CN113125570A

CN113125570A - Method for detecting micro-particle dose of Wumei ammonium bromide vilanterol inhalation powder aerosol

Info

Publication number: CN113125570A
Application number: CN201911391912.5A
Authority: CN
Inventors: 张克霞; 夏俊; 郭志强
Original assignee: Tianjin Pharmaceutical Research Institute Co Ltd
Current assignee: Tianjin Pharmaceutical Research Institute Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2021-07-16

Abstract

The invention provides a method for detecting the dose of fine particles of an Wumei ammonium bromide vilanterol inhalation powder aerosol, and relates to the technical field of analytical chemistry. The method for detecting the fine particle dose of the Wumei ammonium bromide vilanterol inhalation powder aerosol adopts high performance liquid chromatography, and the chromatographic conditions comprise that: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: a fluorescence detector is used for Vilantelo, and an ultraviolet detector is used for Umamizium bromide; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, and gradient elution. The invention can rapidly and accurately detect the micro-particle doses of the wumei ammonium bromide and the vilanterol tritetate in the wumei ammonium bromide vilanterol inhalation powder aerosol by using a mobile phase condition; simple operation, accurate and reliable measuring result, strong specificity, good durability, high sensitivity and short detection time.

Description

Method for detecting micro-particle dose of Wumei ammonium bromide vilanterol inhalation powder aerosol

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a method for detecting the dose of fine particles of a Umamizumab vilanterol inhalation powder aerosol.

Background

Respiratory diseases are common diseases, high morbidity, mortality rate second to tumor and cardiovascular diseases, and asthma and Chronic Obstructive Pulmonary Disease (COPD) are worldwide recognized medical problems. In China, over 3000 thousands of asthma patients and over 8000 thousands of COPD patients exist, inhalation preparations are internationally accepted as the first choice medicine for treating asthma and COPD, but at present, 70 percent of patients never use inhalation therapy, inhalation drug therapists used for a long time in rural areas only account for 4.46 percent, and most of patients treat asthma by injection and medication, so cardiovascular side effects are frequent.

The inhalation preparation is mainly divided into aerosol and powder inhalation, the powder inhalation is easier to use than the aerosol, and the patient actively inhales the powder; the propellant Freon is not used, so that the pollution to the atmospheric environment is avoided; the metering is accurate, and the risk of overdose administration is avoided; the gel does not contain solvents such as antiseptic, ethanol and the like, and has no irritation to pathological mucosa; the medicine is in a dry powder shape, and has the advantages of good stability, less interference factors and the like.

Wumei ammonium bromide vilanterol inhalation powder aerosol is a 4-class imitation drug developed by my company. Has long-acting bronchodilatory effect, is suitable for long-term maintenance therapy of Chronic Obstructive Pulmonary Disease (COPD), and can be used for relieving symptoms of COPD patients once a day. Wumei ammonium bromide is a long-acting muscarinic receptor antagonist, and plays a role in bronchiectasis mainly by competitively inhibiting the binding of acetylcholine to M3-type muscarinic receptors on respiratory smooth muscle. Vilanterol is a selective long-acting β 2 adrenoceptor agonist with activation of intracellular adenylate cyclase, which catalyzes the conversion of ATP to cyclic-3 ', 5' -phosphoadenosine cAMP levels, relaxes bronchial smooth muscle and inhibits the release of immediate hypersensitivity mediators by cells, especially mast cells. The systemic exposure (AUC and Cmax) of the single agent administered by inhalation of the combination of umei bromide and vilanterol tritoate was similar or lower than that of the single agent administered alone, and there was no increasing effect of the single agent on the exposure of the other components in the combination. The combined administration can realize mechanism complementation through a dual-action passage when taking effect, achieves the effect of optimizing airway comfort, takes effect quickly, can achieve the characteristic of long-acting performance for 24 hours, can quickly improve the uncomfortable condition of a patient, can stably improve the lung function, and relieves a series of uncomfortable symptoms caused by chronic obstructive pneumonia.

The molecular formula of the trityl acetate vilanterol is C₂₄H₃₃Cl₂NO₅·C₂₀H₁₆O₂The structural formula (I) is as follows:

the molecular formula of the Wumei ammonium bromide is C₂₉H₃₄BrNO₂The structural formula (II) is as follows:

through literature research, no method for simultaneously measuring the fine particle doses of the effective components of the ume ammonium bromide and the vilanterol trithionate in the ume ammonium bromide vilanterol inhalation powder inhalation agent in any literature and pharmacopoeia is found.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The main object of the present invention is to provide a method for detecting the fine particle dose of an inhaled powder aerosol of umei ammonium bromide vilanterol, which is intended to at least partially solve at least one of the above technical problems.

The invention provides a method for detecting the dose of fine particles of an Wumei ammonium bromide vilanterol inhalation powder aerosol, which adopts a high performance liquid chromatography, and the chromatographic conditions comprise that: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: a fluorescence detector is used for Vilantelo, and an ultraviolet detector is used for Umamizium bromide; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, gradient elution according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	A1	B1
3.5-4.0	A2	B2

Wherein, A1 is 63-67%, A2 is 28-32; b1-33%, B2-68-72%.

Further, high performance liquid chromatography is adopted, and the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: vilantelo uses a fluorescence detector, Umamizium bromide uses an ultraviolet detector; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, gradient elution according to the following table:

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

Wherein, A1 is 63-67%, A2 is 28-32; b1-33%, B2-68-72%.

Further, the mobile phase A is 0.05-0.15 vol% trifluoroacetic acid solution.

Further, the mobile phase A is 0.1 vol% trifluoroacetic acid solution.

Further, the mobile phase B is acetonitrile solution containing 0.05-0.15 vol% of trifluoroacetic acid.

Further, the mobile phase B is acetonitrile solution containing 0.1 vol% of trifluoroacetic acid.

Furthermore, the Vilantelo uses a fluorescence detector, the excitation wavelength is 220-230nm, and the detection wavelength is 310-320 nm.

Further, the vilanterol uses a fluorescence detector, the excitation wavelength is 225nm, and the detection wavelength is 315 nm.

Further, the detection wavelength of the Wumei ammonium bromide is 210-230nm by using an ultraviolet detector.

Further, the Wumei ammonium bromide uses an ultraviolet detector, and the detection wavelength is 220 nm.

Further, the column temperature used was 28-32 ℃.

Further, the column temperature used was 30 ℃.

Further, the sample to be detected is dissolved by using a mixed solution of methanol and water.

Further, the volume ratio of the methanol to the water is 60: 40.

further, the flow rate used was 0.8-1.2 mL/min.

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

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

Further, the amount of the sample to be used is 90 to 110. mu.L.

Further, the amount of the sample used was 100. mu.L.

Further, high performance liquid chromatography is adopted, and the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: the excitation wavelength of the Vilantelo is 230nm plus 220nm, the detection wavelength of the Vilantelo is 320nm plus 310nm, the detection wavelength of the Uume ammonium bromide is 230nm plus 210 nm; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, gradient elution according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	65	35
4.0	30	70

。

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	65	35
4.0	30	70
			4.1	65	35
6.0	65	35

。

Further, high performance liquid chromatography is adopted, and the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: the Vilantelo uses a fluorescence detector, the excitation wavelength is 225nm, the detection wavelength is 315nm, the Uume ammonium bromide uses an ultraviolet detector, and the detection wavelength is 220 nm; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: 0.1 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile containing 0.1 vol% trifluoroacetic acid, gradient elution according to the following table:

。

Compared with the prior art, the invention has the following beneficial effects:

according to the detection method of the micro-particle dose of the wumei ammonium bromide vilanterol inhalation powder aerosol, a set of liquid chromatography system is developed by optimizing a detector, a mobile phase and an elution gradient according to the physicochemical properties of the wumei ammonium bromide and the vilanterol tritetate, and the micro-particle dose of two components of the wumei ammonium bromide and the vilanterol tritetate in the wumei ammonium bromide vilanterol inhalation powder aerosol can be rapidly and accurately detected by using a mobile phase condition; the detection method has the advantages that: simple operation, accurate and reliable measuring result, strong specificity, good durability, high sensitivity and short detection time.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is an HPLC chromatogram of a fluorescent channel tolnaftate blank adjuvant;

FIG. 2 is an HPLC chromatogram of a blank auxiliary material of ultraviolet channel Umei ammonium bromide;

FIG. 3 is an HPLC chromatogram of a fluorescent channel tolnaftate blank solvent;

FIG. 4 is an HPLC chromatogram of a blank solvent of Ume ammonium bromide in an ultraviolet channel;

FIG. 5 is an HPLC chromatogram of a fluorescent channel tolnaftate control solution;

FIG. 6 is an HPLC chromatogram of a control solution of ultraviolet channel ammonium umei bromide;

FIG. 7 is an HPLC chromatogram of a fluorescent channel tolytron NGI assay;

FIG. 8 is an HPLC chromatogram of an ultraviolet channel Umamizumab NGI test;

FIG. 9 is an HPLC chromatogram of a system suitability test for Umamizumab;

FIG. 10 is an HPLC chromatogram of a system suitability test of vilanterol tritoate.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples were carried out under the conventional conditions, unless otherwise specified. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Water, from Dreches;

acetonitrile (chromatographically pure), from TEDIA;

trifluoroacetic acid (analytically pure) from chemical reagents ltd, miuiou, department of tianjin;

umami ammonium bromide working reference (purity 99.63%) from Tianjin pharmaceutical research institute, Inc.;

vilanterol trithionate working control (purity 99.11%) from Tianjin pharmaceutical research institute, ltd;

uumei ammonium bromide vilanterol inhalation powder aerosol from Tianjin pharmaceutical research institute, Inc.

Example 1 specificity test

Test solution: according to the method, a device is connected (0951 device 3 of the four general rules of the Chinese pharmacopoeia 2015 edition), before use, 4mL of 19.25% (w/v) polyethylene glycol 200 acetone solution is added into a collection cup 1, 2mL of 19.25% (w/v) polyethylene glycol 200 acetone solution is added into the other collection cups, the collection cups are placed until acetone is volatilized, 15mL of methanol-water (60: 40) is added into a preseparator, a special filter paper is placed on an external filter, the flow rate is adjusted to 60L/min, the pressure drop ratio of a control valve is checked, and P3/P2 is not more than 0.5. Taking a box of the product 1, pulling a switch, inserting an adapter, starting a vacuum pump, exhausting for 4S, repeatedly sucking out the 1 st, 8 th, 16 th, 23 th and 30 th sucked doses (the rest sucked doses are discarded in a special device, and the flow rate is adjusted to (60 +/-2) L/min), waiting for at least 30S between every two suctions, closing the pump, taking down the suction device, weighing, recording the reading, and testing for 5 suctions in total. The respective parts were washed separately with the corresponding volumes of methanol-water (60: 40) according to the table to extract samples, shaken to dissolve, filtered, and the subsequent filtrate was taken as the NGI test sample solution.

Mixing the reference solution: taking a proper amount of the Umamizumab and a proper amount of a vilanterol trithionate reference substance, precisely weighing, adding methanol-water (60: 40) for dissolving, and quantitatively diluting to prepare a mixed reference substance solution containing 1.1 mu g of Umamizumab and 1.25 mu g of vilanterol in every 1 mL;

taking a proper amount of Ume ammonium bromide reference substance, and preparing a Ume ammonium bromide reference substance solution containing about 1.1 mu g of Ume ammonium bromide in each 1mL by the same method;

then taking a proper amount of the trithioacetate vilanterol reference substance, and preparing trithioacetate vilanterol reference substance solution containing 0.25 mu g of vilanterol in every 1mL by the same method.

Blank adjuvant solution: preparing an adjuvant solution in proportion to the main drug amount by the same method to obtain a blank adjuvant solution.

Blank solvent: methanol-water (60: 40).

Examples 1 to 1

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

mobile phase: mobile phase A: 0.1 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing 0.1 vol% trifluoroacetic acid;

detection wavelength: the Vilantelo uses a fluorescence detector, the excitation wavelength is 225nm, the detection wavelength is 315nm, and the ultraviolet detector for Wumei ammonium bromide is 220 nm;

column temperature: 30 ℃;

flow rate: 1.0 mL/min;

sample introduction amount: 100 mu L of the solution;

gradient elution was performed according to the following table:

。

Precisely measuring 100 μ L of each solution, injecting into liquid chromatograph, and recording chromatogram. An HPLC chromatogram of the fluorescent channel vilanterol triphenylacetate blank auxiliary material is shown in figure 1, an HPLC chromatogram of the ultraviolet channel vilanterol bromide blank auxiliary material is shown in figure 2, an HPLC chromatogram of the fluorescent channel vilanterol triphenylacetate blank solvent is shown in figure 3, an HPLC chromatogram of the ultraviolet channel vilanterol bromide blank solvent is shown in figure 4, an HPLC chromatogram of the fluorescent channel vilanterol triphenylacetate reference substance solution is shown in figure 5, an HPLC chromatogram of the ultraviolet channel vilanterol bromide to the reference substance solution is shown in figure 6, an HPLC chromatogram of the fluorescent channel vilanterol NGI test is shown in figure 7, and an HPLC chromatogram of the ultraviolet channel vilanterol bromide NGI test is shown in figure 8.

The mass of each grade of the Umamizumab and the Vilandiolo of the NGI is respectively calculated according to the external standard method by using the peak areas of the Umamizumab and the Vilandiolo, and the amount of the fine particle medicine smaller than 5.0 mu m is calculated by using the Software of Copley Inhaler Testing DataAnalyss Software according to the mass of each grade of the Umamizumab and the Vilandiolo, and the result is shown in Table 1.

TABLE 1 Fine particle dosimetry results

As can be seen from fig. 1 to 8, under the detection method of the present embodiment, the blank auxiliary materials and the solvent do not interfere with the main drug, the separation degrees of the wumei ammonium bromide and the vilanterol are good, and no adjacent impurity peak exists.

Examples 1 to 2

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

mobile phase: mobile phase A: 0.05 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing 0.15 vol% trifluoroacetic acid;

detection wavelength: the Vilantelo uses a fluorescence detector, the excitation wavelength is 220nm, the detection wavelength is 320nm, and the ultraviolet detector for Umamizumab is 210 nm;

column temperature: 31 ℃;

flow rate: 0.9 mL/min;

sample introduction amount: 110 mu L of the solution;

gradient elution was performed according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	63	37
3.5	32	68
			4.6	63	37
5.5	63	37

。

Precisely measuring 100 μ L of each solution, injecting into liquid chromatograph, and recording chromatogram. Since the chromatogram of this example is not much different from the chromatogram of example 1-1, it is not provided here.

The mass of each grade of the NGI Ume ammonium bromide and the mass of each grade of the NGI Viland Row are respectively calculated by the peak areas of the Ume ammonium bromide and the Viland Row according to an external standard method, and the amount of the fine particle medicine with the particle size of less than 5.0 mu m is calculated by using 'Copley Inhaler Testing DataAnalyss Software' according to the mass of each grade of the Ume ammonium bromide and the Viland Row, and the result is shown in Table 2.

TABLE 2 Fine particle dosimetry results

Under the chromatographic conditions of the detection method, the blank auxiliary materials and the solvent have no interference on the main drug, the separation degrees of the wumei ammonium bromide and the vilanterol are good, and no adjacent impurity peak exists.

Examples 1 to 3

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

mobile phase: mobile phase A: 0.15 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing 0.05 vol% trifluoroacetic acid;

detection wavelength: the Vilantelo uses a fluorescence detector, the excitation wavelength is 230nm, the detection wavelength is 310nm, and the ultraviolet detector for Wumei ammonium bromide is 230 nm;

column temperature: 29 ℃;

flow rate: 1.1 mL/min;

sample introduction amount: 90 mu L of the solution;

gradient elution was performed according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	67	33
3.8	28	72
			4.4	67	33
5.8	67	33

。

The mass of each grade of the NGI Ume ammonium bromide and the mass of each grade of the NGI Viland Row are respectively calculated by the peak areas of the Ume ammonium bromide and the Viland Row according to an external standard method, and the amount of the fine particle medicine with the particle size of less than 5.0 mu m is calculated by using 'Copley Inhaler Testing DataAnalyss Software' according to the mass of each grade of the Ume ammonium bromide and the Viland Row, and the result is shown in Table 3.

TABLE 3 Fine particle dosimetry results

Examples 1 to 4

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

mobile phase: mobile phase A: 0.08 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing 0.12 vol% trifluoroacetic acid;

detection wavelength: the Vilantelo uses a fluorescence detector, the excitation wavelength is 223nm, the detection wavelength is 317nm, and the ultraviolet detector for Wumei ammonium bromide is 218 nm;

column temperature: 28 ℃;

flow rate: 0.8 mL/min;

sample introduction amount: 95 mu L of the solution;

gradient elution was performed according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	66	34
4.0	29	71

。

The mass of each grade of the Umamizumab and the Vilandiolo of the NGI is respectively calculated according to the external standard method by using the peak areas of the Umamizumab and the Vilandiolo, and the amount of the fine particle medicine smaller than 5.0 mu m is calculated by using the Software of Copley Inhaler Testing Data Analysis Software according to the mass of each grade of the Umamizumab and the Vilandiolo, and the result is shown in Table 4.

TABLE 4 Fine particle dosimetry results

Examples 1 to 5

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

mobile phase: mobile phase A: 0.12 vol% trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing 0.08 vol% trifluoroacetic acid;

detection wavelength: the Vilantelo uses a fluorescence detector, the excitation wavelength is 227nm, the detection wavelength is 312nm, and the ultraviolet detector for Wumei ammonium bromide is 222 nm;

column temperature: at 32 ℃;

flow rate: 1.2 mL/min;

sample introduction amount: 105 mu L of the solution;

gradient elution was performed according to the following table:

time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	64	36
4.0	31	69

。

The mass of each grade of the Umamizumab and the Vilandiolo of the NGI is respectively calculated according to the external standard method by using the peak areas of the Umamizumab and the Vilandiolo, and the amount of the fine particle medicine smaller than 5.0 μm is calculated by using the Software of Copley Inhaler Testing Data Analysis Software according to the mass of each grade of the Umamizumab and the Vilandiolo, and the result is shown in Table 5.

TABLE 5 Fine particle dosimetry results

Example 2 System suitability test

Control solution: accurately weighing 15mg of Ume ammonium bromide contrast, placing the Ume ammonium bromide contrast in a 100mL measuring flask, dissolving and diluting the Ume ammonium bromide contrast to a scale mark by using methanol-water (60: 40), shaking up to obtain a Ume ammonium bromide stock solution, accurately weighing 1mL of the Ume ammonium bromide stock solution, placing the Ume ammonium bromide stock solution in a 25mL measuring flask, dissolving and diluting the Ume ammonium bromide stock solution to the scale mark by using methanol-water (60: 40), and shaking up to obtain a stock solution a;

accurately weighing 20mg of a vilanterol trithionate reference substance, placing the control substance into a 100mL measuring flask, dissolving the control substance with methanol-water (60: 40) and diluting the control substance to a scale, shaking the control substance uniformly to serve as a vilanterol trithionate stock solution, accurately weighing 1mL of the vilanterol trithionate stock solution, placing the stock solution into a 50mL measuring flask, dissolving the stock solution with methanol-water (60: 40) and diluting the stock solution to a scale, and shaking the stock solution uniformly to serve as a stock solution b;

precisely measuring 1mL of each of the stock solutions a and b, placing in a 10mL measuring flask, dissolving with methanol-water (60: 40), diluting to scale, and shaking to obtain system applicability solution.

Chromatographic conditions are as follows:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

column temperature: 30 ℃;

flow rate: 1.0 mL/min;

sample introduction amount: 100 mu L of the solution;

gradient elution was performed according to the following table:

。

Taking a solution with system applicability, continuously feeding a sample for 6 needles, and inspecting the system applicability of chromatographic conditions. The HPLC chromatogram of the Umamizumab system applicability test is shown in FIG. 9, and the detection data is shown in Table 6.

Table 6 Umami ammonium bromide system suitability results

The HPLC chromatogram for the systematic suitability test of vilanterol tritoate is shown in FIG. 10, and the detection data is shown in Table 7.

TABLE 7 System suitability results for vilanterol tritoate

As can be seen from tables 6-7, the RSD value of the Wumei ammonium bromide peak area is less than 0.23% and less than 2.0% when 6 needles are repeatedly injected; the RSD of the Wumei ammonium bromide retention time is 0.51 percent and less than 1.0 percent; the RSD value of the area of the Vilantelo peak is 0.60 percent and less than 2.0 percent; RSD of Retention time of Vilandiolo is 0.20% < 1.0%; the theoretical plate numbers of the Wumei ammonium bromide and the Vilantelo peaks are more than 3000, which shows that the method for measuring the fine particle dose has better system applicability.

EXAMPLE 3 durability test

Location of a body part	Volume of solvent (mL)	Mode of shaking	Shaking time (min)
				Suction nozzle adapter	30	Hand-operated sealing bag	Shaking for 1min, standing for 10min
L-shaped connecting pipe	50	Swinging device	Front and back sides for 10min respectively
				Pre-separator	200	Swinging device	Front and back sides for 10min respectively
1-stage collecting cup	15	Oscillator	10min
				2-stage collecting cup	15	Oscillator	10min
3-stage collecting cup	15	Oscillator	10min
				4-stage collecting cup	15	Oscillator	10min
5-stage collecting cup	15	Oscillator	10min
				6-stage collecting cup	15	Oscillator	10min
7-grade collecting cup	15	Oscillator	10min
				MOC-level collecting cup	100	Oscillator	10min

precisely measuring 1mL of each of the stock solutions a and b, placing in a 10mL measuring flask, dissolving with methanol-water (60: 40), diluting to scale, and shaking to obtain control solution.

Original chromatographic conditions:

a chromatographic column: ZORBAX SB-C18 (50X 4.6mm, 1.8 μm) in Agilent;

column temperature: 30 ℃;

flow rate: 1.0 mL/min;

sample introduction amount: 100 mu L of the solution;

gradient elution procedure:

。

The detection wavelength, the column temperature, the flow rate and the initial proportion of the mobile phase are slightly changed, and the change conditions of the detection wavelength, the column temperature, the flow rate and the initial proportion of the mobile phase are as follows:

each of the solutions was measured 100. mu.L precisely, and the measurement was performed under various chromatographic conditions. Respectively calculating the mass of each grade of the Umamizumab and the Vilandiolo of the NGI according to the external standard method by using the peak areas of the Umamizumab and the Vilandiolo, and calculating the drug amount of the fine particles smaller than 5.0 mu m by using 'Copley Inhaler Testing Data Analysis Software' according to the mass of each grade of the Umamizumab and the Vilandiolo. The results of the measurements were observed for consistency before and after slight changes in the original chromatographic conditions, and the results are shown in tables 8 to 11.

TABLE 8 flow Rate variation Fine particle dosimetry results

TABLE 9 measurement results of fine particle dose in accordance with the change in column temperature

TABLE 10 measurement results of the dose of the fine particles for detecting wavelength changes

TABLE 11 measurement results of fine particle dose for change of flow phase ratio

As can be seen from tables 8-11, when the flow rate, the column temperature, the detection wavelength and the initial phase ratio of the flow are slightly changed, the measured fine particle doses of Umamizumab and Vilandiolo in the test solution are not obviously different, and the RSD values are all less than 2.0%, which indicates that the measuring method provided by the research has good durability and can meet the requirement of fine particle dose measurement.

Example 4 sensitivity test

Wumei ammonium bromide and vilanterol detection limits: taking reference substance solutions containing Umamizumab and Vilandiolo with the concentrations of 0.511 mug/mL and 0.049 mug/mL respectively, diluting step by step, carrying out sample injection in sequence, recording a chromatogram, recording the sample injection concentration when the signal-to-noise ratios of Umamizumab and Vilandiolo in the chromatogram are 2-4 as a detection limit, and observing that the signal-to-noise ratio of a main Umamizumab peak is 4.12 and the signal-to-noise ratio of a main Vilandiolo peak is 3.86, so that the minimum detection limits of Umamizumab and Vilandiolo are 0.0025 mug/mL and 0.00024 mug/mL respectively.

Wumei ammonium bromide and vilanterol limits: taking reference substance solutions containing Uume ammonium bromide and vilanterol with the concentrations of 0.511 mug/mL and 0.049 mug/mL respectively, diluting step by step, sequentially injecting samples, recording a chromatogram, recording the sample injection concentration when the signal-to-noise ratio of Uume ammonium bromide and vilanterol in the chromatogram is 9-11 as a detection limit, and observing that the signal-to-noise ratio of a main Uume ammonium bromide peak is 11.98 and the signal-to-noise ratio of a main vilanterol peak is 11.42, so that the quantitative limits of Uume ammonium bromide and vilanterol are 0.0511 mug/mL and 0.0049 mug/mL respectively.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the dose of fine particles of an Wumei ammonium bromide vilanterol inhalation powder aerosol is characterized in that a high performance liquid chromatography is adopted, and the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: a fluorescence detector is used for Vilantelo, and an ultraviolet detector is used for Umamizium bromide; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, gradient elution according to the following table:

time (min) Mobile phase A (%) Mobile phase B (%) 0 A1 B1 3.5-4.0 A2 B2

Wherein, A1 is 63-67%, A2 is 28-32; b1-33%, B2-68-72%.

2. The method for detecting the fine particle dose of the wumei ammonium bromide vilanterol inhalation powder cloud agent according to claim 1, wherein the high performance liquid chromatography is adopted, and the chromatographic conditions comprise: a chromatographic column of octadecylsilane bonded silica gel filler is adopted; a detector: a fluorescence detector is used for Vilantelo, and an ultraviolet detector is used for Umamizium bromide; the mobile phase consists of a mobile phase A and a mobile phase B, wherein the ratio of the mobile phase A: trifluoroacetic acid solution, mobile phase B: acetonitrile solution containing trifluoroacetic acid, gradient elution according to the following table:

Wherein, A1 is 63-67%, A2 is 28-32; b1-33%, B2-68-72%.

3. The method for detecting fine particle dose of Umamizumab vilanterol inhalation aerosol powder dose according to any of claims 1 to 3, wherein the mobile phase A is 0.05-0.15 vol% trifluoroacetic acid solution.

4. The method for detecting the fine particle dose of the umeclidinium hydrobromide vilanterol inhalation powder cloud agent according to claim 4, wherein said mobile phase B is acetonitrile solution containing 0.05-0.15 vol% trifluoroacetic acid.

5. The method for detecting the fine particle dose of vilanterol ume bromide inhalation powder aerosol according to any one of claims 1-3, wherein the vilanterol uses a fluorescence detector, the excitation wavelength is 220-230nm, and the detection wavelength is 310-320 nm.

6. The method for detecting the fine particle dose of vilanterol ammonium bromide inhalation powder aerosol according to any one of claims 1-3, wherein the detection wavelength of the vilanterol ammonium bromide is 210-230nm by using an ultraviolet detector.

7. The method for detecting the fine particle dose of Umamizumab vilanterol inhalation powder cloud according to any of claims 1 to 3, wherein the column temperature used is 28-32 ℃.

8. The method for detecting the fine particle dose of Umamizumab vilanterol inhalation powder cloud according to any one of claims 1 to 3, wherein the sample to be detected is dissolved with a mixture of methanol and water.

9. The method for detecting the fine particle dose of the umei ammonium bromide vilanterol inhalation powder cloud agent according to claim 8, wherein the volume ratio of methanol to water is 60: 40.

10. the method for detecting fine particle dose of Umamizumab vilanterol in a powder inhalation formulation according to any of claims 1 to 3, wherein the sample volume is 90 to 110 μ L.