A method for separating and detecting related substances in arformoterol tartrate intermediates by high performance liquid chromatography

Abstract

The invention discloses a method for separating and detecting related substances in arformoterol tartrate intermediates by high performance liquid chromatography, using high performance liquid chromatography, chromatographic column filler cellulose tris (3-chloro-4-methylphenylcarbamate) bonded silica gel as filler; mobile phase is n-hexane ethanol solution. The detection method provided by the present invention effectively solves the problem that the enantiomers and impurity II-9 in the arformoterol tartrate intermediate are similar to the intermediate structure, and the chromatogram of the conventional method is completely overlapped and difficult to separate. Compared with other chromatographic columns, including C8 chromatographic columns, reverse-phase chiral isomer chromatographic columns of different fillers, and normal-phase chiral isomer chromatographic columns of different fillers, it is found that the use of normal-phase chiral isomer chromatographic columns has the advantage of low back pressure, and the arformoterol tartrate intermediates, enantiomers and impurity II-9 are effectively separated, and the above two impurities can be accurately quantitatively analyzed, and the quality of the arformoterol tartrate intermediate is better controlled.

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 an arformoterol tartrate intermediate by using a high performance liquid chromatography method. Background Art

[0002] Arformoterol tartrate is chemically named N-[2-hydroxy-5-[(1R)-1-hydroxy-2-[(1R)-2-(4-methoxyphenyl)-1-methylethyl]amino]ethyl]phenyl]formamide-L-(+)-tartrate. It was developed by Sepracor in the United States, approved by the FDA in October 2006, and first launched in the United States in April 2007 under the trade name BROVANA. This product is suitable for long-term maintenance treatment of bronchoconstriction in patients with chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema. The synthesis of arformoterol tartrate is mainly divided into racemic method and asymmetric synthesis method.

[0003] The synthetic route of arformoterol tartrate is (Study on the synthetic process of arformoterol tartrate, Chen Jiao et al., Guangzhou Chemical Industry):

[0004]

[0005] Among them, intermediate 4 (named as intermediate II in this application) is a structure with therapeutic activity, and its isomer (S)-N-[2-(benzyloxy)-5-(2-bromo-1-hydroxyethyl)phenyl]formamide (named as impurity II-5 in this application) is an inactive isomer and is used as an impurity for quality control; in addition, the source of N-[2-(benzyloxy)-5-(2-bromo-1-hydroxyethyl)phenyl]acetamide (named as impurity II-9 in this application) is the acetylated impurity generated during the preparation of intermediate II.

[0006] The structural formula of impurity II-5 is:

[0007]

[0008] The structural formula of impurity II-9 is:

[0009]

[0010] After searching the literature and patents, no literature and patents for separating enantiomer II-5 and impurity II-9 in arformoterol tartrate intermediates have been found, and the technology is vacant. Since enantiomers and arformoterol tartrate intermediates are difficult to separate, and impurity II-9 and arformoterol tartrate intermediates have similar structures, the two are also difficult to separate, and the existing technology cannot effectively monitor arformoterol tartrate intermediates. Summary of the invention

[0011] In order to overcome the shortcomings of the prior art, the present invention provides a method for separating and detecting related substances in arformoterol tartrate intermediates by high performance liquid chromatography, which can effectively separate isomeric impurities and impurity II-9 in arformoterol tartrate intermediate II. The technical solution provided by the present invention is as follows:

[0012] A method for separating and detecting related substances in an arformoterol tartrate intermediate by high performance liquid chromatography, wherein the intermediate is intermediate II, and the structural formula is:

[0013]

[0014] High performance liquid chromatography was used, and chromatographic column filler was cellulose-tris (3-chloro-4-methylphenylcarbamate) bonded silica gel as filler;

[0015] The mobile phase was hexane-ethanol solution.

[0016] Furthermore, in the mobile phase, the volume ratio of n-hexane to ethanol is 87:13 to 89:11, preferably 88:12.

[0017] Preferably, the chromatographic column is Daicel OZ-3, 4.6 mm×250 mm, 3 μm or a chromatographic column with equivalent performance.

[0018] Preferably, the column temperature of the chromatographic column is 20-30°C, preferably 25°C.

[0019] Preferably, the flow rate of the mobile phase is 0.9 to 1.1 ml / min, preferably 1.0 ml / min.

[0020] Preferably, the detector is an ultraviolet detector with a detection wavelength of 214 nm.

[0021] Preferably, the related substances in the arformoterol tartrate intermediate include isomeric impurities II-5 and II-9.

[0022] Preferably, the method further comprises sample preparation:

[0023] Test solution: Dissolve the product in diluent and dilute to a concentration of 0.4 mg / ml.

[0024] Reference solution: Take appropriate amount of II-5 reference substance and II-9 reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing 10μg of II-5 and 10μg of II-9 per 1ml.

[0025] Preferably, the method further comprises determining:

[0026] Accurately measure the diluent, test solution and reference solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram; calculate the content of impurity II-5 and impurity II-9 in the test solution by peak area according to the external standard method, %;

[0027] Calculation formula:

[0028]

[0029] Where: A 供试 is the peak area of ​​Ⅱ-5 or Ⅱ-9 in the chromatogram of the test solution;

[0030] A 对照 is the peak area of ​​Ⅱ-5 or Ⅱ-9 in the chromatogram of the reference solution;

[0031] m 供试 is the sample weight of the test product, mg;

[0032] m 对照 is the sample weight of II-5 or II-9 reference substance, mg;

[0033] V 供试 is the dilution volume of the test solution, ml;

[0034] V 对照 is the dilution volume of II-5 or II-9 reference solution, ml;

[0035] N is the content of II-5 or II-9 reference substance.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] 1. The detection method provided by the present invention effectively solves the problem that the enantiomers and impurity II-9 in the arformoterol tartrate intermediate are similar to the intermediate in structure, causing the chromatograms of the conventional method to completely overlap and be difficult to separate. Compared with other chromatographic columns, including C8 chromatographic columns, reverse-phase chiral isomer chromatographic columns with different fillers, and normal-phase chiral isomer chromatographic columns with different fillers, it is found that the use of the normal-phase chiral isomer chromatographic column has the advantage of low back pressure, and the arformoterol tartrate intermediate, enantiomers and impurity II-9 are effectively separated; at the same time, the chromatographic column needs to be matched with a mobile phase with a specific composition and ratio, and the above two impurities can be accurately quantitatively analyzed, thereby better controlling the quality of the arformoterol tartrate intermediate.

[0038] 2. The method of the present invention has strong specificity and applicability, high detection sensitivity, good linear relationship, high precision and accuracy, good durability of the detection method, high detection efficiency, and does not affect the accurate detection and quantification of impurities when there is a certain degree of fluctuation in the chromatographic conditions, thereby effectively controlling the quality of the product.

[0039] 3. In the prior art, there is a lack of effective detection methods for enantiomers and impurity II-9 in arformoterol tartrate intermediates. The present invention provides a detection method for impurities that have long lacked effective detection methods, making up for the inability of the industry to conduct more comprehensive quality control of arformoterol tartrate intermediates.

[0040] 4. The method provided by the present invention can achieve effective detection and accurate quantification of enantiomers and impurity II-9 without affecting the presence of other impurities. Therefore, the present invention provides a complete analytical method for arformoterol tartrate intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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.

[0042] Figure 1 The chromatogram of the test solution in Example 1;

[0043] Figure 2 It is a chromatogram overlay of the specific property test in Example 2, wherein the chromatograms are, from top to bottom, impurity II-6 interfering solution, impurity II-3 interfering solution, impurity II-7 interfering solution, impurity I-8 interfering solution, impurity II-1 interfering solution, impurity II-8 interfering solution, impurity II-5 interfering solution, impurity II-9 interfering solution, mixed solution, test solution and blank solvent;

[0044] Figure 3 is a separate chromatogram of the mixed solution in the specificity test in Example 2;

[0045] Figure 4 Overlapping chromatograms of the diluent, impurity localization solution, test solution and mixed solution of Method 1 in Comparative Example 1;

[0046] Figure 5 It is the chromatogram of method 2 in comparative example 1;

[0047] Figure 6 This is the chromatogram of method 3 in comparative example 1;

[0048] Figure 7 This is the chromatogram of method 4 in Comparative Example 1;

[0049] Figure 8 It is the chromatogram of method 5 in comparative example 1;

[0050] Fig. 9 This is the chromatogram of method 6.1 in Comparative Example 1;

[0051] Fig.10 This is the chromatogram of method 6.2 in Comparative Example 1;

[0052] Fig.11 This is the chromatogram of method 6.3 in Comparative Example 1;

[0053] Fig.12 This is the chromatogram of method 7 in Comparative Example 1;

[0054] Fig.13 This is the chromatogram of method 8 in comparative example 1. DETAILED DESCRIPTION

[0055] 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.

[0056] 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.

[0057] During the method development process, the relevant impurity structures of concern are as follows:

[0058]

[0059]

[0060] Impurity II-1: Intermediate I. If the reaction is incomplete, it may remain in intermediate II and needs to be studied.

[0061] Impurity Ⅰ-8: impurities remain in intermediate Ⅰ, and if the formylation reaction of the nitro reduction product of intermediate Ⅰ is incomplete, impurity Ⅰ-8 may also be generated and remain in intermediate Ⅱ, which needs to be studied.

[0062] Impurity II-3: Intermediate I contains impurity I-2 which participates in the reaction to generate impurity II-3. In addition, debromination reaction may also occur during the preparation of intermediate II, and the resulting impurity II-3 needs to be studied.

[0063] Impurity II-5: An isomeric impurity generated during the preparation of intermediate II may remain in intermediate II and needs to be studied.

[0064] Impurity II-6: Impurity I-6 contained in intermediate I participates in subsequent nitro reduction and formylation reactions to generate impurity II-6, and intermediate II may also undergo a cyclization reaction. The resulting impurity II-6 needs to be studied.

[0065] Impurity II-7: Impurity I-6 contained in intermediate I can participate in the reaction to generate impurity II-7. The nitro reduced product of intermediate I may also undergo a cyclization reaction, and the resulting impurity II-7 needs to be studied.

[0066] Impurity II-8: The debenzylated impurity generated during the preparation of Intermediate II may remain in Intermediate II and needs to be studied.

[0067] Impurity II-9: Acetylated impurity generated during the preparation of intermediate II may remain in intermediate II and needs to be studied.

[0068] The above impurities may remain in the intermediates. In order to eliminate the possibility that these impurities may affect the detection and quantification of impurities II-5 and II-9, the inventors deliberately investigated the peak conditions of these impurities in this method to avoid their influence on the impurities to be detected.

[0069] Example 1, a method for separating and detecting related substances in arformoterol tartrate intermediates by high performance liquid chromatography

[0070] High performance liquid chromatography

[0071] Detector: UV detector

[0072] Chromatographic column: Daicel OZ-3, 4.6mm×250mm, 3μm

[0073] Mobile phase: n-hexane-ethanol (88:12);

[0074] The flow rate was 1.0 ml per minute; the detection wavelength was 214 nm; the column temperature was 25°C; and the injection volume was 20 μl.

[0075] Diluent: Ethanol

[0076] Test solution: Take about 20 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0077] II-5 reference substance stock solution: Take about 12.5 mg of II-5 reference substance, weigh accurately, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0078] II-9 reference substance stock solution: Take about 12.5 mg of II-9 reference substance, weigh accurately, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0079] Reference substance solution: Accurately measure 1 ml of II-5 reference substance stock solution and 1 ml of II-9 reference substance stock solution, place them in the same 50 ml volumetric flask, dilute to the scale with ethanol, and shake well.

[0080] Determination method:

[0081] Accurately measure ethanol, test solution and reference solution, inject them into high performance liquid chromatography, and record the chromatogram; calculate the content of impurity II-5 and impurity II-9 in the test solution by peak area according to the external standard method;

[0082] Calculation formula:

[0083]

[0084] Where: A 供试 is the peak area of ​​Ⅱ-5 or Ⅱ-9 in the chromatogram of the test solution;

[0085] A 对照 is the peak area of ​​Ⅱ-5 or Ⅱ-9 in the chromatogram of the reference solution;

[0086] m 供试 is the sample weight of the test product, mg;

[0087] m 对照 is the sample weight of II-5 or II-9 reference substance, mg;

[0088] V 供试 is the dilution volume of the test solution, ml;

[0089] V 对照 is the dilution volume of II-5 or II-9 reference solution, ml;

[0090] N is the content of II-5 or II-9 reference substance.

[0091] The test results are shown in Table 1. Figure 1 .

[0092] Table 1 Sample test results

[0093] Sample batch number 2103926 2108910 Ⅱ-5(％) 0.07 0.11 Ⅱ-9(％) 1.08 1.13

[0094] The results showed that the contents of impurity II-5 and impurity II-9 in batch 2108910 were 0.11% and 1.13% respectively, and the contents of impurity II-5 and impurity II-9 in batch 2103926 were 0.07% and 1.08% respectively.

[0095] Example 2, method verification test

[0096] 2.1 System suitability test

[0097] II-5 reference substance stock solution: Take about 12.5 mg of II-5 reference substance, weigh accurately, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0098] II-9 reference substance stock solution: Take about 12.5 mg of II-9 reference substance, weigh accurately, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0099] System suitability solution: Take about 20 mg of intermediate II, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately measure 1 ml of the stock solution of II-5 and II-9 reference substances, place them in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0100] Accurately measure ethanol and system suitability solution, inject them into the high performance liquid chromatograph respectively, inject the system suitability solution 5 times continuously, record the chromatogram, record the separation degree, theoretical plate number, tailing factor, signal-to-noise ratio, retention time and peak area of ​​Ⅱ-5 and Ⅱ-9, and calculate the RSD value of retention time and peak area. The test results are shown in Table 2.

[0101] Table 2 System suitability test results

[0102]

[0103] Verification results: The system suitability solution was injected 5 times continuously, the RSDs of retention times of Ⅱ-5 and Ⅱ-9 were 0.3% and 0.3% respectively, which were less than 1%; the RSDs of peak areas were 0.3% and 0.2% respectively, which were less than 2%, the separation degrees between intermediate Ⅱ and Ⅱ-5 and Ⅱ-9 were all greater than 1.5, the theoretical plate numbers of intermediate Ⅱ peaks were greater than 1500, and the tailing factor did not affect the detection of Ⅱ-5 and Ⅱ-9. The results showed that the system had good applicability.

[0104] 2.2 Specificity test

[0105] Prepare II-5 reference substance stock solution and II-9 reference substance stock solution according to the method under "System suitability test".

[0106] Stock solutions of reference substances Ⅱ-1, Ⅱ-6, Ⅱ-7, and Ⅰ-8: Take about 2.5 mg of each reference substance Ⅱ-1, Ⅱ-6, Ⅱ-7, and Ⅰ-8, place them in 4 different 50 ml volumetric flasks, add ethanol to dissolve and dilute to the scale, and shake well.

[0107] Stock solutions of II-8 and II-3 reference substances: Take about 5 mg of II-8 reference substance and 3 mg of II-3 reference substance, place them in two different 10 ml volumetric flasks respectively, add ethanol to dissolve and dilute to the scale, and shake well.

[0108] Each impurity interference solution: Accurately measure 1 ml each of Ⅱ-1 reference substance stock solution, Ⅱ-6 reference substance stock solution, Ⅱ-7 reference substance stock solution, Ⅰ-8 reference substance stock solution, Ⅱ-8 reference substance stock solution, and Ⅱ-3 reference substance stock solution, place them in 6 different 50 ml volumetric flasks, dilute to the scale with ethanol, and shake well.

[0109] II-5 reference substance solution: Accurately measure 1 ml of II-5 reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0110] II-9 reference substance solution: Accurately measure 1 ml of II-9 reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0111] Test solution: Take about 20 mg of intermediate II, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0112] Mixed solution: Take about 20 mg of intermediate II, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately measure 1 ml each of Ⅱ-5 reference substance stock solution and Ⅱ-9 reference substance stock solution, place them in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0113] Precisely measure the blank solvent, impurity positioning solution, each impurity interference solution, test solution and mixed solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram. The test results are shown in Table 3. Figure 2 , Figure 3 .

[0114] Table 3 Specificity test results

[0115]

[0116] Verification results: Blank solvent, main component and Ⅱ-1, Ⅱ-3, Ⅱ-6, Ⅱ-7, Ⅱ-8, Ⅰ-8 do not interfere with the detection of Ⅱ-5 and Ⅱ-9. The separation degree of intermediates Ⅱ, Ⅱ-5 and Ⅱ-9 is greater than 1.5. The results show good specificity.

[0117] 2.3 Limit of quantitation and limit of detection test

[0118] Prepare II-5 reference substance stock solution and II-9 reference substance stock solution according to the method under "System suitability test".

[0119] Intermediate II reference substance stock solution: Take about 12.5 mg of intermediate II reference substance, accurately weigh, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0120] II-5 reference substance solution: Accurately measure 1 ml of II-5 reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0121] Intermediate II reference solution: Accurately measure 1 ml of intermediate II reference stock solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0122] II-9 reference substance solution: Accurately measure 1 ml of II-9 reference substance stock solution, place it in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0123] Quantitation limit solution: Accurately measure 1 ml of II-5 reference solution, 1 ml of intermediate II reference solution, and 0.5 ml of II-9 reference solution, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0124] Detection limit solution: Accurately measure 5 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0125] Report the lowest quantitative and detection concentration (μg / ml) and the percentage relative to the test sample concentration. Prepare 6 quantitation limit concentration solutions and inject them in sequence, record the chromatograms, and examine the repeatability of the quantitation limit solution. The test results are shown in Tables 4 and 5.

[0126] Table 4 Results of quantitation limit and detection limit tests

[0127]

[0128] Table 5 Results of repeatability test of limit of quantitation

[0129]

[0130]

[0131] Verification results: The minimum quantitative concentrations of Ⅱ-5, intermediate Ⅱ and Ⅱ-9 were 0.098μg / ml, 0.099μg / ml and 0.048μg / ml, respectively; the minimum quantitative limits of Ⅱ-5, intermediate Ⅱ and Ⅱ-9 were 1.96ng, 1.98ng and 0.96ng, respectively; when the test sample concentration was 0.4mg / ml, the quantitative limits of Ⅱ-5, intermediate Ⅱ and Ⅱ-9 were equivalent to 0.02%, 0.02% and 0.01% of the test sample concentration, respectively; the signal-to-noise ratios of the quantitative limits of Ⅱ-5, intermediate Ⅱ and Ⅱ-9 were 12.1, 13.2 and 11.6, respectively.

[0132] The minimum detection concentrations of Ⅱ-5, intermediate Ⅱ, and Ⅱ-9 were 0.049μg / ml, 0.050μg / ml, and 0.020μg / ml, respectively. The minimum detection limits of Ⅱ-5, intermediate Ⅱ, and Ⅱ-9 were 1.0ng, 1.0ng, and 0.4ng, respectively. When the concentration of the test sample was 0.4mg / ml, the detection limit concentrations of Ⅱ-5, intermediate Ⅱ, and Ⅱ-9 were equivalent to 0.01%, 0.01%, and 0.005% of the test sample concentration, respectively. The signal-to-noise ratios of the detection limits of Ⅱ-5, intermediate Ⅱ, and Ⅱ-9 were 4.8, 3.2, and 3.8, respectively. The RSD values ​​of the repeatability of the quantitative limit of Ⅱ-5, intermediate Ⅱ, and Ⅱ-9 were 0.4%, 0.2%, and 0.07%, respectively, which were all less than 1%, and the RSD values ​​of the peak area were 7.3%, 7.8%, and 13.8%, respectively, which were all less than 15%. The results all met the requirements, and the results showed that the quantitative limit repeatability was good.

[0133] 2.4 Linearity and range tests

[0134] Prepare II-5 reference substance stock solution and II-9 reference substance stock solution according to the “System suitability test”.

[0135] Intermediate II reference substance stock solution: Take about 12.5 mg of intermediate II reference substance, accurately weigh, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, shake well, and obtain.

[0136] Accurately measure 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.5ml, 2.0ml of Ⅱ-9 reference substance stock solution, Ⅱ-5 reference substance stock solution and intermediate Ⅱ reference substance stock solution, respectively, and place them in 6 50ml volumetric bottles, dilute to the scale with ethanol, shake well, and use them as 20%, 50%, 80%, 100%, 150%, 200% linear solutions (respectively as the 2nd to 7th points of the linear test). Take the quantitative limit solution as the first point of linearity.

[0137] Accurately measure the quantitative limit solution, 20%, 50%, 80%, 100%, 150%, and 200% linear solutions, inject them into the high performance liquid chromatograph, and record the chromatogram. Perform linear regression with concentration C (μg / ml) as the abscissa and peak area A as the ordinate. The test results are shown in Tables 6 to 8.

[0138] Table 6 Impurity II-5 linearity and range test results

[0139]

[0140] Table 7 Impurity II-9 linearity and range test results

[0141]

[0142] Table 8 Intermediate II linearity and range test results

[0143]

[0144] Verification results: The linear equations of Ⅱ-5 in the concentration range of 0.098μg / ml~19.600μg / ml, intermediate Ⅱ in the range of 0.099μg / ml~19.760μg / ml, and Ⅱ-9 in the range of 0.048μg / ml~19.320μg / ml were A=114168.8017C+29617.2743, A=115099.8106C+21501.0491, and A=116271.0045C+16431.7961 respectively; r were 0.9995, 0.9998, and 0.9999 respectively, all greater than 0.99. The results showed that Ⅱ-5, intermediate Ⅱ and Ⅱ-9 had good linear relationships in the range of quantitative limit to limit concentration 200%.

[0145] 2.5 Solution stability test

[0146] Prepare II-5 reference substance stock solution and II-9 reference substance stock solution according to the “System suitability test”.

[0147] Reference substance solution: Accurately measure 1 ml of Ⅱ-5 reference substance stock solution and Ⅱ-9 reference substance stock solution, place in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0148] Test solution: Take about 20 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0149] Spiked test solution: Take about 20 mg of this product, accurately weigh it, and place it in a 50 ml volumetric flask. Accurately measure 1 ml of II-5 reference stock solution and place it in the same 50 ml volumetric flask. Add ethanol to dissolve and dilute to the scale, shake well. Since the content of impurity II-5 in the test sample is relatively small (refer to the detection results and Figure 1 Therefore, in the method validation test, the solution stability, repeatability, intermediate precision and durability were tested using a spiked test solution with impurity II-5 added to verify the reliability of the method.

[0150] The reference solution, the test solution and the spiked test solution were placed at room temperature, and the samples were analyzed at 0h, 3h, 6h, 9h, 12h and 24h, and the chromatogram was recorded. The ratio of the impurity peak area at each time point in the reference solution to the peak area at 0h was calculated, and the difference between the impurity content at each time point in the test solution and the spiked test solution and the content at 0h was calculated. The test results are shown in Tables 9 and 10.

[0151] Table 9 Results of stability test of reference solution

[0152]

[0153] Table 10 Stability test results of test solution and spiked test solution

[0154]

[0155] Verification results: In the chromatogram of the reference solution, the ratio of the peak area of ​​Ⅱ-5 at each time point within 24 hours to the peak area at 0 hour was between 93.3% and 100.0%, all within the range of 92% to 105%; the ratio of the peak area of ​​Ⅱ-9 at each time point within 24 hours to the peak area at 0 hour was between 97.8% and 100.0%, all within the range of 92% to 105%; but at 9 hours, an impurity peak appeared after Ⅱ-5. The results showed that the reference solution had good stability at room temperature for at least 6 hours.

[0156] The differences between the contents of Ⅱ-5 and Ⅱ-9 in the test solution and the spiked test solution within 24 hours and those at 0 hours were no more than 0.01%. The results showed that the test solution and the spiked test solution had good stability at room temperature for at least 24 hours.

[0157] 2.6 Repeatability test

[0158] Prepare II-5 reference substance stock solution and II-9 reference substance stock solution according to the “System suitability test”.

[0159] Reference substance solution: Accurately measure 1 ml of Ⅱ-5 reference substance stock solution and Ⅱ-9 reference substance stock solution, place in a 50 ml volumetric flask, dilute to the mark with ethanol, and shake well.

[0160] Test solution: Take about 20 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0161] Spiked test solution: Take about 20 mg of this product, weigh accurately, and place in a 50 ml volumetric flask. Accurately measure 1 ml of II-5 reference stock solution, place in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the mark, shake well. Prepare 6 copies in parallel.

[0162] Accurately measure ethanol, reference solution, test solution and spiked test solution, inject them into high performance liquid chromatograph respectively, and record the chromatogram. Calculate the contents of impurities II-5 and II-9 in the spiked test solution by peak area according to the external standard method and calculate the recovery rate of impurity II-5. The test results are shown in Table 11.

[0163] Table 11 Repeatability test results

[0164] Sample No. Ⅱ-9 content (%) Ⅱ-5 Recovery rate (%) 1 1.16 98.76 2 1.17 99.04 3 1.18 98.66 4 1.18 99.75 5 1.17 99.47 6 1.17 98.74 Mean (%, n=6) 1.17 99.1 RSD (%, n = 6) 0.7 0.5

[0165] Verification results: The recovery of Ⅱ-5 and the content of Ⅱ-9 were calculated by external standard method for 6 spiked test solutions. The recovery of Ⅱ-5 was in the range of 98.66% to 99.75%, the RSD of the recovery was 0.5%, and the RSD of the content of Ⅱ-9 was 0.7%, both less than 2%. The results show that the method has good repeatability.

[0166] 2.7 Intermediate precision test

[0167] Six spiked test solutions were prepared by different analysts at different times according to the repeatability test method, and the solutions were determined by the same method on different liquid chromatographs. The repeatability test results were combined for a total of 12 samples to examine the intermediate precision. The test results are shown in Table 12.

[0168] Table 12 Intermediate precision test results

[0169]

[0170] Verification results: In 12 spiked test solutions, the recovery of Ⅱ-5 and the content of Ⅱ-9 were calculated by peak area according to the external standard method. The recovery of Ⅱ-5 was in the range of 98.43% to 100.83%, the RSD of the recovery was 0.9%, and the RSD of the content of Ⅱ-9 was 1.0%, both less than 4%. The results show that the intermediate precision of this method is good.

[0171] 2.8 Accuracy test

[0172] II-5 reference substance stock solution: Take about 12.5 mg of II-5 reference substance, weigh accurately, place in a 25 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0173] II-9 reference substance stock solution: Take about 12.5 mg of II-9 reference substance, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0174] Reference substance solution: Accurately measure 1 ml of II-5 reference substance stock solution and 2 ml of II-9 reference substance stock solution, place them in the same 50 ml volumetric flask, dilute to the scale with ethanol, and shake well.

[0175] Test solution: Take about 20 mg of the product, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0176] Low concentration recovery solution: Take about 20 mg of intermediate II, weigh accurately, and accurately measure 0.5 ml of II-5 reference substance stock solution and 0.5 ml of II-9 reference substance stock solution, place in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, shake well. Prepare 3 copies in parallel.

[0177] Medium concentration recovery solution: Take about 20 mg of intermediate II, weigh accurately, and accurately measure 1 ml of II-5 reference substance stock solution and 1 ml of II-9 reference substance stock solution, place in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, shake well. Prepare 3 copies in parallel.

[0178] High concentration recovery solution: Take about 20 mg of intermediate II, weigh accurately, and accurately measure 1.5 ml of II-5 reference substance stock solution and 2 ml of II-9 reference substance stock solution, put them in the same 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, shake well. Prepare 3 copies in parallel.

[0179] Accurately measure ethanol, reference solution, test solution and three concentrations of sample recovery solutions, inject them into the liquid chromatograph respectively, and calculate the recovery of impurities II-5 and II-9 by peak area according to the external standard method. The test results are shown in Tables 13 and 14.

[0180] Calculation formula:

[0181]

[0182] Table 13Ⅱ-5 Accuracy test results

[0183]

[0184]

[0185] Table 14Ⅱ-9 Accuracy test results

[0186]

[0187] Verification results: Within the limit concentration range of 50% to 150%, the recoveries of Ⅱ-5 and Ⅱ-9 were in the range of 99.12% to 103.47% and 94.15% to 99.12%, respectively, and both were between 92% and 105%. The results showed that the method had good accuracy.

[0188] 2.9 Durability test

[0189] Prepare reference solution, spiked test solution and test solution according to the method under "Repeatability test".

[0190] Accurately measure ethanol, reference solution, spiked test solution and test solution, inject them into the liquid chromatograph respectively, and record the chromatogram. According to the conditions of Table 15-Table 16, the samples were injected and tested respectively to investigate the influence of slight changes in chromatographic conditions on the impurity detection results, relative retention time, theoretical plate number, tailing factor and separation degree. The test results are shown in Table 17-Table 18.

[0191] Table 15 Summary of durability inspection items

[0192]

[0193] Table 16 Durability Information of Chromatographic Columns Used

[0194]

[0195] Table 17 Durability test system suitability results

[0196]

[0197] Table 18 Durability test results

[0198]

[0199] Verification results: The recovery of Ⅱ-5 under each condition is in the range of 98.2% to 104.0%, the ratio of the content of Ⅱ-9 to the content of Ⅱ-9 under standard conditions is in the range of 100.0% to 104.3%, the separation between intermediate Ⅱ and Ⅱ-5 and Ⅱ-9 should be greater than 1.5, the theoretical plate number of intermediate Ⅱ peak is not less than 1500, the tailing factor does not affect the detection of impurities, and the system suitability requirements are met. The results show that the method has good durability.

[0200] Comparative Example 1, Detection of related substances under different chromatographic conditions

[0201] Method 1: Use Kromasil C8, 4.6mm×250mm, 3.5μm for separation, the effect is poor.

[0202] Diluent: acetonitrile-water (50:50).

[0203] Stock solution of impurity reference substance: Take about 2 mg of each of the impurity reference substances Ⅱ-8, Ⅱ-7, Ⅱ-6, Ⅱ-3, Ⅰ-8, and Ⅱ-1, place them in 6 10 ml volumetric flasks, add diluent to dissolve and dilute to the scale, and shake well.

[0204] Mixed solution: Take about 10 mg of the intermediate, and accurately measure 1 ml, 0.25 ml, 0.25 ml, 0.5 ml, 0.25 ml, and 0.25 ml of the impurity reference stock solutions of Ⅱ-8, Ⅱ-7, Ⅱ-6, Ⅱ-3, Ⅰ-8, and Ⅱ-1 respectively, place them in the same 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0205] Impurity localization solution: Accurately measure 1 ml, 0.25 ml, 0.25 ml, 0.5 ml, 0.25 ml and 0.25 ml of the stock solutions of impurity reference substances Ⅱ-8, Ⅱ-7, Ⅱ-6, Ⅱ-3, Ⅰ-8 and Ⅱ-1 respectively and place them in 6 100 ml volumetric flasks respectively. Add diluent to dissolve and dilute to the scale and shake well.

[0206] Ⅱ-5 positioning solution: Take about 10 mg of intermediate Ⅱ-5 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0207] Ⅱ-9 positioning solution: Take about 10 mg of intermediate Ⅱ-9 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, accurately measure 1 ml, place it in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0208] Test solution: Take about 10 mg of the intermediate, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0209] Chromatographic conditions: octylsilane bonded silica gel was used as filler (Kromasil C8, 4.6 mm × 250 mm, 3.5 μm); phosphate buffer (3.7 g of sodium dihydrogen phosphate monohydrate was dissolved in 1000 ml of water and the pH value was adjusted to 3.1 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B; the flow rate was 1.0 ml per minute, and linear gradient elution was performed according to the table below; the column temperature was 50°C; the detection wavelength was 214 nm; and the injection volume was 20 μl.

[0210]

[0211] Accurately measure the diluent, impurity location solution, test solution and mixed solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Figure 4 .

[0212] The results showed that the enantiomer (impurity II-5) completely overlapped with the intermediate, and the separation between impurity II-9 and the intermediate was poor, making it unsuitable for detecting enantiomers and impurity II-9.

[0213] Method 2: Using Merck KGaA Chiradex, 4.0 mm × 250 mm, 5 μm column

[0214] Diluent: acetonitrile-water (50:50).

[0215] II-5 reference substance stock solution: Take about 10 mg of intermediate II-5 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0216] II-9 reference substance stock solution: Take about 10 mg of intermediate II-9 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0217] Spiked test solution: Take about 10 mg of the intermediate and place it in a 25 ml volumetric flask. Accurately measure 1 ml of the II-5 and II-9 reference stock solutions and place them in the same 25 ml volumetric flask. Add diluent to dissolve and dilute to the scale, and shake well.

[0218] Chromatographic conditions: β-cyclodextrin bonded silica gel was used as filler (Merck KGaA Chiradex,

[0219] 4.0mm×250mm, 5μm); phosphate buffer (1.2g of sodium dihydrogen phosphate was taken and dissolved in 1000ml of water)-acetonitrile (70:30) was used as the mobile phase; the flow rate was 1.0ml per minute; the column temperature was 30℃; the detection wavelength was 214nm; and the injection volume was 10μl.

[0220] Accurately measure the diluent and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 5 .

[0221] The results showed that the enantiomer (impurity II-5) and impurity II-9 completely overlapped with the intermediate and were not suitable for detecting enantiomers and impurity II-9.

[0222] Method 3: Use Shodex Asahipak ODP-50-4D, 250mm×4.6mm, 5μm column to spike the sample solution: Use the spiked sample solution of “Method 2”.

[0223] Chromatographic conditions: octadecyl polyvinyl alcohol was used as filler (Shodex Asahipak ODP-50-4D, 250 mm × 4.6 mm, 5 μm); phosphate buffer (2.3 g of tripotassium phosphate was dissolved in 1000 ml of water and the pH value was adjusted to 12.0 with potassium hydroxide solution)-acetonitrile (60:40) was used as mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 15°C; the detection wavelength was 214 nm; and the injection volume was 10 μl.

[0224] Accurately measure the diluent and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 6 .

[0225] The results showed that the peak shapes of the enantiomer (impurity II-5) and impurity II-9 were poor, and the enantiomer (impurity II-5) completely overlapped with the intermediate, making it unsuitable for detecting the enantiomer and impurity II-9.

[0226] Method 4: Using IG-3, 4.6mm×250mm, 3μm column

[0227] Spiked test solution: Use the spiked test solution of "Method 2".

[0228] Chromatographic conditions: amylose-tris (3-chloro-5-methylphenylcarbamate) bonded silica gel was used as the filler (Daikolu IG-3, 4.6 mm × 250 mm, 3 μm); phosphate buffer (1.2 g of diammonium hydrogen phosphate was dissolved in 1000 ml of water)-methanol-acetonitrile (50:10:40) was used as the mobile phase; the flow rate was 0.3 ml per minute; the column temperature was 25°C; the detection wavelength was 214 nm; and the injection volume was 10 μl.

[0229] Accurately measure the diluent and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 7 .

[0230] The results showed that impurity II-9 completely overlapped with the intermediate and was not suitable for detecting impurity II-9.

[0231] Method 5: Using CHIRALCEL OJ, 4.6 mm × 250 mm, 10 μm column

[0232] Diluent: ethanol.

[0233] II-5 reference substance stock solution: Take about 10 mg of intermediate II-5 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0234] II-9 reference substance stock solution: Take about 10 mg of intermediate II-9 reference substance, place it in a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0235] Spiked test solution: Take about 10 mg of the intermediate and place it in a 25 ml volumetric flask. Accurately measure 1 ml of the II-5 and II-9 reference stock solutions and place them in the same 25 ml volumetric flask. Add diluent to dissolve and dilute to the scale, and shake well.

[0236] Chromatographic conditions: cellulose-tri(4-methylbenzoate) bonded silica gel as filler (CHIRALCEL OJ, 4.6 mm×250 mm, 10 μm); n-hexane-ethanol (70:30) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 30°C; detection wavelength of 214 nm; injection volume of 20 μl.

[0237] Accurately measure the diluent and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Figure 8 .

[0238] The results showed that the enantiomer (impurity II-5) completely overlapped with the intermediate and was not suitable for detecting the enantiomer (impurity II-5).

[0239] Method 6: Using OD-H, 4.6mm×250mm, 5μm column

[0240] 6.1, the mobile phase is n-hexane-isopropanol (97:3)

[0241] Diluent: ethanol.

[0242] Spiked test solution: Use the spiked test solution of "Method 5".

[0243] Chromatographic conditions: cellulose-tris (3,5-dimethylphenylcarbamate) bonded silica gel as filler (Daikolu OD-H, 4.6 mm × 250 mm, 5 μm); n-hexane-isopropanol (97:3) as mobile phase; flow rate of 0.6 ml per minute; column temperature of 35 ° C; detection wavelength of 214 nm; injection volume of 20 μl.

[0244] Accurately measure the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. Fig. 9 .

[0245] The results showed that the intermediate, impurity II-9 and enantiomer (impurity II-5) had no obvious main peaks, indicating that the elution ability of the isopropanol system was too weak. When n-hexane-isopropanol (97:3) was used as the mobile phase, it was not enough to elute the three substances from the chromatographic column, and was not suitable for detecting impurity II-9 and enantiomer (impurity II-5). Increasing the proportion of isopropanol can improve the elution ability of the mobile phase.

[0246] 6.2, n-hexane-isopropanol (85:15), change the mobile phase ratio

[0247] Diluent: ethanol.

[0248] Spiked test solution: Use the spiked test solution of "Method 5".

[0249] Chromatographic conditions: cellulose-tris (3,5-dimethylphenylcarbamate) bonded silica gel as filler (Daikolu OD-H, 4.6 mm × 250 mm, 5 μm); n-hexane-isopropanol (85:15) as mobile phase; flow rate of 0.6 ml per minute; column temperature of 35 ° C; detection wavelength of 214 nm; injection volume of 20 μl.

[0250] Accurately measure the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. Fig.10 .

[0251] The results showed that the intermediate, impurity II-9 and enantiomer (impurity II-5) had no obvious main peaks, indicating that the elution ability of the isopropanol system was too weak. When n-hexane-isopropanol (85:15) was used as the mobile phase, it was not enough to elute the three substances from the chromatographic column, and was not suitable for the detection of impurity II-9 and enantiomer (impurity II-5).

[0252] 6.3, n-hexane-ethanol (88:12), except for the chromatographic column, other conditions are consistent with the example method. Diluent: ethanol.

[0253] Spiked test solution: Use the spiked test solution of "Method 5".

[0254] II-5 positioning solution: Accurately measure 1 ml of II-5 reference substance stock solution, place it in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0255] II-9 positioning solution: Accurately measure 1 ml of II-9 reference substance stock solution, place it in a 25 ml volumetric flask, dilute to the scale with diluent, and shake well.

[0256] Chromatographic conditions: cellulose-tris (3,5-dimethylphenylcarbamate) bonded silica gel as filler (Daikolu OD-H, 4.6 mm × 250 mm, 5 μm); n-hexane-ethanol (88:12) as mobile phase; flow rate of 1.0 ml per minute; column temperature of 25 ° C; detection wavelength of 214 nm; injection volume of 20 μl.

[0257] Accurately measure the Ⅱ-5 positioning solution, Ⅱ-9 positioning solution and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram. Fig.11 .

[0258] The results showed that the intermediate and impurity Ⅱ-9 completely overlapped, and the intermediate and the enantiomer (impurity Ⅱ-5) did not achieve baseline separation, indicating that using n-hexane-ethanol system as the mobile phase and OD-H as the chromatographic column was not sufficient to effectively separate the three substances, and the OD-H chromatographic column was not suitable for detecting impurity Ⅱ-9 and enantiomer (impurity Ⅱ-5).

[0259] Method 7: Using n-hexane-ethanol (85:15) as the mobile phase

[0260] Diluent: ethanol.

[0261] Spiked test solution: Use the spiked test solution of "Method 5".

[0262] Chromatographic conditions: Cellulose-tris (3-chloro-4-methylphenylcarbamate) bonded silica gel was used as the filler (Daikolu OZ-3, 4.6 mm × 250 mm, 3 μm); n-hexane-ethanol (85:15) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 214 nm; and the injection volume was 20 μl.

[0263] Accurately measure the spiked test solution, inject it into the liquid chromatograph, and record the chromatogram. Fig.12 .

[0264] The results showed that in this test, except for the mobile phase ratio, the other conditions were consistent with the method in Example 1; in the chromatogram of the spiked test solution in this test, the separation degree between the intermediate and impurity II-9 was 3.0, and the separation degree between the intermediate and the enantiomer (impurity II-5) was 1.8, and the separation degree between the intermediate and the enantiomer (impurity II-5) was low.

[0265] Example 1 Figure 1 and method 8 below Fig.13 In comparison, there is no small unknown impurity in front of impurity II-9, and it is suspected that the small unknown impurity completely overlaps with impurity II-9. Therefore, this method cannot accurately detect the related substances.

[0266] Method 8: Using n-hexane-ethanol (86:14) as the mobile phase

[0267] Diluent: ethanol.

[0268] Test solution: Take about 20 mg of intermediate II, weigh accurately, place in a 50 ml volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0269] Chromatographic conditions: Cellulose-tris (3-chloro-4-methylphenylcarbamate) bonded silica gel was used as the filler (Daikolu OZ-3, 4.6 mm × 250 mm, 3 μm); n-hexane-ethanol (86:14) was used as the mobile phase; the flow rate was 1.0 ml per minute; the column temperature was 25°C; the detection wavelength was 214 nm; and the injection volume was 20 μl.

[0270] Accurately measure the test solution, inject it into the liquid chromatograph, and record the chromatogram. Fig.13 .

[0271] The results showed that except for the mobile phase ratio, the other conditions were consistent with those in Example 1. In the chromatogram of the test solution, impurity II-9 and the adjacent peak did not reach baseline separation, and was not suitable for detecting impurity II-9.

[0272] 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 an arformoterol tartrate intermediate by high performance liquid chromatography, wherein the intermediate is intermediate II, and the structural formula is: It is characterized in that High performance liquid chromatography was used, and the chromatographic column filler was cellulose-tris (3-chloro-4-methylphenylcarbamate) bonded silica gel as filler; The mobile phase was hexane-ethanol solution; In the mobile phase, the volume ratio of n-hexane to ethanol was 87:13 to 89:11; The column temperature is 20-30°C; The detector uses an ultraviolet detector with a detection wavelength of 214nm; The related substances in the arformoterol tartrate intermediate include isomeric impurities II-5 and impurity II-9, and the structural formula of the impurity II-5 is: The structural formula of impurity II-9 is: The method also includes sample preparation: Test solution: Dissolve the product in diluent and dilute to a concentration of 0.4 mg / ml. The diluent is ethanol. Reference solution: Take appropriate amount of impurity II-5 reference substance and impurity II-9 reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing 10μg of impurity II-5 and 10μg of impurity II-9 per 1ml.

2. The method according to claim 1, characterized in that In the mobile phase, the volume ratio of n-hexane to ethanol was 88:

12.

3. The method according to claim 1, characterized in that The chromatographic column used is Daicel OZ-3, 4.6 mm×250 mm, 3 μm.

4. The method according to claim 1, wherein the flow rate of the mobile phase is 0.9 to 1.1 ml / min.

5. The method according to claim 1, characterized in that The method also includes determining: Accurately measure the diluent, test solution and reference solution, inject them into the high performance liquid chromatograph respectively, and record the chromatogram; calculate the content of impurity II-5 and impurity II-9 in the test solution by peak area according to the external standard method, %; Calculation formula: Where: A 供试 is the peak area of ​​impurity II-5 or impurity II-9 in the chromatogram of the test solution; A 对照 The peak area of ​​impurity II-5 or impurity II-9 in the chromatogram of the reference solution; m 供试 is the sample weight of the test product, mg; m 对照 is the sample weight of impurity II-5 or impurity II-9 reference substance, mg; V 供试 is the dilution volume of the test solution, ml; V 对照 is the dilution volume of the impurity II-5 or impurity II-9 reference solution, ml; N is the content of impurity II-5 or impurity II-9 reference substance.

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