Method for detecting contents of budesonide, formoterol and glycopyrronium bromide in budigegfu inhalation aerosol

Abstract

The invention discloses a method for detecting the content of budesonide, formoterol and glycopyrronium bromide in budeagford inhalation aerosol, and relates to the technical field of drug detection and analysis, and the method comprises the following steps: preparing a test solution and a reference solution; detecting the contents of budesonide, formoterol and glycopyrronium bromide in the test solution and the reference solution by adopting a high performance liquid chromatography ultraviolet detector method; the detection conditions are as follows: octadecyl bonded silica gel is used as a filler, absolute ethyl alcohol-sodium dihydrogen phosphate solution is used as a mobile phase A, absolute ethyl alcohol-sodium dihydrogen phosphate solution-acetonitrile is used as a mobile phase B, and gradient elution is performed; the detection wavelength of the formoterol and the glycopyrronium bromide is 214 nm, and the detection wavelength of the budesonide is 254 nm. The detection method can simultaneously detect the contents of budesonide, formoterol fumarate and glycopyrronium bromide, has good specificity and high accuracy, and can effectively reduce the subsequent detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug detection and analysis, and particularly relates to a detection method for the contents of budesonide, formoterol and glycopyrronium bromide in budesonide / formoterol / glycopyrronium bromide inhalation aerosol. BACKGROUND

[0002] Budesonide / formoterol / glycopyrronium bromide inhalation aerosol is a compound preparation composed of budesonide, glycopyrronium bromide and formoterol. Budesonide / formoterol / glycopyrronium bromide inhalation aerosol is mainly used for the maintenance treatment of patients with chronic obstructive pulmonary disease (COPD). Budesonide is a glucocorticoid, which has a dose-dependent anti-inflammatory effect after inhalation through the airway, can reduce the symptoms of COPD, and reduce the acute exacerbation of COPD; formoterol has the effect of relaxing bronchial smooth muscle and relieving bronchial spasm; and glycopyrronium bromide is a long-acting acetylcholine receptor antagonist, which can inhibit the receptors of bronchial smooth muscle, thereby dilating the airway. The combination of three drugs with different mechanisms of action can increase the efficacy and help to further improve lung function.

[0003] At present, the quality standards of budesonide / formoterol / glycopyrronium bromide inhalation aerosol are not collected in various pharmacopoeias (such as USP, EP, BP and ChP), and only the content determination method of the raw material drug is collected. The method only controls the content of a single component, and is not suitable for compound preparations, that is, it cannot realize the detection of the contents of budesonide, formoterol and glycopyrronium bromide in budesonide / formoterol / glycopyrronium bromide inhalation aerosol.

[0004] Therefore, since the current determination method cannot quantitatively detect the three components in budesonide / formoterol / glycopyrronium bromide inhalation aerosol, it is difficult to ensure the controllability of the quality of the product, and therefore it is urgent to develop a detection method for the determination of the contents of the three main components in budesonide / formoterol / glycopyrronium bromide inhalation aerosol. SUMMARY

[0005] Based on the problem that the current determination method cannot quantitatively detect the three components in budesonide / formoterol / glycopyrronium bromide inhalation aerosol, it is difficult to ensure the controllability of the quality of the product, the purpose of the present application is to provide a detection method for the contents of budesonide, formoterol and glycopyrronium bromide in budesonide / formoterol / glycopyrronium bromide inhalation aerosol, which can simultaneously detect the contents of budesonide, formoterol and glycopyrronium bromide, has good specificity, high accuracy, and can effectively reduce the subsequent detection cost.

[0006] The present application realizes the following technical scheme:

[0007] The present application provides a detection method for the contents of budesonide, formoterol and glycopyrronium bromide in budesonide / formoterol / glycopyrronium bromide inhalation aerosol, which comprises the following steps:

[0008] Preparation of test sample solution and control solution;

[0009] The content of budesonide, formoterol and glycopyrronium bromide in the test solution and the control solution is detected by high performance liquid chromatography ultraviolet detector method; the detection conditions are as follows: octadecyl bonded silica is used as the filler, anhydrous ethanol-sodium dihydrogen phosphate solution is used as the mobile phase A, and anhydrous ethanol-sodium dihydrogen phosphate solution-acetonitrile is used as the mobile phase B for gradient elution; the detection wavelength of formoterol and glycopyrronium bromide is 210nm-220nm, preferably 214nm, and the detection wavelength of budesonide is 210nm-260nm, preferably 254nm.

[0010] The preparation method of the test solution is as follows: 1 bottle of budesonide formoterol inhalation aerosol is taken, the outer package is removed, the surface is cleaned with ethanol, and then accurately weighed (W1); a small hole is drilled on the aluminum cap, and a syringe needle is inserted (without contacting the liquid surface); after the propellant is volatilized and exhausted, the bottle is separated from the valve, and then placed in a 250ml conical flask; 100ml of solvent (the solvent is 30mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) is added, sealed, ultrasonically treated for 10min, centrifuged at 10000rpm and 5℃ for 5min, and then the supernatant is taken as the test solution; the aluminum cap and the medicine tank are rinsed with methanol, dried, and then accurately weighed (W2); and W1-W2 is the sampling amount of the test solution.

[0011] The preparation method of the control solution is as follows: appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide control substances are respectively accurately weighed, dissolved in the solvent and quantitatively diluted to prepare a control solution containing about 0.31mg of budesonide, 0.009mg of formoterol fumarate and 0.018mg of glycopyrronium bromide per 1ml. In the chromatogram of the control solution, the theoretical plate number of formoterol fumarate is not less than 7000, the theoretical plate number of glycopyrronium bromide is not less than 20000, the theoretical plate number of the two epimers of budesonide is not less than 15000, and the separation degree of the two epimers of budesonide should be not less than 1.5.

[0012] According to the polarity difference of each component (formoterol fumarate and glycopyrronium bromide are high-polarity, and budesonide is medium-polarity), the analysis method of the reversed-phase chromatography system is selected, and the conventional octadecyl bonded silica chromatographic column is used to achieve the effective separation of each component. Since there are unsaturated carbonyl (C=O) and benzene ring structures in the three compounds, they all have ultraviolet absorption, so the ultraviolet detector is selected, and the content test is completed by using double wavelengths according to the ultraviolet absorption band difference of each component. Meanwhile, combined with the different pKa of each component (formoterol is about 8.61, glycopyrronium bromide is about 11.53, and budesonide is about 13.75, the data is from Chemaxon), the appropriate mobile phase is selected to realize the simultaneous detection of the content of budesonide, formoterol fumarate and glycopyrronium bromide. The detection method has good specificity, high accuracy, and can effectively reduce the subsequent detection cost.

[0013] In a specific embodiment, the pH of the sodium dihydrogen phosphate solution in the mobile phase A and the mobile phase B is 2.9-3.3.

[0014] In a specific embodiment, the concentration of the sodium dihydrogen phosphate solution in the mobile phase A and the mobile phase B is 28-32 mmol / L, preferably 30 mmol / L.

[0015] In a specific embodiment, the volume ratio of the anhydrous ethanol to the sodium dihydrogen phosphate solution in the mobile phase A is 0:100-4:100, preferably 2:100.

[0016] In a specific embodiment, the mobile phase B is a combination of anhydrous ethanol, sodium dihydrogen phosphate solution and acetonitrile, and the volume ratio of the anhydrous ethanol is not more than 4%, the volume ratio of the sodium dihydrogen phosphate solution is not more than 30%, and the volume ratio of the acetonitrile is not more than 80%, preferably 2:25:75.

[0017] In a specific embodiment, the column temperature is controlled at 40-52°C.

[0018] In a specific embodiment, the flow rate is controlled at 0.7-1.2 ml / min.

[0019] In a specific embodiment, the injection volume is controlled at 5-15 μl.

[0020] In a specific embodiment, the injection temperature is controlled at 20°C.

[0021] In a specific embodiment, the elution procedure of the gradient elution is as follows:

[0022] .

[0023] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0024] (1) The detection method of the present application can simultaneously detect the content of budesonide, formoterol fumarate and glycopyrronium bromide, has good specificity, high accuracy, and can effectively reduce the subsequent detection cost.

[0025] (2) The present application selects the analysis method of the reversed-phase chromatography system according to the polarity difference of each component (formoterol fumarate and glycopyrronium bromide are high-polarity, and budesonide is medium-polarity), and uses a conventional octadecyl-bonded silica chromatographic column to achieve effective separation of each component.

[0026] (3) According to the difference of ultraviolet absorption wavelength bands of the components, the content test is completed by using double wavelengths, and meanwhile, the content of budesonide, formoterol fumarate and glycopyrronium bromide is detected by selecting a suitable mobile phase according to the difference of pKa (pKa of formoterol fumarate is about 8.61, pKa of glycopyrronium bromide is about 11.53, and pKa of budesonide is about 13.75, and the data is from Chemaxon). BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the example embodiments of the present application, the drawings needed in the examples will be briefly introduced as follows, and it should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope, and for those skilled in the art, other related drawings can also be obtained without creative labor on the basis of the drawings. In the drawings:

[0028] Figure 1 The chromatogram of the control solution in Example 1 of the present application;

[0029] Figure 2 The chromatogram of the control solution in Example 2 of the present application;

[0030] Figure 3 The chromatogram of the control solution in Example 3 of the present application;

[0031] Figure 4 The chromatogram of the control solution in Example 4 of the present application;

[0032] Figure 5 The chromatogram of the control solution in Example 5 of the present application;

[0033] Figure 6 The chromatogram of the control solution in Example 6 of the present application;

[0034] Figure 7 The chromatogram of the control solution in Example 7 of the present application;

[0035] Figure 8 The chromatogram of the control solution in Example 8 of the present application;

[0036] Figure 9 The chromatogram of the control solution in Comparative Example 1 of the present application;

[0037] Figure 10 The chromatogram of the control solution in Comparative Example 2 of the present application;

[0038] Figure 11 The chromatogram of the control solution in Comparative Example 3 of the present application. DETAILED DESCRIPTION

[0039] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with embodiments. The illustrative embodiments of the present application and their description are only used to explain the present application and not as a limitation of the present application.

[0040] In the following description, numerous specific details are set forth to provide a thorough understanding of the present application. However, it will be apparent to one of ordinary skill in the art that the present application can be practiced without the specific details. In other instances, well-known materials or methods have not been described in detail in order to avoid obscuring the present application.

[0041] Throughout this specification, the term "one embodiment," "an embodiment," "one example," or "an example" means that a particular feature, structure, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. Therefore, the appearance of the phrases "in one embodiment," "in an embodiment," "in one example," or "in an example" in various places in the specification are not necessarily referring to the same embodiment or example. Furthermore, the particular features, structures, or characteristics can be combined in any suitable

[0042] "ranges" disclosed herein are defined with endpoints between the lower and the upper limit and include the endpoints. Ranges created by combining these open-ended range listings have the same endpoints as the ranges from which they were created. It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. There is no intention to limit the application to the specific embodiments disclosed, but the specific embodiments disclosed are supplied solely by way of example. Any reference citations, including patents and scientific literature, cited herein are hereby incorporated by reference in their entirety.

[0043] If not otherwise specified, all steps of the present application can be carried out in sequence or randomly, preferably in sequence. For example, the method comprises steps (a) and (b) means that the method can comprise steps (a) and (b) in sequence, or steps (b) and (a) in sequence. For example, the method also comprises step (c) means that step (c) can be added to the method in any order, for example, the method can comprise steps (a), (b) and (c), or steps (a), (c) and (b), or steps (c), (a) and (b), etc.

[0044] Example 1

[0045] The present example provides a method for detecting the content of budesonide, formoterol and glycopyrronium bromide in budesonide-formoterol-glycopyrronium bromide inhalation aerosol, the specific steps are as follows:

[0046] S1, preparing test solution: taking one bottle of budesonide-formoterol-glycopyrronium bromide inhalation aerosol (Jiangsu Dimai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing budesonide 160 μg, glycopyrronium bromide 7.2 μg and formoterol fumarate 4.8 μg), removing the outer packaging, cleaning the surface with ethanol, accurately weighing (W1), drilling a small hole on the aluminum cap, inserting a syringe needle (not in contact with the liquid surface), after the propellant gas is evaporated and exhausted, separating the bottle from the valve, placing it in a 200 ml conical flask, adding 100 ml of solvent (30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), sealing, ultrasonic for 10 min, centrifuging at 10000 rpm for 5 min at 5℃, taking the supernatant as the test solution, rinsing the aluminum cap and the medicine tank with methanol, drying and accurately weighing (W2), taking W1-W2 as the sample amount.

[0047] S2, preparing control solution: taking appropriate amount of budesonide, formoterol fumarate and glycopyrronium bromide control substances respectively, accurately weighing and dissolving in solvent (30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and quantitatively diluting to prepare a control solution containing about 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per 1 ml.

[0048] S3, accurately measuring the test solution and the control solution, and injecting them into the liquid chromatograph respectively, and recording the chromatogram.

[0049] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0050] .

[0051] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0052] like Figure 1 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0053] Example 2

[0054] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol. The difference from Embodiment 1 is that the pH value of the sodium dihydrogen phosphate solution in both mobile phase A and mobile phase B is 3.1. Other processes are the same as in Embodiment 1.

[0055] The specific steps are as follows:

[0056] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0057] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0058] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0059] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 3.1) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 3.1)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0060] .

[0061] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0062] like Figure 2 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0063] Example 3

[0064] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol. The difference from Embodiment 1 is that the pH value of the sodium dihydrogen phosphate solution in both mobile phase A and mobile phase B is 3.3. Other processes are the same as in Embodiment 1.

[0065] The specific steps are as follows:

[0066] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0067] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0068] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0069] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 3.3) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 3.3)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0070] .

[0071] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0072] like Figure 3The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0073] Example 4

[0074] This embodiment provides a method for detecting the content of budesonide, formoterol, and glycopyrronium bromide in budesonide / formoterol inhaler aerosol. The difference from Embodiment 1 is that the column temperature in step S3 of this embodiment is controlled at 40°C. Other processes are the same as in Embodiment 1.

[0075] The specific steps are as follows:

[0076] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0077] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0078] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0079] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0080] .

[0081] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0082] like Figure 4 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0083] Example 5

[0084] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol. The difference from Embodiment 1 is that the flow rate in step S3 of this embodiment is controlled at 0.8 ml per minute. Other processes are the same as in Embodiment 1.

[0085] The specific steps are as follows:

[0086] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff contains 160 μg of budesonide, 7.2 μg of glycopyrronium bromide and 4.8 μg of formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (the solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry and weigh accurately (W2). The sample amount is W1-W2.

[0087] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0088] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0089] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution according to the table below; column temperature was 40℃; flow rate was 0.8 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0090] .

[0091] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0092] like Figure 5 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0093] Example 6

[0094] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol. The difference from Embodiment 1 is that the flow rate in step S3 of this embodiment is controlled at 1.2 ml per minute. Other processes are the same as in Embodiment 1.

[0095] The specific steps are as follows:

[0096] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0097] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0098] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0099] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.2 mL / min; injection volume was 10 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide was detected at 254 nm). The gradient elution program was as follows:

[0100] .

[0101] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0102] like Figure 6The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0103] Example 7

[0104] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in budesonide / formoterol inhalation aerosol. The difference from Embodiment 1 is that the injection volume in step S3 of this embodiment is controlled at 5 μl. Other processes are the same as in Embodiment 1.

[0105] The specific steps are as follows:

[0106] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff contains 160 μg of budesonide, 7.2 μg of glycopyrronium bromide and 4.8 μg of formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (the solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry and weigh accurately (W2). The sample amount is W1-W2.

[0107] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0108] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0109] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 5 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0110] .

[0111] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0112] like Figure 7 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0113] Example 8

[0114] This embodiment provides a method for detecting the contents of budesonide, formoterol, and glycopyrronium bromide in budesonide / formoterol inhalation aerosol. The difference from Embodiment 1 is that the injection volume in step S3 of this embodiment is controlled at 15 μl. Other processes are the same as in Embodiment 1.

[0115] The specific steps are as follows:

[0116] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0117] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0118] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0119] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution as shown in the table below; column temperature was 40℃; flow rate was 1.0 mL / min; injection volume was 15 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0120] .

[0121] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0122] like Figure 8 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peaks of formoterol fumarate, glycopyrronium bromide and budesonide are well-defined, and the two peaks of budesonide are clearly well separated.

[0123] Comparative Example 1

[0124] This comparative example provides a method for detecting the content of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol. The difference from Example 1 is that anhydrous ethanol was not added to the sodium dihydrogen phosphate solution in mobile phases A and B in this comparative example. Other processes are the same as in Example 1.

[0125] The specific steps are as follows:

[0126] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0127] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0128] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0129] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; 30 mmol / L sodium dihydrogen phosphate solution (pH: 2.5) was used as mobile phase A, and 30 mmol / L sodium dihydrogen phosphate solution (pH: 2.5)-acetonitrile (25:75) was used as mobile phase B, with gradient elution according to the table below; column temperature was 40℃; flow rate was 0.8 mL / min; injection volume was 5 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide was detected at 254 nm). The gradient elution program was as follows:

[0130] .

[0131] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0132] like Figure 9As shown, the chromatogram formed by the method of this embodiment shows that the peak shape of budesonide is good, but there is no baseline separation between 11-ketobudesonide and budesonide.

[0133] Place in a 250ml Erlenmeyer flask, add 100ml of solvent (Comparative Example 2)

[0134] This comparative example provides a method for detecting the content of budesonide, formoterol, and glycopyrronium bromide in a budesonide / glucosamine inhaler aerosol, which differs from Example 1 in that the proportion of the organic phase in the elution process is increased. Other processes are the same as in Example 1.

[0135] The specific steps are as follows:

[0136] S1. Preparation of the test solution: Take one bottle of budesonide / formoterol inhaler (Jiangsu Demai Pharmaceutical Co., Ltd., 120 puffs per bottle, each puff containing 160 μg budesonide, 7.2 μg glycopyrronium bromide and 4.8 μg formoterol fumarate), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve, place it in a 250 ml conical flask, add 100 ml of solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10 min, centrifuge at 10000 rpm at 5℃ for 5 min, and take the supernatant as the test solution. Rinse the aluminum cap and the container with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0137] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0138] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0139] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution according to the table below; column temperature was 35℃; flow rate was 0.8 mL / min; injection volume was 5 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide was detected at 254 nm). The gradient elution program was as follows:

[0140] .

[0141] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0142] like Figure 10 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that the peak shape of budesonide is good, and 11-ketobudesonide and budesonide completely overlap.

[0143] Comparative Example 3

[0144] This comparative example provides a method for detecting the content of budesonide, formoterol, and glycopyrronium bromide in a budesonide / formoterol inhaler aerosol, which differs from Example 1 in that the proportion of the organic phase in the elution gradient is reduced. Other processes are the same as in Example 1.

[0145] The specific steps are as follows:

[0146] S1. Preparation of test solution: Take one bottle of Budegene-Foam inhaler (Jiangsu Demai Pharmaceutical Co., Ltd.), remove the outer packaging, clean the surface with ethanol, and weigh accurately (W1). Drill a small hole in the aluminum cap and insert the injection needle (do not contact the liquid surface). After the propellant has vaporized and evaporated, separate the bottle from the valve and place it in a 250ml conical flask. Add 100ml of solvent (the solvent is 30mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)), seal the bottle, sonicate for 10min, centrifuge at 10000rpm at 5℃ for 5min, and take the supernatant as the test solution. Rinse the aluminum cap and the canister with methanol, dry, and weigh accurately (W2). The sample amount is W1-W2.

[0147] S2. Preparation of reference solutions: Take appropriate amounts of budesonide, formoterol fumarate and glycopyrronium bromide reference standards, weigh them accurately, dissolve them in solvent (solvent is 30 mmol / L sodium dihydrogen phosphate (pH=3.1)-methanol (1:1)) and dilute quantitatively to prepare a reference solution containing approximately 0.31 mg of budesonide, 0.009 mg of formoterol fumarate and 0.018 mg of glycopyrronium bromide per ml.

[0148] S3. Accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0149] The chromatographic conditions were as follows: Octadecyl-bonded silica gel was used as the stationary phase; mobile phase A was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9) (2:100), and mobile phase B was anhydrous ethanol-30 mmol / L sodium dihydrogen phosphate solution (pH: 2.9)-acetonitrile (2:25:75), with gradient elution according to the table below; column temperature was 55℃; flow rate was 0.8 mL / min; injection volume was 5 μL; injection plate temperature was 20℃; and detection wavelengths were 214 nm / 254 nm (formoterol fumarate and glycopyrronium bromide were detected at 214 nm, and budesonide at 254 nm). The gradient elution program was as follows:

[0150] .

[0151] System suitability requirements: In the chromatogram of the reference solution, the theoretical plate number of formoterol fumarate shall not be less than 7000, the theoretical plate number of glycopyrronium bromide shall not be less than 20000, the theoretical plate number of the two epimers of budesonide shall not be less than 15000, and the peak resolution of the two epimers of budesonide shall not be less than 1.5.

[0152] like Figure 11 The figure shows a chromatogram formed using the method of this embodiment. It can be seen from the figure that extending the elution time of budesonide peaks results in poor economic benefits.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions 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, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for the determination of the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol, characterized in that, The method comprises the following steps: Preparation of test sample solution and control solution; The content of budesonide, formoterol and glycopyrronium bromide in the test sample solution and the control solution is detected by high performance liquid chromatography ultraviolet detector method; the detection condition is that octadecyl bonded silica gel is used as the filler, and anhydrous ethanol-sodium dihydrogen phosphate solution is used as the mobile phase A, and anhydrous ethanol-sodium dihydrogen phosphate solution-acetonitrile is used as the mobile phase B for gradient elution; the detection wavelength of formoterol and glycopyrronium bromide is 210nm~220nm, and the detection wavelength of budesonide is 210nm~260nm.

2. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The pH value of the sodium dihydrogen phosphate solution in the mobile phase A and the mobile phase B is 2.9~3.

3.

3. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The concentration of the sodium dihydrogen phosphate solution in the mobile phase A and the mobile phase B is 28mmol / L~32mmol / L.

4. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The volume ratio of anhydrous ethanol to sodium dihydrogen phosphate solution in the mobile phase A is 0:100~4:

100.

5. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The mobile phase B is a combination of anhydrous ethanol, sodium dihydrogen phosphate solution and acetonitrile, and the volume ratio of anhydrous ethanol is not more than 4%, the volume ratio of sodium dihydrogen phosphate solution is not more than 30%, and the volume ratio of acetonitrile is not more than 80%.

6. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The column temperature is controlled at 40℃~52℃.

7. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The flow rate is controlled at 0.7ml / min~1.2ml / min.

8. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The injection volume is controlled at 5μl~15μl.

9. A method for detecting the content of budesonide, formoterol and glycopyrronium bromide in a budesonide, formoterol and glycopyrronium bromide inhalation aerosol according to claim 1, characterized in that, The elution program of the gradient elution is as follows: 。

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