Method for detecting genotoxic impurities in viloxin hydrochloride

Abstract

The invention relates to a method for detecting genotoxic impurities in viloxin hydrochloride, in particular to a method for detecting the genotoxic impurities 2-hydroxymethyl morpholine-4-toluenesulfonate or hydrochloride thereof in the viloxin 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 separation degree and good in detection accuracy, and effectively solves the problem that genotoxic impurities in the viloxin hydrochloride are difficult to separate and the content of the genotoxic impurities in the viloxin hydrochloride is difficult to detect.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for detecting genotoxic impurities in viloxazine hydrochloride, and more particularly to a method for detecting 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in viloxazine hydrochloride. Background Art

[0002] Viloxazine is a morpholine derivative and a selective norepinephrine reuptake inhibitor. It was developed by Supernus Pharma and 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 trade name is Qelbree. The active ingredient is viloxazine hydrochloride and the drug preparation 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] Methylsulfonate compounds are common genotoxic impurities. Trace amounts of genotoxic impurities 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride may be produced during the synthesis of the viloxazine API. Therefore, it is necessary to control the residues of 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in viloxazine hydrochloride. The structure of 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride is shown in Formula A below:

[0005]

[0006] The maximum daily dose of Viloxaquin is no more than 600mg. According to the limit value specified in the guidelines for the limitation of genotoxic impurities, the acceptable limit of 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in Viloxaquin raw materials shall not be higher than 0.00025%. Since the content limit of 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in Viloxaquin is low, and the concentration of the main component is large, it will pollute the instrument or interfere with the baseline. 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is similar to the main component in structure and properties, and separation is more difficult. The sensitivity of conventional liquid chromatography cannot meet the detection requirements, and there is no literature report that can meet the detection requirements. Therefore, the development of a method for detecting 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in Viloxaquin hydrochloride is of great significance to the quality control of Viloxaquin hydrochloride. Summary of the invention

[0007] In order to solve the above problems existing in the prior art, the present invention aims to provide a method for detecting genotoxic impurities in viloxazine hydrochloride having one or more advantages such as high detection sensitivity, strong specificity, high accuracy, and good reproducibility. The method can meet the detection requirements of the content of 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in viloxazine hydrochloride and effectively control the product quality.

[0008] Specifically, the present invention provides a method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the genotoxic impurity is 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, and the method comprises:

[0009] Prepare reference solution and test solution;

[0010] Liquid chromatography-mass spectrometry was used to detect the reference solution and the test solution;

[0011] The chromatographic column in the liquid chromatography is selected from poroshell 120EC-C18 or ZORBAX Eclipse PlusC18; the mobile phase A is an aqueous solution of ammonium acetate or an aqueous solution of formic acid, and the mobile phase B is acetonitrile, or a mixture of acetonitrile and one or more of methanol, ethanol, and isopropanol.

[0012] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the mobile phase A is a 1-10 mmol / L aqueous solution of ammonium acetate, preferably a 3-7 mmol / L aqueous solution of ammonium acetate; in another embodiment, the mobile phase A is a 0.01%-0.2% (v / v) aqueous solution of formic acid; further preferably, the pH of the 0.01%-0.2% (v / v) aqueous solution of formic acid is adjusted to 3-5 with aqueous ammonia.

[0013] In one embodiment, in the method for detecting genotoxic impurities in viloxazine hydrochloride, the mobile phase B is acetonitrile.

[0014] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the liquid chromatography adopts isocratic elution, wherein the volume ratio of mobile phase A and mobile phase B is (70-90): (30-10), for example 70:30, 75:25, 80:20, 85:15, 90:10 and any value therebetween, preferably (78-80): (22-20).

[0015] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the liquid chromatography adopts gradient elution, and the gradient elution conditions are shown in Table E7.1 or Table E8.1:

[0016] Table E7.1: Gradient elution conditions:

[0017]

[0018] Table E8.1: Gradient elution conditions:

[0019]

[0020] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the flow rate of the mobile phase in the liquid chromatography is 0.1 to 0.6 ml / min, such as 0.4 ml / min, 0.5 ml / min, 0.6 ml / min and any value therebetween; preferably 0.4 to 0.6 ml / min, more preferably 0.5 ml / min;

[0021] More preferably, the injection volume is 1 μl to 10 μl, for example, 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl and any value therebetween, preferably 3 to 5 μl.

[0022] More preferably, the column temperature of the chromatographic column is 20 to 50°C, for example, 23°C, 24°C, 25°C, 26°C, 27°C, 30°C, 35°C, 40°C, 45°C, 50°C and any value therebetween; preferably 37 to 43°C;

[0023] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride of the present invention, wherein the chromatographic column of the liquid chromatography is preferably an Agilent poroshell 120EC-C18 chromatographic column; the specifications of the chromatographic column are: the column length is between 100mm and 200mm, the inner diameter of the chromatographic column is between 1mm and 10mm, the particle size is between 3μm and 5μm, and the specifications of the chromatographic column are preferably 100mm×4.6mm×2.7μm. Further preferably, the chromatographic column is selected from the Agilent poroshell 120EC-C18 series chromatographic columns, and particularly preferably, the Agilent poroshell 120EC-C18, the specification is 100mm×4.6mm, and the filler particle size is 2.7μm.

[0024] In one embodiment, in the method for detecting genotoxic impurities in viloxazine hydrochloride, the mass spectrometry adopts an electrospray ion source and positive ion mode acquisition, and the quantitative ion pair of the genotoxic impurities is 100 / 272.06.

[0025] In one embodiment, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the ion transmission temperature in the mass spectrometry is 320-380°C, preferably 350°C±50°C, the spray voltage is 3500V±100V, and the atomization temperature is 350°C±50°C.

[0026] In an embodiment of the present invention, the method for detecting genotoxic impurities in viloxazine hydrochloride further includes the preparation of a reference solution and a test solution; further preferably, the reference solution and the test solution are prepared using a diluent comprising water, and in a more preferred embodiment, the reference solution and the test solution are prepared using water as a diluent.

[0027] In an embodiment of the present invention, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the preparation of the reference solution comprises: taking a certain amount of genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or a salt thereof (e.g., 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride), accurately weighing, placing in a volumetric bottle, and diluting with water to prepare; preferably, first preparing a reference stock solution, then accurately measuring an appropriate amount of the reference stock solution, placing in a volumetric bottle, and gradually diluting with water to prepare a reference solution of the target concentration.

[0028] In an embodiment of the present invention, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the preparation of the test solution comprises: taking an appropriate amount of viloxazine hydrochloride, accurately weighing, placing in a volumetric bottle, and diluting with water to prepare a test solution of the target concentration.

[0029] In an embodiment of the present invention, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the concentration of the reference solution is 0.5 to 10.0 ng / ml, preferably 0.5 to 7.7 ng / ml, such as 0.5 ng / ml, 1 ng / ml, 1.5 ng / ml, 2 ng / ml, 2.5 ng / ml, 3 ng / ml, 3.5 ng / ml, 4 ng / ml, 4.5 ng / ml, 5 ng / ml, 7 ng / ml, 7.7 ng / ml, 10.0 ng / ml and any value therebetween;

[0030] In an embodiment of the present invention, the method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the concentration of the test solution is 0.1-2 mg / ml, for example, 0.1 mg / ml, 0.5 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml and any value therebetween; preferably 1 mg / ml.

[0031] In an embodiment of the present invention, in the method for detecting genotoxic impurities in viloxazine hydrochloride, the content of the genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is calculated by peak area using an external standard method.

[0032] The present invention can realize the quantitative detection of trace genotoxic impurities 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride in viloxazine hydrochloride by selecting a specific chromatographic column in combination with a specific type of buffer, and the blank reagent has no interference with the sample, and the separation, accuracy and sensitivity in the spectrum are all good. In addition, the method provided by the present invention also overcomes the problem that due to the low limit of 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, a high concentration of the test solution needs to be prepared to achieve the required detection limit, but the high concentration of the test solution may face the problems of blocking the chromatographic column, contaminating the ion source, and matrix effect.

[0033] Other features and advantageous effects of the present invention are further described and illustrated through the following embodiments and drawings.

[0034] Definition of terms

[0035] The present invention is intended to cover all substitutions, modifications and equivalent technical solutions, which are all included in 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. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents and similar materials are different from or contradictory to the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

[0036] It should be further appreciated that certain features of the invention, which for clarity are described in multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in a single embodiment, may also be provided separately or in any suitable sub-combination.

[0037] The term “comprise” or “include” is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

[0038] In the context of the present invention, all numbers disclosed herein are approximate values, regardless of whether the words "about" or "approximately" are used. The value of each number may vary by less than 10% or by a reasonable difference considered by those skilled in the art, such as 1%, 2%, 3%, 4% or 5%. "V / V" as used herein refers to volume ratio.

[0039] 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℃.

[0040] 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 of mass spectrometry (abbreviation: MS) to separate complex chemical components; and uses mass spectrometry as a detector for quantitative and qualitative analysis.

[0041] The "viloxazine bulk drug" described in the context of the present invention specifically includes viloxazine or a pharmaceutically acceptable salt thereof. Preferably, in the present invention, the "viloxazine bulk drug" is viloxazine hydrochloride.

[0042] The viloxazine hydrochloride toxic impurity described in the context of the present invention refers to 2-hydroxymethylmorpholine-4-toluenesulfonate or its salt, wherein the 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride is referred to as impurity A and has a structure shown in the following formula A:

[0043]

[0044] The "room temperature" in the context of the present invention refers to a temperature of 25°C ± 5°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 : MS graph showing the diluent (blank solution) detected by the method of Example 1;

[0046] Figure 2 : The MS graph of the reference solution (STD1) detected by the method of Example 1 is shown, wherein the peak position of the genotoxic impurity A is at 15.011 min, and S / N=187.8;

[0047] Figure 3 : The MS graph of the viloxazine hydrochloride test solution detected by the method of Example 1 is shown, wherein the peak position of the genotoxic impurity A is at 14.971 min, and S / N=14.9;

[0048] Figure 4 : The chromatogram of the mixed solution of the reference solution and the test solution detected by the method of Example 1 is shown, wherein the peak position of viloxazine hydrochloride is at 9.910min, and the peak position of genotoxic impurity A is at 15.250min, and the separation degree between the two is 11.25;

[0049] Figure 5 : Display the linear regression graph of Example 2;

[0050] Figure 6 : The MS graph of the sample detected by the method of Comparative Example 1 is shown. The molecular weight extracted from MS by this method shows that only viloxazine peaks, and the genotoxic impurity A does not peak;

[0051] Figure 7: Shows the MS graph of the sample tested by the method of Comparative Example 2. The test results show that the genotoxic impurity A cannot be separated from viloxazine hydrochloride;

[0052] Figure 8 : The MS graph of the sample detected by the comparative example 3 method is shown. The detection results show that the separation degree of impurity A and viloxazine is relatively Figure 6 and 7 It has improved, but still cannot meet the testing needs;

[0053] Fig. 9 : The chromatogram of the sample detected by the method of Example 7 is shown. The ultraviolet display has good separation. The first peak is viloxazine and the second peak is genotoxic impurity A.

[0054] Fig.10 : The MS graph of the sample detected by the method of Example 8 is shown. The detection results show that viloxazine and genotoxic impurity A can be effectively separated. DETAILED DESCRIPTION

[0055] The technical scheme and beneficial effects of the present invention are further explained below in conjunction with specific embodiments. Unless otherwise specified, all scientific and technological terms used in the present invention have the same meaning as those generally understood by technicians in the field to which the present invention belongs. All patents and publications to which the present invention relates are incorporated herein by reference as a whole. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under 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 used are all chromatographically pure reagents; the detection instrument used is a liquid chromatography-mass spectrometry (Quantis) instrument from Thermo Fisher; the Viloxacin API used is Viloxacin hydrochloride (purity not less than 99%) produced by Suzhou Enhua Biopharmaceutical Technology Co., Ltd.

[0056] The present invention provides a method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the genotoxic impurities are 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, and the method comprises:

[0057] Prepare a reference solution and a test solution, preferably the reference solution and the test solution are prepared using water as a diluent; further preferably, the concentration of the reference solution is 0.5 to 10.0 ng / ml, preferably 0.5 to 7.7 ng / ml; the concentration of the test solution is 0.1 to 2 mg / ml, preferably 1 mg / ml;

[0058] Liquid chromatography-mass spectrometry was used to detect the reference solution and the test solution;

[0059] Wherein, the chromatographic column in the liquid chromatography is selected from poroshell 120EC-C18 or ZORBAX Eclipse PlusC18, and the preferred model is Agilent poroshell 120EC-C18, with a specification of 100mm×4.6mm×2.7μm;

[0060] Mobile phase A is an aqueous solution of ammonium acetate or an aqueous solution of formic acid, and mobile phase B is acetonitrile, or a mixture of one or more of methanol, ethanol, and isopropanol with acetonitrile. Preferably, mobile phase A is an aqueous solution of ammonium acetate of 3 to 7 mmol / L; or the mobile phase A is an aqueous solution of formic acid of 0.01% to 0.2% (v / v); further preferably, the pH of the aqueous solution of formic acid of 0.01% to 0.2% (v / v) is adjusted to 3 to 5 with ammonia water; mobile phase B is acetonitrile, and isocratic elution or gradient elution is adopted. Preferably, the volume ratio of mobile phase A to mobile phase B during isocratic elution is (70 to 90): (30 to 10), more preferably (78 to 80): (22 to 20); during gradient elution, the elution conditions are as shown in Table E7.1 or Table E8.1.

[0061] Table E7.1: Gradient elution conditions:

[0062]

[0063] Table E8.1: Gradient elution conditions:

[0064]

[0065] More preferably, the flow rate of the liquid chromatography is 0.1-0.6 ml / min, preferably 0.4-0.6 ml / min; the injection volume is 1-10 μl, preferably 3-5 μl; and the column temperature is 20-50°C, preferably 37-43°C.

[0066] Further preferably, the mass spectrometry method adopts an electrospray ion source and collects in positive ion mode, and the quantitative ion pair of the genotoxic impurity is 100 / 272.06; the ion transmission temperature in the mass 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.

[0067] More preferably, the content of the genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is calculated by peak area using an external standard method.

[0068] In a specific embodiment of the present invention, a method for detecting genotoxic impurities in viloxazine hydrochloride is provided, wherein the genotoxic impurity is 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, and the method comprises:

[0069] Prepare a reference solution and a test solution, preferably the reference solution and the test solution are prepared using water as a diluent; further preferably, the concentration of the reference solution is 0.5 to 10.0 ng / ml, preferably 0.5 to 7.7 ng / ml; the concentration of the test solution is 0.1 to 2 mg / ml, preferably 1 mg / ml;

[0070] Liquid chromatography-mass spectrometry was used to detect the reference solution and the test solution;

[0071] The chromatographic column in the liquid chromatography is selected from Agilent poroshell 120EC-C18, with a specification of 100 mm×4.6 mm×2.7 μm; the mobile phase A is a 3-7 mmol / L aqueous solution of ammonium acetate, and the mobile phase B is acetonitrile; isocratic elution is adopted, and the volume ratio of the mobile phase A to the mobile phase B is (70-90): (30-10), and more preferably (78-80): (22-20).

[0072] The flow rate of the liquid chromatography method is 0.1-0.6 ml / min, preferably 0.4-0.6 ml / min; the injection volume is 1-10 μl, preferably 3-5 μl; the temperature of the chromatographic column is 20-50° C., preferably 37-43° C.

[0073] The mass spectrometry method adopts an electrospray ion source and positive ion mode collection, and the quantitative ion pair of the genotoxic impurity is 100 / 272.06; the ion transmission temperature in the mass 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.

[0074] The content of the genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is calculated by peak area using an external standard method.

[0075] In another specific embodiment of the present invention, a method for detecting genotoxic impurities in viloxazine hydrochloride is provided, wherein the genotoxic impurity is 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, and the method comprises:

[0076] Prepare a reference solution and a test solution, preferably the reference solution and the test solution are prepared using water as a diluent; further preferably, the concentration of the reference solution is 0.5 to 10 ng / ml, preferably 0.5 to 7.7 ng / ml; the concentration of the test solution is 0.1 to 2 mg / ml, preferably 1 mg / ml;

[0077] Liquid chromatography-mass spectrometry was used to detect the reference solution and the test solution;

[0078] Wherein, the chromatographic column in the liquid chromatography is selected from the chromatographic column of model Agilent poroshell 120EC-C18, with specifications of 100mm×4.6mm×2.7μm; the mobile phase A is 0.01% to 0.2% (v / v) formic acid aqueous solution, preferably the 0.01% to 0.2% (v / v) formic acid aqueous solution is adjusted to pH 3 to 5 with ammonia water, the mobile phase B is acetonitrile, and gradient elution is adopted,

[0079] The gradient elution conditions are shown in Table E7.1 and Table E8.1.

[0080] Table E7.1: Gradient elution conditions:

[0081]

[0082] Table E8.1: Gradient phase elution conditions:

[0083]

[0084] The flow rate of the mobile phase in the liquid chromatography method is 0.1-0.6 ml / min, preferably 0.4-0.6 ml / min; the injection volume is 1-10 μl, preferably 3-5 μl; and the temperature of the chromatographic column is 20-50° C., preferably 37-43° C.

[0085] The mass spectrometry method adopts an electrospray ion source and positive ion mode collection, and the quantitative ion pair of the genotoxic impurity is 100 / 272.06; the ion transmission temperature in the mass 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.

[0086] The content of the genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is calculated by peak area using an external standard method.

[0087] Example 1: Detection method of 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride (impurity A) in viloxazine hydrochloride:

[0088] 1.1 Solution preparation:

[0089] (1) Preparation of reference solution

[0090] Stock solution of reference substance: accurately weigh 25.84 mg of 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride reference substance, place in a 1000 ml volumetric flask, dissolve and dilute to the mark with water by ultrasonication, and shake well.

[0091] Reference substance solution (STD1): Accurately measure 2 ml of reference substance stock solution, place in a 200 ml volumetric flask, dilute to the mark with water, shake well, then accurately measure 4 ml of the shaken solution, place in a 200 ml volumetric flask, dilute to the mark with water, shake well, filter with a 0.2 μm filter membrane to obtain 5.168 ng of 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride per 1 ml of solution.

[0092] (2) Preparation of test solution

[0093] Test solution (REC): Take about 20 mg of viloxazine hydrochloride, accurately weigh it, put it in a 20 ml volumetric flask, dissolve it with water ultrasonically and dilute it to the scale, shake it well to make each 1 ml of solution contain about 1 mg of viloxazine hydrochloride, filter it with a 0.2 μm filter membrane, and obtain REC.

[0094] (3) Preparation of mixed solution of reference substance and test substance

[0095] (3.1) Take 5.04 mg of impurity A and place it in a 250 ml volumetric flask, dissolve it in water and dilute to the mark;

[0096] (3.2) Take 10.64 mg of impurity A and place it in a 100 ml volumetric flask, dissolve it in water and dilute to the mark;

[0097] Take 500 μl of the solution prepared in (3.1) and 500 μl of (3.2) and mix them evenly to obtain a mixed solution of the reference substance and the test substance containing 0.05 mg of viloxazine hydrochloride and 0.01 mg of impurity A in 1 ml of the solution.

[0098] 1.2 Chromatographic conditions:

[0099] Column: Agilent poroshell 120EC-C18, 100 × 4.6 mm, 2.7 μm;

[0100] Mobile phase A: 5mmol / L ammonium acetate aqueous solution;

[0101] Mobile phase B: acetonitrile;

[0102] Isocratic elution: mobile phase A-mobile phase B = 80:20;

[0103] Column temperature: 40°C;

[0104] Flow rate: 0.5ml / min;

[0105] Injection volume: 5 μl;

[0106] Blank solution: water;

[0107] MS parameters: electrospray ion source (H-ESI), positive ion mode acquisition, ion transmission temperature of 350°C, spray voltage of 3000 V, nebulization temperature of 350°C, quantitative ion pair of 100 / 272.06;

[0108] Running time: 20 minutes, MS test start time: 13 minutes.

[0109] Accurately measure 5 μl of the diluent (blank solution), reference solution, and test solution respectively, inject them into the liquid chromatography-mass spectrometer, and record the spectrum.

[0110] Figure 1 It shows that the diluent has no interference with the peak detection of impurity A.

[0111] Figure 2 This is the MS graph of the reference solution (STD1), in which the peak position of genotoxic impurity A is at 15.011 min, S / N=187.8.

[0112] Figure 3 This is the MS graph of the test solution, in which the peak position of genotoxic impurity A is at 14.971min, S / N=14.9.

[0113] Accurately measure 20 μl of the mixed solution of the reference substance and the test substance described in (3) and inject it into the liquid chromatography. Use an ultraviolet detector to detect the separation of the two. The detection wavelength is 200 nm. The chromatogram is as follows: Figure 4 shown.

[0114] from Figure 4 It can be seen that the separation degree of impurity A and viloxazine hydrochloride is 11.25, and the separation effect of the two is good.

[0115] The reference solution was injected continuously for 5 times, and the peak area and relative standard deviation (RSD) data of impurity A are shown in Table 1.1.

[0116] Table 1.1: Peak area and relative standard deviation (RSD) data for 5-pronged reference solution (STD1)

[0117]

[0118] From the test results in Table 1.1, it can be seen that the relative standard deviation of the peak area of ​​genotoxic impurity A measured by 5 injections does not exceed 3.0%, indicating that the detection accuracy is good and the system specificity is good.

[0119] Example 2: Linear relationship and linear range:

[0120] Prepare linear solutions of impurity A reference solution within the range of quantitative limit to 300% of limit concentration, and record them as samples L-20%, L-50%, L-100%, L-200%, L-300% (as shown in Table 1); accurately measure 5 μl of diluent and samples L-20%, L-50%, L-100%, L-200%, L-300% and inject them into liquid chromatography-mass spectrometer for liquid chromatography-mass spectrometry detection. The LC chromatography conditions and MS parameters are the same as those in Example 1; the diluent is water. Linear regression is performed with concentration as the horizontal axis and peak area as the vertical axis. The results are shown in Tables 2.1 and Figure 5 shown.

[0121] Table 2.1: Linear sample concentration and test results

[0122]

[0123] From Table 2.1 and Figure 5 It can be seen that impurity A shows good linearity in the concentration range of 0.5155 ng / ml (quantification limit) to 7.7318 ng / ml (approximately 300% limit level), and the linear correlation coefficient r value between the concentration of impurity A and the response is 0.9996.

[0124] Example 3: Limit of detection and limit of quantitation test

[0125] (1) Preparation of quantitative limit solution:

[0126] Accurately weigh the impurity A reference substance and place it in a volumetric bottle, dissolve it in water to prepare a reference substance stock solution, gradually dilute the reference substance stock solution to prepare a quantitative limit solution (LOQ), then accurately weigh the quantitative limit solution, dilute it with water, and prepare a detection limit solution (LOD). Liquid chromatography-mass spectrometry is used for detection, and the LC chromatographic conditions and MS parameters are the same as in Example 1.

[0127] Accurately measure 5 μl of the detection limit (LOD) solution and inject it into the liquid chromatography-mass spectrometer, test 3 needles continuously, and record the spectrum;

[0128] Accurately measure 5 μl of the quantification limit solution (LOQ) and inject it into the liquid chromatography-mass spectrometer, test 6 injections continuously, and record the chromatogram. The signal-to-noise ratios of the detection of impurity A in the 3-injection detection limit solution and the 6-injection quantification limit solution are shown in Table 3.1.

[0129] Table 3.1 Detection limit and quantification limit test results

[0130]

[0131] The results showed that when the quantitative limit concentration of impurity A was 0.5168 ng / ml, the signal-to-noise ratio was not less than 10, and when the detection limit concentration was 0.2584 ng / ml, the signal-to-noise ratio was not less than 3, indicating that the detection sensitivity of the method of the present invention was high and the content of impurity A could be accurately controlled.

[0132] Example 4: Solution stability:

[0133] Solution preparation

[0134] (1) Preparation of reference solution STD2

[0135] Take 25.51 mg of 2-hydroxymethylmorpholine-4-toluenesulfonate hydrochloride reference substance, accurately weigh it, place it in a 1000 ml volumetric flask, dissolve it with water by ultrasonic and dilute it to the mark, shake it well, prepare 2 portions in parallel as reference substance stock solution; accurately measure 2 ml of reference substance stock solution, place it in a 200 ml volumetric flask, dilute it to the mark with water, shake it well, accurately measure 4 ml of the shaken solution, place it in a 200 ml volumetric flask, dilute it to the mark with water, shake it well, filter it with a 0.2 μm filter membrane, and obtain impurity A reference substance solution STD2 with a concentration of 5.102 ng / ml.

[0136] (2) Preparation of test solutions REC1 and REC2

[0137] Impurity stock solution A: Accurately weigh 20.44 mg of impurity A, dissolve it in water ultrasonically and gradually dilute it to obtain about 20.44 ng of impurity compound A per 1 ml of solution.

[0138] Impurity stock solution B: Accurately weigh 20.58 mg of impurity A, dissolve it in water ultrasonically and gradually dilute it to obtain about 20.58 ng of impurity compound A per 1 ml of solution.

[0139] Take about 20 mg of the viloxazine hydrochloride test sample, accurately weigh it, and place it in a 20 ml volumetric flask. Accurately measure 2 ml of the impurity stock solution A or B, place it in the same volumetric flask, dissolve it with water ultrasonically and dilute it to the scale, shake it well, and use them as the test solutions REC1 and REC2 respectively.

[0140] Investigation method: Place the reference solution STD2 and the test solutions REC1 and REC2 at room temperature (RT) and 2-8°C, respectively, take samples at 0h and 24h, and detect the content of impurity A.

[0141] The content of impurity A was detected by the same liquid chromatography-mass spectrometry as in Example 1, and the LC chromatography conditions and MS parameters were the same as in Example 1, and the peak area was calculated by the external standard method. The detection results are shown in Table 4.1.

[0142] Table 4.1 Solution stability test results

[0143]

[0144] Note: The ratio in the table refers to the ratio of the impurity A content detected at 24h to the impurity A content detected at 0h for the same sample.

[0145] As can be seen from Table 4.1, when the impurity A reference substance and the test solution were placed at 2-8°C and room temperature for 24 hours respectively, there was no significant change in the impurity A content, indicating that the solution had good stability.

[0146] Example 5: Recovery test

[0147] Preparation of accuracy solution A (Rec-20%): Take about 20 mg of the viloxazine hydrochloride test sample, accurately weigh it, and place it in a 20 ml volumetric bottle. Accurately measure 4 ml of the impurity A reference solution with a concentration of 2.5 ng / ml, place it in the same volumetric bottle, dissolve it with water ultrasonically and dilute it to the scale, shake it well, and prepare 3 copies in parallel;

[0148] Preparation of accuracy solution B (Rec-100%): take about 20 mg of viloxazine hydrochloride test sample, accurately weigh it, and place it in a 20 ml volumetric bottle. Accurately measure 2 ml of impurity A reference solution with a concentration of 25 ng / ml, place it in the same volumetric bottle, dissolve it with water by ultrasonic and dilute it to the scale, shake it well, and prepare 3 copies in parallel;

[0149] Preparation of accuracy solution C (Rec-300%): Take about 20 mg of the viloxazine hydrochloride test sample, accurately weigh it, and place it in a 20 ml volumetric bottle. Accurately measure 6 ml of the impurity A reference solution with a concentration of 25 ng / ml, place it in the same volumetric bottle, dissolve it with water by ultrasonication and dilute it to the scale, shake it well, and prepare 3 copies in parallel;

[0150] Detection method:

[0151] The same liquid chromatography-mass spectrometry as in Example 1 was used for detection. The LC chromatographic conditions and MS parameters were the same as in Example 1. The peak area was calculated by the external standard method. 5 μl of each accuracy solution A to C was accurately measured and injected into the liquid chromatography-mass spectrometer to record the spectrum.

[0152] Table 5.1: Recovery test results of spiked samples

[0153]

[0154] 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 20% to about 300%, and the recovery rate RSD of 9 spiked samples is ≤10%, indicating that the method has high detection accuracy and reliable measurement results.

[0155] Example 6: Method Robustness:

[0156] To investigate the tolerance of the detection method when the chromatographic conditions change.

[0157] The preparation of the reference solution is the same as in Example 1.

[0158] Preparation of test solution: Take about 20 mg of the test sample (viloxazine hydrochloride), accurately weigh it, and place it in a 20 ml volumetric bottle. Accurately measure 2 ml of the impurity stock solution in Example 3 and place it in the same volumetric bottle. Dissolve it with water ultrasonically and dilute it to the scale, and shake it well. Prepare 3 portions in parallel.

[0159] 5 μl of diluent (water), reference solution and test solution were measured, respectively injected into liquid phase-mass spectrometer, spectrum was recorded, and peak area was calculated according to external standard method. Taking the chromatogram strip of Example 1 as normal condition, while keeping other conditions unchanged, the durability of the method was investigated when the mobile phase, column temperature, flow rate, and mobile phase A:B ratio were changed. The test results showed that when the column temperature was 37-43°C, the flow rate was 0.4-0.6 ml / min, and the ratio of mobile phase A to B was between 78:22 and 82:28, the impurity A content measured in the same test solution was compared with the initial point, and the ratio should be between 0.9 and 1.1. The test sample did not interfere with the detection of impurity A, and the separation effect was good, indicating that the durability of the method was good.

[0160] Comparative Examples 1 to 3 and Examples 5 to 6 Study the Effects of Different Mobile Phases and Chromatographic Columns on the Detection Results

[0161] Solution preparation of Comparative Example 1:

[0162] Take 4.10 mg of the test sample (viloxazine hydrochloride), dissolve it in 4 ml of methanol and mix it evenly, record it as solution 01;

[0163] Take 2.29 mg of impurity A reference substance, dissolve it in 2.3 ml of methanol and mix well, and record it as solution 02.

[0164] Take 01 of the above solution, add water and gradually dilute it, mix it evenly, and prepare it to contain 10 ng of viloxazine hydrochloride in each 1 ml of the solution, which is used as the LC-MS sample to be detected in Comparative Example 1.

[0165] Solution preparation of Comparative Examples 2-3 and Examples 7-8:

[0166] Solution 01 and solution 02 in Comparative Example 1 were taken separately, gradually diluted with water and then mixed to prepare a solution containing 5 ng of viloxazine hydrochloride and 5 ng of impurity A per 1 ml, as LC-MS samples to be detected in Comparative Examples 2 and 3 and Examples 7 to 8.

[0167] Solution 01 and solution 02 in Comparative Example 1 were taken separately, gradually diluted with water and then mixed to prepare a solution containing 1000 ng of viloxazine hydrochloride and 1000 ng of impurity A per 1 ml, as Comparative Example 3 and the samples to be tested when the MS was disconnected for separation detection in Examples 7 to 8.

[0168] The LC-MS method was used for detection. The liquid chromatography conditions are shown in Table D1. The MS parameters are as follows: MS parameters: electrospray ion source (H-ESI) was used, positive ion mode was used for acquisition, ion transmission temperature was 350°C, spray voltage was 3000V, nebulization temperature was 350°C, and quantitative ion pair was 100 / 272.06; the LC-MS detection injection volume was 5μl, MS was disconnected, and high concentration samples were used for separation detection, the UV detection wavelength was 200nm, and the injection volume was 20μl.

[0169] The liquid chromatography conditions are shown in Table D1 below:

[0170]

[0171] Table D1.1: Mobile phase elution conditions:

[0172]

[0173] Table D2.1: Mobile phase elution conditions:

[0174]

[0175] Table D3.1: Mobile phase elution conditions:

[0176]

[0177] Table E7.1: Mobile phase elution conditions:

[0178]

[0179] Table E8.1: Mobile phase elution conditions:

[0180]

[0181] Test results such as Figures 6 to 10 As shown:

[0182] When the method of Comparative Example 1 was used for detection, only viloxazine peaked in the MS spectrum, and the genotoxic impurity A did not peak.

[0183] When the method of Comparative Example 2 is used for detection, it can be seen from the MS spectrum that the genotoxic impurity A and viloxazine cannot be separated.

[0184] When the method of comparative example 3 is used for detection, it can be seen from the MS spectrum that although the separation degree of genotoxic impurity A and viloxazine is relatively Figure 6 and 7 There is improvement, but it cannot fail to meet the detection requirements; when the flow rate in Comparative Example 3 is adjusted to 0.3 ml / min and the elution gradient is changed to Table D3.2, MS separation still cannot meet the detection requirements.

[0185] Table D3.2: Gradient elution conditions:

[0186]

[0187] The mobile phase was replaced and the method of Example 7 was used for detection. The MS was disconnected and the separation degree was detected. The concentrations of viloxazine hydrochloride and impurity A in the sample were both 1000 ng / ml. The injection volume was 20 μl and the UV detection wavelength was 200 nm. The UV spectrum showed that the separation degree of impurity A and viloxazine hydrochloride was good, the former peak was the peak of viloxazine, and the latter was the peak of impurity A.

[0188] The pH of mobile phase A was adjusted, and the method of Example 8 was used for detection. The UV display showed good separation, the first peak was the peak of viloxazine, and the second peak was the peak of impurity A. MS determination showed that viloxazine and impurity A could be effectively separated.

[0189] From the above test results, 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 impurity A in viloxazine hydrochloride. In addition, the method of the present invention has good separation and can accurately detect impurity A in viloxazine hydrochloride.

Claims

1. A method for detecting genotoxic impurities in viloxazine hydrochloride, wherein the genotoxic impurity is 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride, characterized in that: The method comprises: Prepare reference solution and test solution; Liquid chromatography-mass spectrometry was used to detect the reference solution and the test solution; The chromatographic column in the liquid chromatography is selected from poroshell 120EC-C18 or ZORBAX Eclipse Plus C18; The mobile phase A is an aqueous solution of ammonium acetate or an aqueous solution of formic acid, and the mobile phase B is acetonitrile, or a mixture of acetonitrile and one or more of methanol, ethanol, and isopropanol.

2. The method according to claim 1, characterized in that: The mobile phase A is a 1-10 mmol / L aqueous solution of ammonium acetate, preferably a 3-7 mmol / L aqueous solution of ammonium acetate; or The mobile phase A is a 0.01% to 0.2% (v / v) formic acid aqueous solution; more preferably, the 0.01% to 0.2% (v / v) formic acid aqueous solution is adjusted to a pH of 3 to 5 with aqueous ammonia.

3. The method according to claim 1, characterized in that: The mobile phase B is acetonitrile.

4. The method according to claim 1, characterized in that: The liquid chromatography adopts isocratic elution, wherein the volume ratio of mobile phase A to mobile phase B is (70-90): (30-10), preferably (78-80): (22-20).

5. The method according to claim 1, characterized in that: The liquid chromatography adopts gradient elution, and the gradient elution conditions include: during the period of 0 to 25 minutes: the volume ratio of mobile phase A to mobile phase B is (80-50): (20-50), during the period of 25 to 30 minutes: the volume ratio of mobile phase A to mobile phase B is (20-80): (80-20); Further preferably, the gradient elution conditions are as shown in Table E7.1 or Table E8.1: Table E7.1: Gradient elution conditions: Table E8.1: Gradient elution conditions:

6. The method according to any one of claims 1 to 5, characterized in that: The flow rate of the mobile phase in the liquid chromatography method is 0.1 to 0.6 ml / min, preferably 0.4 to 0.6 ml / min; and / or, The injection volume is 1 to 10 μl, preferably 3 to 5 μl; and / or, The temperature of the chromatographic column is 20-50°C, preferably 37-43°C.

7. The method according to any one of claims 1 to 5, characterized in that: The specification of the chromatographic column in the liquid chromatography is 100 mm×4.6 mm×2.7 μm; more preferably, the model of the chromatographic column is Agilent poroshell 120EC-C18.

8. The method according to any one of claims 1 to 5, characterized in that: The concentration of the reference solution is 0.5 to 10.0 ng / ml, preferably 0.5 to 7.7 ng / ml; and / or The concentration of the test solution is 0.1-2 mg / ml, preferably 1 mg / ml.

9. The method according to claim 1, characterized in that: The mass spectrometry method uses an electrospray ion source and positive ion mode acquisition, and the quantitative ion pair of the genotoxic impurity is 100 / 272.06; Further preferably, in the mass spectrometry method, the ion transmission temperature is 320-380°C, preferably 350°C±50°C, the spray voltage is 3500V±100V, and the atomization temperature is 350°C±50°C.

10. The method according to any one of claims 1 to 5, characterized in that: The reference and test solutions were prepared using a diluent containing water.

11. The method according to any one of claims 1 to 5, characterized in that: The content of the genotoxic impurity 2-hydroxymethylmorpholine-4-toluenesulfonate or its hydrochloride is calculated by peak area using an external standard method.

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