A method for detecting maraviroc and its metabolites in serum and application

Abstract

The application belongs to the technical field of analysis and detection, and particularly relates to a detection method and application of maraviroc and metabolites thereof in serum. The method comprises adding an internal standard solution and an extractant to a serum sample to be detected or a standard curve working solution, solid-liquid separation, then taking supernatant for nitrogen blowing, after nitrogen blowing, using a redissolving agent for redissolution, taking supernatant for liquid chromatography tandem mass spectrometry analysis, and calculating the content of maraviroc and metabolites thereof in the serum sample to be detected; the liquid chromatography tandem mass spectrometry method comprises: using 0.15-0.25 mM ammonium fluoride aqueous solution as mobile phase A and methanol as mobile phase B for gradient elution. The detection method can quickly, simply and accurately detect the concentration level of maraviroc and metabolites thereof in serum at the same time, and realizes the simultaneous therapeutic drug monitoring of maraviroc and metabolites thereof in serum.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology, and specifically relates to a method and application for detecting marsurasavirin and its metabolites in serum. Background Technology

[0002] In recent years, seasonal influenza and influenza pandemics caused by influenza viruses have posed a serious threat to public health. Currently, influenza viruses are generally resistant to matrix protein 2 ion channel (M2) inhibitors, and resistance to neuraminidase inhibitors (such as oseltamivir) has also been reported. Therefore, marsurasavir, as an anti-influenza drug that targets the PA subunit of influenza virus RNA polymerase to achieve its antiviral purpose, is of great significance for influenza patients, especially those with drug resistance.

[0003] Masuraxavir, chemical formula: C 29 H 25 F2N3O7S is an orally administered small molecule drug that exerts its antiviral effect by inhibiting the PA subunit of influenza virus RNA polymerase. Preclinical studies have shown that marsuravir has nanomolar inhibitory activity against both influenza A and B viruses. Clinical trials have shown that a single dose of marsuravir significantly shortens the time to symptom relief and rapidly reduces viral load. Furthermore, marsuravir has a good safety profile, with most adverse events being mild or moderate, and a low incidence of drug resistance mutations. Although the clinical efficacy and safety of marsuravir have been validated, further evaluation of its pharmacokinetic characteristics in humans is still needed. After oral administration, marsuravir is rapidly converted to the active metabolite GP1707D07 (marsuravir metabolite GP1707D07, chemical formula: C). 26 H 21 (F2N3O4S) has a long plasma elimination half-life; even 5 days after administration, the average concentration of GP1707D07 in patient plasma remains higher than its 90% effective concentration. However, standardized detection methods for marsurasavir and its metabolites are currently lacking, particularly quantitative analysis techniques in biological matrices such as serum. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for detecting marsurasavir and its metabolites in serum, so as to establish an analytical method that can rapidly and accurately detect the concentration of marsurasavir and its metabolites in serum.

[0005] The first aspect of this invention provides a method for detecting marsurasavir and its metabolites in serum, comprising the following steps: 1) Mix the standard stock solutions of marsurasavir and marsurasavir metabolites with blank serum to obtain a series of standard curve working solutions; 2) Add the internal standard solution and extractant to the serum sample to be tested and the standard curve working solution respectively, separate the solid and liquid, and then take the supernatant for nitrogen blowing. After nitrogen blowing, reconstitute with a reconstituter and take the supernatant for liquid chromatography-tandem mass spectrometry analysis to calculate the content of marsurasavir and marsurasavir metabolites in the serum sample to be tested. The liquid chromatography-tandem mass spectrometry analysis used a Synergi™ 4 µm Fusion-RP 80 Å column, with gradient elution using 0.15–0.25 mM ammonium fluoride aqueous solution as mobile phase A and methanol as mobile phase B.

[0006] The blank serum sample refers to a serum sample that does not contain masuraxavir or its metabolites; the serum sample to be tested is a serum sample taken after taking a drug preparation containing masuraxavir.

[0007] The metabolite of masuraxavir of this invention is GP1707D07, with the chemical formula: C 26 H 21 F2N3O4S, the specific structural formula is as follows: .

[0008] The supernatant of the present invention is treated with nitrogen blowing. After nitrogen blowing, only a small amount of substance remains on the bottom of the tube. This substance contains the analyte. Then, a reconstitution solution is directly added, and this substance will dissolve in the reconstitution solution.

[0009] In some optional embodiments, the liquid chromatography conditions described in step 2) further include a Synergi™ 4 µm Fusion-RP 80 Å column with an inner diameter of 2.0 mm, a column length of 50 mm, a particle size of 4 μm, a column temperature of 35-45 °C, and an injection volume of 15-25 μL. And / or, in step 2), the liquid chromatography uses a gradient elution process by mixing different volumes of mobile phase A and mobile phase B. The gradient elution process includes: From 0 to 1.0 min, the volume content of mobile phase A was maintained at 90%, the volume content of mobile phase B was maintained at 10%, and the flow rate was 0.8 mL / min; From 1.0 min to 1.5 min, the volume content of mobile phase A was adjusted from 90% to 2%, and the volume content of mobile phase B was adjusted from 10% to 98%, with a flow rate of 0.8 mL / min. From 1.5 min to 3.0 min, the volume content of mobile phase A was maintained at 2%, the volume content of mobile phase B was maintained at 98%, and the flow rate was 0.8 mL / min; From 3.0 min to 3.1 min, the volume content of mobile phase A was adjusted from 2% to 90%, and the volume content of mobile phase B was adjusted from 98% to 10%, with a flow rate of 0.8 mL / min. From 3.1 min to 4.0 min, the volume content of mobile phase A was maintained at 90%, the volume content of mobile phase B was maintained at 10%, and the flow rate was 0.8 mL / min.

[0010] In some alternative implementations, The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: Under positive ion monitoring, the ion spray voltage is 5000-5500 V, the temperature is 350-450℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 50-70 psi, the air curtain gas pressure is 10-30 psi, and the collision gas pressure is 5-15 psi; under negative ion monitoring, the ion spray voltage is -4000 to -5000 V, the temperature is 350-450℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 50-70 psi, the air curtain gas pressure is 10-30 psi, and the collision gas pressure is 5-15 psi.

[0011] In some optional embodiments, the standard stock solution of masuraxavir and masuraxavir metabolites mentioned in step 1) is obtained by mixing masuraxavir solution and masuraxavir metabolite solution with methanol aqueous solution; Optionally, the standard stock solution of masuraxavir and masuraxavir metabolites is a series of standard stock solutions of masuraxavir and masuraxavir metabolites at different concentrations. Optionally, the concentration range of masuraxavir or masuraxavir metabolites in the series of concentrations of masuraxavir and masuraxavir metabolite standard stock solutions is 10 ng / mL to 10000 ng / mL; Optionally, the concentrations of methuraxavir or methuraxavir metabolites in the series of methuraxavir and methuraxavir metabolite standard stock solutions are respectively 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, 1000 ng / mL, 5000 ng / mL, and 10000 ng / mL.

[0012] Optionally, the volume percentage of methanol in the methanol-water solution is 40-60%; In some alternative embodiments, the marsurasavir solution is formed by dissolving marsurasavir in dimethyl sulfoxide, and the marsurasavir metabolite solution is formed by dissolving marsurasavir metabolites in dimethyl sulfoxide.

[0013] Optionally, the mass concentration of maturaxavir in the maturaxavir solution is 50 ng / mL, and the mass concentration of maturaxavir metabolites in the maturaxavir metabolite solution is 100 ng / mL.

[0014] In some optional embodiments, the internal standard solution in step 2) is a solution containing ritonavir-D6; The concentration of ritonavir-D6 in the internal standard solution was 0.5-2 μg / ml; And / or, the extractant is a mixed solution of ethyl acetate and n-hexane in a volume ratio of (80-90):(10-20); And / or, the complex solvent is a methanol aqueous solution with a methanol volume percentage of 40-60%; And / or, the volume ratio of the resolvent to the supernatant before nitrogen blowing is 1:(7-9).

[0015] In some optional embodiments, the volume ratio of the serum sample to be tested or the standard curve working solution, the internal standard solution and the extractant in step 2) is (270-330):(9-11):(900-1100). And / or, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: Under positive ion monitoring, the ion spray voltage was 5500 V, the temperature was 400 ℃, the atomizing gas pressure was 50 psi, the auxiliary heating gas pressure was 60 psi, the air curtain gas pressure was 20 psi, and the collision gas pressure was 10 psi; under negative ion monitoring, the ion spray voltage was -4500 V, the temperature was 400 ℃, the atomizing gas pressure was 50 psi, the auxiliary heating gas pressure was 60 psi, the air curtain gas pressure was 20 psi, and the collision gas pressure was 10 psi.

[0016] In some optional implementations, calculating the levels of marsurasavir and its metabolites in the serum sample to be tested includes: The peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the standard curve working solution were collected; the peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the serum samples to be tested were also collected. A standard curve for masuraxavir was plotted with the concentration of masuraxavir in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​masuraxavir in the working solution to the peak area of ​​the internal standard as the ordinate. The linear regression equation of masuraxavir was then obtained. The ratio of the peak area of ​​masuraxavir to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation of masuraxavir to obtain the content of masuraxavir in the serum sample to be tested. A standard curve for rasurasavir metabolites was plotted with the concentration of rasurasavir metabolites in the standard curve working solution as the x-axis and the ratio of the peak area of ​​rasurasavir metabolites in the standard curve working solution to the peak area of ​​the internal standard as the y-axis. The linear regression equation for rasurasavir metabolites was then obtained. The ratio of the peak area of ​​rasurasavir metabolites to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation for rasurasavir metabolites to obtain the content of rasurasavir metabolites in the serum sample to be tested.

[0017] In some alternative implementations, the series of concentrations in step 1) includes at least 8 concentrations; And / or, the concentration range of masuraxavir or masuraxavir metabolites in the standard curve working solutions of the series of concentrations is 1-1000 ng / mL.

[0018] In some optional embodiments, the concentrations of masuraxavir or masuraxavir metabolites in the standard curve working solutions of the series of concentrations sequentially include 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL.

[0019] In some optional embodiments, step 1) further includes mixing a blank serum sample with a marsuravir solution and a marsuravir metabolite solution to prepare quality control working solutions with marsuravir or marsuravir metabolite concentrations of 10 ng / mL, 50 ng / mL, and 500 ng / mL. The quality control working solutions are then treated in the same way as the standard curve working solutions, and the supernatant is then taken for liquid chromatography-tandem mass spectrometry analysis.

[0020] A second aspect of the present invention also provides an application of the above-described method, the application comprising detecting marsurasavir and / or marsurasavir metabolites in human serum.

[0021] Compared with the prior art, the technical solution of the present invention has at least the following advantages: The present invention provides a method for detecting marsurasavir and its metabolites in serum. It utilizes the high separation performance of liquid chromatography and the high selectivity and high sensitivity of mass spectrometry to establish a quantitative analysis method of liquid chromatography-tandem mass spectrometry. This method can rapidly, simply, and accurately detect the concentration levels of marsurasavir and its metabolites in serum, realize therapeutic drug monitoring (TDM) of marsurasavir and its metabolites, and achieve personalized precision treatment.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the standard curve of masuraxavir (GP681) in serum in Example 1; Figure 2 This is a schematic diagram of the standard curve of the metabolite of marsurasavirin (GP1707D07) in serum in Example 1; Figure 3 In Example 1 of this invention, when the final concentrations of masuraxavir and its metabolites in the standard curve working solution were 1 ng / mL, the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 were obtained from the standard curve working solution at the limit of quantitation (LoQ) point. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0025] Figure 4 When the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Example 1 were 1000 ng / mL, the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0026] Figure 5 To obtain the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 when the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Comparative Example 1 were 1000 ng / mL, the following chromatograms were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0027] Figure 6 To obtain the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 when the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Comparative Example 2 were 1000 ng / mL, the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0028] Figure 7 To obtain the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 when the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Comparative Example 3 were 1000 ng / mL, the following chromatograms were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0029] Figure 8 To obtain the chromatograms of the peak substances of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 when the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Comparative Example 4 were 1000 ng / mL, the chromatograms of the peak substances of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6.

[0030] Figure 9 To obtain the chromatograms of the peaks of masuraxavir and its metabolites and the internal standard peak of ritonavir-D6 when the final concentrations of masuraxavir and its metabolites in the standard curve working solution of Comparative Example 5 were 1000 ng / mL, the following chromatograms were obtained. Figure (A) shows the chromatogram of the peaks of masuraxavir, Figure (B) shows the chromatogram of the peaks of the metabolites of masuraxavir, and Figure (C) shows the chromatogram of the internal standard peak of ritonavir-D6. Detailed Implementation

[0031] Exemplary embodiments of this disclosure will now be described in more detail. It should be understood that this disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this disclosure and to fully convey its scope to those skilled in the art. Any product identical or similar to this invention, derived by any person based on the teachings of this invention or by combining features of this invention with other prior art, falls within the protection scope of this invention.

[0032] The technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are all conventional methods.

[0033] In recent years, seasonal influenza and influenza pandemics caused by influenza viruses have posed a serious threat to public health. Currently, influenza viruses are generally resistant to M2 ion channel inhibitors, and resistance to neuraminidase inhibitors (such as oseltamivir) has also been reported from time to time. Therefore, marsurasavir, as a PA inhibitor anti-influenza drug, achieves its antiviral purpose by targeting the PA subunit of influenza virus RNA polymerase, and it is of great significance for influenza patients, especially those with drug resistance.

[0034] Masuraxavir, chemical formula: C 29 H 25 F2N3O7S is an orally administered small molecule drug that exerts its antiviral effect by inhibiting the PA subunit of influenza virus RNA polymerase. Preclinical studies have shown that marsurazavir has nanomolar inhibitory activity against both influenza A and B viruses. Clinical trials have shown that a single dose of marsurazavir significantly shortens the time to symptom relief and rapidly reduces viral load. Furthermore, marsurazavir has a good safety profile, with most adverse events being mild or moderate, and a low incidence of drug resistance mutations. Although the clinical efficacy and safety of marsurazavir have been validated, further evaluation of its pharmacokinetic characteristics in humans is still needed. After oral administration, marsurazavir is rapidly converted to the active metabolite GP1707D07 (GP1707D07, chemical formula: C...). 26 H 21 GP1707D07 (F2N3O4S) has a long plasma elimination half-life; even 5 days after administration, the average concentration of GP1707D07 in patient plasma remains higher than its 90% effective concentration. However, standardized detection methods for marsurasavir and its metabolites are currently lacking, particularly quantitative analysis techniques for marsurasavir and its metabolites in biological matrices such as serum.

[0035] Therapeutic drug monitoring (TDM), which involves detecting drug concentrations in bodily fluids, is of great significance in clinical treatment. TDM guides clinicians to adjust dosing regimens based on changes in blood drug concentrations and pharmacokinetic parameters. This helps improve drug efficacy while reducing or avoiding toxic reactions. TDM is particularly important for drugs with a narrow therapeutic index and for drugs whose toxic symptoms are easily confused with the disease itself. Through TDM, clinicians can more accurately determine the dosage and frequency of administration, thereby improving treatment efficacy and success rates. For marsurasavir, a highly sensitive and specific detection method needs to be established for TDM to support its clinical use optimization and resistance management.

[0036] Currently, liquid chromatography (LC) is considered the gold standard for total molecular weight microscopy (TDM), typically combined with tandem mass spectrometry (MS / MS). LC-MS / MS is a combined detection method using liquid chromatography as the separation system and mass spectrometry as the detection system. The sample is separated from the mobile phase in the mass spectrometer, ionized, and then the mass spectrometer separates the ion fragments according to their mass-to-charge ratio. The mass spectrum is then obtained by the detector. LC-MS / MS offers high selectivity and sensitivity and is increasingly widely used in clinical applications, capable of detecting many biomarkers that are undetectable by traditional methods.

[0037] Therefore, this invention establishes an analytical method based on LC-MS / MS technology that enables the simultaneous, rapid, and accurate determination of the concentrations of marsurasavirin and its metabolites in human serum. This method, through liquid-liquid extraction pretreatment combined with optimized chromatographic and mass spectrometric conditions, achieves precise quantification of the target drug, providing crucial evidence for personalized clinical drug administration.

[0038] The preparation methods for the mobile phase solution, standard solution, internal standard solution, standard stock solution, standard curve working solution, and quality control working solution used in all embodiments and comparative examples of this invention include: I. Preparation of mobile phase solution 1. Prepare mobile phase A.

[0039] Add 2 mL of 100 mM ammonium fluoride to a 1000 mL volumetric flask and dilute to volume with ultrapure water (taking the preparation of 1 L of mobile phase A as an example; when reducing the volume, the amount of each component added must be reduced proportionally). After ultrasonically shaking for 5 minutes to remove the gas, it is ready for use. Store at 20℃~25℃ and the shelf life is one week.

[0040] 2. Prepare mobile phase B.

[0041] Take 1000 mL of methanol (taking the preparation of 1 L of mobile phase B as an example, reduce the amount of each component added proportionally when preparing the reduced volume), and use it after ultrasonic vibration for 5 minutes to remove the gas. Store at 20℃~25℃, and the shelf life is one month.

[0042] II. Preparation of working solution 1. Prepare standard solutions and internal standard solutions.

[0043] Mesuravir and its metabolites were dissolved in dimethyl sulfoxide to prepare initial standard solutions for each. The concentration of GP681 in the initial standard solution of mesuravir was 50 μg / mL, and the concentration of GP1707D07 in the initial standard solution of mesuravir metabolites was 100 μg / mL. An internal standard solution (ritonavir-D6) with a concentration of 1 μg / mL was prepared using 50% (v / v) methanol solution as a diluent.

[0044] 2. Prepare stock solutions (S1-S8) of masuraxavir and its metabolites at various concentrations.

[0045] S8: 80 μL of initial standard solution of marsurasavir (GP681 concentration of 50 μg / mL) + 40 uL of initial standard solution of marsurasavir metabolite (GP1707D07 concentration of 100 μg / mL) + 280 uL of 50% (v / v) methanol solution, with final concentrations of marsurasavir and marsurasavir metabolite of 10000 ng / mL.

[0046] S7: 200 μL S8 + 200 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 5000 ng / mL.

[0047] S6: 100 μL S7 + 400 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 1000 ng / mL.

[0048] S5: 200 μL S6 + 200 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 500 ng / mL.

[0049] S4: 200 μL S5 + 300 μL 50% (v / v) methanol solution, with final concentrations of 200 ng / mL for both marsurasavir and its metabolites.

[0050] S3: 200 μL S4 + 200 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 100 ng / mL.

[0051] S2: 200 μL S3 + 200 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 50 ng / mL.

[0052] S1: 100 μL S2 + 400 μL 50% (v / v) methanol solution, with final concentrations of masuraxavir and masuraxavir metabolites of 10 ng / mL.

[0053] 3. Prepare the standard curve working solution. Add 30 μL of standard stock solution (S1-S8) to 270 μL of normal serum sample to obtain 300 μL of standard curve working solution (STD1-STD8). The final concentrations of masuraxavir and masuraxavir metabolites in the standard curve working solution are 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively.

[0054] 4. Prepare the quality control working solution.

[0055] Add 30 μL of standard stock solution (S3 / S5 / S7) to 270 μL of normal serum sample to obtain 300 μL of low, medium, and high quality control working solutions (QCL / QCM / QCH). The concentrations of marsurasavirin and its metabolites in the low, medium, and high quality control working solutions are 10 ng / mL, 50 ng / mL, and 500 ng / mL, respectively.

[0056] In this embodiment of the invention, the linearity r of the standard curve should be greater than 0.99, and the accuracy of the quality control working solution should be within ±15% of the theoretical value.

[0057] Example 1 This embodiment provides a method for detecting marsurasavir and its metabolites in serum, comprising the following steps: Take a 2 mL centrifuge tube and add 300 μL of standard curve working solution, 10 μL of internal standard solution, and 1000 μL of a mixed solution of ethyl acetate and n-hexane (volume ratio 85:15) in sequence. Vortex for 10 min and centrifuge at 12000 rpm for 10 min. Transfer 800 μL of supernatant to a 96-well plate and blow with nitrogen. Then add 100 μL of 50% (v / v) methanol solution, vortex for 10 min, centrifuge at 12000 rpm for 10 min, and transfer 90 μL of supernatant to a 96-well plate for analysis by liquid chromatography-tandem mass spectrometry.

[0058] Take a 2 mL centrifuge tube and add 300 μL of quality control working solution, 10 μL of internal standard solution, and 1000 μL of a mixed solution of ethyl acetate and n-hexane (volume ratio 85:15) in sequence. Vortex for 10 min and centrifuge at 12000 rpm for 10 min. Transfer 800 μL of supernatant to a 96-well plate and blow with nitrogen. Then add 100 μL of 50% (v / v) methanol solution, vortex for 10 min, centrifuge at 12000 rpm for 10 min, and transfer 90 μL of supernatant to a 96-well plate for analysis by liquid chromatography-tandem mass spectrometry.

[0059] Take a 2 mL centrifuge tube and add 300 μL of the serum to be tested, 10 μL of the internal standard solution, and 1000 μL of a mixed solution of ethyl acetate and n-hexane (volume ratio 85:15). Vortex for 10 min and centrifuge at 12000 rpm for 10 min. Transfer 800 μL of the supernatant to a 96-well plate and blow it with nitrogen. Then add 100 μL of 50% (v / v) methanol solution, vortex for 10 min, centrifuge at 12000 rpm for 10 min, and transfer 90 μL of the supernatant to a 96-well plate for analysis by liquid chromatography-tandem mass spectrometry.

[0060] The conditions for liquid chromatography-tandem mass spectrometry are as follows: 1. Liquid chromatography conditions are shown in Table 1.

[0061] Table 1 Summary of Liquid Chromatography Conditions

[0062] 2. Mass spectrometry conditions are shown in Table 2.

[0063] Table 2 Summary of mass spectrometry conditions

[0064] 3. Ion pair parameters are shown in Table 3.

[0065] Table 3 Summary of ion pair parameters

[0066] For quantitative ion pairs The calculation of the levels of marsurasavir and its metabolites in the serum sample to be tested includes: The peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the standard curve working solution were collected; the peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the serum samples to be tested were also collected. A standard curve for masuraxavir was plotted with the concentration of masuraxavir in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​masuraxavir in the working solution to the peak area of ​​the internal standard as the ordinate. The linear regression equation of masuraxavir was then obtained. The ratio of the peak area of ​​masuraxavir to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation of masuraxavir to obtain the content of masuraxavir in the serum sample to be tested. A standard curve for rasurasavir metabolites was plotted with the concentration of rasurasavir metabolites in the standard curve working solution as the x-axis and the ratio of the peak area of ​​rasurasavir metabolites in the standard curve working solution to the peak area of ​​the internal standard as the y-axis. The linear regression equation for rasurasavir metabolites was then obtained. The ratio of the peak area of ​​rasurasavir metabolites to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation for rasurasavir metabolites to obtain the content of rasurasavir metabolites in the serum sample to be tested.

[0067] This embodiment also provides an instrument operation procedure for a liquid chromatography-mass spectrometry system, including the following steps: Analysis steps: 1. Turn on the computer, enter the Windows operating system, check if the instrument is in good condition, log in to the Analyst software, and enter the workstation main interface.

[0068] 2. Double-click the Hardware configuration button, select LCMS, and then click Activate Profile on the right to activate the instrument. The LCMS module will display a green checkmark, indicating that the connection is successful.

[0069] 3. Double-click "Build Acquisition Batch" in the Acquire tab, select the established method, and edit the sample sequence (sample number, sample tray position, injection volume, etc.) in the sequence editing menu.

[0070] 4. Click Equilibrate in the menu bar, select the previously created liquid chromatography-mass spectrometry method to be run, and set the equilibration time to 10-20 minutes.

[0071] 5. In the Batch Editor, go to the Submit interface on the right, select the sample sequence to be analyzed, and click the Submit button to submit.

[0072] 6. After the equilibration time is over, the instrument status in the lower right corner of the main interface will show "Ready". Click "Start sample" in the menu bar to begin sample analysis and data acquisition.

[0073] 7. Double-click the "Open Data File" option in the "Explore" menu on the left to view the chromatographic images during the detection process at any time.

[0074] 8. After sample collection and analysis, use Mμltiquant to create a quantitative list and perform quantitative analysis.

[0075] Standby steps: 1. Click the Standby button in the menu bar. You will see the flow rate slowly decrease to zero. After the system pressure drops to 0, double-click Hardware configuration. In the pop-up dialog box, select LCMS and then click Deactivateprofile on the right. The LCMS module will display a cross, indicating that the connection can be disconnected.

[0076] 2. Exit the Analyst software and shut down the computer.

[0077] Comparative Example 1 This comparative example provides a method for detecting marsurasavirin and its metabolites in serum: the only difference from Example 1 is that the chromatographic column used is replaced with the Ultimate XB column. C18 column (4.6mm×50mm, 5μm, Welch), the remaining steps are the same as in Example 1.

[0078] Comparative Example 2 This comparative example provides a method for detecting marsurasavir and its metabolites in serum: the only difference from Example 1 is that the chromatographic column used is replaced with an XBridge C18 column (2.1 mm × 100 mm, 3.5 μm, Waters), and the other steps are the same as in Example 1.

[0079] Comparative Example 3 This comparative example provides a method for detecting marsurasavir and its metabolites in serum: the only difference from Example 1 is that the mobile phase A is replaced with 0.1% formic acid aqueous solution, and the remaining steps are the same as in Example 1.

[0080] Comparative Example 4 This comparative example provides a method for detecting marsurasavirin and its metabolites in serum: the only difference from Example 1 is that the mobile phase B is 1 mM ammonium acetate methanol solution, and the other steps are the same as in Example 1.

[0081] Comparative Example 5 This comparative example provides a method for detecting marsurasavir and its metabolites in serum: the only difference from Example 1 is that the mobile phase B is 0.1% formic acid methanol solution, and the other steps are the same as in Example 1.

[0082] Performance verification results of the method 1. Linear Prepared a series of serially diluted standard solutions of known concentrations (masuraxavir and its metabolites at concentrations of 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 50 ng / ml, 100 ng / ml, 500 ng / ml, and 1000 ng / ml, respectively). An equal volume of internal standard solution was added. A calibration curve was formed by fitting the calibration points using the least squares method, with linearity requirements r>0.99, deviations from theoretical values ​​at each concentration point less than 15%, and deviations from theoretical values ​​at the limit of quantification (LoQ) point less than 20%. The linear range of masuraxavir and its metabolites in serum was found to be 1 ng / mL to 1000 ng / mL, with LoQ = 1 ng / mL. The masuraxavir standard curve was formed as follows: Figure 1 As shown, the standard curve of masuravir metabolites is as follows: Figure 2 As shown, the standard curve for marsurasavir has r=0.99874, and the standard curve for marsurasavir metabolites has r=0.99925; marsurasavir and its metabolites all exhibit good linearity, and the LoQ point quantification is accurate, as shown in the figure. Figure 3 As shown, this invention demonstrates good detection sensitivity and linearity, meeting the needs of clinical testing and scientific research.

[0083] 2. Analysis of test results To achieve more sensitive and faster detection, Example 1 of this invention uses a Synergi™ 4 µm Fusion-RP 80 Å column (2.0 mm × 50 mm, 4.0 μm, Phenomenex) and gradient elution is performed under the chromatographic conditions shown in Table 1. Comparative experiments were also conducted using the same sample to strongly illustrate the technical effects of this invention.

[0084] Under the chromatographic conditions shown in Table 1, after the samples underwent the same pretreatment, it was found that: if the chromatographic column was Ultimate XB... C18 column (4.6mm × 50mm, 5μm, Welch), results are shown in [link to results]. Figure 5 Compared to a Synergi™ 4 µm Fusion-RP 80 Å column, the response indices of GP681, GP1707D07, and ritonavir-D6 were extremely low, making accurate quantification impossible. However, when using an XBridge C18 column (2.1 mm × 100 mm, 3.5 μm, Waters), the results are as follows: Figure 6 Compared to a Synergi™ 4 µm Fusion-RP 80 Å column, the peak shape of GP1707D07 was poor, and the response intensities of GP681 and ritonavir-D6 were both low, affecting quantification. Therefore, the Synergi™ 4 µm Fusion-RP 80 Å column (2.0 mm × 50 mm, 4.0 μm, Phenomenex) yielded better results. Figure 4 ).

[0085] With a Synergi™ 4 µm Fusion-RP 80 Å column (2.0 mm × 50 mm, 4.0 μm, Phenomenex) and under the chromatographic conditions shown in Table 1, after the same sample pretreatment, it was found that if mobile phase A was 0.1% formic acid aqueous solution, the results are as follows: Figure 7 Compared to mobile phase A, which is a 0.2 mM ammonium fluoride aqueous solution, the peak shape of GP1707D07 is poor and the response intensity is low, affecting quantification. GP1707D07 is an active metabolite of GP681, and its concentration is lower than that of GP681. Considering better sensitivity, mobile phase A of 0.2 mM ammonium fluoride aqueous solution is more effective in this invention. Figure 4 ).

[0086] With a Synergi™ 4 µm Fusion-RP 80 Å column (2.0 mm × 50 mm, 4.0 μm, Phenomenex), mobile phase A being 0.2 mM ammonium fluoride aqueous solution, and under the chromatographic conditions shown in Table 1, after the same sample pretreatment, it was found that if mobile phase B was 1 mM ammonium acetate methanol solution, the results are as follows: Figure 8 Compared to mobile phase B, which is a methanol solution, the peak shape of GP1707D07 is poor and the response intensity is low, affecting quantification. If mobile phase B is a 0.1% formic acid methanol solution, the results are as follows: Figure 9Compared to mobile phase B, which is a methanol solution, the peak shape of GP1707D07 is poor and the response intensity is low, affecting quantification. Furthermore, ritonavir-D6 exhibits a delayed peak, also affecting quantification. GP1707D07 is the active metabolite of GP681, and its concentration is lower than that of GP681. Considering better sensitivity, mobile phase B, a methanol solution, is more effective in this invention. Figure 4 ).

[0087] 3. Precision and carryover contamination The prepared serum samples of masuraxavir and its metabolites were continuously tested three times over five consecutive days using low, medium, and high quality control working solutions. The intra-day coefficients of variation (CV) for precision of masuraxavir in serum were calculated to be 4.31%, 1.40%, and 0.50%, respectively, and the inter-day coefficients of variation for precision were 5.51%, 3.89%, and 6.04%, respectively, all less than 15%, demonstrating good precision (see Table 4). The intra-day coefficients of variation for precision of masuraxavir metabolites in serum were calculated to be 3.05%, 3.24%, and 3.63%, respectively, and the inter-day coefficients of variation for precision were 9.45%, 7.24%, and 9.31%, respectively, all less than 15%, demonstrating good precision (see Table 5). Blank samples were measured immediately after the highest point of the calibration curve. The peak area of ​​the blank samples was less than 0.1% of the highest point of the calibration curve (see Table 6), demonstrating that the detection method of this invention has no significant carryover contamination.

[0088] Table 4. Precision of Detection of Masurasavir

[0089] Table 5. Precision of Detection of Maturaxavir Metabolites

[0090] Table 6. Contamination rate of masuraxavir and its metabolites

[0091] 4. Accuracy Spike recovery rate: At least 5 population samples from different sources were taken, and equal amounts of serum were selected from each sample and mixed. Then, 3 equal amounts of the mixed serum were selected and low / medium / high concentration stock solutions of known concentrations were added to each sample. The same pretreatment and injection analysis procedures were performed, and the analysis was carried out in parallel three times. The spike recovery rate % was calculated using the formula: (actual spiked sample concentration - actual matrix sample concentration) / theoretical spiked concentration * 100%. The data obtained are shown in Table 7. The spike recovery rate of marsurasavirin was 97.04%~106.74%, and the spike recovery rate of marsurasavirin metabolites was 95.66%~103.15%, indicating that the detection method of this invention has good accuracy.

[0092] Table 7 Recovery rates of masuraxavir and its metabolites

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting marsurasavirin and its metabolites in serum, characterized in that, Includes the following steps: 1) Prepare standard curve working solutions of a series of concentrations by mixing stock solutions of masuraxavir and masuraxavir metabolites with blank serum; 2) Add the internal standard solution and extractant to the serum sample to be tested and the standard curve working solution respectively, separate the solid and liquid, and then take the supernatant for nitrogen blowing. After nitrogen blowing, reconstitute with a reconstituter and take the supernatant for liquid chromatography-tandem mass spectrometry analysis to calculate the content of marsurasavir and marsurasavir metabolites in the serum sample to be tested. The liquid chromatography-tandem mass spectrometry analysis used a Synergi column for liquid chromatography-tandem mass spectrometry. TM A gradient elution was performed using a 4 µm Fusion-RP 80 Å laminator with 0.15–0.25 mM ammonium fluoride aqueous solution as mobile phase A and methanol as mobile phase B.

2. The method for detecting marsurasavir and its metabolites in serum according to claim 1, characterized in that, The liquid chromatography conditions described in step 2) also include: a Synergi column. TM 4 µm Fusion-RP 80 Å, inner diameter 2.0 mm, column length 50 mm, particle size 4 μm, column temperature 35-45 ℃, injection volume 15-25 μL; And / or, the liquid chromatography described in step 2) uses a gradient elution by mixing different volumes of mobile phase A and mobile phase B; The gradient elution process includes: The flow rate was 0.8 mL / min, with the volume content of mobile phase A maintained at 90% and the volume content of mobile phase B maintained at 10%. From 1.0 min to 1.5 min, the volume content of mobile phase A was adjusted from 90% to 2%, and the volume content of mobile phase B was adjusted from 10% to 98%, with a flow rate of 0.8 mL / min. From 1.5 min to 3.0 min, the volume content of mobile phase A was maintained at 2%, the volume content of mobile phase B was maintained at 98%, and the flow rate was 0.8 mL / min; From 3.0 min to 3.1 min, the volume content of mobile phase A was adjusted from 2% to 90%, and the volume content of mobile phase B was adjusted from 98% to 10%, with a flow rate of 0.8 mL / min. From 3.1 min to 4.0 min, the volume content of mobile phase A was maintained at 90%, the volume content of mobile phase B was maintained at 10%, and the flow rate was 0.8 mL / min.

3. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, The mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: Under positive ion monitoring, the ion spray voltage is 5000-5500 V, the temperature is 350-450℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 50-70 psi, the air curtain gas pressure is 10-30 psi, and the collision gas pressure is 5-15 psi. Under negative ion monitoring, the ion spray voltage is -4000 to -5000 V, the temperature is 350-450℃, the atomizing gas pressure is 40-60 psi, the auxiliary heating gas pressure is 50-70 psi, the air curtain gas pressure is 10-30 psi, and the collision gas pressure is 5-15 psi.

4. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, The internal standard solution mentioned in step 2) is a solution containing ritonavir-D6; The concentration of ritonavir-D6 in the internal standard solution was 0.5-2 μg / ml; And / or, the extractant is a mixed solution of ethyl acetate and n-hexane in a volume ratio of (80-90):(10-20); And / or, the complex solvent is a methanol aqueous solution with a methanol volume percentage of 40-60%; And / or, the volume ratio of the resolvent to the supernatant before nitrogen blowing is 1:(7-9).

5. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, In step 2), the volume ratio of the serum sample to be tested or the standard curve working solution, the internal standard solution, and the extraction solvent is (270-330):(9-11):(900-1100). And / or, the mass spectrometry conditions in the liquid chromatography-tandem mass spectrometry analysis described in step 2) include: Under positive ion monitoring, the ion spray voltage was 5500 V, the temperature was 400 ℃, the atomizing gas pressure was 50 psi, the auxiliary heating gas pressure was 60 psi, the air curtain gas pressure was 20 psi, and the collision gas pressure was 10 psi; under negative ion monitoring, the ion spray voltage was -4500 V, the temperature was 400 ℃, the atomizing gas pressure was 50 psi, the auxiliary heating gas pressure was 60 psi, the air curtain gas pressure was 20 psi, and the collision gas pressure was 10 psi.

6. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, The calculation of the levels of marsurasavir and its metabolites in the serum sample to be tested includes: The peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the standard curve working solution were collected; the peak areas of masuraxavir, masuraxavir metabolites, and internal standards in the serum samples to be tested were also collected. A standard curve for masuraxavir was plotted with the concentration of masuraxavir in the working solution of the standard curve as the abscissa and the ratio of the peak area of ​​masuraxavir in the working solution to the peak area of ​​the internal standard as the ordinate. The linear regression equation of masuraxavir was then obtained. The ratio of the peak area of ​​masuraxavir to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation of masuraxavir to obtain the content of masuraxavir in the serum sample to be tested. A standard curve for rasurasavir metabolites was plotted with the concentration of rasurasavir metabolites in the standard curve working solution as the x-axis and the ratio of the peak area of ​​rasurasavir metabolites in the standard curve working solution to the peak area of ​​the internal standard as the y-axis. The linear regression equation for rasurasavir metabolites was then obtained. The ratio of the peak area of ​​rasurasavir metabolites to the peak area of ​​the internal standard in the serum sample to be tested was then substituted into the linear regression equation for rasurasavir metabolites to obtain the content of rasurasavir metabolites in the serum sample to be tested.

7. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, The series of concentrations mentioned in step 1) includes at least 8 concentrations; And / or, the concentration range of masuraxavir or masuraxavir metabolites in the standard curve working solutions of the series of concentrations is 1-1000 ng / mL.

8. The method for detecting marsurasavir and its metabolites in serum according to claim 7, characterized in that, The concentrations of methuraxavir or its metabolites in the standard curve working solutions of the aforementioned series of concentrations are 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 100 ng / mL, 500 ng / mL, and 1000 ng / mL, respectively.

9. The method for detecting marsurasavir and its metabolites in serum according to claim 1 or 2, characterized in that, Step 1) also includes mixing blank serum samples with masuraxavir solution and masuraxavir metabolite solution to prepare quality control working solutions with concentrations of 10 ng / mL, 50 ng / mL, and 500 ng / mL of masuraxavir or masuraxavir metabolite. The quality control working solutions are then treated in the same way as the standard curve working solutions, and the supernatant is then taken for liquid chromatography-tandem mass spectrometry analysis.

10. An application of the method according to any one of claims 1-9, characterized in that, The applications include the detection of marsurasavir and / or marsurasavir metabolites in human serum.

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