A method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets and its application

Abstract

The invention discloses a method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets. The method comprises the following steps: mixing phosphate buffer and methanol, wherein the volume ratio of the phosphate buffer to the methanol is 1-20:80-100, to obtain a solvent; grinding the vortioxetine hydrobromide orally disintegrating tablets, adding the mixture into the solvent for dissolution, filtering, and obtaining a test solution; then detecting the test solution by an HPLC method to obtain the content of the genotoxic impurities in the flutioxetine hydrobromide orally disintegrating tablets; the structural formula of the genotoxic impurities is shown in formula (I): The detection method of the invention has accurate and reliable results, is simple and convenient to operate, is rapid, has high stability, good repeatability and durability, can effectively break the difference between solubility and stability, has a recovery rate close to 100%, and has no significant degradation trend of nitrosamine impurities.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, and in particular to a method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets and an application thereof. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] The main dosage form of vortioxetine hydrobromide is oral tablets, which are used to treat depression in adults. The active secondary amine -NH2 in the structure of the vortioxetine hydrobromide raw material reacts with nitrite under certain conditions (especially high temperature) to generate warning impurities (nitrosamine impurities) with genotoxicity, as shown in the following formula:

[0004]

[0005] According to the instructions for the reference preparation (Xindayue), the maximum daily dose of this product is 20 mg, and the initial dose and recommended dose are both 10 mg, once a day. Adjustments can be made based on individual patient responses, and the minimum can be reduced to 5 mg, once a day. Referring to the ICH-M7 genotoxic impurity evaluation and control guidelines and the limit requirements issued by EMA, based on the limit of 400 ng / day, the limit of a single genotoxic impurity should be 0.002% (20 ppm).

[0006] At present, the detection method of nitrosamine impurities is not included in the pharmacopoeias of various countries, and the detection method of nitrosamine impurities is not found in the patents. When determining nitrosamine impurities, due to the low limit, the high concentration of vortioxetine hydrobromide, and the presence of a large number of excipients in the tablets, which have a certain degree of wrapping, the accuracy is low, which causes a very large interference to the measurement results. Therefore, how to provide an efficient, convenient, fast, specific, stable and accurate method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets to control the quality of vortioxetine hydrobromide is an urgent problem to be solved. Summary of the invention

[0007] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets and its application, which makes up for the lack of a method for detecting nitrosamine impurities in vortioxetine hydrobromide, and the detection method is convenient and efficient, has strong specificity, good stability and high accuracy.

[0008] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0009] A method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets comprises the following steps:

[0010] Mixing phosphate buffer and methanol, wherein the volume ratio of phosphate buffer to methanol is 1-20:80-100, to obtain a solvent;

[0011] Grind the vortioxetine hydrobromide orally disintegrating tablets, add them into the solvent to dissolve, and filter to obtain a test solution;

[0012] Then, the test solution was tested by HPLC method to obtain the content of genotoxic impurities in flutioxetine hydrobromide orally disintegrating tablets;

[0013] The structural formula of the genotoxic impurity is shown in formula (I):

[0014]

[0015] The inventors have found through experiments that the solubility and stability of nitrosamine impurities vary to a certain extent in different solvents, which will cause great interference to the detection of nitrosamine impurities. In tablets, methanol alone cannot completely disintegrate them and cannot extract all nitrosamine impurities; therefore, it is necessary to add a certain amount of water phase to disintegrate the tablets in order to extract all nitrosamine impurities. If water-methanol is used, the pH value of water is generally 5.5-7, and the stability of nitrosamine impurities is poor, which affects the accuracy of the data. Therefore, buffer salts are added to the water phase to give the water phase a constant pH value (about 4-4.5) to improve the stability of nitrosamine impurities and improve the accuracy of the determination. When phosphate buffer (1.36g potassium dihydrogen phosphate is taken, 1000ml of water is added)-methanol (10:90) (methanol ratio is greater than 60%) as solvent, it can be completely dissolved. When phosphate buffer (1.36g potassium dihydrogen phosphate is taken, 1000ml of water is added)-methanol (10:90) (methanol ratio is greater than 80%) as solvent, it is better. Other ratios will affect the results. The present invention adds buffer salt and methanol to the solvent to break the difference between solubility and stability, thereby improving the stability of nitrosamine impurity determination and improving the accuracy of detection. The present invention adopts HPLC method (liquid chromatography) for detection, and the operation is simple.

[0016] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0017] The detection method of the present invention can quickly and effectively detect the content of genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets, and HPLC can be used to determine the presence and content of genotoxic impurities. The detection method of the present invention is accurate and reliable, easy to operate, fast, highly stable, repeatable and durable, and can effectively break the difference between solubility and stability, with a recovery rate of nearly 100%, and nitrosamine impurities have no significant degradation trend. The detection method of the present invention makes up for the vacancy of the detection method of nitrosamine impurities in vortioxetine hydrobromide orally disintegrating tablets, and can be used in the quality control of vortioxetine hydrobromide drug production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] Figure 1 This is a typical spectrum of the genotoxic impurities of Example 1 of the present invention.

[0020] Figure 2 It is a concentration-peak area linear curve of the genotoxic impurities in Example 1 of the present invention. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0022] A method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets comprises the following steps:

[0023] Mixing phosphate buffer and methanol, wherein the volume ratio of phosphate buffer to methanol is 1-20:80-100, to obtain a solvent;

[0024] Grind the vortioxetine hydrobromide orally disintegrating tablets, add them into the solvent to dissolve, and filter to obtain a test solution;

[0025] Then, the test solution was tested by HPLC method to obtain the content of genotoxic impurities in flutioxetine hydrobromide orally disintegrating tablets;

[0026] The structural formula of the genotoxic impurity is shown in formula (I):

[0027]

[0028] In some embodiments, the concentration of the phosphate buffer is 1.3-1.4 g / ml based on potassium dihydrogen phosphate.

[0029] In some embodiments, the volume percentage of methanol in the solvent is 80%-98%, preferably 85%-95%, further preferably 88%-92%, and more preferably 90%.

[0030] In some embodiments, the dosage ratio of vortioxetine hydrobromide orally disintegrating tablet to solvent is 700-800:8-12, mg:ml.

[0031] Preferably, the dosage ratio of vortioxetine hydrobromide orally disintegrating tablets to the solvent is 760:8-12, mg:ml.

[0032] In some embodiments, the liquid chromatography conditions are as follows: mobile phase A is a phosphate buffer, to which 0.4-0.6% by mass of triethylamine is added, and its pH value is adjusted to 2-4;

[0033] Mobile phase B was acetonitrile.

[0034] Adding triethylamine to mobile phase A can reduce the tailing factor of impurities. Without triethylamine, the tailing factor is larger (above 2), acetonitrile has strong elution ability and good peak shape, while methanol has poor elution ability and poor peak shape.

[0035] Preferably, in the mobile phase A, the mass percentage of triethylamine is 0.5%, and the pH value of the mobile phase A is 3-3.5.

[0036] Further preferably, the mobile phase A uses phosphoric acid to adjust the pH value.

[0037] Preferably, the chromatographic column of the liquid chromatography uses octadecylsilane bonded silica gel as a filler.

[0038] Preferably, the elution mode of liquid chromatography is gradient elution, and the gradient elution program is:

[0039] 0-2min, mobile phase A is 80%; 2-30min, mobile phase A changes from 80% to 30%; 20-45min, mobile phase A is 30%; 45-46min, mobile phase A changes from 30% to 80%; 46-80min, mobile phase A is 80%, % is volume percentage.

[0040] Preferably, during the liquid chromatography detection process, the flow rate of the mobile phase is 1.4-1.6 mL / min, preferably 1.5 mL / min; the column temperature is 25-35°C, preferably 30°C.

[0041] In a second aspect, the present invention provides application of the detection method in quality control of flutioxetine hydrobromide orally disintegrating tablets.

[0042] In some embodiments, the content of genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets is controlled within 20 pp to avoid adverse effects of genotoxic impurities on the human body. According to the instructions, the maximum daily dose of this product is 20 mg. Referring to the ICH-M7 guidelines for the evaluation and control of genotoxic impurities and the limit requirements published by EMA, based on the limit of 400 ng / day, the limit of a single genotoxic impurity should be 0.002% (20 ppm).

[0043] Mutagenic impurities are mainly process impurities (halogenated benzene derivatives, nitrosamine impurities). Halogenated benzene derivatives refer to the ICHM7 guidelines for the evaluation and control of genotoxic impurities, and the maximum acceptable intake per person per day is 1.5 μg.

[0044]

[0045]

[0046] The present invention will be further described below in conjunction with the embodiments.

[0047] Example 1 Linearity Experiment

[0048] Linear stock solution: Take about 1 mg of nitrosamine impurity, accurately weigh it, place it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well (1 μg / ml).

[0049] Accurately measure 2ml, 3.2ml, 4ml, 4.8ml, and 6ml of the linear stock solution, respectively, and place them in a 50ml volumetric flask. Dilute to the mark with solvent, shake well, and use them as linear series solutions. Separately take the quantitative limit solution as the quantitative limit concentration linear solution. Accurately measure 50μl of the above solution, inject it into the liquid chromatograph, and record the chromatogram.

[0050] The peak area was linearly regressed with concentration, and the relevant parameters and results are shown in Table 1. Figure 1 The concentration-peak area linear curve of the genotoxic impurities is shown in Figure 1. The linear relationship is good within the concentration range of 0.016 to 0.122 μg / mL, the correlation coefficient is 0.9999, and the ratio of the intercept to the 100% response value is 2.35%.

[0051] Table 1 Linearity test and results

[0052]

[0053] Example 2

[0054] Solvent: phosphate buffer (take 1.36 g of potassium dihydrogen phosphate, add 1000 ml of water)-methanol, the volume ratio of phosphate buffer to methanol is 10:90.

[0055] Test solution: Take 760 mg (equivalent to 40 mg of vortioxetine) of ground flutioxetine hydrobromide oral tablets (trade name: Xindayue), accurately weigh, place in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve, cool, dilute to the scale with solvent, shake well, and filter.

[0056] Reference solution: Take 1 mg of nitrosamine impurity, weigh accurately, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 4 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent, shake well.

[0057] Chromatographic conditions: octadecylsilane bonded silica gel is used as filler (XB-C18, 150 mm × 4.6 mm, 5 μm or chromatographic column of equivalent performance produced by Yuexu Technology (Shanghai) Co., Ltd.); phosphate buffer (1.36 g of potassium dihydrogen phosphate, 1000 ml of water, 0.5% triethylamine, and phosphoric acid are added to adjust the pH value to 3.5) is used as mobile phase A; acetonitrile is used as mobile phase B; linear gradient elution is performed according to the gradient elution program in Table 2.

[0058] Table 2 Gradient elution program

[0059] Time (min) Mobile phase A (%) Mobile phase B (%) 0 60 40 2 60 40 20 30 70 45 30 70 46 60 40 60 60 40

[0060] The flow rate was 1.5 ml per minute; the detection wavelength was 250 nm; and the injection volume was 50 μl.

[0061] Determination method: Accurately measure 50 μl of the solvent and the above solution, inject into the liquid chromatograph, and record the chromatogram. Determine by liquid chromatography (HPLC, Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512).

[0062] 1. Limit of Quantitation Test

[0063] Take an appropriate amount of genotoxic impurities, add solvent to dissolve and dilute to make a series of concentration solutions, with the quantification limit being when S / N (signal-to-noise ratio) ≈ 10, and the detection limit being when S / N (signal-to-noise ratio) ≈ 3. Table 3 shows the genotoxic impurity inspection - quantification limit and detection limit - test results.

[0064] Table 3 Genotoxic impurities inspection - quantitative limit and detection limit test results

[0065]

[0066] The quantitative limit concentration solution was sampled 6 times continuously and the chromatogram was recorded. Table 4 shows the results of the genotoxic impurity inspection-quantitative limit repeatability test (t R represents retention time).

[0067] Table 4 Genotoxic impurities inspection - quantitative limit repeatability test results

[0068]

[0069] The test results showed that the RSD of retention time was 0.10%, the RSD of peak area was 5.40%, and the quantitative limit had good repeatability.

[0070] 2. Repeatability test of reference substance

[0071] Take about 1 mg of nitrosamine impurity, weigh it accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 4 ml, put it in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, as the reference solution.

[0072] Accurately measure 50 μl of the solvent and the above solution, inject into the liquid chromatograph, and record the chromatogram. Repeat the injection 6 times. Table 5 shows the results of the genotoxic impurity inspection-repeatability test.

[0073] Table 5 Genotoxic impurities inspection - repeatability test results

[0074]

[0075] Note: 95% LCI (Lower limit of confident interval) is the lower limit of 95% confidence interval, and 95% HCI (High limit of confident interval) is the upper limit of 95% confidence interval.

[0076] The test results showed that the RSD% of the peak area of ​​the reference solution was 0.77%, with good repeatability.

[0077] 3. Stability test

[0078] Solvent:

[0079] 1) Water-methanol (volume ratio of water to methanol is 10:90);

[0080] 2) Phosphate buffer (1.36 g potassium dihydrogen phosphate, add 1000 ml water)-methanol (the volume ratio of phosphate buffer to methanol is 10:90);

[0081] 3) Phosphate buffer (1.36 g potassium dihydrogen phosphate, add 1000 ml water)-methanol (the volume ratio of phosphate buffer to methanol is 20:80);

[0082] 4) Methanol.

[0083] Reference solution: Take about 1 mg of nitrosamine impurity, accurately weigh, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 4 ml, place in a 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0084] Take about 80 mg of this product from each group, for a total of four groups, accurately weigh, place in a 10 ml volumetric flask, add an appropriate amount of reference solution to dissolve, cool, dilute each group to the scale with solvent, shake well, filter, and use as the test product stability solution.

[0085] The stability solution of the test product was placed at room temperature for 8 hours. During this period, samples were taken and measured at 0 hours, 2 hours, 4 hours, 6 hours and 8 hours.

[0086] Accurately measure 50 μl of the solvent and the above solution, inject them into the liquid chromatograph, and record the chromatogram. The stability test results are shown in Table 6.

[0087] Table 6 Vortioxetine hydrobromide - Genotoxic impurity determination - stability test results

[0088]

[0089]

[0090] The test results show that among different solvents, the stability of nitrosamine impurities in water is not as good as that in buffer salt. Considering the solubility and stability, phosphate buffer (1.36g potassium dihydrogen phosphate, add 1000ml water)-methanol (the volume ratio of phosphate buffer to methanol is 10:90) is preferred as the solvent of this method.

[0091] 5. Accuracy test

[0092] Take about 1 mg of nitrosamine impurity, accurately weigh it, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, and use it as the accuracy stock solution.

[0093] Take 760 mg of fine powder of this product (equivalent to 40 mg of vortioxetine), weigh accurately, put into a 10 mL volumetric flask, accurately measure 0.2 mL of the accuracy stock solution, dilute to the scale with solvent, shake well, filter, and use as the 25% recovery rate solution. Prepare three copies in the same way.

[0094] Take 760 mg of fine powder of this product (equivalent to 40 mg of vortioxetine), weigh accurately, put into a 10 mL volumetric flask, accurately measure 0.4 mL of the accuracy stock solution, dilute to the scale with solvent, shake well, filter, and use as the 50% recovery rate solution. Prepare three copies in the same way.

[0095] Take 760 mg of fine powder of this product (equivalent to 40 mg of vortioxetine), weigh accurately, put into a 10 mL volumetric bottle, accurately measure 0.8 mL of the accuracy stock solution, dilute to the scale with solvent, shake well, filter, and use as the 100% recovery rate solution. Prepare three copies in the same way.

[0096] Take 760 mg of fine powder of this product (equivalent to 40 mg of vortioxetine), weigh accurately, put into a 10 mL volumetric flask, accurately measure 1.2 mL of the accuracy stock solution, dilute to the scale with solvent, shake well, filter, and use as the 120% recovery rate solution. Prepare three copies in the same way.

[0097] Take about 1 mg of nitrosamine impurity, weigh it accurately, put it in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, put it in a 100 ml volumetric flask, dilute to the scale with solvent, shake well, accurately measure 4 ml, put it in a 50 ml volumetric flask, dilute to the scale with solvent, shake well, as the reference solution.

[0098] Accurately measure 50 μl of the solvent and the above solution, inject them into the liquid chromatograph, and record the chromatogram. The recovery test results are shown in Table 7.

[0099] Table 7 Recovery determination results

[0100] Group Water-methanol (10:90, v / v) Buffer saline-methanol (10:90, v / v) Methanol (100, v / v) 25％-1 100.24 100.22 94.47 25％-2 100.15 98.25 93.44 25％-3 99.25 99.4 95.47 50％-1 97.35 99.44 92.11 50％-2 96.58 99.71 95.95 50％-3 96.67 101.39 94.19 100％-1 94.35 98.77 94.11 100％-2 95.28 99.16 92.03 100％-3 93.21 99.32 94.47 150％-1 92.28 98.01 95.57 150％-2 91.34 98.72 93.77 150％-3 91.62 98.02 93.93 average 95.69 99.20 94.13 RSD(%) 3.35 0.98 1.31

[0101] The test results show that the recovery rate of genotoxic impurities determined by high performance liquid chromatography (HPLC) is different in different solvents due to the difference in solubility and stability. The present invention significantly improves and enhances the recovery rate by adding different proportions of organic phase to the solvent to break the difference in solubility and stability. In methanol, due to the presence of auxiliary materials in the tablet, no water phase is added, and the auxiliary materials are wrapped to a certain extent, so that the overall recovery rate is low. No buffer salt is added to the water, and the stability is poor. The recovery rate is best in the solvent of buffer salt-methanol (10:90, v / v).

[0102] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets, characterized in that: The steps include: Mixing phosphate buffer and methanol, wherein the volume ratio of phosphate buffer to methanol is 1-20:80-100, to obtain a solvent; Grind the vortioxetine hydrobromide orally disintegrating tablets, add them into the solvent to dissolve, and filter to obtain a test solution; Then, the test solution was tested by HPLC method to obtain the content of genotoxic impurities in flutioxetine hydrobromide orally disintegrating tablets; The structural formula of the genotoxic impurity is shown in formula (I):

2. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 1, characterized in that: The concentration of the phosphate buffer is 1.3-1.4 g / ml based on potassium dihydrogen phosphate.

3. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 1, characterized in that: In the solvent, the volume percentage of methanol is 80%-98%, preferably 85%-95%, more preferably 88%-92%, and more preferably 90%.

4. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 1, characterized in that: The dosage ratio of vortioxetine hydrobromide orally disintegrating tablets to the solvent is 700-800:8-12, mg:ml; Preferably, the dosage ratio of vortioxetine hydrobromide orally disintegrating tablets to the solvent is 760:8-12, mg:ml.

5. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 1, characterized in that: The liquid chromatography conditions are as follows: mobile phase A is a phosphate buffer, to which 0.4-0.6% by mass of triethylamine is added, and its pH value is adjusted to 2-4; Mobile phase B was acetonitrile.

6. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 5, characterized in that: In the mobile phase A, the mass percentage of triethylamine is 0.5%, and the pH value of the mobile phase A is 3-3.

5.

7. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 5, characterized in that: The chromatographic column of liquid chromatography is filled with octadecylsilane bonded silica gel.

8. The method for detecting genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets according to claim 5, characterized in that: The elution mode of liquid chromatography is gradient elution, and the gradient elution program is: 0-2min, mobile phase A is 80%; 2-30min, mobile phase A changes from 80% to 30%; 20-45min, mobile phase A is 30%; 45-46min, mobile phase A changes from 30% to 80%; 46-80min, mobile phase A is 80%, % is volume percentage; Preferably, during the liquid chromatography detection process, the flow rate of the mobile phase is 1.4-1.6 mL / min, preferably 1.5 mL / min; the column temperature is 25-35°C, preferably 30°C.

9. Use of the method for detecting genotoxic impurities in vortioxetine hydrobromide orodisintegrating tablets according to any one of claims 1 to 8 in quality control of flutioxetine hydrobromide orodisintegrating tablets.

10. The use according to claim 9, characterized in that: The content of genotoxic impurities in vortioxetine hydrobromide orally disintegrating tablets was controlled within 20 pp.

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