Mass spectrometric detection and analysis method for three genotoxic impurities in lamidetan hemisuccinate

Abstract

The invention provides a mass spectrometric detection and analysis method for three genotoxic impurities in lamidetan hemisuccinate, which adopts a mass spectrometric detector, selects proper mobile phase, column temperature, flow velocity, chromatographic column and ion pair, performs quantitative analysis on the three impurities in lamidetan hemisuccinate, improves the detection means for quality control of a lamidetan hemisuccinate product, and improves the detection accuracy of the lamidetan hemisuccinate product. The method is convenient and feasible, has high accuracy, can effectively control the quality of lamidetan hemisuccinate, and improves the safety of drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a method for analyzing and determining impurities in lasmiditan hemisuccinate. Background Art

[0002] Lasmiditan (Las. trade name: Reyvow) is a 5-HT1f receptor agonist developed by Eli Lilly Pharmaceutical Company and was approved by the US Food and Drug Administration in October 2019 for the treatment of acute migraine. Clinical studies have shown that it has a satisfactory effect in treating migraine, can significantly increase the proportion of patients without headache, and reduce the incidence of headache aura symptoms. Lasmiditan has no obvious vasoconstrictive effect and is suitable for patients with cardiovascular diseases. Lasmiditan has good tolerance, is safe and reliable for long-term use, and common adverse reactions include dizziness, fatigue, paresthesia, etc. The marketed product of lasmiditan is the hemisuccinate salt, and its structural formula is shown in Formula (I): It provides an important treatment option for the treatment of acute migraine attacks.

[0003] In order to better control the quality of lasmiditan hemisuccinate and improve the safety of the drug, it is necessary to strictly monitor the impurities (including degradation impurities and process impurities) that may be generated in the synthesis process of lasmiditan hemisuccinate. The structural formulas of the impurities are shown as follows:

[0004] WHN2006-Z15:

[0005] WHN2006-Z19:

[0006] WHN2006-Z34:

[0007] The existing preparation process of lasmiditan raw material drug is prone to generate genotoxic impurities with the above structures, and the impurity limits are low. It is difficult to detect their contents by ordinary HPLC methods, and there is currently no relevant report on the analytical detection method for 3 genotoxic impurities in lasmiditan hemisuccinate. Summary of the Invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a detection method for 3 genotoxic impurities in lasmiditan hemisuccinate with good stability, good reproducibility, simple operation and high precision; specifically, it is a mass spectrometry method that uses a mass spectrometry detector and selects appropriate mobile phase, column temperature, flow rate, chromatographic column and ion pair to quantitatively analyze 3 impurities in lasmiditan hemisuccinate.

[0009] The technical solution adopted by the present invention to solve the above technical problems is:

[0010] An analytical method for three genotoxic impurities in ramelteon hemisuccinate, and the genotoxic impurities are respectively:

[0011] WHN2006-Z15:

[0012] WHN2006-Z19:

[0013] WHN2006-Z34:

[0014] The method includes the following steps:

[0015] (1) Prepare a test solution:

[0016] Dissolve and dilute the ramelteon hemisuccinate sample to be detected with a solvent to a solution containing 0.2 mg to 2 mg per 1 ml as the test solution; preferably, the concentration of the test solution is 0.5 - 1.5 mg / ml or 0.8 - 1.2 mg / ml; more preferably, the concentration of the test solution is 1 mg / ml.

[0017] (2) Prepare an impurity reference solution:

[0018] Take the reference substances of impurities WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34, dissolve them with a solvent, and prepare solutions containing 3 - 20 ng / ml of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34 respectively; preferably, the impurity reference solution contains 4 - 15 ng / ml of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34, and more preferably 5 - 10 ng / ml or 6.5 ng / ml.

[0019] (3) Set the mass spectrometry conditions:

[0020] Use a mass spectrometry detector.

[0021] The Fragmentor voltage value is 30 V - 150 V.

[0022] The collision energy is 10 V - 40 V.

[0023] Set quantitative and / or qualitative ion pairs for genotoxic impurities.

[0024] (4) Set the chromatographic conditions:

[0025] Chromatographic column: A chromatographic column filled with octadecylsilyl-bonded silica gel.

[0026] Mobile phase: One or a mixture of two of formic acid aqueous solution or ammonium formate aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; preferably, the mobile phase A is formic acid aqueous solution;

[0027] Mobile phase flow rate: 0.4 - 0.6 ml / min;

[0028] Injection volume: 5 μl - 10 μl;

[0029] Column temperature: 28°C - 32°C;

[0030] Sample tray temperature: 5°C - room temperature;

[0031] Gradient elution program is as follows:

[0032]

[0033] (5) Determination:

[0034] Take the test solution in step (1) and the reference solution in step (2), inject them into the liquid chromatography - mass spectrometry instrument, record the mass spectrometry diagram, and determine whether the lamotrigine hemisuccinate sample to be detected contains genotoxic impurities and / or calculate the content of genotoxic impurities in the lamotrigine hemisuccinate sample to be detected by the external standard method.

[0035] Furthermore, the solvents in steps (1) and (2) are one or a mixture of two of water and acetonitrile.

[0036] Furthermore, the solvents in steps (1) and (2) are acetonitrile - water with a volume ratio of 10:90.

[0037] Furthermore, the Fragmentor voltage in step (3) is WHN2006 - Z15: 70 V; WHN2006 - Z19: 50 V; WHN2006 - Z34: 130 V.

[0038] Furthermore, the ion pairs and collision energy of WHN2006 - Z15 in step (3) are: quantitative ion pair 110.1 → 93.1, collision energy 20 V; qualitative ion pair 110.1 → 66.1, collision energy 40 V;

[0039] The ion pairs and collision energy of WHN2006 - Z19 are: quantitative ion pair 95.0 → 78.1, collision energy 20 V;

[0040] The ion pairs and collision energy of WHN2006 - Z34 are: quantitative ion pair 187.2 → 126.1, collision energy 40 V; qualitative ion pair 187.2 → 153.1, collision energy 40 V.

[0041] Further, the concentration of the formic acid aqueous solution or ammonium formate aqueous solution as mobile phase A in step (4) is 0.1% - 0.5%;

[0042] Preferably, the concentration of the formic acid aqueous solution or ammonium formate aqueous solution as mobile phase A is 0.1%.

[0043] Further, the flow rate of the mobile phase in step (4) is 0.5 ml / min.

[0044] Further, the injection volume in step (4) is 10 μl.

[0045] Further, the column temperature in step (4) is 30 °C.

[0046] Further, the temperature of the sample tray in step (4) is 5 °C.

[0047] The beneficial effects of the present invention are:

[0048] The present invention uses a convenient and fast mass spectrometry method to detect 3 genotoxic impurities in ramelteon hemisuccinate, improving the detection means for the quality control of ramelteon hemisuccinate products. This method is convenient, feasible, has high accuracy, can effectively control the quality of ramelteon hemisuccinate, and improve the safety of the drug. Description of the Drawings

[0049] Figure 1 is the mass spectrometry diagram of the blank solvent;

[0050] Figure 2 is the mass spectrometry diagram of the reference substance solution;

[0051] Figure 3 is the mass spectrometry diagram of the test sample of ramelteon hemisuccinate. Specific Embodiments

[0052] The following examples are used to further explain the present invention, but the examples do not limit the present invention in any form.

[0053] Unless otherwise specified, the following instruments and reagents are used in the examples of the present invention:

[0054] Example 1:

[0055] Instrument: Triple quadrupole liquid chromatography - mass spectrometry, including a liquid phase pump, a mass spectrometry detector, and an injector

[0056] Reagents: Formic acid (chromatographically pure), acetonitrile

[0057] Chromatographic column: Octadecylsilane bonded silica gel chromatographic column

[0058] Determined according to the mass spectrometry method (General Principles 0431, Volume IV, Chinese Pharmacopoeia 2020 Edition).

[0059] Blank solution: Solvent, water - acetonitrile (90:10).

[0060] (1) Test solution: Take an appropriate amount of ramelteon hemisuccinate, dissolve and dilute it with the solvent to prepare a solution containing about 1 mg per 1 ml.

[0061] (2) Reference solution:

[0062] (2 - 1) Reference stock solution

[0063] Take about 26 mg of impurity WHN2006 - Z15, accurately weigh it, place it in a 200 - ml volumetric flask, dissolve and dilute it to the mark with the solvent, shake well, and use it as the WHN2006 - Z15 reference stock solution;

[0064] Take about 26 mg of impurity WHN2006 - Z19, accurately weigh it, place it in a 200 - ml volumetric flask, dissolve and dilute it to the mark with the solvent, shake well, and use it as the WHN2006 - Z19 reference stock solution;

[0065] Take about 26 mg of impurity WHN2006 - Z34, accurately weigh it, place it in a 200 - ml volumetric flask, dissolve and dilute it to the mark with the solvent, shake well, and use it as the WHN2006 - Z34 reference stock solution;

[0066] (2 - 2) Accurately measure 1 ml each of the WHN2006 - Z15 reference stock solution, WHN2006 - Z19 reference stock solution, and WHN2006 - Z34 reference stock solution, place them in a 20 - ml volumetric flask, dilute to the mark with the solvent, shake well, accurately measure 1 ml, place it in a 50 - ml volumetric flask, dilute to the mark with the solvent, shake well, accurately measure 1 ml, place it in a 20 - ml volumetric flask, dilute to the mark with the solvent, shake well, and use it as the reference solution.

[0067] (3) Chromatographic conditions and mass spectrometric conditions

[0068] Use octadecylsilane chemically bonded silica gel as the filler (ACE Excel 3C18 - AR, 4.6 mm × 150 mm); use 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B, and perform linear gradient elution according to the following table; the flow rate is 0.5 ml per minute; the column temperature is 30 °C; the injection volume is 10 μl; the sample tray temperature is 5 °C.

[0069] A triple quadrupole mass spectrometer detector was used, and the electrospray positive ion mode (ESI+) was adopted for multiple reaction monitoring (MRM). The selected mass-to-charge ratios (m / z) were 110.1 (double charge) → 93.1 with a collision energy of 20 V and 110.1 (double charge) → 66.1 with a collision energy of 40 V as the detection ion pairs for WHN2006-Z15, the mass-to-charge ratio (m / z) 95.0 → 78.1 with a collision energy of 20 V as the detection ion pair for WHN2006-Z19, and the mass-to-charge ratios (m / z) 187.2 (double charge) → 126.1 with a collision energy of 40 V and 187.2 (double charge) → 153.1 with a collision energy of 40 V as the detection ion pairs for WHN2006-Z34.

[0070]

[0071] For the system suitability requirements, in the chromatogram of the reference solution, the signal-to-noise ratios of the peak heights of the chromatographic peaks of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34 should be greater than 30.

[0072] (4) Determination method: Accurately measure the reference solution and the test solution, inject them into the liquid chromatography-mass spectrometry combined instrument, and record the chromatogram.

[0073] Limit: If there are impurity peaks in the chromatogram of the test solution, calculate the content by the external standard method based on the peak area. The contents of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34 shall not exceed 6.5 ppm, and the sum of the contents shall not exceed 6.5 ppm.

[0074] The results showed that:

[0075] The detected content data of the test sample in this example were: neither WHN2006-Z15 nor WHN2006-Z34 was detected, and the content of WHN2006-Z19 was 0.02 ppm.

[0076] Through a complete methodological verification of this detection method, including accuracy verification, the verification results are as follows

[0077] Table 1 Accuracy results of WHN2006-Z15

[0078]

[0079] Table 2 Accuracy results of WHN2006-Z19

[0080]

[0081]

[0082] Table 3 Accuracy results of WHN2006-Z34

[0083]

[0084] The results show that the recoveries of impurities WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34 in 12 accuracy solutions are all between 70% and 130%, and the RSD (n = 12) are 5.2%, 8.9%, and 9.3% respectively, not greater than 15%, indicating that the accuracy of this method is good.

[0085] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An analytical method for three genotoxic impurities in lasmiditan semi-succinate, characterized in that, the genotoxic impurities are respectively: WHN2006-Z15: WHN2006-Z19: WHN2006-Z34: the method comprises the following steps: (1) Preparation of test solution: Dissolve and dilute the lasmiditan semi-succinate sample to be detected with a solvent to a solution containing 0.2 mg to 2 mg per 1 ml as the test solution; preferably, the concentration of the test solution is 0.5 - 1.5 mg / ml or 0.8 - 1.2 mg / ml; more preferably, the concentration of the test solution is 1 mg / ml; (2) Preparation of impurity reference solution: Take the reference substances of impurities WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34, dissolve them with a solvent, and prepare solutions containing 3 - 20 ng / ml of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34 respectively; Preferably, the impurity reference solution contains 4 - 15 ng / ml of WHN2006-Z15, WHN2006-Z19, and WHN2006-Z34; More preferably, it is 5 - 10 ng / ml; Even more preferably, it is 6.5 ng / ml; (3) Setting of mass spectrometry conditions: Using a mass spectrometry detector, The Fragmentor voltage value is 30 V - 150 V, The collision energy is 10 V - 40 V, Set quantitative and / or qualitative ion pairs for genotoxic impurities; (4) Setting of chromatographic conditions: Chromatographic column: A chromatographic column filled with octadecylsilyl-bonded silica gel; Mobile phase: A mixture of one or both of formic acid aqueous solution or ammonium formate aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B; preferably, the mobile phase A is formic acid aqueous solution; Mobile phase flow rate: 0.4 - 0.6 ml / min; Injection volume: 5 μl - 10 μl; Column temperature: 28°C - 32°C; Sample tray temperature: 5°C - room temperature; The gradient elution program is: (5) Determination: Inject the test solution in step (1) and the reference solution in step (2) into the liquid chromatography - mass spectrometry instrument, record the mass spectrometry diagram, and determine whether the lasmiditan semi-succinate sample to be detected contains genotoxic impurities and / or calculate the content of genotoxic impurities in the lasmiditan semi-succinate sample to be detected by the external standard method.

2. The analytical method according to claim 1, characterized in that, the solvents in steps (1) and (2) are one or a mixture of two of water and acetonitrile.

3. The analytical method according to claim 1, characterized in that, the solvents in steps (1) and (2) are acetonitrile - water with a volume ratio of 10:

90.

4. The analytical method according to claim 1, characterized in that, the Fragmentor voltage in step (3) is WHN2006-Z15: 70 V; WHN2006-Z19: 50 V; WHN2006-Z34: 130 V.

5. The analytical method according to claim 1, characterized in that, The ion pairs and collision energies of WHN2006-Z15 described in step (3) are: quantitative ion pair 110.1 → 93.1, collision energy 20 V; qualitative ion pair 110.1 → 66.1, collision energy 40 V; The ion pairs and collision energies of WHN2006-Z19 described are: quantitative ion pair 95.0 → 78.1, collision energy 20 V; The ion pairs and collision energies of WHN2006-Z34 described are: quantitative ion pair 187.2 → 126.1, collision energy 40 V; qualitative ion pair 187.2 → 153.1, collision energy 40 V.

6. The analysis method according to claim 1, characterized in that, the concentration of the formic acid aqueous solution or ammonium formate aqueous solution as mobile phase A in step (4) is 0.1% - 0.5%; Preferably, the concentration of the formic acid aqueous solution or ammonium formate aqueous solution as mobile phase A is 0.1%.

7. The analysis method according to claim 1, characterized in that, the flow rate of the mobile phase in step (4) is 0.5 ml / min.

8. The analysis method according to claim 1, characterized in that, the injection volume in step (4) is 10 μl.

9. The analysis method according to claim 1, characterized in that, the column temperature in step (4) is 30 °C.

10. The analysis method according to claim 1, characterized in that, the sample disk temperature in step (4) is 5 °C.

Images