Analytical method for detecting related substances in umeclidinium bromide and vilanterol compound preparations

Abstract

The invention discloses an analytical method for detecting related substances in umeclidinium bromide and vilanterol compound preparations, using high performance liquid chromatography, a phenyl column whose filler is phenyl-hexyl chemically bonded silica gel is used as a chromatographic column, and a mobile phase adopts a sodium perchlorate solution and an acetonitrile system, and a sodium perchlorate solution concentration is 0.1-0.3 mol / L, with a sodium perchlorate solution as a mobile phase A, and acetonitrile as a mobile phase B, using gradient elution. The present invention makes up for the drawbacks of the lack of umeclidinium bromide and vilanterol compound preparation related substance inspection methods in the prior art, using a commonly used detector, by a single injection can simultaneously realize the inspection of the impurities introduced by two active ingredients, the method has low detection cost and high efficiency; strong specificity, good separation between each impurity peak and between the active ingredient and the impurity peak, and no mutual influence; the detection limit and quantitative limit of impurities are reasonable, and the sensitivity is high; accurate detection and quantification of impurities can be achieved.

Description

Technical Field

[0001] The invention relates to the technical field of drug analysis, in particular to an analysis method for detecting related substances in a umeclidinium bromide and vilanterol compound preparation. Background Art

[0002] Umeclidinium-vilanterol inhalation powder is a dual long-acting bronchodilator, in which Umeclidinium is a reversible long-acting cholinergic receptor antagonist with a strong affinity for M3 receptors; Vilanterol triphenylacetate is a new type of long-acting β2 receptor agonist with strong lipid solubility. The bronchial dilation effect can last for more than 12 hours after administration, and the reversibility and selectivity for β2 adrenergic receptors are high. The structures of Umeclidinium and Vilanterol triphenylacetate are as follows:

[0003]

[0004] As a part of product quality control, it is necessary to measure the related substances of the product. However, after searching public literature including the Chinese, British, European and Japanese Pharmacopoeias, no inspection methods for related substances in umeclidinium bromide and vilanterol combination preparations were found. The public patent "Method for Detecting Fine Particle Dosage of Umeclidinium and Vilanterol Inhalation Powder" (application number: CN201911391912.5) adopts high performance liquid chromatography, using a fluorescence detector for vilanterol and a UV detector for umeclidinium; gradient elution is performed with mobile phase A (trifluoroacetic acid solution) and mobile phase B (acetonitrile solution containing trifluoroacetic acid). This method uses one mobile phase to detect the fine particle dose of two components, umeclidinium and vilanterol triphenylacetate, in umeclidinium and vilanterol inhalation powder. However, a fluorescence detector is used to detect vilanterol in the chromatographic conditions. The detector is a non-universal detector with a narrow application range and high cost. At the same time, it is easily interfered by background fluorescence and quenching effects, and the response value is extremely sensitive to temperature. If it is used for detecting impurities with low content in related substances, on the one hand, the accuracy cannot be guaranteed, and on the other hand, the operating conditions are high.

[0005] Therefore, there is still an urgent need for an analytical method that is easy to operate and can accurately detect related substances in umeclidinium bromide and vilanterol compound preparations. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides an analytical method for detecting related substances in a umeclidinium bromide-vilanterol compound preparation, which can simultaneously detect impurities from umeclidinium bromide and triphenylacetate vilanterol in the compound preparation, and the method is easy to operate and has high accuracy. The specific technical scheme of the present invention is as follows:

[0007] The analytical method for detecting related substances in the umeclidinium bromide and vilanterol compound preparation adopts high performance liquid chromatography, the detector is an ultraviolet detector, the chromatographic column adopts a phenyl column filled with phenyl-hexyl chemically bonded silica gel, and the mobile phase adopts a perchlorate solution and acetonitrile system. The structures of umeclidinium bromide, triphenylacetate and vilanterol, and their related substances are as follows:

[0008]

[0009]

[0010] Preferably, the perchlorate is potassium perchlorate or sodium perchlorate, which acts as a dissociation agent and has a buffering effect. In the embodiment of the present invention, sodium perchlorate is used to prepare the mobile phase.

[0011] Preferably, the concentration of the sodium perchlorate solution is 0.1 to 0.3 mol / L, preferably 0.2 mol / L.

[0012] Preferably, the pH value of the sodium perchlorate solution is in the range of 3.2 to 3.6, and perchloric acid is used to adjust the pH value.

[0013] Preferably, sodium perchlorate solution is used as mobile phase A, acetonitrile is used as mobile phase B, and gradient elution is adopted, wherein the elution procedure is as follows: within 0 to 30 minutes, the volume percentage of mobile phase A is linearly changed from 72% to 78% to 55% to 61%, and the volume percentage of mobile phase B is linearly changed from 22% to 28% to 39% to 45%; within 30 to 45 minutes, the volume percentage of mobile phase A is linearly changed from 55% to 61% to 27% to 33%, and the volume percentage of mobile phase B is linearly changed from 22% to 28% to 39% to 45%. The volume percentage of mobile phase B linearly changes from 39% to 45% to 67% to 73%; within 45 to 45.1 minutes, the volume percentage of mobile phase A linearly changes from 27% to 33% to 72% to 78%, and the volume percentage of mobile phase B linearly changes from 67% to 73% to 22% to 28%; within 45.1 to 55 minutes, the volume percentage of mobile phase A remains at 72% to 78%, and the volume percentage of mobile phase B remains at 22% to 28%.

[0014] Preferably, the chromatographic column is Xselect CHS Phenyl-Hexyl, 150 mm×4.6 mm, 2.5 μm or a chromatographic column with equivalent performance.

[0015] Preferably, the column temperature of the chromatographic column is in the range of 35 to 45°C, preferably 40°C.

[0016] Preferably, the flow rate of the mobile phase is 0.8 to 1.2 ml / min, preferably 1.0 ml / min.

[0017] Furthermore, the present invention also includes the preparation of a sample solution, and the preparation method of the test solution is: transfer all the contents of the product to a volumetric bottle with a diluent, dilute to the scale with the diluent, and shake well. Specifically, take an appropriate amount of the contents of the product and quantitatively dilute with a diluent to a solution containing 0.47 mg of umeclidinium bromide and 0.25 mg of vilanterol triphenylacetate per 1 ml.

[0018] Preferably, the diluent is a methanol-water solution 50:50 in a volume ratio of 50:50.

[0019] Preferably, the detection wavelength is 220 nm.

[0020] Preferably, the injection volume is 25 μl.

[0021] Preferably, an ultraviolet detector or a diode array detector is used.

[0022] The method provided by the public patent "Method for Detecting Fine Particle Dosage of Umeclidinium and Vilanterol Inhalation Powder Aerosol" (Application Number: CN201911391912.5) mentioned in the background technology is used to detect the fine particle dose. The concentration of the solution to be tested is about 1.1 μg and 0.25 μg of Umeclidinium and Vilanterol triphenylacetate per 1 mL, respectively. However, in the determination of related substances, the concentration of general impurities is much lower than this concentration. In this method, the use of fluorescence detectors in particular can easily cause the problem that trace impurities cannot be accurately quantified or even cannot be detected. The quantitative limit and detection limit of impurities in the method provided by the present invention are low, and the lower detection limit of each impurity is no higher than 0.02% of the test sample concentration, which is lower than the reporting limit (0.1%).

[0023] According to the method provided by the present invention, the separation degree between active component peaks and between active component peaks and impurity peaks is as low as 1.69, and the separation is good to avoid mutual influence.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention makes up for the drawback of the lack of related substance inspection methods for umeclidinium bromide and vilanterol compound preparations in the prior art, adopts a commonly used ultraviolet detector, and can simultaneously realize the inspection of impurities introduced by two components through one injection. The method has low detection cost and high efficiency;

[0026] 2. The analytical method provided by the present invention has strong specificity, and the impurity peaks and the active ingredients and impurity peaks are well separated and do not affect each other; the detection limit and quantitative limit of impurities are reasonable and highly sensitive; accurate detection and quantification of impurities can be achieved;

[0027] 3. The present invention has good durability and high robustness, can adapt to the situation of fine-tuning conditions, and can still achieve accurate detection when conditions change. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 The chromatogram of the mixed solution in the method verification test of Example 2 of the present invention;

[0030] Among them, ① is a diluent, ② is a bromide ion, ③ is an impurity J of umeclidinium bromide, ④ is an impurity A of umeclidinium bromide, ⑤ is vilanterol, ⑥ is an impurity H of umeclidinium bromide, ⑦ is an impurity 1 of vilanterol, ⑧ is umeclidinium, ⑨ an impurity E of umeclidinium bromide, and ⑩ is triphenylacetic acid, Vilanterol impurity 2, It is impurity N of umeclidinium bromide, It is umeclidinium bromide impurity L;

[0031] Figure 2 This is the main component chromatogram under the C8 chromatographic column conditions in Example 3 of the present invention;

[0032] Figure 3 This is the main component chromatogram under the conditions of Waters, Symmetry C18 chromatographic column in Example 3 of the present invention;

[0033] Figure 4 This is the main component chromatogram under the conditions of Kromasil C18 chromatographic column and trifluoroacetic acid mobile phase in Example 3 of the present invention;

[0034] Figure 5 This is the main component chromatogram under the conditions of Dima, C18 chromatographic column and trifluoroacetic acid mobile phase in Example 3 of the present invention;

[0035] Figure 6 This is the main component chromatogram under the conditions of Shimadzu, Shim-Pack VP-ODS chromatographic column and trifluoroacetic acid mobile phase in Example 3 of the present invention;

[0036] Figure 7 This is the main component chromatogram under the conditions of Agela, venusil MP C18 chromatographic column and sodium perchlorate solution mobile phase in Example 3 of the present invention;

[0037] Figure 8 This is the main component chromatogram under the conditions of Welch, ultimate AQ-C18 chromatographic column and sodium perchlorate solution mobile phase in Example 3 of the present invention;

[0038] Fig. 9 This is the main component chromatogram under the conditions of Agilent Eclipse XDB-C18 chromatographic column and sodium perchlorate solution mobile phase in Example 3 of the present invention;

[0039] Fig.10 This is the chromatogram of the test sample under the conditions of Kromasil C18 chromatographic column and sodium perchlorate solution mobile phase in Example 3 of the present invention;

[0040] Fig.11 This is the main component chromatogram under the conditions of Agilent, ZORBAX NH2 chromatographic column in Example 3 of the present invention;

[0041] Fig.12 This is the main component chromatogram under the conditions of Waters, xselect HSS PFP chromatographic column and trifluoroacetic acid mobile phase in Example 3 of the present invention;

[0042] Fig.13 This is the main component chromatogram under the conditions of Agilent Zorbax SB-Pheny1 chromatographic column and sodium perchlorate solution mobile phase in Example 3 of the present invention;

[0043] Fig.14 The main component chromatogram is provided under the phosphate system mobile phase conditions in Example 3 of the present invention;

[0044] Fig.15 This is a chromatogram of the test sample under the conditions of mobile phase gradient elution program 1 in Example 3 of the present invention;

[0045] Fig.16 This is the chromatogram of the test sample under the conditions of mobile phase gradient elution procedure 2 in Example 3 of the present invention;

[0046] Fig.17 This is a chromatogram of the test sample under the conditions of mobile phase gradient elution program 3 in Example 3 of the present invention;

[0047] Fig.18 It is the chromatogram of the test sample in the comparative example of the present invention.

[0048] In the chromatogram, the horizontal axis is time, the unit is min, and the vertical axis is peak height, the unit is mAU. DETAILED DESCRIPTION

[0049] 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.

[0050] 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.

[0051] The impurity structures introduced by the two raw materials of umeclidinium bromide and vilanterol triphenylacetate are as follows:

[0052]

[0053]

[0054] In view of the fact that the prior art does not disclose a method for detecting impurities in the compound preparation, the inventors of the present application have provided a method for detecting related substances through research and development experiments, which has good impurity separation, high sensitivity, high repeatability, high accuracy, and strong durability.

[0055] In the detection test of the present application, after umeclidinium bromide is dissolved, it exists separately as bromide ion and umeclidinium. After the sample enters the high performance liquid chromatograph, bromide ion and umeclidinium emit separate peaks. Similarly, after triphenylacetate and vilanterol enter the high performance liquid chromatograph, triphenylacetate and vilanterol emit separate peaks.

[0056] Example 1, Analytical method for detecting related substances in umeclidinium bromide and vilanterol compound preparation

[0057] 1. Chromatographic Conditions

[0058] Instrument: High performance liquid chromatography

[0059] Detector: UV detector

[0060] Column: Waters, Xselect CHS Phenyl-Hexyl, 150 mm × 4.6 mm, 2.5 μm

[0061] Trapping column: Yuexu, 50 mm × 4.6 mm, installed between the gradient mixer and the injection valve;

[0062] Mobile phase A: 0.2 mol / L sodium perchlorate solution (pH 3.4);

[0063] Mobile phase B: acetonitrile;

[0064] The detection wavelength was 220 nm, the flow rate was 1.0 ml / min, the column temperature was 40 °C, and the injection volume was 25 μl;

[0065] Diluent: methanol-water (50:50);

[0066] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 30 58 42 45 30 70 45.1 75 25 55 75 25

[0067] As an alternative, the detector may be a diode array detector.

[0068] 2. Solution Preparation

[0069] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.47 mg of umeclidinium bromide and approximately 0.25 mg of vilanterol triphenylacetate per 1 ml. Shake well to obtain the solution.

[0070] The compound preparation referred to in the present invention can be in the form of an inhalation preparation such as an inhalation aerosol, an inhalation powder aerosol, an inhalation spray, an inhalation liquid preparation, etc. Here, the inhalation powder aerosol is taken as an example to illustrate the analysis process.

[0071] Control solution: Accurately measure 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with diluent, shake well, and obtain.

[0072] Sensitivity solution: Accurately measure 1 ml of the control solution into a 20 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0073] System suitability solution: Take about 1 mg of vilanterol impurity 1 reference substance, about 2 mg of umeclidinium impurity J and umeclidinium impurity E, respectively place them in different 10 ml volumetric flasks, add methanol to dissolve, dilute to the scale with methanol, shake well, as the stock solution of each impurity, accurately measure 2 ml of each impurity stock solution into a 100 ml volumetric flask containing 6 mg of umeclidinium reference substance and 3 mg of vilanterol triphenylacetate reference substance, add diluent to dissolve by ultrasonication and dilute to the scale, shake well, and obtain.

[0074] Respectively measure the diluent, system suitability solution, sensitivity solution, control solution, and test solution and inject them into the liquid chromatograph to record the chromatogram.

[0075] 3. Calculation method

[0076]

[0077]

[0078]

[0079] Total impurities of Vilanterol (%) = ∑ other impurities (%) + ∑ impurities of Vilanterol (%)

[0080] Total impurities of umeclidinium bromide (%) = ∑ impurities of umeclidinium bromide (%)

[0081] Where: A i is the peak area of ​​impurities in the test solution;

[0082] A r维 is the main peak area of ​​vilanterol in the control solution;

[0083] A r乌 is the main peak area of ​​umeclidinium in the control solution;

[0084] f is the correction factor.

[0085] 4. Results

[0086] 4.1, in the chromatogram of the sensitivity solution, the signal-to-noise ratios of the vilanterol peak and the umeclidinium peak were both greater than 10. In the chromatogram of the system suitability solution, the separation of each impurity was greater than 1.5, indicating that the system suitability of this method was good.

[0087] 4.2. Two batches of compound preparation samples were tested for related substances, and the results are as follows.

[0088] limit Batch 1 Batch 2 Umeclidinium Bromide Impurity J≤1.0% ND ND Vilanterol Impurity 1 ≤ 1.0% ND ND Vilanterol Impurity 2 ≤ 1.0% <0.1％ <0.1％ Other individual impurities (calculated as vilanterol) ≤ 1.0% <0.1％ <0.1％ Total impurities of umeclidinium bromide ≤ 1.5% <0.1％ <0.1％ Vilanterol total impurities ≤ 2.5% <0.1％ <0.1％

[0089] Conclusion: This method can detect related substances in compound preparation samples normally.

[0090] Example 2, method verification test

[0091] 2.1 Specificity test:

[0092] This method has strong specificity: each impurity is located, and the diluent and blank excipient are completely separated from the two active ingredients and known impurities.

[0093] Triphenylacetic acid solution: Take an appropriate amount of triphenylacetic acid and quantitatively dilute it to make a solution containing 0.1 mg of triphenylacetic acid per 1 ml of diluent.

[0094] Potassium bromide solution: Take an appropriate amount of potassium bromide and quantitatively dilute it to make a solution containing 0.1 mg of potassium bromide per 1 ml of diluent.

[0095] Umeclidinium Impurity A Solution: Take an appropriate amount of Umeclidinium Impurity A reference substance, dissolve it in methanol and dilute it to make a stock solution containing 0.5 mg of Umeclidinium Impurity A per 1 ml; accurately measure 1 ml into a 100 ml volumetric flask, dilute to the scale with a diluent, shake well, and obtain.

[0096] The preparation method of umeclidinium bromide impurity E, H, J, L, and N solutions is the same as that of umeclidinium bromide impurity A solution.

[0097] Vilanterol Impurity 1 Solution: Take an appropriate amount of Vilanterol Impurity 1 reference substance, dissolve it in methanol and dilute it to make a stock solution containing 0.2 mg of Vilanterol Impurity 1 per 1 ml; accurately measure 2 ml into a 20 ml volumetric flask, dilute to the scale with a diluent, shake well, and obtain.

[0098] The preparation method of vilanterol impurity 2 solution is the same as that of vilanterol impurity 1 solution.

[0099] Mixed impurity reference substance stock solution: Accurately measure 2 ml of each of umeclidinium bromide impurity A, E, H, J, L, N stock solution and vilanterol triphenylacetate impurity 1 and 2 stock solution into a 20 ml volumetric flask, dilute to the scale with methanol, shake well, and use as the mixed impurity reference substance stock solution.

[0100] Mixed impurity reference solution: Accurately measure 2 ml of mixed impurity reference stock solution, place it in a 20 ml volumetric flask, dilute to the scale with diluent, shake well, and obtain.

[0101] Umeclidinium bromide reference solution: Take about 12 mg of umeclidinium bromide reference, weigh accurately, add diluent to dissolve and dilute to make a solution containing 0.6 mg of umeclidinium bromide per 1 ml.

[0102] Vilanterol triphenylacetate reference solution: Take about 6 mg of vilanterol triphenylacetate reference substance, dissolve it in a diluent and dilute it to make a solution containing 0.3 mg of vilanterol triphenylacetate per 1 ml.

[0103] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.47 mg of umeclidinium bromide and approximately 0.25 mg of vilanterol triphenylacetate per 1 ml. Shake well to obtain the solution.

[0104] Blank excipient solution: Take blank preparation and prepare it in the same way as the test solution.

[0105] Mixed solution: Take this product, rinse the contents with diluent and transfer to a 20ml volumetric flask containing 2ml of mixed impurity reference stock solution, dilute to the scale with diluent, shake well, and you have it.

[0106] The diluent, blank excipient, triphenylacetic acid, potassium bromide, umeclidinium impurities A, E, H, J, L, N, vilanterol impurities 1, 2, umeclidinium reference substance, and triphenylacetic acid vilanterol reference substance solutions were sampled respectively for peak location and peak retention time were recorded; the mixed solution was sampled and the retention time, separation degree and main peak purity factor were recorded, which are summarized in the following table:

[0107] Specificity results

[0108]

[0109]

[0110] Conclusion: The diluent and blank excipients do not produce peaks at the known impurities or main peaks; the chromatogram of the mixed solution is as follows Figure 1As shown, the minimum separation degree between each impurity and the main peak and gradient peak is 1.69, which is greater than 1.5, and the separation degree meets the requirements; the retention time of bromide ion is 1.132min, and the retention time of triphenylacetic acid is 31.829min; the purity of the main peak in the mixed solution meets the requirements.

[0111] 2.2 Sensitivity test:

[0112] High sensitivity: The detection limit of each impurity is no higher than 0.02% of the test sample concentration and lower than the reporting limit (0.1%);

[0113] The limit of quantification of impurity J of umeclidinium bromide is 2.4703 ng (accounting for 0.02% of the test sample concentration);

[0114] The limit of quantification of impurity A of umeclidinium bromide is 2.1963 ng (accounting for 0.03% of the test sample concentration);

[0115] The limit of quantification of impurity H of umeclidinium bromide is 2.2332 ng (accounting for 0.03% of the test sample concentration);

[0116] The limit of quantification of impurity E of bromobiscarbamide is 5.3620 ng (accounting for 0.04% of the test sample concentration);

[0117] The limit of quantification of impurity N of umeclidinium bromide is 2.1576 ng (accounting for 0.03% of the test sample concentration);

[0118] The limit of quantification of impurity L of umeclidinium bromide was 2.2389 ng (accounting for 0.03% of the test sample concentration);

[0119] The limit of quantification of impurity 1 of vilanterol was 0.9072 ng (accounting for 0.02% of the test sample concentration);

[0120] The limit of quantification of impurity 2 of vilanterol is 2.9472 ng (accounting for 0.05% of the test sample concentration).

[0121] The detection limit of impurity J of umeclidinium bromide was 0.7411 ng (accounting for 0.01% of the test sample concentration);

[0122] The detection limit of impurity A of umeclidinium bromide is 0.7321 ng (accounting for 0.01% of the test sample concentration);

[0123] The detection limit of impurity H of umeclidinium bromide is 0.7444 ng (accounting for 0.01% of the test sample concentration);

[0124] The detection limit of impurity E of bromobiscarbamide is 2.4540 ng (accounting for 0.02% of the test sample concentration);

[0125] The detection limit of impurity N of umeclidinium bromide is 0.7198 ng (accounting for 0.01% of the test sample concentration);

[0126] The detection limit of impurity L of umeclidinium bromide was 0.7463 ng (accounting for 0.01% of the test sample concentration);

[0127] The detection limit of impurity 1 of vilanterol was 0.2910 ng (accounting for 0.01% of the test sample concentration);

[0128] The detection limit of vilanterol impurity 2 was 0.9824 ng (accounting for 0.02% of the test sample concentration).

[0129] 2.3 Linearity test:

[0130] The linear relationship is good: each impurity is within the range of quantification limit to 150%, and the correlation coefficient is greater than 0.99. The impurity J of umeclidinium bromide is within the range of 0.09 μg / ml to 7.4109 μg / ml (quantification limit to 150%), the linear equation is y=21.5607x+0.1808, and the correlation coefficient is 0.9999.

[0131] The impurity 1 of Vilanterol was in the range of 0.0388 μg / ml to 2.9098 μg / ml (limit of quantification ~150%), the linear equation was y=50.8730x-0.3015, and the correlation coefficient was 0.9999.

[0132] 2.4 Accuracy test:

[0133] Accuracy: The recovery rate of impurity J of umeclidinium bromide was 99.9% to 102.1%, the average recovery rate was 101.3%, and the RSD was 0.8%; the recovery rate of impurity 1 of vilanterol was 101.3% to 101.7%, the average recovery rate was 101.0%, and the RSD was 0.5%. The recovery rates of each impurity met the requirements of 90% to 108%.

[0134] 2.5 Precision test

[0135] Precision: In 6 test solutions, the RSD of the impurity J content of umeclidinium bromide was 1.2%, and the RSD of the impurity 1 content of vilanterol was 1.2%, indicating that the repeatability of this method for the inspection of related substances of this product is good; in 12 test solutions, the RSD of the impurity J content of umeclidinium bromide was 1.7%, and the RSD of the impurity 1 content of vilanterol was 2.2%, indicating that the intermediate precision of this method for the inspection of related substances of this product is good.

[0136] In summary, the RSD of each impurity content detection in repeatability and intermediate precision was less than 3%, and the precision was good.

[0137] 2.6 Durability test

[0138] Strong durability: fine-tuning of chromatographic conditions, column temperature (35°C, 45°C), different flow rates (0.8ml / min, 1.2ml / min), pH (3.2, 3.6), different batches of chromatographic columns and changing the mobile phase ratio (see the table below), the separation degree can meet the requirements.

[0139] Time (min) Mobile phase A (%) Mobile phase B (%) 0 72％～78％ 22％～28％ 30 55％～61％ 39％～45％ 45 27％～33％ 67％～73％ 45.1 72％～78％ 22％～28％ 55 72％～78％ 22％～28％

[0140] Each chromatographic condition was fine-tuned. In the sensitivity solution chromatogram, the S / N of the vilanterol peak and the S / N of the umeclidinium peak were both greater than 10. In the system suitability solution chromatogram, umeclidinium impurity J, vilanterol, vilanterol impurity 1, umeclidinium, umeclidinium impurity E, and triphenylacetic acid appeared in sequence, and the minimum separation between adjacent peaks was 1.90, which was greater than 1.5. The system suitability under the fine-tuning chromatographic conditions met the requirements. The minimum separation between the main peak and adjacent impurities in the mixed solution was 1.07. The ratio of the content of umeclidinium impurity J to that under normal conditions was 98.7% to 100.5%, and the ratio of the content of vilanterol impurity 1 to that under normal conditions was 99.0% to 101.6%, both between 90% and 108%.

[0141] Example 3, detection of related substances using its chromatographic conditions

[0142] In the process of method exploration, various chromatographic columns, mobile phase components and gradient programs were used to detect related substances of umeclidinium bromide and vilanterol triphenylacetate. The test results under different chromatographic conditions are as follows:

[0143] 3.1. Chromatographic column test

[0144] Based on the structures of the two components, the inventors preferentially selected C8 column, C18 column, amino column, and phenyl column for method development respectively:

[0145] 3.1.1, C8 column

[0146] The chromatographic conditions are:

[0147] Chromatographic column: GL C8 250×4.6mm, 5μm;

[0148] Mobile phase A: 0.1 mol / L sodium perchlorate solution (adjust pH to 3.4 with perchloric acid);

[0149] Mobile phase B: acetonitrile;

[0150] Detection wavelength: 220nm;

[0151] Flow rate: 1.0ml / min;

[0152] Column temperature: 25°C;

[0153] Injection volume: 25 μl;

[0154] Gradient Program:

[0155] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 60 40 40 45 55 50 10 90 51 90 10 65 90 10

[0156] Diluent: methanol-water (50:50)

[0157] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0158] Vilanterol triphenylacetate solution: Take about 7 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0159] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 2 As shown, the umeclidinium peak overlaps with the vilanterol impurity peak.

[0160] 3.1.2, C18 column

[0161] 1)Waters, Symmetry C18 column,

[0162] Chromatographic column: Waters, Symmetry C18 250×4.6mm, 5μm

[0163] Mobile phase A: 0.05% trifluoroacetic acid solution

[0164] Mobile phase B: 0.05% trifluoroacetic acid in acetonitrile

[0165] Detection wavelength: 220nm;

[0166] Flow rate: 1.0ml / min;

[0167] Column temperature: 30°C;

[0168] Injection volume: 25 μl;

[0169] Gradient Program:

[0170] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 5 80 20 25 40 60 40 20 80 41 80 20 55 80 20

[0171] Diluent: methanol-water (50:50)

[0172] Umeclidinium bromide solution: Take about 6 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0173] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0174] Take umeclidinium bromide solution and vilanterol triphenylacetate solution and inject them separately, overlap the chromatograms, and the results are as follows Figure 3 As shown, the umeclidinium peak affects the detection of the vilanterol impurity peak.

[0175] 2) Kromasil C18

[0176] Column: Kromasil C18, 250×4.6mm, 5μm

[0177] The mobile phase composition, elution gradient program and other chromatographic conditions were the same as 1) Waters, Symmetry C18 column.

[0178] Diluent: methanol-water (50:50);

[0179] Umeclidinium bromide solution: Take about 2 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0180] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0181] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 4 As shown, the peak of umeclidinium overlaps with the peak of vilanterol impurity, and normal related substance detection cannot be performed.

[0182] 3) Dima, C18 column,

[0183] Chromatographic column: Dima, C18 column, 250×4.6mm, 5μm

[0184] Mobile phase components, elution gradient program and other chromatographic conditions are the same as 1) Waters, Symmetry C18 column. Diluent: methanol-water (50:50)

[0185] Umeclidinium bromide solution: Take about 2 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0186] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0187] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 5 As shown, the peaks of umeclidinium and vilanterol affect the detection of various impurity peaks.

[0188] 4) Shimadzu, Shim-Pack VP-ODS column

[0189] Chromatographic column: Shimadzu, Shim-Pack VP-ODS250×4.6mm, 5μm

[0190] Mobile phase components, elution gradient program and other chromatographic conditions are the same as 1) Waters, Symmetry C18 column. Diluent: methanol-water (50:50)

[0191] Umeclidinium bromide solution: Take about 2 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0192] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0193] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 6 As shown, the umeclidinium peak and the impurity peak can be separated, but the detection of the vilanterol impurity peak is significantly reduced, that is, it cannot be detected completely.

[0194] 5) Agela, venusil MP C18 column

[0195] Chromatographic column: Agela, venusil MP C18, 250×4.6mm, 5μm

[0196] Mobile phase A: 0.1 mol / L sodium perchlorate solution (adjust pH to 3.4 with perchloric acid)

[0197] Mobile phase B: acetonitrile

[0198] Detection wavelength: 220nm;

[0199] Flow rate: 1.0ml / min;

[0200] Column temperature: 30°C;

[0201] Injection volume: 25 μl;

[0202] Gradient Program:

[0203] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 25 60 40 40 45 55 50 10 90 51 90 10 65 90 10

[0204] Diluent: methanol-water (50:50)

[0205] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0206] Vilanterol triphenylacetate solution: Take about 7 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0207] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 7 As shown in the figure, the peak of umeclidinium overlapped with the impurity peak of vilanterol. During the method development process, fine-tuning of the mobile phase ratio, gradient elution program, and screening of chromatographic columns failed to achieve separation.

[0208] 6)Welch, ultimate AQ-C18 column,

[0209] Chromatographic column: Welch, ultimate AQ-C18 250×4.6mm, 3μm

[0210] The mobile phase components, elution gradient program and other chromatographic conditions are the same as 5) Agela, venusil MP C18 column. Diluent: methanol-water (50:50)

[0211] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0212] Vilanterol triphenylacetate solution: Take about 7 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0213] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Figure 8 As shown, the peak shapes of umeclidinium and vilanterol are poor and are not suitable for related substance inspection.

[0214] 7)Agilent Eclipse XDB-C18 column

[0215] Chromatographic column: Agilent Eclipse XDB-C18 250×4.6mm, 5μm

[0216] The mobile phase components, elution gradient program and other chromatographic conditions are the same as 5) Agela, venusil MP C18 column. Diluent: methanol-water (50:50)

[0217] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0218] Vilanterol triphenylacetate solution: Take about 7 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0219] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Fig. 9As shown, the peak of umeclidinium overlaps with the impurity peak of vilanterol. Adjustments to the chromatographic conditions, gradient, mobile phase ratio, and pH, all resulted in interference between other impurities and the main peak of the active ingredient. This chromatographic column is not suitable for the inspection of related substances in this project.

[0220] 8) Kromasil C18 column

[0221] Column: Kromasil C18 250×4.6mm, 5μm

[0222] The mobile phase components, elution gradient program and other chromatographic conditions are the same as 5) Agela, venusil MP C18 column. Diluent: methanol-acetonitrile-water (60:10:30)

[0223] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.47 mg of umeclidinium bromide and approximately 0.25 mg of vilanterol triphenylacetate per 1 ml. Shake well to obtain the solution.

[0224] Take the test solution and inject it. The result is as follows Fig.10 As shown, umeclidinium, umeclidinium bromide impurity E, and vilanterol impurity 1 are close to each other, and the separation is poor under the ruggedness condition.

[0225] 2.1.3, Amino column, Agilent, ZORBAX NH2 column

[0226] Chromatographic column: Agilent, ZORBAX NH2 250×4.6mm, 5μm

[0227] The mobile phase components, elution gradient program and other chromatographic conditions are the same as 5) Agela, venusil MP C18 column. Diluent: methanol-water (50:50)

[0228] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0229] Vilanterol triphenylacetate solution: Take about 6 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0230] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Fig.11 As shown, the peak shapes of umeclidinium and vilanterol are poor and are not suitable for related substance inspection.

[0231] 2.1.4, Phenyl column

[0232] 1)Waters, xselect HSS PFP column

[0233] Chromatographic column: Waters, xselect HSS250×4.6mm, 5μm

[0234] Mobile phase components, elution gradient program and other chromatographic conditions are the same as 1) Waters, Symmetry C18 column. Diluent: methanol-water (50:50)

[0235] Umeclidinium bromide solution: Take about 6 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0236] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0237] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Fig.12 As shown, the impurities of umeclidinium bromide and vilanterol triphenylacetate could not be detected, and the sensitivity was low.

[0238] 2) Agilent Zorbax SB-Pheny1

[0239] Chromatographic column: Agilent Zorbax SB-Pheny1 250×4.6mm, 5μm

[0240] The mobile phase components, elution gradient program and other chromatographic conditions are the same as 5) Agela, venusil MP C18 column. Diluent: methanol-water (50:50)

[0241] Umeclidinium bromide solution: Take about 12 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0242] Vilanterol triphenylacetate solution: Take about 6 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0243] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Fig.13 As shown, the umeclidinium peak overlaps with the vilanterol impurity peak.

[0244] Compared with Example 1 and Example 2 of the method verification test, it can be seen that the phenyl-hexyl column is more suitable for the detection of related substances in this project. The trifunctionally bonded C6 phenyl ligand in the XSelect CSH phenyl-hexyl column is a stable low-leakage adsorbent that selectively retains polyaromatic compounds through π-π interactions, complementing the linear C18 column and providing better selectivity for the separation of aromatic analytes.

[0245] 2.2. Exploration of mobile phase

[0246] 2.2.1 Phosphate system

[0247] Column: YMC-Pack ODS-AQ, 150×3.0mm, 3μm

[0248] Mobile phase A: 0.1 mol / L phosphoric acid solution

[0249] Mobile phase B: acetonitrile

[0250] Detection wavelength: 220nm;

[0251] Flow rate: 1.0ml / min;

[0252] Column temperature: 50°C;

[0253] Injection volume: 25 μl;

[0254] Gradient Program:

[0255]

[0256]

[0257] Diluent: methanol-water (50:50);

[0258] Umeclidinium bromide solution: Take about 4 mg of Umeclidinium bromide, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and the solution is ready.

[0259] Vilanterol triphenylacetate solution: Take about 4 mg of vilanterol triphenylacetate, weigh accurately, add diluent to dissolve and dilute to the scale, shake well, and obtain.

[0260] Take the umeclidinium bromide solution and the vilanterol triphenylacetate solution and inject them separately. The results are as follows Fig.14 As shown, the peak of umeclidinium overlaps with the peak of vilanterol impurity, and normal related substance detection cannot be performed.

[0261] Compared with Example 1 and Example 2 of the method verification test, it can be seen that perchlorate is more suitable for the detection of related substances in this project. Sodium perchlorate, as a chaotropic agent, forms a weaker ion pair buffer in the mobile phase, which has less baseline noise and drift when used for gradient elution, and can effectively change the structure of molecules, reduce the polarity of molecules, and effectively increase the polarity of organic matter in the liquid mobile phase, thereby increasing the adsorption of its molecules and improving the separation degree.

[0262] 2.3. Experimental test of gradient elution procedure

[0263] 2.3.1, Elution procedure 1:

[0264] Column: Xselect CHS Phenyl-Hexyl, 150 mm × 4.6 mm, 2.5 μm

[0265] Mobile phase A: 0.1 mol / L sodium perchlorate solution (adjust pH to 3.4 with perchloric acid)

[0266] Mobile phase B: acetonitrile

[0267] Detection wavelength: 220nm;

[0268] Flow rate: 1.0ml / min;

[0269] Column temperature: 45°C;

[0270] Injection volume: 25 μl;

[0271] Diluent: methanol-water (50:50);

[0272] The mobile phase gradient is:

[0273]

[0274]

[0275] Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the scale to prepare a solution containing approximately 0.47 mg of umeclidinium bromide and approximately 0.25 mg of vilanterol triphenylacetate per 1 ml. Shake well to obtain the solution.

[0276] Take the test solution and inject it. The result is as follows Fig.15 As shown, the separation degree of umeclidinium impurity E and triphenylacetic acid is poor, and the peak around 46 min is vilanterol impurity 2 and umeclidinium impurity N, and the two impurity peaks overlap.

[0277] 2.3.2, Elution procedure 2:

[0278] Column: Xselect CHS Phenyl-Hexyl, 150 mm × 4.6 mm, 2.5 μm

[0279] Mobile phase A: 0.1 mol / L sodium perchlorate solution (adjust pH to 3.4 with perchloric acid)

[0280] Mobile phase B: acetonitrile

[0281] Detection wavelength: 220nm;

[0282] Flow rate: 1.0ml / min;

[0283] Column temperature: 45°C;

[0284] Injection volume: 25 μl;

[0285] Diluent: methanol-water (50:50);

[0286] The mobile phase gradient is:

[0287] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 80 20 45 55 45 55 55 45 55.1 80 20 60 80 20

[0288] Test solution: Prepare in the same manner as in “2.3.1, Elution Procedure 1”.

[0289] Take the test solution and inject it. The result is as follows Fig.16 As shown, Umeclidinium bromide impurity J and Umeclidinium bromide impurity A, and triphenylacetic acid impurity 2 and Umeclidinium bromide impurity N cannot be separated.

[0290] 2.3.3, Elution procedure 3:

[0291] Column: Xselect CHS Phenyl-Hexyl, 150 mm × 4.6 mm, 2.5 μm

[0292] Mobile phase A: 0.1 mol / L sodium perchlorate solution (adjust pH to 3.4 with perchloric acid)

[0293] Mobile phase B: acetonitrile

[0294] Detection wavelength: 220nm;

[0295] Flow rate: 1.0ml / min;

[0296] Column temperature: 45°C;

[0297] Injection volume: 25 μl;

[0298] Diluent: methanol-water (50:50);

[0299] The mobile phase gradient is:

[0300] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 45 55 45 50 55 45 50.1 75 25 55 75 25

[0301] Test solution: Prepare in the same manner as in “2.3.1, Elution Procedure 1”.

[0302] Take the test solution and inject it. The result is as follows Fig.17 As shown, the umeclidinium impurity E and triphenylacetic acid peaks cannot be separated.

[0303] By comparing the results of the method exploration test with those of Example 1 and Example 2, it can be seen that the type of chromatographic column, the composition of the mobile phase and the gradient program have a significant impact on the detection of umeclidinium bromide, vilanterol and the impurities introduced by each.

[0304] Comparative Example

[0305] The comparison chart of adding a capture column and not adding a capture column under the same conditions is as follows Fig.18 As shown, it is obvious that after adding the capture column, the diluent ghost peak disappears and the baseline is smooth, which is conducive to the detection of impurities without interference.

[0306] 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. The analytical method for detecting related substances in the umeclidinium bromide and vilanterol compound preparation adopts high performance liquid chromatography, which is characterized by: Using UV detector; Column: Waters, Xselect CHS Phenyl-Hexyl, 150 mm × 4.6 mm, 2.5 μm Trapping column: Yuexu, 50 mm × 4.6 mm, installed between the gradient mixer and the injection valve; Mobile phase A: 0.2 mol / L sodium perchlorate solution, pH 3.4; Mobile phase B: acetonitrile; The detection wavelength is 220nm; Diluent: methanol: water = 50:50; Test solution: Take the contents of this product, transfer all of it to a volumetric flask with a diluent, and dilute to the mark to prepare a solution containing 0.47 mg of umeclidinium bromide and 0.25 mg of vilanterol triphenylacetate per 1 ml. Shake well to obtain the solution. Elution gradient: The structures of umeclidinium bromide, vilanterol triphenylacetate and their related substances are as follows:

2. The analytical method for detecting related substances in the umeclidinium bromide and vilanterol compound preparation according to claim 1, characterized in that: The column temperature range is 35~45℃.

3. The analytical method for detecting related substances in the umeclidinium bromide and vilanterol compound preparation according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8~1.2ml / min.

4. The analytical method for detecting related substances in the umeclidinium bromide and vilanterol compound preparation according to claim 1, characterized in that: Elution gradient:

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