LC-MS / MS analysis method for determining concentrations of fluticasone furoate, vilanterol and imequat in plasma

Abstract

The invention belongs to the field of in-vivo quantitative drug concentration analysis, and particularly discloses an LC-MS / MS analysis method for determining the concentrations of fluticasone furoate, vilanterol and imequat in plasma. According to the method disclosed by the invention, the contents of fluticasone furoate, vilanterol and umepiride in the blood sample can be accurately and quantitatively detected at one 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 determining the concentrations of fluticasone propionate, vilanterol and umclidinium in blood plasma. BACKGROUND

[0002] Fluticasone propionate is an adrenocortical hormone drug with local vasoconstriction and anti-inflammatory activity, which mainly reduces the inflammatory response in the nasal cavity by inhibiting the activity and number of inflammatory cells (such as eosinophils, monocytes, mast cells, etc.) and reducing the production of inflammatory mediators; umclidinium is a long-acting muscarinic receptor antagonist that exerts a bronchodilatory effect by competitively inhibiting the binding of acetylcholine to M3-type muscarinic receptors on the smooth muscle of the respiratory tract; vilanterol is a long-acting β2 adrenergic receptor agonist that relaxes the smooth muscle by activating the β2 adrenergic receptor on the airway smooth muscle, thereby dilating the airway and improving respiration.

[0003] Fluticasone propionate, umclidinium and vilanterol are often used in combination to treat chronic obstructive pulmonary disease (COPD), including chronic bronchitis and emphysema. During the research and development stage, blood drug concentration detection of fluticasone propionate + umclidinium + vilanterol triple inhalation preparation can verify whether the formulation optimization leads to significant changes in systemic exposure, providing data support for new drug bioequivalence verification and subsequent dosage form improvement; in the clinical application stage, it can objectively evaluate whether the patient uses the inhalation device as specified, and if the blood concentration is significantly lower than expected, it may indicate poor inhalation technique or insufficient use frequency; for patients with impaired liver and kidney function, elderly patients or pediatric patients, blood drug concentration monitoring can help determine whether to reduce the dosage or change the treatment regimen, thereby achieving more precise respiratory disease management. However, there are currently only reports of LC-MS / MS analysis methods for blood concentration detection of any one of fluticasone propionate, umclidinium and vilanterol, and there is no research on a method that can simultaneously monitor the concentrations of fluticasone propionate, vilanterol and umclidinium (in the form of umclidinium in blood) in blood, which results in the need to reprocess samples and prepare detection reagents and set instrument parameters according to the different components to be detected, resulting in low detection efficiency. SUMMARY

[0004] To solve the above problems, the present application provides a LC-MS / MS analysis method for determining the concentrations of fluticasone propionate, vilanterol and umclidinium in blood plasma, which comprises the following steps:

[0005] (1) Establishment of standard curve

[0006] a. Preparation of series concentration standard curve samples: Take fluticasone furoate, vilanterol trifenatate and umclidinium bromide, add dimethyl sulfoxide to dissolve, and then add acetonitrile aqueous solution to dilute into series concentrations;

[0007] Take the mixed solution of series concentrations respectively, add matrix, internal standard working solution and ethyl acetate in turn, mix well, centrifuge, take the supernatant, blow dry, add acetonitrile aqueous solution to dissolve and mix well, centrifuge, and take the supernatant to obtain;

[0008] b. Inject the series concentration standard samples into LC-MS / MS instrument respectively, measure the peak area to obtain the standard curve of fluticasone furoate, vilanterol and umclidinium;

[0009] The chromatographic conditions are as follows:

[0010] Chromatographic column: BEH silica gel particles as filler; formic acid-ammonium acetate aqueous solution as mobile phase A, and formic acid acetonitrile solution as mobile phase B;

[0011] The gradient elution program for detecting umclidinium and vilanterol is as follows:

[0012]

[0013]

[0014] The gradient elution program for detecting fluticasone furoate is as follows:

[0015]

[0016] Mass spectrometry conditions: ion source is electrospray ionization source; scanning type: multiple ion reaction monitoring;

[0017] (2) Determination of drug content in the sample to be tested:

[0018] c. Treatment of biological samples

[0019] Take the plasma sample to be tested, add internal standard working solution and ethyl acetate, mix well, centrifuge, take the supernatant, blow dry, add acetonitrile aqueous solution to dissolve and mix well, centrifuge, and take the supernatant.

[0020] d. Determination of biological samples

[0021] Take the supernatant obtained in step c, inject it into the LC-MS / MS instrument for detection, and detect under the same conditions as step b. According to the standard curve in step (1), the content of fluticasone furoate, vilanterol and umclidinium in the plasma sample to be tested is obtained.

[0022] Further, the content determination process in step (2) also includes a quality control procedure, and the specific steps are as follows:

[0023] 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, and take the supernatant to obtain the blank sample test solution; take the dimethyl sulfoxide solution containing fluticasone furoate, vilanterol and umemonium 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, and take the supernatant to obtain the quality control sample test solution; inject the test solutions of the blank sample and quality control sample into the LC-MS / MS instrument respectively to monitor the interference in the measurement process;

[0024] 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 100:30:600:500:70.

[0025] The volume ratio of the quality control sample to the internal standard working solution, ethyl acetate, supernatant for drying, and acetonitrile aqueous solution was 100:30:600:500:70.

[0026] Further, the volume ratio of the series of concentrations of the mixed solution, matrix, internal standard solution, ethyl acetate, supernatant for drying, and acetonitrile aqueous solution in step a is 10:90:30:600:500:70; after adding the matrix, the concentration of fluticasone furoate in the series of concentrations of the mixed solution is 20-4000 pg / mL, the concentration of vilanterol is 10-2000 pg / mL, and the concentration of umemet is 3-600 pg / mL.

[0027] Furthermore, the matrix is ​​EDTA-K2 anticoagulated blank plasma from Beagle dogs.

[0028] Furthermore, the mixing is vortex mixing; the centrifugation temperature is 4℃, the rotation speed is 18000×g, and the time is 1~5min.

[0029] Furthermore, the internal standard working solution is an aqueous solution of acetonitrile containing 4 ng / mL Lluticasone Furoate-d3, 1.0 ng / mL Vilanterol-d4 and 1.0 ng / mL Llmepium-d10 standard substances;

[0030] Furthermore, the concentration of the acetonitrile aqueous solution is 50%.

[0031] Furthermore, the chromatographic column is an ACQUITYUPLC BEH C18 1.7um 2.1*100mm column; the injection volume is 5-10μL; the column temperature is 50℃; the mobile phase A is 0.2% formic acid-5mM ammonium acetate aqueous solution, and the mobile phase B is 0.2% formic acid-acetonitrile solution.

[0032] Further, the mass spectrometry conditions in step b are as follows: polarity mode: positive ion mode; curtain gas: 30 psi; collision gas: 9 psi; ion source gas 1: 50 psi; ion source gas 2: 50 psi; inlet potential: 10 V; collision cell exit potential: 10 V.

[0033] Further, the mass spectrometry conditions are as follows: ion spray voltage (IS) for detecting ulmepium and vilanterol: 5500 V; temperature (TEM): 550℃; vilanterol detection ion pair m / z 486.2→m / z 159.0, declustering voltage (DP) 90 V, collision energy 35 eV; ulmepium detection ion pair m / z 428.3→m / z 96.2, declustering voltage (DP) 120 V, collision energy 80 eV; vilanterol-d4 detection ion pair m / z 490.2→m / z 159.0, declustering voltage (DP) 60 V, collision energy 45 eV; ulmepium-d10 detection ion pair m / z 438.2→m / z 96.2, declustering voltage (DP) 85 V, collision energy 80 eV.

[0034] ion spray voltage (IS) for detecting fluticasone furate: 5000 V; temperature (TEM): 380℃; fluticasone furate detection ion pair m / z 539.1→m / z 313.3, declustering voltage (DP) 80 V, collision energy 21 eV; fluticasone furate-d3 detection ion pair m / z 542.3→m / z 313.0, declustering voltage (DP) 80 V, collision energy 20 eV.

[0035] The LC-MS / MS analysis method for determining the concentrations of fluticasone furate, vilanterol and ulmepium in blood plasma provided by the present application can accurately and quantitatively detect the contents of fluticasone furate, vilanterol and ulmepium in blood samples at one time through specific sample processing and chromatography and mass spectrometry conditions, has high sensitivity and low detection limit, and provides a new practically applicable method for the pharmacokinetic study of the three components, and has application value.

[0036] 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.

[0037] The above content of the present application will be further described 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

[0038] Figure 1 Fluticasone Furorate and Fluticasone Furoate-d3 chromatograms in blank sample;

[0039] Figure 2 Fluticasone Furorate and Fluticasone Furoate-d3 chromatograms in zero concentration sample;

[0040] Figure 3 Fluticasone Furorate and Fluticasone Furoate-d3 chromatograms in standard curve lowest point sample (STD1);

[0041] Figure 4 Fluticasone Furorate and Fluticasone Furoate-d3 chromatograms in standard curve highest point sample (STD8);

[0042] Figure 5 Fluticasone Furorate and Fluticasone Furoate-d3 chromatograms in carry over;

[0043] Figure 6 Vilanterol and Vilanterol-d4 chromatograms in blank sample;

[0044] Figure 7 Vilanterol and Vilanterol-d4 chromatograms in zero concentration sample;

[0045] Figure 8 Vilanterol and Vilanterol-d4 chromatograms in standard curve lowest point sample (STD1);

[0046] Figure 9 Vilanterol and Vilanterol-d4 chromatograms in standard curve highest point sample (STD8);

[0047] Figure 10 Vilanterol and Vilanterol-d4 chromatograms in carry over;

[0048] Figure 11 Ulmepium and Ulmepium-d10 chromatograms in blank sample;

[0049] Figure 12 Ulmepium and Ulmepium-d10 chromatograms in zero concentration sample;

[0050] Figure 13 Chromatograms of Ulmepium and Ulmepium-d10 in sample (STD1) at the lowest point of the standard curve;

[0051] Figure 14 Chromatograms of Ulmepium and Ulmepium-d10 in sample (STD8) at the highest point of the standard curve;

[0052] Figure 15 Chromatograms of Ulmepium and Ulmepium-d10 in carryover;

[0053] Figure 16 Typical standard curve for determining Fluticasone Furorate in Beagle dog plasma by LC-MS / MS;

[0054] Figure 17 Typical standard curve for the determination of Vilanterol in Beagle dog plasma by LC-MS / MS

[0055] Figure 18 Typical standard curve for the determination of Ulmepium in Beagle dog plasma by LC-MS / MS. Detailed Implementation

[0056] Example 1: Detection of Fluticasone Furoate, Vilanterol, and Umeprazole Concentrations in Plasma

[0057] (I) Preparation of solution

[0058] ① Standard curve stock solution

[0059] Standards of fluticasone furoate, vilanterol triphenylacetic acid, and umembranone bromide were prepared by adding dimethyl sulfoxide to prepare stock solutions of fluticasone furoate, vilanterol, and umembranone bromide with a concentration of 1.00 mg / mL.

[0060] ② Internal standard working solution

[0061] Standards of Fluticasone Furoate-d3, Vilanterol-d4 Triphenylacetate, and Umeclidinium Bromide-d10 were prepared with dimethyl sulfoxide to form internal standard stock solutions of Fluticasone Furoate-d3, Vilanterol-d4, and Umeclidinium Bromide-d10 with a concentration of 1.00 mg / mL.

[0062] Take Fluticasone Furoate-d3 internal standard stock solution, Vilanterol-d4 internal standard stock solution and Ulmepium-d10 internal standard stock solution, dilute with 50% acetonitrile aqueous solution to obtain an internal standard working solution containing 4 ng / mL Fluticasone Furoate-d3, 1.0 ng / mL Vilanterol-d4 and 1.0 ng / mL Ulmepium-d10;

[0063] (ii) Establishment of standard curve

[0064] a. Preparation of series concentration standard curve samples:

[0065] Take the stock solution of Fluticasone Furoate, the stock solution of Vilanterol and the stock solution of Ulmepium, dilute with 50% acetonitrile aqueous solution to obtain a series concentration mixed solution with Fluticasone Furoate concentration of 0.2-40 ng / mL, Vilanterol concentration of 0.1-20 ng / mL and Ulmepium concentration of 0.03-6 ng / mL;

[0066] Take 10 μL of the series concentration mixed solution, mix with 90 μL of blank matrix (EDTA-K2 anticoagulated Beagle dog blank plasma), then add 30 uL of internal standard working solution, finally add 600 uL of ethyl acetate and mix for 5 min, put into the centrifuge, 4°C, 18000xg centrifugation for 5 min, take 500 μL of supernatant, nitrogen blowing dry, add 70 μL of 50% acetonitrile aqueous solution, mix for 5 min, put into the centrifuge, 4°C, 18000xg centrifugation for 1 min, take the supernatant to obtain;

[0067] b. Inject the series concentration standard curve samples into the LC-MS / MS instrument respectively, measure the peak area, take the concentration of each drug component in the series concentration standard curve working solution as the abscissa (X), 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 calculate the linear regression equation, which is the standard curve;

[0068] The chromatographic conditions are as follows:

[0069] The chromatographic column is ACQUITY UPLC BEH C18 1.7um 2.1*100mm Column; the column temperature is 50°C; 0.2% formic acid-5mM ammonium acetate aqueous solution is used as mobile phase A, and 0.2% formic acid acetonitrile solution is used as mobile phase B; the injection volume is 5-10 μL;

[0070] The gradient elution program for detecting Ulmepium and Vilanterol is as follows:

[0071]

[0072] The gradient elution procedure for the detection of fluticasone furate was as follows:

[0073]

[0074] The mass spectrometry conditions were as follows:

[0075] Ion source: electrospray ionization source; scan type: multiple ion reaction monitoring; polarity mode: positive ion mode; curtain gas (CUR): 30 psi; collision gas (CAD): 9 psi; ion source gas 1 (GS1): 50 psi; ion source gas 2 (GS2): 50 psi; entrance potential (EP): 10 V; collision cell exit potential (CXP): 10 V;

[0076] Ion spray voltage (IS) for the detection of ulmepium and vilanterol: 5500 V; temperature (TEM): 550℃; vilanterol detection ion pair m / z 486.2→m / z 159.0, declustering voltage (DP) 90 V, collision energy 35 eV; ulmepium detection ion pair m / z 428.3→m / z 96.2, declustering voltage (DP) 120 V, collision energy 80 eV; vilanterol-d4 detection ion pair m / z 490.2→m / z 159.0, declustering voltage (DP) 60 V, collision energy 45 eV; ulmepium-d10 detection ion pair m / z 438.2→m / z 96.2, declustering voltage (DP) 85 V, collision energy 80 eV;

[0077] Ion spray voltage (IS) for the detection of fluticasone furate: 5000 V; temperature (TEM): 380℃; fluticasone furate detection ion pair m / z 539.1→m / z 313.3, declustering voltage (DP) 80 V, collision energy 21 eV; fluticasone furate-d3 detection ion pair m / z 542.3→m / z 313.0, declustering voltage (DP) 80 V, collision energy 20 eV;

[0078] (III) Determination of the contents of fluticasone furate, vilanterol, and ulmepium in the sample to be tested

[0079] c. Treatment of biological samples, blank samples, and quality control samples

[0080] Take 100 μL of the plasma sample in a polypropylene tube, add 30 uL of the internal standard working solution, then add 600 uL of ethyl acetate, vortex to mix, and place in a centrifuge, 4℃, 18000 x g centrifugation for 5 min, take 500 μL of the supernatant in a polypropylene tube, dry under nitrogen, then add 70 μL of 50% acetonitrile aqueous solution, mix, and finally place in a centrifuge, 4℃, 18000 x g centrifugation for 1 min, take the supernatant to obtain the biological sample test solution;

[0081] Take blank matrix (EDTA-K2 anticoagulant Beagle dog blank plasma) 100 μL in a polypropylene tube, add 30 μL of 50% aqueous acetonitrile solution, then add 600 uL of ethyl acetate, vortex, put into centrifuge, 4°C, 18000xg centrifugation for 5 min, take 500 μL supernatant in a polypropylene tube, nitrogen blowing dry, add 70 μL of 50% aqueous acetonitrile solution, mix, finally put into centrifuge, 4°C, 18000xg centrifugation for 1 min, take the supernatant to obtain the blank sample test solution;

[0082] Take fluticasone furoate stock solution, vilanterol stock solution and umclidinium stock solution, dilute with 50% aqueous acetonitrile solution to obtain quality control samples containing 0.2-160 ng / mL fluticasone furoate, 0.1-80 ng / mL vilanterol and 0.03-24 ng / mL umclidinium, take 100 μL quality control sample in a polypropylene tube, add 30 uL of internal standard working solution, then add 600 uL of ethyl acetate, vortex, put into centrifuge, 4°C, 18000xg centrifugation for 5 min, take 500 uL supernatant in a polypropylene tube, nitrogen blowing dry, add 70 uL of 50% aqueous acetonitrile solution, mix, finally put into centrifuge, 4°C, 18000xg centrifugation for 1 min, take the supernatant to obtain the quality control sample test solution;

[0083] d. Determination of biological samples

[0084] Take the test solution of blank sample, quality control sample and biological sample respectively, inject into LC-MS / MS instrument for detection, detect under the same conditions as step b, monitor the operation process with blank sample to determine whether there is interference, obtain the content of fluticasone furoate, vilanterol and umclidinium in the biological sample according to the standard curve of step (two).

[0085] The beneficial effects of the present application are illustrated by the following test examples.

[0086] There are many reports on the LC-MS / MS detection method of the concentration of one of the drug components of fluticasone propionate, vilanterol, umclidinium in the blood, which cannot realize the simultaneous accurate quantification of the three components in the blood after testing one by one. In view of the problem that the existing reports are difficult to realize the simultaneous accurate quantification of fluticasone propionate, vilanterol and umclidinium, the four key links of chromatographic column, mobile phase, gradient elution program and biological sample pretreatment are systematically optimized: the chromatographic column is selected, Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 x 100 mm) is selected because it has excellent separation degree and signal-to-noise ratio for the three compounds; the optimal system is determined by investigating different proportions of formic acid-buffered saline phase and formic acid-methanol / acetonitrile / isopropanol organic phase combination; the gradient elution program is optimized by flow rate, starting gradient and time gradient, so that the three compounds have good response and signal-to-noise ratio, and the residual is minimized, and the analysis time is shortened to 3.5 minutes / needle; after comprehensive evaluation of protein precipitation method, liquid-liquid extraction method and solid phase extraction method, the liquid-liquid extraction method is selected, which can significantly improve the cleanliness of the sample and reduce the interference peak compared with the protein precipitation method, and can better balance the extraction recovery rate of the three components to meet the low lower limit of quantification.

[0087] Test Example 1 Methodology verification of the application

[0088] 1. Method overview

[0089] After liquid-liquid extraction with ethyl acetate, 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:

[0090] Table 1. Verification information of the detection method

[0091]

[0092] 2. Instruments and chromatography-mass spectrometry conditions

[0093] 2.1 Main instruments and equipment

[0094]

[0095] 2.2 Chromatography conditions

[0096] 2.2.1 Suitable for detecting umclidinium and vilanterol.

[0097]

[0098]

[0099] Gradient elution program

[0100]

[0101] 2.2.2 Suitable for the detection of fluticasone furoate.

[0102]

[0103] Gradient elution program

[0104]

[0105]

[0106] 2.3 Mass spectrometry conditions

[0107] 2.4.1 Suitable for the detection of umeclidinium and vilanterol.

[0108]

[0109] 2.4.2 Suitable for the detection of fluticasone furoate.

[0110]

[0111]

[0112] 3 Standard substances, reagents, solution preparation and blank matrix

[0113] 3.1 Standard substance of the analyte

[0114] Fluticasone furoate, content 99.8%, from MedChenExpress; Vilanterol trifenatate, content 99.68%, from MedChenExpress; Umeclidinium bromide, content 99.7%, from MedChenExpress;

[0115] 3.2 Standard substance of internal standard (IS)

[0116] Fluticasone furoate-d3, content 98.3%, from TLC Pharmaceutical Standards; Vilanterol-d4 Triphenylacetate, content 97.4%, from TLC Pharmaceutical Standards; Umeclidinium bromide-d10, content 93.49%, from MedChenExpress.

[0117] 3.3 Solution preparation

[0118] The preparation of the solution only needs to keep the same proportion, and the volume of the preparation can be adjusted according to the actual situation.

[0119]

[0120]

[0121] 3.4 Blank matrix

[0122] EDTA-K2 anticoagulated Beagle dog plasma, from our institute, thawed at 10-30℃ before use.

[0123] EDTA-K2 anticoagulated Beagle dog whole blood at our institute, transferred in sample transport box after collection, stored at 2-8℃.

[0124] Hemolyzed blank matrix: Take 50 μL EDTA-K2 anticoagulated Beagle dog whole blood in a polypropylene tube, freeze at -60℃ and below for at least 30 min, thaw at 10-30℃, 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.

[0125] 4. Preparation of stock solution, working solution and sample

[0126] 4.1 Preparation of stock solution of fluticasone furoate with concentration of about 1.00 mg / mL

[0127] Weigh about 3 mg (2.7-3.3 mg) fluticasone furoate standard substance in a polypropylene tube at 10-30℃ under yellow light, add a certain volume of dimethyl sulfoxide to prepare 1.00 mg / mL. Mark as SS-YyMmDd-x. Weigh two portions, one as standard curve stock solution, the other as quality control stock solution, store the stock solution at -30--10℃ in a polypropylene tube. When calculating the volume, you need to convert according to the following formula:

[0128] Volume = m x content ÷ concentration of stock solution

[0129] 4.2 Preparation of stock solution of vilanterol with concentration of about 1.00 mg / mL

[0130] Weigh about 3 mg (2.7-3.3 mg) vilanterol trifenatate standard substance in a polypropylene tube at 10-30℃ under yellow light, add a certain volume of dimethyl sulfoxide to prepare 1.00 mg / mL. Mark as SS-YyMmDd-x. Weigh two portions, one as standard curve stock solution, the other as quality control stock solution, store the stock solution at -30--10℃ in a polypropylene tube. When calculating the volume, you need to convert according to the following formula:

[0131] Volume = m x content x (486.43 / 774.78) ÷ concentration of stock solution

[0132] 4.3 Preparation of about 1.00 mg / mL Ume Bromide Stock Solution

[0133] At yellow light 10-30 °C, weigh about 3 mg (2.7-3.3 mg) of Ume Bromide Standard Material into a polypropylene tube, add a certain volume of dimethyl sulfoxide to prepare 1.00 mg / mL. Marked as SS-YyMmDd-x. Weigh double, one as standard curve stock solution, the other as quality control stock solution, stored at -30-10 °C in polypropylene tubes. When calculating the volume, it needs to be converted according to the following formula.

[0134] Volume = m x content x (428.49 / 508.49) ÷ concentration of stock solution

[0135] 4.4 Preparation of working solution

[0136] At yellow light 10-30 °C, prepare the standard curve working solution and quality control working solution according to the following table, vortex mix well, the dilution is 50% acetonitrile aqueous solution. Stored at -30-10 °C in polypropylene tubes.

[0137] Table 2 Preparation of standard curve working solution

[0138]

[0139]

[0140] Table 3 Preparation of quality control working solution

[0141]

[0142] 4.5 Preparation of plasma samples

[0143] Prepare the standard curve and quality control plasma samples according to Table 4 at yellow light 10-30 °C, mix well after preparation. The storage condition is -60 °C and below, in polypropylene tubes.

[0144] Table 4 Preparation of standard curve plasma samples

[0145]

[0146] Table 5 Preparation of quality control plasma samples

[0147]

[0148] 4.6 Preparation of internal standard stock solution and working solution

[0149] 4.6.1 Preparation of about 1.00 mg / mL Fluticasone Furoate-d3 Internal Standard Stock Solution

[0150] At 10-30 °C under yellow light, about 2 mg (1.8-2.2 mg) of Fluticasone Furoate-d3 standard substance was weighed into a polypropylene tube, a certain volume of dimethyl sulfoxide was added, and a 1.00 mg / mL stock solution was prepared. The stock solution was stored in a polypropylene tube at -30 to -10 °C. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0151] Volume = m x content ÷ concentration of stock solution

[0152] 4.6.2 Preparation of Vilanterol-d4 internal standard stock solution with a concentration of about 1.00 mg / mL

[0153] At 10-30 °C under yellow light, about 2 mg (1.8-2.2 mg) of Vilanterol-d4 Triphenylacetate standard substance was directly dispensed into a polypropylene tube, a certain volume of dimethyl sulfoxide was added, and a 1.00 mg / mL stock solution was prepared. The stock solution was stored in a polypropylene tube at -30 to -10 °C. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0154] Volume = m x content x (490.46 / 778.81) ÷ concentration of stock solution

[0155] 4.6.3 Preparation of Ulmepium-d10 internal standard stock solution with a concentration of about 1.00 mg / mL

[0156] At 10-30 °C under yellow light, 2 mg of Ulmepium-d10 standard substance was directly taken, a certain volume of dimethyl sulfoxide was added, and a 1.00 mg / mL stock solution was prepared. The stock solution was stored in a polypropylene tube at -30 to -10 °C. Record as ISSS-YyMmDd-x. When calculating the concentration, it needs to be converted according to the following formula:

[0157] Volume = m x purity x (438.49 / 538.49) ÷ concentration of stock solution

[0158] 4.6.4 Preparation of internal standard working solution

[0159] At 10-30 °C under yellow light, the mixed internal standard working solution was prepared according to the following table, vortexed and diluted with 50% acetonitrile aqueous solution.

[0160] Stored at -30 to -10 °C in a polypropylene tube.

[0161]

[0162] 5 Sample treatment

[0163] (1) All matrix samples were thawed at 10-30 °C under yellow light and vortexed well;

[0164] (2) Take 100 μL of plasma sample in a polypropylene tube or directly to the polypropylene tube containing 100 μL of plasma sample, except for the blank sample or ULOQ sample, add 30 μL of internal standard working solution (Fluticasone Furoate-d3 / Vilanterol-d4 / Ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL), add 30 μL of 50% acetonitrile solution to the blank sample;

[0165] (3) Add 600 μL of ethyl acetate solution and vortex well;

[0166] (4) Centrifuge at 4 °C, 18000 x g for 5 min;

[0167] (5) Take 500 μL of supernatant in a polypropylene tube and dry with nitrogen;

[0168] (6) After drying with nitrogen, add 70 μL of 50% acetonitrile solution and mix well;

[0169] (7) Centrifuge at 4 °C, 18000 x g for 1 min, take the supernatant solution for sample analysis.

[0170] 6 Preparation of samples for verification items (applicable to method validation, all under yellow light)

[0171] 6.1 Preparation of samples for selectivity and specificity

[0172] (1) Blank sample: take 6 different sources of blank plasma samples, process according to the steps under the item "5 sample processing";

[0173] (2) Zero sample: take 6 different sources of blank plasma samples, process according to the steps under the item "5 sample processing", and replace 30 μL of internal standard working solution (Fluticasone Furoate-d3 / Vilanterol-d4 / Ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL) in step (2) with 30 μL of ISWS1-YyMmDd-x, ISWS2-YyMmDd-x, ISWS3-YyMmDd-x, respectively, each prepared in parallel for 6 times;

[0174] (3) LLOQ sample: take 6 different sources of blank plasma, process according to the steps under the item "5 sample processing";

[0175] (4)ULOQ samples Take 6 different sources of blank plasma, parallel preparation of ULOQ1-YyMmDd-x, ULOQ2-YyMmDd-x, ULOQ3-YyMmDd-x samples each 6 copies, and according to the steps of "5 sample processing" item processing.

[0176] 6.2 Preparation of extraction recovery samples

[0177] Extraction samples: take 100 μL of low, medium and high concentration quality control samples in polypropylene tubes, respectively, according to the steps of "5 sample processing" item processing.

[0178] Non-extraction samples: (1) take 120 μL of blank Beagle dog plasma in polypropylene tubes, parallel preparation of 6 copies, add 36 μL of 50% acetonitrile aqueous solution, then add 720 μL of ethyl acetate, mix for 10 min;

[0179] (2) 4℃, 18000×g centrifugation for 5 min;

[0180] (3) take out 690 μL of supernatant, add 10 μL of low, medium or high concentration working solution, and then add 30 μL of internal standard working solution (Fluticasone Furoate-d3 / Vilanterol-d4 / Ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL);

[0181] (4) transfer 500 μL of (3) to polypropylene tubes, parallel preparation of 6 copies, nitrogen blowing dry;

[0182] (5) after nitrogen blowing dry, add 70 μL of 50% acetonitrile aqueous solution, mix for 10 min;

[0183] (6) 4℃, 18000×g centrifugation for 1 min, take 60 μL of solution for analysis.

[0184] 6.3 Preparation of whole blood stability samples

[0185] Take 5 μL of fluticasone furoate, vilanterol, umclidinium stock solution respectively in three polypropylene tubes, add 995 μL of 50% acetonitrile aqueous solution respectively to obtain intermediate solutions containing fluticasone furoate, vilanterol, umclidinium at a concentration of 5.00 μg / mL respectively; take 60 μL of fluticasone furoate, 30 μL of vilanterol, 9 μL of umclidinium intermediate solution in a polypropylene tube, add 901 μL of 50% acetonitrile aqueous solution to dilute to obtain a mixed solution containing fluticasone furoate, vilanterol, umclidinium at a concentration of 300 / 150 / 45.0 ng / mL; take 35 μL of the mixed solution in a polypropylene tube, add 3465 μL of freshly collected Beagle dog EDTA-K2 anticoagulant whole blood to prepare a high concentration sample of whole blood quality control; take 35 μL of WS-QC M working solution in a polypropylene tube, add 3465 μL of Beagle dog EDTA-K2 anticoagulant whole blood to prepare a low concentration sample of whole blood quality control. After gently inverting the whole blood sample up and down for 10 times, equilibrate at 37°C under the condition of 400 rpm for 10 min. The low and high concentration quality control samples are divided into two parts, each containing 1500 μL. One part is immediately centrifuged (4°C, 2200g) for 10 min, and the upper plasma is taken as the 0h sample and stored at yellow light at 10-30°C or below -60°C. The remaining 1 part of the whole blood sample is stored in the test environment (ice box) for about 4 h (which can be adjusted according to the actual situation, but should not be lower than the interval time from the collection of the whole blood to the centrifugation process), then centrifuged (4°C, 2200g) for 10 min. The obtained plasma and 0h plasma are treated according to the method under the "5 sample treatment" item, and then detected. Each of the low and high concentration quality control samples is processed in parallel for 5 times.

[0186] 6.4 Preparation of stability samples of stock solutions and working solutions

[0187] 6.4.1 Preparation method of stability samples of stock solutions

[0188] (1) Take 5 μL of fluticasone furoate, vilanterol, umclidinium stock solution in three polypropylene tubes, respectively, and add 995 μL of 50% acetonitrile aqueous solution to dilute to a solution containing fluticasone furoate, vilanterol, umclidinium at a concentration of 5.00 μg / mL respectively;

[0189] (2) Take 10 μL of 5.00 μg / mL umclidinium intermediate solution in a polypropylene tube, add 990 μL of 50% acetonitrile aqueous solution to dilute to a concentration of 50.0 ng / mL;

[0190] (3) Respectively, 6 μL, 3 μL, 90 μL of the solution of fluticasone furoate 5.00 μg / mL, vilanterol 5.00 μg / mL, ulmepium 50.0 ng / mL were taken in a polypropylene tube, 1401 μL of 50% acetonitrile aqueous solution was added, and diluted to 20.0 / 10.0 / 3.00 ng / mL of fluticasone furoate, vilanterol, ulmepium concentration mixed intermediate solution;

[0191] (4) 50 μL (3) was taken in a polypropylene tube, 150 μL of internal standard working solution (fluticasone furoate-d3 / vilanterol-d4 / ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL) was added, and 310 μL of 50% acetonitrile aqueous solution was added, and mixed well;

[0192] (5) 200 μL (4) was taken for sample analysis.

[0193] 6.4.2 Working solution stability sample preparation method:

[0194] (1) 25 μL of the highest point working solution of the calibration curve was taken in a polypropylene tube, 150 μL of internal standard working solution (fluticasone furoate-d3 / vilanterol-d4 / ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL) was added, and 335 μL of 50% acetonitrile aqueous solution was added, and mixed well for 5 min;

[0195] (2) 50 μL of the lowest point working solution of the calibration curve was taken in a polypropylene tube, 75 μL of internal standard working solution (fluticasone furoate-d3 / vilanterol-d4 / ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL) was added, and 130 μL of 50% acetonitrile aqueous solution was added, and mixed well for 5 min;

[0196] 200 μL (1) or (2) was taken for sample analysis.

[0197] 6.5 System suitability sample

[0198] The system suitability sample was prepared at LLOQ concentration at 10-30°C under yellow light. The system suitability sample can be prepared by the following method:

[0199] (1) Take 50 μL of the lower limit of quantification working solution in a polypropylene tube, add 150 μL of the internal standard working solution (Fluticasone Furoate-d3 / Vilanterol-d4 / Ulmepium-d10 = 4.00 / 1.00 / 1.00 ng / mL), and finally add 310 μL of 50% acetonitrile aqueous solution, mix well;

[0200] (2) Take 200 μL of (1) for sample analysis. The remaining analysis batch system suitability sample preparation is the same as the "5 sample processing" item.

[0201] 6.6 Preparation of dilution reliability samples

[0202] Take 5 μL of QC-dilution quality control plasma sample into a polypropylene tube, add 95 μL of blank matrix (diluted 20 times),

[0203] Prepare 5 in parallel, mix well, and process under the "5 sample processing" item.

[0204] 7. Data processing

[0205] Bias (%) = (measured value - theoretical value) ÷ theoretical value * 100%

[0206]

[0207] Accuracy is expressed as bias, and precision is expressed as relative standard deviation (RSD) or coefficient of variation (CV).

[0208] Peak area is reported as an integer, and full precision is involved in calculation; concentration retains three significant digits, bias, coefficient of variation (CV), and ratio (Ratio) retain one digit after the decimal point.

[0209] 8. Validation results

[0210] 8.1 Selectivity and specificity

[0211] Prepare 6 blank plasma (non-hemolytic and non-high-fat) samples (Blank) of different sources of Beagle dogs 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.

[0212] Prepare 6 ULOQ plasma samples containing only the analyte (without internal standard) in parallel, investigate the interference of the analyte on the internal standard, as well as the interference between the analytes.

[0213] The hemolytic matrix was prepared by adding 2% whole blood cell lysate into at least one source of blank plasma. Six blank plasma samples (Blank) were prepared in parallel using the hemolytic blank matrix to investigate the interference of hemolytic blank plasma to the analyte and internal standard.

[0214] The peak area of the analyte measured in the Blank and Zero samples compared to the mean peak area of the LLOQ analyte sample should not have significant (<20% of the analyte peak area) interference affecting the determination of the analyte. The peak area of the internal standard measured in the Blank and ULOQ samples compared to the mean peak area of the LLOQ sample should not have significant (<5% of the internal standard peak area) interference. The peak area of the interference measured in the ULOQ samples of different analytes should be less than 5% of the mean peak area of the analyte in the ULOQ sample of each other, and the peak area of the interference between internal standards should be less than 5% of the mean peak area of the internal standard in the LLOQ sample of each other.

[0215] The calculation formula is as follows:

[0216]

[0217]

[0218] The results show that: the endogenous substances in the blank plasma of the six different sources of Beagle dogs have no interference with the three test substances fluticasone furorate, vilanterol and ulmepium, and the isotope internal standards of the three test substances; the interference of Fluticasone Furorate-d3, Vilanterol-d4 and Ulmepium-d10 on fluticasone furorate is within 2.6%; Fluticasone Furorate-d3, Vilanterol-d4 and Ulmepium-d10 have no interference with vilanterol; the interference of Fluticasone Furorate-d3, Vilanterol-d4 and Ulmepium-d10 on ulmepium is within 7.5%; the interference of fluticasone furorate, vilanterol and ulmepium on Fluticasone Furorate-d3 is within 3.8%; the interference of fluticasone furorate, vilanterol and ulmepium on Vilanterol-d4 is within 3.5%; fluticasone furorate, vilanterol and ulmepium have no interference with Ulmepium-d10; the mutual interference among fluticasone furorate, vilanterol and ulmepium is within 0.1%; there is no interference among Fluticasone Furorate-d3, Vilanterol-d4 and Ulmepium-d10. The interference of the endogenous substances in the hemolyzed blank plasma on fluticasone furorate, vilanterol and ulmepium is within 2.7%, and the interference on Fluticasone Furorate-d3, Vilanterol-d4 and Ulmepium-d10 is within 3.5%. All meet the requirements of the plan. The typical chromatograms are shown in Figures 1-15 .

[0219] 8.2 Extraction recovery rate

[0220] Extraction samples: Prepare 6 QC samples of low, medium and high concentrations as extraction samples.

[0221] Unextracted samples: Take 6 blank matrices of different sources, add low, medium and high concentrations of test substances and internal standards after pretreatment as unextracted samples, 6 for each concentration.

[0222] The calculation formula is as follows:

[0223]

[0224] Compare the peak area of the test substance or internal standard of the extraction sample with the average value of the peak area of the test substance or internal standard of the unextracted sample, calculate the extraction recovery rate of the test substance or internal standard, and the precision (RSD) of the extraction recovery rate of the test substance or internal standard should be within 15%.

[0225] The results showed that the precision (RSD) of the extraction recovery of fluticasone fururate, vilanterol, ulmepium in all samples were within 9.6%, and the precision (RSD) of the extraction recovery of fluticasone fururate-d3, vilanterol-d4, ulmepium-d10 internal standard were within 5.4%. Both met the requirements of the plan.

[0226] 8.3 Matrix effect

[0227] 6 different sources / batches of matrix, each with 6 replicates of low or high concentration quality control samples, were analyzed with freshly prepared standard curve and quality control samples. It was required that for each source / batch of matrix, the mean accuracy should be within ± 15% of the labeled value, and the precision should not exceed 15%. Hemolytic matrix effect: 2% whole blood cell lysate was added to the blank plasma to prepare hemolytic matrix, and 6 low and high concentration quality control samples were prepared and analyzed with freshly prepared standard curve and quality control samples of normal matrix for hemolytic effect investigation. The precision (RSD) of each concentration should not exceed 15%, and the bias of the mean should be within ± 15%.

[0228] The results showed that for each source / batch of matrix, the bias of the mean of fluticasone fururate at low and high concentrations was between -1.2% and 2.5% and -5.8% and 1.2%, and the precision (RSD) was ≤ 4.7% and ≤ 5.8%; the bias of the mean of vilanterol at low and high concentrations was between -1.0% and 6.2% and 3.5% and 4.7%, and the precision (RSD) was ≤ 3.6% and ≤ 4.8%; the bias of the mean of ulmepium at low and high concentrations was between -3.8% and 1.3% and 3.6% and 5.8%, and the precision (RSD) was ≤ 6.7% and ≤ 3.2%; the precision (RSD) of fluticasone fururate at low and high concentrations in hemolytic matrix effect was 8.1% and 4.7%, and the bias of the mean was -2.3% and -4.6%; the precision (RSD) of vilanterol at low and high concentrations in hemolytic matrix effect was 3.8% and 2.3%, and the bias of the mean was -3.5% and 2.4%; the precision (RSD) of ulmepium at low and high concentrations in hemolytic matrix effect was 3.3% and 0.9%, and the bias of the mean was -1.8% and 2.1%; all met the requirements of the plan.

[0229] 8.4 Linear range of standard curve

[0230] Prepare two replicates of each of the standard curve samples containing eight concentrations to establish the standard curve. Respectively, with 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), the weighted least squares method (w = 1 / x2) is used for regression calculation to obtain the linear regression equation, which is the standard curve. The concentration of each standard curve sample is calculated by the regression equation, but the blank and zero concentration samples are not involved in the calculation, and the deviation [(calculated value-theoretical value) / theoretical value x 100%] of the back calculation concentration should be within ±15% (LLOQ is ±20%), and 75% of the samples and at least six concentration levels should meet the above requirements. The sample should be discarded and the regression calculation should be performed again.

[0231] The results showed that the plasma concentration of fluticasone propionate was linear in the range of 20.0-4000 pg / mL, the plasma concentration of vilanterol was linear in the range of 10.0-2000 pg / mL, and the plasma concentration of umclidinium was linear in the range of 3.00-600 pg / mL, and the results met the planned requirements. The typical standard curve of fluticasone propionate, vilanterol, and umclidinium in plasma is shown in Figures 16-18 .

[0232] 8.5 Residual

[0233] 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.

[0234] The results showed that fluticasone propionate, vilanterol, and umclidinium had no residual, and the residual of each isotope internal standard was within 0.1%. They all met the planned requirements.

[0235] 8.6 Accuracy, precision and sensitivity

[0236] Prepare six replicates of LLOQ, low, medium and high concentration quality control samples (QC) respectively, calculate the concentration of QC samples by the standard curve of the batch, and calculate the precision and accuracy of the method (prepare and determine three batches, and perform in three days). The within-batch and between-batch 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-batch and between-batch average value 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 the LLOQ sample should be ≥5.

[0237] The results show that the LLOQ batch precision (RSD) of fluticasone furoate, vilanterol, and umclidinium is within 20%, the mean accuracy (Bias) is within ±20%; the batch precision (RSD) of low, medium, and high concentration QC samples is within 15%, the mean accuracy (Bias) is within ±15%; the LLOQ batch precision (RSD) is within 20%, the mean accuracy (Bias) is within ±20%; the batch precision (RSD) of low, medium, and high concentration QC samples is within 15%, the mean accuracy (Bias) is within ±15%. All meet the plan requirements.

[0238] The signal-to-noise ratio (S / N) of LLOQ samples in the precision and accuracy of fluticasone furoate is ≥55; the signal-to-noise ratio (S / N) of LLOQ samples in the precision and accuracy of vilanterol is ≥32; the signal-to-noise ratio (S / N) of LLOQ samples in the precision and accuracy of umclidinium is ≥64, all meeting the plan requirements.

[0239] 8.7 Dilution reliability

[0240] The reliability of diluting blank plasma by a corresponding multiple of the plasma sample higher than the upper limit of quantification is investigated. The concentration mean value after multiplying the measured value by the dilution multiple should be within ±15% of the prepared concentration value, and the precision (RSD) should be within 15%.

[0241] The results show that the mean accuracy (Bias) of fluticasone furoate plasma sample diluted by 20 times is 1.2%, and the precision (RSD) is 6.6%; the mean accuracy (Bias) of vilanterol plasma sample diluted by 20 times is 4.9%, and the precision (RSD) is 2.6%; the mean accuracy (Bias) of umclidinium plasma sample diluted by 20 times is 3.3%, and the precision (RSD) is 2.2%.

[0242] 8.8 Stability of stock solution and working solution

[0243] The stability of fluticasone furoate, vilanterol, and umclidinium stock solutions is investigated at 10-30°C under yellow light for about 52.5 hours; the stability of fluticasone furoate, vilanterol, and umclidinium stock solutions is investigated at -30 to -10°C for 38 days. The stability of fluticasone furoate, vilanterol, and umclidinium working solutions is investigated at 10-30°C under yellow light for about 52.5 hours; the stability of fluticasone furoate, vilanterol, and umclidinium working solutions is investigated at -30 to -10°C for 38 days.

[0244] The precision (RSD) of the average peak area of the stock solution or working solution stored for a period of time compared with the average peak area of the freshly prepared solution is required to be within 10%, and the deviation (Bias) of the two responses is required to be within ±10%.

[0245] The results show that the fluticasone furoate, vilanterol, and umclidinium stock solutions are stable at 10-30 °C for about 52.5 hours under yellow light; the fluticasone furoate, vilanterol, and umclidinium stock solutions are stable at -30 to -10 °C for about 38 days. The fluticasone furoate, vilanterol, and umclidinium working solutions are stable at 10-30 °C for about 52.5 hours under yellow light; the fluticasone furoate, vilanterol, and umclidinium working solutions are stable at -30 to -10 °C for about 38 days. The precision (RSD) of all the stability samples is within 10%, and the bias of the average peak area is within ±10%, and the results meet the requirements.

[0246] 8.9 Stability of the samples after treatment

[0247] The low and high concentration samples (n=5) are stored in the sample manager (6 °C) for about 62.4 hours after treatment, and then injected (the storage time is not less than the actual storage time of the samples in the environment). The precision (RSD) of fluticasone furoate at each concentration is not more than 15% by using the fresh standard curve, and the bias (Bias) should be within ±15%. The low and high concentration samples (n=5) are stored in the sample manager (6 °C) for about 68.3 hours after treatment, and then injected (the storage time is not less than the actual storage time of the samples in the environment). The precision (RSD) of vilanterol and umclidinium at each concentration is not more than 15% by using the fresh standard curve, and the bias (Bias) should be within ±15%.

[0248] The results show that the samples are stable in the sample manager (6 °C) for about 62.4 hours after treatment. The precision (RSD) of fluticasone furoate at low and high concentrations is 9.9% and 7.2%, respectively, and the bias (Bias) is -9.2% and 3.3%, respectively. The samples are stable in the sample manager (6 °C) for about 68.3 hours after treatment. The precision (RSD) of vilanterol at low and high concentrations is 7.7% and 1.3%, respectively, and the bias (Bias) is -0.4% and 8.8%, respectively. The samples are stable in the sample manager (6 °C) for about 68.3 hours after treatment. The precision (RSD) of umclidinium at low and high concentrations is 9.2% and 1.5%, respectively, and the bias (Bias) is 5.5% and 9.5%, respectively. All meet the planned requirements.

[0249] 8.10 Injection reproducibility

[0250] The precision (RSD) of each concentration of fluticasone furoate should not be more than 15% and the bias should be within ± 15% after the sample was stored in the sample manager (6°C) for about 51.5 hours after pre-treatment. Meanwhile, 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 should be within ± 15%. The precision (RSD) of each concentration of vilanterol and umeliene should not be more than 15% and the bias should be within ± 15% after the sample was stored in the sample manager (6°C) for about 42.4 hours after pre-treatment. Meanwhile, 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 should be within ± 15%.

[0251] The results showed that the precision (RSD) of fluticasone furoate at low, medium and high concentrations were all within 3.7% and the bias was between -5.2% and 2.3% after the sample was stored in the sample manager (6°C) for about 51.5 hours after pre-treatment. The precision (RSD) of vilanterol at low, medium and high concentrations were all within 3.6% and the bias was between -2.1% and 1.2% after the sample was stored in the sample manager (6°C) for about 42.4 hours after pre-treatment. The precision (RSD) of umeliene at low, medium and high concentrations were all within 5.1% and the bias was between -3.3% and 1.1% after the sample was stored in the sample manager (6°C) for about 42.4 hours after pre-treatment, all of which met the requirements of the plan.

[0252] 8.11 Stability of whole blood

[0253] The bias of the mean of the peak area ratio of the analyte to the internal standard of the sample stored in the ice box for about 4.0 hours to that of the sample immediately after separation of the plasma (0h) should be within ± 15% and the precision (RSD) of the peak area ratio should be within 15% after the stability of the whole blood sample under ice box conditions for about 4.0 hours was investigated.

[0254] The results showed that the bias of the mean of the peak area ratio of fluticasone furoate of the low and high concentration whole blood samples stored in the ice box for about 4.0 hours to that of the sample at 0h was -2.5% and -1.1% respectively, the bias of the mean of the peak area ratio of vilanterol was -0.9% and 0.8% respectively, and the bias of the mean of the peak area ratio of umeliene was 1.2% and -0.1% respectively. The precision (RSD) of the peak area ratio of all whole blood samples was within 2.5%, which met the requirements of the plan.

[0255] 8.12 Batch maximum sample number

[0256] QC samples of low, medium and high concentrations (25 replicates) were prepared and injected repeatedly. The maximum number of injections that could be carried out by a batch of analysis was determined using QC samples, provided that the standard curve met the requirements.

[0257] The precision (RSD) of QC samples of the same concentration should not exceed 15%, the bias of the mean should be within ±15%, and at least two-thirds of the QC samples should meet the requirements. At least 50% of the QC samples at the same concentration level should meet the accuracy (Bias) requirements, which should be within ±15%.

[0258] The results showed that the precision (RSD) of the low, medium and high concentration QC samples of fluticasone propionate, vilanterol and umclidinium was within 6.7%, and the bias of the mean was between -2.8% and 9.6%, meeting the planned requirements. The total number of samples in a batch (including system suitability, standard curve, residual, and QC samples) was stable within 177 samples.

[0259] 8.13 Sample stability before processing

[0260] The stability of low and high concentration plasma samples (n=5) was investigated, including the stability of plasma samples at 10-30°C for about 22.4 hours, the stability after 5 freeze-thaw cycles, and the stability after long-term storage at -60°C or below for 44 days and 102 days. The concentration of the analyte in the samples was calculated based on the standard curve on the same day, and the precision (RSD) of each concentration should not exceed 15%, and the bias (Bias) of the mean should be within ±15%.

[0261] The results showed that the low and high concentration plasma samples were stable when placed at 10-30°C for about 22.4 hours before processing, the precision (RSD) of fluticasone propionate, vilanterol and umclidinium was within 8.1%, and the bias (Bias) of the mean was between -2.3% and 9.6%. The low and high concentration plasma samples were stable after 5 freeze-thaw cycles, the precision (RSD) of fluticasone propionate, vilanterol and umclidinium was within 2.5%, and the bias (Bias) of the mean was between 1.5% and 9.4%. The low and high concentration plasma samples were stable after long-term storage at -60°C or below for 44 days, the precision (RSD) of fluticasone propionate, vilanterol and umclidinium was within 5.1%, and the bias (Bias) of the mean was between 0.3% and 5.9%. The low and high concentration plasma samples were stable after long-term storage at -60°C or below for 102 days, the precision (RSD) of fluticasone propionate, vilanterol and umclidinium was within 7.6%, and the bias (Bias) of the mean was between -1.6% and 2.1%. All met the planned requirements.

[0262] 8.14 Analysis batch evaluation

[0263] 8.14.1 Quality control samples

[0264] Quality control samples and standard curve samples are prepared with different stock solutions. Low, medium and high concentrations of quality control samples are determined, at least two 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, the batch result is rejected.

[0265] The results show that the low, medium and high concentration quality control samples of fluticasone furoate, vilanterol and umclidinium of all qualified analysis batches meet the acceptance criteria, meeting the above requirements.

[0266] 8.14.2 Blank sample and zero concentration sample

[0267] The sample peak area measured by the blank sample of the analysis batch compared with the average value of the sample peak area of the qualified standard curve lowest point sample needs to be <20% of the peak area of the measured substance, and the sample peak area measured by the zero concentration sample compared with the average value of the sample peak area of the qualified standard curve lowest point sample needs to be <20% of the peak area of the measured substance.

[0268] The results show that the peak area of fluticasone furoate, vilanterol and umclidinium measured by the blank sample is within 20% of the average value of the peak area of fluticasone furoate, vilanterol and umclidinium of the qualified standard curve lowest point sample; the peak area of fluticasone furoate, vilanterol and umclidinium measured by the zero sample is within 20% of the average value of the peak area of fluticasone furoate, vilanterol and umclidinium of the qualified standard curve lowest point sample, all meeting the above requirements

[0269] 9. Conclusion

[0270] 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 fluticasone furoate, vilanterol and umclidinium in plasma, and is suitable for preclinical pharmacokinetic and toxicokinetic studies.

Claims

1. An LC-MS / MS analytical method for the determination of fluticasone propionate, vilanterol and umclidinium concentrations in plasma, characterized in that: It comprises the following steps: (1) Establishment of standard curve a. Preparation of standard curve samples of serial concentrations: Take fluticasone furoate, vilanterol trifenatate and umclidinium bromide, add dimethyl sulfoxide to dissolve, and then dilute with acetonitrile aqueous solution to serial concentrations; Take the mixed solution of serial concentrations respectively, add matrix, internal standard working solution and ethyl acetate in turn, mix well, centrifuge, take supernatant, blow dry, dissolve with acetonitrile aqueous solution, centrifuge, and take supernatant, to obtain; b. Take the standard samples of serial concentrations respectively, inject into LC-MS / MS instrument, measure peak area, and obtain standard curves of fluticasone furoate, vilanterol and umclidinium; Chromatographic conditions are as follows: chromatographic column: BEH silica gel particles as filler; formic acid-ammonium acetate aqueous solution as mobile phase A, formic acid acetonitrile solution as mobile phase B; Gradient elution program for detecting umclidinium and vilanterol is as follows: Gradient elution program for detecting fluticasone furoate is as follows: Mass spectrometry conditions: ion source is electrospray ionization source; scanning type: multiple ion reaction monitoring; (2) Determination of drug content in sample to be tested: c. Processing of biological sample Take the sample to be tested, add internal standard working solution and ethyl acetate, mix well, centrifuge, take supernatant, blow dry, dissolve with acetonitrile aqueous solution, mix well, centrifuge, and take supernatant, to obtain; d. Determination of biological sample Take supernatant obtained in step c, inject into LC-MS / MS instrument, detect under the same conditions in step b, and obtain fluticasone furoate, vilanterol and umclidinium content in the sample to be tested according to the standard curve in step (1).

2. The LC-MS / MS analytical method according to claim 1, characterized in that: During the determination of content in step (2), a quality control procedure is also included, and the specific steps are as follows: Take matrix, mix with acetonitrile aqueous solution and ethyl acetate, centrifuge, take supernatant, blow dry, dissolve with acetonitrile aqueous solution, mix well, centrifuge, take supernatant, to obtain blank sample test solution; take dimethyl sulfoxide solution containing fluticasone furoate, vilanterol and umclidinium obtained in step a, dilute with acetonitrile aqueous solution, to obtain quality control sample, take quality control sample, mix with internal standard working solution and ethyl acetate, centrifuge, take supernatant, blow dry, dissolve with acetonitrile aqueous solution, mix well, centrifuge, and take supernatant, to obtain quality control sample test solution; Take blank sample test solution and quality control sample test solution respectively, inject into LC-MS / MS instrument, and monitor interference existing in the determination process; The volume ratio of the matrix to acetonitrile aqueous solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution for dissolving is 100:30:600:500:70; The volume ratio of the quality control sample to internal standard working solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution is 100:30:600:500:

70.

3. The LC-MS / MS analytical method according to claim 1, characterized in that: The volume ratio of the mixed solution of serial concentrations, matrix, internal standard solution, ethyl acetate, supernatant for blowing dry and acetonitrile aqueous solution in step a is 10:90:30:600:500:70; the concentration of fluticasone furoate after adding matrix to the mixed solution of serial concentrations is 20-4000 pg / mL, the concentration of vilanterol is 10-2000 pg / mL, and the concentration of umclidinium is 3-600 pg / mL.

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

5. The LC-MS / MS analytical method according to claim 1, wherein: The mixing is vortex mixing; the temperature of centrifugation is 4℃, the speed is 18000xg, and the time is 1-5min.

6. The LC-MS / MS analytical method according to claim 1, wherein: The internal standard working solution is an acetonitrile aqueous solution containing 4ng / mL Fluticasone Furoate-d3, 1.0ng / mL Vilanterol-d4 and 1.0ng / mL Ulmepium-d10 standard substance.

7. The LC-MS / MS analytical method according to claims 1 to 3 or 6, characterized in that: The concentration of the acetonitrile aqueous solution is 50%.

8. The LC-MS / MS analytical method of claim 1, wherein: The chromatographic column is ACQUITY UPLC BEH C18 1.7um 2.1*100mm Column; the injection volume is 5-10uL; the column temperature is 50℃; the mobile phase A is 0.2% formic acid-5mM ammonium acetate aqueous solution, and the mobile phase B is 0.2% formic acid acetonitrile solution.

9. The LC-MS / MS analytical method according to claim 1, characterized in that: In the mass spectrometry condition of step b, the polarity mode is positive ion mode; curtain gas: 30psi; Collision gas: 9psi; ion source gas 1: 50psi; ion source gas 2: 50psi; inlet potential: 10V; Collision cell exit potential: 10V.

10. The LC-MS / MS analysis method of claim 9, wherein: In the mass spectrometry condition, the ion spray voltage when detecting ulmepium and vilanterol is 5500V; Temperature: 550℃; vilanterol detection ion pair m / z 486.2→m / z 159.0, declustering voltage 90V, collision energy 35eV; ulmepium detection ion pair m / z 428.3→m / z 96.2, declustering voltage 120V, collision energy 80eV; vilanterol-d4 detection ion pair m / z 490.2→m / z 159.0, declustering voltage 60V, collision energy 45eV; ulmepium-d10 detection ion pair m / z 438.2→m / z 96.2, declustering voltage 85V, collision energy 80eV; The ion spray voltage when detecting fluticasone furfuryl alcohol is 5000V; temperature: 380℃; fluticasone furfuryl alcohol detection ion pair m / z 539.1→m / z 313.3, declustering voltage 80V, collision energy 21eV; fluticasone furfuryl alcohol-d3 detection ion pair m / z 542.3→m / z 313.0, declustering voltage 80V, collision energy 20eV. The ion spray voltage when detecting fluticasone furfuryl alcohol is 5000V; temperature: 380℃; fluticasone furfuryl alcohol detection ion pair m / z 539.1→m / z 313.3, declustering voltage 80V, collision energy 21eV; fluticasone furfuryl alcohol-d3 detection ion pair m / z 542.3→m / z 313.0, declustering voltage 80V, collision energy 20eV.

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