A HPLC detection method for related substances in vortioxetine hydrobromide tablets

Abstract

The invention relates to an HPLC detection method for related substances of vortioxetine hydrobromide tablets. The test solution of the HPLC detection method is a methanol solution containing vortioxetine impurities, the mobile phase A is a mixed solution of diammonium hydrogen phosphate buffer and methanol, and the mobile phase B is methanol; the elution procedure is: #imgabs0# The method can detect many impurities and can quickly, effectively and accurately monitor related substances in vortioxetine hydrobromide tablets. The separation between a main peak of vortioxetine hydrobromide and adjacent impurity peaks is greater than 1.5, and the impurities can be effectively separated from the main peak. The method is simple to operate, has good specificity, linearity and precision, and the solution is stable. The quantitative limit and the detection limit are both small, the accuracy is high, and the method is durable.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drug analysis methods, and particularly relates to an HPLC detection method for related substances of vortioxetine hydrobromide tablets. Background Art

[0002] Vortioxetine hydrobromide is an antidepressant. Unlike other first-line antidepressants, vortioxetine hydrobromide is a novel antidepressant with a multi-receptor mechanism of action. It exerts its antidepressant effects through a combination of receptor activity modulation and neurotransmitter reuptake inhibition. It is effective in treating depression in adults, especially in improving patients' cognitive function. It has a low incidence of adverse reactions and good short-term and long-term safety and tolerability, with a particularly low impact on sexual function. Vortioxetine hydrobromide is a promising new antidepressant.

[0003] The chemical name of vortioxetine hydrobromide is 1-[2-(2,4-dimethylphenylthio)phenyl]piperazine hydrobromide, its molecular formula is C18H22N2S·HBr, its molecular weight is 379.36, its CAS number is 960203-27-4, and its structural formula is as follows:

[0004]

[0005] Vortioxetine hydrobromide tablets are prepared by mixing vortioxetine hydrobromide raw materials and excipients. The impurities in the vortioxetine hydrobromide raw materials are significantly different from those in the vortioxetine hydrobromide raw materials. The impurities in the vortioxetine hydrobromide raw materials are mainly by-products and isomeric impurities, while the impurities in the vortioxetine hydrobromide tablets are mainly degradation impurities. As a result, the HPLC detection method using the vortioxetine hydrobromide raw materials cannot effectively detect the impurities in the vortioxetine hydrobromide tablets. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide an HPLC detection method for related substances in vortioxetine hydrobromide tablets. The present invention can detect multiple impurities and can quickly, effectively and accurately monitor related substances in vortioxetine hydrobromide tablets. The present invention is simple to operate, has good specificity, linearity and precision, and has a stable solution, high sensitivity, high accuracy and good method durability.

[0007] The present invention provides an HPLC detection method for related substances in vortioxetine hydrobromide tablets. The structures of the related substances in vortioxetine hydrobromide tablets are as follows:

[0008] See the table below.

[0009]

[0010] Among the above impurities, vortioxetine impurity 5 and vortioxetine impurity 9 are process impurities, and vortioxetine impurity D, vortioxetine impurity 31 and vortioxetine impurity 41 are degradation impurities.

[0011] The test solution of the HPLC detection method is a methanol solution containing vortioxetine impurities, the mobile phase A is a mixed solution of diammonium hydrogen phosphate buffer and methanol, and the mobile phase B is methanol;

[0012] The elution procedure is:

[0013]

[0014] Preferably, in the mobile phase A, the volume ratio of diammonium hydrogen phosphate buffer to methanol is 9:1.

[0015] Preferably, the pH value of the diammonium hydrogen phosphate buffer is 5.5.

[0016] Preferably, the flow rate of the HPLC detection method is 1.0 ml / min.

[0017] Preferably, the column temperature of the HPLC detection method is 30°C.

[0018] Preferably, the detection wavelength of the HPLC detection method is 226 nm.

[0019] Preferably, in the HPLC detection method, the concentration of the test sample is 0.5 mg / mL.

[0020] Preferably, the filler of the HPLC detection method is octadecylsilane bonded silica gel.

[0021] Preferably, the filler is Waters X-Bridge Shield RP18.

[0022] The beneficial effects of the present invention are that the present invention can detect a large number of impurities and can quickly, effectively and accurately monitor related substances in vortioxetine hydrobromide tablets. The separation between the main peak of vortioxetine hydrobromide and the adjacent impurity peaks is greater than 1.5, and the impurities and the main peak can be effectively separated. The method is simple to operate, has good specificity, linearity and precision, and the solution is stable. The quantitative limit and the detection limit are both small, the accuracy is high, and the method is durable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 HPLC chart of the sample solution under normal conditions in Example 2;

[0024] Figure 2 HPLC chart of the sample solution with mobile phase pH 5.3 in Example 2;

[0025] Figure 3HPLC chart of the sample solution with mobile phase pH 5.7 in Example 2;

[0026] Figure 4 HPLC chart of the sample solution with the mobile phase ratio (88:12) in Example 2;

[0027] Figure 5 HPLC chart of the sample solution with the mobile phase ratio (92:8) in Example 2;

[0028] Figure 6 This is the HPLC chart of the sample solution at a column temperature of 28° C. in Example 2;

[0029] Figure 7 This is the HPLC chart of the sample solution at a column temperature of 32° C. in Example 2;

[0030] Figure 8 HPLC chart of the sample solution at a wavelength of 224 nm in Example 2;

[0031] Figure 9 This is the HPLC graph of the sample solution at a wavelength of 228 nm in Example 2;

[0032] Figure 10 HPLC chart of the system suitability solution in Comparative Example 1;

[0033] Figure 11 HPLC chart of the test solution (spiked) in Comparative Example 1;

[0034] Figure 12 HPLC chart of the system suitability solution in Comparative Example 2;

[0035] Figure 13 HPLC chart of the test solution (spiked) in Comparative Example 2;

[0036] Figure 14 HPLC chart of the system suitability solution in Comparative Example 3;

[0037] Figure 15 This is the HPLC chart of the test solution (spiked) in Comparative Example 3. DETAILED DESCRIPTION

[0038] Example 1

[0039] 1. Drugs and reagents

[0040] Reagents / standard substances Level / content batch number factory phosphoric acid HPLC 20220110 Comeau Diammonium hydrogen phosphate HPLC 20220110 West Asia Reagent Methanol HPLC 16220515 Hipure Chem Vortioxetine hydrobromide 98.6％ 0816-RA-0010 CATO Vortioxetine Impurity D 94.36％ U40-230210-03 OST Vortioxetine Impurity 5 87.17％ U40-23021110-03 OST Vortioxetine Impurity 9 99.05％ U40-2302115-03 OST Vortioxetine Impurity 31 100％ 0809-RA-0007 CATO Vortioxetine Impurity 41 98.2％ 1211-RB-0026 CATO

[0041] 2. Main instruments

[0042] Instrument name model factory Liquid chromatograph Agilent II Agilent Liquid chromatograph LC2050C Shimadzu electronic balance EX125DZH Ohaus electronic balance AR224CN Ohaus pH meter FE28 Mettler-Toledo

[0043] 3. Detection Methods

[0044] This example is used to investigate the specificity of the method.

[0045] Individual impurity stock solutions: accurately weigh approximately 10 mg of vortioxetine impurity D, approximately 10 mg of vortioxetine hydrobromide, approximately 10 mg of vortioxetine impurity 5, approximately 10 mg of vortioxetine impurity 9, approximately 25 mg of vortioxetine impurity 31, and approximately 25 mg of vortioxetine impurity 41, place in different 100 ml volumetric flasks, dissolve in methanol, dilute to the mark, and shake well to obtain the product.

[0046] Single impurity localization solution: Accurately measure 1 ml each of vortioxetine hydrobromide, vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, place them into different 100 ml volumetric flasks, dilute to the scale with diluent, shake well, and filter to obtain.

[0047] System suitability solution: Accurately measure 1 ml each of the stock solutions of vortioxetine hydrobromide, vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, shake well, and filter to obtain the solution.

[0048] Liquid chromatography conditions: HPLC: Agilent 1260II; chromatographic column: Waters X-BridgeShield RP18, 4.6 mm × 150 mm, 3.5 μm; wavelength: 226 nm; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 5 μl.

[0049] Mobile phase: Mobile phase A is 10 mmol / L phosphate buffer (pH adjusted to 3.5 with phosphoric acid)-methanol (90:10), and mobile phase B is methanol. The elution procedure is as follows:

[0050]

[0051] 4. Results

[0052] High performance liquid chromatography (HPLC) was performed under the above chromatographic conditions. The results are shown in Table 1. The separation degree of each impurity met the requirements, and the specificity of the related substance detection method was good.

[0053] Table 1 Specificity test results

[0054]

[0055] Example 2

[0056] In this example, the durability of the chromatographic method was investigated by changing some of the parameters of the chromatographic conditions. The drugs, reagents, and main instruments were the same as in Example 1, and the following chromatographic conditions were used as the basis: octadecylsilane bonded silica gel was used as the filler (Waters X-Bridge Shield RP18, 4.6 mm × 150 mm, 3.5 μm), phosphate buffer (1.32 g of diammonium hydrogen phosphate was dissolved in 1000 ml of water and the pH was adjusted to 5.5 with phosphoric acid)-methanol (9:1) was used as the mobile phase A, and methanol was used as the mobile phase B. Gradient elution was performed as in Example 1; the flow rate was 1.0 ml per minute; the detection wavelength was 226 nm; the column temperature was 30° C.; and the injection volume was 5 μl (i.e., normal conditions).

[0057] This example examines the effects of varying chromatographic conditions, such as phosphate buffer pH values, phosphate buffer to methanol volume ratios, column temperatures, and wavelengths, on the detection system suitability solution. As shown in Table 2, a mobile phase pH of 5.3 refers to a phosphate buffer pH of 5.3 in mobile phase A compared to normal conditions. Similarly, all other conditions are single-factor changes under normal conditions. The primary effect of varying chromatographic conditions on the resolution of vortioxetine impurity D, vortioxetine impurity 5, vortioxetine hydrobromide, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41 in the system suitability solution was examined.

[0058] System suitability solution: same as Example 1.

[0059] Under each chromatographic condition, 5 μl of the system suitability solution was injected into the liquid chromatograph for detection. The detection results are shown in Table 2, and the chromatograms are shown in Table 2. Figure 1-9 The elution order is vortioxetine impurity D, vortioxetine impurity 5, vortioxetine hydrobromide, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41.

[0060] The results showed that the system suitability solution resolution was significantly affected by changes in some chromatographic parameters, such as a phosphate buffer pH of 5.3–5.7, a phosphate buffer / methanol volume ratio of 88:12–92:8, a column temperature of 28°C–32°C, and a detection wavelength of 224–226 nm. Under these conditions, the method remained suitable for the detection of related substances in vortioxetine hydrobromide tablets. The method demonstrated good robustness.

[0061] Table 2 Durability test results

[0062]

[0063]

[0064] Example 3

[0065] This example investigates the quantification limit and detection limit of each impurity. The drugs, reagents, and main instruments are as in Example 1, and the following chromatographic conditions are used as the basis: octadecylsilane bonded silica gel is used as the filler (Waters X-Bridge Shield RP18, 4.6 mm × 150 mm, 3.5 μm), phosphate buffer (1.32 g of diammonium hydrogen phosphate is dissolved in 1000 ml of water and the pH is adjusted to 5.5 with phosphoric acid)-methanol (9:1) is used as the mobile phase A, and methanol is used as the mobile phase B. Gradient elution is performed as in Example 1; the flow rate is 1.0 ml per minute; the detection wavelength is 226 nm; the column temperature is 30°C; and the injection volume is 5 μl.

[0066] Test solution: Dilute each impurity reference solution with methanol to prepare a solution with a signal-to-noise ratio ≥10, which is used as the quantification limit solution (prepare 6 portions in parallel); dilute with methanol to prepare a solution with a signal-to-noise ratio ≥3, which is used as the detection limit solution.

[0067] The results are shown in Tables 3 and 4, indicating that the detection method has high sensitivity.

[0068] Table 3 Quantitation limit test results

[0069] name Limit of quantification (μg / ml) Equivalent to test sample concentration% Equivalent to limit concentration% Vortioxetine Impurity D 0.0749 0.0150 14.98 Vortioxetine Impurity 5 0.0518 0.0104 6.90 Vortioxetine hydrobromide 0.0408 0.0082 5.44 Vortioxetine Impurity 9 0.0633 0.0127 8.44 Vortioxetine Impurity 31 0.0734 0.0147 2.94 Vortioxetine Impurity 41 0.1412 0.0282 5.65

[0070] Table 4 Detection limit test results

[0071] name Detection limit concentration (μg / ml) Equivalent to test sample concentration% Equivalent to limit concentration% Vortioxetine Impurity D 0.0702 0.0140 14.04 Vortioxetine Impurity 5 0.0324 0.0065 4.31 Vortioxetine hydrobromide 0.0383 0.0077 5.10 Vortioxetine Impurity 9 0.0396 0.0079 5.28 Vortioxetine Impurity 31 0.0367 0.0073 1.47 Vortioxetine Impurity 41 0.0706 0.0141 2.82

[0072] Comparative Example 1

[0073] Instrument conditions

[0074] High performance liquid chromatograph: Agilent 1260II; chromatographic column: Waters X-Bridge Shield RP18, 4.6 mm × 150 mm, 3.5 μm; wavelength: 226 nm; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 5 μl.

[0075] Mobile phase: Mobile phase A was 0.1% triethylamine solution (pH adjusted to 3.0 with phosphoric acid)-acetonitrile (90:10), and mobile phase B was acetonitrile.

[0076] Individual impurity stock solutions: accurately weigh approximately 10 mg of vortioxetine impurity D, approximately 10 mg of vortioxetine hydrobromide, approximately 10 mg of vortioxetine impurity 5, approximately 10 mg of vortioxetine impurity 9, approximately 25 mg of vortioxetine impurity 31, and approximately 25 mg of vortioxetine impurity 41, place in different 100 ml volumetric flasks, dissolve in methanol, dilute to the mark, and shake well to obtain the product.

[0077] System suitability solution: Accurately measure 1 ml each of the stock solutions of vortioxetine hydrobromide, vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, shake well, and filter to obtain the solution.

[0078] Test solution (spiked): Weigh 20 vortioxetine hydrobromide tablets, grind them into powder, accurately weigh the fine powder (equivalent to 50 mg of vortioxetine), place it into a 100 ml volumetric flask, accurately pipette 1 ml of the stock solutions of vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, dilute to the mark with methanol, shake well, and filter to obtain the test solution.

[0079] Table 5 Comparative Example 1 Elution Procedure

[0080]

[0081]

[0082] The HPLC test was carried out according to the above conditions. The results were as follows: Figure 10 、 Figure 11 As shown in Table 6. Figure 10-11 As can be seen from Table 6, in the spiked solution of the test sample, the separation degree between vortioxetine impurity 5 and the main peak is less than 1.5, and the analytical method needs to be optimized.

[0083] Table 6 Retention time and separation of different substances

[0084]

[0085] Comparative Example 2

[0086] Instrument conditions

[0087] High performance liquid chromatograph: Agilent 1260II; chromatographic column: Waters X-Bridge Shield RP18, 4.6 mm × 150 mm, 3.5 μm; wavelength: 226 nm; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 5 μl.

[0088] Mobile phase: Mobile phase A was 0.01 mol / L sodium dihydrogen phosphate buffer (pH adjusted to 3.0 with phosphoric acid)-acetonitrile (90:10), and mobile phase B was acetonitrile.

[0089] Individual impurity stock solutions: accurately weigh approximately 10 mg of vortioxetine impurity D, approximately 10 mg of vortioxetine hydrobromide, approximately 10 mg of vortioxetine impurity 5, approximately 10 mg of vortioxetine impurity 9, approximately 25 mg of vortioxetine impurity 31, and approximately 25 mg of vortioxetine impurity 41, place in different 100 ml volumetric flasks, dissolve in methanol, dilute to the mark, and shake well to obtain the product.

[0090] System suitability solution: Accurately measure 1 ml each of the stock solutions of vortioxetine hydrobromide, vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, shake well, and filter to obtain the solution.

[0091] Test solution (spiked): Weigh 20 vortioxetine hydrobromide tablets, grind them into powder, accurately weigh the fine powder (equivalent to 50 mg of vortioxetine), place it into a 100 ml volumetric flask, accurately pipette 1 ml of the stock solutions of vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, dilute to the mark with methanol, shake well, and filter to obtain the test solution.

[0092] Table 7 Comparative Example 2 Elution Procedure

[0093]

[0094]

[0095] The HPLC test was carried out according to the above conditions. The results were as follows: Figure 12 、 Figure 13 As shown in Table 8. Figure 12 、 Figure 13 As can be seen from Table 8, the separation degree between vortioxetine impurity 5 and the main peak does not reach baseline separation, and the analytical method needs to be adjusted and optimized.

[0096] Table 8 Retention time and separation of different substances

[0097]

[0098] Comparative Example 3

[0099] Instrument conditions

[0100] High performance liquid chromatograph: Agilent 1260II; chromatographic column: Waters X-Bridge Shield RP18, 4.6 mm × 150 mm, 3.5 μm; wavelength: 226 nm; flow rate: 1.0 ml / min; column temperature: 30°C; injection volume: 5 μl.

[0101] Mobile phase: Mobile phase A was 10 mmol / L diammonium hydrogen phosphate buffer (pH adjusted to 5.5 with phosphoric acid)-acetonitrile (90:10), and mobile phase B was acetonitrile.

[0102] Individual impurity stock solutions: accurately weigh approximately 10 mg of vortioxetine impurity D, approximately 10 mg of vortioxetine hydrobromide, approximately 10 mg of vortioxetine impurity 5, approximately 10 mg of vortioxetine impurity 9, approximately 25 mg of vortioxetine impurity 31, and approximately 25 mg of vortioxetine impurity 41, place in different 100 ml volumetric flasks, dissolve in methanol, dilute to the mark, and shake well to obtain the product.

[0103] System suitability solution: Accurately measure 1 ml each of the stock solutions of vortioxetine hydrobromide, vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, place them in the same 100 ml volumetric flask, dilute to the mark with diluent, shake well, and filter to obtain the solution.

[0104] Test solution (spiked): Weigh 20 vortioxetine hydrobromide tablets, grind them into powder, accurately weigh the fine powder (equivalent to 50 mg of vortioxetine), place it into a 100 ml volumetric flask, accurately pipette 1 ml of the stock solutions of vortioxetine impurity D, vortioxetine impurity 5, vortioxetine impurity 9, vortioxetine impurity 31, and vortioxetine impurity 41, dilute to the mark with methanol, shake well, and filter to obtain the test solution.

[0105] Table 9 Comparative Example 3 Elution Program

[0106]

[0107] The HPLC test was carried out according to the above conditions. The results were as follows: Figure 14 、 Figure 15 As shown in Table 10. Figure 14 、 Figure 15 As can be seen from Table 10, the separation degree between vortioxetine impurity 5 and the main peak does not reach baseline separation, and the analytical method needs to be adjusted and optimized.

[0108] Table 10 Retention time and separation of different substances

[0109]

[0110] In summary, the analytical method of the present invention has the characteristics of good specificity, durability, high sensitivity, etc. in the determination of related substances in vortioxetine hydrobromide tablets.

[0111] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.

[0112] The one or more embodiments of this application are intended to encompass all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application should be included in the scope of protection of this application.

Claims

1. A HPLC method for detecting related substances in vortioxetine hydrobromide tablets, characterized in that: The structures of the related substances of the vortioxetine hydrobromide tablets are as follows: 、 、 、 、 ； The test solution of the HPLC detection method is a methanol solution containing vortioxetine impurities, the mobile phase A is a mixed solution of diammonium hydrogen phosphate buffer and methanol, and the mobile phase B is methanol; The elution procedure is: In the mobile phase A, the volume ratio of diammonium hydrogen phosphate buffer to methanol is 9:1; The pH of the diammonium hydrogen phosphate buffer is 5.5; the filler of the HPLC detection method is octadecylsilane bonded silica gel.

2. The HPLC detection method for related substances of vortioxetine hydrobromide tablets as claimed in claim 1, characterized in that: The flow rate of the HPLC detection method was 1.0 ml / min.

3. The HPLC detection method for related substances of vortioxetine hydrobromide tablets as claimed in claim 1, characterized in that: The column temperature of the HPLC detection method was 30°C.

4. The HPLC detection method for related substances of vortioxetine hydrobromide tablets as claimed in claim 1, characterized in that: The detection wavelength of the HPLC detection method is 226 nm.

5. The HPLC detection method for related substances of vortioxetine hydrobromide tablets as claimed in claim 1, characterized in that: In the HPLC detection method, the concentration of the test sample is 0.5 mg / mL.

6. The HPLC detection method of related substances of vortioxetine hydrobromide tablets as claimed in claim 1, characterized in that, The filler is Waters X-Bridge Shield RP18.

Images