A method for separating and detecting related substances in umeclidinium bromide starting material benzyl-2-bromoethyl ether based on gas chromatography and liquid chromatography

Abstract

The invention discloses a method for separating and detecting related substances in umeclidinium bromide starting material benzyl-2-bromoethyl ether based on gas chromatography and liquid chromatography, including gas chromatography and liquid chromatography detection, and the test sample of liquid chromatography detection is prepared by dilution using gas chromatography detection test sample. The present invention respectively realizes the simultaneous detection of dichloromethane, impurity iii-1, impurity iii-7, impurity iii-9, impurity iii-13 and impurity iii-15 in umeclidinium bromide starting material (benzyl-2-bromoethyl ether), and impurity iii-2, impurity iii-4, impurity iii-5, impurity iii-8, impurity iii-10, impurity iii-11 and impurity iii-12, and confirms that the present method is completely separated from impurities by method verification test, has strong specificity, high sensitivity, good linear relationship of each impurity, high precision, high accuracy and good durability, and can effectively control the quality of product.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for separating and detecting related substances in benzyl-2-bromoethyl ether, a starting material of umeclidinium bromide, based on gas chromatography and liquid chromatography. Background Art

[0002] The synthetic route of umeclidinium bromide starting material (benzyl-2-bromoethyl ether) is as follows,

[0003]

[0004] Combined with the impurity detection of the purchased materials, the impurity spectrum of benzyl-2-bromoethyl ether was determined as follows:

[0005]

[0006]

[0007] Impurity ⅲ-5 is a process impurity generated by the reaction of the raw material benzyl chloride with N-bromosuccinimide (NBS);

[0008]

[0009] Impurity ⅲ-7 may be an impurity produced by the bromination reaction of ethylene glycol, the raw material for producing this product;

[0010]

[0011] Impurity ⅲ-8 may be produced by the reaction of ethylene glycol with benzyl chloride or by the reaction of the intermediate 2-benzyloxyethanol with benzyl chloride;

[0012]

[0013] Impurity ⅲ-9 may be produced by the bromination reaction of diethylene glycol contained in the raw material ethylene glycol, or by the reaction of impurity ⅲ-13 and impurity ⅲ-7;

[0014]

[0015] Impurity iii-10 is an impurity generated by the reaction of the intermediate 2-benzyloxyethanol with N-chlorosuccinimide (NCS) which may be contained in the starting material N-bromosuccinimide (NBS);

[0016]

[0017] Impurity ⅲ-11 is produced by hydrolysis of the raw material benzyl chloride used to produce this product under alkaline conditions;

[0018]

[0019] Impurity ⅲ-12 is a reaction byproduct of the raw material triphenylphosphine;

[0020]

[0021] Impurity ⅲ-13 is an impurity produced by the bromination reaction of ethylene glycol, the raw material for producing this product;

[0022]

[0023] Impurity ⅲ-15 may be produced by the reaction of impurity ⅲ-7 with residual ethanol in the raw material ethylene glycol, or by the reaction of impurity ⅲ-13 with impurity ⅲ-16.

[0024]

[0025] After searching the literature and patents, no literature or patents on separating impurities in benzyl-2-bromoethyl ether were found, and there is a technical gap. Analysis of the impurity structure shows that impurity ⅲ-4, impurity ⅲ-11 and impurity ⅲ-12 have poor stability under gas chromatography conditions; dichloromethane, impurity ⅲ-1, impurity ⅲ-7, impurity ⅲ-9, impurity ⅲ-13 and impurity ⅲ-15 have no ultraviolet absorption and cannot be detected by conventional liquid chromatography; since impurity ⅲ-2 and impurity ⅲ-10 have similar polarities, it is predicted that the two impurities will be poorly separated, and impurity ⅲ-5 and benzyl-2-bromoethyl ether have similar structures and are poorly separated, making it impossible to effectively monitor benzyl-2-bromoethyl ether. Summary of the invention

[0026] In order to solve the above problems in the prior art, the present invention provides a method for separating and detecting related substances in benzyl-2-bromoethyl ether, a starting material of umeclidinium bromide, based on gas chromatography and liquid chromatography, which can realize accurate detection of impurities in benzyl-2-bromoethyl ether. The technical solution of the present invention is as follows:

[0027] It includes two modules: gas chromatography detection and liquid chromatography detection;

[0028] The sample preparation method is also included:

[0029] Take an appropriate amount of this product, dissolve it in acetonitrile and quantitatively dilute it to make a solution with a concentration of 25-100 mg / ml, which is used as the test solution for gas chromatography detection; the concentration of the test solution is more preferably 50 mg / ml;

[0030] Dilute the sample solution for gas chromatography 100 times as the sample solution for liquid chromatography.

[0031] Further, the gas chromatography detection chromatographic conditions are:

[0032] The chromatographic column is a capillary column with 100% polyethylene glycol or a filler with similar polarity as the stationary liquid; the starting temperature is 35-45°C, the injection port temperature is 225-235°C; the detector temperature is 225-235°C; and the carrier gas flow rate is 1.4-1.6 ml per minute.

[0033] Preferably, in gas chromatography detection, the chromatographic column is Agilent DB-WAX, 30m×0.320mm×0.50μm.

[0034] Preferably, in the gas chromatography detection, the starting temperature is 40°C, maintained for 5 minutes, raised to 150°C at a rate of 5°C per minute, and then raised to 230°C at a rate of 50°C per minute, and maintained for 20 minutes;

[0035] Preferably, in gas chromatography detection, the split ratio is 10:1; and the injection volume is 1.0 μl.

[0036] Preferably, the gas chromatography detection also includes sample injection detection and result calculation:

[0037] Accurately measure the test solution and the control solution, inject them into the gas chromatograph respectively, and record the chromatogram;

[0038] If there are impurity peaks in the chromatogram of the test solution, each impurity is calculated using the principal component self-reference method with the addition of a correction factor.

[0039] Calculation formula:

[0040]

[0041] Where: A 杂质 is the peak area of ​​the known impurities in the test solution;

[0042] A 对照 is the main peak area in the chromatogram of the reference solution;

[0043] f is the correction factor of the known impurities, where the relative retention time of dichloromethane is 0.22, the correction factor is 3.9, the relative retention time of impurity iii-7 is 0.56, the correction factor is 3.7, and the relative retention time of impurity iii-13 is 0.78, and the correction factor is 2.9.

[0044] Furthermore, the chromatographic conditions for liquid chromatography detection are: the chromatographic column uses octadecylsilane bonded silica gel as a filler; 18-22 mmol / L phosphate buffer is used as mobile phase A, acetonitrile is used as mobile phase B, the flow rate is 0.9-1.1 ml per minute, and the gradient elution program is:

[0045] Within 0 to 5 minutes, the volume percentage of mobile phase A is 78 to 82%. Within 5 to 45 minutes, the volume percentage of mobile phase A linearly changes from 78 to 82% to 20%. Within 45 to 50 minutes, the volume percentage of mobile phase A linearly changes from 20% to 78 to 82%. After 50 minutes, the volume percentage of mobile phase A remains unchanged at 78 to 82%.

[0046] Preferably, in the liquid chromatography detection, the chromatographic column is Waters C18, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance; the column temperature is 40~50℃.

[0047] Preferably, in liquid chromatography detection, the pH value of the phosphate buffer is 2.9 to 3.1.

[0048] Furthermore, 20mmol / L phosphate buffer was used as the mobile phase, and the preparation method was as follows: 2.4g of anhydrous sodium dihydrogen phosphate was taken, 1000ml of water was added to dissolve it, and the pH value was adjusted to 3.0 with phosphoric acid.

[0049] Preferably, in liquid chromatography detection, the column temperature is 45° C.; the detection wavelength is 210 nm; and the injection volume is 20 μl.

[0050] Preferably, in liquid chromatography detection, the injector temperature is 5°C.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The present invention realizes the simultaneous detection of dichloromethane, impurity iii-1, impurity iii-7, impurity iii-9, impurity iii-13 and impurity iii-15, as well as impurity iii-2, impurity iii-4, impurity iii-5, impurity iii-8, impurity iii-10, impurity iii-11 and impurity iii-12 in benzyl-2-bromoethyl ether, a starting material of umeclidinium bromide, based on gas chromatography and liquid chromatography detection methods. The method validation test confirms that the method has complete impurity separation, strong specificity, high sensitivity, good linear relationship of each impurity, high precision, high accuracy and good durability, and can effectively control the quality of the product;

[0053] 2. In the present invention, the gas phase detection sample is further diluted for liquid chromatography detection, and all impurities can be detected simultaneously in one test, which improves the detection efficiency and reduces the detection workload;

[0054] 3. The present invention uses liquid chromatography for detection and controls the temperature of the sample injector to be 5°C, thereby solving the problem of poor stability of impurities ⅲ-4, ⅲ-11 and ⅲ-12 under gas chromatography conditions;

[0055] 4. The present invention adopts gas chromatography to solve the problem that impurities ⅲ-1, ⅲ-7, ⅲ-9, ⅲ-13 and ⅲ-15 have no ultraviolet absorption and cannot be controlled by liquid chromatography, and controls them together with the residual solvent dichloromethane, effectively solving the detection problems of 5 impurities and 1 residual solvent, saving costs;

[0056] 5. The present invention solves the problem of poor separation of impurity ⅲ-13, impurity ⅲ-15 and impurity ⅲ-7 by screening chromatographic columns and optimizing gas chromatography conditions;

[0057] 6. The detection method of the present invention adopts dual mobile phases and more than three gradient elution procedures to solve the problem of poor separation between ⅲ-2 and impurity ⅲ-10, and poor separation between impurity ⅲ-5 and benzyl-2-bromoethyl ether;

[0058] 7. The present invention provides a detection scheme for impurities that have long lacked effective detection means, making up for the deficiency that the industry cannot conduct more comprehensive quality control on benzyl-2-bromoethyl ether. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0060] Figure 1 This is the chromatogram of the specificity test in 1.2 in Example 2.

[0061] Figure 2 It is the 2.2 specificity test chromatogram in Example 2; wherein, from top to bottom, they are impurity Ⅲ-8 localization solution, impurity Ⅲ-5 localization solution, impurity Ⅲ-10 localization solution, impurity Ⅲ-2 localization solution, impurity Ⅲ-12 localization solution, impurity Ⅲ-7 localization solution, impurity Ⅲ-4 localization solution, impurity Ⅲ-11 localization solution, reference solution, test solution, mixed solution, mixed impurity interference solution, and solvent.

[0062] Figure 3 This is the chromatogram of Method 1 in Comparative Example 1.

[0063] Figure 4 This is the chromatogram of method 2 in comparative example 1.

[0064] Figure 5 This is the chromatogram of method 3 in comparative example 1.

[0065] Figure 6 This is the chromatogram of Method 1 in Comparative Example 2.

[0066] Figure 7 This is the chromatogram of Method 2 in Comparative Example 2. DETAILED DESCRIPTION

[0067] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0068] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0069] Example 1, a method for separating and detecting related substances in umeclidinium bromide starting material benzyl-2-bromoethyl ether based on gas chromatography and liquid chromatography

[0070] It includes two modules: gas chromatography detection and liquid chromatography detection;

[0071] Gas chromatography testing includes:

[0072] Solution preparation:

[0073] Test solution: Take an appropriate amount of the product, dissolve it in acetonitrile and quantitatively dilute it to make a solution containing about 50 mg per 1 ml.

[0074] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0075] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0076] Chromatographic conditions and system suitability:

[0077] The chromatographic column is a capillary column with 100% polyethylene glycol (or similar polarity) as the stationary liquid (Agilent DB-WAX, 30m×0.320mm×0.50μm is recommended); the starting temperature is 40°C, maintained for 5 minutes, raised to 150°C at a rate of 5°C per minute, and then raised to 230°C at a rate of 50°C per minute, and maintained for 20 minutes; the injection port temperature is 230°C; the detector temperature is 230°C; the split ratio is 10:1; the carrier gas flow rate is 1.5ml per minute, and the injection volume is 1.0μl.

[0078] System suitability requires that the signal-to-noise ratio of the main component peak height in the sensitivity solution chromatogram should be greater than 10.

[0079] Determination method

[0080] Accurately measure the test solution and control solution, inject them into the gas chromatograph respectively, and record the chromatogram.

[0081] If there are impurity peaks in the chromatogram of the limit test solution, the relative retention time and correction factor of each impurity peak are shown in the table below.

[0082] Impurity Name Relative retention time Correction Factor Dichloromethane 0.22 3.9 Impurity iii-7 0.56 3.7 Impurity iii-13 0.78 2.9

[0083] Calculation formula:

[0084]

[0085] Where: A 杂质 is the peak area of ​​the known impurities in the test solution;

[0086] A 对照 is the main peak area in the chromatogram of the reference solution;

[0087] f is the correction factor for known impurities.

[0088] The test results are shown in Table 1.

[0089] Table 1 Sample test results

[0090]

[0091]

[0092] Note: ND stands for Not Detected.

[0093] Liquid chromatography testing includes:

[0094] Solution preparation:

[0095] Solvent: water-acetonitrile (45:55)

[0096] Test solution: Accurately measure 1 ml of the test solution under gas chromatography conditions, place it in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0097] Control solution: Accurately measure 2 ml of the test solution, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, shake well; accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute it to the mark with solvent, shake well.

[0098] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0099] Mixed reference substance stock solution: Take about 15.6 mg of impurity III-5 reference substance and about 5 mg of impurity III-10 reference substance, place in a 25 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0100] System suitability solution: Take about 25 mg of this product and place it in a 50 ml volumetric flask. Accurately measure 1 ml of the mixed reference stock solution and place it in the same 50 ml volumetric flask. Dilute to the scale with solvent and shake well.

[0101] Chromatographic conditions and system suitability:

[0102] Use octadecylsilane bonded silica gel as filler (Waters C18, 4.6mm×250mm, 5μm or chromatographic column with equivalent performance); 20mmol / L phosphate buffer (take 2.4g of anhydrous sodium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) as mobile phase A, acetonitrile as mobile phase B, flow rate of 1.0ml per minute, linear gradient elution according to the table below; column temperature of 45℃; detection wavelength of 210nm; injection volume of 20μl; injector temperature of 5℃. If the gradient peak interferes with the detection of impurities, it is recommended to install a ghost peak capture column (Mr. Chromatography, Ghost-Sniper Column, 4.0mm×30mm) between the mixer and injector of the liquid chromatograph pump.

[0103]

[0104] System suitability requirements: In the system suitability solution chromatogram, impurity III-10, impurity III-5 and benzyl-2-bromoethyl ether appear in sequence, and the separation between the peaks of each component should meet the requirements; in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be greater than 10.

[0105] Determination method

[0106] Accurately measure the test solution and control solution, inject them into the liquid chromatograph respectively, and record the chromatogram.

[0107] Limit: If there are impurity peaks in the chromatogram of the test solution, deduct the chromatographic peak at a relative retention time of approximately 0.69 (impurity ⅲ-7, detected by gas phase method). The relative retention time and correction factor of each impurity peak are shown in the table below.

[0108]

[0109] Calculation formula:

[0110]

[0111] Where A 杂质 is the peak area of ​​the known impurity in the chromatogram of the test solution;

[0112] A 对照 is the main peak area in the chromatogram of the reference solution;

[0113] f is the correction factor for known impurities.

[0114]

[0115] Where A 杂质 is the peak area of ​​the unknown impurity in the test solution;

[0116] A 对照 is the main peak area in the chromatogram of the reference solution.

[0117] Total impurities (%) = ∑ known impurities % + ∑ unknown impurities %

[0118] Two batches of samples were taken for testing, and the test results are shown in Table 2.

[0119] Table 2 Sample test results (%)

[0120]

[0121] Note: ND stands for Not Detected.

[0122] Example 2, Methodology Verification

[0123] 1. Gas chromatography detection

[0124] 1.1 System suitability test

[0125] Mixed reference stock solution: Take appropriate amounts of dichloromethane, impurity III-1 reference substance, impurity III-9 reference substance, impurity III-15 reference substance, impurity III-7 reference substance and impurity III-13 reference substance, dissolve them in acetonitrile and dilute them to concentrations of approximately 0.6 mg / ml, 2 mg / ml, 2 mg / ml, 10 mg / ml and 6 mg / ml, respectively.

[0126] Reference substance solution: Mix reference substance stock solution 1ml → 20ml.

[0127] Sensitivity solution: Take an appropriate amount of this product, dissolve it in acetonitrile and dilute it to a concentration of about 0.025 mg / ml.

[0128] Precisely measure the blank solvent, sensitivity solution and reference solution, inject them into the gas chromatograph respectively, record the chromatogram, record the separation degree of each known impurity peak and adjacent peak, the retention time, peak area, theoretical plate number and signal-to-noise ratio of each known impurity. The test results are shown in Table 3.

[0129] Table 1 System suitability test results

[0130]

[0131] Verification results: The theoretical plate number of each impurity peak in the mixed reference solution is not less than 5000, the separation between each known impurity and the adjacent peak is greater than 1.5, and in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height is 694.0, which is greater than 10 and meets the requirements, indicating that the method has good system applicability.

[0132] 1.2 Specificity test

[0133] Take the mixed reference stock solution prepared under "System Suitability Test". The solvent is acetonitrile.

[0134] Solutions for localizing each impurity:

[0135] Dichloromethane: about 30mg→10ml, 1ml→100ml.

[0136] Impurity ⅲ-1: about 10 mg → 100 ml.

[0137] Impurity ⅲ-9: about 10 mg → 100 ml.

[0138] Impurity ⅲ-15: about 10 mg → 100 ml.

[0139] Impurity ⅲ-7: about 50mg→100ml.

[0140] Impurity ⅲ-13: about 30mg→100ml.

[0141] Impurity interference stock solution: impurity ⅲ-2 about 50 mg, impurity ⅲ-8 about 50 mg, impurity ⅲ-10 about 50 mg, impurity ⅲ-11 about 50 mg, impurity ⅲ-4 about 125 mg and impurity ⅲ-12 about 125 mg → 25 ml.

[0142] Impurity interference solution: impurity ⅲ-5 about 30 mg, impurity interference stock solution 1 ml → 20 ml.

[0143] Test solution: Take about 1g of this product → 20ml.

[0144] Reference solution: Take the reference solution prepared under “System suitability test”.

[0145] Mixed solution: Take about 1g of this product and about 30mg of impurity III-5 reference substance, 1ml of mixed reference substance stock solution, and 1ml→20ml of impurity interference stock solution.

[0146] Precisely measure the blank solvent, reference solution, each impurity location solution, impurity interference solution, test solution and mixed solution, inject them into the gas chromatograph respectively, and record the chromatogram. The test results are shown in Table 4 and Figure 1 .

[0147] Table 2 Specificity test results

[0148]

[0149] Note: N / A stands for not applicable. Validation results: Blank solvent, main component, unknown impurities in the main component and impurities studied in other liquid chromatography methods (impurity III-2, impurity III-4, impurity III-5, impurity III-8, impurity III-10, impurity III-11, impurity III-12) do not interfere with the detection of known impurities studied in this method. The separation between each known impurity and adjacent peaks is greater than 1.5, which meets the requirements, indicating that the method has good specificity.

[0150] 1.3 Limit of Quantitation and Limit of Detection Test

[0151] The concentration at which the signal-to-noise ratio (S / N) is not less than 10:1 is taken as the quantitative limit concentration, and its percentage (sensitivity) relative to the test sample concentration (1 mg / ml) is calculated.

[0152] Take the dichloromethane localization solution and each impurity localization solution prepared under the "Specificity Test" respectively.

[0153] Benzyl-2-bromoethyl ether reference substance stock solution: Take about 50 mg of benzyl-2-bromoethyl ether reference substance, accurately weigh it, place it in a 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0154] Benzyl-2-bromoethyl ether quantitative limit stock solution: Accurately measure 1 ml of benzyl-2-bromoethyl ether reference stock solution, place it in a 100 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0155] Quantitation limit solution: Accurately measure 3 ml of impurity ⅲ-1 localization solution, 2 ml of impurity ⅲ-7 localization solution, 3 ml of impurity ⅲ-9 localization solution, 2 ml of impurity ⅲ-13 localization solution, 5 ml of impurity ⅲ-15 localization solution and 2 ml of benzyl-2-bromoethyl ether quantitation limit stock solution, place them in the same 20 ml volumetric flask, dilute to the scale with acetonitrile, shake well, accurately measure 1 ml, place it in a 20 ml volumetric flask, accurately measure 2 ml of dichloromethane localization solution, place it in the same 20 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0156] Detection limit solution: Accurately measure 4 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with acetonitrile, and shake well.

[0157] Report the lowest quantitative and detection concentration (μg / ml), limit of quantitation and limit of detection (ng), percentage of test sample concentration and percentage of limit concentration respectively.

[0158] Results: The signal-to-noise ratios (S / N) of the peak heights of dichloromethane, impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1, impurity ⅲ-9 and benzyl-2-bromoethyl ether in the quantitative limit solution were 24.7, 19.8, 24.5, 17.0, 14.7, 19.9 and 21.9, respectively. The minimum quantitative concentrations were 3.473 μg / ml, 1.242 μg / ml, 2.659 μg / ml, 1.290 μg / ml, 0. .757μg / ml, 0.688μg / ml and 0.102μg / ml, which are approximately equivalent to 0.0069%, 0.0025%, 0.0053%, 0.0026%, 0.0015%, 0.0014% and 0.0002% of the test solution concentration, respectively, and approximately equivalent to 11.58%, 1.24%, 0.66%, 0.43%, 0.76%, 0.69% and 0.02% of the limit concentration, respectively.

[0159] The S / N ratios of the peak heights of dichloromethane, impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1, impurity ⅲ-9 and benzyl-2-bromoethyl ether in the detection limit solution were 9.8, 8.1, 9.8, 7.4, 6.6, 7.3 and 9.6, respectively, and the minimum detection concentrations were 1.389 μg / ml, 0.497 μg / ml, 1.064 μg / ml, 0.516 μg / ml, 0.303 μg / ml, 0.275μg / ml and 0.041μg / ml, which are approximately equivalent to 0.0028%, 0.0010%, 0.0021%, 0.0010%, 0.0006%, 0.0006% and 0.0001% of the test solution concentration, respectively, and approximately equivalent to 4.63%, 0.50%, 0.27%, 0.17%, 0.30%, 0.28% and 0.01% of the limit concentration, respectively.

[0160] 1.4 Linearity and range tests

[0161] Mixed reference substance stock solution ①: Take approximately 50 mg each of benzyl-2-bromoethyl ether reference substance, impurity ⅲ-1 reference substance, impurity ⅲ-9 reference substance and impurity ⅲ-15 reference substance, accurately weigh, place in the same 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0162] Mixed reference substance stock solution ②: Take approximately 150 mg each of benzyl-2-bromoethyl ether reference substance and impurity ⅲ-13 reference substance, accurately weigh, place in the same 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0163] Mixed reference substance stock solution ③: Take about 250 mg of benzyl-2-bromoethyl ether reference substance and impurity ⅲ-7 reference substance, accurately weigh them, place them in the same 25 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0164] Mixed reference substance stock solution ④: Take about 60 mg of benzyl-2-bromoethyl ether reference substance and dichloromethane reference substance, accurately weigh, place in the same 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0165] Linear solution 1:

[0166] 40% linear solution: Accurately measure ①1 ml of the mixed reference stock solution, place it in a 50 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and use this as the second linear point.

[0167] Accurately measure 1 ml, 2 ml, 3 ml, and 4 ml of the mixed reference stock solution ①, place them in 4 different 20 ml volumetric bottles, dilute to the mark with acetonitrile, shake well, and use them as 100%, 200%, 300%, and 400% linear solutions (as the 3rd to 6th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0168] Linear solution 2:

[0169] 40% linear solution: Accurately measure 1 ml of the mixed reference stock solution ②, place it in a 50 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and use this as the second linear point.

[0170] Accurately measure 1 ml, 2 ml, 3 ml, and 4 ml of the mixed reference stock solution ②, place them in 4 different 20 ml volumetric bottles, dilute to the mark with acetonitrile, shake well, and use them as 100%, 200%, 300%, and 400% linear solutions (as the 3rd to 6th points of the linear test, respectively). Use the quantitative limit solution as the first point of linearity.

[0171] Linear solution 3:

[0172] 40% linear solution: Accurately measure 1 ml of the mixed reference stock solution ③, place it in a 50 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and use this as the second linear point.

[0173] Accurately measure 1 ml, 2 ml, 3 ml, and 4 ml of the mixed reference stock solution ③, place them in 4 different 20 ml volumetric bottles, dilute to the mark with acetonitrile, shake well, and use them as 100%, 200%, 300%, and 400% linear solutions (as the 3rd to 6th points of the linear test, respectively). Use the quantitative limit solution as the first point of linearity.

[0174] Linear solution 4:

[0175] 40% linear solution: Accurately measure 1 ml of the mixed reference stock solution ④, place it in a 50 ml volumetric flask, dilute to the scale with acetonitrile, shake well, and use this as the second linear point.

[0176] Accurately measure 1 ml, 2 ml, 3 ml, and 4 ml of the mixed reference stock solution ④, respectively, and place them in 4 different 20 ml volumetric bottles, dilute to the scale with acetonitrile, shake well, and use them as 100%, 200%, 300%, and 400% linear solutions (respectively as the 3rd to 6th points of the linear test). Use the quantitative limit solution as the first point of linearity.

[0177] Take linear solution 1, linear solution 2, linear solution 3 and linear solution 4 respectively, inject them into gas chromatograph, and record chromatograms. Take the concentration C (μg / ml) of each component as the horizontal coordinate, and take the impurity peak area or the peak area of ​​benzyl-2-bromoethyl ether after deducting the peak area response value of each impurity in the test solution as the vertical coordinate to perform linear regression. The correction factor of each impurity is calculated by the ratio of the slope of the main component standard curve to the slope of the impurity standard curve.

[0178] Results: In the range of quantitation limit to 400% of limit concentration, the linear equations of impurity ⅲ-15, impurity ⅲ-1, impurity ⅲ-9 and benzyl-2-bromoethyl ether in linear 1 were A=0.7612C+4.8450, A=0.8565C+6.7244, A=0.5699C+4.0638 and A=1.6008C+12.4462, respectively. The correlation coefficients r were 0.9971, 0.9959, 0.9959 and 0.9960, all greater than 0.99. The absolute values ​​of the Y-axis intercepts were equivalent to the percentages of 100% response values, which were 6.3%, 6.9%, 7.3% and 7.2%, respectively, all less than 25%.

[0179] The linear equations of impurity ⅲ-13 and benzyl-2-bromoethyl ether in Linear 2 are A=0.6231C-6.8534 and A=1.7888C-22.3887, respectively, and the correlation coefficients r are 0.9979 and 0.9975, respectively, both greater than 0.99, and the percentages of the absolute values ​​of the Y-axis intercepts equivalent to 100% response values ​​are 3.7% and 4.1%, respectively, both less than 25%;

[0180] The linear equations of impurity ⅲ-7 and benzyl-2-bromoethyl ether in linear 3 are A=0.4719C+12.4606 and A=1.7569C+47.0669, respectively, and the correlation coefficients r are 0.9906 and 0.9911, respectively, both greater than 0.99, and the percentages of the absolute values ​​of the Y-axis intercepts equivalent to 100% response values ​​are 5.0% and 5.3%, respectively, both less than 25%;

[0181] The linear equations of dichloromethane and benzyl-2-bromoethyl ether in Linear 4 are A=0.5543C-2.0562 and A=2.1627C-5.0183, respectively, and the correlation coefficients r are 0.9975 and 0.9963, respectively, both greater than 0.99, and the percentages of the absolute values ​​of the Y-axis intercepts equivalent to 100% response values ​​are 12.3% and 8.5%, respectively, both less than 25%, which are in compliance with the regulations, indicating that the linear relationship is good within this concentration range.

[0182] The correction factors for dichloromethane, impurity Ⅲ-15, impurity Ⅲ-7, impurity Ⅲ-13, impurity Ⅲ-1 and impurity Ⅲ-9 were calculated by the ratio of the slope of the main component standard curve to the slope of the impurity standard curve, which were 3.9, 2.1, 3.7, 2.9, 1.9 and 2.8 respectively.

[0183] 1.5 Solution stability test

[0184] Prepare the mixed reference stock solution according to the method under "System suitability test".

[0185] Reference substance solution: Accurately measure 1 ml of the mixed reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0186] Test solution: Take about 1g of the product, weigh accurately, place in a 20ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0187] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0188] Spiked test solution: Take about 1g of the product, accurately weigh it, and place it in a 20ml volumetric flask. Accurately measure 1ml of the mixed reference stock solution and place it in the same 20ml volumetric flask. Add acetonitrile to dissolve and dilute to the scale, and shake well.

[0189] Accurately measure the reference solution, control solution, test solution and spiked test solution and place them at room temperature. Inject and analyze them at 0h, 4h, 8h, 12h, 16h and 24h after preparation, record the chromatogram, and calculate the ratio of the peak area of ​​each impurity at each time point to the peak area at 0h. The test results are shown in Tables 5 to 7.

[0190] Table 5 Peak area results of stability test of reference solution and control solution

[0191]

[0192] Table 6 Impurity peak area results of the spiked test solution stability test

[0193] Time / h Dichloromethane Impurity ⅲ-15 Impurity ⅲ-7 Impurity ⅲ-13 Impurity ⅲ-1 Impurity ⅲ-9 0 7.35 48.52 196.07 109.15 53.62 34.66 2 7.41 47.64 192.53 107.47 52.80 33.92 4 7.33 47.70 193.15 107.84 52.85 33.97 8 7.36 48.50 196.01 109.14 53.46 34.48 12 7.64 49.03 197.71 110.00 53.79 34.60 24 7.23 48.01 193.78 108.68 53.33 34.57

[0194] Table 37 Impurity peak area results of the test solution stability test

[0195] Time / h Dichloromethane Impurity ⅲ-15 Impurity ⅲ-7 Impurity ⅲ-13 Impurity ⅲ-1 Impurity ⅲ-9 0 ND ND 50.97 5.27 ND 2.94 4 ND ND 51.76 5.37 ND 2.94 8 ND ND 51.13 5.25 ND 2.89 12 ND ND 51.05 5.29 ND 2.96 16 ND ND 51.13 5.22 ND 2.97 24 ND ND 51.58 5.38 ND 2.99

[0196] Results: After standing at room temperature for 24 hours, in the chromatograms of the reference solution and the spiked test solution, the ratios of the peak areas of dichloromethane, impurity ⅲ-1, impurity ⅲ-9, impurity ⅲ-15 and impurity ⅲ-13 to the peak areas at 0 hours were all in the range of 90% to 108%, and the ratios of the peak areas of impurity ⅲ-7 to the peak areas at 0 hours were all in the range of 92% to 105%, indicating that the reference solution and the spiked test solution had good stability within 24 hours;

[0197] After being placed at room temperature for 24 hours, in the chromatogram of the control solution, the ratio of the peak area of ​​benzyl-2-bromoethyl ether at each time point to the peak area at 0 hour was in the range of 92% to 105%, indicating that the control solution had good stability within 24 hours;

[0198] After being placed at room temperature for 24 hours, in the chromatogram of the test solution, the ratios of the peak areas of impurity Ⅲ-7, impurity Ⅲ-13 and impurity Ⅲ-9 at each time point to the peak area at 0 hour were all in the range of 90% to 108%, and no new impurities greater than the reporting limit that would affect the detection of known impurities were produced, indicating that the test solution had good stability within 24 hours.

[0199] 1.6 Repeatability test

[0200] Prepare the mixed reference stock solution according to the method under "System suitability test".

[0201] Reference substance solution: Accurately measure 1 ml of the mixed reference substance stock solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0202] Spiked test solution: Take about 1g of this product, weigh accurately, and place in a 20ml volumetric flask. Accurately measure 1ml of the mixed reference stock solution, place in the same 20ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, shake well. Prepare 6 copies in parallel.

[0203] Control solution: Accurately measure 1 ml of the spiked test solution, place it in a 100 ml volumetric flask, dilute to the mark with acetonitrile, and shake well. Prepare 6 copies in parallel.

[0204] Precisely measure blank solvent, reference solution, spiked test solution and reference solution, inject them into gas chromatograph respectively, record chromatograms. Calculate the content of each impurity by external standard method and main component self-reference method with correction factor.

[0205] Six spiked test solutions were prepared in parallel. The contents of each known impurity in the spiked test solution were calculated by the external standard method. The RSD value of dichloromethane content was 1.7%, not more than 5%. The RSD values ​​of impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1 and impurity ⅲ-9 were 0.9%, 0.8%, 0.8%, 1.0% and 0.9%, respectively, all less than 3%. The contents of each known impurity in the spiked test solution were calculated by the principal component self-reference method with correction factor. The RSD value of dichloromethane content was 1.7%, not more than 5%. The RSD values ​​of impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1 and impurity ⅲ-9 were 0.9%, 0.8%, 0.8%, 1.0% and 0.9%, respectively, all less than 3%. The RSD value of the content is 1.3%, not more than 5%. The RSD values ​​of the impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1 and impurity ⅲ-9 are 0.7%, 0.7%, 0.7%, 0.8% and 0.8% respectively, all less than 3%, which is in line with the regulations, indicating that the method has good repeatability. The impurity contents calculated by the main component self-reference method with correction factors are slightly larger than those calculated by the external standard method, indicating that impurities can be strictly controlled by the self-reference method with correction factors.

[0206] 1.7 Reproducibility test:

[0207] Different operators used different gas chromatographs on different dates to prepare 6 spiked test solutions and calculate the contents in the same way according to the repeatability test method. According to the results of the combined repeatability test, the RSD value of the dichloromethane content in the 12 spiked test solutions was 7.8%, no more than 10%, and the RSD values ​​of the impurity ⅲ-1, impurity ⅲ-9, impurity ⅲ-15, impurity ⅲ-7 and impurity ⅲ-13 contents were 3.7%, 3.0%, 2.4%, 2.6% and 2.9%, respectively, all no more than 6%, indicating that the method has good reproducibility.

[0208] 1.8 Accuracy test

[0209] Prepare mixed reference substance stock solution and reference substance solution according to the method under "System suitability test".

[0210] Test solution: Take about 1g of this product → 20ml.

[0211] 50% recovery solution: Take about 1g of this product and mix it with 0.5ml→20ml of the reference stock solution.

[0212] 100% recovery solution: Take about 1g of this product and mix it with 1ml of reference stock solution → 20ml.

[0213] 200% recovery solution: Take about 1g of this product and mix it with 2ml of reference stock solution → 20ml.

[0214] Precisely measure blank solvent, reference solution, test solution and each recovery solution, inject them into gas chromatograph respectively, record chromatograms. Calculate the measured amount of each impurity and the recovery rate by peak area according to the external standard method.

[0215] Calculation formula:

[0216]

[0217] Results: Within the limit concentration ranges of 50%, 100% and 200%, the average recoveries of impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1, dichloromethane and impurity ⅲ-9 were 100.8%, 100.6%, 100.4%, 100.0%, 101.9% and 93.8%, respectively, all between 90% and 108%, indicating that the method has high accuracy.

[0218] 1.9 Durability test

[0219] Prepare spiked test solution and control solution according to the method under "Repeatability test".

[0220] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 20 ml volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0221] Precisely measure blank solvent, sensitivity solution, control solution and spiked test solution, and inject them into the gas chromatograph under the chromatographic conditions in Tables 8 to 9, respectively, and record the chromatograms. Repeat the injection of the control solution 5 times to investigate the system repeatability test. Investigate the impact of slight changes in chromatographic conditions on the system suitability test results and impurity detection results. Calculate the contents of impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1 and impurity ⅲ-9 by the main component self-reference method with correction factors. The test results are shown in Tables 8 to 12.

[0222] Table 4 Summary of durability inspection items

[0223]

[0224] Table 9 Durability Information of the Chromatographic Column Used

[0225] Chromatographic columns 1 2 Column specifications 30m×0.320mm,0.50μm 30m×0.320mm,0.50μm Chromatographic column model Agilent DB-WAX Agilent DB-WAX Column batch number US2536116H US2595333H Column No. GCC221003 GCC221004

[0226] Table 10 Durability test results of the influence of different changing conditions on the separation degree of each impurity

[0227]

[0228] Table 11 Durability test results of the effects of different changing conditions on the relative retention time of each impurity

[0229]

[0230] Table 512 Durability test results of the influence of different changing conditions on the determination results of each impurity

[0231]

[0232]

[0233] Verification results: Under each condition, the control solution was injected 5 times repeatedly, and the RSD value of the retention time of the main component (or impurity ⅲ-7 in the reference solution under standard conditions) was no more than 1%, and the RSD value of the peak area was no more than 2%. In the chromatogram of the spiked test solution, the separation between each known impurity and the adjacent peak was greater than 1.5, and the theoretical plate number of each impurity was no less than 5000. In the chromatogram of the sensitive solution, the signal-to-noise ratio of the main component peak height was above 500 and greater than 10. The difference between the content of each impurity and the standard conditions was no more than ±20% of the standard test result, which met the requirements. It shows that the method has good durability when the initial column temperature (35℃~45℃), the carrier gas flow rate (1.4ml / min~1.6ml / min), the injection port temperature (225℃~235℃), the detector temperature (225℃~235℃) and the replacement of chromatographic columns of the same brand, model, specification and different batches are changed.

[0234] The relative retention times of dichloromethane, impurity ⅲ-15, impurity ⅲ-7, impurity ⅲ-13, impurity ⅲ-1 and impurity ⅲ-9 are 0.22, 0.44, 0.56, 0.78, 0.87 and 0.93, respectively.

[0235] 2. Liquid chromatography detection:

[0236] 2.1 System suitability test:

[0237] System suitability test 1

[0238] Solvent: water-acetonitrile (45:55).

[0239] Mixed reference substance stock solution: take approximately 12.5 mg each of impurity Ⅲ-2 reference substance, impurity Ⅲ-8 reference substance and impurity Ⅲ-10 reference substance, approximately 31 mg each of impurity Ⅲ-4 reference substance and impurity Ⅲ-12 reference substance, approximately 187.5 mg of impurity Ⅲ-5 reference substance → 25 ml, 2 ml → 100 ml.

[0240] System suitability solution: Take about 25 mg of benzyl-2-bromoethyl ether reference substance and mix the reference substance stock solution from 5 ml to 50 ml.

[0241] System suitability test 2

[0242] Impurity III-11 reference substance stock solution: take about 12.5 mg of impurity III-11 reference substance → 25 ml, 2 ml → 100 ml.

[0243] System suitability solution: Take about 25 mg of benzyl-2-bromoethyl ether reference substance and 5 ml → 50 ml of impurity III-11 reference substance stock solution.

[0244] Sensitivity solution: system suitability solution 2ml→20ml, 1ml→20ml, 1ml→10ml.

[0245] Verification results: The system suitability solution was injected 5 times repeatedly, and the separation between each known impurity and the adjacent peak was greater than 1.5. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main component peak height was greater than 10. The results showed that the method had good system applicability.

[0246] 2.2 Specificity test

[0247] Solvent: water-acetonitrile (45:55).

[0248] Stock solutions of each impurity reference substance:

[0249] Impurity ⅲ-2: about 25 mg → 50 ml;

[0250] Impurity ⅲ-4: about 25 mg → 20 ml;

[0251] Impurity ⅲ-5: about 150mg→20ml;

[0252] Impurity ⅲ-7: about 25 mg → 20 ml;

[0253] Impurity ⅲ-8: about 25 mg → 50 ml;

[0254] Impurity ⅲ-10: about 25 mg → 50 ml;

[0255] Impurity ⅲ-11: about 25 mg → 50 ml;

[0256] Impurity ⅲ-12: about 25 mg → 20 ml;

[0257] Mixed reference substance stock solution: Accurately measure 1 ml of each impurity reference substance stock solution of impurity ⅲ-2, impurity ⅲ-4, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-11, impurity ⅲ-12, and impurity ⅲ-5, place them in the same 50 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0258] Impurity localization solution: Take the stock solution of each impurity reference substance and dilute it in two steps: 1ml→50ml, 2ml→20ml.

[0259] Test solution: 25mg→50ml.

[0260] Reference substance solution: Accurately measure and mix 2 ml → 20 ml of reference substance stock solution.

[0261] Stock solution of impurity interference reference substance: take approximately 10 mg each of impurity Ⅲ-1 reference substance, impurity Ⅲ-9 reference substance, impurity Ⅲ-15 reference substance, approximately 25 mg of impurity Ⅲ-7 reference substance, approximately 30 mg of impurity Ⅲ-13 reference substance → 20 ml; then dilute 1 ml → 50 ml.

[0262] Mixed impurity interference solution: impurity interference reference stock solution 2ml→20ml.

[0263] Mixed solution: Take about 25 mg of this product, 5 ml of mixed reference stock solution, and 5 ml of impurity interference reference stock solution → 50 ml.

[0264] Accurately measure the solvent, test solution, reference solution, mixed solution, mixed impurity interference solution and each known impurity location solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Figure 2 , Table 13.

[0265] Table 63 Specificity test results

[0266]

[0267]

[0268] Verification results: Solvents, main components, and impurities studied by other methods (impurity ⅲ-1, impurity ⅲ-7, impurity ⅲ-9, impurity ⅲ-13, impurity ⅲ-15) do not interfere with the detection of impurities ⅲ-2, impurity ⅲ-4, impurity ⅲ-5, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-11, impurity ⅲ-12 and other unknown impurities, and the separation between each known impurity and the adjacent peak is greater than 1.5, indicating that the method has good specificity.

[0269] 2.3 Limit of quantification and limit of detection

[0270] 2.3.1 Limit of Quantitation and Limit of Detection Test 1

[0271] Solvent: water-acetonitrile (45:55).

[0272] According to the method under "Specificity Test", prepare impurity III-5 reference stock solution, impurity III-2 reference stock solution, impurity III-8 reference stock solution, impurity III-10 reference stock solution, impurity III-11 reference stock solution, impurity III-4 reference stock solution and impurity III-12 reference stock solution.

[0273] Benzyl-2-bromoethyl ether reference substance stock solution: Benzyl-2-bromoethyl ether reference substance approximately 25 mg → 50 ml.

[0274] Impurity III-2 reference solution: Impurity III-2 reference stock solution 1ml→50ml.

[0275] Impurity III-4 reference solution: Impurity III-4 reference stock solution 1ml→50ml.

[0276] Impurity III-5 reference solution: Impurity III-5 reference stock solution was diluted in two steps: 1ml→25ml, 1ml→100ml.

[0277] Impurity III-8 reference solution: Impurity III-8 reference stock solution 1ml→50ml.

[0278] Impurity III-10 reference solution: Impurity III-10 reference stock solution 1ml→50ml.

[0279] Impurity III-12 reference solution: Impurity III-12 reference stock solution 1ml→25ml, 1ml→100ml.

[0280] Benzyl-2-bromoethyl ether reference solution: Benzyl-2-bromoethyl ether reference stock solution 1ml→50ml.

[0281] Quantitation limit solution ①: Take 0.5 ml of impurity Ⅲ-2 reference solution, 0.3 ml of impurity Ⅲ-4 reference solution, 2 ml of impurity Ⅲ-5 reference solution, 0.4 ml of impurity Ⅲ-8 reference solution, 0.5 ml of impurity Ⅲ-10 reference solution, 3 ml of impurity Ⅲ-12 reference solution and 0.9 ml of benzyl-2-bromoethyl ether reference solution, place them in the same 50 ml volumetric flask, dilute to the scale with solvent and shake well.

[0282] Detection limit solution: Measure the quantitative limit solution ①2.5ml→10ml.

[0283] 2.3.2 Limit of Quantitation and Limit of Detection Test 2

[0284] Impurity III-11 reference solution: Impurity III-11 reference stock solution 2ml→100ml, 5ml→50ml.

[0285] Quantitative limit solution ②: Impurity ⅲ-11 reference solution 1ml→20ml.

[0286] Detection limit solution: Quantitation limit solution ②2.5ml→10ml.

[0287] Verification results: The minimum quantitative concentrations of impurity Ⅲ-11, impurity Ⅲ-4, impurity Ⅲ-12, impurity Ⅲ-2, impurity Ⅲ-10, impurity Ⅲ-5, impurity Ⅲ-8 and benzyl-2-bromoethyl ether were 0.0516μg / ml, 0.1518μg / ml, 0.0328μg / ml, 0.0979μg / ml, 0.0915μg / ml, 0.1153μg / ml, 0.0763μg / ml and 0.1732μg / ml, respectively, and the quantitative limits were 1.0320ng, 3.0360ng, 0.6560ng, 1.9580ng, 1.8300ng, 2.3060ng, 1.526 0ng and 3.4640ng, which are approximately equivalent to 0.01%, 0.03%, 0.01%, 0.02%, 0.02%, 0.02%, 0.02% and 0.03% of the concentration of the test solution, respectively, and approximately equivalent to 5.2%, 6.1%, 1.3%, 9.8%, 2.3%, 0.9%, 7.6%, and 17.3% of the limit concentration, respectively. The signal-to-noise ratios of the peak heights of impurity Ⅲ-11, impurity Ⅲ-4, impurity Ⅲ-12, impurity Ⅲ-2, impurity Ⅲ-10, impurity Ⅲ-5, impurity Ⅲ-8 and benzyl-2-bromoethyl ether are 24.2, 23.4, 15.8, 20.6, 15.2, 17.9, 18.2 and 29.0, respectively.

[0288] The minimum detection concentrations of impurity Ⅲ-11, impurity Ⅲ-4, impurity Ⅲ-12, impurity Ⅲ-2, impurity Ⅲ-10, impurity Ⅲ-5, impurity Ⅲ-8 and benzyl-2-bromoethyl ether were 0.0129μg / ml, 0.0380μg / ml, 0.0082μg / ml, 0.0245μg / ml, 0.0229μg / ml, 0.0288μg / ml, 0.0191μg / ml and 0.0433μg / ml, respectively, and the detection limits were 0.2580ng, 0.7600ng, 0.1640ng, 0.4900ng, 0.4580ng, 0.5760ng, 0.3820ng, respectively. g and 0.8660ng, which are approximately equivalent to 0.003%, 0.008%, 0.002%, 0.005%, 0.005%, 0.006%, 0.004% and 0.009% of the concentration of the test solution, respectively, and approximately equivalent to 1.3%, 1.5%, 0.3%, 2.5%, 0.6%, 0.2%, 1.9% and 4.3% of the limit concentration, respectively. The signal-to-noise ratios of the impurity Ⅲ-11, impurity Ⅲ-4, impurity Ⅲ-12, impurity Ⅲ-2, impurity Ⅲ-10, impurity Ⅲ-5, impurity Ⅲ-8 and the peak height of benzyl-2-bromoethyl ether are 3.7, 2.9, 3.5, 4.3, 2.5, 3.2, 3.1 and 6.8, respectively.

[0289] 2.4 Linearity and range

[0290] Solvent: water-acetonitrile (45:55).

[0291] Prepare impurity III-5 reference stock solution, impurity III-2 reference stock solution, impurity III-8 reference stock solution, impurity III-10 reference stock solution, impurity III-11 reference stock solution, impurity III-4 reference stock solution and impurity III-12 reference stock solution under the "Specificity Test" item.

[0292] Benzyl-2-bromoethyl ether reference substance stock solution ①: Take about 25 mg of benzyl-2-bromoethyl ether reference substance → 50 ml.

[0293] Benzyl-2-bromoethyl ether reference substance stock solution ②: Take about 25 mg of benzyl-2-bromoethyl ether reference substance → 20 ml.

[0294] Benzyl-2-bromoethyl ether reference substance stock solution ③: Take about 150 mg of benzyl-2-bromoethyl ether reference substance → 20 ml.

[0295] Linear solution 1:

[0296] Mixed reference substance stock solution ①: Accurately measure 2 ml each of impurity ⅲ-11 reference substance stock solution, impurity ⅲ-2 reference substance stock solution, impurity ⅲ-10 reference substance stock solution, impurity ⅲ-8 reference substance stock solution and benzyl-2-bromoethyl ether reference substance stock solution ①, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0297] Accurately measure ①1ml, 3ml, 5ml, 7ml, 10ml, 20ml of the mixed reference stock solution and place them in 6 different 50ml volumetric bottles, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 140%, 200%, 400% linear solutions (as the 2nd to 7th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0298] Linear solution 2:

[0299] Mixed reference substance stock solution ②: Accurately measure 2 ml each of impurity ⅲ-4 reference substance stock solution, impurity ⅲ-12 reference substance stock solution and benzyl-2-bromoethyl ether reference substance stock solution ②, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0300] Accurately measure ②1ml, 3ml, 5ml, 7ml, 10ml, 20ml of the mixed reference stock solution and place them in 6 different 50ml volumetric bottles, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 140%, 200%, 400% linear solutions (as the 2nd to 7th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0301] Linear solution 3:

[0302] Mixed reference substance stock solution ③: Accurately measure 2 ml of impurity ⅲ-5 reference substance stock solution and benzyl-2-bromoethyl ether reference substance stock solution ③, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0303] Accurately measure 1ml, 3ml, 5ml, 7ml, 10ml, and 20ml of the mixed reference stock solution into 6 different 50ml volumetric bottles, dilute to scale with solvent, shake well, and use them as 20%, 60%, 100%, 140%, 200%, and 400% linear solutions (as the 2nd to 7th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0304] Linear solution 4:

[0305] Mixed reference stock solution ④: Accurately measure 2 ml of impurity ⅲ-11 reference stock solution and benzyl-2-bromoethyl ether reference stock solution ①, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0306] Accurately measure 1ml, 3ml, 5ml, 7ml, 10ml, and 20ml of the mixed reference stock solution into 6 different 50ml volumetric bottles, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 140%, 200%, and 400% linear solutions (as the 2nd to 7th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0307] Linear solution 5:

[0308] Impurity III-10 reference substance stock solution: Take about 125 mg of impurity III-10 reference substance, accurately weigh, place in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0309] Benzyl-2-bromoethyl ether reference substance stock solution ④: Take about 125 mg of benzyl-2-bromoethyl ether reference substance, accurately weigh it, put it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0310] Mixed reference stock solution ⑤: Accurately measure 2 ml each of impurity ⅲ-10 reference stock solution and benzyl-2-bromoethyl ether reference stock solution ④, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0311] Accurately measure ⑤1ml, 3ml, 5ml, 7ml, 10ml, 20ml of the mixed reference stock solution and place them in 6 different 50ml volumetric bottles, dilute to the scale with solvent, shake well, and use them as 20%, 60%, 100%, 140%, 200%, 400% linear solutions (as the 2nd to 7th points of the linear test respectively). Use the quantitative limit solution as the first point of linearity.

[0312] Accurately measure the quantitative limit solution, 20%, 60%, 100%, 140%, 200%, and 400% linear solutions, inject them into the liquid chromatograph, and record the chromatogram. Perform linear regression with concentration C (μg / ml) as the horizontal coordinate and peak area as the vertical coordinate. The correction factor of each impurity is calculated by the ratio of the slope of the main component standard curve to the slope of the impurity standard curve. The test results are shown in Table 14.

[0313] Table 14 Results of correction factors for various impurities

[0314]

[0315]

[0316] Note: N / A means not applicable

[0317] Verification results: In the first group of linear tests, the peak area A and concentration C of impurity ⅲ-2 in the range of 0.0979μg / ml to 3.9156μg / ml, impurity ⅲ-8 in the range of 0.0763μg / ml to 3.8137μg / ml, and benzyl-2-bromoethyl ether in the range of 0.1732μg / ml to 3.8490μg / ml showed a good linear relationship. The linear equation of impurity ⅲ-2 was A=62342.2221C+218.6970, r was 1.0000, greater than 0.99, and the absolute value of the Y-intercept was The value is equivalent to 0.4% of the 100% response value, which is less than 25%; the linear equation of impurity ⅲ-8 is A=65969.5652C+186.4973, r is 1.0000, which is greater than 0.99, and the absolute value of the Y-axis intercept is equivalent to 0.3% of the 100% response value, which is less than 25%; the linear equation of benzyl-2-bromoethyl ether is A=49559.9829C+121.7803, r is 1.0000, which is greater than 0.99, and the absolute value of the Y-axis intercept is equivalent to 0.3% of the 100% response value, which is less than 25%.

[0318] In the second group of linear tests, the peak area A and concentration C of impurity ⅲ-4 in the range of 0.1518μg / ml to 10.1214μg / ml, impurity ⅲ-12 in the range of 0.0328μg / ml to 10.9407μg / ml, and benzyl-2-bromoethyl ether in the range of 0.1732μg / ml to 10.1423μg / ml showed a good linear relationship. The linear equation of impurity ⅲ-4 was A=56593.3581C-520.5223, r was 1.0000, greater than 0.99, and the absolute value of the Y-axis intercept was It is equivalent to 0.4% of the 100% response value and less than 25%; the linear equation of impurity ⅲ-12 is A=117700.8769C+1303.5507, r is 1.0000, which is greater than 0.99, and the absolute value of the Y-axis intercept is equivalent to 0.4% of the 100% response value and less than 25%; the linear equation of benzyl-2-bromoethyl ether is A=43020.7488C+176.2705, r is 1.0000, which is greater than 0.99, and the absolute value of the Y-axis intercept is equivalent to 0.2% of the 100% response value and less than 25%.

[0319] In the third group of linear tests, the peak area A and concentration C of impurity Ⅲ-5 in the range of 0.1153μg / ml to 57.6436μg / ml and benzyl-2-bromoethyl ether in the range of 0.1732μg / ml to 59.1002μg / ml showed a good linear relationship. The linear equation of impurity Ⅲ-5 was A=47162.8705C+15173.4119, r was 0.9999, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 2.2% of the 100% response value, less than 25%; the linear equation of benzyl-2-bromoethyl ether was A=42364.6840C+7482.7507, r was 1.0000, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 1.2% of the 100% response value, less than 25%.

[0320] In the fourth group of linear tests, the peak area A and concentration C of impurity Ⅲ-11 in the range of 0.0516μg / ml to 4.1300μg / ml and benzyl-2-bromoethyl ether in the range of 0.1732μg / ml to 4.4030μg / ml showed a good linear relationship. The linear equation of impurity Ⅲ-11 was A=78189.0463C+481.9170, r was 1.0000, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 0.6% of the 100% response value, which was less than 25%; the linear equation of benzyl-2-bromoethyl ether was A=42175.4268C+497.0645, r was 0.9999, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 1.1% of the 100% response value, which was less than 25%.

[0321] In the fifth group of linear tests, the peak area A and concentration C of impurity Ⅲ-10 in the range of 0.0968μg / ml to 19.3659μg / ml and benzyl-2-bromoethyl ether in the range of 0.1848μg / ml to 20.5314μg / ml showed a good linear relationship. The linear equation of impurity Ⅲ-10 was A=50255.6195C+2046.3687, r was 1.0000, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 0.8% of the 100% response value, which was less than 25%; the linear equation of benzyl-2-bromoethyl ether was A=43584.0522C+3349.2112, r was 1.0000, greater than 0.99, and the absolute value of the Y-axis intercept was equivalent to 1.5% of the 100% response value, which was less than 25%. The results showed that the method had a good linear relationship in the range of the limit of quantification to 400% of the limit concentration.

[0322] The correction factors of impurity ⅲ-2, impurity ⅲ-8, impurity ⅲ-4, impurity ⅲ-12, impurity ⅲ-5, impurity ⅲ-11, and impurity ⅲ-10 are 0.8, 0.8, 0.8, 0.37, 0.9, 0.5, and 0.9, respectively. The correction factors of each impurity are used in this validation report to calculate the content of each impurity. Impurity ⅲ-2, impurity ⅲ-8, and impurity ⅲ-11 are not included in the quality standards and are controlled as non-specific impurities, which is equivalent to strict control. The correction factor of impurity ⅲ-4 is 0.8, which is included in the quality standards as specific impurities. The correction factors of impurity ⅲ-5, impurity ⅲ-10, and impurity ⅲ-12 are 1.0, 1.0, and 0.4, respectively, which are included in the quality standards as specific impurities.

[0323] 2.5 Solution stability

[0324] Solution stability test 1

[0325] Solvent: water-acetonitrile (45:55).

[0326] Mixed reference substance stock solution: take approximately 12.5 mg each of impurity Ⅲ-2 reference substance, impurity Ⅲ-8 reference substance, impurity Ⅲ-11 reference substance and impurity Ⅲ-10 reference substance, approximately 31 mg each of impurity Ⅲ-4 reference substance and impurity Ⅲ-12 reference substance, approximately 187.5 mg of impurity Ⅲ-5 reference substance → 25 ml, 2 ml → 100 ml.

[0327] Reference substance solution: Mix 2 ml of reference substance stock solution → 20 ml.

[0328] Test solution: Take about 25 mg of the product → 50 ml.

[0329] Control solution: test solution 2ml→20ml, 1ml→20ml.

[0330] Spiked test solution: Take about 25 mg of this product and mix it with 5 ml of reference stock solution → 50 ml.

[0331] Place the control solution at low temperature (injector temperature is 5°C), inject and analyze at the end of 0h, 4h, and 13h after preparation, and record the chromatogram. Place the reference solution, test solution, and spiked test solution at low temperature (injector temperature is 5°C), inject and analyze at the end of 0h, 3h, 6h, 9h, 12h, and 24h after preparation, record the chromatogram, and calculate the ratio of the impurity peak area at each time point to the peak area at 0h.

[0332] Solution stability test 2

[0333] Impurity III-11 reference substance stock solution: take about 12.5 mg of impurity III-11 reference substance → 25 ml, 2 ml → 100 ml.

[0334] Impurity III-11 reference solution: Accurately measure 2 ml → 20 ml of the impurity III-11 reference stock solution.

[0335] Spiked test solution: take about 25 mg of this product and 5 ml of impurity III-11 reference stock solution → 50 ml.

[0336] Take the reference solution of impurity ⅲ-11 and the spiked test solution and place them at low temperature (injector temperature is 5°C), inject and analyze them at the end of 0h, 2h, 4h, 6h, 8h, 10h, 12h and 16h after preparation, record the chromatogram, and calculate the ratio of the impurity peak area at each time point to the peak area at 0h. Test results are shown in Tables 15 to 19.

[0337] Table 715 Peak area results of stability test of reference substance solution

[0338]

[0339] Table 16 Stability test results of spiked test solution 1

[0340]

[0341] Table 17 Stability test results of impurity ⅲ-11 reference solution and spiked test solution

[0342]

[0343]

[0344] Table 18 Control solution stability test results

[0345] Time / h Main component peak area Ratio of peak area to 0h (%) 0 98379 N / A 4 97921 99.5 13 97794 99.4

[0346] Table 819 Test solution stability test content results (%)

[0347]

[0348] Verification results: When placed under low temperature 5°C conditions, in the chromatogram of the reference solution, the ratio of the peak area of ​​impurity ⅲ-5 at each time point to the peak area at 0 hour was in the range of 92% to 105%, and the ratio of the peak area of ​​impurity ⅲ-2, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-4 and impurity ⅲ-12 to the peak area at 0 hour was in the range of 90% to 108%. The results showed that the reference solution was stable for at least 24 hours under low temperature 5°C conditions; in the chromatogram of the impurity ⅲ-11 reference solution, the ratio of the peak area of ​​impurity ⅲ-11 at each time point to the peak area at 0 hour was in the range of 90% to 108%. The results showed that the impurity ⅲ-11 reference solution was stable for at least 16 hours under low temperature 5°C conditions.

[0349] When placed at low temperature of 5°C, in the chromatogram of the control solution, the ratio of the peak area of ​​benzyl-2-bromoethyl ether at each time point to the peak area at 0 hour was in the range of 90% to 108%, indicating that the control solution was stable at low temperature of 5°C for at least 13 hours;

[0350] Placed at low temperature of 5°C, in the chromatogram of the test solution, the difference between the content of each impurity within 12 hours and the content at 0 hour was less than 0.05%, the difference between the total amount of impurities and the total amount of impurities at 0 hour was less than 0.1%, and no new impurities greater than the reporting limit were generated. The results showed that the test solution was stable at low temperature of 5°C for at least 12 hours. It is recommended that the test solution should be injected within 6 hours after preparation.

[0351] When placed under low temperature 5°C, in the chromatogram of the spiked test solution, the ratio of the peak area of ​​impurity ⅲ-5 at each time point to the peak area at 0 hour was in the range of 92% to 105%, and the ratio of the peak area of ​​impurity ⅲ-2, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-4 and impurity ⅲ-12 to the peak area at 0 hour was in the range of 90% to 108%; in the chromatogram of the impurity ⅲ-11 spiked test solution, the ratio of the peak area of ​​impurity ⅲ-11 within 10 hours to the peak area at 0 hour was in the range of 90% to 108%. The results showed that the spiked test solution was stable for at least 24 hours under low temperature 5°C, and the impurity ⅲ-11 spiked test solution was stable for at least 10 hours.

[0352] 2.6 Repeatability test

[0353] Prepare 6 spiked test solutions in parallel, and calculate the content of each known impurity by the external standard method and the principal component self-reference method with correction factor, respectively. The RSD value of the impurity ⅲ-5 content is not greater than 2%, the RSD values ​​of the impurity ⅲ-2, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-11, and impurity ⅲ-4 contents are all not greater than 3%, the RSD value of the impurity ⅲ-12 content is not greater than 6%, the RSD of the other largest single impurity content is not greater than 10%, and the RSD of the total impurity content is not greater than 3%.

[0354] 2.7 Reproducibility test

[0355] Different operators used different liquid chromatographs on different dates to prepare 6 spiked test solutions according to the repeatability test method, and calculated the content of each known impurity according to the principal component self-control method with correction factors. Based on the results of the combined repeatability test, among the 12 spiked test solutions, the RSD value of the impurity Ⅲ-5 content was not greater than 4%, the RSD values ​​of the impurity Ⅲ-2, impurity Ⅲ-8, impurity Ⅲ-10, impurity Ⅲ-11, and impurity Ⅲ-4 contents were all not greater than 6%, the RSD value of the impurity Ⅲ-12 content was not greater than 12%, the RSD of the other largest single impurity content was not greater than 15%, and the RSD of the total impurity content was not greater than 6%.

[0356] 2.8 Accuracy test

[0357] Solvent: water-acetonitrile (45:55).

[0358] 2.8.1 Accuracy Test 1

[0359] Prepare the mixed reference stock solution as described in “System suitability test”.

[0360] Reference substance solution: Mix 2 ml of reference substance stock solution → 20 ml.

[0361] Test solution: Take about 25 mg of the product → 50 ml.

[0362] Low concentration recovery solution: Take about 25 mg of this product and mix it with 2 ml of reference stock solution → 50 ml.

[0363] Medium concentration recovery solution: Take about 25 mg of this product and mix it with 5 ml of reference stock solution → 50 ml.

[0364] High concentration recovery solution: Take about 25 mg of this product and mix it with 10 ml → 50 ml of the reference stock solution.

[0365] 2.8.2 Accuracy Test 2

[0366] Prepare the impurity III-11 reference stock solution according to the "System Suitability Test".

[0367] Impurity III-11 reference solution: Accurately measure 2 ml → 20 ml of the impurity III-11 reference stock solution.

[0368] Test solution: Take about 25 mg of the product → 50 ml.

[0369] Refer to the preparation method of low, medium and high concentration recovery rate solutions in "Accuracy Test 1", take about 25 mg of this product, and then measure 2 ml, 5 ml, 10 ml → 50 ml of the stock solution of ⅲ-11 reference substance respectively.

[0370] 2.8.2 Accuracy Test 3

[0371] Prepare the impurity III-10 reference stock solution according to the "System Suitability Test".

[0372] Impurity III-10 reference solution: Accurately measure 2 ml of the impurity III-10 reference stock solution → 100 ml, 2 ml → 20 ml.

[0373] Test solution: Take about 25 mg of the product → 50 ml.

[0374] Refer to the preparation method of low, medium and high concentration recovery rate solutions in "Accuracy Test 1", take about 25 mg of this product, and then measure 2 ml, 5 ml, 10 ml → 50 ml of the III-10 reference substance stock solution respectively.

[0375] Accurately measure the solvent, reference solution, impurity III-10 reference solution, impurity III-10 reference solution, test solution and low, medium and high concentration recovery solutions, respectively inject them into liquid chromatograph, record the chromatogram, calculate the detection amount of each known impurity and the recovery rate by peak area according to the external standard method.

[0376] Calculation formula:

[0377]

[0378] Verification results:

[0379] Within the low, medium and high concentration ranges, the recovery rates of impurities ⅲ-5 and ⅲ-10 should be between 92% and 105%, and the recovery rates of impurity ⅲ-2, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-11, impurity ⅲ-4 and impurity ⅲ-12 should all be between 90% and 108%.

[0380] 2.9 Durability test

[0381] Prepare spiked test solution and control solution according to the method under "Repeatability test".

[0382] Sensitivity solution: Accurately measure 1 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0383] Accurately measure the solvent, sensitivity solution, control solution and spiked test solution, and inject them into the liquid chromatograph under the chromatographic conditions in Tables 20 and 21, and record the chromatograms. Repeat the injection of the control solution 5 times to investigate the system repeatability test. Investigate the impact of slight changes in chromatographic conditions on the system suitability test results and impurity detection results. Calculate the contents of impurity Ⅲ-5, impurity Ⅲ-2, impurity Ⅲ-8, impurity Ⅲ-10, impurity Ⅲ-11, impurity Ⅲ-4 and impurity Ⅲ-12 by the principal component self-reference method with correction factors, and calculate the content of unknown impurities by the principal component self-reference method. The test results are shown in Tables 20 to 25.

[0384] Table 20 Summary of durability inspection items

[0385]

[0386] Table 21 Durability Information of Chromatographic Columns Used

[0387]

[0388] Table 22 Durability test results of the effects of different changing conditions on the separation of each component

[0389]

[0390] Table 9 Durability test results of the influence of different changing conditions on the relative retention time of each impurity

[0391]

[0392]

[0393] Table 10 Durability test results of the influence of different changing conditions on the system suitability test results

[0394]

[0395] Table 25 Durability test results of the influence of different changing conditions on the determination results of each impurity content 1

[0396]

[0397]

[0398] Verification results: Under each condition, the control solution was injected 5 times repeatedly, and the RSD value of the main component retention time was less than 1%, and the RSD value of the main component peak area was less than 2%. In the chromatogram of the spiked test solution, the separation between each known impurity and the adjacent peak was greater than 1.5, the theoretical plate number of the main peak was greater than 5000, and the tailing factor of the main peak was less than 1.5. In the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main component peak height was greater than 10. The difference between the content of each impurity and the standard conditions was less than ±20% of the standard test result, indicating that the method has good durability when the flow rate (0.9ml / min~1.1ml / min), column temperature (40℃~50℃), buffer-acetonitrile (78:22~82:18), buffer salt concentration (18mmol / L~22mmol / L), buffer pH (2.9~3.1) changes and when replacing different batches of chromatographic columns of the same brand, model, and specification.

[0399] Using the relative retention time under standard conditions, the relative retention times of impurity Ⅲ-11, impurity Ⅲ-4, impurity Ⅲ-12, impurity Ⅲ-2, impurity Ⅲ-10, impurity Ⅲ-5 and impurity Ⅲ-8 were 0.25, 0.36, 0.82, 0.92, 0.94, 0.97 and 1.19, respectively.

[0400] Comparative Example 1, gas chromatography detection:

[0401] 1-1, Selection of chromatographic column:

[0402] Method 1, Agilent HP-5, 50m×0.320mm, 0.52μm column

[0403] Chromatographic conditions: capillary column (Agilent HP-5, 50m×0.320mm, 0.52μm) with 5% phenylmethylpolysiloxane as the stationary phase; starting temperature of 40°C, maintained for 5 minutes, heated to 150°C at a rate of 5°C per minute, then heated to 300°C at a rate of 50°C per minute, maintained for 10 minutes; injection port temperature of 300°C; detector temperature of 300°C; split ratio of 10:1; carrier gas flow rate of 2.0ml per minute, injection volume of 1.0μl.

[0404] Spiked test solution: Take about 200 mg of this product and place it in a 20 ml volumetric flask. Prepare the reference stock solution and take appropriate amount and place it in the same volumetric flask. The concentrations of impurity ⅲ-1, impurity ⅲ-7, impurity ⅲ-9, impurity ⅲ-13 and impurity ⅲ-15 are all 0.05 mg / ml, and the concentration of dichloromethane is 0.03 mg / ml. The chromatogram is shown in the figure below. Figure 3 shown.

[0405] Analysis: The dichloromethane peak overlaps with the solvent peak, impurity ⅲ-1 has severe tailing and poor peak shape, which affects the accurate quantification of the impurity; impurity ⅲ-9 and the previously unknown impurity peak do not achieve baseline separation and are not suitable for the detection of related substances of benzyl-2-bromoethyl ether.

[0406] Method 2, Agilent DB-624, 30m×0.53mm×3μm column

[0407] Take the spiked test solution prepared in "Method 1".

[0408] Chromatographic conditions: a capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary phase (Agilent DB-624, 30m×0.53mm×3μm); the starting temperature was 40°C, maintained for 5 minutes, raised to 150°C at a rate of 5°C per minute, and then raised to 250°C at a rate of 50°C per minute, maintained for 15 minutes; the injection port temperature was 250°C; the detector temperature was 250°C; the split ratio was 10:1; the carrier gas flow rate was 2.0ml per minute, and the injection volume was 1.0μl.

[0409] Accurately measure the spiked test solution, inject it into the gas chromatograph, and record the chromatogram. Figure 4 .

[0410] Analysis: The order of impurity peaks is dichloromethane, impurity ⅲ-13, impurity ⅲ-15, impurity ⅲ-7 and impurity ⅲ-9. Impurity ⅲ-1 overlaps with the solvent peak and no obvious impurity ⅲ-1 peak is observed. Impurities ⅲ-15 and impurity ⅲ-7 fail to achieve baseline separation. The peak shape of impurity ⅲ-13 is poor and is not suitable for the detection of related substances of benzyl-2-bromoethyl ether.

[0411] Method 3, Agilent DB-WAX, 30m×0.32mm×0.5μm column

[0412] Take the spiked test solution prepared in "Method 1".

[0413] Chromatographic conditions: a capillary column with 100% polyethylene glycol as the stationary liquid (Agilent DB-WAX, 30m×0.32mm×0.5μm); the starting temperature was 40°C, maintained for 5 minutes, raised to 150°C at a rate of 5°C per minute, then raised to 230°C at a rate of 50°C per minute, and maintained for 20 minutes; the injection port temperature was 230°C; the detector temperature was 230°C; the split ratio was 10:1; the carrier gas flow rate was 2.0 ml per minute, and the injection volume was 1.0μl.

[0414] Accurately measure the spiked test solution, inject it into the gas chromatograph, and record the chromatogram. Figure 5 .

[0415] Analysis: The separation between impurity Ⅲ-1 and the adjacent unknown impurity peak in the spiked test solution is poor; the separation between impurity Ⅲ-13 and the adjacent unknown impurity peak is poor, which is not suitable for the detection of related substances of benzyl-2-bromoethyl ether.

[0416] 1-2. Solution stability and solvent selection

[0417] Solvents: acetonitrile, ethyl acetate, n-butanol, methanol, n-propanol, tetrahydrofuran, n-hexane and n-heptane

[0418] Test solution: Take about 300 mg of the product, accurately weigh it, place it in a 10 ml volumetric flask, add acetonitrile to dissolve and dilute to the scale, and shake well.

[0419] Chromatographic conditions: capillary column (Agilent HP-5, 50m×0.320mm, 0.52μm) with 5% phenylmethylpolysiloxane as the stationary phase; starting temperature of 40°C, maintained for 5 minutes, heated to 150°C at a rate of 5°C per minute, then heated to 300°C at a rate of 50°C per minute, maintained for 10 minutes; injection port temperature of 300°C; detector temperature of 300°C; split ratio of 10:1; carrier gas flow rate of 2.0ml per minute, injection volume of 1.0μl.

[0420] Accurately measure each blank solvent and test solution, inject them into the gas chromatograph, and record the chromatogram. The stability results are shown in Table 26.

[0421] Table 26 Stability results

[0422]

[0423] The results showed that at each time point, the ratio of the peak area of ​​impurities ⅲ-4, ⅲ-11 and ⅲ-12 to the peak area of ​​0h exceeded the range of 90.0-108.0%, and the remaining impurities were all within the range of 90.0-108.0%. This indicates that impurities ⅲ-4, ⅲ-11 and ⅲ-12 have poor stability under gas chromatography conditions and are not suitable for gas phase detection.

[0424] Since there is a high-temperature gasification process in the gas phase, it may be a factor that causes the instability of each impurity. Impurity ⅲ-2, impurity ⅲ-4, impurity ⅲ-5, impurity ⅲ-8, impurity ⅲ-10, impurity ⅲ-11 and impurity ⅲ-12 have ultraviolet absorption. Liquid phase is used for method optimization, and a low-temperature injector can be used to stabilize each impurity. Since impurity ⅲ-1, impurity ⅲ-7, impurity ⅲ-9, impurity ⅲ-13 and impurity ⅲ-15 have no ultraviolet absorption, it is impossible to control them using liquid chromatography. Therefore, a gas chromatograph is used for method development.

[0425] Through the investigation of solvents, it was found that acetonitrile did not interfere with the detection of impurity iii-1, impurity iii-7, impurity iii-9, impurity iii-13, impurity iii-15 and dichloromethane. Ethyl acetate interfered with the detection of impurity iii-7 and impurity iii-15. n-Butanol interfered with the detection of impurity iii-1 and impurity iii-13. Methanol interfered with the detection of impurity iii-1. n-Propanol interfered with the detection of impurity iii-1, impurity iii-7 and impurity iii-15. Tetrahydrofuran interfered with the detection of impurity iii-7. n-Hexane did not interfere with the detection of impurity iii-1, impurity iii-7, impurity iii-9, impurity iii-13, impurity iii-15 and dichloromethane, but when n-hexane was used as the solvent, impurity iii-1 did not dissolve, and it was not suitable for the detection of benzyl-2-bromoethyl ether. n-Heptane interferes with the detection of impurities iii-1, iii-7, iii-13 and iii-15. Therefore, acetonitrile was used as the solvent.

[0426] Comparative Example 2, Liquid Chromatography Conditions

[0427] 2-1, Gradient selection

[0428] Method 1, spiked test solution: take about 10 mg of the product and place it in a 20 ml volumetric flask, prepare the stock solution of each reference substance and measure an appropriate amount, and place it in the same volumetric flask. The concentrations of impurity ⅲ-2, impurity ⅲ-4, impurity ⅲ-5, impurity ⅲ-8, impurity ⅲ-10, and impurity ⅲ-11 are all 0.002 mg / ml.

[0429] Impurity localization solution: prepare impurity localization solutions with a concentration of 0.007 mg / ml for impurity ⅲ-2, impurity ⅲ-4, impurity ⅲ-5, impurity ⅲ-8, impurity ⅲ-10 and impurity ⅲ-11 respectively.

[0430] The same chromatographic conditions as in Example 1 were used, except that the gradient elution procedure was different:

[0431]

[0432] Accurately measure the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. Figure 6 The results showed that in the spiked test solution, the separation effect of impurity ⅲ-2 and impurity ⅲ-10 was improved, but the baseline separation was not achieved, and it was not suitable for the detection of related substances of benzyl-2-bromoethyl ether.

[0433] 2-2, Selection of column temperature

[0434] Method 1 was used, the test solution was spiked, and the chromatographic conditions were the same as in Example 1, except that the column temperature was 30°C. The test results are shown in Figure 7 .

[0435] The results showed that when the column temperature was 30°C, the separation of impurity ⅲ-2 and impurity ⅲ-10 in the spiked test solution was poor and did not reach baseline separation, making it unsuitable for the detection of related substances of benzyl-2-bromoethyl ether.

[0436] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for separating and detecting related substances in benzyl-2-bromoethyl ether, a starting material of umeclidinium bromide, based on gas chromatography and liquid chromatography, characterized in that: It includes two modules: gas chromatography detection and liquid chromatography detection; Gas chromatography detection of impurities includes: Dichloromethane, ethylene glycol, 1,2-dibromoethane, 2,2'-dibromodiethyl ether, bromoethanol, 2-bromoethyl ethyl ether; Impurities detected by liquid chromatography include: Benzyl chloride, 2-benzyloxyethanol, benzyl bromide, ethylene glycol dibenzyl ether, benzyl-2-chloroethyl ether, benzyl alcohol, triphenylphosphine oxide; The sample preparation method is as follows: take an appropriate amount of the product, dissolve it in acetonitrile and quantitatively dilute it to make a solution with a concentration of 50 mg / ml, which is used as the test solution for gas chromatography detection; Dilute the sample solution for gas chromatography 100 times as the sample solution for liquid chromatography; The chromatographic conditions for gas chromatography detection were as follows: a capillary column with 100% polyethylene glycol filler as the stationary liquid as the chromatographic column; a starting temperature of 35-45°C, an injection port temperature of 225-235°C; a detector temperature of 225-235°C; a carrier gas flow rate of 1.4-1.6 ml per minute; In the gas chromatography test, the starting temperature was 40°C, maintained for 5 minutes, raised to 150°C at a rate of 5°C per minute, and then raised to 230°C at a rate of 50°C per minute and maintained for 20 minutes; The liquid chromatography conditions are as follows: the column is filled with octadecylsilane bonded silica gel; 18-22 mmol / L phosphate buffer is used as mobile phase A, acetonitrile is used as mobile phase B, and the flow rate is 0.9-1.1 ml per minute; the gradient elution program is: From 0 to 5 minutes, the volume percentage of mobile phase A was 78-82%. From 5 to 45 minutes, the volume percentage of mobile phase A linearly changed from 78-82% to 20%. From 45 to 50 minutes, the volume percentage of mobile phase A linearly changed from 20% to 78-82%. After 50 minutes, the volume percentage of mobile phase A remained unchanged at 78-82%. In liquid chromatography detection, the pH value of phosphate buffer is 2.9~3.

1.

2. The method according to claim 1, characterized in that In the gas chromatography detection, the chromatographic column used was Agilent DB-WAX, 30m×0.320mm×0.50µm.

3. The method according to claim 1, characterized in that In the liquid chromatography detection, the chromatographic column is WatersXBridge® C18, 4.6mm×250mm, 5μm chromatographic column; the column temperature is 40~50℃.

4. The method according to claim 1, characterized in that: Use 20mmol / L phosphate buffer as the mobile phase. The preparation method is: take 2.4g of anhydrous sodium dihydrogen phosphate, add 1000ml of water to dissolve it, and adjust the pH value to 3.0 with phosphoric acid.

5. The method according to claim 1, characterized in that In the liquid chromatography detection, the column temperature was 45°C; the detection wavelength was 210 nm; and the injection volume was 20 µl.

6. The method according to claim 1, characterized in that During the liquid chromatography test, the injector temperature was 5 °C.

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