Analysis method of related substances of 4-(4-methoxyphenyl)-2-butanone

Abstract

The invention discloses an analysis method for detecting related substances of 4-(4-methoxyphenyl)-2-butanone. The analysis method comprises the following steps: detecting a test solution, a 4-(4-methoxyphenyl)-2-butanone contrast solution and a system applicability solution by adopting a high performance liquid chromatography, and recording chromatograms; according to the system applicability solution, qualitative detection chromatographic peaks of related impurities in the test sample solution are obtained; calculating according to a self-control method to obtain the concentration of each impurity in the test solution; and analyzing the concentration of the test solution to obtain the content of each known impurity in the test solution. The method disclosed by the invention has good specificity, system applicability, sensitivity, linearity, repeatability, stability, accuracy and durability, so that the quality control of the 4-(4-methoxyphenyl)-2-butanone is more comprehensively realized.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and specifically relates to an analysis method for 4-(4-methoxyphenyl)-2-butanone-related substances. Background Art

[0002] At present, the quality control of 4-(4-methoxyphenyl)-2-butanone (structure as shown in Formula I) in traditional chemical enterprises is mostly carried out by GC-FID detection, and some of the potential impurities in the material have a high boiling point and cannot be completely vaporized in the GC injection port, so the impurity control of this product is not perfect, especially 4-(4-methoxyphenyl)-2-butanone is used in the synthesis of dobutamine hydrochloride raw materials, and its quality attributes are crucial to the quality of raw materials. Therefore, considering the safety and effectiveness of the drug, it is necessary to comprehensively control the content of various potential impurities in 4-(4-methoxyphenyl)-2-butanone. The prior art is obviously insufficient in controlling the impurities of 4-(4-methoxyphenyl)-2-butanone, so it is of great practical significance to develop an analytical method that takes into account various potential impurities for the analysis of 4-(4-methoxyphenyl)-2-butanone related substances.

[0003] Summary of the invention

[0004] The purpose of the present invention is to provide an analytical method for related substances of 4-(4-methoxyphenyl)-2-butanone. The method of the present invention has good specificity, system applicability, sensitivity, repeatability, accuracy and durability, thereby more comprehensively realizing the quality control of 4-(4-methoxyphenyl)-2-butanone.

[0005] The method for analyzing related substances of 4-(4-methoxyphenyl)-2-butanone provided by the present invention comprises the following steps:

[0006] (1) Solution preparation

[0007] Take 4-(4-methoxyphenyl)-2-butanone test sample and use diluent as solvent to prepare test sample solution;

[0008] Diluting the test solution with the diluent to prepare a control solution;

[0009] The diluent is used as a solvent to prepare a system suitability solution; the system suitability solution is prepared from an impurity SM2-A standard, an impurity SM2-B standard, an impurity SM2-C standard, an impurity SM2-D standard, an impurity SM2-E standard and a 4-(4-methoxyphenyl)-2-butanone standard;

[0010] (2) Qualitative detection: The system suitability solution, the control solution, and the test solution are respectively detected by high performance liquid chromatography, and the chromatograms are recorded; based on the chromatographic peaks in the chromatograms of the system suitability solution and the control solution, the qualitative detection chromatographic peaks of 4-(4-methoxyphenyl)-2-butanone and its related substances in the test solution are obtained;

[0011] (3) Quantitative detection: The system suitability solution, the control solution and the test solution are respectively detected by high performance liquid chromatography, and the chromatograms are recorded; if there are chromatographic peaks with the same retention time as impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D and / or impurity SM2-E in the obtained chromatogram of the test solution, the content of each impurity in the test solution is calculated according to the peak area.

[0012] The diluent in the above method is a mixed solvent of methanol and water. Specifically, the volume ratio of methanol to water in the mixed solvent can be 1:1.

[0013] In the above analysis method, the chromatographic conditions used in the high performance liquid chromatography method are as follows:

[0014] The detector is an ultraviolet detector or a diode array detector;

[0015] The detection wavelength is 279nm~281nm;

[0016] The stationary phase is octadecylsilane bonded silica gel;

[0017] Mobile phase A is 0.08% to 0.12% phosphoric acid aqueous solution, and mobile phase B is methanol;

[0018] The linear elution program is:

[0019] The initial volume ratio of mobile phase A:B was 65:35, which was maintained for 5 min. From 5 min to 20 min, the volume percentage of mobile phase A decreased from 65% to 30%. From 20 min to 25 min, the volume ratio of mobile phase A:B was maintained at 30:70. From 25 min to 25.1 min, the volume percentage of mobile phase A increased from 30% to 65%. From 25.1 min to 30 min, the volume ratio of mobile phase A:B was maintained at 65:35.

[0020] Flow rate: 0.9 mL / min to 1.1 mL / min;

[0021] The column temperature is 28℃~32℃.

[0022] Injection volume: 10 μl.

[0023] According to an embodiment of the present invention, the specific chromatographic conditions are as follows:

[0024] The detector is an ultraviolet detector;

[0025] The detection wavelength is 280nm;

[0026] The stationary phase is octadecylsilane bonded silica gel;

[0027] Mobile phase A was 0.1% phosphoric acid aqueous solution, mobile phase B was methanol, and the linear elution program was:

[0028] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 5 65 35 20 30 70 25 30 70 25.1 65 35 30 65 35

[0029] The flow rate is 1.0 mL / min;

[0030] The column temperature is 30°C;

[0031] Injection volume: 10 μl.

[0032] In the above analytical method, the separation degree between each known impurity and between the impurity and the main component in the chromatogram of the system suitability solution is greater than 1.5;

[0033] The peak area of ​​4-(4-methoxyphenyl)-2-butanone in the reference solution was measured repeatedly 3 to 6 times, and the RSD was no greater than 2.0%.

[0034] In the above analytical method, the concentration of 4-(4-methoxyphenyl)-2-butanone in the test solution is 1.0 mg to 3.0 mg / mL;

[0035] The preparation method of the test solution is as follows: take an appropriate amount of the test sample, accurately weigh it, add the diluent to dissolve it and quantitatively dilute it to prepare a solution containing 1.0 mg to 3.0 mg (specifically 2.0 mg) of 4-(4-methoxyphenyl)-2-butanone per 1 mL.

[0036] In the above analysis method, the concentration of 4-(4-methoxyphenyl)-2-butanone in the control solution is 2.0 to 100 μg / mL;

[0037] The preparation method of the control solution is as follows: take an appropriate amount of the test solution, add the diluent to dissolve and quantitatively dilute to prepare a solution containing 2.0 to 100 μg (specifically 20.0 μg) of 4-(4-methoxyphenyl)-2-butanone per 1 mL.

[0038] In the above analysis method, in the system suitability solution, the concentrations of impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, impurity SM2-E and 4-(4-methoxyphenyl)-2-butanone are all 1 to 2000 μg / mL.

[0039] The concentrations of impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, and impurity SM2-E are all 3.0 to 9.0 μg / mL, and the concentration of 4-(4-methoxyphenyl)-2-butanone is 1000 to 3000 μg / mL.

[0040] The preparation method of the system suitability solution is as follows: take appropriate amounts of the impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, impurity SM2-E and 4-(4-methoxyphenyl)-2-butanone reference substance, accurately weigh them, add the diluent to dissolve and quantitatively dilute them to prepare a solution containing 3.0 to 9.0 μg of each impurity (specifically 6.0 μg) and 1000 to 3000 μg of 4-(4-methoxyphenyl)-2-butanone (specifically 2000 μg) per 1 mL.

[0041] In the above analysis method, the method for calculating the content of the impurities adopts the self-control method.

[0042] In the above analysis method, step (2) further comprises preparing an impurity localization solution and using the high performance liquid chromatography method to detect so as to exclude interfering peaks of each known impurity;

[0043] The impurity positioning is prepared using impurity SM2-A standard, impurity SM2-B standard, impurity SM2-C standard, impurity SM2-D standard, impurity SM2-E standard and 4-(4-methoxyphenyl)-2-butanone standard, respectively, and the solvent is the diluent;

[0044] The impurity SM2-A has a chemical name of (4-methoxyphenyl)methanol and a molecular formula of C8H 10 O2, the structural formula is shown in Formula I:

[0045]

[0046] The impurity SM2-B, chemical name is 4-(4-hydroxyphenyl)-2-butanol (also known as rhododendron alcohol), molecular formula is C 10 H 14 O2, the structural formula is shown in Formula II:

[0047]

[0048] The SM2-C, chemical name is 4-methoxybenzaldehyde (p-anisaldehyde), molecular formula is C8H8O2, and structural formula is shown in formula III:

[0049]

[0050] The impurity SM2-D has a chemical name of 1-(p-methoxyphenyl)-1-butene-3-one and a molecular formula of C11H 12 O2, the structural formula is shown in Formula IV:

[0051]

[0052] In the present invention, the impurity SM2-E has a chemical name of 4-(4-methoxyphenyl)butane-2-ol and a molecular formula of C 11 H 16 O2, the structural formula is shown in Formula V:

[0053]

[0054] The present invention has the following beneficial effects:

[0055] The present invention makes up for the lack of HPLC detection technology for related substances of 4-(4-methoxyphenyl)-2-butanone. The present invention explores and optimizes the chromatographic conditions of high-performance liquid phase, and realizes the qualitative and quantitative detection of 5 potential impurities; and the analytical method of the present invention has good specificity and high sensitivity (in specific experiments, the concentrations of SM2-A, SM2-B, SM2-C, SM2-D and SM2-E quantitative limit solutions are 0.63 μg / mL, 0.62 μg / mL, 0.061 μg / mL, 0.18 μg / mL and 0.70 μg / mL, respectively, and the signal-to-noise ratio is not less than 10, and the peak area RSDs of repeated injections for 6 times are 2.6%, 2.2%, 1.8%, and 0.70 μg / mL, respectively). 1.2% and 2.2%, less than 10.0%; the concentrations of the detection limit solutions are 0.25μg / mL, 0.25μg / mL, 0.024μg / mL, 0.070μg / mL and 0.28μg / mL, respectively, and the signal-to-noise ratio is not less than 3), it has repeatability, high accuracy (9 accuracy solutions at 3 concentration levels are used in the specific experiment, the recoveries are all between 85% and 110%, and the RSD values ​​are all less than 15%), good precision, and good durability (it is applicable to a wide range of chromatographic conditions and is stable under room temperature conditions for 60 hours); the analytical method of the invention is of great significance to the quality control of 4-(4-methoxyphenyl)-2-butanone and the key quality attributes of the starting materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 HPLC spectrum of blank solvent in the system suitability and specificity experiment in Example 1 of the present invention;

[0057] Figure 2 This is the HPLC spectrum for localizing impurity SM2-A in the system suitability and specificity experiments in Example 1 of the present invention;

[0058] Figure 3 This is the HPLC spectrum for localizing impurity SM2-B in the system suitability and specificity experiments in Example 1 of the present invention;

[0059] Figure 4 This is the HPLC spectrum for localizing impurity SM2-C in the system suitability and specificity experiments in Example 1 of the present invention;

[0060] Figure 5 This is the HPLC spectrum for localizing impurity SM2-D in the system suitability and specificity experiments in Example 1 of the present invention;

[0061] Figure 6 This is the HPLC spectrum for localizing impurity SM2-E in the system suitability and specificity experiments in Example 1 of the present invention;

[0062] Figure 7 HPLC spectrum of the system suitability solution in the system suitability and specificity experiment in Example 1 of the present invention;

[0063] Figure 8 HPLC spectrum of the sensitivity solution in the system suitability and specificity experiment in Example 1 of the present invention;

[0064] Fig. 9 The HPLC spectrum of the impurity quantitative limit solution in the sensitivity experiment in Example 2 of the present invention;

[0065] Fig.10 The HPLC spectrum of the impurity detection limit solution in the sensitivity experiment in Example 2 of the present invention;

[0066] Fig.11 It is the linear relationship diagram of SM2-A in Example 3 of the present invention;

[0067] Fig.12 This is a linear relationship diagram of SM2-B in Example 3 of the present invention;

[0068] Fig.13 It is a linear relationship diagram of SM2-C in Example 3 of the present invention;

[0069] Fig.14 SM2-D linear relationship diagram in Example 3 of the present invention;

[0070] Fig.15 It is the linear relationship diagram of SM2-E in Example 3 of the present invention;

[0071] Fig.16 This is a linear relationship diagram of 4-(4-methoxyphenyl)-2-butanone in Example 3 of the present invention;

[0072] Fig.17 HPLC spectrum of system suitability under the mobile phase ratio (67:33) of Example 6 of the present invention;

[0073] Fig.18HPLC spectrum of system suitability under the mobile phase ratio (63:77) of Example 6 of the present invention;

[0074] Fig.19 HPLC spectrum of system suitability under the flow rate (1.1 mL / min) condition of Example 6 of the present invention;

[0075] Fig. 20 HPLC spectrum of system suitability under the flow rate (0.9 mL / min) condition of Example 6 of the present invention;

[0076] Fig.21 HPLC spectrum of system suitability under column temperature (32° C.) conditions of Example 6 of the present invention;

[0077] Fig. 22 HPLC spectrum of system suitability under flow rate (28° C.) conditions of Example 6 of the present invention;

[0078] Fig.23 This is the HPLC spectrum of system suitability when the phosphoric acid content in mobile phase A is 0.08% in Example 6 of the present invention;

[0079] Fig.24 This is the HPLC spectrum of system suitability when the phosphoric acid content in mobile phase A is 0.12% in Example 6 of the present invention;

[0080] Fig.25 The HPLC spectrum of system suitability under chromatographic columns of the same brand but different batch numbers in Example 6 of the present invention. DETAILED DESCRIPTION

[0081] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0082] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0083] Impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, impurity SM2-E and 4-(4-methoxyphenyl)-2-butanone used in the following examples were purchased from:

[0084]

[0085] The structural formula of the impurity SM2-A [(4-methoxyphenyl)methanol] is shown in Formula I:

[0086]

[0087] The structural formula of the impurity SM2-B [4-(4-hydroxyphenyl)-2-butanol] is shown in Formula II:

[0088]

[0089] The structural formula of the impurity SM2-C [4-methoxybenzaldehyde] is shown in Formula III:

[0090]

[0091] The structural formula of the impurity SM2-D (1-(p-methoxyphenyl)-1-butene-3-one) is shown in Formula IV:

[0092]

[0093] The structural formula of the impurity SM2-E (4-(4-methoxyphenyl)butane-2-ol) is shown in Formula V:

[0094]

[0095] Example 1. System Applicability and Specificity

[0096] (1) Instruments and conditions:

[0097] High performance liquid chromatograph: Waters e2695 liquid chromatograph

[0098] Detector: UV detector

[0099] Detection wavelength: 280nm

[0100] Chromatographic column: Octadecylsilane bonded silica gel column (Supelcosil LC 18-DB, 150×4.6mm 5μm)

[0101] Column temperature: 30°C

[0102] Mobile phase A: 0.1% phosphoric acid aqueous solution (v / v)

[0103] Mobile phase B: methanol

[0104] The linear gradient elution program was:

[0105] Time (minutes) Mobile phase A (%, v / v) Mobile phase B (%, v / v) 0 65 35 5 65 35 20 30 70 25 30 70 25.1 65 35 30 65 35

[0106] Flow rate: 1.0mL / min

[0107] Injection volume: 10 μL

[0108] (2) Preparation of solution:

[0109] Diluent: methanol:water (1:1, v / v).

[0110] System suitability solution: Accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 60 mg of impurity SM2-E respectively, place them in the same 100 mL volumetric flask, add methanol to sonicate to dissolve and dilute to volume with methanol as the impurity stock solution; accurately weigh 100 mg of 4-(4-methoxyphenyl)-2-butanone (reference substance), place it in a 50 mL volumetric flask, add 0.5 mL of the impurity stock solution, add 25 mL of methanol to dissolve and dilute to volume with water.

[0111] Sensitivity solution (0.05%): Accurately weigh 100 mg of 4-(4-methoxyphenyl)-2-butanone (reference substance), place in a 50 mL volumetric flask, dilute to volume with methanol, accurately measure 1.0 mL, place in a 100 mL volumetric flask, dilute to volume with diluent, then transfer 1.0 mL to a 20 mL volumetric flask, and dilute to volume with diluent.

[0112] Test solution: Take 100 mg of the product (4-(4-methoxyphenyl)-2-butanone sample), accurately weigh it, place it in a 50 mL volumetric flask, add diluent to dissolve it and dilute it to volume.

[0113] Control solution: Accurately measure 1.0 mL of the test solution, place it in a 100 mL volumetric flask, and dilute to volume with diluent.

[0114] SM2-A localization solution: Accurately weigh 60 mg of impurity SM2-A, add diluent to dissolve and dilute to make a solution containing 6 μg of SM2-A per mL.

[0115] SM2-B localization solution: Accurately weigh 60 mg of impurity SM2-B, add diluent to dissolve and dilute to make a solution containing 6 μg of SM2-B per mL.

[0116] SM2-C localization solution: Accurately weigh 60 mg of impurity SM2-C, add diluent to dissolve and dilute to make a solution containing 6 μg of SM2-C per mL.

[0117] SM2-D localization solution: Accurately weigh 60 mg of impurity SM2-D, add diluent to dissolve and dilute to make a solution containing 6 μg of SM2-D per mL.

[0118] SM2-E localization solution: Accurately weigh 60 mg of impurity SM2-E, add diluent to dissolve and dilute to make a solution containing 6 μg of SM2-E per mL.

[0119] 4-(4-methoxyphenyl)-2-butanone localizing solution: Accurately weigh 100 mg of 4-(4-methoxyphenyl)-2-butanone (reference substance), dissolve it in a diluent and dilute it to make a solution containing 2000 μg of 4-(4-methoxyphenyl)-2-butanone per mL.

[0120] (3) Detection:

[0121] Take 10 μL of the diluent, system suitability solution, each impurity and 4-(4-methoxyphenyl)-2-butanone positioning solution respectively and inject them into the liquid chromatograph to record the chromatogram.

[0122] The results are shown in Table 1. The color spectrum is shown in Figures 1 to 8 shown.

[0123] Table 1 Example 1 Results

[0124]

[0125]

[0126] Example 2: Sensitivity experiment

[0127] (1) Instruments and conditions:

[0128] Same as Example 1.

[0129] (2) Preparation of solution:

[0130] Diluent: methanol:water (1:1, v / v).

[0131] Quantitation limit solution: Take an appropriate amount of each positioning solution and dilute it with a diluent to a solution of corresponding concentration with a signal-to-noise ratio of about 10:1, which is used as the quantitation limit solution.

[0132] Detection limit solution: Take an appropriate amount of the quantitative limit solution and dilute it with a diluent to a solution of corresponding concentration with a signal-to-noise ratio of about 3:1, which is used as the detection limit solution.

[0133] (3) Implementation:

[0134] Take 10 μL of the detection limit solution and the quantification limit solution respectively and inject them into the liquid chromatograph, record the chromatogram, and inject the quantification limit solution continuously for 6 times.

[0135] The concentrations of the quantification limit solutions of SM2-A, SM2-B, SM2-C, SM2-D and SM2-E were 0.63μg / mL, 0.62μg / mL, 0.061μg / mL, 0.18μg / mL and 0.70μg / mL (not more than 0.05% of the test sample concentration), the signal-to-noise ratio was not less than 10, and the peak area RSD of 6 repeated injections was not more than 10.0%; the concentrations of the detection limit solutions of SM2-A, SM2-B, SM2-C, SM2-D and SM2-E were 0.25μg / mL, 0.25μg / mL, 0.024μg / mL, 0.070μg / mL and 0.28μg / mL, and the signal-to-noise ratio was not less than 3. The results showed that the analytical method for detecting related substances of 4-(4-methoxyphenyl)-2-butanone has sufficient sensitivity. The quantitative limit is as follows Fig. 9 As shown, the detection limit is Fig.10 shown.

[0136] Example 3: Linearity and range

[0137] (1) Instruments and conditions:

[0138] Same as Example 1.

[0139] (2) Preparation of solution:

[0140] Diluent: methanol:water (1:1, v / v).

[0141] Reference substance linear stock solution: accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D, 60 mg of impurity SM2-E and 60 mg of 4-(4-methoxyphenyl)-2-butanone respectively, place them in the same 100 mL volumetric flask, add 20 mL of methanol to dissolve by ultrasound and dilute to volume with methanol as the reference substance reserve solution; accurately measure 5.0 mL of the reference substance reserve solution, place it in a 50 mL volumetric flask, add diluent to dilute to volume as the reference substance linear stock solution.

[0142] Linear solution: Accurately measure 1.0mL, 0.5mL, 1.0mL, 1.5mL and 2.0mL of the reference linear stock solution and place them in 50mL, 10mL, 10mL, 10mL and 10mL volumetric flasks respectively. Dilute to the scale with diluent and shake well. The obtained solution concentrations are 1.2μg / mL, 3.0μg / mL, 6.0μg / mL, 9.0μg / mL and 12.0μg / mL respectively.

[0143] (3) Implementation:

[0144] Take 10 μL of the linear solution and inject it into the liquid chromatograph to record the chromatogram.

[0145] A linear regression was performed using peak area (Y) versus concentration (X). The linear regression equation for impurity SM2-A was Y=5087.5781X+226.5611. The correlation coefficient (r) was 0.9999, which was greater than 0.990. The Y-axis intercept accounted for 0.7% of the 100% response value, which was within 25%.

[0146] The linear regression equation of impurity SM2-B is Y=5040.0266X+792.2823, the correlation coefficient (r) is 0.9993, which is greater than 0.990, and the Y-axis intercept accounts for 2.7% of the 100% response value, which is within 25%;

[0147] The linear regression equation for impurity SM2-C is Y=70714.0154X+3001.8250, the correlation coefficient (r) is 0.9999, which is greater than 0.990, and the Y-axis intercept accounts for 0.7% of the 100% response value, which is within 25%;

[0148] The linear regression equation for impurity SM2-D is Y=29701.3904X+540.4495, the correlation coefficient (r) is 0.9999, which is greater than 0.990, and the Y-intercept accounts for 0.3% of the 100% response value, which is within 25%;

[0149] The linear regression equation for impurity SM2-E is Y=4942.3706X+41.2286, the correlation coefficient (r) is 0.9999, which is greater than 0.990, and the Y-axis intercept accounts for 0.1% of the 100% response value, which is within 25%;

[0150] This indicates that the five impurities in this analytical method have a good linear relationship within the range of LOQ to 150%. Figures 11 to 15 shown.

[0151] The linear regression equation for 4-(4-methoxyphenyl)-2-butanone was Y=17236.7763X+712.0187, the correlation coefficient (r) was 0.9999, which was greater than 0.999, and the Y-intercept accounted for 1.3% of the 100% response value, which was within 25%;

[0152] This indicates that the analytical method for 4-(4-methoxyphenyl)-2-butanone has a good linear relationship within the range of LOQ to 150%.

[0153] The linear relationship diagram of 4-(4-methoxyphenyl)-2-butanone is as follows Fig.16 shown.

[0154] Example 4: Accuracy Experiment

[0155] (1) Instruments and conditions:

[0156] Same as Example 1.

[0157] (2) Preparation of solution:

[0158] Diluent: methanol:water (1:1, v / v).

[0159] Reference substance spiked stock solution: accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 100 mg of impurity SM2-E respectively and place them in the same 100 mL volumetric flask, add 20 mL of methanol by ultrasonic dissolution and dilute to volume with methanol as the reference substance stock solution; 5.0 mL of the reference substance stock solution was placed in the same 20 mL volumetric flask, diluted to volume with diluent as the reference substance spiked stock solution.

[0160] Test solution: Prepare according to Table 2 below:

[0161] Table 2 Preparation of test solution

[0162]

[0163] (3) Implementation:

[0164] Take 10 μL of each spiked test solution and inject it into the liquid chromatograph, record the chromatogram, and calculate the content of each impurity by peak area according to the self-control method.

[0165] The calculation formula of the self-control method is as follows:

[0166]

[0167] A i : Peak area of ​​impurities in the test solution;

[0168] C d : Control solution concentration;

[0169] A d : Main peak area in control solution;

[0170] C a : Concentration of test solution.

[0171] The recoveries of the nine accuracy solutions at three concentration levels were all between 85% and 110%, and the RSD values ​​were all less than 10%, as shown in Table 3. This indicates that the analytical method is accurate and feasible for detecting the five impurities.

[0172] Table 3 Example 4 Results - Accuracy

[0173]

[0174]

[0175] Example 5: Precision Experiment

[0176] (1) Instruments and conditions:

[0177] Same as Example 1.

[0178] (2) Preparation of solution:

[0179] Diluent: methanol:water (1:1, v / v).

[0180] Reference substance spiked stock solution: accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 100 mg of impurity SM2-E respectively and place them in the same 100 mL volumetric flask, add 20 mL of methanol by ultrasonic dissolution and dilute to volume with methanol as the reference substance stock solution; 5.0 mL of the reference substance stock solution was placed in the same 20 mL volumetric flask, diluted to volume with diluent as the reference substance spiked stock solution.

[0181] 100% spiked test solution: Take about 100 mg of the test sample, weigh accurately, and place in a 50 mL volumetric flask. Accurately measure 2.0 mL of the reference spiked stock solution, place in the same volumetric flask, add diluent to dissolve to the mark, and shake well. Prepare 6 portions in parallel.

[0182] Control solution: Accurately measure 1.0 mL of 100% spiked test solution, place it in a 100 mL volumetric flask, dilute to volume with diluent, and shake well.

[0183] (3) Implementation:

[0184] The above tests were conducted by different analysts at different times using different instruments as Experiment 1 (repeatability) and Experiment 2 (intermediate precision). 10 μL of the control solution and 100% spiked test solution were injected into the liquid chromatograph and the chromatogram was recorded. The content of each impurity was calculated by peak area according to the self-control method.

[0185] The results are shown in Table 4.

[0186] Table 4 Example 5 Results - Precision

[0187]

[0188] From the above experimental results, it can be seen that the average contents of SM2-A, SM2-B, SM2-C, SM2-D, SM2-E, RRT0.79 impurities and RRT1.32 impurities in 12 spiked test solutions in Experiment 1 and Experiment 2 are 0.27%, 0.36%, 0.30%, 0.24%, 0.75%, 0.06% and 0.09% respectively, and the RSD is no more than 30%. It shows that the repeatability (measurement results of the same analyst) and intermediate precision (measurement results of changing conditions in the same laboratory) of the related substance analysis method meet the detection requirements and have good precision.

[0189] Example 6. Durability - Chromatographic Condition Variation

[0190] (1) Instruments and conditions:

[0191] The column temperature, initial ratio of mobile phase, flow rate, concentration of phosphoric acid solution (mobile phase A), and different batches of chromatographic columns were changed, and the other conditions were the same as in Example 1.

[0192] (2) Preparation of solution:

[0193] Diluent: methanol:water (1:1, v / v).

[0194] System suitability solution: Accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 60 mg of impurity SM2-E respectively, place them in the same 100 mL volumetric flask, add methanol to sonicate to dissolve and dilute to volume with methanol as the impurity stock solution; accurately weigh 100 mg of 4-(4-methoxyphenyl)-2-butanone (reference substance), place it in a 50 mL volumetric flask, add 0.5 mL of the impurity stock solution, add 25 mL of methanol to dissolve and dilute to volume with water.

[0195] Reference substance spiked stock solution: accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 100 mg of impurity SM2-E respectively and place them in the same 100 mL volumetric flask, add 20 mL of methanol to dissolve by ultrasonication and dilute to volume with methanol as the reference substance stock solution; 5.0 mL of the reference substance stock solution was placed in the same 20 mL volumetric flask, diluted to volume with diluent as the reference substance spiked stock solution.

[0196] 100% spiked test sample solution: Take about 100 mg of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Accurately measure 2.0 mL of the reference sample spiked stock solution, place it in the same volumetric flask, add diluent to dissolve it to the scale, and shake well.

[0197] Control solution: Accurately measure 1.0 mL of 100% spiked test solution, place it in a 100 mL volumetric flask, dilute to volume with diluent, and shake well.

[0198] (3) Implementation:

[0199] On the basis of the established chromatographic conditions (same as in Example 1), a small change was made to the chromatographic system to examine the tolerance level without affecting the measurement results.

[0200] Take 10 μL of diluent and system suitability solution respectively, inject into liquid chromatograph, record chromatograms using established chromatographic conditions and changed chromatographic conditions respectively; take 10 μL of 100% spiked test solution and inject into liquid chromatograph, record chromatograms. Calculate the content of each impurity by peak area according to the self-control method.

[0201] The results are shown in Table 5. The system suitability under different chromatographic conditions is as follows Figures 17 to 25 shown.

[0202] Table 5 Example 6 Results

[0203]

[0204]

[0205] The results show that when there are slight changes in column temperature, initial proportion of mobile phase, flow rate, concentration of phosphoric acid solution, and chromatographic columns of different batches, the separation between impurities and between impurities and the main peak is greater than 1.5. For 100% spiked test solution, RRT0.79 and RRT1.32 (≤0.20% impurities), the change values ​​of the results under different durability conditions relative to the standard chromatographic conditions are no more than ±0.05% (absolute difference); for the five impurities SM2-A, SM2-B, SM2-C, SM2-D, and SM2-E (addition level is 0.5%), the change values ​​of the results under different durability conditions relative to the standard chromatographic conditions are no more than ±20% (relative difference). Slight changes in chromatographic conditions can still meet the detection requirements, and the method is robust and reliable.

[0206] Example 7. Durability-Solution Stability

[0207] (1) Instruments and conditions:

[0208] Same as Example 1.

[0209] (2) Preparation of solution:

[0210] Diluent: methanol:water (1:1, v / v).

[0211] System suitability solution: Accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 60 mg of impurity SM2-E respectively, place them in the same 100 mL volumetric flask, add methanol to sonicate to dissolve and dilute to volume with methanol as the impurity stock solution; accurately weigh 100 mg of 4-(4-methoxyphenyl)-2-butanone, place it in a 50 mL volumetric flask, add 0.5 mL of the impurity stock solution, add 25 mL of methanol to dissolve and dilute to volume with water.

[0212] Reference substance spiked stock solution: accurately weigh 60 mg of impurity SM2-A, 60 mg of impurity SM2-B, 60 mg of impurity SM2-C, 60 mg of impurity SM2-D and 100 mg of impurity SM2-E respectively and place them in the same 100 mL volumetric flask, add 20 mL of methanol to dissolve by ultrasonication and dilute to volume with methanol as the reference substance stock solution; 5.0 mL of the reference substance stock solution was placed in the same 20 mL volumetric flask, diluted to volume with diluent as the reference substance spiked stock solution.

[0213] 100% spiked test sample solution: Take about 100 mg of the test sample, accurately weigh it, and place it in a 100 mL volumetric flask. Accurately measure 2.0 mL of the reference stock solution and place it in the same volumetric flask. Add diluent to dissolve it to the scale and shake well.

[0214] Control solution: Accurately measure 1.0 mL of 100% spiked test solution, place it in a 100 mL volumetric flask, dilute to volume with diluent, and shake well.

[0215] Test sample solution: Take about 100 mg of the test sample, weigh accurately, place in a 100 mL volumetric flask, add diluent to dissolve to the scale, and shake well.

[0216] (3) Implementation:

[0217] The control solution and the 100% spiked test solution were placed at room temperature for 15 hours, 39 hours, and 60 hours, and 10 μL of each solution was accurately measured and injected into the liquid chromatograph to record the chromatogram. The content of each impurity was calculated by the peak area according to the self-control method.

[0218] The results are shown in Table 6.

[0219] Table 6 Example 7 Results

[0220]

[0221] The results showed that the control solution and the test solution were stable within 60 hours at room temperature, and the method was robust and reliable.

[0222] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A method for analyzing related substances of 4-(4-methoxyphenyl)-2-butanone, comprising the following steps: (1) Solution preparation Take 4-(4-methoxyphenyl)-2-butanone as a test sample and use the diluent as a solvent to prepare a test sample solution; Diluting the test solution with the diluent to prepare a control solution; The diluent is used as a solvent to prepare a system suitability solution; the system suitability solution is prepared from an impurity SM2-A standard, an impurity SM2-B standard, an impurity SM2-C standard, an impurity SM2-D standard, an impurity SM2-E standard and a 4-(4-methoxyphenyl)-2-butanone standard; The structural formulas of the impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, and impurity SM2-E are as follows: (2) Qualitative detection: The system suitability solution, the control solution, and the test solution are respectively detected by high performance liquid chromatography, and the chromatograms are recorded; based on the chromatographic peaks in the chromatograms of the system suitability solution and the control solution, the qualitative detection chromatographic peaks of 4-(4-methoxyphenyl)-2-butanone and its related substances in the test solution are obtained; (3) Quantitative detection: The system suitability solution, the control solution and the test solution are respectively detected by high performance liquid chromatography, and the chromatograms are recorded; if there are chromatographic peaks in the chromatogram of the test solution with the same retention time as impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D and / or impurity SM2-E, the content of each impurity in the test solution is calculated according to the peak area; The diluent is a mixed solvent of methanol and water; The chromatographic conditions adopted by the high performance liquid chromatography are as follows: The detector is an ultraviolet detector or a diode array detector; The detection wavelength is 279nm~281nm; The stationary phase is octadecylsilane bonded silica gel; Mobile phase A is 0.08% to 0.12% phosphoric acid aqueous solution, and mobile phase B is methanol; The linear elution program was as follows: the initial volume ratio of mobile phase A:B was 65:35, maintained for 5 min, and from 5 min to 20 min, the volume percentage of mobile phase A decreased from 65% to 30%; from 20 min to 25 min, the volume ratio of mobile phase A:B was maintained at 30:70, and from 25 min to 25.1 min, the volume percentage of mobile phase A increased from 30% to 65%; from 25.1 min to 30 min, the volume ratio of mobile phase A:B was maintained at 65:

35.

2. The analysis method according to claim 1, characterized in that: The detection wavelength is 280 nm; the mobile phase A is a 0.1% by volume phosphoric acid aqueous solution.

3. The analysis method according to claim 1 or 2, characterized in that: The chromatographic conditions include a flow rate of 0.9 mL / min to 1.1 mL / min and a column temperature of 28° C. to 32° C.

4. The analysis method according to claim 3, characterized in that: The chromatographic conditions include a flow rate of 1.0 mL / min and a column temperature of 30°C.

5. The analysis method according to any one of claims 1 to 4, characterized in that: The injection volume of each solution in the chromatographic conditions was 10 μl.

6. The analysis method according to any one of claims 1 to 5, characterized in that: In the diluent, the volume ratio of methanol to water is 1:

1.

7. The analysis method according to any one of claims 1 to 6, characterized in that: In the test solution, the concentration of 4-(4-methoxyphenyl)-2-butanone is 1.0 mg to 3.0 mg / mL; and / or, in the control solution, the concentration of 4-(4-methoxyphenyl)-2-butanone is 2 to 100 μg / mL; and / or, in the system suitability solution, the concentrations of impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, impurity SM2-E and 4-(4-methoxyphenyl)-2-butanone are all 1 to 2000 μg / mL; The concentrations of impurity SM2-A, impurity SM2-B, impurity SM2-C, impurity SM2-D, and impurity SM2-E are all 3.0 to 9.0 μg / mL, and the concentration of 4-(4-methoxyphenyl)-2-butanone is 1000 to 3000 μg / mL.

8. The analysis method according to any one of claims 1 to 7, characterized in that: In the quantitative detection step, the impurity content is calculated by a self-control method.

9. The analysis method according to any one of claims 1 to 8, characterized in that: The analysis method further comprises preparing an impurity localization solution and using the high performance liquid chromatography method for detection to eliminate the interference peaks of the gradient change on the impurities and 4-(4-methoxyphenyl)-2-butanone.

10. The analysis method according to claim 9, characterized in that: The impurity localization solution is prepared using impurity SM2-A standard, impurity SM2-B standard, impurity SM2-C standard, impurity SM2-D standard, impurity SM2-E standard and 4-(4-methoxyphenyl)-2-butanone standard, and the solvent is the diluent.

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