LC-MS / MS analysis method for simultaneously quantifying concentrations of budesonide, formoterol and glycopyrronium in plasma

Abstract

The invention belongs to the field of in-vivo quantitative drug concentration analysis, and particularly discloses an LC-MS / MS (liquid chromatography-mass spectrometry / mass spectrometry) analysis method for simultaneously quantifying the concentrations of budesonide, formoterol and glycopyrronium in plasma. According to the method, the contents of budesonide, formoterol and glycopyrronium in the blood sample can be accurately and quantitatively detected at the same time through specific sample treatment and chromatography-mass spectrometry conditions, the sensitivity is high, the detection limit is low, a novel method capable of being practically popularized and applied is provided for pharmacokinetic research of the three components, and the method has popularization and application values.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vivo quantitative drug concentration analysis, in particular to a LC-MS / MS analysis method for simultaneously quantifying the concentrations of budesonide, formoterol and glycopyrronium in blood plasma. BACKGROUND

[0002] Budesonide is a glucocorticoid that reduces airway inflammation by inhibiting the activation and migration of inflammatory cells and reducing the release of inflammatory mediators. It is one of the basic drugs for asthma treatment and can also reduce the symptoms of chronic obstructive pulmonary disease (COPD). Glycopyrronium is a long-acting anticholinergic drug that relaxes the bronchial smooth muscle by antagonizing acetylcholine receptors, thereby dilating the airway and relieving bronchospasm. Its effect lasts for a long time, providing sustained bronchodilation. Formoterol is a selective β2-adrenergic receptor agonist that relaxes bronchial smooth muscle by stimulating β2 receptors, thereby relieving bronchospasm. Budesonide, glycopyrronium and formoterol are usually used in combination to form a triple preparation for maintenance therapy of COPD and asthma. This combination preparation has a synergistic effect through multiple mechanisms, which can more effectively control symptoms and reduce the risk of acute exacerbation. However, excessive blood drug concentrations can cause adverse reactions. For example, budesonide can cause adrenal suppression, formoterol can cause arrhythmia, and glycopyrronium can cause dry mouth and blurred vision. By simultaneously quantifying the concentrations of these three drugs in blood, we can detect drug overdose in a timely manner and adjust the treatment regimen to reduce the occurrence of adverse reactions.

[0003] However, there is currently no analysis method for simultaneously quantifying the concentrations of budesonide, formoterol and glycopyrronium in blood. Although the European Medicines Agency stated in its 2020 evaluation report that it had achieved accurate monitoring of these three components in blood at the same time, the specific detection method was not disclosed, which made it difficult to effectively monitor the concentrations of budesonide, formoterol and glycopyrronium in blood at the same time in clinical practice, and further hindered the study of the pharmacokinetic characteristics of the three drugs when they acted together. SUMMARY

[0004] To solve the above problems, the present application provides a LC-MS / MS analysis method for simultaneously quantifying the concentrations of budesonide, formoterol and glycopyrronium in blood plasma, which comprises the following steps:

[0005] (1) Establishment of standard curve

[0006] a. Preparation of series concentration standard curve samples: Budesonide was dissolved in acetonitrile to obtain a budesonide solution; Formoterol and glycopyrronium were dissolved in dimethyl sulfoxide to obtain a formoterol solution and a glycopyrronium solution, respectively; the budesonide solution, the formoterol solution and the glycopyrronium solution were diluted with acetonitrile water solution to obtain a series of concentrations;

[0007] The series concentration solutions were mixed with the matrix, and the mixture was mixed with the internal standard working solution and ethyl acetate. After centrifugation, the supernatant was blown dry, and then dissolved in acetonitrile water solution, centrifuged, and the supernatant was obtained.

[0008] b. The series concentration standard curve samples were injected into the LC-MS / MS instrument, and the peak area was measured to obtain the standard curve of budesonide, formoterol and glycopyrronium.

[0009] The chromatographic conditions are as follows:

[0010] The chromatographic column was filled with ethylene bridge hybrid particles; formic acid-ammonium acetate aqueous solution was used as mobile phase A, and isopropyl alcohol acetonitrile solution containing formic acid was used as mobile phase B, and the gradient elution program was as follows:

[0011]

[0012] The mass spectrometry conditions are as follows: the ion source is an electrospray ionization source; the scanning type is multiple ion reaction monitoring.

[0013] (2) Determination of the content of the drug components in the sample to be tested:

[0014] c. Treatment of biological samples

[0015] The sample to be tested was mixed with the internal standard working solution and ethyl acetate, and then centrifuged. The supernatant was blown dry, and then dissolved in acetonitrile water solution, centrifuged, and the supernatant was obtained.

[0016] d. Determination of biological samples

[0017] The supernatant obtained in step c was injected into the LC-MS / MS instrument for detection, and the same conditions as in step b were used for detection. According to the standard curve in step (1), the content of budesonide, formoterol and glycopyrronium in the sample to be tested was obtained.

[0018] Further, the content determination process in step (2) also includes a quality control procedure, and the specific steps are as follows:

[0019] Take the matrix, mix with acetonitrile aqueous solution and ethyl acetate, after centrifugation, take the supernatant and blow dry, then add acetonitrile aqueous solution to dissolve and mix, centrifuge, take the supernatant to obtain the blank sample test solution; take the budesonide solution, formoterol solution and glononium solution obtained in step a, dilute with acetonitrile aqueous solution to obtain the quality control sample, take the quality control sample, mix with internal standard working solution and ethyl acetate, after centrifugation, take the supernatant and blow dry, then add acetonitrile aqueous solution to dissolve and mix, centrifuge, take the supernatant to obtain the quality control sample test solution; inject the test solution of the blank sample and the quality control sample into the LC-MS / MS instrument respectively to monitor the interference existing in the determination process;

[0020] The volume ratio of the matrix to the mixed acetonitrile aqueous solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution for dissolving is 100:20:600:500:65;

[0021] The volume ratio of the quality control sample to the internal standard working solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution is 100:20:600:500:65.

[0022] Further, the volume ratio of the series concentration solution to the matrix in step a is 15:135, the concentration of budesonide after adding the series concentration solution to the matrix is 0.1-40 ng / mL, the concentration of formoterol is 5-2000 pg / mL, and the concentration of glononium is 10-4000 pg / mL; the volume ratio of the mixed solution, internal standard solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution is 100:20:600:500:65.

[0023] Further, the matrix is EDTA-K2 anticoagulated Beagle dog blank plasma.

[0024] Further, the mixing time is 10 min; the centrifugation temperature is 4℃, the rotation speed is 18000xg, and the time is 1-5 min.

[0025] Further, the internal standard working solution is acetonitrile aqueous solution containing 50.0 ng / mL Budesonide-d8, 0.5 ng / mL Formoterol-13C-d3 and 5 ng / mL Glononium-d5 standard substance;

[0026] Further, the concentration of the acetonitrile aqueous solution is 50%.

[0027] Further, the chromatographic column is Xbridge C18 5um 4.6*150mm Column; the injection amount is 5μL; the column temperature is 50℃; the mobile phase A is 0.1% formic acid-2mM ammonium acetate aqueous solution, and the mobile phase B is isopropanol and acetonitrile containing 0.1% formic acid with a volume ratio of 1:9.

[0028] Further, the mass spectrometry conditions in step b are as follows: polarity mode: positive ion mode; curtain gas (CUR): 30 psi; collision gas (CAD): 9 V; ion spray voltage (IS): 5500 V; temperature (TEM): 550℃; ion source gas 1 (GS1): 40 psi; ion source gas 2 (GS2): 40 psi; entrance potential (EP): 10 V; collision cell exit potential (CXP): 10 V.

[0029] Further, the mass spectrometry conditions are as follows: budesonide detection ion pair m / z 431.2→m / z 322.7, declustering voltage (DP) 60 V, collision energy 18 eV; formoterol detection ion pair m / z 344.9→m / z 149.0, declustering voltage (DP) 60 V, collision energy 26 eV; glycopyrronium detection ion pair m / z 318.2→m / z 116.1, declustering voltage (DP) 90 V, collision energy 34 eV; Budesonide-d8 detection ion pair m / z 439.2→m / z 323.2, declustering voltage (DP) 100 V, collision energy 25 eV; Formoterol-13C-d3 detection ion pair m / z 349.0→m / z 153.0, declustering voltage (DP) 60 V, collision energy 27 eV; Glononium-d5 detection ion pair m / z 323.2→m / z 116.1, declustering voltage (DP) 90 V, collision energy 34 eV.

[0030] The LC-MS / MS analysis method for quantifying the concentrations of budesonide, formoterol and glycopyrronium in blood plasma according to the present application can accurately detect the contents of budesonide, formoterol and glycopyrronium in blood samples by specific sample processing and chromatography-mass spectrometry conditions, and has high sensitivity and low detection limit, thereby providing a new practically applicable method for the pharmacokinetic study of the three components and having the value of popularization and application.

[0031] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the present application.

[0032] The above content of the present application will be further explained in detail through the following embodiment. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 Chromatograms of budesonide, formoterol, glycopyrronium and internal standard in blank samples;

[0034] Figure 2 Budesonide, Formoterol, Glycopyrronium and Internal Standard chromatograms in zero concentration sample;

[0035] Figure 3 Budesonide, Formoterol, Glycopyrronium and Internal Standard chromatograms in standard curve lowest point sample (STD1);

[0036] Figure 4 Budesonide, Formoterol, Glycopyrronium and Internal Standard chromatograms in standard curve highest point sample (STD8);

[0037] Figure 5 Budesonide, Formoterol, Glycopyrronium and Internal Standard chromatograms in carry over;

[0038] Figure 6 Budesonide standard curve;

[0039] Figure 7 Formoterol standard curve;

[0040] Figure 8 Glycopyrronium standard curve. DETAILED DESCRIPTION

[0041] Example 1, Determination of Budesonide, Formoterol, Glycopyrronium concentration in plasma

[0042] (I) Preparation of solutions

[0043] ① Standard curve stock solution

[0044] Take Budesonide, add acetonitrile to make a Budesonide stock solution with a concentration of 1.00 mg / mL; take Formoterol and Glycopyrronium respectively, add dimethyl sulfoxide to make Formoterol stock solution and Glycopyrronium stock solution with a concentration of 1.00 mg / mL;

[0045] ② Internal standard working solution

[0046] Take Budesonide-d8 standard substance, add acetonitrile to make a Budesonide-d8 internal standard stock solution with a concentration of 1.00 mg / mL; take Formoterol-13C-d3 standard substance and Glononium-d5 standard substance respectively, add dimethyl sulfoxide to make Formoterol-13C-d3 internal standard stock solution and Glononium-d5 internal standard stock solution with a concentration of 1.00 mg / mL;

[0047] Take Budesonide-d8 internal standard stock solution, Formoterol-13C-d3 internal standard stock solution and Glononium-d5 internal standard stock solution, and dilute with 50% acetonitrile aqueous solution to obtain an internal standard working solution containing 50.0 ng / mL Budesonide-d8, 0.5 ng / mL Formoterol-13C-d3 and 5 ng / mL Glononium-d5;

[0048] (ii) Establishment of standard curve

[0049] a. Preparation of series concentration standard curve samples:

[0050] Take Budesonide stock solution, Formoterol-d stock solution and Glononium stock solution, and dilute with 50% acetonitrile aqueous solution to obtain a series of concentration solutions with Budesonide concentration of 1-400 ng / mL, Formoterol-d concentration of 0.05-20 ng / mL and Glononium concentration of 0.1-40 ng / mL;

[0051] Take 15 μL of the series of concentration solutions, respectively, and mix with 135 μL of blank matrix (EDTA-K2 anticoagulated Beagle dog blank plasma) to obtain standard curve plasma samples; take 100 μL of the standard curve plasma samples, add 20 uL of internal standard working solution and 600 uL of ethyl acetate, mix for 10 min, and place in a centrifuge at 4°C, 18000 x g for 5 min; take 500 uL of supernatant, dry under nitrogen, add 65 uL of 50% acetonitrile aqueous solution, mix for 10 min, and place in a centrifuge at 4°C, 18000 x g for 1 min; take the supernatant to obtain the standard curve plasma samples;

[0052] b. Inject the series of concentration standard curve samples into the LC-MS / MS instrument, respectively, to determine the peak area; take the concentration of each drug component in the series of concentration standard curve samples as the abscissa (X), and take the peak area ratio of each drug component to the corresponding internal standard as the ordinate (Y), and use weighted least squares method (W = 1 / X 2 ) to perform regression calculation to obtain the linear regression equation, which is the standard curve;

[0053] The chromatographic conditions are as follows:

[0054] The chromatographic column is Xbridge C18 5um 4.6*150mm Column; the column temperature is 50°C; the flow rate is 1.5 mL / min; and 0.1% formic acid-2mM ammonium acetate aqueous solution is used as mobile phase A, and 0.1% formic acid isopropanol: acetonitrile (1:9) is used as mobile phase B, and the gradient elution program is as follows:

[0055]

[0056] Mass spectrometry conditions: ion source: electrospray ionization source; scan type: multiple ion reaction monitoring; polarity mode: positive ion mode; curtain gas (CUR): 30; collision gas (CAD): 9; ion spray voltage (IS): 5500; temperature (TEM): 550; ion source gas 1 (GS1): 40; ion source gas 2 (GS2): 40; inlet potential (EP): 10; collision cell exit potential (CXP): 10; Budesonide detection ion pair m / z 431.2→m / z 322.7, declustering voltage (DP) 60V, collision energy 18eV; Formoterol detection ion pair m / z 344.9→m / z 149.0, declustering voltage (DP) 60V, collision energy 26eV; Glononium detection ion pair m / z 318.2→m / z 116.1, declustering voltage (DP) 90V, collision energy 34eV; Budesonide-d8 detection ion pair m / z 439.2→m / z 323.2, declustering voltage (DP) 100V, collision energy 25eV; Formoterol-13C-d3 detection ion pair m / z 349.0→m / z 153.0, declustering voltage (DP) 60V, collision energy 27eV; Glononium-d5 detection ion pair m / z 323.2→m / z 116.1, declustering voltage (DP) 90V, collision energy 34eV;

[0057] (III) Determination of the contents of budesonide, formoterol and glononium in the sample to be tested

[0058] c. Treatment of biological samples, blank samples and quality control samples

[0059] Take 100 μL of the plasma sample in a polypropylene tube, add 20 uL of the internal standard working solution, then add 600 uL of ethyl acetate and mix for 10 min, then put it in a centrifuge, 4°C, 18000xg centrifugation for 5 min, take 500 uL of supernatant in a polypropylene tube, nitrogen blowing dry, then add 65 uL of 50% acetonitrile aqueous solution, mix for 10 min, finally put it in a centrifuge, 4°C, 18000xg centrifugation for 1 min, take the supernatant to obtain the biological sample test solution;

[0060] Take 100 μL of the blank matrix (EDTA-K2 anticoagulated Beagle dog blank plasma) in a polypropylene tube, add 20 uL of 50% acetonitrile aqueous solution, then add 600 uL of ethyl acetate and mix for 10 min, then put it in a centrifuge, 4°C, 18000xg centrifugation for 5 min, take 500 uL of supernatant in a polypropylene tube, nitrogen blowing dry, then add 65 uL of 50% acetonitrile aqueous solution, mix for 10 min, finally put it in a centrifuge, 4°C, 18000xg centrifugation for 1 min, take the supernatant to obtain the blank sample test solution;

[0061] Take budesonide stock solution, formoterod stock solution and glycopyrronium stock solution, and dilute with 50% acetonitrile aqueous solution to prepare quality control samples containing 1-300 ng / mL budesonide, 0.05-15 ng / mL formoterod and 0.1-30 ng / mL glycopyrronium. Take 100 μL of quality control sample into a polypropylene tube, add 20 μL of internal standard working solution, then add 600 μL of ethyl acetate and mix for 10 min. Place in a centrifuge and centrifuge at 4℃, 18000×g for 5 min. Take 500 μL of supernatant into a polypropylene tube, blow dry with nitrogen, add 65 μL of 50% acetonitrile aqueous solution, mix for 10 min, and finally place in a centrifuge and centrifuge at 4℃, 18000×g for 1 min. Take the supernatant to obtain the quality control sample test solution.

[0062] d. Determination of biological samples

[0063] Take 5 μL of the test solution of blank sample, quality control sample and biological sample respectively, inject it into LC-MS / MS instrument for detection, and detect under the same conditions as in step b. Use quality control sample and blank sample to monitor whether there is any interference in the operation process. According to the standard curve in step (ii), obtain the contents of budesonide, formoterol and glycopyrronium in biological sample.

[0064] The following experimental examples illustrate the beneficial effects of the present invention.

[0065] Experimental Example 1: Screening of Detection Conditions in the Method of the Invention

[0066] Existing reports contain numerous LC-MS / MS methods for detecting the concentration of one of the drug components (budesonide, formoterol, and glycopyrronium) in blood. However, after testing each method, none could accurately quantify all three components simultaneously in blood. The main issues lie in the chromatographic conditions: the chromatographic column, mobile phase, gradient elution program, and the method of biological sample pretreatment. Therefore, these conditions were screened. The following brand and model of chromatographic column was selected: Phenomenx Kinetex C18 2.6µm.

[0067] 2.1*100mm, Waters ACQUITYUPLC BEH C181.7um 2.1*100mm Column, Waters Xbridge C185um 4.6*150mm Column, among which the Waters Xbridge C185um 4.6*150mm Column showed good separation and signal-to-noise ratio for the three compounds and their surrounding interfering peaks;

[0068] The mobile phase is screened for different proportions of formic acid and buffer salt proportion combination for aqueous phase, and screened for different proportions of formic acid and methanol, acetonitrile, isopropanol combination for organic phase, and finally the mobile phase system of 0.1% formic acid-2mM ammonium acetate aqueous solution as the aqueous phase, and 0.1% formic acid isopropanol: acetonitrile (1:9) as the organic phase is determined;

[0069] The gradient elution program is optimized for flow rate, initial gradient, time gradient change, etc., and finally determined that the organic phase is gradually increased to 95% at two different rates, and then isocratic elution for 0.3 min, and finally reduced to 40% for isocratic elution for 0.7 min, so that the three compounds have good response and signal-to-noise ratio in 3.5 minutes per needle.

[0070] The biological sample pretreatment is compared by using protein precipitation method, liquid-liquid extraction method and solid phase extraction method, and the extraction recovery of the three compounds and the cleanliness of the sample are combined, finally, compared with the protein precipitation method, the sample is cleaner and the interference of the compound peak is smaller by using the liquid-liquid extraction method; compared with the solid phase extraction method, the extraction recovery of the three compounds can be better considered to meet the demand of low quantitative lower limit, and finally the liquid-liquid extraction method is determined as the biological sample pretreatment method.

[0071] Test Example 2 Methodology verification of the application

[0072] 1. Method overview

[0073] After liquid-liquid extraction with ethyl acetate, analysis is performed, and the standard curve is calculated by weighted least squares method (w = 1 / x 2 ) for regression, and the ratio of the peak area of the measured substance to the peak area of the internal standard is quantified. The relevant information is shown in Table 1:

[0074] Table 1 Verification information of detection method

[0075]

[0076] Note: The English name of budesonide is Budesonide; the English name of formoterol is Formoterol; the English name of glononium is Glononium.

[0077] 2. Instruments and chromatography-mass spectrometry conditions

[0078] 2.1 Main instruments and equipment

[0079]

[0080]

[0081] 2.2 Chromatography conditions

[0082]

[0083] Gradient elution program

[0084]

[0085] 2.3 Mass spectrometry conditions

[0086]

[0087]

[0088] 3 Preparation of standard substances, reagents, solutions and blank matrix

[0089] 3.1 Standard substance of the analyte

[0090] Budesonide, content 99.5%; Formoterol fumarate, content 95.0%; Glycopyrrolate, content 99.9%, all from China Institute for Drug Control;

[0091] 3.2 Standard substance of internal standard (IS)

[0092] Budesonide-d8 (Mixture of Diastereomers), content 97.5%; Formoterol-13C-d3 Hemifumarate, content 96.5%; Glycopyrrolate-d5 Bromide, content 98.0%, all from TLC Pharmaceutical Standards.

[0093] 3.3 Preparation of solutions

[0094] The preparation of solutions only needs to keep the same proportion, and the volume of preparation can be adjusted according to the actual situation.

[0095]

[0096]

[0097] 3.4 Blank matrix

[0098] EDTA-K2 anticoagulated Beagle dog plasma, from our institute, thawed at 10-30℃ before use.

[0099] EDTA-K2 anticoagulated Beagle dog whole blood in our institute, transferred in sample transport box after collection, stored at 2-8℃.

[0100] Hemolytic blank matrix: Take 50 μL EDTA-K2 anticoagulated Beagle dog whole blood in a polypropylene tube, freeze at -60°C and below for at least 30 min, thaw at 10-30°C, transfer 20 μL to another polypropylene tube, add 980 μL EDTA-K2 anticoagulated blank Beagle dog plasma (2% V / V), mix for about 1 min to obtain.

[0101] 4. Preparation of stock solution, working solution and sample

[0102] 4.1 Preparation of budesonide stock solution with concentration of about 1.00 mg / mL

[0103] At yellow light 10-30°C, weigh about 3 mg (2.7-3.3 mg) of budesonide standard substance in a polypropylene tube, add a certain volume of acetonitrile to prepare 1.00 mg / mL. Marked as SS-YyMmDd-x. Weigh in duplicate, one as standard curve stock solution, the other as quality control stock solution, stock solution stored at -30 to -10°C in a polypropylene tube. When calculating the volume, it needs to be converted according to the following formula:

[0104] Volume = m x content + concentration of stock solution

[0105] 4.2 Preparation of formoterol stock solution with concentration of about 1.00 mg / mL

[0106] At yellow light 10-30°C, weigh about 3 mg (2.7-3.3 mg) of formoterol fumarate standard substance in a polypropylene tube, add a certain volume of dimethyl sulfoxide to prepare 1.00 mg / mL. Marked as SS-YyMmDd-x. Weigh in duplicate, one as standard curve stock solution, the other as quality control stock solution, stock solution stored at -30 to -10°C in a polypropylene tube. When calculating the volume, it needs to be converted according to the following formula:

[0107] Volume = m x content x (344.4*2 / 804.88) + concentration of stock solution

[0108] 4.3 Preparation of glycopyrronium stock solution with concentration of about 1.00 mg / mL

[0109] At yellow light 10-30°C, weigh about 3 mg (2.7-3.3 mg) of glycopyrronium bromide standard substance in a polypropylene tube, add a certain volume of dimethyl sulfoxide to prepare 1.00 mg / mL. Marked as SS-YyMmDd-x. Weigh in duplicate, one as standard curve stock solution, the other as quality control stock solution, stock solution stored at -30 to -10°C in a polypropylene tube. When calculating the volume, it needs to be converted according to the following formula:

[0110] Volume = m x content x (317.42 / 398.37) + concentration of stock solution

[0111] 4.4 Preparation of working solutions

[0112] Prepare the standard curve working solutions and quality control working solutions according to the following table at yellow light 10-30°C, vortex mix well, and dilute with 50% acetonitrile aqueous solution. Store at -30 to -10°C in polypropylene tubes.

[0113] Table 2 Preparation of standard curve working solutions

[0114]

[0115] Table 3 Preparation of quality control working solutions

[0116]

[0117]

[0118] 4.5 Preparation of plasma samples

[0119] Prepare the standard curve and quality control plasma samples according to Table 4 at yellow light 10-30°C, mix well after preparation. Store at -60°C or below in polypropylene tubes.

[0120] Table 4 Preparation of standard curve plasma samples

[0121]

[0122] Table 5 Preparation of quality control plasma samples

[0123]

[0124] 4.6 Preparation of internal standard stock solution and working solution

[0125] 4.6.1 Preparation of Budesonide-d8 internal standard stock solution with a concentration of about 1.00 mg / mL

[0126] At yellow light 10-30°C, weigh about 2 mg (1.8-2.2 mg) of Budesonide-d8 (Mixture of Diastereomers) standard substance into a polypropylene tube, add a certain volume of acetonitrile to prepare 1.00 mg / mL stock solution, and store the stock solution at -30 to -10°C in a polypropylene tube. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0127] Volume = m x content ÷ concentration of stock solution

[0128] 4.6.2 Preparation of Formoterol-13C-d3 internal standard stock solution with a concentration of about 1.00 mg / mL

[0129] Take Formoterol-13C-d3 Hemifumarate standard substance which has been about 2 mg (1.8-2.2 mg) in polypropylene tube under yellow light 10-30℃, add a certain volume of dimethyl sulfoxide, prepare 1.00 mg / mL, stock solution in polypropylene tube at-30-10℃. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0130] Volume = m x content x (348.42 / 406.46) ÷ concentration of stock solution

[0131] 4.6.3 Preparation of Glononium-d5 internal standard stock solution with a concentration of about 1.00 mg / mL

[0132] Take Glycopyrrolate-d5 Bromide standard substance which has been about 2 mg (1.8-2.2 mg) in polypropylene tube under yellow light 10-30℃, add a certain volume of dimethyl sulfoxide, prepare 1.00 mg / mL, stock solution in polypropylene tube at-30-10℃. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0133] Volume = m x content x (322.45 / 403.37) ÷ concentration of stock solution

[0134] 4.6.4 Preparation of internal standard working solution

[0135] Prepare mixed internal standard working solution under yellow light 10-30℃ according to the following table, vortex mix, dilute solution is 50% acetonitrile aqueous solution.

[0136] Store at-30-10℃ in polypropylene tube.

[0137]

[0138] 5 Sample processing

[0139] (1) All matrix samples were thawed and mixed well at 10-30℃ under yellow light before processing;

[0140] (2) Take 100 μL of plasma sample in a polypropylene tube, add 20 μL of internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL) except for blank samples, and add 20 μL of 50% acetonitrile aqueous solution to blank samples and ULOQ samples;

[0141] (3) Add 600 μL of ethyl acetate and mix for 10 min;

[0142] (4) 4°C, 18000 x g centrifugation for 5 min;

[0143] (5) Take 500 μL supernatant in a polypropylene tube, nitrogen blowing dry;

[0144] (6) After nitrogen blowing dry, add 65 μL of 50% acetonitrile aqueous solution, mix for 10 min;

[0145] (7) 4°C, 18000 x g centrifugation for 1 min, take 60 μL solution for sample analysis.

[0146] 6 Preparation of samples for verification of investigation items (applicable to method validation, all under yellow light)

[0147] 6.1 Preparation of selective and specific samples

[0148] (1) Blank sample: take 6 blank plasma samples from different sources, process according to the steps under "5 sample processing";

[0149] (2) Zero sample: take 6 blank plasma samples from different sources, process according to the steps under "5 sample processing", and replace 20 μL of internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL) in step (2) with 20 μL of ISWS1-YyMmDd-x, ISWS2-YyMmDd-x, ISWS3-YyMmDd-x, respectively, each prepared in parallel for 6 times;

[0150] (3) LLOQ sample: take 6 blank plasma samples from different sources, process according to the steps under "5 sample processing";

[0151] (4) ULOQ sample: take 6 blank plasma samples from different sources, prepare ULOQ1-YyMmDd-x, ULOQ2-YyMmDd-x, ULOQ3-YyMmDd-x samples each for 6 times, and process according to the steps under "5 sample processing".

[0152] 6.2 Preparation of extraction recovery rate samples

[0153] Extraction sample: take 100 μL of low, medium and high concentration quality control samples in a polypropylene tube, and process according to the steps under "5 sample processing".

[0154] Non-extraction sample: (1) take 120 μL of blank Beagle dog plasma in a polypropylene tube, prepare 6 times in parallel, add 24 μL of 50% acetonitrile aqueous solution, then add 720 μL of ethyl acetate, mix for 10 min;

[0155] (2) Centrifuge at 4℃, 18000×g for 5 min;

[0156] (3) Take out 552 μL of supernatant, add 8 μL of low, medium or high concentration working solution for quality control, and then add 16 μL of internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL);

[0157] (4) Transfer 500 μL (3) into a polypropylene tube and dry it with nitrogen.

[0158] (5) After drying with nitrogen, add 65 μL of 50% acetonitrile aqueous solution and mix for 10 min.

[0159] (6) Centrifuge at 4℃, 18000×g for 1 min, and take 60μL of solution for analysis.

[0160] 6.3 Preparation of whole blood stability samples

[0161] 5 μL of budesonide, formoterol, and glycopyrronium stock solutions were separately pipetted into three polypropylene tubes, and 995 μL of 50% acetonitrile aqueous solution was added to each to obtain intermediate solutions containing 5.00 μg / mL of budesonide, formoterol, and glycopyrronium. 600 μL of the intermediate solutions of budesonide, 30 μL of formoterol, and 60 μL of glycopyrronium were pipetted into a polypropylene tube, and 310 μL of 50% acetonitrile aqueous solution was added to dilute and obtain a mixed solution containing 3000 / 150 / 300 ng / mL of budesonide, formoterol, and glycopyrronium. 20 μL of the mixed solution was pipetted into a polypropylene tube, and 1980 μL of freshly collected EDTA-K2 anticoagulated whole blood from Beagle dogs was added to prepare a high-concentration whole blood quality control sample. WS-QC... 25 μL of working solution M was added to a polypropylene tube, and 1975 μL of Beagle canine EDTA-K2 anticoagulated whole blood was added to prepare a low-concentration whole blood quality control sample. The whole blood sample was gently inverted 10 times and equilibrated at 37°C for 400 rpm for 10 min. The low and high concentration control samples were aliquoted into two 900 μL portions. One portion was immediately centrifuged (4°C, 2200×g) for 10 min, and the supernatant plasma was collected as the 0h sample and stored under yellow light at 10–30°C or -60°C and below for later use. The remaining whole blood sample was stored in the environment to be tested (ice box, yellow light) for about 4 h (this can be adjusted according to the actual situation, but should not be less than the interval between whole blood collection and centrifugation), and then centrifuged (4°C, 2200×g) for 10 min. The obtained plasma and the 0h plasma were processed according to the method in section "7" and then analyzed. Six low and six high concentration control samples were processed in parallel.

[0162] 6.4 Preparation of stock solution and working solution stability samples

[0163] 6.4.1 Preparation method of stock solution stability sample

[0164] (1) Take 5 μL of budesonide, formoterol and glycopyrronium stock solutions in three polypropylene tubes, respectively, add 995 μL of 50% acetonitrile aqueous solution to dilute to a solution containing budesonide, formoterol and glycopyrronium at a concentration of 5.00 μg / mL.

[0165] (2) Take 120 μL, 6 μL and 12 μL of the solution containing budesonide, formoterol and glycopyrronium at a concentration of 5.00 μg / mL, respectively, in a polypropylene tube, add 1362 μL of 50% acetonitrile aqueous solution to dilute to a mixed intermediate solution containing budesonide, formoterol and glycopyrronium at a concentration of 400 / 20.0 / 40.0 ng / mL.

[0166] (3) Take 25 μL of (2) in a polypropylene tube, add 100 μL of internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL), and then add 343 μL of 50% acetonitrile aqueous solution, mix for 5 min.

[0167] (4) Take 200 μL of (3) for injection analysis.

[0168] 6.4.2 Preparation method of internal standard stock solution stability sample

[0169] (1) Take 100 μL of Budesonide-d8 stock solution, 5 μL of Formoterol-13C-d3 stock solution and 10 μL of Glononium-d5 stock solution into three polypropylene tubes, respectively, add 900 μL, 5000 μL and 990 μL of 50% acetonitrile aqueous solution to dilute to internal standard intermediate solutions containing Budesonide-d8, Formoterol-13C-d3 and Glononium-d5 at a concentration of 100 μg / mL, 1.00 μg / mL and 10.0 μg / mL, respectively.

[0170] (2) Take 5 μL of Budesonide-d8, Formoterol-13C-d3 and Glononium-d5 internal standard intermediate solutions into a polypropylene tube, add 10 mL of 50% acetonitrile aqueous solution to dilute to an internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL).

[0171] (3) Take 100 μL (2) in a polypropylene tube, add 368 μL of 50% acetonitrile aqueous solution, mix for 5 min.

[0172] (4) Take 200 μL (3) for sample analysis.

[0173] 6.4.3 Preparation method of working solution stability sample

[0174] (1) Take 25 μL of the highest point working solution of the calibration curve in a polypropylene tube, add 100 μL of the internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL), and then add 343 μL of 50% acetonitrile aqueous solution, mix for 5 min;

[0175] (2) Take 50 μL of the lowest point working solution of the calibration curve in a polypropylene tube, add 25 μL of the internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL), and then add 42 μL of 50% acetonitrile aqueous solution, mix for 5 min;

[0176] (3) Take 100 μL (1) or (2) for sample analysis.

[0177] 6.4.4 Preparation method of internal standard working solution stability sample

[0178] (1) Take 100 μL of the internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL) in a polypropylene tube, add 368 μL of 50% acetonitrile aqueous solution, mix for 5 min;

[0179] (2) Take 200 μL (1) for sample analysis.

[0180] 6.5 System suitability sample

[0181] The system suitability sample is prepared separately at the LLOQ concentration under yellow light at 10-30°C. The system suitability sample can be prepared by the following method:

[0182] (1) Take 50 μL of the lower limit of quantification working solution in a polypropylene tube, add 100 μL of the internal standard working solution (Budesonide-d8 / Formoterol-13C-d3 / Glononium-d5 = 50.0 / 0.500 / 5.00 ng / mL), and then add 318 μL of 50% acetonitrile aqueous solution, mix for 5 min;

[0183] (2) Take 200 μL (1) for analysis. The rest of the analysis batch system suitability sample preparation is the same as "5 sample processing" under the processing.

[0184] 7. Data processing

[0185] Bias (%) = (measured value - theoretical value) ÷ theoretical value * 100%

[0186]

[0187]

[0188] Accuracy is expressed as Bias, and precision is expressed as relative standard deviation (RSD) or coefficient of variation (CV).

[0189] Peak area is reported as an integer, with full precision in the calculation; concentration retains three significant figures, bias (Bias), coefficient of variation (CV) and ratio (Ratio) retain one decimal place.

[0190] 8. Validation results

[0191] 8.1 Selectivity and specificity

[0192] Prepare 6 different sources of Beagle dog blank plasma (non-hemolytic and non-high fat) samples (Blank) respectively, prepare 6 LLOQ samples in parallel, investigate the interference of blank plasma on the analyte and internal standard; prepare 6 zero concentration samples (Zero) in parallel, investigate the interference of internal standard on the analyte, as well as the interference between internal standards.

[0193] Prepare 6 ULOQ plasma samples containing only the analyte (without internal standard) in parallel, investigate the interference of the analyte on the internal standard.

[0194] Select one source of blank plasma, add 2% whole blood cell lysate to prepare hemolytic matrix, use the hemolytic blank matrix to prepare 6 blank plasma samples (Blank) in parallel, investigate the interference of hemolytic blank plasma on the analyte and internal standard.

[0195] The measured analyte peak area of the above Blank, Zero samples compared with the average LLOQ analyte peak area needs no obvious (<20% analyte peak area) interference to affect the determination of the analyte. The measured internal standard peak area of the Blank, ULOQ samples compared with the average LLOQ sample internal standard peak area needs no obvious (<5% internal standard peak area) interference.

[0196] The measured analyte peak area of the different ULOQ samples compared to the mean of the ULOQ sample analyte peak area should not have significant (<5% of analyte peak area) interference, and the interfering peak area between the internal standards should be less than 5% of the peak area of each other's Zero peak.

[0197] The calculation formula is as follows:

[0198]

[0199] The results show that the endogenous substances in the blank plasma of the six different sources of Beagle dogs have a disturbance of ≤12.1% on budesonide, formoterol has no disturbance on budesonide, glononium has no disturbance on budesonide, the internal standard Budesonide-d8 has a disturbance of ≤7.9% on budesonide, the internal standard Formoterol-13C-d3 has a disturbance of ≤5.4% on budesonide, and the internal standard Glononium-d5 has no disturbance on budesonide.

[0200] The endogenous substances in the blank plasma of the six different sources of Beagle dogs have no disturbance on formoterol, budesonide has no disturbance on formoterol, glononium has no disturbance on formoterol, the internal standard Budesonide-d8 has a disturbance of ≤7.8% on formoterol, the internal standard Formoterol-13C-d3 has no disturbance on formoterol, and the internal standard Glononium-d5 has no disturbance on formoterol.

[0201] The endogenous substances in the blank plasma of the six different sources of Beagle dogs have a disturbance of ≤7.2% on glononium, budesonide has a disturbance of ≤0.1% on glononium, formoterol has a disturbance of ≤0.1% on glononium, the internal standard Budesonide-d8 has a disturbance of ≤13.7% on glononium, the internal standard Formoterol-13C-d3 has a disturbance of ≤12.7% on glononium, and the internal standard Glononium-d5 has a disturbance of ≤11.9% on glononium.

[0202] The endogenous substances in the blank plasma of the six different sources of Beagle dogs have a disturbance of ≤0.1% on the internal standard Budesonide-d8, budesonide has a disturbance of ≤0.1% on the internal standard Budesonide-d8, formoterol has a disturbance of ≤0.1% on the internal standard Budesonide-d8, glononium has a disturbance of ≤0.2% on the internal standard Budesonide-d8, the internal standard Formoterol-13C-d3 has a disturbance of ≤0.1% on the internal standard Budesonide-d8, and the internal standard Glononium-d5 has no disturbance on the internal standard Budesonide-d8.

[0203]

[0204] ​6 different sources of Beagle dog blank plasma endogenous substances had no interference with the internal standard Formoterol-13C-d3, Budesonide had an interference of ≤0.6% with the internal standard Formoterol-13C-d3, Formoterol had an interference of ≤1.2% with the internal standard Formoterol-13C-d3, Glononium had no interference with the internal standard Formoterol-13C-d3, the internal standard Budesonide-d8 had an interference of ≤0.2% with the internal standard Formoterol-13C-d3, and the internal standard Glononium-d5 had an interference of ≤2.1% with the internal standard Formoterol-13C-d3.

[0205] 6 different sources of Beagle dog blank plasma endogenous substances had no interference with the internal standard Formoterol-13C-d3, Budesonide had an interference of ≤0.6% with the internal standard Formoterol-13C-d3, Formoterol had an interference of ≤1.2% with the internal standard Formoterol-13C-d3, Glononium had no interference with the internal standard Formoterol-13C-d3, the internal standard Budesonide-d8 had an interference of ≤0.2% with the internal standard Formoterol-13C-d3, and the internal standard Glononium-d5 had an interference of ≤2.1% with the internal standard Formoterol-13C-d3.

[0206] ≤0.1%, Budesonide had an interference of ≤0.1% with the internal standard Glononium-d5, Formoterol had an interference of ≤0.1% with the internal standard Glononium-d5, Glononium had an interference of ≤0.1% with the internal standard Glononium-d5, the internal standard Budesonide-d8 had an interference of ≤0.2% with the internal standard Glononium-d5, and the internal standard Formoterol-13C-d3 had an interference of ≤0.2% with the internal standard Glononium-d5.

[0207] ≤0.2%.

[0208] Hemolyzed blank plasma endogenous substances had an interference of ≤9.1% with Budesonide, no interference with Formoterol, an interference of ≤8.9% with Glononium, an interference of ≤0.1% with the internal standard Budesonide-d8, an interference of ≤0.4% with the internal standard Formoterol-13C-d3, and an interference of ≤0.1% with the internal standard Glononium-d5. All met the planned requirements, and a typical chromatogram is shown in Figures 1-5 .

[0209] 8.2 Extraction recovery rate

[0210] Extraction samples: Prepare 6 QC samples of low, medium, and high concentrations each as extraction samples.

[0211] Unextracted samples: Take the blank matrix after pretreatment, add low, medium, and high concentrations of the analyte and internal standards respectively as unextracted samples, 6 each for low, medium, and high concentrations. The calculation formula is as follows:

[0212]

[0213] The peak area of the extracted sample was compared with the average peak area of the unextracted sample, and the extraction recovery of the analyte or internal standard was calculated. The precision (RSD) of the extraction recovery of the analyte or internal standard should be within 15%.

[0214] The results show that the precision (RSD) of the extraction recovery of budesonide is 4.3%, the precision (RSD) of the extraction recovery of formoterol is 9.2%, the precision (RSD) of the extraction recovery of glononium is 7.5%, the precision (RSD) of the extraction recovery of the internal standard Budesonide-d8 is 6.1%, the precision (RSD) of the extraction recovery of the internal standard Formoterol-13C-d3 is 3.9%, and the precision (RSD) of the extraction recovery of the internal standard Glononium-d5 is 8.1%. All meet the planned requirements.

[0215] 8.3 Matrix effect

[0216] Six different sources / batches of matrix were analyzed, each with six replicates of low and high concentration quality control samples. The samples were analyzed with freshly prepared standard curves and quality control samples. For each source / batch of matrix, the precision should not exceed 15%, and the accuracy of the mean should be within ± 15% of the labeled value.

[0217] Hemolyzed matrix effect: A hemolyzed matrix was prepared by adding 2% whole blood cell lysate to blank plasma. Six low and high concentration quality control samples were prepared for hemolyzed effect investigation. The samples were analyzed with freshly prepared standard curves and quality control samples. The RSD of each concentration should not exceed 15%, and the bias of the mean should be within ± 15%.

[0218] The results show that for each source / batch of matrix, the mean accuracy (Bias) of budesonide at low and high concentrations is between -3.2% and 10.7% and between 0.4% and 7.5%, respectively, and the precision is ≤ 10.0% and ≤ 4.8%, respectively. The precision (RSD) of the hemolyzed matrix effect at low and high concentrations is 6.9% and 7.8%, respectively, and the mean bias is -1.1% and 5.2%, respectively. The mean accuracy (Bias) of formoterol at low and high concentrations is between -5.2% and 6.0% and between -2.6% and 5.1%, respectively, and the precision is ≤ 10.5% and ≤ 6.2%, respectively. The precision (RSD) of the hemolyzed matrix effect at low and high concentrations is 6.5% and 6.7%, respectively, and the mean bias is 2.7% and -3.9%, respectively. The mean accuracy (Bias) of glononium at low and high concentrations is between -0.8% and 11.2% and between -2.7% and 2.5%, respectively, and the precision is ≤ 7.1% and ≤ 4.1%, respectively. The precision (RSD) of the hemolyzed matrix effect at low and high concentrations is 7.5% and 3.5%, respectively, and the mean bias is -2.5% and 3.7%, respectively. All meet the planned requirements.

[0219] 8.4 Linearity range of standard curve

[0220] Prepare two replicates of standard curve samples containing 8 concentrations to establish the standard curve. Take the concentration (C) of the analyte in the biological sample as the abscissa (x) and the peak area ratio of the analyte to the internal standard as the ordinate (y), respectively, and use the weighted least squares method (w = 1 / x2) to perform regression calculation to obtain the linear regression equation, which is the standard curve. Calculate the concentration of each standard curve sample by the regression equation, but the blank and zero concentration samples are not involved in the calculation, and the deviation of the back calculation concentration [(calculated value - theoretical value) / theoretical value x 100%] should be within ± 15% (LLOQ is ± 20%), 75% of the samples and at least 6 concentration levels should meet the above requirements, and unqualified samples should be discarded and regression calculation should be performed again; if all repeated data below the lower limit of quantification or above the upper limit of quantification are unqualified, the results of this batch should be rejected.

[0221] The results show that the plasma concentration of budesonide is linear within the range of 0.100-40.0 ng / mL, the plasma concentration of formoterol is linear within the range of 5.00-2000 pg / mL, and the plasma concentration of glononium is linear within the range of 10.0-4000 pg / mL, and the results all meet the planned requirements. The typical standard curve of budesonide, formoterol and glononium in plasma is shown in Figure 1. Figures 6-8 .

[0222] 8.5 Residual

[0223] A blank biological sample is injected after the STD8 sample for analysis, and the residual peak area of the analyte should not exceed 20% of the average peak area of the analyte of the qualified STD1 sample, and the residual peak area of the internal standard should not exceed 5% of the average peak area of the internal standard of the qualified STD1 sample.

[0224] The results show that the residual of budesonide is within 3.2%, the residual of the internal standard Budesonide-d8 is within 0.1%, the residual of formoterol is within 5.8%, the residual of the internal standard Formoterol-13C-d3 is within 0.4%, the residual of glononium is within 10.8%, and the residual of the internal standard Glononium-d5 is within 0.4%. All meet the planned requirements.

[0225] 8.6 Accuracy, precision and sensitivity

[0226] Prepare LLOQ, low, medium and high concentration quality control samples (QCs) each 6, to calculate the concentration of QC samples with the standard curve of the batch, and to calculate the precision and accuracy of the method. The within-run and between-run precision (RSD) of low, medium and high concentration QC samples should not exceed 15%, and the LLOQ should not exceed 20%; the accuracy (Bias) of the within-run and between-run average of low, medium and high concentration QC samples should be within ± 15%, and the LLOQ should be within ± 20%; at the same time, at least two-thirds of the quality control samples, at least 50% of the samples at the same concentration level should meet the accuracy (Bias) of low, medium and high concentration QC samples within ± 15%, and the LLOQ within ± 20%. The signal-to-noise ratio of LLOQ sample should be ≥5.

[0227] The results showed that the within-run precision (RSD) of LLOQ of budesonide, formoterol and glycopyrronium was within 20%, and the mean accuracy (Bias) was within ± 20%; the within-run precision (RSD) of low, medium and high concentration QC samples was within 15%, and the mean accuracy (Bias) was within ± 15%; the between-run precision (RSD) of LLOQ was within 20%, and the mean accuracy (Bias) was within ± 20%; the between-run precision (RSD) of low, medium and high concentration QC samples was within 15%, and the mean accuracy (Bias) was within ± 15%. All meet the requirements of the plan.

[0228] The signal-to-noise ratio (S / N) of LLOQ sample in the precision and accuracy of budesonide was ≥97, the signal-to-noise ratio (S / N) of LLOQ sample in the precision and accuracy of formoterol was ≥53, and the signal-to-noise ratio (S / N) of LLOQ sample in the precision and accuracy of glycopyrronium was ≥18, which met the requirements of the plan.

[0229] 8.7 Stability of stock solution and working solution

[0230] The stability of budesonide, formoterol, glycopyrronium and internal standard stock solution was investigated at 10-30℃ under yellow light for about 52.1 hours, and the stability of budesonide, formoterol, glycopyrronium and internal standard stock solution was investigated under storage conditions of-30 to-10℃ for 42 days. The stability of budesonide, formoterol, glycopyrronium and internal standard working solution was investigated at 10-30℃ under yellow light for about 52.1 hours, and the stability of budesonide, formoterol, glycopyrronium and internal standard working solution was investigated under storage conditions of-30 to-10℃ for 42 days.

[0231] Compare the average peak area of the stock solution or working solution stored for a period of time with the average peak area of the freshly prepared one, and the precision (RSD) should be within 10%, and the bias (Bias) of the two responses should be within ± 10%.

[0232] The results show that the budesonide, formoterol, glycopyrronium and internal standard stock solutions are stable at 10-30°C, yellow light for about 52.1 hours; the budesonide, formoterol, glycopyrronium and internal standard stock solutions are stable at -30 to -10°C for 42 days. The budesonide, formoterol, glycopyrronium and internal standard working solutions are stable at 10-30°C, yellow light for about 52.1 hours; the budesonide, formoterol, glycopyrronium and internal standard working solutions are stable at -30 to -10°C for 42 days. The precision (RSD) of all stability samples is within 10%, the bias of average peak area is within ±10%, and the results meet the requirements.

[0233] 8.8 Stability of the samples after treatment

[0234] The low and high concentration samples (n=6) are stored in the sample manager (6°C) for about 68.0 hours before being injected (the storage time is not less than the actual storage time of the samples in the actual storage environment), and the precision (RSD) of each concentration is calculated by using the freshly prepared standard curve, and the bias (Bias) should be within ±15%.

[0235] The results show that the samples are stable after being stored in the sample manager (6°C) for about 68.0 hours, the RSD of the low and high concentrations of budesonide is 7.7% and 6.5% respectively, and the bias is -3.4% and -2.8% respectively; the RSD of the low and high concentrations of formoterol is 5.3% and 4.6% respectively, and the bias is -1.5% and -0.8% respectively; the RSD of the low and high concentrations of glycopyrronium is 2.5% and 3.6% respectively, and the bias is 6.3% and -1.3% respectively, which meet the planned requirements.

[0236] 8.9 Injection reproducibility

[0237] The injection reproducibility of the standard curve and the low, medium and high concentration samples (n=6) after being stored in the sample manager (6°C) for about 71.5 hours is investigated, the precision (RSD) of the low, medium and high concentration samples should be less than 15%, and the bias should be within ±15%; at the same time, at least two-thirds of the quality control samples, at least 50% of the samples at the same concentration level should meet the accuracy (Bias) of the low, medium and high concentration QC samples within ±15%.

[0238] The results show that: the sample is stable after treatment and placed in the sample manager (6°C) for about 71.5 hours, the RSD of budesonide at low, medium and high concentrations is within 4.7%, the mean deviation is between -3.4% and 1.5%; the RSD of formoterol at low, medium and high concentrations is within 7.9%, the mean deviation is between -4.6% and 2.2%; the RSD of glycopyrronium at low, medium and high concentrations is within 5.8%, the mean deviation is between -10.4% and 2.1%, all meeting the planned requirements.

[0239] 8.10 Stability of whole blood

[0240] The stability of budesonide, glycopyrronium and formoterol whole blood samples under ice box and yellow light conditions for about 4.0 hours is investigated, the mean of the peak area ratio of the analyte to the internal standard of the sample placed under ice box and yellow light conditions for about 4.0 hours is compared with the mean of the peak area ratio of the analyte to the internal standard obtained after the whole blood sample is immediately separated into plasma (0h), the deviation is within ±15%, and the precision (RSD) of the peak area ratio should be within 15%.

[0241] The results show that: the peak area ratio of budesonide whole blood sample is -1.9% and 6.3% respectively, the RSD is within 8.5%; the peak area ratio of glycopyrronium whole blood sample is -2.1% and 2.2% respectively, the RSD is within 5.8%; the peak area ratio of formoterol whole blood sample is 2.5% and -0.6% respectively, the RSD is within 5.5%; all meeting the planned requirements.

[0242] 8.11 Maximum number of samples in a batch

[0243] QC samples (n=15) of low, medium and high concentrations are prepared and injected repeatedly multiple times, under the premise that the standard curve meets the requirements, the maximum number of injection needles that can be carried by one analysis batch is investigated.

[0244] The precision (RSD) of QC samples of the same concentration should not exceed 15%, the mean deviation should be within ±15%, and at least two-thirds of the QC samples, at least 50% of the QC samples at the same concentration level should meet the accuracy (Bias) requirements, which should be within ±15%.

[0245] The results show that the RSD of the low, medium and high concentration quality control samples of budesonide is within 5.8%, the deviation of the mean values of each concentration is between 2.8% and 7.6%, the RSD of the low, medium and high concentration quality control samples of formoterol is within 4.5%, the deviation of the mean values of each concentration is between -3.0% and 5.2%, the RSD of the low, medium and high concentration quality control samples of glycopyrronium is within 5.9%, the deviation of the mean values of each concentration is between 1.8% and 4.6%, which meets the plan requirements, and the total number of samples (including system suitability, standard curve, residual, quality control samples, etc.) is stable within 162 samples.

[0246] 8.12 Stability of the samples before treatment

[0247] The stability of the low and high concentration quality control plasma samples (n=5) is investigated, including the stability of the plasma samples at 10-30°C under yellow light for about 28.8 hours, the stability of the samples after 5 times of freeze-thaw cycles at -60°C and below / 10-30°C under yellow light, and the stability of the samples after long-term storage at -60°C and below for 35 days and 92 days. The concentration of the analyte in the samples is calculated according to the standard curve on the same day, the precision (RSD) of each concentration is not more than 15%, and the mean deviation is within ±15%.

[0248] The results show that: low, high concentration of plasma samples before 10-30℃, yellow light placed about 28.8 hours stable, budesonide RSD were 3.1% and 6.4%, the mean deviation were -3.8% and 6.5%, formoterol RSD were 3.8% and 2.9%, the mean deviation were -3.6% and 7.7%, glycopyrronium RSD were 4.5% and 6.6%, the mean deviation were 3.9% and 3.2%. Low, high concentration of plasma samples repeated freeze-thaw 5 times stable, budesonide RSD were 7.7% and 2.5%, the mean deviation were -9.7% and 5.6%, formoterol RSD were 5.8% and 3.2%, the mean deviation were -1.2% and 5.4%, glycopyrronium RSD were 3.0% and 6.7%, the mean deviation were -9.5% and 12.5%. Low, high concentration of plasma samples-60℃ and below long-term storage 35 days stable, budesonide RSD were 4.1% and 2.2%, the mean deviation were -5.5% and -2.3%, formoterol RSD were 9.5% and 2.3%, the mean deviation were 8.8% and -1.1%, glycopyrronium RSD were 7.5% and 4.6%, the mean deviation were 2.2% and 5.6%; Low, high concentration of plasma samples-60℃ and below long-term storage 92 days stable, budesonide RSD were 2.4% and 1.9%, the mean deviation were 3.3% and 1.5%, formoterol RSD were 2.9% and 3.5%, the mean deviation were -2.6% and -3.1%, glycopyrronium RSD were 4.4% and 6.1%, the mean deviation were -1.5% and -1.9%. All meet the plan requirements.

[0249] 8.13 Analysis batch evaluation

[0250] 8.13.1 Quality control samples

[0251] Quality control samples and standard curve samples are prepared with different stock solutions, low, medium and high concentration of quality control samples are determined, at least double samples, at least 2 / 3 of the quality control samples, at least 50% of the samples at each concentration meet the deviation within ± 15%. If it does not meet the acceptance criteria, reject the batch results.

[0252] The results show that: all qualified analysis batch of low, medium and high concentration of quality control samples meet the acceptance criteria, meet the above requirements.

[0253] 8.13.2 Blank samples and zero concentration samples

[0254] The sample peak area measured by the blank sample of the analysis batch compared with the average of the sample peak area of the lowest point of the qualified standard curve should be less than 20% of the peak area of the analyte and less than 5% of the peak area of the internal standard, and the sample peak area measured by the zero concentration sample compared with the sample peak area of the lowest point of the qualified standard curve should be less than 20% of the peak area of the analyte.

[0255] The results show that: the peak area of budesonide, formoterol and glycopyrronium measured by the blank sample is within 20% of the average of the peak area of budesonide, formoterol and glycopyrronium of the lowest point of the qualified standard curve; the peak area of the internal standard measured by the blank sample is within 5% of the average of the peak area of the internal standard of the lowest point of the qualified standard curve; the peak area of budesonide, formoterol and glycopyrronium measured by the zero sample is within 20% of the average of the peak area of budesonide, formoterol and glycopyrronium of the lowest point of the qualified standard curve, which meets the above requirements.

[0256] 9Conclusion

[0257] In summary, the analysis method of the present application has high specificity and accuracy, good reproducibility, and can be used for the concentration detection of budesonide, formoterol and glycopyrronium in plasma, and is suitable for preclinical pharmacokinetics and toxicokinetics research.

Claims

1. An LC-MS / MS method for simultaneously quantifying the concentrations of budesonide, formoterol, and glycopyrronium in plasma, characterized in that: It includes the following steps: (1) Establishment of the standard curve a. Preparation of a series of concentration standard curve samples: Take budesonide, dissolve it in acetonitrile to obtain budesonide solution; take formoterol and glycopyrronium, dissolve them in dimethyl sulfoxide to obtain formoterol solution and glycopyrronium solution respectively; take budesonide solution, formoterol solution and glycopyrronium solution, and dilute them with acetonitrile aqueous solution to obtain a series of concentrations; Take solutions of a series of concentrations, add matrix and mix well. Add internal standard working solution and ethyl acetate to the mixture and mix well. After centrifugation, take the supernatant and dry it. Add acetonitrile aqueous solution to dissolve and mix well. Centrifuge and take the supernatant to obtain the solution. b. Inject the series of concentration standard curve samples into the LC-MS / MS instrument, measure the peak area, and obtain the standard curves of budesonide, formoterol, and glycopyrronium; The chromatographic conditions are as follows: Column: Ethylene-bridged hybrid particles as packing material; formic acid-ammonium acetate aqueous solution as mobile phase A, isopropanol-acetonitrile solution containing formic acid as mobile phase B, gradient elution program as follows: Mass spectrometry conditions: Ion source: electrospray ionization source; Scan type: multiple ion reaction monitoring; (2) Determination of drug component content in the sample to be tested: c. Processing of biological samples Take the plasma sample to be tested, add internal standard working solution and ethyl acetate, mix well, centrifuge, take the supernatant and blow dry, then add acetonitrile aqueous solution to dissolve and mix well, centrifuge, take the supernatant; d. Determination of biological samples Take the supernatant obtained in step c and inject it into the LC-MS / MS instrument for detection. Under the same conditions as in step b, the contents of budesonide, formoterol, and glycopyrronium in the plasma sample to be tested are obtained according to the standard curve of step (1).

2. The LC-MS / MS analysis method according to claim 1, characterized in that: Step (2) in the content determination process also includes a quality control procedure, the specific steps of which are as follows: Take the matrix, add acetonitrile aqueous solution and ethyl acetate, mix well, centrifuge, take the supernatant and dry it, then add acetonitrile aqueous solution to dissolve and mix well, centrifuge, take the supernatant to obtain the blank sample test solution; take the budesonide solution, formoterod solution and glycopyrronium solution obtained in step a, dilute with acetonitrile aqueous solution to obtain the quality control sample, take the quality control sample, add internal standard working solution and ethyl acetate, mix well, centrifuge, take the supernatant and dry it, then add acetonitrile aqueous solution to dissolve and mix well, centrifuge, take the supernatant to obtain the quality control sample test solution; The test solutions of blank samples and quality control samples were injected into the LC-MS / MS instrument to monitor the interference in the measurement process. The volume ratio of the matrix to the acetonitrile aqueous solution for mixing, ethyl acetate, the supernatant for drying, and the acetonitrile aqueous solution for dissolving is 100:20:600:500:

65. The volume ratio of the quality control sample to the internal standard working solution, ethyl acetate, supernatant for drying, and acetonitrile aqueous solution was 100:20:600:500:

65.

3. The LC-MS / MS analysis method according to claim 1, characterized in that: The volume ratio of the series of solutions and the matrix in step a is 15:

135. After adding the matrix, the concentration of budesonide in the series of solutions is 0.1-40 ng / mL, the concentration of formoterod is 5-2000 pg / mL, and the concentration of glycopyrronium is 10-4000 pg / mL. The volume ratio of the mixture, internal standard solution, ethyl acetate, supernatant for drying, and acetonitrile aqueous solution is 100:20:600:500:

65.

4. The LC-MS / MS analysis method according to any one of claims 1 to 3, characterized in that: The matrix is ​​blank plasma from Beagle dogs anticoagulated with EDTA-K2.

5. The LC-MS / MS analysis method according to claim 1, characterized in that: The mixing time is 10 min; the centrifugation temperature is 4℃, the rotation speed is 18000×g, and the time is 1-5 min.

6. The LC-MS / MS analysis method according to claim 1 or 3, characterized in that: The internal standard working solution is an aqueous solution of acetonitrile containing 50.0 ng / mL Budesonide-d8, 0.5 ng / mL Formoterol-13C-d3 and 5 ng / mL Glononium-d5 standards.

7. The LC-MS / MS analysis method according to claim 1, characterized in that: The concentration of the acetonitrile aqueous solution is 50%.

8. The LC-MS / MS analysis method according to claim 1, characterized in that: The chromatographic column was an Xbridge C185um 4.6*150mm column; the injection volume was 5μL; the column temperature was 50℃; the mobile phase A was 0.1% formic acid-2mM ammonium acetate aqueous solution, and the mobile phase B was isopropanol and acetonitrile containing 0.1% formic acid in a volume ratio of 1:

9.

9. The LC-MS / MS analysis method according to claim 1, characterized in that: In step b, the mass spectrometry conditions are: polar mode: positive ion mode; curtain gas: 30 psi. Collision gas: 9V; Ion spray voltage: 5500V; Temperature: 550℃; Ion source gas 1: 40psi; Ion source gas 2: 40psi; Inlet potential: 10V; Collision pool outlet potential: 10V.

10. The LC-MS / MS analysis method according to claim 9, characterized in that: The mass spectrometry conditions described are as follows: Budesonide detection ion pair m / z 431.2→m / z 322.7, declustering voltage 60V, collision energy 18eV; Formoterol detection ion pair m / z 344.9→m / z 149.0, declustering voltage 60V, collision energy 26eV; Glycolone bromide detection ion pair m / z 318.2→m / z 116.1, declustering voltage 90V, collision energy 34eV; Budesonide-d8 detection ion pair m / z 439.2→m / z 323.2, declustering voltage 100V, collision energy 25eV; Formoterol-13C-d3 detection ion pair m / z 349.0→m / z 153.0, declustering voltage 60V, collision energy 27eV; Glononium-d5 detection ion pair m / z 323.2→m / z 153.

0. 116.1, declustering voltage 90V, collision energy 34eV.

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