Method for detecting content of nitrosamine impurity in viloxacin hydrochloride

Abstract

The invention relates to a method for detecting the content of nitrosamine impurities in viloxacin hydrochloride, in particular to a method for detecting the content of N-nitroso viloxacin in viloxacin hydrochloride by adopting a UPLC-MS (Ultra Performance Liquid Chromatography-Mass Spectrometry) method. The method provided by the invention is good in system durability, high in detection sensitivity and good in accuracy, and effectively solves the problems that the content of nitrosamine impurities in the vinloxin hydrochloride is extremely low and the content is difficult to detect.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis, and specifically relates to a method for detecting the content of nitrosamine impurities in viloxazine hydrochloride, and more particularly to a method for detecting the content of N-nitrosoviloxazine in viloxazine hydrochloride. Background Art

[0002] Viloxazine (trade name: Qelbree) is a morpholine derivative and a selective norepinephrine reuptake inhibitor. Developed by Supernus Pharma, it was approved by the U.S. Food and Drug Administration (FDA) in April 2021 for the treatment of attention deficit hyperactivity disorder (ADHD) in adolescents and adults aged 6 years and above. The active ingredient is viloxazine hydrochloride, and the drug formulation is a sustained-release capsule. Qelbree is the first new non-stimulant drug for the treatment of ADHD in children and adolescents in more than 10 years. The structure of viloxazine hydrochloride is as follows:

[0003]

[0004] NDSRIs (Nitrosamine Drug Substance-Related Impurities) refer to nitrosamine impurities generated by nitrosation reactions of secondary amines, tertiary amine groups or fragments thereof in the structure of the active pharmaceutical ingredient (API) itself under specific conditions (such as residual nitrite, acidic conditions). The structure of viloxazine contains secondary amine groups, which is a high-risk structure for the formation of nitrosamine impurities. The FDA requires that all APIs containing secondary amine / tertiary amine structures must be evaluated for nitrosation. The inventors have found through research that N-nitrosoviloxazine impurities are formed in viloxazine hydrochloride APIs and preparations. Therefore, it is necessary to control the content of N-nitrosoviloxazine (impurity J) in viloxazine hydrochloride APIs and preparations to ensure the quality and safety of the products. The structure of N-nitrosoviloxazine is shown in Formula J below:

[0005]

[0006] The maximum daily dose of viloxazine does not exceed 600 mg. According to the provisions of ICH M7 (R1) and the calculation of the maximum daily dose of viloxazine, the nitrosamine impurity limit in viloxazine hydrochloride sustained-release capsules should not exceed 0.16 ppm, and the nitrosamine impurity limit in API should not exceed 0.14 ppm. The content of this nitrosamine impurity in API should be controlled at a lower level.

[0007] Currently, no literature or data discloses a method for determining the content of N-nitrosoviloxazine in viloxazine hydrochloride. To control the quality of viloxazine hydrochloride products, improve the safety of viloxazine hydrochloride sustained-release capsules, meet the requirements of regulatory agencies, and benefit patients, it is necessary to develop a method for detecting N-nitrosoviloxazine in viloxazine hydrochloride. Summary of the Invention

[0008] During the research process, the inventors found that N-nitrosoviloxazine has a very low response in chromatographic detection. Since the content of N-nitrosoviloxazine in the viloxazine hydrochloride raw material and pharmaceutical preparation is required to be controlled at an extremely low level, the detection limit of quantification is required to reach at least 0.014ppm, which requires very high detection sensitivity. Conventional detection methods and equipment cannot achieve such high sensitivity requirements, resulting in very great difficulties in detecting the content of this impurity. After a large amount of experimental exploration and research, the inventors finally developed a method for detecting the content of N-nitrosoviloxazine in viloxazine hydrochloride that is suitable for industrial application.

[0009] Specifically, the present invention provides a method for detecting the content of N-nitrosoviloxazine in viloxazine hydrochloride, the method comprising:

[0010] (1) Preparation of test solution and reference solution, including: pre-treating the test sample and N-nitrosoviloxazine reference substance with a solvent and an extractant, respectively, and then re-dissolving them with a diluent to prepare the test solution and reference solution at the target concentration;

[0011] (2) Liquid chromatography-mass spectrometry was used to test the reference solution and the test solution;

[0012] The solvent in step (1) is water or an acidic aqueous solution, preferably water or an aqueous hydrochloric acid solution; the extractant is preferably ethyl acetate or methyl isobutyl ketone. In a preferred embodiment, the solvent in step (1) is water or an aqueous hydrochloric acid solution, and the extractant is preferably ethyl acetate; or the solvent is water or an aqueous hydrochloric acid solution, and the extractant is methyl isobutyl ketone.

[0013] The solution provided by the present invention pre-treats the impurity reference and test samples using water or an acidic aqueous solution as a solvent and ethyl acetate or methyl isobutyl ketone as an extractant before testing, which helps improve detection sensitivity. The test sample and reference solution prepared after pre-treatment can meet detection sensitivity requirements using conventional equipment. Furthermore, the pre-treatment of the sample before reconstitution into the corresponding solution also helps improve the instrument's anti-contamination capability. This pre-treatment method can address matrix effects caused by high test sample concentrations and improve the accuracy of impurity detection.

[0014] In a preferred embodiment, in the preparation of the test solution in step (1), the solvent is water or aqueous hydrochloric acid, and the extractant is ethyl acetate or methyl isobutyl ketone.

[0015] In a preferred embodiment, in the preparation of the reference solution in step (1), the solvent is water or aqueous hydrochloric acid, and the extractant is ethyl acetate or methyl isobutyl ketone.

[0016] In one embodiment, the preparation of the test sample and the reference substance in step (1) comprises: dissolving the test sample and the reference substance with a solvent and an extractant, respectively, then removing the solvent and the extractant, and then re-dissolving with a diluent to prepare a test sample solution and a reference substance solution of target concentration.

[0017] In a preferred embodiment of the present invention, the preparation of the test solution in step (1) comprises: taking an appropriate amount of viloxazine hydrochloride test sample, accurately weighing it, placing it in a volumetric flask, adding a solvent and an extractant, shaking it, and then centrifuging it, taking an appropriate amount of the supernatant, removing the solvent and the extractant under nitrogen, and then adding a diluent to redissolve it to prepare a test solution of the target concentration.

[0018] In a preferred embodiment of the present invention, the preparation of the reference solution in step (1) comprises: first preparing the N-nitrosoviloxazine reference substance with acetonitrile, water or a mixture thereof to prepare a reference substance stock solution, then accurately measuring an appropriate amount of the reference substance stock solution, placing it in a volumetric flask, adding a solvent and an extractant, shaking, and then centrifuging, taking an appropriate amount of the supernatant, removing the solvent and the extractant under nitrogen conditions, and then adding a diluent to redissolve to prepare a reference substance solution of the target concentration.

[0019] In a specific embodiment, the pretreatment of the sample in step (1) includes: taking the sample, accurately weighing it, adding a solvent to dissolve it, then adding an extractant to dissolve it, centrifuging it, taking the supernatant, placing it under nitrogen conditions (such as a nitrogen blowdown apparatus) at room temperature to remove the solvent and extractant (for example, volatilize it for at least 30 minutes), and setting it aside.

[0020] In a specific embodiment, the pretreatment of the reference substance in step (1) includes: taking the reference substance, accurately weighing it, dissolving it with water, acetonitrile or a mixture thereof to obtain a reference substance solution I, accurately measuring the reference substance solution I, mixing the reference substance solution I with water, and then adding ethyl acetate to mix, centrifuging, taking the supernatant, placing it under nitrogen conditions (such as a nitrogen blowdown instrument) at room temperature to remove the solvent (for example, volatilize for at least 30 minutes), and setting it aside.

[0021] In a specific embodiment, the preparation of the reference solution in step (1) comprises: taking an N-nitrosoviloxazine reference substance, accurately weighing it, placing it in a volumetric flask, gradually diluting it with water, acetonitrile or a mixture thereof to prepare a reference substance stock solution, then accurately measuring an appropriate amount of the reference substance stock solution, adding water, shaking, then adding ethyl acetate and continuing to shake, centrifuging, taking the supernatant, placing it under nitrogen conditions (such as a nitrogen blowdown apparatus) at room temperature to remove the solvent (for example, evaporating it for at least 30 minutes), and then adding an appropriate amount of diluent to re-dissolve it to prepare a reference substance solution of the target concentration.

[0022] In the preparation of the test sample and reference solution in step (1) of the present invention, the amount of the solvent and extractant can fluctuate within a wide range. Since the solvent and extractant will be removed in the subsequent steps, as long as the amount of solvent and extractant can meet the purpose of dissolving the test sample or reference and extracting the impurity J, it is sufficient. For example, the weight-to-volume ratio of the test sample to the extractant can be (1-200):1, preferably (25-100):1, and the unit of the weight-to-volume ratio is mg / ml; the weight-to-volume ratio of the test sample to the solvent can be (1-200):(1-100), preferably (20-100):(1-50), and the unit of the weight-to-volume ratio is mg / ml. The concentration of the impurity J reference solution is relatively low. Generally, the volume of solvent such as water used for pretreatment to prepare a 2 ml reference solution with a concentration within the linear range can be 0.1 ml to 10 ml of solvent, preferably 1-5 ml, and the volume of the extraction solvent used is 0.1-10 ml, preferably 1-5 ml.

[0023] In one embodiment, the diluent in step (1) is a mixture of acetonitrile and water, and the volume ratio of acetonitrile to water can fluctuate within a relatively large range, for example, the preferred range is 0.5 to 10:1, more preferably 1 to 5:1.

[0024] In a preferred embodiment of the present invention, the concentration of N-nitrosoviloxazine in the reference solution prepared in step (1) is 0.56 to 17.3 ng / ml, for example, 17.3 ng / ml, 16.8 ng / ml, 11.2 ng / ml, 5.6 ng / ml, 2.8 ng / ml, 1.4 ng / ml, 0.58 ng / ml or 0.56 ng / ml, and values between any two numbers. It is particularly preferred that the concentration of N-nitrosoviloxazine in the reference solution is 5.6 ng / ml.

[0025] In a preferred embodiment, the concentration of viloxazine hydrochloride in the test solution prepared in step (1) is 1mg / ml~80mg / ml, such as 1mg / ml, 5mg / ml, 10mg / ml, 15mg / ml, 20mg / ml, 25mg / ml, 30mg / ml, 35mg / ml, 40mg / ml, 45mg / ml, 50mg / ml, 55mg / ml, 60mg / ml, 65mg / ml, 70mg / ml, 75mg / ml or 80mg / ml and the value between any two numbers. In this embodiment, the detection sensitivity is high, and the concentration of the test sample can fluctuate widely within a relatively low concentration range. However, the high concentration test solution may face problems such as blocking the chromatographic column, contaminating the ion source, and matrix effect. Therefore, in the present invention, the concentration of viloxazine hydrochloride in the preferred test solution is 1mg / ml~80mg / ml, particularly preferably 20mg / ml~80mg / ml.

[0026] In one embodiment, in the liquid chromatography of step (2), in the liquid chromatography conditions, mobile phase A is selected from an aqueous formic acid solution, the aqueous formic acid solution contains a sodium salt, preferably sodium formate, sodium acetate, sodium methoxide, or sodium chloride, and mobile phase B is selected from one or more of acetonitrile, methanol, or a mixture thereof. The addition of sodium salts to the mobile phase of liquid chromatography conditions is not common in the art. In the technical solution of the present invention, the applicant has found through extensive experimental studies that the addition of sodium formate, sodium chloride, etc. to mobile phase A can significantly improve the sensitivity and stability of the instrument and reduce drift during MS detection.

[0027] Further preferably, the mobile phase A is selected from a formic acid aqueous solution, the mass percentage of the formic acid aqueous solution is 0.05% to 0.3%, and the formic acid aqueous solution contains 0.05 to 0.5 μg / ml sodium salt, more preferably the sodium salt is sodium formate or sodium chloride.

[0028] In a preferred embodiment, the mobile phase A is selected from a 0.05% to 0.3% by mass formic acid aqueous solution containing 0.05 to 0.5 μg / ml sodium formate.

[0029] In a preferred embodiment, the mobile phase A is selected from a 0.05% to 0.3% by mass formic acid aqueous solution containing 0.05 to 0.5 μg / ml sodium chloride.

[0030] In a preferred embodiment, the mobile phase A is selected from a 0.1% by mass formic acid aqueous solution containing 0.1 μg / ml sodium formate or sodium chloride.

[0031] In one embodiment, the liquid chromatography conditions in step (2) are such that the mobile phase B is acetonitrile.

[0032] In one embodiment, the liquid chromatography in step (2), wherein the chromatographic column in the liquid chromatography conditions is selected from a reverse phase chromatography column with octadecylsilane bonded silica gel as a filler; the chromatographic column has a length of 50 to 100 mm, a diameter of 2.1 to 3 mm, and a filling particle size of 1.0 to 3 μm; further preferably, the chromatographic column is a Shim-Pack Scepter C18, 1.9 μm, 50 mm × 2.1 mm, or a chromatographic column with equivalent performance; or the chromatographic column is a Welch Xtimate C18, 3 μm, 100 mm × 3.0 mm, or a chromatographic column with equivalent performance.

[0033] In one embodiment, the liquid chromatography of step (2), wherein the flow rate of the mobile phase in the liquid chromatography conditions is 0.1 to 0.5 ml / min; for example, 0.1 ml / min, 0.2 ml / min, 0.3 ml / min, 0.4 ml / min or 0.5 ml / min, etc. and the range between two numbers; preferably, the flow rate is 0.3 ml / min.

[0034] In one embodiment, in the liquid chromatography of step (2), the column temperature in the liquid chromatography conditions is 30 to 50°C; preferably 40±5°C.

[0035] In one embodiment, the liquid chromatography in step (2), wherein the injection volume in the liquid chromatography conditions is 1 to 10 μl, for example, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl or 10 μl, etc., and the range between any two numbers.

[0036] In one embodiment, the liquid chromatography of step (2), wherein the elution mode is linear gradient elution; preferably, the linear gradient elution conditions are: the initial volume ratio and the final volume ratio of the mobility A and the mobility B are independently (90-70): (10-30), preferably (85-75): (15-25), further preferably (81-79): (19-21), and the running time is 12-15 min; in a preferred embodiment, the linear gradient elution conditions are: during the 0-7 min: the volume ratio of mobile phase A to mobile phase B is reduced from (70-90): (30-10) to (50-40): (50-60), during the 7-12 min: the volume ratio of mobile phase A to mobile phase B is reduced from (40-50): (60-50) to (10-5):

[0037] (90-95), during the 12th to 15th minute: the volume ratio of mobile phase A to mobile phase B is (70-90): (30-10);

[0038] More preferably, the linear gradient elution conditions are:

[0039]

[0040]

[0041] In one embodiment, the method for detecting the content of N-nitrosoviloxazine in viloxazine hydrochloride, wherein the mass spectrometry in step (2) adopts an electrospray ion source and is collected in positive ion mode, and the quantitative ion pair of N-nitrosoviloxazine is 289m / z and 160.125m / z.

[0042] More preferably, in the mass spectrometry method, the ion transmission temperature is 350°C±50°C, the spray voltage is 800V±50V, and the atomization temperature is 350°C±50°C.

[0043] In an embodiment of the present invention, the viloxazine hydrochloride includes viloxazine hydrochloride bulk drug (abbreviated as API) and viloxazine hydrochloride pharmaceutical preparations.

[0044] In an embodiment of the present invention, in the method for detecting N-nitrosoviloxazine in viloxazine hydrochloride, the content of N-nitrosoviloxazine is calculated by peak area using an external standard method.

[0045] The present invention significantly improves the sample detection sensitivity and accuracy by pre-treating the test sample and the reference sample before preparation, particularly by pre-treating with water and ethyl acetate, and then redissolving and preparing with a diluent. Secondly, the present invention creatively adds sodium salt to the fluidity, which not only improves the detection sensitivity of the sample, but also improves the stability of the instrument and avoids MS detection drift. The method provided by the present invention has extremely high detection sensitivity. For example, the concentration of the quantitative limit has very good accuracy in the range of 0.57ng / ml to 17.3ng / ml, and the detection sensitivity can reach 0.014ppm, which solves the problem of difficulty in detecting the content of extremely small amounts of N-nitrosoviloxazine in viloxazine or its salts, and can effectively control the quality of viloxazine hydrochloride products. In addition, the solution provided by the present invention is highly adaptable and can be operated using conventional chromatographic instruments, making it very suitable for industrial applications.

[0046] Other features and advantageous effects of the present invention are further described and illustrated through the following embodiments and drawings.

[0047] Definition of terms

[0048] The present invention is intended to cover all replacements, modifications and equivalent technical solutions, which are included within the scope of the present invention as defined in the claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention.

[0049] It will be further appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

[0050] Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

[0051] The terms "include", "comprising", "having", "containing", "selected from", or "involving" and their variations herein are inclusive or open-ended and do not exclude other unlisted elements or method steps. That is, they include the contents specified in the present invention but do not exclude other aspects.

[0052] The terms "selected from...", "preferably..." and "more preferably..." refer to one or more elements in the groups listed thereafter, selected independently, and may include combinations of two or more elements.

[0053] In the context of the present invention, all numerical values disclosed herein are approximate, regardless of whether the word "about" or "approximately" is used. The numerical value of each number may vary by less than 10% or by amounts deemed reasonable by one skilled in the art, such as 1%, 2%, 3%, 4%, or 5%. As used herein, "v / v" refers to a volume ratio.

[0054] In the context of the present invention, the numerical value X±Y refers to any value between the numerical value "XY" and the numerical value "X+Y", including the endpoint values. For example, "350℃±50℃" mentioned in the present invention refers to any value between 300℃ and 400℃, including the endpoint values 300℃ and 400℃.

[0055] The "liquid chromatography-mass spectrometry" (abbreviation: UPLC-MS) described in the context of the present invention is a technology that uses ultra-high performance liquid chromatography (abbreviation: UPLC) as the injection system for mass spectrometry (abbreviation: MS) to separate complex chemical components; and uses mass spectrometry as a detector for quantitative and qualitative analysis.

[0056] In the context of the present invention, the "viloxazine pharmaceutical preparation" or "viloxazine hydrochloride pharmaceutical preparation" refers to a drug obtained by combining the viloxazine hydrochloride raw material with a pharmaceutically acceptable pharmaceutical excipient or excipient in accordance with the "Drug Administration Law of the People's Republic of China" and / or the Pharmacopoeia, such as the viloxazine sustained-release capsule Qelbree or a viloxazine pharmaceutical preparation that is identical or similar to Qelbree in composition, the active ingredient of which is viloxazine hydrochloride. The viloxazine pharmaceutical preparation can also be prepared by referring to the methods disclosed in existing documents such as WO2010028207, IN202341001788A, US20240041893A, and CN119679757A, and the contents disclosed in these documents are incorporated into the present invention by reference.

[0057] The viloxazine nitrosamine impurity in the context of the present invention refers to N-nitrosoviloxazine, wherein the N-nitrosoviloxazine is referred to as impurity J and has a structure shown in the following formula J:

[0058]

[0059] The "room temperature" in the context of the present invention refers to a temperature of 25°C ± 5°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 : Shows the MS graph of the blank solution detected by the method of Example 1;

[0061] Figure 2 : Shows the MS graph of the sensitive solution detected by the method of Example 1, wherein the retention time of impurity J is 15.940 min, S / N=169, and S / N is much greater than 10;

[0062] Figure 3 : Shows the MS graph of the reference solution detected by the method of Example 1, wherein the retention time of impurity J is 15.900 min, S / N=2276

[0063] Figure 4 : Shows the MS spectrum of the test solution of viloxazine hydrochloride spiked (100% level) detected by the method of Example 1, wherein the retention time of impurity J is 15.860 min, S / N=1871; DETAILED DESCRIPTION

[0064] The following is a further explanation of the technical solutions and beneficial effects of the present invention in conjunction with specific examples. The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or under the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products. The mobile phase solvents are all chromatographically pure reagents; the water used to prepare the solution is deionized water; the detection instrument used is a liquid chromatography-mass spectrometry (Quantis) instrument from Thermo Fisher Scientific; the viloxazine raw material used is viloxazine hydrochloride (purity not less than 99%) produced by Suzhou Enhua Biopharmaceutical Technology Co., Ltd.; N-nitrosoviloxazine is referred to as impurity J. The viloxazine hydrochloride pharmaceutical preparation used is a sustained-release capsule that is identical or similar in composition to Qelbree and is provided by Suzhou Enhua Biopharmaceutical Technology Co., Ltd.

[0065] The present invention provides a method for detecting N-nitrosoviloxazine (impurity J) in viloxazine hydrochloride, the method comprising:

[0066] The preparation of the test solution comprises: taking an appropriate amount of a test sample of viloxazine hydrochloride (raw material or pharmaceutical preparation), accurately weighing it, placing it in a volumetric flask, adding water and ethyl acetate, shaking it, and then centrifuging it, taking an appropriate amount of the supernatant, removing the solvent under nitrogen, and then adding a diluent to redissolve it to prepare a test solution of the target concentration; the concentration of viloxazine hydrochloride in the test solution is 1 mg / ml to 80 mg / ml, preferably 20 mg / ml to 80 mg / ml;

[0067] The preparation of the reference solution comprises: first preparing an impurity J reference substance with acetonitrile, water, or a mixture thereof to prepare a reference substance stock solution; then accurately measuring an appropriate amount of the reference substance stock solution, placing it in a volumetric flask, adding water and ethyl acetate, shaking, and then centrifuging; taking an appropriate amount of the supernatant, removing the solvent under nitrogen, and then adding a diluent for re-dissolution to prepare a reference solution of a target concentration; wherein the concentration of N-nitrosoviloxazine in the reference solution is 0.56 to 17.3 ng / ml;

[0068] Liquid chromatography-mass spectrometry was used to detect N-nitrosoviloxazine in the reference solution and the test solution.

[0069] The test solution in step (1) is prepared in a volume ratio of water to ethyl acetate of 1 to 3:1, preferably 2:1, and a weight-volume ratio of the test sample to ethyl acetate of 20 to 40:1 (mg / ml); the reference solution in step (1) is prepared in a volume ratio of water to ethyl acetate of 1 to 3:1, preferably 2:1, and a weight-volume ratio of the N-nitrosoviloxazine reference substance to ethyl acetate of 20 to 40:1 (mg / ml).

[0070] The liquid chromatography conditions are as follows: mobile phase A is selected from an aqueous solution of formic acid and a sodium salt (preferably sodium formate or sodium chloride), preferably, mobile phase A is selected from a 0.1% formic acid aqueous solution, and the 0.1% formic acid solution contains 0.1 μg / ml sodium formate; mobile phase B is selected from one or more of acetonitrile, methanol, or a mixture thereof, preferably, mobile phase B is selected from acetonitrile; the flow rate of the mobile phase is 0.1 to 0.5 ml / min, preferably 0.3 ml / min, and gradient elution is adopted; the chromatographic column is selected from a reverse phase chromatographic column filled with octadecylsilane bonded silica gel, preferably a Shim-Pack Scepter C 18-120 , 1.9μm, 50mm×2.1mm, or a chromatographic column with equivalent performance; column temperature 30~50℃, preferably 40±5℃; injection volume 3~6μl, preferably 5μl;

[0071] Preferably, the gradient elution conditions are as follows: during the 0-7 min period, the volume ratio of mobile phase A to mobile phase B decreases from (70-90): (30-10) to (50-40): (50-60); during the 7-12 min period, the volume ratio of mobile phase A to mobile phase B decreases from (40-50): (60-50) to (10-5): (90-95); during the 12-15 min period, the volume ratio of mobile phase A to mobile phase B is (70-90): (30-10);

[0072] The mass spectrometry method uses an electrospray ion source and is collected in positive ion mode. The quantitative ion pairs of N-nitrosoviloxazine are 289 m / z and 160.125 m / z. The ion transmission temperature in the spectrometry method is 320-380°C, preferably 350°C±50°C, the spray voltage is 3500V±100V, and the atomization temperature is 350°C±50°C.

[0073] The content of N-nitrosoviloxazine is calculated using the peak area using the external standard method.

[0074] The viloxazine hydrochloride is a viloxazine hydrochloride bulk drug or a viloxazine hydrochloride pharmaceutical preparation.

[0075] Example 1: Detection of the content of nitrosamine impurity J in viloxazine hydrochloride

[0076] Chromatographic conditions:

[0077] Chromatographic column: Shim-Pack Scepter C18-120, 1.9 μm, 50 mm × 2.1 mm, or column with equivalent performance;

[0078] Mobile phase A: 0.1% formic acid-0.1 μg / ml sodium formate in water;

[0079] Mobile phase B: acetonitrile;

[0080] Isocratic elution: mobile phase A-mobile phase B = 80:20;

[0081] Column temperature: 40°C;

[0082] Flow rate: 0.3 ml / min;

[0083] Injection volume: 5 μl;

[0084] Diluent: 50% acetonitrile aqueous solution;

[0085] Perform gradient elution according to the following table:

[0086]

[0087] Run time: 15 minutes, MS test time: 3.8 minutes to 7.2 minutes; 10.5 minutes to 12.0 minutes, which can be adjusted according to needs.

[0088] MS parameters: electrospray ionization source (H-ESI), positive ion mode acquisition, ion transmission temperature of 345 ° C, spray voltage of 800 V, nebulization temperature of 380 ° C, quantitative ion pairs of 289 m / z and 160.125 m / z.

[0089] Solution preparation:

[0090] (1) Preparation of mobile phase:

[0091] Mobile phase A: Take 100 μl of 1 mg / ml sodium formate aqueous solution and 1 ml of formic acid, add 1000 ml of water, and degas by ultrasonication to obtain a 0.1% formic acid-0.1 μg / ml sodium formate aqueous solution.

[0092] Mobility B: acetonitrile;

[0093] Diluent: Take 50 ml of acetonitrile and 50 ml of water, mix well, and ultrasonicate to obtain a 50% acetonitrile aqueous solution.

[0094] Blank solution: Accurately measure 4 ml of water and place it in a 20 ml screw-cap bottle. Oscillate on an oscillator for 10 minutes. Add 2 ml of ethyl acetate and continue to oscillate for 20 minutes. After oscillation, transfer the solution to a centrifuge tube and centrifuge at 5000 rpm for (at least) 5 minutes. Take 1 ml of the supernatant and evaporate to dryness at room temperature under nitrogen blow (at least 30 minutes). Add 1 ml of diluent to reconstitute the solution and vortex on a vortexer for 2 minutes to obtain a blank solution.

[0095] (2) Preparation of reference solution

[0096] Reference substance stock solution: Take an appropriate amount of N-nitrosoviloxazine (impurity J) reference substance, accurately weigh it, place it in a volumetric flask, and dilute it to volume with acetonitrile to obtain the reference substance stock solution.

[0097] Reference substance solution: Accurately measure an appropriate amount of reference substance stock solution and 2 ml of water into the same volumetric flask and oscillate on an oscillator for about 10 minutes; add 2 ml of ethyl acetate and continue oscillating for about 20 minutes. After oscillation, transfer the solution to a centrifuge tube and centrifuge at 5000 rpm or above for (at least) 5 minutes. Take 1 ml of the supernatant and evaporate to dryness at instrument temperature by nitrogen blowdown (at least 30 minutes). Add 1 ml of diluent to reconstitute and vortex on a vortexer for about 2 minutes to obtain a solution containing approximately 5.6 ng of impurity J per 1 ml.

[0098] (3) Preparation of sensitivity solution

[0099] Sensitivity solution: Accurately measure an appropriate amount of reference solution and dilute it with diluent to prepare a solution containing approximately 0.56 ng of impurity J per 1 ml.

[0100] (4) Preparation of test solution

[0101] Test solution: Take an appropriate amount of viloxazine hydrochloride raw material or drug preparation test sample, accurately weigh it, place it in a volumetric flask, add 4 ml of water, and oscillate on an oscillator for 10 minutes; add 2 ml of ethyl acetate and continue to oscillate for 20 minutes. After oscillation, transfer the solution to a centrifuge tube and centrifuge at 5000 rpm (at least) for 5 minutes. Take 1 ml of the supernatant and evaporate it to dryness at room temperature under nitrogen (at least 30 minutes). Add 1 ml of diluent to reconstitute it, vortex it on a vortexer for 2 minutes, and filter it to obtain a solution containing approximately 40 mg of viloxazine hydrochloride per 1 ml.

[0102] (5) Preparation of test sample spiked solution

[0103] Take an appropriate amount of the raw material test sample of viloxazine hydrochloride, accurately weigh it, and add it to a volumetric flask. Then add the impurity J reference substance and water, and oscillate on a shaker for 10 minutes; add ethyl acetate and continue to oscillate for 20 minutes. After oscillation, transfer the solution to a centrifuge tube and centrifuge at 5000 rpm (at least) for 5 minutes. Take 1 ml of the supernatant and place it in an injection vial to evaporate to dryness at room temperature under a nitrogen blowdown device for 30 minutes. Add 1 ml of diluent to re-dissolve, and vortex on a vortexer for 2 minutes to obtain 40 mg of viloxazine hydrochloride and 11.4 ng of impurity J reference substance per 1 ml of solution.

[0104] Detection method:

[0105] Accurately measure 5 μl of blank solution, sensitivity solution, reference solution, test solution, and test spiked solution, respectively, and inject 5 μl of each into high performance liquid chromatography-mass spectrometer to record the spectrum.

[0106] Figure 1 The spectrum of the blank solution is shown.

[0107] Figure 2The spectrum of the sensitive solution is shown. The retention time of impurity J is 15.940 min, and the S / N ratio is 169, which is much greater than 10. This indicates that the method of the present invention has good sensitivity.

[0108] Figure 3 This is the MS spectrum of the reference solution, in which the retention time of impurity J is 15.900 min and S / N=2276.

[0109] like Figure 1 and 3 The results show that the blank solution has no peak at the retention time of impurity J, which does not interfere with the detection, indicating that the method of the present invention has good specificity.

[0110] Figure 4 This is the MS spectrum of the spiked solution of the viloxazine hydrochloride test sample, where the retention time of impurity J is 15.860 min.

[0111] The test results of the spiked solution of the test sample showed that the detection specificity was good and there was no interference at the peak position of impurity J.

[0112] The reference solution was injected 5 times continuously, and the peak area and relative standard deviation (RSD) data of impurity J are shown in Table 1.1.

[0113] Table 1.1: Peak area and relative standard deviation (RSD) data for 5 reference solutions

[0114]

[0115] From the test results in Table 1.1, it can be seen that the relative standard deviation of the peak area of genotoxic impurity J measured by 5 injections does not exceed 3.0%, indicating that the precision of the detection method is good.

[0116] Example 2: Linear relationship and linear range:

[0117] A linear solution of the impurity J reference solution was prepared within the range of the limit of quantitation (LOQ) to 300% of the limiting concentration using the same preparation method as in Example 1. The theoretical concentrations of impurity J were 0.56 ng / ml, 1.4 ng / ml, 2.8 ng / ml, 5.6 ng / ml, 11.2 ng / ml, and 16.8 ng / ml, respectively, designated as L-10% (LOQ), L-25%, L-50%, L-100%, L-200%, and L-300% (as shown in Table 2.1). Accurately measure 5 μl of each of the blank solution and the linear solution and inject them into a high-performance liquid chromatography-mass spectrometer to record the spectrum. The chromatographic conditions and MS parameters were the same as in Example 1. Linear regression was performed using concentration as the abscissa and peak area as the ordinate. The results are shown in Table 2.1.

[0118] Table 2.1: Linear sample concentration and test results

[0119]

[0120] As can be seen from Table 2.1, impurity J shows good linearity in the concentration range of (limit of quantification) to 17.3 ng / ml (approximately 300% limit level), and the linear correlation coefficient r value between the concentration of impurity J and the response is 0.999.

[0121] Example 3: Limit of Detection and Limit of Quantitation Test

[0122] According to the preparation method of the reference solution in Example 1, the impurity J reference substance was taken to prepare the detection limit solution and the quantification limit solution, respectively, wherein the impurity J concentration in the detection limit (LOD) solution was 0.2887 ng / ml, and the impurity J concentration in the quantification limit solution (LOQ) was 0.57775 ng / ml. The results were detected by high performance liquid chromatography-mass spectrometry, and the chromatographic conditions and MS parameters were the same as in Example 1.

[0123] Accurately measure 5 μl of the limit of detection (LOD) solution and inject it into the HPLC-MS / MS instrument. Test three times in a row and record the spectrum.

[0124] Accurately measure 5 μl of the limit of quantitation (LOQ) solution and inject it into the high performance liquid chromatography-mass spectrometry instrument. Test 6 needles continuously and record the chromatogram.

[0125] The signal-to-noise ratios for detecting impurity J in 3-injection detection limit solution and 6-injection quantification limit solution are shown in Table 3.1.

[0126] Table 3.1 Results of detection limit and quantification limit tests

[0127]

[0128] The results showed that when the detection limit concentration of impurity J was 0.2887 ng / ml, the minimum signal-to-noise ratio was 155, which was much greater than 3. When the quantitative limit concentration was 0.57775 ng / ml, the minimum signal-to-noise ratio was 315, which was much greater than 10. This shows that the method of the present invention has high detection sensitivity and can detect impurity J with low content.

[0129] Example 4: Solution stability:

[0130] The reference solution and the test solution were prepared according to the method of Example 1, and placed at 2-8°C and room temperature, respectively. Samples were taken at different time points to test the sample stability.

[0131] Measure 5 μl of the reference solution and the test solution, inject them into the high performance liquid chromatography-mass spectrometry instrument, record the spectrum, calculate the peak area according to the external standard method, and compare the impurity J content of the reference solution and the test solution at each stability test point with the initial point (T: 0h).

[0132] The experimental results show that, when the reference solution and the test solution are stored at 2-8°C for 3 days, the ratio of impurity J to the initial point is between 0.87 and 0.92, indicating that the content of impurity J in the reference solution and the test solution is basically unchanged when stored at 2-8°C for 3 days, and the solution has good stability.

[0133] Example 5: Recovery test

[0134] Preparation of 50%, 100% and 150% accuracy solutions: According to the preparation method of the test solution in Example 1, take an appropriate amount of the test sample, accurately weigh it, add an appropriate amount of impurity J reference substance, then add water and ethyl acetate to dissolve and shake, centrifuge, take the supernatant and evaporate it to dryness at room temperature under a nitrogen blower for 30 minutes, add diluent to re-dissolve, vortex on a vortex instrument for 2 minutes, and prepare 50% horizontal accuracy solution, 100% horizontal accuracy solution, and 150% horizontal accuracy solution, respectively. Prepare 3 replicates for each spiked sample.

[0135] Detection Method: Detection was performed using the same liquid chromatography-mass spectrometry method as in Example 1. The HPLC conditions and MS parameters were the same as in Example 1. Accurately measure 5 μl of each level of accuracy solution and inject it into the HPLC-MS instrument, and record the spectrum. Impurity J content was calculated using the peak area using the external standard method.

[0136] Table 5.1: Recovery test results of spiked samples

[0137]

[0138] The above results show that the method provided by the present invention has a recovery rate of 70%-130% for spiked samples at an impurity limit level of about 50% to about 150%, and the recovery rate RSD of 9 spiked samples is ≤10%, indicating that the method has high detection accuracy and the measurement results are accurate and reliable.

[0139] Example 6: Method Robustness:

[0140] The blank solution diluent, sensitivity solution, reference solution and test solution were prepared in the same manner as in Example 1.

[0141] Investigation of changes in chromatographic parameters: Under the condition that other conditions remain unchanged, the effects of changes in different chromatographic columns, column flow rates, column temperatures and initial fluidity ratios on the test results were investigated.

[0142] 5 μl of each blank solution, diluent, sensitivity solution, reference solution, and test solution were injected into a HPLC-MS instrument. Chromatograms were recorded and calculated using the external standard method based on peak area. Using the chromatogram strips of Example 1 as standard, while maintaining all other conditions unchanged, the robustness of the method was investigated with variations in mobile phase, column temperature, flow rate, and the mobile phase A:B ratio. The results are shown in Table 6.1 below.

[0143] Table 6.1 Durability results

[0144]

[0145]

[0146] The experimental results show that under various conditions, the ratio of the impurity J content measured in the same test solution to the content measured under the chromatographic conditions of Example 1 is between 0.7 and 1.3. The specificity and sensitivity of the method meet the requirements, indicating that the method provided by the present invention has good durability.

[0147] Comparative Example 1: Effect of sample treatment on test results

[0148] An appropriate amount of Impurity J reference substance was added to a volumetric flask and diluted with water to prepare a 7 ng / ml Impurity J stock solution. 5 mg, 10 mg, 20 mg, and 50 mg of the Viloxacin Hydrochloride raw material were weighed into small vials, and 1 ml of the Impurity J stock solution was added to each vial. The Viloxacin Hydrochloride was dissolved by ultrasound and then sampled for analysis. Chromatographic conditions were the same as in Example 1. The experimental results are shown in Table D1.1 below.

[0149] Table D1.1:

[0150] Sample name Sample weight (mg) Dilution volume (ml) Impurity J peak area Recovery rate (%) 7ng / ml-Impurity J / / 394.366 / 5mg / ml-7ng / ml 5.40 1 298.929 75.87 10mg / ml-7ng / ml 10.79 1 236.998 60.15 20mg / ml-7ng / ml 20.02 1 190.906 48.45 50mg / ml-7ng / ml 50.02 1 126.503 32.11

[0151] Table D1.1 shows that as the test sample concentration increases, the amount of impurity J detected gradually decreases, and the recovery rate also decreases, indicating that the concentration of viloxazine hydrochloride affects the detection of impurity J. When detected using the conventional method, when the 100% level of impurity J is 7 ng / ml and the test sample concentration is 50 mg / ml, the recovery rate is only 32.11%, indicating that the recovery rate of the nitrosamine impurity J in viloxazine hydrochloride does not meet the requirements, and the conventional method cannot meet the detection requirements.

[0152] Comparative Example 2: Effects of pretreatment solvent and extractant on test results

[0153] Accurately weigh the test sample and place it in a volumetric flask. Add an appropriate amount of reference substance impurity J solution, then add water or other solvent and shake on a shaker for 10 minutes. Then, add ethyl acetate or other organic solvent and continue shaking for 20 minutes. After shaking, transfer the solution to a centrifuge tube and centrifuge at 5000 rpm for at least 5 minutes to prepare the test spiked sample. A 100% spiked sample of the test sample was injected and tested using the same chromatographic conditions as in Example 1 to determine the 100% spiked recovery of the test sample. The test results are shown in Table D2.1.

[0154] Table D2.1:

[0155]

[0156]

[0157] The results showed that using water or an acidic aqueous solution such as hydrochloric acid aqueous solution as a solvent and ethyl acetate or methyl isobutyl ketone as an extractant can effectively improve the accuracy of detection.

[0158] Comparative Example 3: Effect of the ratio of water and ethyl acetate in pretreatment on the test results

[0159] Take the same mass of test sample, accurately weigh it, and place it in a volumetric flask. Add an appropriate amount of reference substance impurity J solution (concentration is 5.6 ng / ml), then add different volumes of water and ethyl acetate for pretreatment, and finally re-dissolve it with diluent to prepare a 100% spiked sample of the test sample.

[0160] Among them, the weight volume ratio of the test sample and ethyl acetate used in the preparation process of Sample 1 was 80:1, and the volume ratio of water to ethyl acetate was 3:1; the recovery rate was 114.4%.

[0161] In the preparation of Sample 2, the weight-to-volume ratio of the test sample to ethyl acetate was 40:1, and the volume ratio of water to ethyl acetate was 2:1; the recovery rate was 93.3%.

[0162] In the preparation of Sample 3, the weight-to-volume ratio of the test sample to ethyl acetate was 100:1, and the volume ratio of water to ethyl acetate was 4:1; the recovery rate was 87.7%.

[0163] In the preparation of Sample 4, the weight-to-volume ratio of the test sample to ethyl acetate was 25:1, and the volume ratio of water to ethyl acetate was 40:1; the recovery rate was 85.3%.

[0164] Samples 1 to 4 were tested using the same chromatographic conditions and methods as in Example 1. The test results showed that the sample spike recovery rates were all between 80% and 120% after pretreatment with the solvent and extractant described in the present invention before testing, meeting the requirements.

[0165] Comparative Example 4: Effect of Fluidity A on Test Results

[0166] Mobile phase A was 0.1% formic acid aqueous solution, i.e., no salt was added to mobile phase A. Other chromatographic conditions were the same as in Example 1. The reference solution prepared in Example 1 was taken and tested using the same method as in Example 1. The results showed that the peak area of the reference solution was 2821;

[0167] In mobile phase A, 0.1% formic acid aqueous solution was added with 0.1 μg / ml sodium chloride solution. Other chromatographic conditions were the same as in Example 1. The reference solution prepared in Example 1 was taken and detected using the same method as in Example 1. The results showed that the peak area of the reference solution was 3894, and the sensitivity was significantly improved.

[0168] When 0.1 μg / ml sodium formate solution was added to mobile phase A, which was 0.1% formic acid aqueous solution, the results showed that the peak area of the reference substance was 10326, and the sensitivity was significantly improved;

[0169] The above test results show that the response of impurity J is significantly improved when sodium salt solution is added to mobile phase A.

[0170] From the above examples and comparative examples, it can be seen that the method provided by the present invention has high detection sensitivity, good accuracy, and good durability. A small change in the chromatographic condition parameters will not affect the detection of the nitrosamine impurity J in viloxazine hydrochloride. In addition, the method of the present invention has good separation and can accurately detect the nitrosamine impurity J in viloxazine hydrochloride.

Claims

1. A method for detecting the content of N-nitrosoviloxazine in viloxazine hydrochloride, characterized in that: The method comprises: (1) Preparation of test solution and reference solution, including: pre-treating the test sample and N-nitrosoviloxazine reference substance with a solvent and an extractant, respectively, and then re-dissolving them with a diluent to prepare the test solution and reference solution at the target concentration; (2) The content of N-nitrosoviloxazine in the reference solution and the test solution was detected by liquid chromatography-mass spectrometry; Wherein, the solvent in step (1) is water or an acidic aqueous solution, and the extractant is ethyl acetate or methyl isobutyl ketone.

2. The method according to claim 1, characterized in that The preparation of the test solution in step (1), wherein the solvent is water or aqueous hydrochloric acid, and the extractant is ethyl acetate or methyl isobutyl ketone; and / or, The reference solution in step (1) is prepared, wherein the solvent is water or aqueous hydrochloric acid solution, and the extractant is ethyl acetate or methyl isobutyl ketone.

3. The method according to claim 1, characterized in that The preparation of the test sample and the reference substance in step (1) includes: dissolving the test sample and the reference substance with a solvent and an extractant, respectively, then removing the solvent and the extractant, and then re-dissolving with a diluent to prepare a test sample solution and a reference substance solution of target concentration.

4. The method according to claim 1, characterized in that The preparation of the test sample in step (1) comprises: taking an appropriate amount of viloxazine hydrochloride, accurately weighing it, placing it in a volumetric flask, adding a solvent and an extractant, shaking it, and then centrifuging it, taking an appropriate amount of the supernatant, removing the solvent and the extractant under nitrogen, and then adding a diluent to redissolve it to prepare a test sample solution of the target concentration; and / or, The preparation of the reference substance solution described in step (1) comprises: first preparing the N-nitrosoviloxazine reference substance into a reference substance stock solution with acetonitrile, water or a mixture thereof, then accurately measuring an appropriate amount of the reference substance stock solution, placing it in a volumetric flask, adding a solvent and an extractant, shaking, and then centrifuging, taking an appropriate amount of the supernatant, removing the solvent and the extractant under nitrogen conditions, and then adding a diluent to redissolve to prepare a reference substance solution of the target concentration.

5. The method according to claim 1, characterized in that: The diluent in step (1) is a mixture of acetonitrile and water, preferably the volume ratio of acetonitrile to water is 0.5 to 10:

1.

6. The method according to claim 1, characterized in that The concentration of N-nitrosoviloxazine in the reference solution of step (1) is 0.56 to 17.3 ng / ml; and / or, The concentration of viloxazine hydrochloride in the test solution is 1 mg / ml to 80 mg / ml.

7. The method according to any one of claims 1 to 6, characterized in that: In the liquid chromatography-mass spectrometry method described in step (2), in the liquid chromatography conditions, mobile phase A is selected from a formic acid aqueous solution containing sodium salt, and mobile phase B is selected from one or more of acetonitrile, methanol or a mixture thereof.

8. The method according to claim 7, characterized in that: In the liquid chromatography-mass spectrometry method of step (2), the mobile phase A is selected from a 0.05% to 0.3% formic acid aqueous solution, and the formic acid aqueous solution contains 0.05 to 0.5 μg / ml sodium salt; Preferably, the sodium salt is sodium formate, sodium acetate, sodium methoxide or sodium chloride.

9. The method according to claim 1, characterized in that: In the liquid chromatography-mass spectrometry method of step (2), the chromatographic column in the liquid chromatography conditions is selected from a reverse phase chromatography column filled with octadecylsilane bonded silica gel; and / or The flow rate of the mobile phase in the liquid chromatography conditions is 0.1 to 0.5 ml / min; and / or, The column temperature in the liquid chromatography condition is 30 to 50°C; and / or, The injection volume in the liquid chromatography conditions is 3 to 6 μl; and / or, The elution method is linear gradient elution; the linear gradient elution conditions are: the initial volume ratio and the final volume ratio of the fluidity A and the fluidity B are independently (90-70): (10-30), preferably (85-75): (15-25), and the running time is 12-15 minutes; The mass spectrometry method uses an electrospray ion source and positive ion mode acquisition, and the quantitative ion pair of the N-nitrosoviloxazine is 289m / z and 160.125m / z; More preferably, the chromatographic column in the liquid chromatography is a C18 column with a length of 50 to 100 mm, a diameter of 2.1 to 3 mm, and a filling particle size of 1.0 to 3 μm; preferably, the liquid chromatography column is a Shim-Pack Scepter C 18-120 , 1.9μm, 50mm×2.1mm, or chromatographic column with equivalent performance.

10. The method according to claim 1, characterized in that: The viloxazine hydrochloride is a viloxazine hydrochloride bulk drug or a viloxazine hydrochloride pharmaceutical preparation.

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