Detection method and application of larotaxel-related substances

Abstract

The present invention discloses a method for detecting larotaxel related substances and application thereof. According to the synthesis process of larotaxel, the related substances I1, I2, I3, I4, I5, I6, I7, I8, I9, I10 and I11 contained in larotaxel are controlled. Through specific high performance liquid chromatography detection conditions, the above eleven related substances can be separated efficiently at one time, and the method has strong specificity, good durability and high sensitivity, and is convenient for establishing a method for studying larotaxel related substances and for quality control of larotaxel raw materials.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical drug analysis methods, and in particular to a detection method and application of larotaxel-related substances. Background Art

[0002] The structure of larotaxel API is shown below:

[0003]

[0004] As a raw material drug, if larotaxel contains impurities (i.e. related substances), then during the preparation production process, impurities or impurity conversion products may also be brought into the later production and affect the quality of the finished preparation. Therefore, it is very important to control the impurities in larotaxel.

[0005] The pharmacopoeias of various countries do not include the detection methods for larotaxel-related substances. Therefore, it is urgent to establish a comprehensive detection method for larotaxel-related substances in order to achieve effective quality control.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a detection method and application of larotaxel-related substances to improve the above technical problems.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a method for detecting larotaxel-related substances, comprising: using high performance liquid chromatography to detect a test solution; the detection conditions of the high performance liquid chromatography include:

[0010] Detection wavelength 213~217nm;

[0011] Mobile phase A: 0.04wt% to 0.06wt% phosphoric acid aqueous solution; Mobile phase B: acetonitrile;

[0012] Perform gradient elution according to the following program:

[0013]

[0014]

[0015] Larotaxel-related substances include: larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10, larotaxel-I11, and the structural formulas are:

[0016] In a second aspect, the present invention provides application of the above-mentioned method for detecting larotaxel-related substances in the quality control of larotaxel.

[0017] The present invention has the following beneficial effects: according to the synthesis process of larotaxel, the related substances I1, I2, I3, I4, I5, I6, I7, I8, I9, I10 and I11 contained in larotaxel are controlled, and the eleven related substances can be separated efficiently at one time through the high performance liquid chromatography detection conditions of the embodiment of the present invention, and the specificity is strong, the durability is good, and the sensitivity is high, which is convenient for establishing a research method for larotaxel related substances and for quality control of larotaxel raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 is the chromatogram of the blank solution of Test Example 1;

[0020] Figure 2 is a chromatogram of the larotaxel-I1 localization solution of Experimental Example 1;

[0021] Figure 3 is a chromatogram of the larotaxel-I2 localization solution of Experimental Example 1;

[0022] Figure 4 is a chromatogram of the larotaxel-I3 localization solution of Experimental Example 1;

[0023] Figure 5 is a chromatogram of the larotaxel-I4 localization solution of Experimental Example 1;

[0024] Figure 6 is a chromatogram of the larotaxel-I5 localization solution of Experimental Example 1;

[0025] Figure 7 is a chromatogram of the larotaxel-I6 localization solution of Experimental Example 1;

[0026] Figure 8 is a chromatogram of the larotaxel-I7 localization solution of Experimental Example 1;

[0027] Fig. 9 is a chromatogram of the larotaxel-I8 localization solution of Experimental Example 1;

[0028] Fig.10 is a chromatogram of the larotaxel-I9 localization solution of Experimental Example 1;

[0029] Fig.11 is a chromatogram of the larotaxel-I10 localization solution of Experimental Example 1;

[0030] Fig.12 is a chromatogram of the larotaxel-I11 localization solution of Experimental Example 1;

[0031] Fig.13 is the chromatogram of the test solution of Test Example 1;

[0032] Fig.14 Provide a linear relationship diagram of larotaxel-I1 for Experimental Example 3 of the present invention;

[0033] Fig.15 Provide a linear relationship diagram of larotaxel-I2 for Experimental Example 3 of the present invention;

[0034] Fig.16 Provide a linear relationship diagram of larotaxel-I3 for Experimental Example 3 of the present invention;

[0035] Fig.17 Provide a linear relationship diagram of larotaxel-I4 for Experimental Example 3 of the present invention;

[0036] Fig.18 Provide a linear relationship diagram of larotaxel-I5 for Experimental Example 3 of the present invention;

[0037] Fig.19 A linear relationship diagram of larotaxel-I6 is provided for Example 10 of the present invention;

[0038] Fig. 20 Provide a linear relationship diagram of larotaxel-I7 for Experimental Example 3 of the present invention;

[0039] Fig.21 Provide a linear relationship diagram of larotaxel-I8 for Experimental Example 3 of the present invention;

[0040] Fig. 22 Provide a linear relationship diagram of larotaxel-I9 for Experimental Example 3 of the present invention;

[0041] Fig.23 Provide a linear relationship diagram of larotaxel-I10 for Experimental Example 3 of the present invention;

[0042] Fig.24 Provide a linear relationship diagram of larotaxel-I11 for Experimental Example 3 of the present invention;

[0043] Fig.25 A linear relationship diagram of larotaxel is provided for Test Example 3 of the present invention. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0045] The verification of specificity, detection limit and quantification limit, precision, linearity and range, accuracy, solution stability and other items in the specific implementation method is carried out in accordance with the "Technical Guidelines for Validation of Analytical Methods for Quality Control of Chemical Drugs", "Technical Guidelines for the Standardized Process for Establishing Quality Standards for Chemical Drugs", "Technical Guidelines for Research on Impurities of Chemical Drugs", "Technical Guidelines for Research on Residual Solvents of Chemical Drugs" and relevant guidelines in the appendix of the current edition of the "Pharmacopoeia of the People's Republic of China" for methodological verification.

[0046] The following is a detailed description of the detection method and application of larotaxel-related substances provided by the present invention.

[0047] Some embodiments of the present invention provide a method for detecting larotaxel-related substances, comprising: using high performance liquid chromatography to detect a test solution; the detection conditions of the high performance liquid chromatography include:

[0048] Detection wavelength 213~217nm;

[0049] Mobile phase A: 0.04wt% to 0.06wt% phosphoric acid aqueous solution; Mobile phase B: acetonitrile;

[0050] Perform gradient elution according to the following program:

[0051]

[0052] Larotaxel-related substances include: larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10, larotaxel-I11, and the structural formulas are:

[0053]

[0054] Among them, the molecular formula of larotaxel-I1 is C 47 H 57 NO 16 , with a molecular weight of 891.96; the molecular formula of larotaxel-I2 is C 45 H 55 NO15 , with a molecular weight of 849.93; the molecular formula of larotaxel-I3 is C 45 H 55 NO 15 , with a molecular weight of 849.93; the molecular formula of larotaxel-I4 is C 47 H 58 N 2 O 15 , with a molecular weight of 890.98; the molecular formula of larotaxel-I5 is C 31 H 36 O 10 , with a molecular weight of 568.62; the molecular formula of larotaxel-I6 is C 59 H 70 N 2 O 18 , with a molecular weight of 1095.21; the molecular formula of larotaxel-I7 is C 43 H 51 NO 13 , with a molecular weight of 789.88; the molecular formula of larotaxel-I8 is C 45 H 53 NO 14 , with a molecular weight of 831.91; the molecular formula of larotaxel-I9 is ​​C 49 H 61 NO 15 , with a molecular weight of 904.02; the molecular formula of larotaxel-I10 is C 33 H 38 O 11 , with a molecular weight of 610.66; the molecular formula of larotaxel-I11 is C 18 H 25 NO 5 , with a molecular weight of 335.40; according to the synthesis process of larotaxel, the related substances larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 contained in larotaxel are controlled, and the eleven related substances can be separated efficiently at one time through the high performance liquid chromatography detection conditions of the present invention, thereby establishing a research method for related substances of larotaxel.

[0055] It should be noted that within 0 to 20 min, the volume ratio of mobile phase A to mobile phase B remains unchanged.

[0056] Furthermore, the elution procedure was optimized, and the optimized elution procedure was:

[0057]

[0058] Furthermore, in some embodiments, the flow rate of gradient elution is 0.9 to 1.1 mL / min, for example, 0.9 mL / min, 1.0 mL / min or 1.1 mL / min, preferably 1.0 mL / min.

[0059] In some embodiments, the HPLC column is an octadecylsilane bonded silica gel column, and the column temperature of the HPLC is 23-27°C, for example, the column temperature can be 23°C, 24°C, 25°C, 26°C or 27°C.

[0060] For example, the chromatographic column is Agilent 5TC-C18(2) 4.6 mm×250 mm, 5 μm or other chromatographic columns with equivalent performance; the column temperature is 25°C.

[0061] In some embodiments, the injection volume of the HPLC is 10-25 μL, for example, 10 μL, 12 μL, 15 μL, 18 μL, 20 μL or 25 μL, etc., and preferably the injection volume is 20 μL.

[0062] Specifically, some embodiments of the present invention calculate the contents of related substances of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 in the test solution by the self-control method.

[0063] Among them, the method for calculating the content of related substances includes: injecting the test solution and the control solution into a high performance liquid chromatograph respectively, determining the corresponding chromatographic peak area, and calculating the content of related substances of larotaxis-I1, larotaxis-I2, larotaxis-I3, larotaxis-I4, larotaxis-I5, larotaxis-I6, larotaxis-I7, larotaxis-I8, larotaxis-I9, larotaxis-I10 and larotaxis-I11 in the test solution by self-control method.

[0064] In some embodiments, the solvent for preparing the test solution and the control solution is acetonitrile. Specifically, the solution for detection can be prepared according to the following process:

[0065] Larotaxel-I1 stock solution: Take about 2 mg of larotaxel-I1, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I1 stock solution (40 μg / ml).

[0066] Larotaxel-I2 stock solution: Take about 2 mg of larotaxel-I2, weigh accurately, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I2 stock solution (40 μg / ml).

[0067] Larotaxel-I3 stock solution: Take about 2 mg of larotaxel-I3, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I3 stock solution (40 μg / ml).

[0068] Larotaxel-I4 stock solution: Take about 2 mg of larotaxel-I4, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I4 stock solution (40 μg / ml).

[0069] Larotaxel-I5 stock solution: Take about 2 mg of larotaxel-I5, weigh accurately, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I5 stock solution (40 μg / ml).

[0070] Larotaxel-I6 stock solution: Take about 2 mg of larotaxel-I6, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I6 stock solution (40 μg / ml).

[0071] Larotaxel-I7 stock solution: Take about 2 mg of larotaxel-I7, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I7 stock solution (40 μg / ml).

[0072] Larotaxel-I8 stock solution: Take about 2 mg of larotaxel-I8, weigh accurately, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I8 stock solution (40 μg / ml).

[0073] Larotaxel-I9 stock solution: Take about 2 mg of larotaxel-I9, weigh accurately, put into a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I9 stock solution (40 μg / ml).

[0074] Larotaxel-I10 stock solution: Take about 2 mg of larotaxel-I10, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I10 stock solution (40 μg / ml).

[0075] Larotaxel-I11 stock solution: Take about 2 mg of larotaxel-I11, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I11 stock solution (40 μg / ml).

[0076] Test solution: Take about 10 mg of the product, weigh accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the test solution (1 mg / ml).

[0077] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0078] (Concentration about 10μg / ml).

[0079] System suitability solution: Weigh about 10 mg of this product and place it in a 10 ml volumetric flask. Accurately measure 0.5 ml of the stock solution of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11, and dissolve and dilute with a solvent to prepare a mixed solution containing approximately 1.0 mg of larotaxel, and 2 μg each of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 per 1 ml.

[0080] Some embodiments of the present invention provide a preferred detection method, wherein the conditions of the high performance liquid chromatography meet the following requirements:

[0081] The detection wavelength is 215nm;

[0082] Mobile phase A: 0.05% phosphoric acid aqueous solution; Mobile phase B: acetonitrile;

[0083] The flow rate of the gradient elution is 1.0 ml / min;

[0084] The gradient elution program was:

[0085]

[0086] The chromatographic column is Agilent 5TC-C18 (2) 4.6 mm × 250 mm, 5 μm, and the column temperature of the chromatographic column is 25°C;

[0087] The injection volume was 20 μL;

[0088] Solvent: acetonitrile.

[0089] Some embodiments of the present invention also provide the use of the method for detecting larotaxel-related substances of the above-mentioned embodiment in the quality control of larotaxel.

[0090] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0091] The test solutions in the following embodiments are mainly prepared with larotaxel, larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11. In actual detection, the larotaxel to be detected is dissolved and diluted to prepare the test solution.

[0092] Example 1

[0093] This embodiment provides a method for detecting larotaxel-related substances, which specifically comprises:

[0094] (1) Sample preparation

[0095] Solvent: acetonitrile;

[0096] Test solution: Take about 10 mg of the product, weigh accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the test solution (1 mg / ml).

[0097] (2) The test solution is tested by high performance liquid chromatography; high performance liquid chromatography test conditions:

[0098] Chromatographic column: Agilent 5TC-C18(2) 4.6 mm × 250 mm, 5 μm;

[0099] Mobile phase A: 0.05% phosphoric acid in water;

[0100] Mobile phase B: acetonitrile;

[0101] Perform gradient elution according to the following table:

[0102]

[0103] Flow rate: 1.0ml / min;

[0104] Detection wavelength: 215nm;

[0105] Column temperature: 25°C;

[0106] Injection volume: 20 μL.

[0107] Example 2

[0108] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection condition of step (2), the column temperature is 23°C.

[0109] Example 3

[0110] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection condition of step (2), the column temperature is 27°C.

[0111] Example 4

[0112] This example refers to the detection method of Example 1, with the only difference being that the wavelength in the HPLC detection condition of step (2) is 213 nm.

[0113] Example 5

[0114] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection condition of step (2), the column temperature is 217 nm.

[0115] Example 6

[0116] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection condition of step (2), the flow rate is 0.9 ml / min.

[0117] Example 7

[0118] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection condition of step (2), the flow rate is 1.1 ml / min.

[0119] Example 8

[0120] This example refers to the detection method of Example 1, with the only difference being that in the HPLC detection conditions of step (2), the chromatographic column is Agilent 5TC-C18 (2) 4.6 mm×250 mm, 5 μm (same manufacturer, different batch number).

[0121] Test Example 1

[0122] System suitability test

[0123] (1) Solution preparation

[0124] Solvent: acetonitrile;

[0125] Blank solution: solvent

[0126] Larotaxel-I5 stock solution: Take about 2 mg of larotaxel-I5, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I5 stock solution (40 μg / ml).

[0127] Larotaxel-I10 stock solution: Take about 2 mg of larotaxel-I10, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I10 stock solution (40 μg / ml).

[0128] Larotaxel-I4 stock solution: Take about 2 mg of larotaxel-I4, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I4 stock solution (40 μg / ml).

[0129] Larotaxel-I7 stock solution: Take about 2 mg of larotaxel-I7, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I7 stock solution (40 μg / ml).

[0130] Larotaxel-I3 stock solution: Take about 2 mg of larotaxel-I3, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I3 stock solution (40 μg / ml).

[0131] Larotaxel-I11 stock solution: Take about 2 mg of larotaxel-I11, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I11 stock solution (40 μg / ml).

[0132] Larotaxel-I8 stock solution: Take about 2 mg of larotaxel-I8, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I8 stock solution (40 μg / ml).

[0133] Larotaxel-I2 stock solution: Take about 2 mg of larotaxel-I2, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I2 stock solution (40 μg / ml).

[0134] Larotaxel-I1 stock solution: Take about 2 mg of larotaxel-I1, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I1 stock solution (40 μg / ml).

[0135] Larotaxel-I9 stock solution: Take about 2 mg of larotaxel-I9, weigh accurately, put into a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I9 stock solution (40 μg / ml).

[0136] Larotaxel-I6 stock solution: Take about 2 mg of larotaxel-I6, accurately weigh it, put it in a 50 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well, and use it as the larotaxel-I6 stock solution (40 μg / ml).

[0137] System suitability solution: Take 10 mg of larotaxel and place it in a 10 ml volumetric flask, add acetonitrile to dissolve, accurately measure 0.5 ml of each of the stock solutions of larotaxel-I5, larotaxel-I10, larotaxel-I4, larotaxel-I7, larotaxel-I11, larotaxel-I3, larotaxel-I8, larotaxel-I2, larotaxel-I1, larotaxel-I9 and larotaxel-I6 and place them in the above volumetric flask, dilute with acetonitrile and make up to the mark, shake well, and obtain.

[0138] LLTS-I5 localization solution: Accurately measure 1 ml of LLTS-I5 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0139] LLTS-I10 positioning solution: Accurately measure 1 ml of LLTS-I10 stock solution, place it in a 20 ml volumetric flask, dilute to the scale with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0140] LLTS-I4 localization solution: Accurately measure 1 ml of LLTS-I4 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0141] LLTS-I7 localization solution: Accurately measure 1 ml of LLTS-I7 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0142] LLTS-I11 positioning solution: Accurately measure 1 ml of LLTS-I11 stock solution, place it in a 20 ml volumetric flask, dilute to the scale with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0143] LLTS-I3 localization solution: Accurately measure 1 ml of LLTS-I3 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0144] LLTS-I8 localization solution: Accurately measure 1 ml of LLTS-I8 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0145] LLTS-I2 positioning solution: Accurately measure 1 ml of LLTS-I2 stock solution, place it in a 20 ml volumetric flask, dilute to the scale with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0146] LLTS-I1 localization solution: Accurately measure 1 ml of LLTS-I1 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0147] LLTS-I9 localization solution: Accurately measure 1 ml of LLTS-I9 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0148] LLTS-I6 localization solution: Accurately measure 1 ml of LLTS-I6 stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 2 μg / ml).

[0149] Test solution: Take about 10 mg of the product, weigh accurately, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well (concentration is about 1 mg / ml).

[0150] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (concentration is about 10 μg / ml).

[0151] (2) Detection steps

[0152] Take 20 μl of blank solution, control solution, system suitability solution, test solution (see Example 1) and each impurity location solution, inject and test, and record the chromatogram. The chromatogram is as follows: Figures 1 to 13 Wherein, the samples were injected in the order of Table 1, and the detection was performed according to the detection conditions in Examples 1-8.

[0153] Table 1 Injection order and requirements

[0154]

[0155]

[0156] 3) Result analysis

[0157] The results of changing different parameters in Examples 1-8 are as follows.

[0158] Table 2 System suitability test results - retention time

[0159]

[0160] Table 3 System suitability test results - theoretical plate number

[0161]

[0162] Table 4 System suitability test results - tailing factor

[0163]

[0164]

[0165] Table 5 System suitability test results - separation

[0166]

[0167] From the above results, we can see that:

[0168] Under each condition, the blank solution did not interfere with the determination of the main components and impurities;

[0169] In the system suitability solution, the separation between the main peak and the known impurities and the adjacent impurity peaks is greater than 1.5, the theoretical plate number of the main peak and each known impurity is not less than 5000, and the tailing factor is greater than 2.0;

[0170] Therefore, the detection method according to the embodiment of the present invention can stably and effectively detect the related substances of larotaxel.

[0171] Test Example 2

[0172] Sensitivity test

[0173] (1) Sample preparation

[0174] Solvent: Acetonitrile

[0175] Blank solution: solvent

[0176] Larotaxel reference substance stock solution: Accurately weigh 10 mg of larotaxel, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well (1 mg / ml).

[0177] Larotaxel reference substance dilution: Accurately measure 1 ml of larotaxel stock solution, place in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (10 μg / ml).

[0178] Larotaxel-I1 mother solution: Weigh 2 mg of larotaxel-I1, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0179] Larotaxel-I1 dilution: Accurately measure 2 ml of larotaxel-I1 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0180] Larotaxel-I2 mother solution: Weigh 2 mg of larotaxel-I2, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0181] Larotaxel-I2 dilution: Accurately measure 2 ml of larotaxel-I2 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0182] Larotaxel-I3 mother solution: Weigh 2 mg of larotaxel-I3, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0183] Larotaxel-I3 dilution: Accurately measure 2 ml of larotaxel-I3 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0184] Larotaxel-I4 mother solution: Weigh 2 mg of larotaxel-I4, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0185] Larotaxel-I4 dilution: Accurately measure 2 ml of larotaxel-I4 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0186] Larotaxel-I5 mother solution: Weigh 2 mg of larotaxel-I5, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0187] Larotaxel-I5 dilution: Accurately measure 2 ml of larotaxel-I5 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0188] Larotaxel-I6 mother solution: Weigh 2 mg of larotaxel-I6, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0189] Larotaxel-I6 dilution: Accurately measure 2 ml of larotaxel-I6 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0190] Larotaxel-I7 mother solution: Weigh 2 mg of larotaxel-I7, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0191] Larotaxel-I7 dilution: Accurately measure 2 ml of larotaxel-I7 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0192] Larotaxel-I8 mother solution: Weigh 2 mg of larotaxel-I5, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, shake well (200 μg / ml).

[0193] Larotaxel-I8 dilution: Accurately measure 2 ml of larotaxel-I8 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0194] Larotaxel-I9 mother solution: Weigh 2 mg of larotaxel-I9, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0195] Larotaxel-I9 dilution: Accurately measure 2 ml of larotaxel-I9 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0196] Larotaxel-I10 mother solution: Weigh 2 mg of larotaxel-I10, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0197] Larotaxel-I10 dilution: Accurately measure 2 ml of larotaxel-I10 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0198] Larotaxel-I11 mother solution: Weigh 2 mg of larotaxel-I11, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to scale, shake well (200 μg / ml).

[0199] Larotaxel-I11 dilution: Accurately measure 2 ml of larotaxel-I11 mother solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (2 μg / ml).

[0200] System suitability solution: Use the “System suitability solution” prepared under “Test Example 1”.

[0201] Quantitation limit solution: Accurately measure 0.1 ml of larotaxel reference dilution, 0.7 ml of larotaxel-I1 dilution, 0.5 ml of larotaxel-I2 dilution, 1 ml of larotaxel-I3 dilution, 0.6 ml of larotaxel-I4 dilution, 0.3 ml of larotaxel-I5 dilution, 1 ml of larotaxel-I6 dilution, 0.5 ml of larotaxel-I7 dilution, 0.5 ml of larotaxel-I8 dilution, 0.5 ml of larotaxel-I9 dilution, 0.4 ml of larotaxel-I10 dilution, and 0.4 ml of larotaxel-I11 dilution, place them in the same 10 ml volumetric flask, dilute to the scale with acetonitrile, and shake well; prepare a solution with a signal-to-noise ratio ≥ 10, which is used as the quantitation limit solution (S / N≈10).

[0202] Preparation of detection limit solution: Accurately measure 3 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and use it as the detection limit solution (S / N≈3).

[0203] (2) HPLC detection conditions

[0204] Same as Example 1

[0205] (3) Detection steps

[0206] Take 20 μl of blank solution, system suitability solution, reference solution, and quantitation limit and detection limit solutions, respectively, and inject them for testing. Record the chromatogram and inject according to the order in the table below.

[0207] Table 6 Injection sequence and number requirements

[0208] order Sample name Number of needles 1 blank solution 1 needle 2 System Suitability Solutions 1 needle 3 Quantitative limit solution 6-pin 4 Detection limit solution 2-pin

[0209] (4) Analysis results

[0210] The sensitivity test results are shown in the table below.

[0211] Table 7 Larotaxel-I5 limit of quantitation test results

[0212] name 1 2 3 4 5 6 average RSD Peak area 1.65 1.62 1.63 1.71 1.67 1.65 1.66 1.93 Retention time 9.126 9.110 9.087 9.083 9.070 9.075 9.092 0.239

[0213] Table 8 Larotaxel-I10 limit of quantitation test results

[0214] name 1 2 3 4 5 6 average RSD(%) Peak area 2.33 2.35 2.45 2.51 2.38 2.37 2.40 2.84 Retention time 15.125 15.098 15.074 15.055 15.038 15.041 15.072 0.228

[0215] Table 9 Larotaxel-I4 limit of quantitation test results

[0216]

[0217] Table 10 Larotaxel-I7 limit of quantitation test results

[0218]

[0219] Table 11 Larotaxel-I11 limit of quantitation test results

[0220]

[0221] Table 12 Results of the quantification limit test of larotaxel-I3

[0222]

[0223] Table 13 Larotaxel limit of quantitation test results

[0224] name 1 2 3 4 5 6 average RSD(%) Peak area 3.56 3.71 3.65 3.59 3.57 3.54 3.60 1.79 Retention time 34.453 34.379 34.304 34.285 34.239 34.218 34.313 0.258

[0225] Table 14 Larotaxel-I8 limit of quantitation test results

[0226] name 1 2 3 4 5 6 average RSD(%) Peak area 3.12 2.70 3.00 2.88 3.01 2.74 2.91 5.67 Retention time 38.392 38.312 38.24 38.221 38.171 38.165 38.250 0.229

[0227] Table 15 Larotaxel-I2 limit of quantitation test results

[0228]

[0229]

[0230] Table 16 Larotaxel-I1 Quantitative Limit Test Results

[0231] name 1 2 3 4 5 6 average RSD(%) Peak area 2.91 2.91 2.81 2.76 2.73 2.84 2.83 2.65 Retention time 44.959 44.892 44.817 44.788 44.755 44.744 44.826 0.188

[0232] Table 17 Larotaxel-I9 limit of quantitation test results

[0233] name 1 2 3 4 5 6 average RSD(%) Peak area 2.99 2.94 3.44 2.94 2.82 2.80 2.99 7.81 Retention time 64.188 64.148 64.079 64.151 64.101 64.107 64.129 0.063

[0234] Table 18 Larotaxel-I6 limit of quantitation test results

[0235] name 1 2 3 4 5 6 average RSD(%) Peak area 5.04 4.80 4.79 4.74 4.73 4.75 4.81 2.43 Retention time (min) 68.282 68.155 68.069 68.00 67.96 67.941 68.069 0.191

[0236] Table 19 Sensitivity test results

[0237]

[0238] Calculation formula:

[0239] Quantitation limit / detection limit concentration (μg / ml) = sample weight × impurity content / dilution factor

[0240] Quantitation limit / detection limit (ng) = concentration × injection volume

[0241] Quantitative limit to test sample ratio (%) = quantitative limit concentration / test sample concentration × 100%

[0242] From the above sensitivity test results, we can know that:

[0243] The quantitative limit solution was tested for 6 consecutive times, and the RSD of the peak area of ​​larotaxel and each impurity was no more than 10.0%, and the RSD of the retention time was no more than 2.0%; the quantitative limits were all less than the reporting limit (0.05% test sample concentration).

[0244] Test Example 3

[0245] Linearity test

[0246] (1) Sample preparation

[0247] Solvent: Acetonitrile

[0248] Blank solution: solvent

[0249] Larotaxel-I1 reference substance stock solution: Take about 2 mg of larotaxel-I1 reference substance, accurately weigh it, put it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well (200 μg / ml).

[0250] The reference stock solutions of larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 were prepared with the “larotaxel-I1 reference stock solution”.

[0251] Larotaxel-I1 reference solution: Accurately measure 2 ml of larotaxel-I1 reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with acetonitrile, and shake well (40 μg / ml).

[0252] The larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10, and larotaxel-I11 reference solutions were prepared with the “larotaxel-I1 reference solution”.

[0253] System suitability solution: Take about 10 mg of larotaxel reference substance, place it in a 10 ml volumetric flask, add acetonitrile to dissolve it, accurately add 0.5 ml each of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10, and larotaxel-I11 reference substance solutions, dilute to the scale with acetonitrile, and shake well (larotaxel: 1 mg / ml, each impurity: 2 μg / ml).

[0254] Larotaxel reference solution: take about 10 mg of larotaxel reference, accurately weigh, place in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well; accurately measure 1 ml, place in a 10 ml volumetric flask, dilute to the scale with acetonitrile, shake well (100 μg / ml).

[0255] Linear stock solution: Accurately measure 5 ml of reference solution, 2.5 ml of larotaxise-I1, larotaxise-I2, larotaxise-I3, larotaxise-I4 and larotaxise-I5 stock solutions, place them in the same 50 ml volumetric flask, dilute to the mark with acetonitrile and shake well.

[0256] Linear solution 1 (quantitation limit solution): prepared as in Example 9 under "Detection limit and quantitation limit".

[0257] Linear solution 2 (equivalent to 0.05% of the test sample concentration): Accurately measure 1 ml of linear stock solution 1, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well (concentration is about 0.5 μg / ml).

[0258] Linear solution 3 (equivalent to 0.1% of the test sample concentration): Accurately measure 12 ml of the linear stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well (concentration is about 1 μg / ml).

[0259] Linear solution 4 (equivalent to 0.2% of the test sample concentration): Accurately measure 14 ml of linear stock solution, place in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well (concentration is about 2 μg / ml).

[0260] Linear solution 5 (equivalent to 0.3% of the test sample concentration): Accurately measure 16 ml of the linear stock solution, place it in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well (concentration is about 3 μg / ml).

[0261] Linear solution 6 (equivalent to 0.5% of the test sample concentration): Accurately measure 10 ml of linear stock solution 1, place in a 20 ml volumetric flask, dilute to the scale with solvent, and shake well (concentration is about 5 μg / ml).

[0262] (2) HPLC detection conditions

[0263] Same as Example 1

[0264] (3) Detection steps

[0265] Take 20 μl of blank solution, system suitability solution and each linear solution, inject and test, and record the chromatogram. Inject in the order shown in the table below.

[0266] Table 20 Injection order and requirements

[0267] order Sample name Number of needles 1 blank solution 1 needle 2 System Suitability Solutions 1 needle 3 Linear solution 1~6 1 needle each

[0268] (4) Analysis results

[0269] The linearity test results are shown in the table below.

[0270] Table 21 Larotaxel-I5 linearity test results

[0271]

[0272] Table 22 Larotaxel-I10 linearity test results

[0273]

[0274] Table 23 Larotaxel-I4 linearity test results

[0275]

[0276] Table 24 Larotaxel-I7 linearity test results

[0277]

[0278]

[0279] Table 25 Larotaxel-I11 linearity test results

[0280]

[0281] Table 26 Larotaxel-I3 linearity test results

[0282]

[0283] Table 27 Larotaxel linearity test results

[0284]

[0285] Table 28 Larotaxel-I8 linearity test results

[0286]

[0287]

[0288] Table 29 Larotaxel-I2 linearity test results

[0289]

[0290] Table 30 Larotaxel-I1 linearity test results

[0291]

[0292] Table 31 Larotaxel-I9 linearity test results

[0293]

[0294] Table 32 Larotaxel-I6 linearity test results

[0295]

[0296] The linear correlation coefficient r was ≥ 0.990; the y-axis intercept was less than 25% of the 100% limit concentration peak area, and the RSD of the response factor was no more than 10.0%. With the concentration C (μg / mL) as the abscissa and the corresponding peak area as the ordinate, the linear regression equation was obtained. The linear relationship diagram is shown in Fig.14 , Fig.15 , Fig.16 , Fig.17 , Fig.18 , Fig.19 , Fig. 20 , Fig.21 , Fig. 22 , Fig.23 , Fig.24 , Fig.25 The linear relationship between the concentration and peak area of ​​larotaxel-I5, larotaxel-I10, larotaxel-I4, larotaxel-I7, larotaxel-I11, larotaxel-I3, larotaxel, larotaxel-I8, larotaxel-I2, larotaxel-I1, larotaxel-I19, and larotaxel-I6 was good, meeting the verification requirements.

[0297] Test Example 4

[0298] The accuracy test includes the following steps:

[0299] (1) Sample preparation

[0300] Solvent: Acetonitrile

[0301] Blank solution: solvent

[0302] System suitability solution: Use the “System suitability solution” prepared under “Test Example 1”.

[0303] Test solution: Take about 10 mg of the product, weigh accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and use it as the test solution (1 mg / ml).

[0304] Reference substance stock solution: take about 2 mg of each of larotaxel-I5, larotaxel-I10, larotaxel-I4, larotaxel-I7, larotaxel-I11, larotaxel-I3, larotaxel, larotaxel-I8, larotaxel-I2, larotaxel-I1, larotaxel-I9, and larotaxel-I6 reference substances, accurately weigh, place in the same 20 ml volumetric flask, add solvent to dissolve and dilute to scale, shake well; accurately measure 5 ml, place in a 50 ml volumetric flask, dilute to scale with solvent, shake well. (Each impurity concentration is about 10 μg / ml, prepare two in parallel).

[0305] Reference substance solution: accurately measure 2 ml of reference substance stock solution, place in a 10 ml volumetric flask, dilute to scale with solvent, and shake well. (The concentration of each impurity is about 2 μg / ml, corresponding to the reference substance stock solution one by one, prepare two in parallel).

[0306] Recovery solution (take reference stock solution -1 to prepare recovery solution).

[0307] 50% solution: Take about 10 mg of this product, weigh accurately, put it in a 10 ml volumetric bottle, add an appropriate amount of solvent to dissolve, accurately add 1 ml of reference substance stock solution, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0308] 100% solution: Take about 10 mg of this product, weigh accurately, place in a 10 ml volumetric bottle, add an appropriate amount of solvent to dissolve, accurately add -1 2 ml of reference substance stock solution, dilute to scale with solvent, shake well. Prepare 3 copies in parallel.

[0309] 150% solution: Take about 10 mg of this product, weigh accurately, put it in a 10 ml volumetric bottle, add an appropriate amount of solvent to dissolve, accurately add 3 ml of reference substance stock solution -1, dilute to the scale with solvent, shake well. Prepare 3 copies in parallel.

[0310] (2) HPLC detection conditions

[0311] Same as Example 1

[0312] (3) Detection steps

[0313] Take 20 μl of blank solution, system suitability solution, reference solution, test solution and recovery solution, inject and test, and record the chromatogram. Inject in the order shown in the table below.

[0314] Table 33 Injection order and requirements

[0315]

[0316]

[0317] Note: Calculation formula: Recovery rate % = (measured amount - content in sample) / added amount × 100% (4) Analysis results

[0318] The accuracy test results are shown in the table below.

[0319] Table 34 Larotaxel-I5 recovery test results

[0320]

[0321] Table 35 Larotaxel-I10 recovery test results

[0322]

[0323] Table 36 Larotaxel-I4 recovery test results

[0324]

[0325]

[0326] Table 37 Larotaxel-I7 recovery test results

[0327]

[0328] Table 38 Larotaxel-I11 recovery test results

[0329]

[0330] Table 39 Larotaxel-I3 recovery test results

[0331]

[0332] Table 40 Larotaxel-I8 recovery test results

[0333]

[0334] Table 41 Larotaxel-I2 recovery test results

[0335]

[0336] Table 42 Larotaxel-I1 recovery test results

[0337]

[0338] Table 43 Larotaxel-I9 recovery test results

[0339]

[0340]

[0341] Table 44 Larotaxel-I6 recovery test results

[0342]

[0343] Taking the impurity limit concentration as 100%, impurities were quantitatively added to the sample at three concentrations of 50%, 100% and 150%. At each concentration level, the impurity recovery rate should be in the range of 90.0% to 110.0%. The RSD of the 9 recoveries was less than 10.0%, with good accuracy.

[0344] Test Example 4

[0345] Repeatability test

[0346] (1) Sample preparation

[0347] Solvent: Acetonitrile

[0348] Blank solution: solvent

[0349] System suitability solution: Use the “System suitability solution” prepared under “Test Example 1”.

[0350] Test solution: Take about 10 mg of the product, weigh accurately, put it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale, shake well, and use it as the test solution (prepare 6 copies in parallel).

[0351] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with solvent, shake well, and use it as the control solution (prepare 6 copies in parallel).

[0352] (2) HPLC detection conditions

[0353] Same as Example 1

[0354] (3) Detection steps

[0355] Take 20 μl of blank solution, system suitability solution, control solution and test solution, inject and test, and record the chromatogram. Inject in the order shown in the table below.

[0356] Table 45 Injection order and requirements

[0357] order sample Number of needles 1 blank solution 1 needle 2 System Suitability Solutions 1 needle 3 Test sample - 1~6 1 needle each 4 Control solution -1~6 1 needle each

[0358] (4) Analysis results

[0359] The repeatability test results are shown in the table below.

[0360] Table 46 Repeatability test results

[0361]

[0362] Note: ND means not detected

[0363] The impurities detected in the 6 test samples were basically consistent, the total impurities and the number of impurities did not change significantly, and the repeatability was good.

[0364] Test Example 5

[0365] Solution stability test,

[0366] (1) Sample preparation

[0367] System suitability solution: Use the “System suitability solution” prepared under “Test Example 1”.

[0368] The preparation of the test solution and the control solution is the same as that of the solution in Test Example 1.

[0369] Solvent: Acetonitrile

[0370] Blank solution: solvent

[0371] (2) HPLC detection conditions

[0372] Same as Example 1

[0373] (3) Detection steps

[0374] Take 20 μl of blank solution, system suitability solution, control solution and test solution, inject and test, and record the chromatogram. Inject in the order shown in the table below.

[0375] Table 47 Injection order and requirements

[0376]

[0377]

[0378] (4) Analysis results

[0379] The solution stability test results are shown in the table below.

[0380] Table 48 Stability results of test solution

[0381]

[0382]

[0383] Table 49 Control solution stability test results

[0384] Time (h) Retention time (min) Peak area 1.5 34.444 288.36 7.5 34.340 288.59 13.5 34.334 288.55 19.5 34.481 288.95 25.5 34.411 289.95 31.5 34.603 290.95 37.5 34.639 290.65 43.5 34.642 291.87 Mean 34.487 289.73 RSD(%) 0.369 0.45

[0385] The test solution was placed at room temperature for 42 hours, and impurities larotaxel-I10, larotaxel-I4, larotaxel-I7, larotaxel-I11, larotaxel-I3, larotaxel-I2, larotaxel-I1, and larotaxel-I6 were not detected; the average content of impurity larotaxel-I5 at each time point was 0.005%, which was relatively consistent; the average content of impurity larotaxel-I8 at each time point was 0.031%, which was relatively consistent; the average content of impurity larotaxel-I9 at each time point was 0.038%, which was relatively consistent; the average content of the largest unknown single impurity at each time point was 0.028%, which was relatively consistent; the average content of total impurities at each time point was 0.119%, and the RSD was 3.32%, which was less than 15.0; there was no obvious change in the number of impurities, indicating that the test solution was stable after being placed at room temperature for 42 hours. The control solution was placed at room temperature for 43.5 hours, and the RSD of the peak area of ​​the main peak was 0.45%, which was no greater than 5.0%, indicating that the control solution was stable after being placed at room temperature for 43.5 hours.

[0386] In summary, the detection method of the embodiment of the present invention can achieve one-time efficient separation of related substances of eleven impurities controlled in larotaxel, and has strong specificity, good durability and high sensitivity, and can be used for quality control of larotaxel raw materials.

[0387] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting larotaxel-related substances, characterized in that: The method comprises: using high performance liquid chromatography to detect the test solution; the detection conditions of the high performance liquid chromatography include: Detection wavelength 213~217nm; Mobile phase A: 0.04wt%~0.06wt% phosphoric acid aqueous solution; Mobile phase B: acetonitrile; Perform gradient elution according to the following program: The larotaxel-related substances include: larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10, and larotaxel-I11, and the structural formulas are: , , , , , , , , , , ; The flow rate of the gradient elution is 0.9-1.1 mL / min; The HPLC column is an octadecylsilane bonded silica gel column, and the column temperature of the HPLC is 23-27°C; The solvent for preparing the test solution and the control solution is acetonitrile.

2. The detection method according to claim 1, characterized in that: The elution procedure is: 。 3. The detection method according to claim 1 or 2, characterized in that: The flow rate of the gradient elution was 1.0 mL / min.

4. The detection method according to claim 1 or 2, characterized in that: The chromatographic column is Agilent 5 TC-C18 (2) 4.6 mm×250 mm, 5 μm; the column temperature is 25°C.

5. The detection method according to claim 1 or 2, characterized in that: The injection volume of the high performance liquid chromatography is 10-25 μL.

6. The detection method according to claim 1 or 2, characterized in that: The injection volume of the HPLC was 20 μL.

7. The detection method according to claim 1 or 2, characterized in that: The contents of related substances of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 in the test solution were calculated by the self-control method.

8. The detection method according to claim 7, characterized in that: The method for calculating the content of the related substances comprises: injecting the test solution and the control solution into a high performance liquid chromatograph respectively, determining the corresponding chromatographic peak areas, and calculating the contents of the related substances of larotaxel-I1, larotaxel-I2, larotaxel-I3, larotaxel-I4, larotaxel-I5, larotaxel-I6, larotaxel-I7, larotaxel-I8, larotaxel-I9, larotaxel-I10 and larotaxel-I11 in the test solution by the self-control method.

9. The detection method according to claim 1 or 2, characterized in that: The conditions of the high performance liquid chromatography meet the following requirements: The detection wavelength is 215nm; Mobile phase A: 0.05% phosphoric acid aqueous solution; Mobile phase B: acetonitrile; The flow rate of the gradient elution is 1.0 ml / min; The chromatographic column is Agilent 5 TC-C18 (2) 4.6 mm × 250 mm, 5 μm, and the column temperature of the chromatographic column is 25°C; The injection volume was 20 μL.

10. Use of the method for detecting larotaxel-related substances according to any one of claims 1 to 9 in the quality detection of larotaxel.

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