Method for detecting concentrations of pronotegravir and ritonavir in blood and alveolar epithelial interlining fluid

Abstract

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting the concentration of pronotegravir and ritonavir in blood and alveolar epithelial interlining fluid. The method comprises the following steps: pretreating a blood and alveolar lavage fluid sample, establishing a liquid chromatography-tandem mass spectrometry measurement method, and further calculating the concentrations of pronotegravir and ritonavir in the blood and alveolar epithelial interlining fluid. The method has the characteristics of rapidness and accuracy, and lays a foundation for drug monitoring, safe use, personalized precise treatment and the like.

Description

Technical Field

[0001] The present application relates to the field of analysis and detection, and specifically to a method for detecting the concentrations of senotevir 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, a combination package of Senotegravir tablets and Ritonavir tablets, its trade name is Senoxin.

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

[0004] In view of the above problems, the present application provides a method for detecting the concentrations of senotetravir and ritonavir in blood and alveolar epithelial lining fluid, which is rapid and accurate.

[0005] On the one hand, the present application provides a method for detecting the concentration of senotevir and ritonavir in blood and alveolar epithelial lining fluid, the detection method comprising the following process: 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 samples to be tested, wherein the samples to be tested include blood to be tested and bronchoalveolar lavage fluid to be tested, adding internal standard substances and precipitants to the blood to be tested and the bronchoalveolar lavage fluid to be tested, respectively, and subjecting the blood to be tested and the bronchoalveolar lavage fluid to be tested to obtain supernatants as injection samples by centrifugation; Detection concentration: Detect the injected sample by liquid chromatography-mass spectrometry, and determine the concentrations of senotevir, 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; 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.

[0006] In some embodiments, the precipitating agent comprises acetonitrile; The volume ratio between the sample to be tested and the precipitant is 1:(1-3).

[0007] In some embodiments, the liquid chromatography conditions include: Mobile phase: comprising phase A and phase B, wherein the volume ratio of phase A to phase B is (40-60): (60-40); The phase A comprises an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L to 12 mmol / L; The phase B comprises a mixture of acetonitrile and isopropanol, and the volume ratio of the acetonitrile to the isopropanol is (40-60): (60-40); Isocratic elution: The flow rate of the mobile phase is 0.3 mL / min to 0.8 mL / min.

[0008] In some embodiments, the liquid chromatography conditions include: The analytical columns used included Phenomenex Kinetex F5; The column temperature of the analytical chromatographic column is 35°C-40°C.

[0009] In some embodiments, the conditions for mass spectrometry include: Atomization temperature is 180℃~220℃; The ion spray voltage was 4500 V (MRM+) and −4500 V (MRM−); The atomizing gas is 45psi~55psi, the auxiliary heating gas is 55psi~65psi, the curtain gas is 35psi~45psi, and the collision gas is 8psi~10psi.

[0010] In some embodiments, the internal standard includes senotevir-D4, ritonavir-D6 and urea- 13 C 15 N2.

[0011] In some embodiments, the injected sample includes the internal standard.

[0012] In some embodiments, the blood to be tested includes plasma or serum.

[0013] In some embodiments, the internal standard solution includes a solution of senotevir-D4, a solution of ritonavir-D6 and a solution of 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.

[0014] In some embodiments, the volume ratio between the sample to be tested and the internal standard solution is (50-150):10.

[0015] In some embodiments, the urea dilution method comprises following the following 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 Refers to the concentration of urea in the blood.

[0016] In some embodiments, the standard curve working solution is obtained by adding PBS buffer to a series of concentrations of 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.

[0017] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below.

[0018] Beneficial technical effects of this application: This application utilizes the high separation performance of liquid chromatography and the high selectivity and 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 sennotevir, ritonavir and urea in blood and bronchoalveolar lavage fluid, and then calculate the concentrations of sennotevir and ritonavir in the alveolar epithelial lining fluid, thereby realizing the monitoring of sennotevir and ritonavir therapeutic drugs, laying the foundation for drug monitoring, safe use and personalized precision treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the detailed description of the preferred embodiments below. The accompanying drawings are only for the purpose of illustrating the preferred embodiments and are not to be considered as limiting the present application. Also, the same reference numerals are used throughout the accompanying drawings to represent the same components. In the accompanying drawings: Figure 1 This is a schematic diagram of the calibration curve of Xiannotervir in this application; Figure 2 This is a schematic diagram of the calibration curve of ritonavir in this application; Figure 3 This is a schematic diagram of the calibration curve of urea in this application; Figure 4 The lowest point STD1 of the standard curve (i.e., the standard curve working solution) in some embodiments of this application is the substance peak of the analyte Xianotevir ( Figure 4 A) and internal standard peak spectra of Senotevir-D4 ( Figure 4 B); Figure 5 The lowest point STD1 of the standard curve (i.e., the standard curve working solution) in some embodiments of this application is the substance peak of ritonavir to be tested ( Figure 5 A) and the internal standard peak spectrum of ritonavir-D6 ( Figure 5 B); Figure 6 The lowest point STD1 of the standard curve (i.e., the standard curve working solution) in some embodiments of this application is the substance peak of the urea to be tested ( Figure 6 A) and urea- 13 C 15 Internal standard peak spectrum of N2 ( Figure 6 B); Figure 7 : Figure 7A is the internal standard peak spectrum of Senotevir-D4 in STD1 sample under the conditions that mobile phase A is 20 mM ammonium acetate aqueous solution and mobile phase B is acetonitrile:isopropanol (50:50) mixed solution; Figure 7 B is the internal standard peak spectrum of Senotevir-D4 in STD1 sample when mobile phase A is 10 mM ammonium acetate aqueous solution and mobile phase B is a mixed solution of acetonitrile:isopropanol (50:50); Figure 8 : Figure 8 A is the internal standard peak spectrum of ritonavir-D6 in STD1 sample under the conditions that mobile phase A is 20 mM ammonium acetate aqueous solution and mobile phase B is acetonitrile:isopropanol (50:50) mixed solution; Figure 8 B is the internal standard peak spectrum of ritonavir-D6 in STD1 sample under the conditions that mobile phase A is 10 mM ammonium acetate aqueous solution and mobile phase B is acetonitrile:isopropanol (50:50) mixed solution; Fig. 9 :10mM ammonium acetate aqueous solution as mobile phase A and acetonitrile:isopropanol (50:50) mixed solution as mobile phase B, the chromatographic column was Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex), 40% mobile phase A and 60% mobile phase B were used for isocratic elution, and the flow rate was 0.5ml / min. Fig. 9 A) Spectrum of Senotegravir-D4 ( Fig. 9 B); Fig.10 :10mM ammonium acetate aqueous solution as mobile phase A and acetonitrile:isopropanol (50:50) mixed solution as mobile phase B, the chromatographic column was Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex), 40% mobile phase A and 60% mobile phase B were used for isocratic elution, and the flow rate was 0.5ml / min. Ritonavir in QCM sample ( Fig.10 A) Spectrum of ritonavir-D6 ( Fig.10 B); Fig.11 :10mM ammonium acetate aqueous solution as mobile phase A and acetonitrile:isopropanol (50:50) mixed solution as mobile phase B, Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex), 40% mobile phase A and 60% mobile phase B for isocratic elution, flow rate of 0.5ml / min, urea in QCM sample ( Fig.11 A) Urea 13 C 15 N 2 The spectrum of Fig.11 B); Fig.12 : When the chromatographic column is XBridge C18 column (2.1×100mm, 3.5μm, Waters), the concentration of senotevir in the QCM sample is Fig.12 A) Spectrum of Senotegravir-D4 ( Fig.12 B); Fig.13 :When the chromatographic column is XBridge C18 column (2.1×100mm, 3.5μm, Waters), the ritonavir in the QCM sample ( Fig.13 A) Spectrum of ritonavir-D6 ( Fig.13 B); Fig.14 : When the chromatographic column is XBridge C18 column (2.1×100mm, 3.5μm, Waters), the urea in the QCM sample ( Fig.14 A) Urea 13 C 15 The spectrum of N2 ( Fig.14 B); Fig.15 :When the chromatographic column is XBridge BEH C8 column (2.1×100mm, 2.5μm, Waters), the concentration of senotevir in the QCM sample is Fig.15 A) Spectrum of Senotegravir-D4 ( Fig.15 B); Fig.16 :When the chromatographic column is XBridge BEH C8 column (2.1×100mm, 2.5μm, Waters), ritonavir in QCM sample ( Fig.16 A) Spectrum of ritonavir-D6 ( Fig.16 B); Fig.17 :Urea in QCM sample when the chromatographic column is XBridge BEH C8 column (2.1×100mm, 2.5μm, Waters) Fig.17 A) Urea 13 C 15 The spectrum of N2 ( Fig.17 B).

[0020] in, Figures 1 to 3 The ordinate of the central axis is the peak area ratio, and the abscissa is the concentration; Figures 4 to 17 The vertical axis is the signal intensity, and the horizontal axis is the retention time. DETAILED DESCRIPTION

[0021] Hereinafter, the embodiments of the method for detecting the concentration of sennotevir and ritonavir in the blood and alveolar epithelial lining fluid of the present application are 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 the same 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 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 described in the claims.

[0022] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a particular range. The range defined in this way can be inclusive or exclusive of end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that the range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0023] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0024] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0025] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0026] If there is no special explanation, the "include" and "comprising" mentioned in this application represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0027] If not specifically stated, in this application, the term "or" is inclusive. 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).

[0028] If not otherwise specified, in this application, the terms "first", "second", etc. are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity, specific order or primary and secondary relationship of the indicated technical features.

[0029] Unless otherwise specified, in this application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple sheets" refers to more than two sheets (including two sheets).

[0030] Unless otherwise specified, the orientations or positional relationships indicated by technical terms such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", and "circumferential" are based on the orientations or positional relationships shown in the accompanying drawings and are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as limiting the embodiments of the present application.

[0031] If there is no special explanation, unless otherwise clearly specified and limited, technical terms such as "install", "connect", "connect", "fix" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral one; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of this application can be understood according to the specific circumstances.

[0032] Simnotrelvir, chemical formula: C 22 H 30 F3N5O4S2, Ritonavir, chemical formula: C 37 H 48 N6O5S2, a combination package of Senotegravir tablets and Ritonavir tablets, its trade name is Senoxin.

[0033] 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. Inhibition of 3CLpro effectively blocks viral RNA replication and transcription, thereby blocking viral 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 worldwide suffer from long-term COVID-19, and more than 200 symptoms affecting multiple organ systems have been identified. Small molecule drugs have received widespread attention due to their cost-effectiveness, ease of transportation, and strong stability, making them very suitable for clinical use as key components in the fight against COVID-19.

[0034] Senotegravir is a highly active, oral, low-toxic anti-COVID-19 drug that not only inhibits wild-type 3CLpro, but also maintains inhibitory activity against variants. Senotegravir is primarily metabolized by cytochrome P450 3A (CYP3A). Ritonavir is a pharmacokinetic enhancer that inhibits CYP3A and can be co-administered to maximize the likelihood of achieving effective concentrations. Therefore, the combination of Senotegravir and low-dose ritonavir can slow down the metabolism of Senotegravir in the body and help exert its antiviral effect.

[0035] Therapeutic drug monitoring (TDM), which is the detection of drug concentrations in the blood, is of great significance in clinical treatment. TDM can guide clinicians to adjust the dosing regimen according to the patient's blood drug concentration and changes in pharmacokinetic parameters. This helps to improve the efficacy of the drug while reducing or avoiding the toxicity of the drug. TDM is particularly important for drugs with a narrow therapeutic index and drugs whose symptoms of poisoning are easily confused with the disease itself. Through TDM, clinicians can more accurately determine the dosage and frequency of administration, thereby improving the effectiveness and success rate of treatment.

[0036] Alveolar epithelial lining fluid (ELF) refers to the fluid between alveolar epithelial cells. It is located in the alveolar cavity and is the interface fluid between alveolar epithelial cells and the alveolar cavity. For lung infections, whether anti-infective drugs can reach an effective concentration in the alveolar epithelial lining fluid affects their actual clinical efficacy and the patient's disease outcome. More and more studies have begun to focus on drug concentrations in target tissues during treatment, and have attempted to optimize drug dosing regimens and predict clinical efficacy by measuring actual drug concentrations at the site of infection, in order to achieve individualized precision drug therapy and ultimately achieve the goal of precision medicine.

[0037] Currently, the most commonly used technique for obtaining alveolar epithelial lining fluid samples to evaluate the drug concentration in the alveolar epithelial lining fluid is bronchoalveolar lavage. The drug concentration in the bronchoalveolar lavage fluid (BALF) after medication is measured by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The actual drug concentration in ELF can be calculated according to the urea dilution method, that is, by detecting the urea concentration in the alveolar lavage fluid and serum of the patient, as well as the concentration of sennotevir and ritonavir in the alveolar lavage fluid, the concentration of sennotevir and ritonavir in the alveolar epithelial lining fluid can be obtained.

[0038] In the urea dilution method, urea (molecular formula: CH4N2O) is a non-polar small molecule that can freely pass through the alveolar wall. Therefore, it is believed that the concentration of urea in the blood and ELF is the same. Therefore, the actual drug concentration in the ELF can be calculated by measuring the urea concentration in the blood and the urea concentration in the BALF as the dilution multiple.

[0039] V ELF =V BALF ×[U] BALF / [U] ELF Where V ELF is the volume of ELF, V BALF is the volume of BALF, [U] BALFis the urea concentration in BALF, [U] ELF is the urea concentration in ELF.

[0040] Drug concentration in ELF ([D] ELF ) is calculated as follows: [D] ELF =[D] BALF ×(V BALF / V ELF ) =[D] BALF ×([U] ELF / [U] BALF ) =[D] BALF ×([U] serum / [U] BALF ) Among them, [D] BALF is the drug concentration in BALF, [U] serum is the concentration of urea in the blood.

[0041] Therefore, by measuring the urea concentration in the blood and recovered BALF during alveolar lavage, as well as the drug concentration in BALF, the actual drug concentration in ELF can be calculated based on the urea dilution ratio.

[0042] According to the above research concept, the present application discloses a method for detecting the concentration of senotevir and ritonavir in blood and alveolar epithelial lining fluid, which comprises the following process: 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 above standard curve working solution, and establishing a standard curve according to the test results; the standard curve includes a sennotvir 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, and performing centrifugation to obtain each supernatant as an injection sample; Detection concentration: Detect the injected samples by liquid chromatography-mass spectrometry, and determine the concentrations of senotevir, ritonavir and urea in the blood and bronchoalveolar lavage fluid according to the detection results and the standard curve; According to the concentrations of sennotevir, ritonavir and urea in the blood and bronchoalveolar lavage fluid to be tested, the concentrations of sennotevir and ritonavir in the alveolar epithelial lining fluid were calculated by urea dilution method.

[0043] In some embodiments, the precipitant comprises acetonitrile; The volume ratio between the sample to be tested and the precipitant is 1:(1-3).

[0044] The present application discloses in these embodiments that the volume ratio between the sample to be tested and the precipitant is any one of 1:1, 1:2, 1:3 or any one of the ranges of any two of the above.

[0045] In some embodiments, the liquid chromatography conditions include: Mobile phase: including phase A and phase B, wherein the volume ratio of phase A to phase B is (40-60): (60-40); The phase A comprises an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L to 12 mmol / L; The phase B comprises a mixture of acetonitrile and isopropanol, and the volume ratio of the acetonitrile to the isopropanol is (40-60): (60-40); Isocratic elution: The flow rate of the above mobile phase is 0.3mL / min~0.8mL / min.

[0046] The present application discloses in these embodiments that the volume ratio of the phase A to the phase B is any one of 40:60, 50:50, 60:40 or any one of the ranges of any two of the above.

[0047] In these embodiments, the present application discloses that the concentration of the aqueous solution of ammonium acetate is any one of 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L or any one of the values ​​satisfying any two of the above ranges.

[0048] The present application discloses in these embodiments that the volume ratio of acetonitrile to isopropanol is any one of 40:60, 50:50, 60:40 or any one of the ranges of any two of the above.

[0049] In these embodiments, the present application discloses that the flow rate of the 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 values ​​within any two ranges.

[0050] In some embodiments, the liquid chromatography conditions include: The analytical columns used included Phenomenex Kinetex F5; The column temperature of the above analytical chromatographic column is 35°C~40°C.

[0051] The present application discloses in these embodiments that 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 values ​​within the range of any two of the above.

[0052] In some embodiments, the conditions for mass spectrometry include: Atomization temperature is 180℃~220℃; The ion spray voltage was 4500 V (MRM+) and −4500 V (MRM−); The atomizing gas is 45psi~55psi, the auxiliary heating gas is 55psi~65psi, the curtain gas is 35psi~45psi, and the collision gas is 8psi~10psi.

[0053] In these embodiments, the present application discloses that the atomization 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 of the ranges of any two of the above.

[0054] In some embodiments, the internal standard includes senotevir-D4, ritonavir-D6 and urea- 13 C 15 N2.

[0055] In some embodiments, the injected sample includes the internal standard.

[0056] In some embodiments, the blood to be tested includes plasma or serum.

[0057] In some embodiments, the internal standard solution includes a solution of senotevir-D4, a solution of ritonavir-D6 and a solution of urea- 13 C 15 N2 solution; The above-mentioned Xianuotewei-D4 solution is obtained by diluting the Xianuotewei-D4 stock solution with methanol aqueous solution; the above-mentioned Xianuotewei-D4 stock solution is a dimethyl sulfoxide solution of Xianuotewei-D4; The above-mentioned ritonavir-D6 solution is obtained by diluting the ritonavir-D6 stock solution with methanol aqueous solution; The above 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 was 50 μg / mL, the concentration of ritonavir-D6 was 0.5 μg / mL, and the concentration of urea- 13 C 15 The concentration of N2 was 100 μg / mL.

[0058] In some embodiments, the volume ratio between the sample to be tested and the internal standard solution is (50-150):10.

[0059] In these embodiments, the present application discloses that the volume ratio between the above-mentioned sample to be tested and the above-mentioned 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 of the ranges of any two of the above.

[0060] In some embodiments, the urea dilution method comprises following the following 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 Refers to the concentration of urea in the blood.

[0061] In some embodiments, the standard curve working solution is obtained by adding PBS buffer to a series of concentrations of standard solutions; The above series of concentrations includes at least 8; In the above standard curve working solution, the concentration of senotevir 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.

[0062] The present application discloses in these embodiments that the concentration of senotevir 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 of the values ​​satisfying any two of the above ranges.

[0063] The present application discloses in these embodiments that 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 of the values ​​satisfying any two of the above ranges.

[0064] In these embodiments, the present application discloses that 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 of the ranges satisfying any two of the above values.

[0065] To make the purpose, technical solution and advantages of the present application clearer, the interference of the secondary battery protected by the present application is described in detail below in conjunction with specific embodiments. The chemical substances, reagents, etc. used in the following embodiments are all of any conventional models and manufacturers in the art.

[0066] Example 1 A method for preparing a standard curve working solution, a horizontal quality control working solution and an internal standard solution is provided, comprising the following process: 1. Preparation of mobile phase solution: 1. Prepare mobile phase A: 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, the amount of each component added must be reduced proportionally when reducing the amount). Use ultrasonic vibration for 5 minutes to expel the gas and then use it. Store at 15℃~25℃ and the shelf life is one week.

[0067] 2. Prepare mobile phase B: Take 500 mL of acetonitrile and 500 mL of isopropanol and mix them evenly (take the preparation of 1 L of mobile phase B as an example, reduce the amount of each component added proportionally when reducing the amount). Use ultrasonic vibration for 5 minutes to expel the gas and then use it. Store at 15℃~25℃ and the shelf life is one month.

[0068] 2. Preparation of working fluid: Simnotrelvir, Simnotrelvir-D4, Ritonavir, Ritonavir-D6, Urea, and Urea- 13 C 15 N2 (Urea- 13 C 15 N2) were purchased from Tianjin Alta Technology Co., Ltd.

[0069] 1. Prepare internal standard solution: Dimethyl sulfoxide was used to dissolve sennotevir-D4 to obtain sennotevir-D4 stock solution, and 50% methanol aqueous solution was used as a diluent to prepare a sennotevir internal standard solution with a concentration of 200 μg / mL; 50% methanol aqueous solution was used as a diluent to prepare a ritonavir internal standard (ritonavir-D6) solution with a concentration of 2 μg / mL; urea- 13 C 15 N2 to urea - 13 C 15 N2 stock solution, and use pure water as a diluent to prepare a urea internal standard solution with a concentration of 200 μg / mL. The sennotevir internal standard solution, ritonavir internal standard solution, and urea internal standard solution are mixed in a ratio of 1:1:2 to obtain an internal standard solution with a final concentration of 50 μg / mL of sennotevir, a final concentration of 0.5 μg / mL of ritonavir, and a concentration of 100 μg / mL of urea.

[0070] 2. Prepare a series of concentrations of sennotevir standard solution (XS1-XS8), a series of concentrations of ritonavir standard solution (RS1-RS8) and a series of concentrations of urea standard solution (US1-US8).

[0071] (1) Prepare a series of concentrations of senotevir standard solutions (XS1-XS8): XS8: 50 μL of 100 μg / mL senotetravir, final concentration is 100 μg / mL.

[0072] XS7: 50 μL XS8 + 200 μL 50% methanol, final concentration is 20 μg / mL.

[0073] XS6: 50 μL XS7 + 50 μL 50% methanol, final concentration is 10 μg / mL.

[0074] XS5: 50 μL XS6 + 200 μL 50% methanol, the final concentration is 2 μg / mL.

[0075] XS4: 50 μL XS5 + 50 μL 50% methanol, final concentration is 1 μg / mL.

[0076] XS3: 50 μL XS4 + 200 μL 50% methanol, final concentration is 200 ng / mL.

[0077] XS2: 50 μL XS3 + 50 μL 50% methanol, final concentration is 100 ng / mL.

[0078] XS1: 50 μL XS2 + 50 μL 50% methanol, final concentration is 50 ng / mL.

[0079] (2) Prepare a series of concentrations of ritonavir standard solutions (RS1-RS8): RS8: 50 μL ritonavir (20 μg / mL), final concentration 20 μg / mL.

[0080] RS7: 50 μL RS8 + 50 μL 50% methanol, the final concentration is 10 μg / mL.

[0081] RS6: 50 μL RS7 + 75 μL 50% methanol, the final concentration is 4 μg / mL.

[0082] RS5: 50 μL RS6 + 150 μL 50% methanol, the final concentration is 1 μg / mL.

[0083] RS4: 50 μL RS5 + 200 μL 50% methanol, the final concentration is 200 ng / mL.

[0084] RS3: 50 μL RS4 + 200 μL 50% methanol, the final concentration is 40 ng / mL.

[0085] RS2: 50 μL RS3 + 150 μL 50% methanol, the final concentration is 10 ng / mL.

[0086] RS1: 50 μL RS2 + 200 μL 50% methanol, the final concentration is 2 ng / mL.

[0087] (3) Prepare urea standard solutions (US1-US8) of different concentrations: US8: 50 μL urea (concentration is 20 mg / mL), the final concentration is 20 mg / mL.

[0088] US7: 50 μL US8+75 μL H2O, final concentration is 8 mg / mL.

[0089] US6: 50 μL US7+150 μL H2O, final concentration is 2 mg / mL.

[0090] US5: 50 μL US6+75 μL H2O, final concentration is 800 μg / mL.

[0091] US4: 50 μL US5 + 150 μL H2O, the final concentration is 200 μg / mL.

[0092] US3: 50 μL US4 + 50 μL H2O, the final concentration is 100 μg / mL.

[0093] US2: 50 μL US3 + 75 μL H2O, the final concentration is 40 μg / mL.

[0094] US1: 50 μL US2 + 50 μL H2O, the final concentration is 20 μg / mL.

[0095] 3. Prepare the standard curve working solution: To 170 μL PBS, add 10 μL of senotevir standard (XS1-XS8) solution, 10 μL of ritonavir standard (RS1-RS8) solution, and 10 μL of urea standard (US1-US8) solution, respectively, to obtain 200 μL of standard curve working solution (STD1-STD8). The concentrations of senotevir in the standard curve working solution were 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, 1000 ng / mL, and 5000 ng / mL, respectively; the concentrations of ritonavir were 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively; and the concentrations of urea were 1 μg / mL, 2 μg / mL, 5 μg / mL, 10 μg / mL, 40 μg / mL, 100 μg / mL, 400 μg / mL, and 1000 μg / mL, respectively.

[0096] 4. Prepare quality control working solution: 10 μL of senotevir standard (XS3 / XS5 / XS7) solution, 10 μL of ritonavir standard (RS3 / RS5 / RS7) solution and 10 μL of urea standard (US3 / US5 / US7) solution were added to 170 μL PBS to obtain 200 μL of quality control (QCL / QCM / QCH) working solution. The concentrations of senotevir in the quality control working solution were 10 ng / mL, 100 ng / mL and 1000 ng / mL, the concentrations of ritonavir were 2 ng / mL, 50 ng / mL and 500 ng / mL, and the concentrations of urea were 5 μg / mL, 40 μg / mL and 400 μg / mL.

[0097] In the embodiments of the present application, the linearity 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.

[0098] Example 2 A sample pretreatment method is provided, comprising the following process: (1) Pretreatment of standard curve working solution: Take a 1.5 mL centrifuge tube, add 200 μL standard curve working solution, 20 μL internal standard solution, and 400 μL 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13,000 rpm for 10 min; take 200 μL of supernatant (1) and transfer it to a 96-well plate for testing; (2) Pretreatment of horizontal quality control working solution: Take a 1.5 mL centrifuge tube, add 200 μL quality control working solution, 20 μL internal standard solution, and 400 μL 100% acetonitrile in sequence, vortex for 10 min, and centrifuge at 13,000 rpm for 10 min; take 200 μL of supernatant (2) and transfer it to a 96-well plate for testing; (3) Pretreatment of serum samples: 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 13,000 rpm for 10 min; take 200 μL of the supernatant (3) and transfer it to a 96-well plate for testing; (4) Pretreatment of bronchoalveolar lavage fluid: Take a 1.5 mL centrifuge tube and 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 13,000 rpm for 10 min. Take 200 μL of the supernatant (4) and transfer it to a 96-well plate for testing.

[0099] Example 3 Provide a liquid chromatography tandem mass spectrometry condition: 1. Liquid chromatography conditions are shown in Table 1: Table 1 Liquid chromatography conditions

[0100] 2. Mass spectrometry conditions are shown in Table 2: Table 2 List of mass spectrometry conditions

[0101] 3. The ion pair parameters are shown in Table 3: Table 3 Ion pair parameter list

[0102] The * in the above table indicates the quantitative ion pair.

[0103] In order to achieve more sensitive detection and faster detection speed, the chromatographic column used in this application is Kinetex F5 column (3 mm × 100 mm, 2.6 μm, Phenomenex) and isocratic elution is performed under the chromatographic conditions shown in Table 1. At the same time, a comparative test description is also carried out in this application.

[0104] The chromatographic column was Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex) and under the chromatographic conditions shown in Table 1, the samples were subjected to the same pretreatment and it was found that when the mobile phase B was a mixed solution of acetonitrile and isopropanol (50:50), compared with the mobile phase A being a 10mM ammonium acetate aqueous solution, when the mobile phase A was a 20mM ammonium acetate aqueous solution, the results are shown in the appendix of the instruction manual. Figure 7 and 8 , it can be found that the response intensity of sennotervir and ritonavir decreased, and the detection sensitivity decreased. The lower concentration of salt in the mobile phase also helps to protect the detection system, so our final detection scheme used 8mmol / L~12mmol / L ammonium acetate aqueous solution as mobile phase A.

[0105] Under the chromatographic conditions (except the chromatographic column) shown in Table 1, where mobile phase A was 10 mM ammonium acetate aqueous solution and mobile phase B was acetonitrile:isopropanol (50:50) mixed solution, the samples were subjected to the same pretreatment and it was found that: if the chromatographic column was XBridge C18column (2.1×100 mm, 3.5 μm, Waters), the results are shown in the attached figure of the specification. Figures 12 to 14 , see the attached figure for comparison Figures 9 to 11(The chromatographic column is Kinetex F5 column). It can be seen that the response intensity of senotevir is reduced, and there is a delayed peak, which affects the quantification; the response intensity of urea is low, the peak shape is poor, and its baseline is high, which cannot be accurately quantified; if the chromatographic column is XBridge BEH C8column (2.1×100mm, 2.5μm, Waters), the results are shown in the attached figure of the specification Figures 15 to 17 , see the attached figure for comparison Figures 9 to 11 (The chromatographic column is Kinetex F5 column). It can be seen that the response intensity of senotevir is reduced; the response intensity of urea is low, the peak shape is poor, and its baseline is high, which cannot be accurately quantified. Therefore, the chromatographic column of this application is Kinetex F5 column (3mm×100mm, 2.6μm, Phenomenex) with better effect (Figure Figures 9 to 11 ).

[0106] Example 4 An instrument analysis procedure is provided, comprising the following steps: 1. Turn on the computer power, enter the Windows operating system, check whether the instrument is in good condition, log in to the Analyst software, and enter the workstation main interface.

[0107] 2. Double-click the Hardware configuration button, click LCMS, and then click Activate profile on the right to activate the instrument. The LCMS module is displayed as a green check mark, indicating that the connection is successful.

[0108] 3. Double-click Build Acquistion Batch in the Acquire column, select the established method, and edit the sample sequence (sample number, sample tray position, injection volume, etc.) in the sequence editing menu bar.

[0109] 4. Click Equilibrate in the menu bar, select the previously established LC / MS method to be run, and set the equilibrium time to 10 to 20 minutes.

[0110] 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.

[0111] 6. After the equilibrium time is over, the instrument status in the lower right corner of the main interface is displayed as Ready. Click Start sample in the menu bar to start sample analysis and data acquisition.

[0112] 7. Double-click the Open Date File option in the Explore option on the left to view the chromatogram image during the detection process at any time.

[0113] 8. After sample analysis and collection, use Mμltiquant to create a quantitative list and perform quantitative analysis.

[0114] Standby steps 1. Click the Standby button in the menu bar, and you can see the flow rate slowly decreases to zero. After the system pressure drops to 0, double-click Hardware configuration, click LCMS in the pop-up dialog box, and then click Deactivateprofile on the right to deactivate the liquid chromatography-mass spectrometry. The LCMS module is displayed as a cross, and you can disconnect.

[0115] 2. Exit the Analyst software and shut down the computer.

[0116] Example 5 A method for verifying the accuracy of the above method is provided, comprising the following process: 1. Linear A series of serial dilutions of standard solutions of known concentrations were performed (the concentrations of senotevir 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, and the concentrations of ritonavir were 1000 ng / mL, 500 ng / mL, 200 ng / mL, 50 ng / mL, 10 ng / mL, 2.5 ng / mL). ng / mL, 0.5ng / mL, 0.1ng / mL, urea concentrations were 1000μg / mL, 400μg / mL, 100μg / mL, 40μg / mL, 10μg / mL, 5μg / mL, 2μg / mL, 1μg / mL), an equal amount of internal standard solution was added, and the calibration points were fitted by the least squares method to form a calibration curve with a linear requirement of r>0.99. The deviation between the value of each concentration point and the theoretical value was less than 15%, and the deviation between the detected value and the theoretical value at the limit of quantification (LoQ) point was less than 20%. The linear range of sennotvir was 2.5~5000 ng / mL, LoQ=2.5 ng / mL, and the linear range of ritonavir was 0.1~1000 ng / mL, LoQ=0.1 ng / mL, the linear range of urea is 1-1000μg / mL, LoQ=1μg / mL, the standard curve of senotevir is r=0.99836, the linearity is good, and the LoQ point is accurate. Figure 1 and Figure 4 As shown; the standard curve of ritonavir is r=0.99982, with good linearity and accurate quantification at the LoQ point. Figure 2 and Figure 5As shown; urea standard curve r = 0.99997, good linearity, LoQ point quantification is accurate, such as Figure 3 and Figure 6 As shown, it is sufficient to show that the detection sensitivity and linearity of the present application are good and can meet the needs of clinical testing and scientific research.

[0117] 2. Precision and carryover The prepared low, medium and high quality control working solutions were tested three times for three consecutive days, and the intra-day precision coefficients of variation of Xiannotervir were calculated to be 4.39%, 4.32% and 0.65%, and the inter-day precision coefficients of variation were 6.17%, 5.99% and 5.18% respectively (see Table 4 for details); the intra-day precision coefficients of variation of ritonavir were 1.85%, 3.51% and 1.32%, and the inter-day precision coefficients of variation were 5.25%, 5.42% and 4.63% respectively (see Table 5 for details); the intra-day precision coefficients of variation of urea were 2.09%, 0.59% and 0.90%, and the inter-day precision coefficients of variation were 4.50%, 1.33% and 2.60% respectively (see Table 6 for details). The intra-day and inter-day precision coefficients of variation of Xiannotervir, 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. The signal responses of the blank samples were all less than 0.1% of the highest point of the calibration curve, as shown in Table 7, which proves that the detection method of this application has no significant carryover contamination.

[0118] Table 4 Detection precision of senotevir

[0119] Table 5 Detection precision of ritonavir

[0120] Table 6 Detection precision of urea

[0121] Table 7 Carryover rate of senotevir, ritonavir and urea

[0122] 3. Accuracy and matrix effects: Spiked recovery rate: Take at least 5 samples from different sources, select an equal amount of serum / alveolar lavage fluid from each sample and mix them, then select 4 equal parts from the mixed serum / alveolar lavage fluid, add low / medium / high value standard solutions of known concentration to 3 of them, respectively, and perform the same pretreatment and injection analysis process, parallel determination three times, and calculate the spiked recovery rate using the formula % = (measured spiked sample concentration - measured matrix sample concentration) / theoretical spiked concentration * 100%, and obtain the data in Table 8. The serum spiked recovery rate was 85.70%~97.62%, and the bronchoalveolar lavage fluid spiked recovery rate was 86.09%~97.39%; the serum spiked recovery rate of ritonavir was 104.90%~107.10%, and the bronchoalveolar lavage fluid spiked recovery rate was 89.71%~97.59%; the serum spiked recovery rate of urea was 95.08%~101.73%, and the bronchoalveolar lavage fluid spiked recovery rate was 98.05%~100.16%, indicating that the detection method of this application has good accuracy.

[0123] Matrix effect: Take at least 5 samples from different sources, select an equal amount of serum / alveolar lavage fluid from each sample and mix them, then select 4 equal parts from the mixed serum / alveolar lavage fluid, add low, medium and high value standard solutions of known concentration to 3 of them, perform the same pretreatment and injection analysis process, and measure three times in parallel to obtain the average signal intensity of each concentration point (A). Replace the serum / alveolar lavage fluid with PBS solution, prepare the corresponding low, medium and high concentration level standard solutions with the same injection reagent, inject and analyze, and obtain the average signal intensity of each concentration point in the pure solution (B). At the same time, prepare blank serum / alveolar lavage fluid samples, measure three times in parallel, and obtain the average signal intensity of blank serum / alveolar lavage fluid samples (C). Calculate the area ratio of the peak area of ​​the analyte to the internal standard in each group of samples. By calculating the formula matrix effect = (area ratio of sample A-area ratio of sample C) / area ratio of sample B*100%, the serum matrix effects of sennotvir were 89.51%, 95.15%, and 85.67%, respectively, and the bronchoalveolar lavage fluid matrix effects were 89.92%, 94.94%, and 89.45%, respectively; the serum matrix effects of ritonavir were 108.93%, 107.04%, and 103.98%, respectively, and the bronchoalveolar 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 bronchoalveolar lavage fluid matrix effects were 98.41%, 93.46%, and 99.61%, respectively, as shown in Table 8, which met the experimental requirements.

[0124] Table 8 Recovery and matrix effect of senotevir and ritonavir

[0125] 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 sennotevir, ritonavir and urea in blood and bronchoalveolar lavage fluid, and then calculate the concentrations of sennotevir and ritonavir in the alveolar epithelial lining fluid, thereby realizing the monitoring of sennotevir and ritonavir therapeutic drugs, laying the foundation for drug monitoring, safe use and personalized precision treatment.

[0126] 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 aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents; 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 included in the scope of the claims and specification of the present application. In particular, as long as there is no structural conflict, the various technical features mentioned in the various embodiments can be combined in any way. The present application is not limited to the specific embodiments disclosed herein, but includes all technical solutions that fall 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 samples to be tested, wherein the samples to be tested include blood to be tested and bronchoalveolar lavage fluid to be tested, adding internal standard substances and precipitants to the blood to be tested and the bronchoalveolar lavage fluid to be tested, respectively, and subjecting the blood to be tested and the bronchoalveolar lavage fluid to be tested to obtain supernatants as injection samples by centrifugation; Detection concentration: Detect the injected sample by liquid chromatography-mass spectrometry, and determine the concentrations of senotevir, 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; 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: The precipitant comprises acetonitrile; The volume ratio between the sample to be tested and the precipitant is 1:(1-3).

3. The detection method according to any one of claims 1 to 2, characterized in that Liquid chromatography conditions included: Mobile phase: comprising phase A and phase B, wherein the volume ratio of phase A to phase B is (40-60): (60-40); The phase A comprises an aqueous solution of ammonium acetate, and the concentration of the aqueous solution of ammonium acetate is 8 mmol / L to 12 mmol / L; The phase B comprises a mixture of acetonitrile and isopropanol, and the volume ratio of the acetonitrile to the isopropanol is (40-60): (60-40); Isocratic elution: The flow rate of the mobile phase is 0.3 mL / min to 0.8 mL / min.

4. The detection method according to any one of claims 1 to 2, characterized in that Liquid chromatography conditions included: The analytical columns used included Phenomenex Kinetex F5; The column temperature of the analytical chromatographic column is 35°C-40°C.

5. The detection method according to any one of claims 1 to 2, characterized in that The conditions for mass spectrometry include: Atomization temperature is 180℃~220℃; The ion spray voltage was 4500 V (MRM+) and −4500 V (MRM−); The atomizing gas is 45psi~55psi, the auxiliary heating gas is 55psi~65psi, the curtain gas is 35psi~45psi, and the collision gas is 8psi~10psi.

6. The detection method according to any one of claims 1 to 2, 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.

7. The detection method according to any one of claims 1 to 2, 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.

8. The detection method according to claim 7, characterized in that: The volume ratio between the sample to be tested and the internal standard solution is (50-150):

10.

9. The detection method according to any one of claims 1 to 2, 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 Refers to the concentration of urea in the blood.

10. The detection method according to any one of claims 1 to 2, 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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