a test sample, placing the test sample in a 10ml measuring flask, adding a proper amount of solvent to dissolve the test sample, precisely adding 5ml of impurity mixed stock solution, diluting the mixed stock solution to a scale by using the solvent, and shaking uniformly (preparing 3 parts in parallel, wherein the weighing quantities are 0.01011g, 0.01010g and 0.01013g respectively). 150% solution: taking about 10mg of a sample, accurately weighing, placing in a 10ml measuring flask, adding a proper amount of solvent to dissolve, accurately adding 7.5ml of impurity mixed stock solution, diluting to scale with the solvent, and shaking up (preparing 3 parts in parallel, weighing 0.01012g, 0.01009g and 0.01005g respectively). ()

(2) High performance liquid chromatography detection conditions

Same as example 12

(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 introduced in the order of the table below.

TABLE 40 sample introduction sequence and requirements

Sequence of	Sample (I)	Number of needles
			1	Blank solution	1 needle
2	Control solution	2 needles
			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: calculating the formula: percent recovery ═ amount measured-content in sample)/amount added × 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 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 11

Repeatability tests (LLTS-M2) comprising the following steps:

(1) sample preparation

Solvent: ethanol-water (90:10)

Blank solution: solvent(s)

Test solution: taking about 10mg of the product, accurately weighing, placing in a 10ml measuring flask, dissolving with solvent, diluting to scale, and shaking up (preparing 6 parts in parallel, 1mg/ml, weighing 0.01015g, 0.01011g, 0.01014g, 0.01002g, 0.01004g, 0.01006 g).

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. (preparation in parallel 6 parts, 10. mu.g/ml)

(2) High performance liquid chromatography detection conditions

Same as example 12

(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 introduced in the order of the table below.

TABLE 43 sample introduction sequence and requirements

Sequence of	Sample (I)	Number of needles
			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 reproducibility test are shown in Table 44.

TABLE 44 repeatability test results

Test article	LLTS-M1	LLTS-M2I1	Maximum unknown simple impurity	Total hetero (%)	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	0.28	1.97	0.62	4.09	/
RSD(％)	2.24	4.51	1.71	1.62	/

Test example 12

(1) Solution stability test (LLTS-M2), comprising the steps of:

system applicability solution the following section of system applicability example 12; the test solution and the control solution were prepared in the same manner as in example 12.

Sample preparation

Solvent: ethanol-water (90:10)

Blank solution: solvent(s)

The test solution and the control solution are prepared in two parts in parallel, the stability of the system is examined by taking the control solution-1, the stability of the control solution is examined by taking the control solution-2, and the stability of the test solution is examined by taking the test solution-2 (the sample weights of the test solution 1 and the test solution 2 are 0.01011g and 0.01013g respectively).

(2) High performance liquid chromatography detection conditions

Same as example 12

(3) Detection step

TABLE 45 sample introduction sequence and requirements

(4) Analysis results

The results of the solution stability tests are shown in tables 46 and 47.

TABLE 46 test article solution stability results

Test result of stability of test 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	14.901	106.57
RSD(％)	0.06	0.61

The sample solution is placed at room temperature for 12 hours, the content average value of the impurity LLTS-M1 at each time point is 0.27%, the RSD is 7.40%, and is less than 10.0%; the content average value of the impurities LLTS-M2I1 at each time point is 2.02 percent, the RSD is 1.90 percent and is less than 5.0 percent; the content average value of the maximum unknown single impurity at each time point is 0.62 percent, the RSD is 1.80 percent and is less than 5.0 percent; the content average value of all the impurities at each time point is 4.14 percent, the RSD is 7.40 percent and is more than 5.0 percent; the test solution is shown to be unstable when left at room temperature for 12 hours.

The sample solution is placed for 4 hours at room temperature, the content average value of the impurity LLTS-M1 at each time point is 0.26 percent, the RSD is 8.45 percent and is less than 10.0 percent; the content average value of the impurities LLTS-M2I1 at each time point is 2.04 percent, the RSD is 1.62 percent and is less than 5.0 percent; the content average value of the maximum unknown single impurity at each time point is 0.62 percent, the RSD is 0.98 percent and is less than 5.0 percent; the content average value of all impurities at each time point is 3.90 percent, the RSD is 4.65 percent and is less than 5.0 percent; the test solution is stable after being placed at room temperature for 4 hours and is applied to new preparation.

The peak area RSD of the 12.5 hour main peak of the control solution is 0.61 percent and is not more than 5.0 percent, which indicates that the control solution is stable when placed at room temperature for 12.5 hours.

In conclusion, the detection method provided by the embodiment of the invention has good specificity, repeatability and accuracy on the basis of ensuring the high-efficiency separation of various related substances and effective components, thereby better realizing the quality control of LLTS-M1 or LLTS-M2.

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

1. A method for detecting LLTS intermediate and related impurities 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-;

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 intermediate is LLTS-M1 or LLTS-M2; the structural formula of the LLTS-M1 is as follows:

the structural formula of the LLTS-M2 is as follows:

preferably, the volume ratio of mobile phase a and mobile phase B is maintained constant during the elution procedure for periods of 0-5min and 25-30 min.

3. The method of claim 2 wherein the step of eluting the LLTS intermediate is LLTS-M1 according to the following steps:

preferably, when the LLTS intermediate is LLTS-M1, the elution procedure of the high performance liquid chromatography is as follows:

or the LLTS intermediate is LLTS-M2, and the elution procedure of the high performance liquid chromatography is as follows:

preferably, when the LLTS intermediate is LLTS-M2, the elution procedure of the high performance liquid chromatography is as follows:

4. the method of claim 2 for detecting LLTS intermediates and related impurities, wherein when the 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 sequentially:

when the LLTS intermediate is LLTS-M2, the related impurities are LLTS-M1 and LLTS-M2I1, and the structural formulas of the LLTS-M1 and the LLTS-M2I1 are sequentially as follows:

5. the method of claim 2, wherein the test solution and the control solution of impurities are acetonitrile-water solution and ethanol-water solution, and when the LLTS intermediate is LLTS-M1, the volume ratio of acetonitrile to water is 45-55: 45-55, preferably 50: 50; when the LLTS intermediate is LLTS-M2, the volume ratio of ethanol to water is 88-92: 12-8, preferably 90: 10.

6. the method for detecting LLTS intermediates and related impurities according to any of claims 1 to 5, wherein said gradient elution has a flow rate of 0.8-1.2 mL/min, preferably 1.0 mL/min.

7. The method for detecting LLTS intermediates and related impurities according to any one of claims 1 to 5, wherein the HPLC uses an octadecylsilane bonded silica gel column, and the column temperature of the column is 25 to 35 ℃ during detection;

preferably, the high performance liquid chromatography adopts Agilent SB-C184.6mm × 250mm, 5 μm of chromatographic column, and the temperature of the chromatographic column is 30 ℃ during detection.

8. The method for detecting LLTS intermediates and related impurities thereof according to any one of claims 1 to 5, wherein the content of related impurities in said test solution is calculated by a self-control method;

preferably, the method for calculating the content of the related impurities 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 impurities in the test solution by using a self-contrast method.

9. The method for detecting LLTS intermediates and related impurities according to any one of claims 1-3, wherein 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, and the flow rate of the gradient elution is 0.8-1.2 mL/min.

10. The use of the LLTS intermediate and its related impurities detection method of any one of claims 1 to 9 in the quality control of LLTS intermediate.