Analysis method for simultaneously separating and detecting various p-toluenesulfonate and epoxy genotoxicity warning structure impurities in landiolol hydrochloride

Abstract

The invention relates to the technical field of analysis and detection, and discloses an analysis method for simultaneously separating and detecting various p-toluenesulfonate and epoxy genotoxicity warning structure impurities in landiolol hydrochloride. The p-toluenesulfonic acid esters comprise methyl p-toluenesulfonate, ethyl p-toluenesulfonate, six p-toluenesulfonic acid ester impurities and three epoxy ring impurities, respectively sucking the same amount of the test solution and the reference solution, and injecting the test solution and the reference solution into a high performance liquid chromatography for determination; according to the method, methyl p-toluenesulfonate, ethyl p-toluenesulfonate, six p-toluenesulfonate impurities and three epoxy ring impurities in landiolol hydrochloride can be detected at the same time, a plurality of different methods for detecting different types of impurities are not needed, and a mass spectrum instrument or complex derivation pretreatment is not needed to meet the requirement for high sensitivity below 10 ppm. Operation is easy, instrument and detection cost is low, and universality is high; all impurities can be effectively separated, the sensitivity can reach 1ppm, and the recovery rate is 90%-110%.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, specifically to an analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxy genotoxicity warning structural impurities in landilol hydrochloride. Background Technology

[0002] The molecular formula of brandylol hydrochloride is C 25 H 39 N3O8·HCl, chemical name 4-[(2 S )-2-hydroxy-3-[[2-[[2-[(4-morpholinoyl)amino]ethyl]amino]propoxy]phenylpropionic acid[(4 S Landiolol hydrochloride, CAS number 144481-98-1, is a 2,2-dimethyl-4-(1,3-dioxolane)-methyl ester. Its chemical structure is shown in Formula 1. Formula 1: Chemical structural formula of brandylol hydrochloride According to publicly available synthetic routes for brandilol hydrochloride, various routes often utilize toluenesulfonic acid or epoxy compounds, potentially involving multiple genotoxic impurities, including p-toluenesulfonates and epoxy rings. These impurities can severely impact the safety and efficacy of brandilol hydrochloride. Because these two types of genotoxic impurities are subject to stricter limits during drug development, their concentration in brandilol hydrochloride must be controlled below 10 ppm. This typically requires specialized and expensive mass spectrometry instruments or complex sample pretreatment processes such as derivatization to achieve the desired detection sensitivity and accuracy. Existing methods for analyzing impurities in brandylolol hydrochloride, including those for p-toluenesulfonate esters and epoxy compounds, cannot simultaneously detect multiple different types of genotoxic impurities in brandylolol hydrochloride at low cost and with convenient operation. For example, Chinese invention patent application number 202411944980.0 discloses a method for detecting related substances in injectable brandylolol hydrochloride, using octadecylsilane-bonded silica gel as filler, and employing a mobile phase A (a solution of potassium dihydrogen phosphate containing pentafluoropropionic acid and methanol). Gradient elution was used to detect related substances in injectable landiolol hydrochloride using a mobile phase B (potassium dihydrogen phosphate solution containing pentafluoropropionic acid and methanol, volume ratio 28:72-32:68). However, this method requires a special mobile phase system and suffers from poor specificity and insufficient sensitivity. Therefore, establishing a highly sensitive, accurate, low-cost, and easy-to-operate analytical method for simultaneously detecting p-toluenesulfonates and epoxy cyclic genotoxic impurities to ensure drug quality is of paramount importance. Currently, no publicly available literature reports a simple, rapid, and accurate HPLC method for the analysis and separation of multiple p-toluenesulfonates and epoxy cyclic genotoxic impurities in landiolol hydrochloride using a single method. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the present invention aims to provide an analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxy genotoxicity warning structural impurities in landilol hydrochloride. This method is characterized by high sensitivity, high accuracy, good specificity, good durability, simple operation, low cost, and strong versatility.

[0004] In order to achieve the above object, the present invention provides the following technical solutions: An analytical method for simultaneously separating and detecting multiple p-toluenesulfonate and epoxy genotoxicity warning structural impurities in brandilol hydrochloride, wherein the genotoxicity warning structural impurities include methyl p-toluenesulfonate (PTSM), ethyl p-toluenesulfonate (PTSE), six p-toluenesulfonate impurities, and three epoxy ring impurities; namely, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, and impurity 11, the structural formulas of which are shown below: The test solution is a diluted solution of brandylol hydrochloride. The reference solution is a diluted solution of reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0005] The analytical method includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 respectively, weigh them accurately, and dilute them with solvent to prepare a solution containing approximately 1 μg each of impurities 1, 2, 4, 5, 6, 7, 8, 9, 10 and 11 and 0.25 μg of impurity 3 per ml; S2 Preparation of test solution: Take an appropriate amount of brandylol hydrochloride, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 25mg of brandylol hydrochloride per 1ml; S3 Detection: Take equal amounts of the test solution and the reference solution and inject them into a high-performance liquid chromatograph for determination.

[0006] Preferably, the solvent in steps S1-S3 is an acetonitrile dilution solution in which water and acetonitrile are mixed in a volume ratio of 1:1.

[0007] As a preferred option, the chromatographic conditions in step S3 are as follows: the chromatographic column is filled with octadecylsilane-bonded silica gel, gradient elution is performed through mobile phase A and mobile phase B, the detection wavelength is 220 nm, the column temperature is 25℃~35℃, and the injection volume is 10~30 μl.

[0008] Preferably, the mobile phase A is an aqueous solution of phosphoric acid, and the mobile phase B is acetonitrile.

[0009] Preferably, the pH of the mobile phase A is 1.8 to 2.2.

[0010] Preferably, the gradient elution procedure is as follows: Isocratic elution for 0-45 to 50 minutes, with mobile phase A comprising 64-70% and mobile phase B comprising 30-36%; Gradient elution for 15 minutes, with mobile phase A comprising 64-70% to 25-30% and mobile phase B comprising 30-36% to 70-75%; Elute isocratically for 5 minutes, with mobile phase A comprising 25-30% and mobile phase B comprising 70-75%; Gradient elution for 5 minutes, with mobile phase A comprising 25-30% to 64-70% and mobile phase B comprising 70-75% to 30-36%; Elute at isocratic rate for 10 minutes, with mobile phase A comprising 64-70% and mobile phase B comprising 30-36%.

[0011] Specifically: Initial stage (isocratic elution 0-45 to 50 minutes): Maintain 64%-70% mobile phase A for column equilibration and separation of weakly retained impurities; Intermediate stage (gradient elution 15 minutes): Reduce mobile phase A from 64%-70% to 25%-30% for separation of moderately retained impurities; Strong elution stage (isocratic elution 5 minutes): Maintain 25%-30% mobile phase A for eluting strongly retained impurities; Reequilibration stage (gradient elution 10 minutes): Restore mobile phase A from 25%-30% to 64%-70% for column reequilibration.

[0012] Preferably, the gradient elution procedure is as follows: Isocratic elution was performed for 0–45 minutes, with mobile phase A comprising 66% and mobile phase B comprising 34%. Gradient elution for 15 minutes, with mobile phase A comprising 66% to 30% and mobile phase B comprising 34% to 70%; Elute at isocratic rate for 5 minutes, with mobile phase A at 30% and mobile phase B at 70%. Gradient elution for 5 minutes, with mobile phase A comprising 30% to 66% and mobile phase B comprising 70% to 34%; Elute at isocratic rate for 10 minutes, with mobile phase A comprising 66% and mobile phase B comprising 34%.

[0013] Preferably, the flow rate of the mobile phase is 0.7~1.2 ml / min.

[0014] Preferably, the flow rate of the mobile phase is 0.7~1.2 ml / min, and the column temperature is 25℃~35℃.

[0015] This invention, by adopting the above technical solutions, has significant technical effects: This invention provides a method for the effective detection of multiple sulfonates and epoxy ring genotoxicity warning structures in landilol hydrochloride using the same method. Specifically, it includes: (1) the ability to simultaneously detect methyl p-toluenesulfonate, ethyl p-toluenesulfonate, six p-toluenesulfonate impurities, and three epoxy ring impurities in landilol hydrochloride without the need for multiple different methods to detect different types of impurities; (2) the elimination of the need for mass spectrometry instruments or complex derivatization pretreatment to meet the high sensitivity requirement of less than 10 ppm, the method is simple to operate, has low instrument and detection costs, and strong versatility; (3) the method has been verified to have high sensitivity, with each impurity sensitivity reaching 1 ppm; good specificity, with each analyte effectively separated; good accuracy, with each impurity recovery rate between 90% and 110%; and good durability, with the durability of each chromatographic condition meeting the requirements of the General Chapter 0512 of the Chinese Pharmacopoeia. Attached Figure Description

[0016] Figure 1 The image shows the high performance liquid chromatography detection results in Example 1.

[0017] Figure 2 The image shows the high performance liquid chromatography detection results in Example 2.

[0018] Figure 3 The image shows the high performance liquid chromatography (HPLC) detection results in Example 3.

[0019] Figure 4 The image shows the high performance liquid chromatography detection results in Example 4. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0021] This invention provides an analytical method for simultaneously separating and detecting multiple p-toluenesulfonate and epoxy genotoxicity warning structural impurities in brandylolol hydrochloride. The genotoxicity warning structural impurities include methyl p-toluenesulfonate (PTSM), ethyl p-toluenesulfonate (PTSE), six p-toluenesulfonate impurities, and three epoxy ring impurities; namely, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, and impurity 11, and their structural formulas are shown below: The test solution is a diluted solution of brandylol hydrochloride. The reference solution is a diluted solution of reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

[0022] In this invention, the preparation methods for the test solution and the reference solution are as follows: (1) Reference solution: Take appropriate amounts of reference standards 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 respectively, weigh them accurately, and dilute them with solvent to prepare a solution containing approximately 1 μg each of impurity 1, 2, 4, 5, 6, 7, 8, 9, 10 and 11 and 0.25 μg of impurity 3 per ml.

[0023] (2) Test solution: Take an appropriate amount of brandylol hydrochloride, accurately weigh it, add solvent to dissolve and dilute it to prepare a solution containing about 25 mg of brandylol hydrochloride per 1 ml.

[0024] Detection: Take equal amounts of the test solution and the reference solution and inject them into a high-performance liquid chromatograph for determination.

[0025] Example 1 Chromatographic column: C18 (250 mm × 4.6 mm, 5 μm), column temperature: 33 ℃, flow rate: 1.1 ml / min, detection wavelength: 220 nm, injection volume: 30 μl, mobile phase A: water (adjusted to pH 2.0 with phosphoric acid), mobile phase B: acetonitrile, elution gradient as shown in Table 1: Table 1 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 66 34 45 66 34 60 30 70 65 30 70 70 66 34 80 66 34 The test results are indicated as follows, see appendix for details. Figure 1 (Note: Impurity 6 and impurity 11 are isomers of each other. They eluted at the same time in the attached figure and are marked as peak 6. The same applies below.)

[0026] (1) The blank solvent and the brandyloer peak of hydrochloric acid do not interfere with the detection of each impurity, and the minimum resolution between each impurity peak is not less than 1.5.

[0027] (2) The quantitation limit concentration of each impurity is no greater than 0.1 μg / ml, which is equivalent to 1 ppm of the sample concentration, and the sensitivity is good.

[0028] (3) With the average recovery rate of each impurity between 93.21% and 102.96%, the present invention can accurately quantify each impurity.

[0029] Example 2 Chromatographic column: C18 (250 mm × 4.6 mm, 5 μm), column temperature: 25 ℃, flow rate: 0.7 ml / min, detection wavelength: 220 nm, injection volume: 30 μl, mobile phase A: water (adjusted to pH 2.0 with phosphoric acid), mobile phase B: acetonitrile, elution gradient as shown in Table 2: Table 2 Time (min) Mobile phase A (%) Mobile phase B (%) 0 70 30 45 70 30 60 30 70 65 30 70 70 70 30 80 70 30 The test results are indicated as follows, see appendix for details. Figure 2 .

[0030] (1) The blank solvent and the brandyloer peak of hydrochloric acid do not interfere with the detection of each impurity, and the minimum resolution between each impurity peak is not less than 1.5.

[0031] (2) The quantitation limit concentration of each impurity is no greater than 0.1 μg / ml, which is equivalent to 1 ppm of the sample concentration, and the sensitivity is good.

[0032] Example 3 Chromatographic column: C18 (250 mm × 4.6 mm, 5 μm), column temperature: 35 ℃, flow rate: 1.2 ml / min, detection wavelength: 220 nm, injection volume: 30 μl, mobile phase A: water (adjusted to pH 2.2 with phosphoric acid), mobile phase B: acetonitrile, elution gradient as shown in Table 3: Table 3 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 66 34 45 66 34 60 30 70 65 30 70 70 66 34 80 66 34 The test results are indicated as follows, see appendix for details. Figure 3 .

[0033] (1) The blank solvent and the brandyloer peak of hydrochloric acid do not interfere with the detection of each impurity, and the minimum resolution between each impurity peak is not less than 1.5.

[0034] (2) The quantitation limit concentration of each impurity is no greater than 0.1 μg / ml, which is equivalent to 1 ppm of the sample concentration, and the sensitivity is good.

[0035] Example 4 Chromatographic column: C18 (250 mm × 4.6 mm, 5 μm), column temperature: 31 ℃, flow rate: 1.0 ml / min, detection wavelength: 220 nm, injection volume: 30 μl, mobile phase A: water (adjusted to pH 1.8 with phosphoric acid), mobile phase B: acetonitrile, elution gradient as shown in Table 4: Table 4 Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 66 34 45 66 34 60 30 70 65 30 70 70 66 34 80 66 34 The test results are indicated as follows, see appendix for details. Figure 4 .

[0036] (1) The blank solvent and the brandyloer peak of hydrochloric acid do not interfere with the detection of each impurity, and the minimum resolution between each impurity peak is not less than 1.5.

[0037] (2) The quantitation limit concentration of each impurity is no greater than 0.1 μg / ml, which is equivalent to 1 ppm of the sample concentration, and the sensitivity is good.

[0038] The above description is only a specific embodiment of the present invention, but the technical features of the present invention are not limited thereto. Any changes or modifications made by those skilled in the art within the scope of the present invention are covered by the patent scope of the present invention.

Claims

1. An analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide-based genotoxicity warning structural impurities in landilol hydrochloride, characterized in that, The genotoxicity warning structural impurities include impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, and impurity 11, and their structural formulas are shown below: The test solution is a diluted solution of brandylol hydrochloride. The reference solution is a diluted solution of reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11.

2. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 1, characterized in that, Includes the following steps: S1 Preparation of reference solution: Take appropriate amounts of reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 respectively, weigh them accurately, and dilute them with solvent to prepare a solution containing approximately 1 μg each of impurities 1, 2, 4, 5, 6, 7, 8, 9, 10 and 11 and 0.25 μg of impurity 3 per ml; S2 Preparation of test solution: Take an appropriate amount of brandylol hydrochloride, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 25mg of brandylol hydrochloride per 1ml; S3 Detection: Take equal amounts of the test solution and the reference solution and inject them into a high-performance liquid chromatograph for determination.

3. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 2, characterized in that, In steps S1-S3, the solvent is an acetonitrile dilution solution in which water and acetonitrile are mixed in a volume ratio of 1:

1.

4. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 2, characterized in that, The chromatographic conditions in step S3 are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel, gradient elution is performed through mobile phase A and mobile phase B, the detection wavelength is 220 nm, the column temperature is 25℃~35℃, and the injection volume is 10~30 μl.

5. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 4, characterized in that, The mobile phase A is an aqueous solution of phosphoric acid, and the mobile phase B is acetonitrile.

6. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 5, characterized in that, The pH of the mobile phase A is 1.8 to 2.

2.

7. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 4, characterized in that, The gradient elution procedure is as follows: Isocratic elution for 0-45 to 50 minutes, with mobile phase A comprising 64-70% and mobile phase B comprising 30-36%; Gradient elution for 15 minutes, with mobile phase A comprising 64-70% to 25-30% and mobile phase B comprising 30-36% to 70-75%; Elute isocratically for 5 minutes, with mobile phase A comprising 25-30% and mobile phase B comprising 70-75%; Gradient elution for 5 minutes, with mobile phase A comprising 25-30% to 64-70% and mobile phase B comprising 70-75% to 30-37%; Elute at isocratic rate for 10 minutes, with mobile phase A comprising 64-70% and mobile phase B comprising 30-36%.

8. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 7, characterized in that, The gradient elution procedure is as follows: Isocratic elution was performed for 0–45 minutes, with mobile phase A comprising 66% and mobile phase B comprising 34%. Gradient elution for 15 minutes, with mobile phase A comprising 66% to 30% and mobile phase B comprising 34% to 70%; Elute at isocratic rate for 5 minutes, with mobile phase A at 30% and mobile phase B at 70%. Gradient elution for 5 minutes, with mobile phase A comprising 30% to 66% and mobile phase B comprising 70% to 34%; Elute at isocratic rate for 10 minutes, with mobile phase A comprising 66% and mobile phase B comprising 34%.

9. An analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to any one of claims 4-8, characterized in that, The flow rate of the mobile phase is 0.7~1.2 ml / min.

10. The analytical method for simultaneously separating and detecting multiple p-toluenesulfonates and epoxide genotoxicity warning structural impurities in landilol hydrochloride according to claim 9, characterized in that, The flow rate of the mobile phase is 0.7~1.2 ml / min, and the column temperature is 25℃~35℃.

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