Detection method of fine particle dosage of umeclidinium bromide and vilanterol inhalation powder

Abstract

The invention provides a method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder, and relates to the technical field of analytical chemistry. The method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder adopts high performance liquid chromatography, and the chromatographic conditions include: a chromatographic column using an octadecylsilane bonded silica gel filler; a detector: a fluorescence detector is used for vilanterol, and an ultraviolet detector is used for umeclidinium; the mobile phase is composed of a mobile phase A and a mobile phase B, and the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is an acetonitrile solution containing trifluoroacetic acid, and gradient elution is performed. The present invention uses a mobile phase condition to quickly and accurately detect the fine particle dosage of two components, umeclidinium bromide and vilanterol triphenylacetate, in the umeclidinium bromide and vilanterol inhalation powder; the operation is simple, the measurement result is accurate and reliable, the specificity is strong, the durability is good, the sensitivity is high, and the detection time is short.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, in particular to a method for detecting the dosage of fine particles of umeclidinium bromide and vilanterol inhalation powder aerosol. Background Art

[0002] Respiratory diseases are common and highly prevalent, with a mortality rate second only to tumors and cardiovascular diseases. Asthma and chronic obstructive pulmonary disease (COPD) are recognized medical problems worldwide. There are more than 30 million asthma patients and more than 80 million COPD patients in my country. Inhaled preparations are internationally recognized as the first choice for the treatment of asthma and COPD, but currently 70% of patients have never used inhaled therapy, and only 4.46% of patients in rural areas have long-term use of inhaled drugs. Most patients treat asthma with injections and medication, which leads to frequent cardiovascular side effects.

[0003] Inhalation preparations are mainly divided into aerosols and powder aerosols. Compared with aerosols, inhalation powder aerosols are easy to use, and patients actively inhale the powder; there is no propellant Freon, which avoids pollution to the atmospheric environment; the dosage is accurate, and there is no risk of overdose; it does not contain preservatives and solvents such as ethanol, and is non-irritating to the diseased mucosa; the drug is in dry powder form, has good stability, and has few interfering factors.

[0004] Umeclidinium and vilanterol inhalation powder is a Class 4 generic drug developed by our company. It has a long-acting bronchodilator effect and is suitable for long-term maintenance treatment of chronic obstructive pulmonary disease (COPD). It is used once a day to relieve the symptoms of COPD patients. Umeclidinium is a long-acting muscarinic receptor antagonist, which mainly exerts a bronchodilator effect by competitively inhibiting the binding of acetylcholine to M3 muscarinic receptors on respiratory smooth muscle. Vilanterol is a selective long-acting β2-adrenergic receptor agonist that activates intracellular adenylate cyclase, which catalyzes the conversion of ATP to cyclic-3',5'-monophosphate adenosine cAMP level, relaxes bronchial smooth muscle and inhibits cells (especially mast cells) from releasing immediate hypersensitivity mediators. Compared with single administration, the systemic exposure (AUC and Cmax) of single drugs of Umeclidinium and vilanterol triphenylacetate combined inhalation is similar or lower, and the single drug in the compound has no effect on increasing the exposure of other components. When combined administration takes effect, it can achieve mechanism complementarity through dual action pathways, achieve the effect of optimizing airway relaxation, take effect relatively quickly, and can achieve 24-hour long-term performance. It can quickly improve the patient's discomfort, stably improve lung function, and relieve a series of uncomfortable symptoms caused by chronic obstructive pneumonia.

[0005] The molecular formula of vilanterol triphenylacetate is C 24 H 33 Cl2NO5·C 20 H 16O2, structural formula (I) is as follows:

[0006]

[0007] The molecular formula of umeclidinium bromide is C 29 H 34 BrNO2, structural formula (II) is as follows:

[0008]

[0009] After literature research, no literature or pharmacopoeia was found that simultaneously determined the fine particle dose of the active ingredients umeclidinium bromide and vilanterol triphenylacetate in umeclidinium bromide and vilanterol inhalation powder.

[0010] In view of this, the present invention is proposed. Summary of the invention

[0011] The main purpose of the present invention is to provide a method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol, so as to at least partially solve at least one of the above-mentioned technical problems.

[0012] The invention provides a method for detecting fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol, adopting high performance liquid chromatography, wherein the chromatographic conditions include: adopting a chromatographic column with an octadecylsilane bonded silica gel filler; a detector: a fluorescence detector is used for vilanterol, and an ultraviolet detector is used for umeclidinium bromide; a mobile phase is composed of a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid solution, and the mobile phase B is an acetonitrile solution containing trifluoroacetic acid, and gradient elution is performed according to the following table:

[0013] Time (min) Mobile phase A (%) Mobile phase B (%) 0 A1 B1 3.5-4.0 A2 B2

[0014] Among them, A1=63-67%, A2=28-32; B1=33-37%, B2=68-72.

[0015] Furthermore, high performance liquid chromatography was used, and the chromatographic conditions included: a chromatographic column using octadecylsilane bonded silica gel filler; a detector: a fluorescence detector was used for vilanterol, and a UV detector was used for umeclidinium bromide; the mobile phase consisted of a mobile phase A and a mobile phase B, wherein the mobile phase A was a trifluoroacetic acid solution, and the mobile phase B was an acetonitrile solution containing trifluoroacetic acid, and the gradient elution was performed according to the following table:

[0016] Time (min) Mobile phase A (%) Mobile phase B (%) 0 A1 B1 3.5-4.0 A2 B2 4.1-4.6 A1 B1 5.5-6.0 A1 B1

[0017] Among them, A1=63-67%, A2=28-32; B1=33-37%, B2=68-72.

[0018] Furthermore, the mobile phase A is a 0.05-0.15 vol% trifluoroacetic acid solution.

[0019] Furthermore, the mobile phase A is a 0.1 vol% trifluoroacetic acid solution.

[0020] Furthermore, the mobile phase B is an acetonitrile solution containing 0.05-0.15 vol% of trifluoroacetic acid.

[0021] Furthermore, the mobile phase B is an acetonitrile solution containing 0.1 vol% of trifluoroacetic acid.

[0022] Furthermore, the vilanterol uses a fluorescence detector with an excitation wavelength of 220-230 nm and a detection wavelength of 310-320 nm.

[0023] Furthermore, the vilanterol uses a fluorescence detector with an excitation wavelength of 225 nm and a detection wavelength of 315 nm.

[0024] Furthermore, the umeclidinium bromide uses an ultraviolet detector with a detection wavelength of 210-230 nm.

[0025] Furthermore, the umeclidinium bromide uses an ultraviolet detector with a detection wavelength of 220 nm.

[0026] Furthermore, the column temperature used was 28-32°C.

[0027] Furthermore, the column temperature used was 30°C.

[0028] Furthermore, the sample to be tested is dissolved in a mixture of methanol and water.

[0029] Furthermore, the volume ratio of methanol to water is 60:40.

[0030] Further, the flow rate used was 0.8-1.2 mL / min.

[0031] Further, the flow rate used was 0.9-1.1 mL / min.

[0032] Further, the flow rate used was 1.0 mL / min.

[0033] Further, the injection volume used was 90-110 μL.

[0034] Furthermore, the injection volume used was 100 μL.

[0035] Further, high performance liquid chromatography was used, and the chromatographic conditions included: a chromatographic column using octadecylsilane bonded silica gel filler; detector: vilanterol used a fluorescence detector with an excitation wavelength of 220-230 nm and a detection wavelength of 310-320 nm, and umeclidinium bromide used an ultraviolet detector with a detection wavelength of 210-230 nm; the mobile phase consisted of mobile phase A and mobile phase B, mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, and gradient elution was performed according to the following table:

[0036] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 .

[0037] Further, high performance liquid chromatography was used, and the chromatographic conditions included: a chromatographic column using octadecylsilane bonded silica gel filler; detector: vilanterol used a fluorescence detector with an excitation wavelength of 220-230 nm and a detection wavelength of 310-320 nm, and umeclidinium bromide used an ultraviolet detector with a detection wavelength of 210-230 nm; the mobile phase consisted of mobile phase A and mobile phase B, mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, and gradient elution was performed according to the following table:

[0038] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 4.1 65 35 6.0 65 35 .

[0039] Further, high performance liquid chromatography was used, and the chromatographic conditions included: a chromatographic column using octadecylsilane bonded silica gel filler; a detector: vilanterol used a fluorescence detector with an excitation wavelength of 225 nm and a detection wavelength of 315 nm, and umeclidinium bromide used an ultraviolet detector with a detection wavelength of 220 nm; the mobile phase consisted of a mobile phase A and a mobile phase B, mobile phase A: a 0.1 vol% trifluoroacetic acid solution, mobile phase B: an acetonitrile solution containing 0.1 vol% trifluoroacetic acid, and gradient elution was performed according to the following table:

[0040] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 4.1 65 35 6.0 65 35 .

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The invention provides a method for detecting fine particle dosage of umeclidinium bromide and vilanterol inhalation powder. According to the physical and chemical properties of umeclidinium bromide and vilanterol triphenylacetate, a set of liquid chromatography system is developed by optimizing a detector, a mobile phase and an elution gradient. The fine particle dosage of two components, umeclidinium bromide and vilanterol triphenylacetate, in the umeclidinium bromide and vilanterol inhalation powder can be quickly and accurately detected by using one mobile phase condition. The detection method has the advantages of simple operation, accurate and reliable measurement results, strong specificity, good durability, high sensitivity and short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 The HPLC chromatogram of the blank auxiliary material of vilanterol triphenylacetate in the fluorescence channel;

[0045] Figure 2 It is the HPLC chromatogram of blank auxiliary material of umeclidinium bromide in UV channel;

[0046] Figure 3 HPLC chromatogram of blank solvent of vilanterol triphenylacetate in the fluorescence channel;

[0047] Figure 4 HPLC chromatogram of blank solvent of umeclidinium bromide in UV channel;

[0048] Figure 5 The HPLC chromatogram of the vilanterol triphenylacetate reference solution in the fluorescence channel;

[0049] Figure 6 It is the HPLC chromatogram of the umeclidinium bromide reference solution in the UV channel;

[0050] Figure 7 HPLC chromatogram of vilanterol triphenylacetate NGI test in the fluorescence channel;

[0051] Figure 8 This is the HPLC chromatogram of the UV channel umeclidinium bromide NGI test;

[0052] Fig. 9 This is the HPLC chromatogram of the system suitability test of umeclidinium bromide;

[0053] Fig.10 This is the HPLC spectrum of the system suitability test of vilanterol triphenylacetate. DETAILED DESCRIPTION

[0054] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, conventional conditions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0055] water, from Watsons;

[0056] Acetonitrile (chromatographic grade), from TEDIA;

[0057] Trifluoroacetic acid (analytical grade) was from Tianjin Kemeiou Chemical Reagent Co., Ltd.;

[0058] Umeclidinium bromide working reference substance (purity 99.63%) was obtained from Tianjin Pharmaceutical Research Institute Co., Ltd.;

[0059] Vilanterol triphenylacetate working reference substance (purity 99.11%) was obtained from Tianjin Pharmaceutical Research Institute Co., Ltd.;

[0060] Umeclidinium bromide and vilanterol inhalation powder spray, from Tianjin Pharmaceutical Research Institute Co., Ltd.

[0061] Example 1 Specificity Test

[0062] Test solution: Connect the device according to the law (Chinese Pharmacopoeia 2015 Edition Part IV General Rules 0951 Device 3*). Before use, add 4 mL of 19.25% (w / v) polyethylene glycol 200 acetone solution to collection cup 1 and 2 mL of 19.25% (w / v) polyethylene glycol 200 acetone solution to the other collection cups. Allow the acetone to evaporate. Add 15 mL of methanol-water (60:40) to the pre-separator. Place special filter paper on the external filter. Adjust the flow rate to 60 L / min. Check the pressure drop ratio of the control valve. P3 / P2 should not be greater than 0.5. Take 1 box of this product, pull the switch, insert the adapter, turn on the vacuum pump, pump for 4 seconds, and repeatedly suck out the 1st, 8th, 16th, 23rd and 30th doses (the remaining doses are discarded in a dedicated device, and the flow rate is adjusted to (60±2) L / min). Wait at least 30 seconds between each suction, turn off the pump, remove the suction device, weigh, record the reading, and test 5 suctions in total. Wash each component with the corresponding volume of methanol-water (60:40) according to the table below to extract the sample, shake to dissolve, filter, and take the filtrate as the NGI test solution.

[0063]

[0064]

[0065] Mixed reference substance solution: Take appropriate amount of umeclidinium bromide and vilanterol triphenylacetate reference substance, weigh accurately, add methanol-water (60:40) to dissolve and quantitatively dilute to make a mixed reference substance solution containing about 1.1 μg and 0.25 μg of umeclidinium bromide and vilanterol per 1 mL respectively;

[0066] Take an appropriate amount of umeclidinium bromide reference substance and prepare a umeclidinium bromide reference substance solution containing about 1.1 μg of umeclidinium bromide per 1 mL in the same manner;

[0067] Take an appropriate amount of vilanterol triphenylacetate reference substance and prepare a vilanterol triphenylacetate reference substance solution containing about 0.25 μg of vilanterol per 1 mL in the same manner.

[0068] Blank excipient solution: Prepare an excipient solution in the same proportion to the above main drug dosage as the blank excipient solution.

[0069] Blank solvent: methanol-water (60:40).

[0070] Example 1-1

[0071] Chromatographic conditions:

[0072] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0073] Mobile phase: Mobile phase A: 0.1 vol% trifluoroacetic acid solution, Mobile phase B: acetonitrile solution containing 0.1 vol% trifluoroacetic acid;

[0074] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 225 nm and a detection wavelength of 315 nm, while umeclidinium uses a UV detector at 220 nm;

[0075] Column temperature: 30°C;

[0076] Flow rate: 1.0 mL / min;

[0077] Injection volume: 100 μL;

[0078] Perform gradient elution according to the following table:

[0079] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 4.1 65 35 6.0 65 35 .

[0080] Accurately measure 100 μL of each of the above solutions, inject into the liquid chromatograph, and record the chromatogram. The HPLC chromatogram of the blank excipient of triphenylacetate vilanterol in the fluorescence channel is as follows: Figure 1 As shown, the HPLC chromatogram of the blank auxiliary material of umeclidinium bromide in the UV channel is as follows Figure 2 As shown, the HPLC chromatogram of the blank solvent of triphenylacetate vilanterol in the fluorescence channel is as follows Figure 3 As shown, the HPLC chromatogram of blank solvent of umeclidinium bromide in UV channel is as follows Figure 4 As shown, the HPLC chromatogram of the vilanterol triphenylacetate reference solution in the fluorescence channel is as follows Figure 5 As shown, the HPLC chromatogram of the UV channel umeclidinium bromide reference solution is as follows Figure 6 As shown, the HPLC chromatogram of the fluorescence channel vilanterol triphenylacetate NGI test is as follows Figure 7 As shown, the HPLC chromatogram of the UV channel umeclidinium bromide NGI test is as follows Figure 8 shown.

[0081] The masses of each level of NGI umeclidinium and vilanterol were calculated using the peak areas of umeclidinium and vilanterol according to the external standard method. Then, based on the masses of each level of umeclidinium and vilanterol, the amount of fine particles less than 5.0 μm was calculated using the "Copley Inhaler TestingDataAnalysis Software" software. The results are shown in Table 1.

[0082] Table 1 Fine particle dose measurement results

[0083]

[0084] Depend on Figure 1-8 It can be seen that under the chromatographic conditions of the detection method of this embodiment, the blank excipient and solvent have no interference with the main drug, and the separation between umeclidinium bromide and vilanterol is good, and there are no adjacent impurity peaks.

[0085] Example 1-2

[0086] Chromatographic conditions:

[0087] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0088] Mobile phase: Mobile phase A: 0.05 vol% trifluoroacetic acid solution, Mobile phase B: 0.15 vol% trifluoroacetic acid in acetonitrile solution;

[0089] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 220 nm and a detection wavelength of 320 nm, while umeclidinium uses a UV detector at 210 nm;

[0090] Column temperature: 31°C;

[0091] Flow rate: 0.9 mL / min;

[0092] Injection volume: 110 μL;

[0093] Perform gradient elution according to the following table:

[0094] Time (min) Mobile phase A (%) Mobile phase B (%) 0 63 37 3.5 32 68 4.6 63 37 5.5 63 37 .

[0095] 100 μL of each of the above solutions was accurately measured and injected into a liquid chromatograph to record the chromatogram. Since the chromatogram of this example is not much different from the chromatogram of Example 1-1, it is not provided here.

[0096] The masses of each level of NGI umeclidinium and vilanterol were calculated using the peak areas of umeclidinium and vilanterol according to the external standard method. Then, based on the masses of each level of umeclidinium and vilanterol, the amount of fine particles of drug less than 5.0 μm was calculated using the "Copley Inhaler TestingDataAnalysis Software" software. The results are shown in Table 2.

[0097] Table 2 Fine particle dose measurement results

[0098]

[0099] Under the chromatographic conditions of the detection method of this embodiment, the blank excipient and solvent have no interference with the main drug, and the separation degree of umeclidinium bromide and vilanterol is good, and there are no adjacent impurity peaks.

[0100] Examples 1-3

[0101] Chromatographic conditions:

[0102] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0103] Mobile phase: Mobile phase A: 0.15 vol% trifluoroacetic acid solution, Mobile phase B: acetonitrile solution containing 0.05 vol% trifluoroacetic acid;

[0104] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 230 nm and a detection wavelength of 310 nm, while umeclidinium uses a UV detector at 230 nm;

[0105] Column temperature: 29°C;

[0106] Flow rate: 1.1 mL / min;

[0107] Injection volume: 90 μL;

[0108] Perform gradient elution according to the following table:

[0109] Time (min) Mobile phase A (%) Mobile phase B (%) 0 67 33 3.8 28 72 4.4 67 33 5.8 67 33 .

[0110] 100 μL of each of the above solutions was accurately measured and injected into a liquid chromatograph to record the chromatogram. Since the chromatogram of this example is not much different from the chromatogram of Example 1-1, it is not provided here.

[0111] The masses of each level of NGI umeclidinium and vilanterol were calculated using the peak areas of umeclidinium and vilanterol according to the external standard method. Then, based on the masses of each level of umeclidinium and vilanterol, the amount of fine particles of drug less than 5.0 μm was calculated using the "Copley Inhaler TestingDataAnalysis Software" software. The results are shown in Table 3.

[0112] Table 3 Fine particle dose measurement results

[0113]

[0114] Under the chromatographic conditions of the detection method of this embodiment, the blank excipient and solvent have no interference with the main drug, and the separation degree of umeclidinium bromide and vilanterol is good, and there are no adjacent impurity peaks.

[0115] Examples 1-4

[0116] Chromatographic conditions:

[0117] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0118] Mobile phase: Mobile phase A: 0.08 vol% trifluoroacetic acid solution, Mobile phase B: 0.12 vol% trifluoroacetic acid in acetonitrile solution;

[0119] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 223 nm and a detection wavelength of 317 nm, while umeclidinium uses a UV detector at 218 nm;

[0120] Column temperature: 28°C;

[0121] Flow rate: 0.8 mL / min;

[0122] Injection volume: 95 μL;

[0123] Perform gradient elution according to the following table:

[0124] Time (min) Mobile phase A (%) Mobile phase B (%) 0 66 34 4.0 29 71 .

[0125] 100 μL of each of the above solutions was accurately measured and injected into a liquid chromatograph to record the chromatogram. Since the chromatogram of this example is not much different from the chromatogram of Example 1-1, it is not provided here.

[0126] The masses of each level of NGI umeclidinium and vilanterol were calculated using the peak areas of umeclidinium and vilanterol according to the external standard method. Then, based on the masses of each level of umeclidinium and vilanterol, the amount of fine particles of drug less than 5.0 μm was calculated using the "Copley Inhaler TestingDataAnalysis Software" software. The results are shown in Table 4.

[0127] Table 4 Fine particle dose measurement results

[0128]

[0129] Under the chromatographic conditions of the detection method of this embodiment, the blank excipient and solvent have no interference with the main drug, and the separation degree of umeclidinium bromide and vilanterol is good, and there are no adjacent impurity peaks.

[0130] Examples 1-5

[0131] Chromatographic conditions:

[0132] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0133] Mobile phase: Mobile phase A: 0.12 vol% trifluoroacetic acid solution, Mobile phase B: acetonitrile solution containing 0.08 vol% trifluoroacetic acid;

[0134] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 227 nm and a detection wavelength of 312 nm, while umeclidinium uses a UV detector at 222 nm;

[0135] Column temperature: 32°C;

[0136] Flow rate: 1.2 mL / min;

[0137] Injection volume: 105 μL;

[0138] Perform gradient elution according to the following table:

[0139] Time (min) Mobile phase A (%) Mobile phase B (%) 0 64 36 4.0 31 69 .

[0140] 100 μL of each of the above solutions was accurately measured and injected into a liquid chromatograph to record the chromatogram. Since the chromatogram of this example is not much different from the chromatogram of Example 1-1, it is not provided here.

[0141] The masses of each level of NGI umeclidinium and vilanterol were calculated using the peak areas of umeclidinium and vilanterol according to the external standard method. Then, based on the masses of each level of umeclidinium and vilanterol, the amount of fine particles of drug less than 5.0 μm was calculated using the "Copley Inhaler TestingDataAnalysis Software" software. The results are shown in Table 5.

[0142] Table 5 Fine particle dose measurement results

[0143]

[0144] Under the chromatographic conditions of the detection method of this embodiment, the blank excipient and solvent have no interference with the main drug, and the separation degree of umeclidinium bromide and vilanterol is good, and there are no adjacent impurity peaks.

[0145] Example 2 System suitability test

[0146] Reference solution: accurately weigh 15 mg of umeclidinium bromide reference substance and place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use this as the umeclidinium bromide stock solution; accurately weigh 1 mL of umeclidinium bromide stock solution and place it in a 25 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use this as the stock solution a;

[0147] Accurately weigh 20 mg of vilanterol triphenylacetate reference substance and place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use it as the vilanterol triphenylacetate stock solution; accurately weigh 1 mL of vilanterol triphenylacetate stock solution and place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use it as the stock solution b;

[0148] Accurately measure 1 mL of stock solution a and b respectively, place them in a 10 mL volumetric flask, dissolve and dilute to the scale with methanol-water (60:40), shake well, and use them as system suitability solutions.

[0149] Chromatographic conditions:

[0150] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0151] Mobile phase: Mobile phase A: 0.1 vol% trifluoroacetic acid solution, Mobile phase B: acetonitrile solution containing 0.1 vol% trifluoroacetic acid;

[0152] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 225 nm and a detection wavelength of 315 nm, while umeclidinium uses a UV detector at 220 nm;

[0153] Column temperature: 30°C;

[0154] Flow rate: 1.0 mL / min;

[0155] Injection volume: 100 μL;

[0156] Perform gradient elution according to the following table:

[0157] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 4.1 65 35 6.0 65 35 .

[0158] Take the system suitability solution and inject 6 injections continuously to examine the system suitability of the chromatographic conditions. The HPLC chromatogram of the system suitability test of umeclidinium bromide is shown in the figure below. Fig. 9 The test data are shown in Table 6.

[0159] Table 6 System suitability results of umeclidinium bromide

[0160]

[0161] The HPLC chromatogram of the system suitability test of vilanterol triphenylacetate is shown in Fig.10 The test data are shown in Table 7.

[0162] Table 7 System suitability results of vilanterol triphenylacetate

[0163]

[0164] As can be seen from Tables 6-7, after 6 repeated injections, the RSD value of the peak area of ​​umeclidinium bromide was 0.23% < 2.0%; the RSD of the retention time of umeclidinium bromide was 0.51% < 1.0%; the RSD value of the peak area of ​​vilanterol was 0.60% < 2.0%; the RSD of the retention time of vilanterol was 0.20% < 1.0%; the theoretical plate numbers of the umeclidinium bromide and vilanterol peaks were both > 3000, indicating that this fine particle dose determination method has good system applicability.

[0165] Example 3 Durability Test

[0166] Test solution: Connect the device according to the law (Chinese Pharmacopoeia 2015 Edition Part IV General Rules 0951 Device 3*). Before use, add 4 mL of 19.25% (w / v) polyethylene glycol 200 acetone solution to collection cup 1 and 2 mL of 19.25% (w / v) polyethylene glycol 200 acetone solution to the other collection cups. Allow the acetone to evaporate. Add 15 mL of methanol-water (60:40) to the pre-separator. Place special filter paper on the external filter. Adjust the flow rate to 60 L / min. Check the pressure drop ratio of the control valve. P3 / P2 should not be greater than 0.5. Take 1 box of this product, pull the switch, insert the adapter, turn on the vacuum pump, pump for 4 seconds, and repeatedly suck out the 1st, 8th, 16th, 23rd and 30th doses (the remaining doses are discarded in a dedicated device, and the flow rate is adjusted to (60±2) L / min). Wait at least 30 seconds between each suction, turn off the pump, remove the suction device, weigh, record the reading, and test 5 suctions in total. Wash each component with the corresponding volume of methanol-water (60:40) according to the table below to extract the sample, shake to dissolve, filter, and take the filtrate as the NGI test solution.

[0167] Location Solvent volume (mL) Shaking method Shaking time (min) Nozzle adapter 30 Sealed bag hand shake Shake for 1 minute and let stand for 10 minutes L-type connecting pipe 50 Rocker 10min for each side Pre-separator 200 Rocker 10min for each side Level 1 Collection Cup 15 Oscillator 10min Level 2 Collection Cup 15 Oscillator 10min Level 3 Collection Cup 15 Oscillator 10min Level 4 Collection Cup 15 Oscillator 10min Level 5 Collection Cup 15 Oscillator 10min Level 6 Collection Cup 15 Oscillator 10min Level 7 Collection Cup 15 Oscillator 10min MOC Grade Collection Cup 100 Oscillator 10min

[0168] Reference solution: accurately weigh 15 mg of umeclidinium bromide reference substance and place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use this as the umeclidinium bromide stock solution; accurately weigh 1 mL of umeclidinium bromide stock solution and place it in a 25 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use this as the stock solution a;

[0169] Accurately weigh 20 mg of vilanterol triphenylacetate reference substance and place it in a 100 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use it as the vilanterol triphenylacetate stock solution; accurately weigh 1 mL of vilanterol triphenylacetate stock solution and place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol-water (60:40), shake well, and use it as the stock solution b;

[0170] Accurately measure 1 mL of stock solution a and b respectively, place them in a 10 mL volumetric flask, dissolve and dilute to the scale with methanol-water (60:40), shake well, and use them as reference solution.

[0171] Original chromatographic conditions:

[0172] Chromatographic column: Agilent ZORBAX SB-C18 (50×4.6 mm, 1.8 μm);

[0173] Mobile phase: Mobile phase A: 0.1 vol% trifluoroacetic acid solution, Mobile phase B: acetonitrile solution containing 0.1 vol% trifluoroacetic acid;

[0174] Detection wavelength: Vilanterol uses a fluorescence detector with an excitation wavelength of 225 nm and a detection wavelength of 315 nm, while umeclidinium uses a UV detector at 220 nm;

[0175] Column temperature: 30°C;

[0176] Flow rate: 1.0 mL / min;

[0177] Injection volume: 100 μL;

[0178] Gradient elution program:

[0179] Time (min) Mobile phase A (%) Mobile phase B (%) 0 65 35 4.0 30 70 4.1 65 35 6.0 65 35 .

[0180] The detection wavelength, column temperature, flow rate and initial ratio of mobile phase were slightly changed. The conditions for the changes of detection wavelength, column temperature, flow rate and initial ratio of mobile phase are as follows:

[0181]

[0182] 100 μL of each of the above solutions was accurately measured and tested under the various chromatographic conditions. The mass of each level of NGI umeclidinium bromide and vilanterol was calculated by the external standard method using the peak area of ​​umeclidinium bromide and vilanterol. Then, based on the mass of each level of umeclidinium bromide and vilanterol, the amount of fine particles less than 5.0 μm was calculated using the "Copley Inhaler Testing DataAnalysis Software" software. The consistency of the test results before and after the original chromatographic conditions were slightly changed was observed, and the results are shown in Tables 8-11.

[0183] Table 8 Flow rate variation fine particle dose measurement results

[0184]

[0185] Table 9 Column temperature variation fine particle dose measurement results

[0186]

[0187] Table 10 Detection wavelength variation fine particle dose measurement results

[0188]

[0189] Table 11: Results of fine particle dose determination with changes in mobile phase ratio

[0190]

[0191] It can be seen from Tables 8-11 that when the flow rate, column temperature, detection wavelength and initial phase ratio of mobile phase change slightly, there is no significant difference in the fine particle dose of umeclidinium bromide and vilanterol in the test solution, and the RSD values ​​are all <2.0%, indicating that the determination method provided in this study has good durability and can meet the requirements of fine particle dose determination.

[0192] Example 4 Sensitivity Test

[0193] Detection limits of umeclidinium and vilanterol: Take the reference solution containing umeclidinium and vilanterol with concentrations of 0.511μg / mL and 0.049μg / mL, respectively, dilute it step by step, inject it in sequence, record the chromatogram, and take the injection concentration when the signal-to-noise ratio of umeclidinium and vilanterol in the chromatogram is 2-4 as the detection limit. After investigation, the signal-to-noise ratio of the main peak of umeclidinium is 4.12, and the signal-to-noise ratio of the main peak of vilanterol is 3.86. Therefore, the minimum detection limits of umeclidinium and vilanterol are 0.0025μg / mL and 0.00024μg / mL, respectively.

[0194] Limit of quantification of umeclidinium and vilanterol: Take the reference solution containing umeclidinium and vilanterol with concentrations of 0.511μg / mL and 0.049μg / mL, respectively, dilute it step by step, inject it in sequence, record the chromatogram, and take the injection concentration when the signal-to-noise ratio of umeclidinium and vilanterol in the chromatogram is 9-11 as the detection limit. After investigation, the signal-to-noise ratio of the main peak of umeclidinium is 11.98, and the signal-to-noise ratio of the main peak of vilanterol is 11.42. Therefore, the limits of quantification of umeclidinium and vilanterol are 0.0511μg / mL and 0.0049μg / mL, respectively.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol, characterized in that: High performance liquid chromatography was used, and the chromatographic conditions included: a chromatographic column with octadecylsilane bonded silica gel filler; a detector: a fluorescence detector was used for vilanterol, with an excitation wavelength of 220-230 nm and a detection wavelength of 310-320 nm, and a UV detector was used for umeclidinium bromide, with a detection wavelength of 210-230 nm; the mobile phase consisted of a mobile phase A and a mobile phase B, wherein the mobile phase A was a 0.05-0.15 vol% trifluoroacetic acid solution, and the mobile phase B was a 0.05-0.15 vol% trifluoroacetic acid acetonitrile solution, and gradient elution was performed according to the following table: Among them, A1=63-67%, A2=28-32%; B1=33-37%, B2=68-72%.

2. The method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol according to claim 1, characterized in that: The column temperature used was 28-32°C.

3. The method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol according to claim 1, characterized in that: The sample to be tested is dissolved in a mixture of methanol and water.

4. The method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder aerosol according to claim 3, characterized in that: The volume ratio of methanol to water is 60:

40.

5. The method for detecting the fine particle dosage of umeclidinium bromide and vilanterol inhalation powder according to any one of claims 1 to 4, characterized in that: The injection volume used was 90-110 μL.

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