Method for detecting N-nitrosavatioxetine
The content of N-nitrosamine vothionetine hydrobromide was detected by liquid mass junction, which solved the problem of lack of detection methods in the prior art, and achieved the quality of vothionetine hydrobromide product effective control and good detection performance.
Patent Information
- Application Number
- CN202411312991.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2024-12-31
AI Technical Summary
The prior art lacks effective methods to detect the content of N-nitrosamine vothionetine in vothionetine hydrobromide, affecting product quality and safety.
Using the liquid mass junction method, the waters XBridge C18 chromatography column, a mixed solution of 10mM ammonium formate solution and acetonitrile was used as the mobile phase, and the detection was carried out in combination with the mass spectrometry parameters. The N-nitrosamine voltsulfide in the test sample was calculated by the external standard method of the reference sample. The content of Etetine.
Accurate detection of N-nitrosamine vothionetine in vothionetine hydrobromide is achieved, which improves product quality control and has good specificity, sensitivity and accuracy.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of chemical drug analysis, and in particular to a method for detecting related substances in vortioxetine hydrobromide by liquid chromatography-mass spectrometry. Background Art
[0002] Vortioxetine hydrobromide is a 5-hydroxytryptamine (5-HT) reuptake inhibitor and an agonist, partial agonist or antagonist of the 5-HT receptor. It was jointly developed by Lundbeck Pharmaceuticals of Denmark and Takeda Pharmaceuticals of Japan for the treatment of depression.
[0003]
[0004] N-nitrosamine vortioxetine is a nitrosamine genotoxic impurity. It is necessary to control its content in raw materials and preparations to ensure the quality and safety of the products. However, there is currently no literature or other information disclosing the determination method of N-nitrosamine vortioxetine in vortioxetine hydrobromide. Therefore, in order to control the quality of vortioxetine hydrobromide products, it is necessary to develop a method for detecting N-nitrosamine vortioxetine in vortioxetine hydrobromide.
[0005] . Summary of the invention
[0006] The present invention provides a method for determining the content of N-nitrosamine vortioxetine in vortioxetine hydrobromide by liquid chromatography-mass spectrometry, which is characterized by comprising the following chromatographic conditions: Column: Waters XBridge C18; Mobile phase: Mobile phase A is 10 mM ammonium formate solution, mobile phase B is acetonitrile, mobile phase A: mobile phase B = 15:85; Elution mode: isocratic elution; Ion source: ESI (+).
[0007] Furthermore, the pH of the mobile phase A is 2.2-2.5.
[0008] Furthermore, the column temperature in the chromatographic conditions is 29-31°C.
[0009] Furthermore, the flow rate in the chromatographic conditions is 0.5-0.7 ml / min.
[0010] Furthermore, the injection volume in the chromatographic conditions is 2-30 μl, preferably 10 μl.
[0011] Furthermore, the chromatographic conditions adopt the following mass spectrometry parameters:
[0012] Furthermore, the detection method further comprises the following steps: (1) Preparation of mobile phase A: Accurately measure 1000 ml of ultrapure water, add 630 mg of ammonium formate, mix well, adjust the pH with formic acid, and sonicate. (2) Preparation of reference solution (fresh preparation for immediate use): Take an appropriate amount of N-nitrosamine vortioxetine reference substance, accurately weigh it, and quantitatively dilute it with 50% acetonitrile to prepare a solution containing about 10 ng of N-nitrosamine vortioxetine per 1 ml; (3) Preparation of test solution: Accurately weigh about 10 mg of vortioxetine hydrobromide and place it in a 10 ml volumetric flask. Add an appropriate amount of 50% acetonitrile and sonicate to dissolve it. Then add 50% acetonitrile to make the volume to the mark to prepare a solution containing about 1 mg of vortioxetine hydrobromide per 1 ml. (4) Detection: Take the above-mentioned reference solution, test solution and 50% acetonitrile (blank solvent) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram.
[0013] The present invention adopts a liquid chromatography-mass spectrometry method, selects a Waters XBridge C18 chromatographic column, adopts a mixed solution of an ammonium formate solution and acetonitrile as a mobile phase, detects N-nitrosamine vortioxetine in vortioxetine hydrobromide, and can use a reference substance external standard method to calculate the content of N-nitrosamine vortioxetine in the test solution. The method of the present invention is simple, has good specificity, sensitivity and accuracy, is suitable for the determination of N-nitrosamine vortioxetine in vortioxetine hydrobromide, is conducive to the control of the content of genotoxic impurities in vortioxetine hydrobromide, and improves the product quality of vortioxetine hydrobromide. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 is the chromatogram of the reference solution when the chromatographic column is Waters XBridge C18 in Example 1; Figure 2 is the chromatogram of the test solution when the chromatographic column is Waters XBridge C18 in Example 1; Figure 3 is the chromatogram of the spiked test solution when the chromatographic column is Waters XBridge C18 in Example 1; Figure 4 is the chromatogram of the blank solvent when the chromatographic column is Waters XBridge C18 in Example 1; Figure 5 is the chromatogram of the reference solution when the chromatographic column is Waters Xbridge Shield RP18 in Example 1; Figure 6is the chromatogram of the test solution when the chromatographic column is Waters Xbridge Shield RP18 in Example 1; Figure 7 is the chromatogram of the blank solvent when the chromatographic column is Waters Xbridge Shield RP18 in Example 1; Figure 8 is a chromatogram of the reference solution when the mobile phase A is 10 mM ammonium formate solution (pH=2.2) in Example 2; Fig. 9 is a chromatogram of a blank solvent when the mobile phase A is a 10 mM ammonium formate solution (pH=2.2) in Example 2; Fig.10 is a chromatogram of the reference solution when the mobile phase A is 10 mM ammonium formate solution (pH=3.0) in Example 2; Fig.11 This is the chromatogram of the blank solvent when the mobile phase A in Example 2 is 10 mM ammonium formate solution (pH=3.0). DETAILED DESCRIPTION
[0015] The content of the present invention is further described in detail below by way of embodiments. However, it should not be understood that the above subject matter of the present invention is limited to the following embodiments. In the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other at will.
[0016] Example 1 Liquid chromatography-mass spectrometry (LC / MS / MS) was used to determine the content of N-nitrosamine vortioxetine in vortioxetine hydrobromide. The chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm) or Waters Xbridge ShieldRP18 (150×4.5mm, 3.5μm); Mobile phase: Mobile phase A is 10 mM ammonium formate solution (pH = 2.5), mobile phase B is acetonitrile, mobile phase A: mobile phase B = 15:85; Elution mode: isocratic elution; Flow rate: 0.6ml / min; Column temperature: 30°C; Injection volume: 10 μl; Collection time: 10min; Ion source: ESI (+); Mass spectrometry parameters:
[0017] Experimental steps: (1) Preparation of mobile phase A: Accurately measure 1000 ml of ultrapure water, add 630 mg of ammonium formate, mix well, adjust the pH to 2.5 with formic acid, and sonicate for 15 min. (2) Preparation of reference solution: Take an appropriate amount of N-nitrosamine vortioxetine reference substance, accurately weigh it, and quantitatively dilute it with 50% acetonitrile to prepare a solution containing about 10 ng of N-nitrosamine vortioxetine per 1 ml; (3) Preparation of test solution: Accurately weigh about 10 mg of vortioxetine hydrobromide and place it in a 10 ml volumetric flask. Add an appropriate amount of 50% acetonitrile and sonicate to dissolve it. Then add 50% acetonitrile to make the volume to the mark to prepare a solution containing about 1 mg of vortioxetine hydrobromide per 1 ml. (4) Preparation of spiked test solution: Take about 2 mg of N-nitrosamine vortioxetine reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and obtain N-nitrosamine vortioxetine stock solution; accurately pipette an appropriate amount of N-nitrosamine vortioxetine stock solution, quantitatively dilute it with 50% acetonitrile to make a solution containing about 100 ng of N-nitrosamine vortioxetine per 1 ml, which is the spiked solution; accurately weigh 10 mg of vortioxetine hydrobromide, place it in a 10 ml volumetric flask, add an appropriate amount of 50% acetonitrile solution, accurately add 0.1 ml of spiked solution, dilute it to the mark with 50% acetonitrile, shake well, and obtain; (5) Detection: Take the above-mentioned reference solution, test solution, spiked test solution and 50% acetonitrile (blank solvent) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram. Calculate the content of N-nitrosamine vortioxetine in the test solution according to the reference external standard method.
[0018] See the detection spectrum Figure 1 to Figure 7 The results showed that the method had weak retention and poor specificity when using the Waters Xbridge Shield RP18 column; the N-nitrosamine vortioxetine impurity in vortioxetine hydrobromide could be detected using the Waters XBridge C18 column.
[0019] Example 2 The content of N-nitrosamine vortioxetine in vortioxetine hydrobromide was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Mobile phase: Mobile phase A is 10mM ammonium formate solution (pH=2.2), 10mM ammonium formate solution (pH=2.5) or 10mM ammonium formate solution (pH=3.0), mobile phase B is acetonitrile, mobile phase A: mobile phase B = 15:85; Other chromatographic conditions were the same as in Example 1.
[0020] Experimental steps: (1) Preparation of mobile phase A: Accurately measure 1000 ml of ultrapure water, add 630 mg of ammonium formate, mix well, adjust the pH to 2.2, 2.5 or 3.0 with formic acid, and sonicate for 15 min to obtain three mobile phases A with different pH values. (2) The preparation of the reference solution and the test solution is the same as in Example 1; (3) Detection: Take the above-mentioned reference solution, test solution and 50% acetonitrile (blank solvent) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram.
[0021] Some of the detection spectra can be seen Figure 8~Figure 11 The results showed that when the mobile phase A was 10 mM ammonium formate solution (pH = 3.0), the specificity of the method was poor.
[0022] Example 3 The content of N-nitrosamine vortioxetine in vortioxetine hydrobromide was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Column temperature: 29°C, 30°C or 31°C; Other chromatographic conditions were the same as in Example 1.
[0023] Experimental steps: (1) The preparation of mobile phase, reference solution and test solution is the same as in Example 1; (3) Detection: Take the above-mentioned reference solution, test solution and 50% acetonitrile (blank solvent) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram.
[0024] The test results are shown in Table 1. The three column temperature conditions can achieve the determination of N-nitrosamine vortioxetine.
[0025] Table 1
[0026] Example 4
[0027] The content of N-nitrosamine vortioxetine in vortioxetine hydrobromide was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Flow rate: 0.5ml / min, 0.6ml / min or 0.7ml / min; Other chromatographic conditions were the same as in Example 1.
[0028] Experimental steps: (1) The preparation of mobile phase, reference solution and test solution is the same as in Example 1; (3) Detection: Take the above-mentioned reference solution, test solution and 50% acetonitrile (blank solvent) and inject them into the liquid chromatography-mass spectrometer respectively, measure under the above-mentioned chromatographic conditions, and record the chromatogram.
[0029] The test results are shown in Table 2. The three flow rate conditions can achieve the determination of N-nitrosamine vortioxetine.
[0030] Table 2
[0031] Example 5
[0032] The content of N-nitrosamine vortioxetine in vortioxetine hydrobromide was determined by liquid chromatography-mass spectrometry, and the chromatographic conditions were as follows: Chromatographic column: Waters XBridge C18 (150×4.6mm, 3.5μm); Other chromatographic conditions were the same as in Example 1.
[0033] Experimental steps: (1) Preparation of mobile phase A: same as in Example 1; (2) Preparation of test solution: Take 10 mg of vortioxetine hydrobromide, place it in a 10 ml volumetric flask, dissolve it in 50% acetonitrile and dilute to the mark, shake well to obtain (containing about 1 mg / ml of vortioxetine hydrobromide), prepare 3 copies in parallel; (3) Preparation of N-nitrosamine vortioxetine stock solution: Take about 2 mg of N-nitrosamine vortioxetine reference substance, accurately weigh it, place it in a 20 ml volumetric flask, dissolve it in acetonitrile and dilute it to the mark, shake well, and the solution is ready; (4) Preparation of spiked solution: Accurately pipette an appropriate amount of N-nitrosamine vortioxetine stock solution and quantitatively dilute it with 50% acetonitrile to make a solution containing about 100 ng of N-nitrosamine vortioxetine per 1 ml; (5) Low level accuracy solution: Accurately weigh 10 mg of vortioxetine hydrobromide and place it in a 10 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution and accurately add 0.1 ml of spiked solution. Dilute to the mark with 50% acetonitrile and shake well. Prepare 3 copies in parallel. (6) Medium accuracy solution: Accurately weigh 10 mg of vortioxetine hydrobromide and place it in a 10 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution and accurately add 1 ml of spiked solution. Dilute to the mark with 50% acetonitrile and shake well. Prepare 3 copies in parallel. (7) High-level accuracy solution: Accurately weigh 10 mg of vortioxetine hydrobromide and place it in a 10 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution and accurately add 1.5 ml of spiked solution. Dilute to the mark with 50% acetonitrile and shake well. Prepare 3 copies in parallel. (8) Detection: Inject the above-mentioned test solution, low-level accuracy solution, medium-level accuracy solution, high-level accuracy solution and blank solvent (50% acetonitrile) into a liquid chromatography-mass spectrometer respectively, and measure under the above-mentioned chromatographic conditions and record the chromatogram.
[0034] The test results are shown in Table 3. The test results show that the recovery rates of low-level accuracy solution, medium-level accuracy solution and high-level accuracy solution are between 88.6% and 118.5%, the average recovery rate is 103.8%, and the RSD of the recovery rate is 7.8%. This method has good accuracy and meets the requirements of pharmacopoeia and relevant regulations.
[0035] Table 3 .
Claims
1. A method for determining the content of N-nitrosamine vortioxetine in vortioxetine hydrobromide by liquid chromatography-mass spectrometry, characterized in that The following chromatographic conditions were included: ， Column: Waters XBridge C18; Mobile phase: Mobile phase A is 10 mM ammonium formate solution, mobile phase B is acetonitrile, mobile phase A: mobile phase B = 15:85; Elution mode: isocratic elution; Ion source: ESI (+).
2. The method according to claim 1, wherein the pH of the mobile phase A is 2.2 to 2.
5.
3. The method according to claim 1, wherein the column temperature in the chromatographic conditions is 29-31°C.
4. The method according to claim 1, wherein the flow rate in the chromatographic conditions is 0.5-0.7 ml / min.
5. The method according to claim 1, wherein the injection volume in the chromatographic conditions is 2 to 30 μl. The method according to claim 5 , wherein the injection volume is 10 μl.
7. The method according to any one of claims 1 to 6, wherein the chromatographic conditions include the following mass spectrometry parameters: 。