Method for Detecting the Concentrations of Xanofivir and Ritonavir in Blood and Alveolar Epithelial Lining Fluid

Abstract

This application belongs to the field of analytical detection technology, and specifically relates to a method for detecting the concentrations of Xanofivir and Ritonavir in blood and alveolar epithelial lining fluid. This method includes preprocessing blood and bronchoalveolar lavage fluid samples, then establishing a liquid chromatography-tandem mass spectrometry measurement method, and further calculating the concentrations of Xanofivir and Ritonavir in blood and alveolar epithelial lining fluid. This method is characterized by being fast and accurate, laying a foundation for drug monitoring, safe use, and personalized precision treatment, etc.

Description

Technical Field

[0001] This application relates to the field of analytical detection, and specifically relates to a method for detecting the concentrations of simnotrelvir and ritonavir in blood and alveolar epithelial lining fluid. Background Art

[0002] Simnotrelvir, chemical formula: C 22 H 30 F3N5O4S2, Ritonavir, chemical formula: C 37 H 48 N6O5S2. Simnotrelvir tablets and ritonavir tablets are packaged in combination, and their trade name is Xiannuoxin.

[0003] As a new drug, it is necessary to establish a method for detecting the drug concentration in blood and alveolar epithelial lining fluid for Xiannuoxin. Summary of the Invention

[0004] In view of the above problems, this application provides a method for detecting the concentrations of simnotrelvir and ritonavir in blood and alveolar epithelial lining fluid, and this detection method is fast and accurate.

[0005] On the one hand, this application provides a method for detecting the concentrations of simnotrelvir and ritonavir in blood and alveolar epithelial lining fluid, and this detection method includes the following processes:

[0006] Establishing a standard curve: providing a standard curve working solution, a level quality control working solution, and an internal standard solution, and detecting the standard curve working solution by liquid chromatography - mass spectrometry, and establishing a standard curve according to the detection results; the standard curve includes a simnotrelvir standard curve, a ritonavir standard curve, and a urea standard curve;

[0007] Pretreatment: providing a sample to be tested, the sample to be tested includes the blood to be tested and the bronchoalveolar lavage fluid to be tested, adding an internal standard and a precipitant to the blood to be tested and the bronchoalveolar lavage fluid to be tested respectively, and centrifuging to obtain each supernatant as an injection sample;

[0008] Detecting the concentration: detecting the injection sample by liquid chromatography - mass spectrometry, and determining the concentrations of simnotrelvir, ritonavir, and urea in the blood to be tested and the bronchoalveolar lavage fluid to be tested according to the detection results and the standard curve;

[0009] According to the concentrations of simnotrelvir, ritonavir, and urea in the blood to be tested and the bronchoalveolar lavage fluid to be tested, the urea dilution method is used to calculate the concentrations of simnotrelvir and ritonavir in the alveolar epithelial lining fluid.

[0010] In some embodiments, the precipitant includes acetonitrile;

[0011] The volume ratio between the sample to be tested and the precipitant is 1:(1 - 3).

[0012] In some embodiments, the liquid chromatography conditions include:

[0013] Mobile phase: including phase A and phase B, the volume ratio of phase A to phase B is (40 - 60):(60 - 40);

[0014] Phase A includes an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L - 12 mmol / L;

[0015] Phase B includes a mixed solution of acetonitrile and isopropanol, and the volume ratio of acetonitrile to isopropanol is (40 - 60):(60 - 40);

[0016] Isocratic elution: the flow rate of the mobile phase is 0.3 mL / min - 0.8 mL / min.

[0017] In some embodiments, the liquid chromatography conditions include:

[0018] The analytical chromatographic column used includes Phenomenex Kinetex F5;

[0019] The column temperature of the analytical chromatographic column is 35°C - 40°C.

[0020] In some embodiments, the mass spectrometry conditions include:

[0021] The nebulization temperature is 180°C - 220°C;

[0022] The ion spray voltage is 4500 V (MRM+) and -4500 V (MRM-);

[0023] The nebulizing gas is 45 psi - 55 psi, the auxiliary heating gas is 55 psi - 65 psi, the curtain gas is 35 psi - 45 psi, and the collision gas is 8 psi - 10 psi.

[0024] In some embodiments, the internal standard includes Xanofos - D4, Ritonavir - D6 and urea - 13 C 15 N2.

[0025] In some embodiments, the injected sample includes the internal standard.

[0026] In some embodiments, the blood sample to be tested includes plasma or serum.

[0027] In some embodiments, the internal standard solution includes Xanofos - D4 solution, Ritonavir - D6 solution and urea - 13 C 15N2 solution;

[0028] The Xanofivir-D4 solution is obtained by diluting the Xanofivir-D4 stock solution with an aqueous methanol solution; the Xanofivir-D4 stock solution is a dimethyl sulfoxide solution of Xanofivir-D4.

[0029] The Ritonavir-D6 solution is obtained by diluting the Ritonavir-D6 stock solution with an aqueous methanol solution.

[0030] The urea- 13 C 15 N2 solution is obtained by diluting the urea- 13 C 15 N2 stock solution with pure water.

[0031] The concentration of Xanofivir-D4 in the internal standard solution is 50 μg / mL, the concentration of Ritonavir-D6 is 0.5 μg / mL, and the concentration of urea- 13 C 15 N2 is 100 μg / mL.

[0032] In some embodiments, the volume ratio between the test sample and the internal standard solution is (50-150):10.

[0033] In some embodiments, the urea dilution method includes the following relationship:

[0034] [D] ELF =[D] BALF ×(V BALF / V ELF )=[D] BALF ×([U] ELF / [U] BALF )=[D] BALF ×([U] serum / [U] BALF );

[0035] V ELF =V BALF ×[U] BALF / [U] ELF ;

[0036] V ELF refers to the volume of alveolar epithelial lining fluid, V BALF refers to the volume of bronchoalveolar lavage fluid, [U] BALF refers to the urea concentration in bronchoalveolar lavage fluid, [U] ELF refers to the urea concentration in alveolar epithelial lining fluid;

[0037] [D] BALF refers to the drug concentration in bronchoalveolar lavage fluid, [U] serumRefers to the urea concentration in the blood.

[0038] In some embodiments, the standard curve working solution is obtained by adding PBS buffer to standard solutions of a series of concentrations respectively;

[0039] The series of concentrations includes at least 8;

[0040] In the standard curve working solution, the concentration of Xanofivir is 2.5 ng / mL to 5000 ng / mL, the concentration of Ritonavir is 0.1 ng / mL to 1000 ng / mL, and the concentration of urea is 1 μg / mL to 1000 μg / mL.

[0041] The above description is only an overview of the technical solution of this application. In order to be able to understand the technical means of this application more clearly, it can be implemented according to the content of the specification. And in order to make the above and other purposes, features and advantages of this application more obvious and understandable, the following specifically illustrates the specific embodiments of this application.

[0042] The beneficial technical effects of this application:

[0043] This application utilizes the high separation performance of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry to establish a liquid chromatography-tandem mass spectrometry quantitative analysis method, which can quickly, simply and accurately detect the concentrations of Xanofivir, Ritonavir and urea in blood and bronchoalveolar lavage fluid, and then can calculate the concentrations of Xanofivir and Ritonavir in alveolar epithelial lining fluid, realizing the monitoring of Xanofivir and Ritonavir therapeutic drugs, and laying a foundation for drug monitoring, safe use and personalized precision treatment, etc. Description of the Drawings

[0044] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of this application. And throughout the drawings, the same reference numerals are used to represent the same components. In the drawings:

[0045] Figure 1 Schematic diagram of the calibration curve of Xanofivir of this application;

[0046] Figure 2 Schematic diagram of the calibration curve of Ritonavir of this application;

[0047] Figure 3 Schematic diagram of the calibration curve of urea of this application;

[0048] Figure 4 Is the mass peak of the analyte Xanofivir at the lowest point STD1 (i.e., the standard curve working solution) of the standard curve in some embodiments of this application Figure 4A) The chromatogram of the internal standard peak of Xiannuotewi-D4 ( Figure 4 B);

[0049] Figure 5 This is the chromatogram of the peak of ritonavir, the analyte in the lowest point STD1 (i.e., the working solution of the standard curve) of the standard curve in some embodiments of this application. ( Figure 5 A) The chromatogram of the internal standard peak of ritonavir-D6 ( Figure 5 B);

[0050] Figure 6 This is the chromatogram of the peak of urea, the analyte in the lowest point STD1 (i.e., the working solution of the standard curve) of the standard curve in some embodiments of this application. ( Figure 6 A) and urea- 13 C 15 The chromatogram of the internal standard peak of N2 ( Figure 6 B);

[0051] Figure 7 : Figure 7 A is the chromatogram of the internal standard peak of Xiannuotewi-D4 in the STD1 sample under the condition that mobile phase A is 20 mM ammonium acetate aqueous solution and mobile phase B is a mixed solution of acetonitrile:isopropanol (50:50); Figure 7 B is the chromatogram of the internal standard peak of Xiannuotewi-D4 in the STD1 sample under the condition that mobile phase A is 10 mM ammonium acetate aqueous solution and mobile phase B is a mixed solution of acetonitrile:isopropanol (50:50);

[0052] Figure 8 : Figure 8 A is the chromatogram of the internal standard peak of ritonavir-D6 in the STD1 sample under the condition that mobile phase A is 20 mM ammonium acetate aqueous solution and mobile phase B is a mixed solution of acetonitrile:isopropanol (50:50); Figure 8 B is the chromatogram of the internal standard peak of ritonavir-D6 in the STD1 sample under the condition that mobile phase A is 10 mM ammonium acetate aqueous solution and mobile phase B is a mixed solution of acetonitrile:isopropanol (50:50);

[0053] Figure 9 : When 10 mM ammonium acetate aqueous solution is used as mobile phase A and a mixed solution of acetonitrile:isopropanol (50:50) is used as mobile phase B, and the chromatographic column is Kinetex F5 column (3 mm×100 mm, 2.6 μm, Phenomenex), isocratic elution is carried out with 40% mobile phase A and 60% mobile phase B, and the flow rate is 0.5 ml / min, the chromatograms of Xiannuotewi ( Figure 9 A), Xiannuotewi-D4 ( Figure 9 B);

[0054] Figure 10: The mobile phase A was an aqueous solution of 10 mM ammonium acetate and the mobile phase B was a mixed solution of acetonitrile: isopropanol (50:50). The chromatographic column was a Kinetex F5 column (3 mm × 100 mm, 2.6 μm, Phenomenex). Isocratic elution was performed with 40% mobile phase A and 60% mobile phase B at a flow rate of 0.5 ml / min. The spectra of ritonavir ( Figure 10 A) and ritonavir-D6 in the QCM sample ( Figure 10 B);

[0055] Figure 11 : The mobile phase A was an aqueous solution of 10 mM ammonium acetate and the mobile phase B was a mixed solution of acetonitrile: isopropanol (50:50). The chromatographic column was a Kinetex F5 column (3 mm × 100 mm, 2.6 μm, Phenomenex). Isocratic elution was performed with 40% mobile phase A and 60% mobile phase B at a flow rate of 0.5 ml / min. The spectra of urea ( Figure 11 A) and urea- 13 C 15 N 2 in the QCM sample ( Figure 11 B);

[0056] Figure 12 : When the chromatographic column was an XBridge C18 column (2.1 × 100 mm, 3.5 μm, Waters), the spectra of nirmatrelvir ( Figure 12 A) and nirmatrelvir-D4 in the QCM sample ( Figure 12 B);

[0057] Figure 13 : When the chromatographic column was an XBridge C18 column (2.1 × 100 mm, 3.5 μm, Waters), the spectra of ritonavir ( Figure 13 A) and ritonavir-D6 in the QCM sample ( Figure 13 B);

[0058] Figure 14 : When the chromatographic column was an XBridge C18 column (2.1 × 100 mm, 3.5 μm, Waters), the spectra of urea ( Figure 14 A) and urea- 13 C 15 N2 in the QCM sample ( Figure 14 B);

[0059] Figure 15 : When the chromatographic column was an XBridge BEH C8 column (2.1 × 100 mm, 2.5 μm, Waters), the spectra of nirmatrelvir ( Figure 15A), the spectrum of Simeprevir-D4 ( Figure 15 B);

[0060] Figure 16 : When the chromatographic column is XBridge BEH C8 column (2.1×100mm, 2.5μm, Waters), ritonavir in the QCM sample ( Figure 16 A), the spectrum of Ritonavir-D6 ( Figure 16 B);

[0061] Figure 17 : When the chromatographic column is XBridge BEH C8 column (2.1×100mm, 2.5μm, Waters), urea in the QCM sample ( Figure 17 A), urea- 13 C 15 N2 spectrum ( Figure 17 B).

[0062] Among them, Figures 1 to 3 the ordinate of the coordinate axis is the peak area ratio, and the abscissa is the concentration; Figures 4 to 17 the ordinate of the coordinate axis is the signal intensity, and the abscissa is the retention time. Specific embodiments

[0063] Hereinafter, the embodiments of the method for detecting the concentrations of simeprevir and ritonavir in blood and alveolar epithelial lining fluid of the present application will be specifically described in detail with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually identical structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0064] The "ranges" disclosed in this application are defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The ranges defined in this way can include or exclude the end values and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a specific parameter, ranges of 60 to 110 and 80 to 120 are also contemplated. In addition, if the minimum range values of 1 and 2 are listed, and if the maximum range values of 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise stated, the numerical range "a to b" represents an abbreviated representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "0 to 5" means that all real numbers between "0 to 5" are fully listed herein, and "0 to 5" is only an abbreviated representation of these numerical combinations. Additionally, when stating that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0065] If there is no special instruction, all embodiments and optional embodiments of this application can be combined with each other to form a new technical solution.

[0066] If there is no special instruction, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0067] If there is no special instruction, all steps of this application can be carried out sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method can include steps (a) and (b) carried out sequentially, or can also include steps (b) and (a) carried out sequentially. For example, it is mentioned that the method may further include step (c), which means that step (c) can be added to the method in any order. For example, the method can include steps (a), (b), and (c), or can also include steps (a), (c), and (b), or can also include steps (c), (a), and (b), etc.

[0068] If there is no special instruction, the "including" and "comprising" mentioned in this application mean open-ended or can also be closed-ended. For example, the "including" and "comprising" can mean that other components not listed can also be included or comprised, or can also mean only including or comprising the listed components.

[0069] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0070] Unless otherwise specified, in this application, terms such as "first", "second", etc. are only used to distinguish different objects, and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity, specific order, or primary-secondary relationship of the indicated technical features.

[0071] Unless otherwise specified, in this application, the term "plural" means two or more (including two). Similarly, "multiple groups" means two or more groups (including two groups), and "multiple pieces" means two or more pieces (including two pieces).

[0072] Unless otherwise specified, for technical terms such as "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., the indicated orientation or positional relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the embodiments of this application and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation on the embodiments of this application.

[0073] Unless otherwise specified, unless otherwise clearly defined and limited, technical terms such as "install", "connect", "join", "fix", etc. should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or integrated; it can also be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and can be the communication inside two elements or the interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of this application can be understood according to specific circumstances.

[0074] Simnotrelvir, chemical formula: C 22 H 30 F3N5O4S2, Ritonavir, chemical formula: C 37 H 48 N6O5S2, The combination package of Simnotrelvir tablets and Ritonavir tablets, and its trade name is Xiannuoxin.

[0075] 3CLpro (3-chymotrypsin-like cysteine protease), also known as main protease (Mpro), is a three-domain (domains I to III) cysteine protease composed of 306 amino acids. 3CLpro plays a crucial role in coronavirus replication and maturation and is highly conserved. Inhibiting 3CLpro can effectively block viral RNA replication and transcription, thereby blocking virus proliferation. The ongoing COVID-19 (novel coronavirus infection) pandemic has produced multiple variants, characterized by enhanced transmissibility, increased disease severity, immune escape, decreased neutralization of antibodies in vaccinated individuals, and increased susceptibility to reinfection. It is estimated that at least 65 million people globally suffer from long-term COVID-19, and more than 200 symptoms affecting multiple organ systems have been identified. Small molecule drugs have received extensive attention due to their cost-effectiveness, ease of transportation, and strong stability, making them very suitable for clinical use as a key component against COVID-19.

[0076] Xinlitaiwei is a highly active, oral, and low-toxic anti-COVID-19 drug that can not only inhibit wild-type 3CLpro but also maintain inhibitory activity against variant strains. Xinlitaiwei is mainly metabolized by cytochrome P450 3A (CYP3A). Ritonavir is a pharmacokinetic enhancer that inhibits CYP3A and can be co-administered to maximize the likelihood of reaching effective concentrations. Therefore, the combination of Xinlitaiwei and low-dose ritonavir can slow down the metabolism of Xinlitaiwei in the body and help exert its antiviral effect.

[0077] Therapeutic drug monitoring (TDM), that is, detecting the drug concentration in the blood, is of great significance in clinical treatment. TDM can guide clinicians to adjust the dosing regimen according to the changes in the patient's blood drug concentration and pharmacokinetic parameters. This helps improve the efficacy of the drug while reducing or avoiding drug toxic reactions. TDM is particularly important for drugs with a narrow therapeutic index and drugs whose poisoning symptoms are easily confused with the disease itself. Through TDM, clinicians can more accurately determine the dosing dose and frequency, thereby improving the efficiency and success rate of treatment.

[0078] Alveolar epithelial lining fluid (ELF) refers to the fluid between alveolar epithelial cells. It is located within the alveolar lumen and serves as the interfacial fluid between alveolar epithelial cells and the alveolar cavity. For pulmonary infections, whether the anti-infective drugs can reach effective concentrations in the alveolar epithelial lining fluid affects their actual clinical efficacy and the prognosis of the patient's condition. An increasing number of studies have begun to focus on the drug concentration in the target tissue during the treatment process and attempt to optimize the drug administration regimen and predict the clinical efficacy by measuring the actual drug concentration at the site of infection, in order to achieve individualized precision drug therapy and ultimately the goal of precision medicine.

[0079] Currently, the most commonly used technique for obtaining alveolar epithelial lining fluid samples to evaluate the drug concentration in ELF is bronchoalveolar lavage. By using liquid chromatography-tandem mass spectrometry (LC-MS / MS), the drug concentration in the bronchoalveolar lavage fluid (BALF) of patients after drug administration is measured. According to the urea dilution method, the actual drug concentration in ELF can be calculated, that is, by detecting the urea concentrations in the patient's alveolar lavage fluid and serum, as well as the concentrations of xiannotew and ritonavir in the alveolar lavage fluid, the concentrations of xiannotew and ritonavir in the alveolar epithelial lining fluid can be obtained.

[0080] In the urea dilution method, urea (with the molecular formula CH4N2O), as a non-polar small molecule, can freely pass through the alveolar wall. Thus, it is considered that the urea concentrations in the blood and ELF are the same. Then, by measuring the urea concentration in the blood and the urea concentration in the BALF, and using this as the dilution factor, the actual drug concentration in ELF can be calculated.

[0081] V ELF =V BALF ×[U] BALF / [U] ELF

[0082] Where V ELF is the volume of ELF, V BALF is the volume of BALF, [U] BALF is the urea concentration in BALF, [U] ELF is the urea concentration in ELF.

[0083] The drug concentration in ELF ([D] ELF ) is calculated as follows:

[0084] [D] ELF =[D] BALF ×(V BALF / V ELF )

[0085] =[D] BALF×([[U]] ELF / [[U]] BALF )

[0086] = [[D]] BALF ×([[U]] serum / [[U]] BALF )

[0087] Wherein, [[D]] BALF is the drug concentration in BALF, and [[U]] serum is the urea concentration in blood.

[0088] Therefore, by measuring the urea concentration in blood and the recovered BALF during alveolar lavage, as well as the drug concentration in BALF, the actual concentration of the drug in ELF can be calculated according to the urea dilution ratio relationship.

[0089] According to the above research concept, the present application discloses a method for detecting the concentrations of xiannuanotew and ritonavir in blood and alveolar epithelial lining fluid, and the detection method includes the following processes:

[0090] Establishing a standard curve: providing a standard curve working solution, a level quality control working solution and an internal standard solution, and detecting the above standard curve working solution by liquid chromatography - mass spectrometry, and establishing a standard curve according to the detection results; the standard curve includes a xiannuanotew standard curve, a ritonavir standard curve and a urea standard curve;

[0091] Pretreatment: providing a sample to be tested, the sample to be tested includes the blood to be tested and the alveolar lavage fluid to be tested, adding an internal standard and a precipitant to the above blood to be tested and alveolar lavage fluid to be tested respectively, and centrifuging to obtain each supernatant as an injection sample;

[0092] Detecting the concentration: detecting the above injection sample by liquid chromatography - mass spectrometry, and determining the concentrations of xiannuanotew, ritonavir and urea in the above blood to be tested and alveolar lavage fluid to be tested according to the detection results and the above standard curve;

[0093] According to the concentrations of xiannuanotew, ritonavir and urea in the above blood to be tested and alveolar lavage fluid to be tested, the urea dilution method is used to calculate the concentrations of xiannuanotew and ritonavir in the alveolar epithelial lining fluid.

[0094] In some embodiments, the above precipitant includes acetonitrile;

[0095] The volume ratio between the above sample to be tested and the above precipitant is 1:(1 - 3).

[0096] In these embodiments of the present application, the volume ratio between the above sample to be tested and the above precipitant is any one of 1:1, 1:2, 1:3 or any one satisfying the range value of any two of the above.

[0097] In some embodiments, the liquid chromatography conditions include:

[0098] Mobile phase: including phase A and phase B, the volume ratio of the above-mentioned phase A to phase B is (40~60):(60~40);

[0099] The above-mentioned phase A includes an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L to 12 mmol / L;

[0100] The above-mentioned phase B includes a mixed solution of acetonitrile and isopropanol, and the volume ratio of the above-mentioned acetonitrile to isopropanol is (40~60):(60~40);

[0101] Isocratic elution: the flow rate of the above-mentioned mobile phase is 0.3 mL / min to 0.8 mL / min.

[0102] In these embodiments of the present application, the volume ratio of the above-mentioned phase A to phase B is any one of 40:60, 50:50, 60:40 or any one of the range values satisfying any two of the above.

[0103] In these embodiments of the present application, the concentration of the aqueous solution of ammonium acetate is any one of 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L or any one of the range values satisfying any two of the above.

[0104] In these embodiments of the present application, the volume ratio of the above-mentioned acetonitrile to isopropanol is any one of 40:60, 50:50, 60:40 or any one of the range values satisfying any two of the above.

[0105] In these embodiments of the present application, the flow rate of the above-mentioned mobile phase is any one of 0.3 mL / min, 0.4 mL / min, 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min or any one of the range values satisfying any two of the above.

[0106] In some embodiments, the liquid chromatography conditions include:

[0107] The analytical chromatographic column used includes Phenomenex Kinetex F5;

[0108] The column temperature of the above-mentioned analytical chromatographic column is 35°C to 40°C.

[0109] In these embodiments of the present application, the column temperature of the analytical chromatographic column is any one of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any one of the range values satisfying any two of the above.

[0110] In some embodiments, the conditions of the mass spectrometry include:

[0111] The nebulization temperature is 180°C to 220°C;

[0112] The ion spray voltage is 4500 V (MRM+) and -4500 V (MRM-);

[0113] The nebulizing gas is 45 psi to 55 psi, the auxiliary heating gas is 55 psi to 65 psi, the curtain gas is 35 psi to 45 psi, and the collision gas is 8 psi to 10 psi.

[0114] In these embodiments of the present application, the nebulization temperature is any one of 180°C, 185°C, 190°C, 195°C, 200°C, 205°C, 210°C, 215°C, 220°C or any one that satisfies any two of the above range values.

[0115] In some embodiments, the internal standard substances include Xanofivir-D4, Ritonavir-D6, and Urea- 13 C 15 N2.

[0116] In some embodiments, the above injection sample includes the above internal standard substances.

[0117] In some embodiments, the above blood sample to be tested includes plasma or serum.

[0118] In some embodiments, the above internal standard substance solution includes Xanofivir-D4 solution, Ritonavir-D6 solution, and Urea- 13 C 15 N2 solution;

[0119] The above Xanofivir-D4 solution is obtained by diluting the Xanofivir-D4 stock solution with a methanol aqueous solution; the above Xanofivir-D4 stock solution is a dimethyl sulfoxide solution of Xanofivir-D4;

[0120] The above Ritonavir-D6 solution is obtained by diluting the Ritonavir-D6 stock solution with a methanol aqueous solution;

[0121] The above Urea- 13 C 15 N2 solution is obtained by diluting the Urea- 13 C 15 N2 stock solution with pure water;

[0122] In the above internal standard substance solution, the concentration of Xanofivir-D4 is 50 μg / mL, the concentration of Ritonavir-D6 is 0.5 μg / mL, and the concentration of Urea- 13 C 15 N2 is 100 μg / mL.

[0123] In some embodiments, the volume ratio between the sample to be tested and the internal standard solution is (50 - 150):10.

[0124] In these embodiments of the present application, the volume ratio between the sample to be tested and the internal standard solution is any one of 50:10, 60:10, 70:10, 80:10, 90:10, 100:10, 110:10, 120:10, 130:10, 140:10, 150:10 or any one within the range of any two of the above.

[0125] In some embodiments, the above urea dilution method is carried out according to the following relationship:

[0126] [D] ELF = [D] BALF × (V BALF / V ELF ) = [D] BALF × ([U] ELF / [U] BALF ) = [D] BALF × ([U] serum / [U] BALF );

[0127] V ELF = V BALF × [U] BALF / [U] ELF ;

[0128] V ELF refers to the volume of alveolar epithelial lining fluid, V BALF refers to the volume of bronchoalveolar lavage fluid, [U] BALF refers to the urea concentration in bronchoalveolar lavage fluid, [U] ELF refers to the urea concentration in alveolar epithelial lining fluid;

[0129] [D] BALF refers to the drug concentration in bronchoalveolar lavage fluid, [U] serum refers to the urea concentration in blood.

[0130] In some embodiments, the above standard curve working solution is obtained by adding PBS buffer to standard solution with a series of concentrations;

[0131] The above series of concentrations includes at least 8;

[0132] In the above standard curve working solution, the concentration of nirmatrelvir is 2.5 ng / mL - 5000 ng / mL, the concentration of ritonavir is 0.1 ng / mL - 1000 ng / mL, and the concentration of urea is 1 μg / mL - 1000 μg / mL.

[0133] In these embodiments of the present application, the concentration of Xanofivir in the standard curve working solution is any one of 2.5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 2000 ng / mL, 3000 ng / mL, 4000 ng / mL, 5000 ng / mL or any one that satisfies any of the above two range values.

[0134] In these embodiments of the present application, the concentration of Ritonavir is any one of 0.1 ng / mL, 1 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL or any one that satisfies any of the above two range values.

[0135] In these embodiments of the present application, the concentration of urea is any one of 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, 100 μg / mL, 500 μg / mL, 1000 μg / mL or any one that satisfies any of the above two range values.

[0136] To make the purpose, technical solution and advantages of the present application clearer, the interference situation of the secondary battery protected by the present application will be described in detail below with specific embodiments. All chemical substances, reagents, etc. used in the following embodiments are of any conventional models and manufacturers in the art.

[0137] Example 1

[0138] A method for preparing a standard curve working solution, a horizontal quality control working solution and an internal standard solution is provided, including the following processes:

[0139] I. Preparation of the mobile phase solution:

[0140] 1. Preparation of mobile phase A:

[0141] Add 10 mL of 1 mol / L ammonium acetate solution to 1000 mL of ultrapure water (taking the preparation of 1 L of mobile phase A as an example, when preparing in a reduced amount, the addition amount of each component must be reduced proportionally). After using ultrasonic oscillation for 5 minutes to remove the gas, it can be used. Store it at 15°C to 25°C, and the validity period is one week.

[0142] 2. Preparation of mobile phase B:

[0143] Take 500 mL of acetonitrile and 500 mL of isopropanol and mix them evenly (taking the preparation of 1 L of mobile phase B as an example, when preparing in a reduced amount, the addition amount of each component is reduced proportionally). After using ultrasonic oscillation for 5 minutes to remove the gas, it can be used. Store it at 15°C to 25°C, and the validity period is one month.

[0144] II. Preparation of the working fluid:

[0145] Simnotrelvir, Simnotrelvir-D4, Ritonavir, Ritonavir-D6, Urea, and Urea- 13 C 15 N2 (Urea- 13 C 15 N2) were all purchased from Tianjin Aladdin Biochemical Technology Co., Ltd.

[0146] 1. Preparation of the internal standard solution:

[0147] Simnotrelvir-D4 was dissolved in dimethyl sulfoxide to obtain the Simnotrelvir-D4 stock solution. A 50% methanol aqueous solution was used as the diluent to prepare a Simnotrelvir internal standard solution with a concentration of 200 μg / mL; a 50% methanol aqueous solution was used as the diluent to prepare a Ritonavir internal standard (Ritonavir-D6) solution with a concentration of 2 μg / mL; Urea- 13 C 15 N2 was dissolved in pure water to obtain the Urea- 13 C 15 N2 stock solution, and pure water was used as the diluent to prepare a Urea internal standard solution with a concentration of 200 μg / mL. The Simnotrelvir internal standard solution, Ritonavir internal standard solution, and Urea internal standard solution were mixed in a ratio of 1:1:2 to obtain an internal standard solution with a final concentration of 50 μg / mL for Simnotrelvir, 0.5 μg / mL for Ritonavir, and 100 μg / mL for Urea.

[0148] 2. Prepare a series of concentrations of Simnotrelvir standard (XS1-XS8) solutions, a series of concentrations of Ritonavir standard (RS1-RS8) solutions, and a series of concentrations of Urea (US1-US8) standards respectively.

[0149] (1) Preparation of a series of concentrations of Simnotrelvir standard (XS1-XS8) solutions:

[0150] XS8: 50 μL of Simnotrelvir (concentration: 100 μg / mL), final concentration: 100 μg / mL.

[0151] XS7: 50 μL of XS8 + 200 μL of 50% methanol, final concentration: 20 μg / mL.

[0152] XS6: 50 μL of XS7 + 50 μL of 50% methanol, final concentration: 10 μg / mL.

[0153] XS5: 50 μL of XS6 + 200 μL of 50% methanol, final concentration is 2 μg / mL.

[0154] XS4: 50 μL of XS5 + 50 μL of 50% methanol, final concentration is 1 μg / mL.

[0155] XS3: 50 μL of XS4 + 200 μL of 50% methanol, final concentration is 200 ng / mL.

[0156] XS2: 50 μL of XS3 + 50 μL of 50% methanol, final concentration is 100 ng / mL.

[0157] XS1: 50 μL of XS2 + 50 μL of 50% methanol, final concentration is 50 ng / mL.

[0158] (2) Prepare a series of concentrations of ritonavir standard (RS1 - RS8) solutions:

[0159] RS8: 50 μL of ritonavir (concentration is 20 μg / mL), final concentration is 20 μg / mL.

[0160] RS7: 50 μL of RS8 + 50 μL of 50% methanol, final concentration is 10 μg / mL.

[0161] RS6: 50 μL of RS7 + 75 μL of 50% methanol, final concentration is 4 μg / mL.

[0162] RS5: 50 μL of RS6 + 150 μL of 50% methanol, final concentration is 1 μg / mL.

[0163] RS4: 50 μL of RS5 + 200 μL of 50% methanol, final concentration is 200 ng / mL.

[0164] RS3: 50 μL of RS4 + 200 μL of 50% methanol, final concentration is 40 ng / mL.

[0165] RS2: 50 μL of RS3 + 150 μL of 50% methanol, final concentration is 10 ng / mL.

[0166] RS1: 50 μL of RS2 + 200 μL of 50% methanol, final concentration is 2 ng / mL.

[0167] (3) Prepare a series of concentrations of urea standard (US1 - US8) solutions:

[0168] US8: 50 μL of urea (concentration is 20 mg / mL), final concentration is 20 mg / mL.

[0169] For US7: 50 μL of US8 + 75 μL of H2O, with a final concentration of 8 mg / mL.

[0170] For US6: 50 μL of US7 + 150 μL of H2O, with a final concentration of 2 mg / mL.

[0171] For US5: 50 μL of US6 + 75 μL of H2O, with a final concentration of 800 μg / mL.

[0172] For US4: 50 μL of US5 + 150 μL of H2O, with a final concentration of 200 μg / mL.

[0173] For US3: 50 μL of US4 + 50 μL of H2O, with a final concentration of 100 μg / mL.

[0174] For US2: 50 μL of US3 + 75 μL of H2O, with a final concentration of 40 μg / mL.

[0175] For US1: 50 μL of US2 + 50 μL of H2O, with a final concentration of 20 μg / mL.

[0176] 3. Prepare the standard curve working solution:

[0177] Add 10 μL of the solution of the Xanofivir standard (XS1 - XS8), 10 μL of the solution of the Ritonavir standard (RS1 - RS8), and 10 μL of the solution of the Urea standard (US1 - US8) to 170 μL of PBS respectively to obtain 200 μL of the standard curve working solution (STD1 - STD8). The concentrations of Xanofivir in this standard curve working solution are 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL in sequence; the concentrations of Ritonavir are 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL in sequence; the concentrations of Urea are 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 40 μg / mL, 100 μg / mL, 400 μg / mL, 1000 μg / mL in sequence.

[0178] 4. Prepare the quality control working solution:

[0179] Add 10 μL of the solution of Xanofivir standard (XS3 / XS5 / XS7), 10 μL of the solution of Ritonavir standard (RS3 / RS5 / RS7), and 10 μL of the solution of Urea standard (US3 / US5 / US7) to 170 μL of PBS respectively to obtain 200 μL of the quality control (QCL / QCM / QCH) working solution. The concentrations of Xanofivir in the quality control working solution are 10 ng / mL, 100 ng / mL, and 1000 ng / mL in sequence, the concentrations of Ritonavir are 2 ng / mL, 50 ng / mL, and 500 ng / mL in sequence, and the concentrations of Urea are 5 μg / mL, 40 μg / mL, and 400 μg / mL in sequence.

[0180] In the examples of this application, the linear r of the standard curve should be greater than 0.99, and the accuracy of the low, medium, and high quality control samples should be within ±15% of the theoretical value.

[0181] Example 2

[0182] Provide a sample pretreatment method, including the following processes:

[0183] (1) Pretreatment of the standard curve working solution: Take a 1.5 mL centrifuge tube, add 200 μL of the standard curve working solution, 20 μL of the internal standard solution, and 400 μL of 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; Take 200 μL of the supernatant (1) and transfer it to a 96-well plate for testing;

[0184] (2) Pretreatment of the horizontal quality control working solution: Take a 1.5 mL centrifuge tube, add 200 μL of the quality control working solution, 20 μL of the internal standard solution, and 400 μL of 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; Take 200 μL of the supernatant (2) and transfer it to a 96-well plate for testing;

[0185] (3) Pretreatment of the serum sample: Take a 1.5 mL centrifuge tube, add 200 μL of the serum sample to be tested, 20 μL of the internal standard solution, and 400 μL of 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; Take 200 μL of the supernatant (3) and transfer it to a 96-well plate for testing;

[0186] (4) Pretreatment of the bronchoalveolar lavage fluid: Take a 1.5 mL centrifuge tube, add 200 μL of the bronchoalveolar lavage fluid sample to be tested, 20 μL of the internal standard solution, and 400 μL of 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13000 rpm for 10 min; Take 200 μL of the supernatant (4) and transfer it to a 96-well plate for testing.

[0187] Example 3

[0188] Provide a liquid chromatography tandem mass spectrometry condition:

[0189] 1. The liquid chromatography conditions are shown in Table 1:

[0190] Table 1 List of liquid chromatography conditions

[0191]

[0192] 2. The mass spectrometry conditions are shown in Table 2:

[0193] Table 2 List of mass spectrometry conditions

[0194]

[0195] 3. The ion pair parameters are shown in Table 3:

[0196] Table 3 List of ion pair parameters

[0197]

[0198] The * in the above table is the quantitative ion pair.

[0199] In order to achieve the purpose of more sensitive detection and faster detection speed, in this application, the chromatographic column used is Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex) and isocratic elution is carried out under the chromatographic conditions shown in Table 1. At the same time, comparative experiments are also described in this application.

[0200] Under the condition that the chromatographic column is Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex) and under the chromatographic conditions shown in Table 1, after the samples are pretreated in the same way, it is found that: under the condition that mobile phase B is an acetonitrile: isopropanol (50:50) mixed solution, compared with mobile phase A being 10 mM ammonium acetate aqueous solution, if mobile phase A is 20 mM ammonium acetate aqueous solution, the results are shown in the attached Figure 7 and 8 , it can be found that the response intensities of nirmatrelvir and ritonavir decrease and the detection sensitivity drops. A lower concentration of salt in the mobile phase also helps to protect the detection system. Therefore, our final detection scheme uses an 8 mmol / L to 12 mmol / L ammonium acetate aqueous solution as mobile phase A.

[0201] Under the conditions that mobile phase A is an aqueous solution of 10 mM ammonium acetate, mobile phase B is a mixed solution of acetonitrile: isopropanol (50:50), and the chromatographic conditions shown in Table 1 (except for the chromatographic column), after the samples are pretreated in the same way, it is found that: if the chromatographic column is XBridge C18 column (2.1×100 mm, 3.5 μm, Waters), the results are shown in the accompanying drawings of the specification Figures 12 to 14 , and the comparison is shown in the accompanying drawings of the specification Figures 9 to 11 (the chromatographic column is Kinetex F5 column). It can be seen that the response intensity of xiannuo tewei decreases, there is a delayed peak, which affects quantification; the response intensity of urea is low, the peak shape is poor, and its baseline is high, so it cannot be accurately quantified; if the chromatographic column is XBridge BEH C8 column (2.1×100 mm, 2.5 μm, Waters), the results are shown in the accompanying drawings of the specification Figures 15 to 17 , and the comparison is shown in the accompanying drawings of the specification Figures 9 to 11 (the chromatographic column is Kinetex F5 column). It can be seen that the response intensity of xiannuo tewei decreases; the response intensity of urea is low, the peak shape is poor, and its baseline is high, so it cannot be accurately quantified. Therefore, the chromatographic column of this application, Kinetex F5 column (3 mm×100 mm, 2.6 μm, Phenomenex), has better effects (accompanying drawings Figures 9 to 11 ).

[0202] Example 4

[0203] Provide an instrumental analysis procedure, including the following steps:

[0204] 1. Turn on the computer power, enter the Windows operating system, check whether the instrument status is good, log in to the Analyst software, and enter the main interface of the workstation.

[0205] 2. Double-click the Hardware configuration button, click to select LCMS and then click Activate profile on the right to activate the instrument. The LCMS module shows a green check mark, indicating successful online connection.

[0206] 3. Double-click Build Acquistion Batch in the Acquire column, select the method that has been established, and edit the sample sequence (sample number, sample tray position, injection volume, etc.) in the sequence editing menu bar.

[0207] 4. Click the Equilibrate menu item, select the previously established liquid chromatography - mass spectrometry method to be run, and set the equilibration time to 10 - 20 min.

[0208] 5. Enter the Submit interface on the right side of the Batch Editor, select the sample sequence to be analyzed, and click the Submit button to submit.

[0209] 6. After the equilibration time ends and the instrument status in the lower right corner of the main interface shows Ready, click Start sample in the menu bar to start sample analysis and data acquisition.

[0210] 7. Double-click the Open Date File option in the Explore option on the left to view the chromatogram images during the detection process at any time.

[0211] 8. After the sample analysis and acquisition are completed, use Mμltiquant to create a quantitative list for quantitative analysis.

[0212] Standby steps

[0213] 1. Click the Standby button in the menu bar. It can be seen that the flow rate slowly decreases to zero. After the system pressure drops to 0, double-click Hardware configuration. In the pop-up dialog box, click to select LCMS, and then click Deactivateprofile on the right to deactivate the liquid chromatography-mass spectrometry instrument. When the LCMS module shows a cross, the online connection can be disconnected.

[0214] 2. Exit the Analyst software and shut down the computer.

[0215] Example 5

[0216] Provide a method for verifying the accuracy of the above method, including the following process:

[0217] 1. Linearity

[0218] A series of serial dilutions were performed on standard solutions of known concentrations (the concentrations of nirmatrelvir were 5000 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL in sequence; the concentrations of ritonavir were 1000 ng / mL, 500 ng / mL, 200 ng / mL, 50 ng / mL, 10 ng / mL, 2 ng / mL, 0.5 ng / mL, 0.1 ng / mL in sequence; the concentrations of urea were 1000 μg / mL, 400 μg / mL, 100 μg / mL, 40 μg / mL, 10 μg / mL, 5 μg / mL, 2 μg / mL, 1 μg / mL in sequence). An equal amount of internal standard solution was added, and the calibration curve was formed by fitting the calibration points using the least squares method. The linearity requirement was r > 0.99, the deviation of the values at each concentration point from the theoretical values was less than 15%, and the deviation of the detected value from the theoretical value at the Limit of Quantification (LoQ) point was less than 20%. The linear range of nirmatrelvir obtained from the experiment was 2.5 - 5000 ng / mL, LoQ = 2.5 ng / mL, the linear range of ritonavir was 0.1 - 1000 ng / mL, LoQ = 0.1 ng / mL, the linear range of urea was 1 - 1000 μg / mL, LoQ = 1 μg / mL. The standard curve of nirmatrelvir had r = 0.99836, showing good linearity and accurate quantification at the LoQ point, as Figure 1 and Figure 4 shown; the standard curve of ritonavir had r = 0.99982, showing good linearity and accurate quantification at the LoQ point, as Figure 2 and Figure 5 shown; the standard curve of urea had r = 0.99997, showing good linearity and accurate quantification at the LoQ point, as Figure 3 and Figure 6 shown, which was sufficient to indicate that the detection sensitivity and linearity of this application were good and could meet the needs of clinical detection and scientific research.

[0219] 2. Precision and carry - over contamination

[0220] The prepared low, medium, and high quality control working fluids were continuously detected 3 times for 3 consecutive days. The within-day precision coefficients of variation of Xiannuo Towvir were calculated to be 4.39%, 4.32%, and 0.65% respectively, and the between-day precision coefficients of variation were 6.17%, 5.99%, and 5.18% respectively (see Table 4 for details); the within-day precision coefficients of variation of Ritonavir were 1.85%, 3.51%, and 1.32% respectively, and the between-day precision coefficients of variation were 5.25%, 5.42%, and 4.63% respectively (see Table 5 for details); the within-day precision coefficients of variation of urea were 2.09%, 0.59%, and 0.90% respectively, and the between-day precision coefficients of variation were 4.50%, 1.33%, and 2.60% respectively (see Table 6 for details). The within-day and between-day precision coefficients of variation of Xiannuo Towvir, Ritonavir, and urea were all less than 10%, indicating good precision. The blank samples were measured immediately after the highest point of the calibration curve, and the signal responses of the blank samples were all less than 0.1% of the highest point of the calibration curve. Please see Table 7, which proves that there is no significant carryover contamination in the detection method of this application.

[0221] Table 4 Detection Precision of Xiannuo Towvir

[0222]

[0223] Table 5 Detection Precision of Ritonavir

[0224]

[0225] Table 6 Detection Precision of Urea

[0226]

[0227] Table 7 Detection Carryover Contamination Rates of Xiannuo Towvir, Ritonavir, and Urea

[0228]

[0229] 3. Accuracy and Matrix Effect:

[0230] Spiked recovery rate: At least 5 population samples from different sources were taken. Equal amounts of serum / alveolar lavage fluid were selected from each sample and mixed. Then, 4 equal amounts were selected from the mixed serum / alveolar lavage fluid. Among them, 3 portions were respectively added with standard solution of low / middle / high values with known concentrations, and the same pretreatment and sample injection analysis procedures were carried out. It was determined in parallel three times. The data in Table 8 was obtained by calculating with the formula spiked recovery rate % = (concentration of the spiked sample measured - concentration of the matrix sample measured) / theoretical spiked concentration * 100%. The serum spiked recovery rate of Xanofivir was 85.70% - 97.62%, and the alveolar lavage fluid spiked recovery rate was 86.09% - 97.39%; the serum spiked recovery rate of Ritonavir was 104.90% - 107.10%, and the alveolar lavage fluid spiked recovery rate was 89.71% - 97.59%; the serum spiked recovery rate of urea was 95.08% - 101.73%, and the alveolar lavage fluid spiked recovery rate was 98.05% - 100.16%, indicating that the accuracy of the detection method of this application was good.

[0231] Matrix effect: At least 5 population samples from different sources were taken. Equal amounts of serum / alveolar lavage fluid were selected from each sample and mixed. Then, 4 equal amounts were selected from the mixed serum / alveolar lavage fluid. Among them, 3 portions were respectively added with standard solution of low, middle, and high values with known concentrations, and the same pretreatment and sample injection analysis procedures were carried out. It was determined in parallel three times to obtain the average signal intensity (A) at each concentration point. The serum / alveolar lavage fluid was replaced with PBS solution, and standard solutions corresponding to low, middle, and high concentration levels were prepared with the same sample injection reagent, and sample injection analysis was carried out to obtain the average signal intensity (B) at each concentration point in the pure solution. At the same time, blank serum / alveolar lavage fluid samples were prepared and determined in parallel three times to obtain the average signal intensity (C) of the blank serum / alveolar lavage fluid samples. Calculation was carried out with the area ratio of the analyte to the internal standard peak in each group of samples. The serum matrix effects of Xanofivir were 89.51%, 95.15%, and 85.67% respectively, and the alveolar lavage fluid matrix effects were 89.92%, 94.94%, and 89.45% respectively, obtained by the calculation formula matrix effect = (area ratio of sample A - area ratio of sample C) / area ratio of sample B * 100%; the serum matrix effects of Ritonavir were 108.93%, 107.04%, and 103.98% respectively, and the alveolar lavage fluid matrix effects were 98.70%, 98.52%, and 87.10% respectively; the serum matrix effects of urea were 95.43%, 95.16%, and 100.93% respectively, and the alveolar lavage fluid matrix effects were 98.41%, 93.46%, and 99.61% respectively. See Table 8 for details, meeting the experimental requirements.

[0232] Table 8 Recovery Rates and Matrix Effects of Xanofivir and Ritonavir

[0233]

[0234] In summary, the present application utilizes the high separation performance of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry to establish a liquid chromatography-tandem mass spectrometry quantitative analysis method, which can quickly, simply, and accurately detect the concentrations of Xanofivir, Ritonavir, and Urea in blood and bronchoalveolar lavage fluid, and then can calculate the concentrations of Xanofivir and Ritonavir in alveolar epithelial lining fluid, realizing the monitoring of Xanofivir and Ritonavir therapeutic drugs, and laying a foundation for drug monitoring, safe use, and personalized precision treatment, etc.

[0235] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should all be covered by the scope of the claims and the description of the present application. In particular, as long as there is no structural conflict, the technical features mentioned in each embodiment can be combined in any way. The present application is not limited to the specific embodiments disclosed in the text, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for detecting the concentration of senotevir and ritonavir in blood and alveolar epithelial lining fluid, characterized in that: The process includes the following: Establishing a standard curve: providing a standard curve working solution, a horizontal quality control working solution and an internal standard solution, and using liquid chromatography-mass spectrometry to detect the standard curve working solution, and establishing a standard curve according to the detection results; the standard curve includes a senotevir standard curve, a ritonavir standard curve and a urea standard curve; Pretreatment: providing a sample to be tested, the sample to be tested includes blood to be tested and bronchoalveolar lavage fluid to be tested, adding an internal standard and a precipitant to the blood to be tested and the bronchoalveolar lavage fluid to be tested respectively, the precipitant is acetonitrile, and performing centrifugation to obtain each supernatant as an injection sample; the volume ratio between the sample to be tested and the precipitant is 1:(1-3); Detection concentration: Detection of the injected sample by liquid chromatography-mass spectrometry; Liquid chromatography conditions included: The mobile phase is a phase A and a phase B, and the volume ratio of the phase A to the phase B is (40-60): (60-40); the phase A is an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L-12 mmol / L; the phase B is a mixed solution of acetonitrile and isopropanol in a volume ratio of (40-60): (60-40); Isocratic elution: the flow rate of the mobile phase is 0.3 mL / min~0.8 mL / min; The analytical columns used included Phenomenex Kinetex F5; The column temperature of the analytical chromatographic column is 35°C to 40°C; The conditions for mass spectrometry include: Atomization temperature is 180℃~220℃; The ion spray voltage was 4500 V (MRM+) and −4500 V (MRM−); Atomizing gas is 45psi~55psi, auxiliary heating gas is 55psi~65psi, curtain gas is 35psi~45psi, collision gas is 8psi~10psi; Determining the concentrations of senotevir, ritonavir and urea in the blood to be tested and the bronchoalveolar lavage fluid to be tested according to the test results and the standard curve; According to the concentrations of sennotevir, ritonavir and urea in the blood to be tested and the bronchoalveolar lavage fluid to be tested, the concentrations of sennotevir and ritonavir in the alveolar epithelial lining fluid are calculated by urea dilution method.

2. The detection method according to claim 1, characterized in that: Internal standards include senotevir-D4, ritonavir-D6 and urea- 13 C 15 N2; and / or; The injected sample includes the internal standard; and / or; The blood to be tested includes plasma or serum.

3. The detection method according to claim 1, characterized in that: The internal standard solution includes senotevir-D4 solution, ritonavir-D6 solution and urea- 13 C 15 N2 solution; The Xianuotewei-D4 solution is obtained by diluting the Xianuotewei-D4 stock solution with methanol aqueous solution; the Xianuotewei-D4 stock solution is a dimethyl sulfoxide solution of Xianuotewei-D4; The ritonavir-D6 solution is obtained by diluting the ritonavir-D6 stock solution with methanol aqueous solution; The urea- 13 C 15 N2 solution is made by diluting urea with pure water. 13 C 15 N2 stock solution is obtained; The concentration of senotevir-D4 in the internal standard solution is 50 μg / mL, the concentration of ritonavir-D6 is 0.5 μg / mL, and the concentration of urea- 13 C 15 The concentration of N2 was 100 μg / mL.

4. The detection method according to claim 3, characterized in that: The volume ratio between the sample to be tested and the internal standard solution is (50-150):

10.

5. The detection method according to claim 1, characterized in that: The urea dilution method includes following the relationship: [D] ELF =[D] BALF ×(V BALF / V ELF )=[D] BALF ×([U] ELF / [U] BALF )=[D] BALF ×([U] serum / [U] BALF ); V ELF =V BALF ×[U] BALF / [U] ELF ; V ELF V refers to the volume of alveolar epithelial lining fluid, BALF refers to the volume of alveolar lavage fluid, [U] BALF Refers to the urea concentration in bronchoalveolar lavage fluid, [U] ELF It refers to the urea concentration in the alveolar epithelial lining fluid; [D] BALF Refers to the drug concentration in bronchoalveolar lavage fluid, [U] serum It refers to the concentration of urea in the blood; [D] ELF It refers to the drug concentration in the alveolar epithelial lining fluid.

6. The detection method according to claim 1, characterized in that: The standard curve working solution is obtained by respectively adding PBS buffer to a series of concentration standard solutions; The series of concentrations includes at least 8; In the standard curve working solution, the concentration of senotevir is 2.5 ng / mL to 5000 ng / mL, the concentration of ritonavir is 0.1 ng / mL to 1000 ng / mL, and the concentration of urea is 1 μg / mL to 1000 μg / mL.

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