Method for detecting N-nitroso compound in vothioxetine hydrobromide

Abstract

The invention discloses a method for detecting an N-nitroso compound in vortioxetine hydrobromide, and the N-nitroso compound is 2, 2 '-(nitroso amino) bis-acetonitrile. According to the method, a high performance liquid chromatography is adopted, an octadecyl silane bonded silica gel filler chromatographic column and an ultraviolet detector are adopted, a mobile phase A is 0.05% phosphoric acid aqueous solution, a mobile phase B is methanol, and gradient elution is adopted. The method has good specificity and sensitivity.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis, and particularly relates to a method for detecting N-nitroso compounds in vortioxetine hydrobromide. Background Art

[0002] Vortioxetine hydrobromide is a drug used to treat major depressive disorder (MDD). 2,2'-(Nitrosoamino)bisacetonitrile is a process impurity derived from the residual impurity iminodiacetonitrile in the starting material SM3 of vortioxetine hydrobromide and trace amounts of nitrite that may remain in the preparation process of the starting material SM2. The derivation pathway is as follows:

[0003]

[0004] Currently, no literature has been found to detect this impurity in vortioxetine hydrobromide. To ensure that the process yields a qualified final product, this impurity must be controlled in vortioxetine hydrobromide. Therefore, it is necessary to develop an analytical method for the detection of 2,2'-(nitrosoamino)bisacetonitrile in vortioxetine hydrobromide to perform quality control for this impurity.

[0005] The present inventors have discovered through extensive research that, in a conventional reversed-phase liquid chromatography mode, by selecting a special type of silica gel column, and a specific mobile phase and solvent composition, 2,2'-(nitrosoamino)bisacetonitrile can have excellent specificity and sensitivity in liquid chromatography, and can accurately determine the content of 2,2'-(nitrosoamino)bisacetonitrile in vortioxetine hydrobromide. Consequently, the present invention was completed. Summary of the Invention

[0006] The object of the present invention is to provide a method for detecting an N-nitroso compound in vortioxetine hydrobromide. The N-nitroso compound is 2,2'-(nitrosoamino)bisacetonitrile, and its chemical structure is as follows:

[0007] This method uses high performance liquid chromatography and has good specificity and sensitivity.

[0008] To achieve the purpose of the present invention, the following embodiments are provided: In one embodiment, the present invention provides a method for detecting N-nitroso compounds in vortioxetine or its salts, characterized in that the method employs high performance liquid chromatography, and the chromatographic conditions are as follows, comprising: Chromatographic column: octadecylsilane bonded silica gel packing column, Detector: UV detector, Mobile phase: Mobile phase A is 0.05% phosphoric acid aqueous solution, mobile phase B is methanol, Using gradient elution, Wherein, the N-nitroso compound is 2,2'-(nitrosoamino)bisacetonitrile.

[0009] Preferably, in the detection method of the present invention, the chromatographic column is Agilent ZORBAX SB-C18, the chromatographic column has a length of 250 mm and a particle size of 5 μm.

[0010] Furthermore, the detection method of the present invention has the following chromatographic conditions: the detection wavelength of the ultraviolet detector is 230 nm, the column temperature is 28-32° C., the flow rate is 0.7-0.9 ml / min, and the injection volume is 15 μl.

[0011] Furthermore, in the detection method of the present invention, the sample preparation solvent is methanol-0.05% phosphoric acid aqueous solution, wherein the volume ratio of methanol-0.05% phosphoric acid aqueous solution is 2:3.

[0012] Preferably, the detection method of the present invention adopts gradient elution, and its elution conditions are as follows:

[0013] Preferably, in the detection method of the present invention, the salt is hydrobromide, i.e., vortioxetine hydrobromide.

[0014] In a specific embodiment, the present invention provides a method for detecting N-nitroso compounds in vortioxetine hydrobromide, wherein the method employs high performance liquid chromatography, and the chromatographic conditions thereof comprise: Chromatographic column packing: octadecylsilane bonded silica gel packing chromatographic column, UV detector, detection wavelength: 230nm, Column temperature: 28~32℃, Flow rate: 0.7~0.9ml / min, Injection volume: 15 μl, Mobile phase: Phase A is 0.05% phosphoric acid aqueous solution, Phase B is methanol, Elution conditions: The initial volume ratio of mobile phase A to mobile phase B is (73~77): (23~27), ​​preferably 75:25.

[0015] Sample preparation solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v).

[0016] Preferably, in the detection method of the present invention, the model of the octadecylsilane bonded silica gel packing chromatographic column is Agilent ZORBAX SB-C18, the column length is 250 mm, the particle size is 5 μm; the column temperature is 30° C.; the flow rate is 0.8 ml / min, and the sample concentration is 10 mg / ml.

[0017] Preferably, the detection method of the present invention, the procedure of gradient elution is:

[0018] The present invention has the following advantages and positive effects: The detection method of the present invention effectively separates and detects N-nitroso compounds (2,2'-(nitrosoamino)bisacetonitrile) with good accuracy, high sensitivity, and a simple operation process, enabling quantitative detection. This method facilitates quality control during the production process and improves the quality of the final product. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : HPLC chromatograms of the positioning solutions under chromatographic conditions 1 and 2 in Example 1; Figure 2 : HPLC chromatograms of the positioning solutions under chromatographic conditions 3 and 4 in Example 2; Figure 3 : HPLC chromatograms of the positioning solution and the test solution under chromatographic condition 5 in Example 2; Figure 4 : HPLC chromatograms of the positioning solution and the test solution under chromatographic condition 6 in Example 2; Figure 5 : HPLC chromatograms of the positioning solution and the test solution under chromatographic condition 7 in Example 2; Figure 6 : HPLC chromatograms of the positioning solution and the test solution under chromatographic condition 8 in Example 2; Figure 7 : HPLC chromatogram of the stability study of the positioning solution under chromatographic condition 8 in Example 2; Figure 8 : HPLC chromatograms of the positioning solution and the test solution under chromatographic condition 9 in Example 2; Figure 9 : HPLC chromatogram of the stability study of the positioning solution under chromatographic condition 9 in Example 2; Figure 10 : HPLC chromatogram of the mixed solution when the flow rate is 0.7 ml / min in Example 3; Figure 11 : HPLC chromatogram of the mixed solution when the flow rate is 0.9 ml / min in Example 3; Figure 12 : HPLC chromatogram of the mixed solution when the column temperature is 28° C. in Example 4; Figure 13 : HPLC chromatogram of the mixed solution when the column temperature is 32° C. in Example 4; Figure 14: HPLC chromatogram of the mixed solution at 1 hour of gradient elution in Example 5; Figure 15 : HPLC chromatogram of the mixed solution at 2 o'clock of gradient elution among embodiment 5. DETAILED DESCRIPTION

[0020] To make the purpose, technical solutions and advantages of this application more clear, the following embodiments describe the technical solutions of the present invention in detail, but do not limit the scope of the present invention. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of this application can be combined with each other in any way.

[0021] Example 1 Investigation of 2,2'-(nitrosoamino)bisacetonitrile detection conditions using a YMC C18 AQ column Chromatographic conditions 1: Chromatographic column: YMC–Pack-ODS-AQ (4.6 mm*150 mm, 3 μm); Mobile phase A: ultrapure water; Mobile phase B: acetonitrile; Detection wavelength: 254nm (high-sensitivity detection cell); Column temperature: 30°C; Flow rate: 0.5 ml / min; Needle washing solution: acetonitrile; Sample tray temperature: 15°C; Injection volume: 5µl; Solvent: methanol; Elution procedure:

[0022] Chromatographic condition 2: Injection volume: 1µl; The rest of the conditions were the same as those in chromatographic condition 1.

[0023] Sample preparation: Positioning solution: Take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and use solvent to prepare a solution containing 0.67 μg of the reference substance per 1 ml.

[0024] Experimental steps: Under chromatographic conditions 1 and 2, inject the above-mentioned positioning solution into a high performance liquid chromatograph and record the chromatogram.

[0025] The results showed that under the above chromatographic conditions, the peak of 2,2'-(nitrosoamino)bisacetonitrile was severely split. After reducing the injection volume, the peak shape did not improve. The influence of solvent effect was preliminarily ruled out, and it was considered to replace other brands of AQ chromatographic columns for further research. Figure 1 .

[0026] Example 2 Investigation of 2,2'-(nitrosoamino)bisacetonitrile detection conditions using an Agilent C18 AQ column Chromatographic conditions 3: Chromatographic column: Agilent ZORBAX SB-Aq (4.6 mm × 250 mm, 5 μm); Mobile phase A: ultrapure water; Mobile phase B: acetonitrile; Detection wavelength: 254nm (high-sensitivity detection cell); Column temperature: 30°C; Flow rate: 0.5 ml / min; Needle washing solution: acetonitrile; Sample tray temperature: 15°C; Injection volume: 15µl; Solvent: methanol; The elution procedure was the same as that of chromatographic condition 1 in Example 1.

[0027] Chromatographic conditions 4: The flow rate is 0.8 ml / min; The rest of the conditions are the same as those in chromatographic condition 3.

[0028] Chromatographic conditions 5: The elution procedure is:

[0029] The rest of the conditions are the same as those in chromatographic condition 4.

[0030] Chromatographic conditions 6: The solvent was 30% methanol, mobile phase A was 0.05% phosphoric acid aqueous solution, the needle wash solution was methanol, the detection wavelength was 230 nm (high-sensitivity detection cell), and the elution procedure was the same as chromatographic condition 1 in Example 1.

[0031] The rest of the conditions are the same as those in chromatographic condition 5.

[0032] Chromatographic conditions 7: Mobile phase B was methanol; other conditions were the same as those in chromatographic condition 6.

[0033] Chromatographic conditions 8: Elution procedure:

[0034] The rest of the conditions are the same as those in chromatographic condition 7.

[0035] Chromatographic conditions 9: The solvent was methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Elution procedure:

[0036] The rest of the conditions were the same as those in chromatographic condition 8.

[0037] Sample preparation: Positioning solution: same as the positioning solution in Example 1; Test solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, and place it in a 20 ml volumetric flask. First add 8 ml of methanol to dissolve it by ultrasonication, then dilute it to the scale with 0.05% phosphoric acid aqueous solution and shake well.

[0038] Experimental steps: (1) Chromatographic condition 3, inject the positioning solution; (2) Chromatographic condition 4, inject the positioning solution; (3) Chromatographic condition 5, inject the positioning solution and the test solution; (4) Chromatographic condition 6, inject the positioning solution and the test solution; (5) Chromatographic condition 7, inject the positioning solution and the test solution; (6) Chromatographic condition 8, inject the positioning solution and the test solution; (7) Chromatographic condition 9, inject the positioning solution and the test solution.

[0039] The results showed that under chromatographic conditions 3 and 4, the peak shape of 2,2'-(nitrosoamino)bisacetonitrile was normal. See the typical figure for details. Figure 2 Under chromatographic conditions 5, 6, and 7, there are interfering peaks in the test solution, and the specificity is poor. Figures 3-5 Under chromatographic condition 8, 2,2'-(nitrosoamino)bisacetonitrile can achieve effective separation, but the solution stability is poor. See the typical figure for details. Figures 6 and 7 Under chromatographic condition 9, 2,2'-(nitrosoamino)bisacetonitrile can achieve effective separation and better solution stability. See the typical figure for details. Figures 8 and 9 .

[0040] Example 3 Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase A: 0.05% phosphoric acid in water; Mobile phase B: methanol; Detection wavelength: 230nm (high-sensitivity detection cell); Column temperature: 30°C; Flow rate: 0.7ml / min, 0.9ml / min; Injection volume: 15µl; Needle wash solution: methanol; Sample tray temperature: no temperature control; Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Elution procedure:

[0041] Experimental steps: Mixed solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, place it in a 20 ml volumetric flask, first add 8 ml of methanol to dissolve it by ultrasonication, then take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance, and use 0.05% phosphoric acid aqueous solution to prepare a mixed solution containing 0.15 μg of reference substance per 1 ml.

[0042] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0043] The mixed solution results are shown in Figures 10-11 The results showed that under the above chromatographic conditions, vortioxetine hydrobromide and 2,2'-(nitrosoamino)bisacetonitrile achieved baseline separation. Example 4

[0044] Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase A: 0.05% phosphoric acid in water; Mobile phase B: methanol; Detection wavelength: 230nm (high-sensitivity detection cell); Column temperature: 28°C, 32°C; Flow rate: 0.8 ml / min; Injection volume: 15µl; Needle wash solution: methanol; Sample tray temperature: no temperature control; Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Elution procedure:

[0045] Sample preparation: Mixed solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, place it in a 20 ml volumetric flask, first add 8 ml of methanol to dissolve it by ultrasonication, then take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance, and use 0.05% phosphoric acid aqueous solution to prepare a mixed solution containing 0.15 μg of reference substance per 1 ml.

[0046] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0047] The mixed solution results are shown in Figures 12-13 The results showed that under the above chromatographic conditions, vortioxetine hydrobromide and 2,2'-(nitrosoamino)bisacetonitrile achieved baseline separation. Example 5

[0048] Chromatographic conditions: Chromatographic column: Agilent ZORBAX SB-C18 (4.6 mm × 250 mm, 5 μm); Mobile phase A: 0.05% phosphoric acid in water; Mobile phase B: methanol; Detection wavelength: 230nm (high-sensitivity detection cell); Column temperature: 30°C; Flow rate: 0.8 ml / min; Injection volume: 15µl; Needle wash solution: methanol; Sample tray temperature: no temperature control; Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Elution procedure: Gradient elution 1:

[0049] Gradient elution 2:

[0050] Sample preparation: Mixed solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, place it in a 20 ml volumetric flask, first add 8 ml of methanol to dissolve it by ultrasonication, then take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance, and use 0.05% phosphoric acid aqueous solution to prepare a mixed solution containing 0.15 μg of reference substance per 1 ml.

[0051] Determination: Take 15µl of the above mixed solution and inject it into the high performance liquid chromatograph and record the chromatogram.

[0052] The mixed solution results are shown in Figures 14-15 The results showed that under the above chromatographic conditions, vortioxetine hydrobromide and 2,2'-(nitrosoamino)bisacetonitrile achieved baseline separation.

[0053] Example 6 System Precision Investigation The chromatographic conditions were the same as those in Example 2, Condition 9.

[0054] Sample preparation: Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Reference substance solution: Take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and use solvent to prepare a solution containing 0.15 μg of the reference substance per 1 ml.

[0055] Determination: Take the reference solution and inject it continuously 6 times.

[0056] The test results are shown in Table 1. The results show that the RSD of the peak area of ​​2,2'-(nitrosoamino)bisacetonitrile is 0.4%, which is less than 10%. The detection method has good system precision and is suitable for the detection of 2,2'-(nitrosoamino)bisacetonitrile.

[0057] Table 1 System precision investigation results

[0058] Example 7 Specificity Investigation The chromatographic conditions were the same as those in Example 2, Condition 9.

[0059] Sample preparation: Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Reference solution: Take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and prepare a solution containing 0.15 μg of reference substance per 1 ml with solvent; Test solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, and place it in a 20 ml volumetric flask. First, add 8 ml of methanol to dissolve it by ultrasonication, then dilute it to the scale with 0.05% phosphoric acid aqueous solution and shake well. Specific solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, place it in a 20 ml volumetric flask, first add 8 ml of methanol to dissolve it by ultrasonication, then take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance, and use 0.05% phosphoric acid aqueous solution to prepare a mixed solution containing 0.15 μg of reference substance per 1 ml.

[0060] Determination: Take the above solvent, reference solution, test solution, and specific solution and inject them once each.

[0061] The test results are shown in Table 2. The results show that the blank solvent does not interfere with the detection of 2,2'-(nitrosoamino)bisacetonitrile; the minimum separation between 2,2'-(nitrosoamino)bisacetonitrile and adjacent peaks in the specific solution is 3.0, which is greater than 1.5, and the method has good specificity.

[0062] Table 2 Results of specificity investigation

[0063] Example 8 Linearity Investigation The chromatographic conditions were the same as those in Example 2, Condition 9.

[0064] Sample preparation: Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Linear solutions 1 to 5: Take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and use solvent to prepare solutions containing 0.015μg, 0.075μg, 0.15μg, 0.30μg, and 0.45μg of the reference substance per 1ml, respectively.

[0065] Determination: Take the above linear solutions and inject them once. Use the peak area as the ordinate and the concentration as the abscissa to calculate the linear regression equation by the least squares method.

[0066] The results showed that in the range of 0.01490 μg / ml~0.4471 μg / ml, the linear equation of 2,2'-(nitrosoamino)bisacetonitrile was y=282719x+460 (y is the peak area, x is the concentration, μg / ml), and the correlation coefficient r was 1.0000, indicating that the method had good linearity. Example 9 Investigation of detection limit and quantification limit

[0067] The chromatographic conditions were the same as those in Example 2, Condition 9.

[0068] Sample preparation: Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Quantitation limit solution: Take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and prepare a solution containing 0.015 μg of the reference substance per 1 ml with solvent; Detection limit solution: Take an appropriate volume of the quantitation limit solution and dilute it with solvent to a solution containing 0.0075 μg of the reference substance per 1 ml.

[0069] Determination: Take the above-mentioned quantitative limit solution and inject it continuously for 6 times, and the detection limit solution and inject it once.

[0070] The results showed that in the 6 injections of quantitative limit solution, the concentration of 2,2'-(nitrosoamino)bisacetonitrile was 0.01490μg / ml (equivalent to the test sample concentration of 1.5ppm), the S / N was 20, 25, 19, 20, 22, and 24, respectively, all greater than 10, and the peak area RSD was 1.7%; in the detection limit solution, the concentration of 2,2'-(nitrosoamino)bisacetonitrile was 0.007451μg / ml (equivalent to the test sample concentration of 0.75ppm), the S / N was 9, greater than 3. Example 10 Accuracy Investigation

[0071] The chromatographic conditions were the same as those in Example 2, Condition 9.

[0072] Sample preparation: Solvent: methanol-0.05% phosphoric acid aqueous solution (2:3, v / v); Blank test solution: Take about 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, and place it in a 20 ml volumetric flask. First, add 8 ml of methanol to dissolve it by ultrasonication, then dilute it to the scale with 0.05% phosphoric acid aqueous solution and shake well (prepare 3 copies in parallel); LOQ accuracy solution: accurately weigh approximately 200 mg of vortioxetine hydrobromide test sample and place in a 20 ml volumetric flask. First, add 8 ml of methanol to dissolve it by ultrasonication. Then, take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and prepare a mixed solution containing 0.015 μg of reference substance per 1 ml with 0.05% aqueous phosphoric acid solution (prepare three replicates). Medium-level accuracy solution: accurately weigh approximately 200 mg of vortioxetine hydrobromide test sample and place in a 20 ml volumetric flask. First, add 8 ml of methanol to dissolve it by ultrasonication. Then, take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and prepare a mixed solution containing 0.15 μg of reference substance per 1 ml with 0.05% aqueous phosphoric acid solution (prepare three replicates). High-concentration horizontal accuracy solution: Take approximately 200 mg of vortioxetine hydrobromide test sample, accurately weigh it, and place it in a 20 ml volumetric flask. First, add 8 ml of methanol to dissolve it by ultrasonication. Then, take an appropriate amount of 2,2'-(nitrosoamino)bisacetonitrile reference substance and use 0.05% phosphoric acid aqueous solution to prepare a mixed solution containing 0.30 μg of reference substance per 1 ml (prepare 3 copies in parallel).

[0073] Determination: Inject the test solution and three accuracy solutions once. Calculate the recovery and RSD of each accuracy solution using the external standard method.

[0074] The test results are shown in Tables 3 and 4: The recoveries of the 9 accuracy solutions were 97.0% to 117.0%, with an average recovery of 104.5%, all within the range of 80.0% to 120.0%. The RSD of the recovery was 7.6%, less than 10.0%, indicating that this method is accurate, reliable, and has good precision, and is suitable for the detection of 2,2'-(nitrosoamino)bisacetonitrile in vortioxetine hydrobromide.

[0075] Table 3 Accuracy test results - blank test solution

[0076] Table 4 Accuracy test results - Accuracy solution

Claims

1. A method for detecting N-nitroso compounds in vortioxetine or its salts, characterized in that: The method uses high performance liquid chromatography, and the chromatographic conditions are as follows, including: Chromatographic column: octadecylsilane bonded silica gel packing column, Detector: UV detector, Mobile phase: Mobile phase A is 0.05% phosphoric acid aqueous solution, mobile phase B is methanol, Gradient elution was used; Wherein, the N-nitroso compound is 2,2'-(nitrosoamino)bisacetonitrile.

2. The detection method according to claim 1, wherein the chromatographic column is an Agilent ZORBAX SB-C18, with a column length of 250 mm and a particle size of 5 μm.

3. detection method as claimed in claim 1, described ultraviolet detector, its detection wavelength is 230nm.

4. The detection method according to claim 1, wherein the column temperature of the chromatographic condition is 28-32°C, preferably 30°C.

5. The detection method according to claim 1, wherein the chromatographic conditions include a flow rate of 0.7 to 0.9 ml / min, preferably 0.8 ml / min. The detection method according to claim 1 , wherein the chromatographic conditions include an injection volume of 15 μl.

7. The detection method according to claim 1, wherein the sample preparation solvent is methanol-0.05% phosphoric acid aqueous solution.

8. The detection method according to claim 7, wherein the volume ratio of methanol to 0.05% phosphoric acid aqueous solution in the sample preparation solvent is 2:

3.

9. The detection method according to claim 1, wherein the gradient elution conditions are as follows:

10. The detection method according to claim 1, wherein the salt is vortioxetine hydrobromide.

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