Method for detecting nitrosamine genotoxic impurities in mirabegron

Abstract

The invention provides an analysis method for detecting nitrosamine impurities in mirabegron. The two nitrosamine impurities, namely an impurity A and an impurity B, can be separated and detected at the same time. The method is high in sensitivity and good in specificity, repeatability and durability, and quantitative detection of nitrosamine impurities A and B in mirabegron can be rapidly and accurately achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting nitrosamine genotoxic impurities in mirabegron. Background Art

[0002] Nitrosamine impurities are a class of highly active mutagenic impurities and are also substances in the "concern cohort" mentioned in the ICH guideline M7 (B1). Drug regulatory agencies in various countries have successively issued documents on the control of nitrosamine impurities. The Center for Drug Evaluation of the National Medical Products Administration issued a notice in May 2020, "Technical Guidelines for the Study of Nitrosamine Impurities in Chemical Drugs (Trial)" (No. 1, 2020), further clarifying the necessity of nitrosamine impurity research. Nitrosamine impurities have become one of the key indicators for drug quality research.

[0003] Mirabegron belongs to β-3 adrenergic agonists, with specifications of 25 mg and 50 mg. It was developed by Astellas Japan to treat the symptoms of frequent urination, urgency, and / or urge incontinence in adult patients with overactive bladder (OAB), and the maximum daily dose is 100 mg. Currently, there are a large number of literature reports on the impurity research and analysis of mirabegron, but little attention has been paid to its nitrosamine genotoxic impurities. According to its maximum daily dose, its limit should not exceed 1.9 ppm.

[0004] The sources of nitrosamine impurities include the self-degradation of the active pharmaceutical ingredient (API) itself and the nitrosation of the secondary amine structure in the API by exogenous nitrites, resulting in the formation of nitrosamine impurities.

[0005] The chemical structure of mirabegron is shown in I,

[0006]

[0007] The literature has reported the synthesis route of this product, and the following synthesis route is commonly used:

[0008]

[0009] Intermediate II is a synthesis intermediate. Both mirabegron I and Intermediate II contain secondary amine structures, and their potential nitrosamine genotoxic impurities are shown as A and B.

[0010]

[0011] Currently, there is no literature reporting the separation and detection methods of the above two nitrosamine impurities in mirabegron. It is urgent to establish a sensitive and accurate method for the quality control of the above impurities to reduce the potential quality and clinical use risks of mirabegron. Summary of the Invention

[0012] The object of the present invention is to provide an analytical method for detecting nitrosamine impurities in mirabegron, which can simultaneously separate and detect two nitrosamine impurities, namely impurity A and impurity B. The technical solution of the present invention is as follows:

[0013] Chromatographic conditions:

[0014] Chromatographic column: An octadecylsilyl-bonded silica gel chromatographic column is used, preferably InfinityLab Poroshell120 EC-C18 250 mm × 4.6 mm, 5 μm;

[0015] The mobile phase includes phase A and phase B. Mobile phase A is an aqueous ammonium acetate solution with an equivalent of 2 - 10 mM, preferably 5 mM; mobile phase B is acetonitrile;

[0016] Elution conditions: Linear gradient elution, with the initial content of phase B being 5% - 15%, preferably 10%. The preferred elution program is as follows:

[0017] Time (min) 0 2 4 6 8.01 10 Phase B 10 90 93 95 10 10

[0018] Flow rate: 0.4 - 0.8 ml / min, preferably 0.6 ml / min;

[0019] Injection volume: 2 - 10 μl, preferably 5 μl;

[0020] Mass spectrometry conditions:

[0021] Ion source: ESI source, positive ion mode;

[0022] Mass spectrometry acquisition time: 2 - 5 min, preferably 2.5 - 3 min;

[0023] Cone voltage: 10 - 50 V, preferably 15 V;

[0024] Collision voltage: 10 - 30 V, preferably 15 V;

[0025] Desolvation temperature: 300 - 450 °C, preferably 350 °C;

[0026] Desolvation flow rate: 800 L / HB;

[0027] Compound parent ions, fragment ions and compound parameters:

[0028]

[0029] Solution preparation method:

[0030] Blank solvent: A mixed solvent of methanol and acetonitrile, where the content of methanol is not higher than 30%, preferably 10% methanol;

[0031] Standard limit reference solution: Accurately weigh impurity reference A and impurity reference B, dissolve them in a solvent and dilute to make a standard reference solution with a concentration of 5 - 10 ng / ml.

[0032] Test solution: Accurately weigh the mirabegron test sample, place it in a 10 mL volumetric flask, add 1.0 mL of methanol, gently shake until the sample is completely dissolved and clarified, dilute to the mark with acetonitrile, shake well to obtain a mirabegron test solution with a concentration of 1 - 10 mg / ml.

[0033] Determination method: Accurately measure blank solution, reference solution and test solution respectively, inject them into the liquid chromatography - mass spectrometer, record the mass spectra, and calculate the contents of impurity A and impurity B in the test solution by the external standard method.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention provides a method for detecting nitrosamine impurities A and B in mirabegron by high - performance liquid chromatography - mass spectrometry, which has high sensitivity, good specificity, repeatability and durability of the method, can quickly and stably achieve the quantitative determination of nitrosamine impurities A and B in mirabegron, effectively control the impurities in mirabegron, and thus is conducive to the quality control of mirabegron preparations and reduces the clinical risk. Description of the Drawings

[0035] Figure 1 : Mass spectra of impurity A and impurity B in the blank solution

[0036] Figure 2 : Mass spectra of impurity A and impurity B in the quantitation limit solution

[0037] Figure 3 : Mass spectra of impurity A and impurity B in the reference solution

[0038] Figure 4 : Mass spectra of impurity A and impurity B in the test solution

[0039] Figure 5 : Mass spectra of impurity A and impurity B in the 100% spiked test solution Detailed Embodiments

[0040] To further illustrate the present invention, the following examples are used to describe in detail the method for determining nitrosamine impurities A and B in mirabegron provided by the present invention, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] (1) Chromatographic conditions

[0043] Chromatographic column: InfinityLab Poroshell 120 EC - C18 250 mm × 4.6 mm, 5 μm;

[0044] The mobile phase consists of phase A and phase B. Mobile phase A is an aqueous solution of 5 mM ammonium acetate, and mobile phase B is acetonitrile;

[0045] Elution conditions:

[0046] Time (min) 0 2 4 6 8.01 10 Phase B 10 90 93 95 10 10

[0047] Flow rate: 0.5 ml / min;

[0048] Injection volume: 5 μl;

[0049] (2) Mass spectrometry conditions

[0050] Ion source: ESI source, positive ion mode;

[0051] Mass spectrometry acquisition time: 2.5 - 3 min;

[0052] Cone voltage: 15 V;

[0053] Collision voltage: 15 V

[0054] Desolvation temperature: 350 °C;

[0055] Desolvation flow rate: 800 L / HB;

[0056] Compound parent ions, fragment ions and compound parameters:

[0057]

[0058] (3) Solution preparation

[0059] Blank solvent: Methanol:Acetonitrile = 2:8

[0060] Quantitation limit solution: Weigh impurity reference substance A and impurity reference substance B accurately, dissolve them accurately, and dilute with solvent to prepare a quantitation limit solution of 0.9 ng / ml;

[0061] Standard limit reference substance solution: Weigh impurity reference substance A and impurity reference substance B accurately, dissolve them accurately, and dilute with solvent to prepare a standard reference substance solution of 9.5 ng / ml;

[0062] Test solution: Weigh the mirabegron test sample accurately, place it in a 10 mL volumetric flask, add 1.0 mL of methanol, shake gently until the sample is completely dissolved and clarified, dilute to the mark with acetonitrile, and shake well to prepare a 5 mg / ml mirabegron test solution.

[0063] (4) Determination method

[0064] Determination method: Accurately measure blank solution, quantitation limit solution, control solution, and test solution respectively, inject them into the liquid chromatography-mass spectrometry instrument, record the mass spectrometry diagram, and calculate the contents of impurity A and impurity B in the test solution by the external standard method.

[0065] (5) Experimental conclusion

[0066] Figure 1 In the blank solution, no mass spectrometry signals of impurity A (quantitative ion: 286 / 120) and impurity B (quantitative ion: 426 / 260) were detected, indicating good specificity.

[0067] Figure 2 In the quantitation limit solution, the retention times of impurity A and impurity B were 2.87 min and 2.78 min respectively, the peak areas were 262 and 374, and the mass spectrometry responses were 1.027e 4 , and the signal-to-noise ratios of impurity A and impurity B were 14.2 and 16.2 respectively. When the concentration of the test substance was 5 mg / ml, the limit concentrations of impurity A and impurity B were 9.5 ng / ml, and the quantitation limit concentration was 0.9 ng / ml, which was 1 / 10 of the limit concentration, i.e., 0.19 ppm, indicating that the analytical method had high sensitivity and could meet the quantitative detection of impurity A and impurity B in mirabegron.

[0068] Figure 3 In the control solution, the retention times of impurity A and impurity B at the limit concentration were 2.87 min and 2.78 min respectively, the peak areas were 2750 and 3665, and the mass spectrometry responses were 9.603e 4 , with good peak shape, strong signal, and meeting the quantitative requirements.

[0069] Figure 4 In the test solution, no mass spectrometry signals of impurity A and impurity B were detected.

[0070] Figure 5 In the chromatogram of the 100% spiked control solution, the retention times of impurity A and impurity B were 2.87 min and 2.78 min respectively, the peak areas were 2502 and 4070, and the mass spectrometry responses were 1.108e 5 , with good peak shape, no matrix interference effect, and strong signal.

[0071] Example 2 Repeatability experiment

[0072] Control solution: Accurately weigh impurity reference substance A and impurity reference substance B, accurately weigh them, and dilute with solvent to prepare a standard control solution of 9.5 ng / ml.

[0073] 100% Spiked Test Solution: Prepared by the same person, taking the same batch of samples, and parallelly preparing 6 portions of standard limit spiked test solutions in accordance with the law. The concentration of the test substance is 5 mg / ml, and the spiked limits of both impurity A and impurity B are 1.9 ppm.

[0074] Precisely pipette 5 μl each of the 100% spiked test solution and the control solution, inject them into the liquid chromatography - mass spectrometer, and calculate by the external standard method. The results of the recovery rate for repeatability investigation are as follows:

[0075] Serial number Impurity A (Recovery rate %) Impurity B (Recovery rate %) 1 100.54 107.27 2 98.43 110.93 3 103.78 112.24 4 97.75 110.33 5 99.41 113.56 6 96.68 113.62 RSD (%) 2.6 2.2

[0076] According to the validation guidelines of the Chinese Pharmacopoeia 2020 edition, for an impurity of 1 ppm, the RSD should not exceed 8%. The research results show that the repeatability of this method is good. The RSD of the content of impurity A in 6 portions of standard limit spiked test solutions is 2.6%, and the RSD of the content of impurity B is 2.2%, indicating that the repeatability of this method is good.

[0077] Example 3 Durability Experiment

[0078] Blank Solution: Methanol: Acetonitrile = 2:8

[0079] Reference Solution: Precisely weigh impurity reference substance A and impurity reference substance B, accurately weigh, and dilute with solvent to prepare a standard reference solution of 9.5 ng / ml;

[0080] 100% Spiked Test Solution for Samples: Take the same batch of samples, and parallelly prepare 6 portions of standard limit spiked test solutions in accordance with the law. The concentration of the test substance is 5 mg / ml, and the spiked limits of both impurity A and impurity B are 1.9 ppm.

[0081] On the basis of the chromatographic conditions in Example 1, investigate the data comparison under 4 conditions of reducing the column flow rate, increasing the column flow rate, reducing the initial proportion of the aqueous phase, and increasing the initial proportion of the aqueous phase compared with the conditions in Example 1:

[0082] Normal Conditions: See the analysis method in Example 1;

[0083] Condition 2: The column flow rate is reduced to 0.58 mL / min, and the rest is the same as the normal conditions;

[0084] Condition 3: The column flow rate is increased to 0.62 mL / min, and the rest is the same as the normal conditions;

[0085] Condition 4: The initial proportion of the aqueous phase is reduced to 88%, and the rest is the same as the normal conditions;

[0086] Condition 5: The initial proportion of water is increased to 92%, and the rest is the same as the normal conditions;

[0087] Precisely measure 5 μl of the spiked test solution and the control solution respectively, inject them into the liquid chromatography-mass spectrometer, and calculate by the external standard method. The results of the recovery rate investigation under different conditions are as follows:

[0088] Condition Impurity A content (%) Impurity B content (%) Normal condition 101.85 111.70 Column flow rate 0.58 mL / min 97.69 108.89 Column flow rate 0.62 mL / min 99.00 106.90 Initial proportion of aqueous phase 88% 102.41 108.12 Initial proportion of aqueous phase 92% 94.89 101.76 RSD (%) 3.2 3.4

[0089] Under different conditions, the RSD of the content of impurity B1 in the spiked test solution at the standard limit is 3.2%, and the RSD of the content of impurity R is 3.4%. The durability of this method is good.

[0090] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements can still be made, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. An analytical method for detecting nitrosamine impurities A and nitrosamine impurities B in mirabegron, wherein the structural formulas of impurities A and B are as follows: It is characterized in that The method comprises the following steps: Step 1) Set up the chromatographic conditions: Column: InfinityLab Poroshell 120EC-C18; Mobile phase: Mobile phase A is ammonium acetate aqueous solution, equivalent to 2-10mM, mobile phase B is acetonitrile; Elution conditions: linear gradient elution, initial 5% to 15% of phase B, gradient elution; Flow rate: 0.4~0.8ml / min; Injection volume: 2-10 μl; Step 2) Set up mass spectrometry conditions: Ion source: ESI source, positive ion mode; Mass spectrometry acquisition time: 2 to 5 minutes Cone voltage: 10~50V; Collision voltage: 10~30V Desolventizing temperature: 300-450°C; Solvent removal flow rate: 800L / HB; The parent ion and fragment ion of compound A are 286 and 120 respectively, and the parent ion and fragment ion of compound B are 426 and 260 respectively; Step 3) Solution preparation Blank solvent: a mixed solvent of methanol and acetonitrile, in which the methanol content is not higher than 30%; Standard limit reference solution: Accurately weigh impurity reference substance A and impurity reference substance B, accurately weigh, and dilute with solvent to make a 5-10 ng / ml standard reference solution; Test solution: Accurately weigh the mirabegron test sample, place it in a 10 mL volumetric flask, add 1.0 mL of methanol, shake gently until the sample is completely dissolved, dilute to the scale with acetonitrile, shake well, and prepare a 1-10 mg / ml mirabegron test solution. Step 4) Determination method Accurately measure the blank solution, control solution, and test solution and inject them into the liquid mass spectrometer, record the mass spectrum, and use the external standard method to calculate the content of impurity A and impurity B in the test solution.

2. The analytical method for detecting nitrosamine impurities A and nitrosamine impurities B in mirabegron according to claim 1, characterized in that: Step 1) Set up the chromatographic conditions: Column: InfinityLab Poroshell 120EC-C18, 250 mm × 4.6 mm, 5 μm; Mobile phase: phase A and phase B, mobile phase A is 5 mM ammonium acetate aqueous solution; mobile phase B is acetonitrile; Elution conditions: linear gradient elution, according to the following elution program: Flow rate: 0.5ml / min; Injection volume: 5 μl; Step 2) Set up mass spectrometry conditions: Ion source: ESI source, positive ion mode; Mass spectrometry acquisition time: 2.5-3 min; Cone voltage: 15V; Collision voltage: 15V; Desolventizing temperature: 350°C; Solvent removal flow rate: 800L / HB; The parent ion and fragment ion of compound A are 286 and 120 respectively, and the parent ion and fragment ion of compound B are 426 and 260 respectively; Step 3) Solution preparation: Blank solvent: methanol and acetonitrile mixed solvent, methanol: acetonitrile = 1:9; Reference solution: Accurately weigh impurity reference substance A and impurity reference substance B, accurately weigh, and dilute with solvent to make a 9.5ng / ml reference solution; Test solution: Accurately weigh the mirabegron test sample, place it in a 10 mL volumetric flask, add 1.0 mL of methanol, shake gently until the sample is completely dissolved, dilute to the scale with acetonitrile, shake well, and prepare a 5 mg / ml mirabegron test solution. Step 4) Determination method: Accurately measure the blank solution, control solution, and test solution and inject them into the liquid mass spectrometer, record the mass spectrum, and use the external standard method to calculate the content of impurity A and impurity B in the test solution.

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