A method for detecting base toxic impurities in mirabegron

Abstract

The present invention discloses a method for detecting genotoxic impurities in mirabegron. The method of the present invention comprises the following steps: taking a solution of a mirabegron test sample and a solution of a genotoxic impurity standard sample, respectively, and performing separation and detection by high performance liquid chromatography-mass spectrometry, and comparing ion peaks in the obtained mass spectrum. If the mass spectrum of the solution of the mirabegron test sample has the same quantitative / qualitative ion peaks as the solution of the genotoxic impurity standard sample, then the genotoxic impurities are present in the mirabegron test sample; otherwise, they are not present. The detection result of the present invention is accurate and has high precision, and is suitable for the quality control of genotoxic impurities in mirabegron finished products.

Description

Technical Field

[0001] The invention relates to a method for detecting base toxic impurities in mirabegron, and belongs to the field of drug analysis. Background Art

[0002] Mirabegron was developed by Astellas and approved for marketing by the Pharmaceuticals and Medical Devices Agency (PMDA) of Japan on July 1, 2011, and then approved for marketing by the U.S. Food and Drug Administration (FDA) on June 28, 2012, and by the European Medicines Agency (EMA) on December 20, 2012. It was approved for marketing by the China Food and Drug Administration (CFDA) on September 29, 2017. It is marketed in Japan by Astellas under the trade name Mirabegron is a selective β3 adrenergic receptor (ADRB3) agonist used to treat overactive bladder (OAB) including urinary incontinence, urgency and frequency of urination. Its synthesis route is shown in the following route 1:

[0003] Route 1:

[0004] During the synthesis process, multiple intermediates and their derived impurities contain warning structures such as nitroaromatic and aminoaromatic (such as compounds IM-1 and IM-2 shown in Scheme 1, compounds IM-1-IM01, IM-2-IM01, IM-3-IM01 shown in Scheme 2, or compounds IM-K shown in Formula VI and compounds IM-L shown in Formula VII). These impurities may have potential mutagenicity and greatly affect the quality of the finished product. Therefore, it is necessary to establish corresponding detection methods to carry out extremely strict control thereof.

[0005] Route 2:

[0006] Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting base toxic impurities in mirabegron.

[0008] The present invention provides a method for detecting base toxic impurities in mirabegron, comprising the following steps: taking a solution of a mirabegron test sample and a solution of a base toxic impurity standard sample, respectively, and performing separation and detection by high performance liquid chromatography-mass spectrometry, and comparing ion peaks in the obtained mass spectra. If the mass spectra of the solution of the mirabegron test sample have the same quantitative / qualitative ion peaks as those of the solution of the base toxic impurity standard sample, then the base toxic impurities are present in the mirabegron test sample; otherwise, the base toxic impurities are not present;

[0009] The base toxic impurity standard includes the following compounds IM-1 shown in Formula I, IM-2 shown in Formula II, IM-1-IM01 shown in Formula III, IM-2-IM01 shown in Formula IV, IM-3-IM01 shown in Formula V, IM-K shown in Formula VI or IM-L shown in Formula VII;

[0010]

[0011] In the above method, the method also includes the step of determining the chromatograms of the solution of the mirabegron test sample and the solution of the base toxic impurity standard by the high performance liquid chromatography, and calculating the peak area by the external standard method to quantify the base toxic impurities in the mirabegron test sample.

[0012] In the method of the present invention, the specific steps of quantifying the base toxic impurities in the mirabegron test sample by the high performance liquid chromatography using the external standard method and calculating the peak area are as follows:

[0013] (1) preparing the mirabegron to be tested into a mirabegron test solution, and preparing the base toxicity impurity standard product into base toxicity impurity standard product solutions with different concentrations;

[0014] (2) The solutions of the base toxic impurity standard substances are respectively subjected to high performance liquid chromatography detection, and the chromatographic peak areas of the base toxic impurity standard substances (specifically including compound IM-1 of formula I, compound IM-2 of formula II, compound IM-1-IM01 of formula III, compound IM-2-IM01 of formula IV, compound IM-3-IM01 of formula V, compound IM-K of formula VI, or compound IM-L of formula VII) are respectively used as the ordinate, and the concentrations of compound IM-1 of formula I, compound IM-2 of formula II, compound IM-1-IM01 of formula III, compound IM-2-IM01 of formula IV, compound IM-3-IM01 of formula V, compound IM-K of formula VI, or compound IM-L of formula VII in the solutions of the base toxic impurity standard substances are respectively used as the abscissa to prepare a standard curve;

[0015] (3) Subjecting the solution of the mirabegron test sample to the HPLC detection to obtain the peak area of ​​the base toxic impurity (compound IM-1 of formula I, compound IM-2 of formula II, compound IM-1-IM01 of formula III, compound IM-2-IM01 of formula IV, compound IM-3-IM01 of formula V, compound IM-K of formula VI or compound IM-L of formula VII) in the test sample solution, and according to the standard curve, the content of the base toxic impurity of formula I in the mirabegron test sample is obtained.

[0016] In the above method, when the base toxic impurity standard includes the compound IM-1 shown in formula I, the compound IM-2 shown in formula II, the compound IM-1-IM01 shown in formula III, and the compound IM-2-IM01 shown in formula IV, in the high performance liquid chromatography-mass spectrometry,

[0017] The detection conditions of HPLC are as follows:

[0018] Chromatographic column: octadecylsilane bonded silica gel as filler column (specifically Waters XBridge 4.6×150mm, 5μm);

[0019] Mobile phase: acetonitrile and 0.05% trifluoroacetic acid aqueous solution, volume ratio 40:60;

[0020] Detection wavelength: 254nm;

[0021] Flow rate: 1.0ml / min;

[0022] Column temperature: 35°C;

[0023] Injection volume: 2 μl;

[0024] Collection time: 15min;

[0025] Blank solvent: methanol;

[0026] The detection conditions of mass spectrometry are as follows:

[0027] Ion source: ESI source (electrospray ionization source);

[0028] Probe temperature: 600℃;

[0029] Acquisition rate: 10 points / second;

[0030] Air flow rate: 20L / min.

[0031] In the above method, the mass spectrometry selected ion monitoring parameters of the base toxic impurity standard are as follows:

[0032] The quantitative / qualitative ion of the compound IM-1 represented by the formula I is 301.01 m / z, positive value, cone voltage is 16V, and capillary voltage is 0.8KV;

[0033] The quantitative / qualitative ion of the compound IM-2 represented by the formula II is 287.07 m / z, positive value, cone voltage is 15 V, and capillary voltage is 0.8 KV;

[0034] The quantitative / qualitative ion of the compound IM-1-IM01 represented by the formula III is 285.06 m / z, positive value, cone voltage is 15 V, and capillary voltage is 0.8 KV;

[0035] The quantitative / qualitative ion of the compound IM-2-IM01 represented by the formula IV is 271.06 m / z, a positive value, the cone voltage is 15 V, and the capillary voltage is 0.8 KV.

[0036] In the above method, when the base toxic impurity standard includes the compound IM-1 shown in formula I, the compound IM-2 shown in formula II, the compound IM-1-IM01 shown in formula III, and the compound IM-2-IM01 shown in formula IV, in the high performance liquid chromatography-mass spectrometry,

[0037] The steps for preparing the mirabegron test solution are as follows: accurately weigh mirabegron, dissolve it in methanol and dilute it to make a solution containing 10 mg per 1 ml;

[0038] The steps for preparing the solution of the base toxic impurity standard are as follows: accurately weigh IM-1, IM-2, IM-1-IM01, and IM-2-IM01, dissolve them in methanol and dilute them to make a mixed solution containing 0.3 μg of each in each ml.

[0039] In the above method, when the base toxic impurity standard includes the compound IM-3-IM01 shown in formula V, the compound IM-K shown in formula VI or the compound IM-L shown in formula VII, in the high performance liquid chromatography-mass spectrometry,

[0040] The detection conditions of HPLC are as follows:

[0041] Chromatographic column: octadecylsilane bonded silica gel as filler (specifically Waters XBridgeShield 4.6×150mm, 3.5μm, No.: 1546-171);

[0042] Mobile phase: acetonitrile and 10 mM ammonium acetate in a volume ratio of 30:70, pH 6.5 adjusted with acetic acid;

[0043] Detection wavelength: 251nm;

[0044] Flow rate: 0.8ml / min;

[0045] Column temperature: 35°C;

[0046] Injection volume: 10 μl;

[0047] Collection time: 12min;

[0048] The detection conditions of mass spectrometry are as follows:

[0049] Ion source: ESI source (electrospray ionization source);

[0050] Probe temperature: 600℃;

[0051] Acquisition rate: 10 points / second;

[0052] Air flow rate: 20L / min.

[0053] In the above method, the mass spectrometry selected ion monitoring parameters of the base toxic impurity standard are as follows:

[0054] The quantitative / qualifier ion of the compound IM-3-IM01 represented by Formula V is 241.20 m / z, positive value, cone voltage is 20 V, and capillary voltage is 0.8 KV;

[0055] The quantitative / qualifier ion of the compound IM-K represented by Formula VI is 397.25 m / z, positive value, cone voltage is 20 V, and capillary voltage is 0.8 KV;

[0056] The quantitative / qualitative ion of the compound IM-L represented by formula VII is 397.25 m / z, a positive value, the cone voltage is 20 V, and the capillary voltage is 0.8 KV.

[0057] In the above method, when the base toxic impurity standard includes the compound IM-3-IM01 shown in formula V, the compound IM-K shown in formula VI or the compound IM-L shown in formula VII, in the high performance liquid chromatography-mass spectrometry,

[0058] The steps for preparing the mirabegron test solution are as follows: accurately weigh mirabegron, dissolve it in methanol and dilute it to make a solution containing 3 mg per 1 ml;

[0059] The steps for preparing the solution of the base toxic impurity standard are as follows: accurately weigh IM-3-IM01, IM-K, and IM-L, add methanol to dissolve and dilute to make a mixed solution containing 0.1 μg of each in each 1 ml.

[0060] The present invention has the following advantages:

[0061] The detection result of the invention is accurate and of high precision, and is suitable for quality control of genotoxic impurities in mirabegron finished products. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the mass spectrum localization map of IM-1-IM01.

[0063] Figure 2 This is the mass spectrum localization map of IM-1.

[0064] Figure 3 This is the mass spectrum positioning map of IM-2-IM01.

[0065] Figure 4 This is the mass spectrum localization map of IM-2.

[0066] Figure 5 This is the mass spectrum positioning map of IM-3-IM01.

[0067] Figure 6 This is the mass spectrum localization map of IM-K+L.

[0068] Figure 7 is the quantification limit of IM-1.

[0069] Figure 8 It is the quantification limit of IM-1-IM01.

[0070] Fig. 9 is the quantification limit of IM-2.

[0071] Fig.10 It is the quantification limit of IM-2-IM01.

[0072] Fig.11 is the detection limit of IM-1.

[0073] Fig.12 It is the detection limit of IM-1-IM01.

[0074] Fig.13 is the detection limit of IM-2.

[0075] Fig.14 It is the detection limit of IM-2-IM01.

[0076] Fig.15 The specificity for IM-1 detection was blank solvent (SIR was 301.01).

[0077] Fig.16 It is the IM-1 detection specificity-reference solution (SIR is 301.01).

[0078] Fig.17 The specificity of the blank solvent was IM-1-IM01 (SIR was 285.06).

[0079] Fig.18 It is the IM-1-IM01 detection specificity-reference solution (SIR is 285.06).

[0080] Fig.19 The specificity for IM-2 detection was blank solvent (SIR was 287.07).

[0081] Fig. 20 It is the IM-2 detection specificity-reference solution (SIR is 287.07).

[0082] Fig.21 The specificity of the blank solvent was IM-2-IM01 (SIR was 271.06).

[0083] Fig. 22 It is the IM-2-IM01 detection specificity-reference solution (SIR is 271.06).

[0084] Fig.23 The linear relationship and range of IM-1.

[0085] Fig.24 This is the linear relationship and range of IM-1-IM01.

[0086] Fig.25 Linear relationship and range for IM-2.

[0087] Fig.26 Linear relationship and range for IM-2-IM01.

[0088] Fig. 27 is the quantification limit of IM-K.

[0089] Fig.28 is the quantification limit of IM-L.

[0090] Fig.29 It is the quantification limit of MLB-3-IM01.

[0091] Fig.30 is the detection limit of IM-K.

[0092] Fig.31 is the detection limit of IM-L.

[0093] Fig.32 It is the detection limit of MLB-3-IM01.

[0094] Fig.33 It is the specific blank solvent for IM-K and IM-L (SIR is 397.25).

[0095] Fig.34 It is the IM-K and IM-L specific reference solution (SIR is 397.25).

[0096] Fig.35 It is IM-3-IM01 specific blank solvent (SIR is 241.20).

[0097] Fig.36 It is the IM-3-IM01 specific reference solution (SIR is 241.20).

[0098] Fig.37 It is the linear relationship and range of IM-K+IM-L.

[0099] Fig.38 This is the linear relationship and range of IM-3-IM01. DETAILED DESCRIPTION

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0101] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0102] Example 1

[0103] 1. Detection method 1

[0104] The detection conditions of HPLC are as follows:

[0105] Chromatographic column: octadecylsilane bonded silica gel as filler column (Waters XBridge 4.6×150mm, 5μm);

[0106] Mobile phase: acetonitrile-water (0.05% trifluoroacetic acid) (40:60);

[0107] Detection wavelength: 254nm;

[0108] Flow rate: 1.0ml / min;

[0109] Column temperature: 35°C;

[0110] Injection volume: 2 μl;

[0111] Collection time: 15min;

[0112] Blank solvent: methanol.

[0113] The ion monitoring parameters of compounds IM-1, IM-2, IM-1-IM01, and IM-2-IM01 are shown in Table 1.

[0114] Table 1 Mass spectrometry selected ion monitoring parameters of four target compounds

[0115] Compound Name Quantitative / qualifier ions (m / z) Positive / negative polarity Cone voltage (V) Capillary voltage(KV) IM-1 301.01 Positive 16 0.8 IM-2 287.07 Positive 15 0.8 IM-1-IM01 285.06 Positive 15 0.8 IM-2-IM01 271.06 Positive 15 0.8

[0116] The detection conditions of mass spectrometry are as follows:

[0117] Ion source: ESI source (electrospray ionization source);

[0118] Probe temperature: 600℃;

[0119] Acquisition rate: 10 points / second;

[0120] Air flow rate: 20L / min.

[0121] Assay: The preparation of the mirabegron test solution is as follows: accurately weigh mirabegron, dissolve it in methanol and dilute it to make a solution containing 3 mg per 1 ml;

[0122] The preparation steps of the base toxic impurity standard solution are as follows: accurately weigh IM-3-IM01, IM-K, and IM-L, dissolve them in methanol and dilute them to make a mixed solution containing 0.1 μg of each in each 1 ml.

[0123] Accurately measure 2 μl of the test solution and reference solution, respectively, and inject them into the liquid chromatograph. Record the chromatogram and calculate the peak area according to the external standard method to obtain the result.

[0124] The mass spectrometry localization diagrams of compounds IM-1-IM01, IM-1, IM-2-IM01, and IM-2 are shown in Figure 1 , Figure 2 , Figure 3 , Figure 4 shown.

[0125] 2. Detection Method 2

[0126] The detection conditions of HPLC are as follows:

[0127] Chromatographic column: octadecylsilane bonded silica gel as filler column (Waters XBridge Shield 4.6×150mm, 3.5μm); (No.: 1546-171)

[0128] Mobile phase: acetonitrile–10 mM ammonium acetate (pH 6.5 adjusted with acetic acid) (30:70);

[0129] Detection wavelength: 251nm;

[0130] Flow rate: 0.8ml / min;

[0131] Column temperature: 35°C;

[0132] Injection volume: 10 μl;

[0133] Collection time: 12min;

[0134] The ion monitoring parameters of compounds IM-3-IM01 and IM-K+L are shown in Table 2.

[0135] Table 2 Mass spectrometry selected ion monitoring parameters for three genotoxic compounds

[0136]

[0137] The detection conditions of mass spectrometry are as follows:

[0138] Ion source: ESI source (electrospray ionization source);

[0139] Probe temperature: 600℃;

[0140] Acquisition rate: 10 points / second;

[0141] Air flow rate: 20L / min.

[0142] Assay: The preparation steps of the mirabegron test solution are as follows: accurately weigh an appropriate amount of mirabegron, dissolve it in methanol and dilute it to make a solution containing 10 mg per 1 ml;

[0143] The steps for preparing the base toxic impurity standard solution are as follows: accurately weigh appropriate amounts of IM-1, IM-2, IM-1-IM01, and IM-2-IM01, dissolve them in methanol, and dilute them to make a mixed solution containing 0.3 μg of each in 1 ml;

[0144] Accurately measure 10 μl of the test solution and the reference solution, respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the peak area according to the external standard method.

[0145] The mass spectrometry localization diagrams of compounds IM-3-IM01 and IM-K+L are shown in Figure 5 , Figure 6 shown.

[0146] 3. Conclusion of analytical method verification

[0147] The detection method of toxic impurities in the finished product of Mirabegron was verified from the aspects of quantitative limit, detection limit, system suitability, specificity, linearity, accuracy, precision and solution stability:

[0148] (I) Methodological investigation experiment on detection method 1

[0149] 1. Limit of Quantitation

[0150] Take appropriate amount of IM-1, IM-2, IM-1-IM01, IM-2-IM01 impurity reference stock solutions respectively, dilute the solutions step by step with methanol to appropriate concentrations, and determine the quantification limit by the amount injected into the chromatograph at a signal-to-noise ratio of 10:1 under the above chromatographic conditions. The quantification limit of IM-1 is 0.250μg / ml; the quantification limit of IM-2 is 0.0027μg / ml; the quantification limit of IM-1-IM01 is 0.017μg / ml; the quantification limit of IM-2-IM01 is 0.001μg / ml.

[0151] The quantitative limit diagrams of compounds IM-1, IM-1-IM01, IM-2, and IM-2-IM01 are shown in Figure 2. Figure 7 , Figure 8 , Fig. 9 , Fig.10 shown.

[0152] 2. Detection limit

[0153] Take appropriate amount of IM-1, IM-2, IM-1-IM01, IM-2-IM01 four kinds of genotoxic impurity reference substance stock solutions, dilute the solutions step by step with methanol to appropriate concentrations, and determine the detection limit under the above chromatographic conditions by the amount injected into the chromatograph when the signal-to-noise ratio is 3:1. The detection limit concentration of IM-1 is 0.0833μg / ml; the detection limit concentration of IM-2 is 0.0009μg / ml; the detection limit concentration of IM-1-IM01 is 0.0057μg / ml; the detection limit concentration of IM-2-IM01 is 0.0003μg / ml.

[0154] The detection limit diagrams of compounds IM-1, IM-1-IM01, IM-2, and IM-2-IM01 are shown in Figure 2. Fig.11 , Fig.12 , Fig.13 , Fig.14 shown.

[0155] 3. System applicability

[0156] After 6 consecutive injections, the retention time RSD values ​​of IM-1, IM-2, IM-1-IM01 and IM-2-IM01 were 0.45%, 0.27%, 0.62% and 0.35%, respectively, and the peak area RSD values ​​were 3.22%, 4.07%, 2.99% and 4.38%, respectively.

[0157] 4. Exclusivity

[0158] The blank solvent did not interfere with the detection of the four impurities IM-1, IM-2, IM-1-IM01, and IM-2-IM01.

[0159] The specificity diagrams of compounds IM-1, IM-1-IM01, IM-2, and IM-2-IM01 are shown in Figure 1. Fig.15 , Fig.16 , Fig.17 , Fig.18 , Fig.19 , Fig. 20 , Fig.21 , Fig. 22 shown.

[0160] 5. Linear

[0161] Mixed mother solution: Accurately weigh appropriate amounts of IM-1, IM-2, IM-1-IM01, and IM-2-IM01, dissolve and dilute with methanol to make a solution containing about 1 μg per 1 ml. Take 0.15, 0.3, 1.5, 2.4, 3.0, 4.5, and 6.0 ml of this mixed solution, place them in 10 ml volumetric bottles, add methanol to the scale, shake well, and inject them in sequence according to the chromatographic conditions of the HPLC of the above-mentioned detection method 1, and record the chromatographic peak area. Use the peak area as the ordinate (y) and the mass concentration of the reference substance as the abscissa (x) to draw a standard curve and perform linear regression. The regression equation and linear range are shown in Table 3.

[0162] Table 3 Linear relationship investigation results

[0163]

[0164] The results in Table 3 show that the HPLC method of the present invention has good linear relationships for each reference substance within its respective mass concentration range.

[0165] The linear graphs of compounds IM-1, IM-1-IM01, IM-2, and IM-2-IM01 are shown in Figure 2. Fig.23 , Fig.24 , Fig.25 , Fig.26 shown.

[0166] 6. Accuracy

[0167] Taking 0.3 μg / ml as the 100% limit concentration, three different concentrations of 50%, 100%, and 150% were designed, and three test solutions were prepared for each concentration for determination. The accuracy of the method was evaluated using the determination results of 9 samples. The accuracy results are shown in Table 4.

[0168] Table 4 Accuracy test results

[0169]

[0170]

[0171] The results in Table 4 show that the recovery rate of the method of the present invention is high. The content determination results of impurities IM-1, IM-2, IM-1-IM01 and IM-2-IM01 under this method are accurate.

[0172] 7. Precision

[0173] Repeatability

[0174] Take mirabegron reference substance, add a certain amount of impurities IM-1, IM-2, IM-1-IM01, IM-2-IM01; prepare 6 copies in parallel. Investigate the repeatability of the method. The results show that the RSDs of the determination results of IM-1, IM-2, IM-1-IM01, and IM-2-IM01 are 4.42%, 4.76%, 2.80%, and 3.78%, respectively.

[0175] Intermediate precision

[0176] Under the repeatability item, 6 samples were prepared in parallel, and the RSDs of the measurement results of 12 samples IM-1, IM-2, IM-1-IM01, and IM-2-IM01 were 7.78%, 18.10%, 3.76%, and 3.29%, respectively.

[0177] 8. Solution stability

[0178] The reference solution was placed at room temperature for 12 hours, and the RSD values ​​of the peak areas of IM-1, IM-2, IM-1-IM01, and IM-2-IM01 were 6.84%, 4.27%, 3.90%, and 6.20%, respectively.

[0179] Test Example 2, Detection Method 2 Methodological Investigation Experiment

[0180] 1. Limit of Quantitation

[0181] Take appropriate amount of IM-3-IM01, IM-K, IM-L three kinds of impurity reference stock solutions, dilute the solutions step by step with methanol to appropriate concentrations, and determine the quantitative limit by the amount injected into the chromatograph when the signal-to-noise ratio is 10:1 under the above chromatographic conditions. The quantitative limit concentration of IM-3-IM01 is 0.01μg / ml; the quantitative limit concentration of IM-K is 0.01μg / ml; the quantitative limit concentration of IM-L is 0.006μg / ml.

[0182] The quantitative limit diagrams of compounds IM-K, IM-L, and IM-3-IM01 are shown in Figure 2. Fig. 27 , Fig.28 , Fig.29 shown.

[0183] 2. Detection limit

[0184] Take appropriate amount of IM-3-IM01, IM-K, IM-L three kinds of genotoxic impurity reference substance stock solution, dilute the solution with methanol to appropriate concentration, and determine the detection limit under the above chromatographic conditions by the amount injected into the chromatograph when the signal-to-noise ratio is 3:1. The detection limit concentration of IM-3-IM01 is 0.00333μg / ml; the detection limit concentration of IM-K is 0.00333μg / ml; the detection limit concentration of IM-L is 0.002μg / ml.

[0185] The detection limit diagrams of compounds IM-K, IM-L, and IM-3-IM01 are shown in Figure 2. Fig.30 , Fig.31 , Fig.32 shown.

[0186] 3. System applicability

[0187] After 6 consecutive injections, the retention time RSD values ​​of IM-3-IM01, IM-K, and IM-L were 0.29% and 0.72%, respectively, and the peak area RSD values ​​were 2.51% and 3.63%, respectively.

[0188] 4. Exclusivity

[0189] The blank solvent did not interfere with the detection of the three impurities IM-3-IM01, IM-K, and IM-L.

[0190] The specificity diagrams of compounds IM-K, IM-L, and IM-3-IM01 are shown in Figure 2. Fig.33 , Fig.34 , Fig.35 , Fig.36 shown.

[0191] 5. Linear

[0192] Mixed mother solution: Accurately weigh appropriate amounts of IM-3-IM01, IM-K, and IM-L, dissolve and dilute with methanol to make a solution containing about 1 μg per 1 ml. Take 0.1, 0.2, 0.5, 0.8, 1.0, 1.5, and 2.0 ml of this mixed solution, place them in 10 ml volumetric bottles, add methanol to the scale, shake well, and inject samples in sequence according to the chromatographic conditions of detection method 2, and record the chromatographic peak area. Use the peak area as the ordinate (y) and the mass concentration of the reference substance as the abscissa (x) to draw a standard curve and perform linear regression. The regression equation and linear range are shown in Table 5.

[0193] Table 5 Linear relationship investigation results

[0194]

[0195] The results in Table 5 show that the HPLC method of the present invention has good linear relationships for each reference substance within its respective mass concentration range.

[0196] The linear graphs of compounds IM-K+IM-L and IM-3-IM01 are shown in Figure 2 Fig.37 , Fig.38 shown.

[0197] 6. Accuracy

[0198] Taking 0.1 μg / ml as the 100% limit concentration, three different concentrations of 50%, 100%, and 150% were designed, and three test solutions were prepared for each concentration for determination. The accuracy of the method was evaluated using the results of the determination of 9 samples. The accuracy results are shown in Table 6.

[0199] Table 6 Accuracy test results

[0200]

[0201]

[0202] The results show that the recovery rate of the method of the present invention is high. The content determination results of impurities IM-3-IM01, IM-K and IM-L under this method are accurate.

[0203] 7. Precision

[0204] Repeatability:

[0205] Take the mirabegron reference substance, add a certain amount of impurities IM-3-IM01, IM-K, and IM-L; prepare 6 copies in parallel. The repeatability of the method was investigated. The results showed that the RSDs of the determination results of IM-3-IM01 and IM-K+IM-L were 1.56% and 6.19%, respectively.

[0206] Intermediate precision:

[0207] Under the repeatability item, 6 samples were prepared in parallel, and the RSDs of the determination results of IM-3-IM01 and IM-K+IM-L were 4.59% and 10.46%, respectively.

[0208] 8. Solution stability

[0209] The reference solution was placed at room temperature for 8 hours, and the RSD values ​​of the peak areas of IM-3-IM01 and IM-K+IM-L were 7.97% and 3.85%, respectively.

[0210] No deviation or change occurred during the test, and the test results all met the established standard requirements, which proves that the detection method of the present invention is suitable for the quality control of genotoxic impurities in mirabegron finished products.

Claims

1. A method for detecting base toxic impurities in mirabegron, characterized in that: The method comprises the following steps: taking a solution of a mirabegron test sample and a solution of a base toxic impurity standard sample, respectively, and performing separation and detection by high performance liquid chromatography-mass spectrometry, and comparing ion peaks in the obtained mass spectra. If the mass spectra of the solution of the mirabegron test sample have the same quantitative / qualitative ion peaks as those of the solution of the base toxic impurity standard sample, the base toxic impurity exists in the mirabegron test sample; otherwise, the base toxic impurity does not exist; The base toxic impurity standard includes the following compounds IM-1 shown in Formula I, IM-2 shown in Formula II, IM-1-IM01 shown in Formula III, IM-2-IM01 shown in Formula IV, IM-3-IM01 shown in Formula V, IM-K shown in Formula VI, and IM-L shown in Formula VII; When the base toxic impurity standard includes the compound IM-1 shown in Formula I, the compound IM-2 shown in Formula II, the compound IM-1-IM01 shown in Formula III and the compound IM-2-IM01 shown in Formula IV, in the high performance liquid chromatography-mass spectrometry, The detection conditions of HPLC are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile and 0.05% trifluoroacetic acid aqueous solution, volume ratio of 40:60; detection wavelength: 254nm; flow rate: 1.0ml / min; column temperature: 35℃; injection volume: 2μl; acquisition time: 15min; blank solvent: methanol; The detection conditions of mass spectrometry are as follows: Ion source: ESI source; Probe temperature: 600°C; Collection rate: 10 points / second; Gas flow: 20L / min; The mass spectrometry selected ion monitoring parameters of the base toxic impurity standard are as follows: The quantitative / qualitative ion of the compound IM-1 represented by the formula I is 301.01 m / z, positive value, cone voltage is 16 V, and capillary voltage is 0.8 KV; The quantitative / qualitative ion of the compound IM-2 represented by the formula II is 287.07 m / z, positive value, cone voltage is 15 V, and capillary voltage is 0.8 KV; The quantitative / qualitative ion of the compound IM-1-IM01 represented by the formula III is 285.06 m / z, positive value, cone voltage is 15V, and capillary voltage is 0.8 KV; The quantitative / qualitative ion of the compound IM-2-IM01 represented by the formula IV is 271.06 m / z, positive value, cone voltage is 15V, and capillary voltage is 0.8 KV; When the base toxic impurity standard includes the compound IM-3-IM01 shown in formula V, the compound IM-K shown in formula VI and the compound IM-L shown in formula VII, in the high performance liquid chromatography-mass spectrometry, The detection conditions of HPLC are as follows: Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: acetonitrile and 10mM ammonium acetate volume ratio of 30:70, acetic acid adjusted to pH 6.5; detection wavelength: 251nm; flow rate: 0.8ml / min; column temperature: 35℃; injection volume: 10μL; acquisition time: 12min; The detection conditions of mass spectrometry are as follows: Ion source: ESI source; Probe temperature: 600°C; Collection rate: 10 points / second; Gas flow: 20L / min; The mass spectrometry selected ion monitoring parameters of the base toxic impurity standard are as follows: The quantitative / qualifier ion of the compound IM-3-IM01 represented by Formula V is 241.20 m / z, positive value, cone voltage is 20 V, and capillary voltage is 0.8 KV; The quantitative / qualifier ion of the compound IM-K represented by Formula VI is 397.25 m / z, positive value, cone voltage is 20 V, and capillary voltage is 0.8 KV; The quantitative / qualifier ion of the compound IM-L represented by formula VII is 397.25 m / z, positive value, the cone voltage is 20 V, and the capillary voltage is 0.8 KV.

2. The method according to claim 1, characterized in that: The method also includes the step of determining the chromatograms of the solution of the mirabegron test sample and the solution of the base toxic impurity standard by high performance liquid chromatography, and calculating the peak area by an external standard method to quantify the base toxic impurities in the mirabegron test sample.

3. The method according to claim 1, characterized in that: The steps for preparing the mirabegron test solution are as follows: accurately weigh mirabegron, dissolve it in methanol and dilute it to make a solution containing 10 mg per 1 ml; The steps for preparing the solution of the base toxic impurity standard are as follows: accurately weigh IM-1, IM-2, IM-1-IM01, and IM-2-IM01, dissolve them in methanol and dilute them to make a mixed solution containing 0.3 μg of each in each ml.

4. The method according to claim 1, characterized in that: The steps for preparing the mirabegron test solution are as follows: accurately weigh mirabegron, dissolve it in methanol and dilute it to make a solution containing 3 mg per 1 ml; The steps for preparing the solution of the base toxic impurity standard are as follows: accurately weigh IM-3-IM01, IM-K, and IM-L, add methanol to dissolve and dilute to make a mixed solution containing 0.1 μg of each in each 1 ml.

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