阅读说明：本技术 大鼠血浆中covid-19潜在治疗药物代谢物的检测方法 (Method for detecting COVID-19 potential therapeutic drug metabolite in rat plasma ) 是由 邢溪溪 刘洁 缪峰 陶庭磊 季中秋 郭文静 周叶兰 耿家豪 陈淑敏 谢一帆 于 2020-05-13 设计创作，主要内容包括：本发明公开了一种大鼠血浆中COVID-19潜在治疗药物代谢物的检测方法。该方法包含以下步骤：采用液相色谱-质谱联用法,对经处理的大鼠血浆进行分析检测,即可；COVID-19潜在治疗药物代谢物为(2R,3R,4S,5R)-2-(4-氨基吡咯并[2,1-f][1,2,4]三嗪-7-基)-3,4-二羟基-5-(羟甲基)四氢呋喃-2-甲腈；经处理的大鼠血浆通过采用蛋白沉淀法对大鼠血浆进行处理得到；蛋白沉淀法中的蛋白沉淀剂为甲醇。本发明中的检测方法,简单、用时短且易操作；所需的样本进样量小,所需的分析时间较短。(The invention discloses a method for detecting a COVID-19 potential therapeutic drug metabolite in rat plasma. The method comprises the following steps: analyzing and detecting the treated rat plasma by adopting a liquid chromatography-mass spectrometry combined method; the potential therapeutic drug metabolite of COVID-19 is (2R,3R,4S,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile; the treated rat plasma is obtained by treating the rat plasma by a protein precipitation method; the protein precipitant in the protein precipitation method is methanol. The detection method is simple, short in time consumption and easy to operate; the required sample amount is small, and the required analysis time is short.)

1. A method for detecting a COVID-19 potential therapeutic drug metabolite in rat plasma, comprising the steps of:

analyzing and detecting the treated rat plasma by adopting a liquid chromatography-mass spectrometry combined method;

the metabolite of the COVID-19 potential therapeutic drug is (2R,3R,4S,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile;

the treated rat plasma is obtained by treating the rat plasma by a protein precipitation method; the protein precipitator in the protein precipitation method is methanol.

2. The assay of claim 1, wherein the rat is an SD rat;

and/or the column temperature of the liquid chromatogram in the liquid chromatogram-mass spectrum combined method is 20-30 ℃, such as 25 ℃;

and/or the sample amount of the liquid chromatogram in the liquid chromatogram-mass spectrum combined method is 1-10 mu L, such as 2 mu L;

and/or, the steps of the protein precipitation method comprise: mixing the rat plasma, the internal standard substance and the protein precipitator, and taking supernatant fluid; the internal standard is preferably tolbutamide.

3. The detection method according to claim 1, wherein a chromatographic column stationary phase of liquid chromatography in the combined liquid chromatography-mass spectrometry is octadecyl bonded silica gel;

and/or the specification of the chromatographic column is 2.1 x 50mm,2.7 μm;

and/or the mobile phase adopted by the liquid chromatography in the liquid chromatography-mass spectrometry combined method is a polar organic solvent and a formic acid aqueous solution; the formic acid aqueous solution contains 0.01 to 0.5 percent of formic acid by volume percentage, such as 0.1 percent of formic acid aqueous solution;

and/or the mass spectrometry equipment in the liquid chromatography-mass spectrometry combined method is model AB SCIEX TRIPLE QUAD^TM4500.

4. The detection method according to claim 3, wherein the polar organic solvent is methanol;

and/or, the liquid chromatography in the combined liquid chromatography-mass spectrometry adopts gradient elution, and the volume ratio of the formic acid aqueous solution to the polar organic solvent in the gradient elution is 95: 5-5: 95;

and/or the total flow rate of the mobile phase is 0.5 mL/min.

5. The detection method according to claim 4, wherein the range of the change of the flow phase ratio with time in the gradient elution is shown in Table 1 below:

TABLE 1

Time (min) Formic acid aqueous solution (%) Polar organic solvent (%) 0～1.5 95 5 1.5 (excluding 1.5) to 2.5 5 95 2.5 (excluding 2.5) to 2.51 5→95 95→5 2.5 (excluding 2.51) to 3 95 5

。

6. The assay of claim 1, wherein the specificity of the assay is further analyzed prior to the assay of the COVID-19 potential therapeutic drug metabolite in the rat plasma, and wherein the step of analyzing the specificity of the assay comprises:

respectively preparing standard working solution and quality control working solution of the COVID-19 potential therapeutic drug metabolite with certain concentration, and then preparing internal standard working solution with certain concentration;

step (2), mixing the standard substance working solution and the quality control working solution with rat blank plasma respectively to obtain a standard substance sample solution and a quality control sample solution;

step (3), sample solution treatment step: respectively mixing the standard sample solution and the quality control sample solution with the internal standard working solution, and then taking supernate;

step (4), the supernatant is injected, liquid chromatography-mass spectrometry detection is carried out, and the peak area ratio of the COVID-19 potential therapeutic drug metabolite to an internal standard substance in the internal standard working solution is measured;

step (5), obtaining a regression equation according to the peak area ratio of the COVID-19 potential therapeutic drug metabolite in the standard working solution and the internal standard substance in the internal standard working solution and the corresponding concentration of the standard working solution;

step (6), substituting the peak area ratio obtained in the step (4) into the regression equation obtained in the step (5), and calculating to obtain the back calculation concentration;

the specificity of the detection method is good when at least 67% of the calculated concentrations (at least 50% of the samples per concentration level) deviate within ± 15.0% from their corresponding actual values.

7. The detection method according to claim 6, wherein the preparation method of the standard working solution comprises the following steps: mixing the COVID-19 potential therapeutic drug metabolite with a first diluent to obtain a standard substance stock solution; mixing the standard substance stock solution with a second diluent to obtain a standard substance working solution with a certain concentration gradient;

and/or, in the step (1), the preparation method of the quality control working solution comprises the following steps: mixing the COVID-19 potential therapeutic drug metabolite with the first diluent to obtain a quality control stock solution; mixing the quality control stock solution with the second diluent to obtain the quality control working solution with a certain concentration gradient;

and/or, in the step (1), the preparation method of the internal standard working solution comprises the following steps: mixing the internal standard substance with the first diluent to obtain an internal standard stock solution; and mixing the internal standard stock solution with the second diluent to obtain the internal standard working solution with a certain concentration.

8. The assay of claim 7, wherein the first diluent is DMSO;

and/or the second diluent is a 30-70% methanol aqueous solution.

9. The detection method according to claim 6, wherein in the step (1), the concentration of the internal standard working solution is 1000 ng/mL;

in the step (1), the concentration gradient of the standard substance working solution is sequentially 2 mug/mL, 5 mug/mL, 25 mug/mL, 50 mug/mL, 125 mug/mL, 250 mug/mL, 500 mug/mL and 2000 mug/mL;

in the step (1), the concentration gradients of the quality control working solution are 75 mug/mL, 1500 mug/mL and 3000 mug/mL in sequence.

10. The detection method according to claim 6, wherein in the step (2), the concentration of the standard sample solution is 100ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL, 6250ng/mL, 12500ng/mL, 25000ng/mL and 100000ng/mL in this order;

in the step (2), the concentration of the quality control sample solution is 300ng/mL, 3750ng/mL and 75000ng/mL in sequence.

Technical Field

The invention relates to the field of drug analysis, in particular to a method for detecting a metabolite of a COVID-19 potential therapeutic drug in rat plasma.

Background

By the end of 2019, outbreaks of new coronavirus pneumonia (Corona Virus Disease 2019, COVID-19) caused by 2019 new coronavirus (2019-nCoV) infection. The virus is rapidly spread, the infectivity is strong, the crowd is generally susceptible, the epidemic situation quickly becomes the focus of global attention, and no approved treatment method aiming at COVID-19 exists in the global scope, and the research and development of anti-new coronavirus medicines are at the forefront.

Reidesciclovir (RDV, GS5734) is an antiviral drug developed by Jilide scientific Inc. in the United states, is originally designed for treating diseases such as hemorrhagic fever caused by Ebola virus, and is of social interest because it shows good broad-spectrum antiviral effect in vivo and in vitro.

Studies have shown that RDV, as a nucleoside analog, after being metabolized by host-active cells into pharmacologically active triphosphate metabolites, competitively inhibits the addition of natural Nucleoside Triphosphates (NTPs) to viral RNA-dependent RNA polymerase (RdRp), preventing viral RNA synthesis to inhibit viral replication. Therefore, most of the COVID-19 potential therapeutic drugs have pharmacological activity through the action of metabolites, and the detection of the blood concentration of the metabolites is particularly important when the blood concentration change of the COVID-19 potential therapeutic drugs is researched.

In recent years, with intensive research on new drug safety evaluation methods by researchers, chromatography has been widely applied to metabolic detection of a large number of small-molecule drugs, and solvents, reagents, substrates and preparation and processing steps used in the development process of the methods are mainly screened, so that a method for accurate determination is formed. Therefore, the development of a detection method for the potential therapeutic drug metabolites of COVID-19 is of great importance.

Disclosure of Invention

The invention provides a method for detecting a COVID-19 potential therapeutic drug metabolite in rat plasma. The method can quickly and simply detect the potential therapeutic drug metabolite of COVID-19 in the plasma of the rat.

The invention solves the technical problems by the following scheme:

a method for detecting COVID-19 potential therapeutic drug metabolites in rat plasma comprising the steps of:

analyzing and detecting the treated rat plasma by adopting a liquid chromatography-mass spectrometry combined method;

the metabolite of the COVID-19 potential therapeutic drug is (2R,3R,4S,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile;

the treated rat plasma is obtained by treating the rat plasma by a protein precipitation method; the protein precipitator in the protein precipitation method is methanol.

Wherein the protein precipitation method may comprise the steps of: and mixing the rat plasma, the internal standard substance and the protein precipitator, and taking supernatant fluid. The internal standard is preferably tolbutamide.

In the present invention, the rat plasma refers to untreated rat plasma; the treated rat plasma became treated rat plasma.In the invention, the rat plasma is generally rat EDTA-K₂Plasma.

In the present invention, the rat may be an SD rat.

In the present invention, the column temperature of the liquid chromatography in the combined liquid chromatography-mass spectrometry is preferably 20 to 40 ℃, for example, 25 ℃.

In the invention, the sample amount of the liquid chromatogram in the liquid chromatogram-mass spectrum combined method can be 1-10 μ L, such as 2 μ L.

In the present invention, the apparatus for liquid chromatography in the combined liquid chromatography-mass spectrometry may be conventional in the art. The chromatographic column stationary phase of the liquid chromatogram in the liquid chromatogram-mass spectrum combined method can be octadecyl bonded silica gel; for example, Poroshell 120 SB-C18. The size of the column may be conventional in the art, for example 2.1X 50mm,2.7 μm.

In the present invention, the mobile phase used in the liquid chromatography-mass spectrometry may be a polar organic solvent and an aqueous solution of formic acid, the volume percentage of formic acid in the aqueous solution of formic acid is 0.01% to 0.5%, for example, 0.1% aqueous solution of formic acid, and the volume percentage of formic acid in the aqueous solution of formic acid is calculated by the ratio of the volume of formic acid to the volume of water.

Wherein, the polar organic solvent is preferably methanol.

In the invention, the liquid chromatography in the combined liquid chromatography-mass spectrometry method can adopt gradient elution, and the volume ratio of the formic acid aqueous solution to the polar organic solvent during the gradient elution can be 95: 5-5: 95.

Wherein, in the gradient elution, the variation range of the flow phase ratio along with time can be shown as table 1:

TABLE 1

Wherein, the gradient elution procedure is as follows: the volume percentage of mobile phase a was 95% and the volume percentage of mobile phase B was 5% in the period from the initial state to 1.5min, and the volume percentage of mobile phase a was changed to 5% and the volume percentage of mobile phase B was changed to 95% in the period from 1.5min to 2.5 min; gradually increasing the volume percentage of the mobile phase A to 95% and gradually decreasing the volume percentage of the mobile phase B to 5% in a time period of 2.5min to 2.51 min; the volume percentage of mobile phase a was maintained at 95% and the volume percentage of mobile phase B was maintained at 5% over a period of 2.51min to 3.00 min. (the volume percentages of mobile phases A and B are based on the total volume of mobile phases A and B).

Wherein the total flow rate of the mobile phase is 0.5 mL/min.

In the present invention, the mass spectrometry equipment in the combined liquid chromatography-mass spectrometry method can be conventional in the art, for example, type AB SCIEX TRIPLE QUAD^TM4500.

In the present invention, preferably, before detecting the COVID-19 potential therapeutic drug metabolite in the rat plasma, the specificity of the detection method needs to be analyzed, and the step of analyzing the specificity of the detection method comprises:

respectively preparing standard working solution and quality control working solution of the COVID-19 potential therapeutic drug metabolite with certain concentration, and then preparing internal standard working solution with certain concentration;

step (2), mixing the standard substance working solution and the quality control working solution with rat blank plasma respectively to obtain a standard substance sample solution and a quality control sample solution;

step (3), sample solution treatment step: respectively mixing the standard sample solution and the quality control sample solution with the internal standard working solution, and then taking supernate;

step (4), the supernatant is injected, liquid chromatography-mass spectrometry detection is carried out, and the peak area ratio of the COVID-19 potential therapeutic drug metabolite to an internal standard substance in the internal standard working solution is measured;

step (5), obtaining a regression equation according to the peak area ratio of the COVID-19 potential therapeutic drug metabolite in the standard working solution and the internal standard substance in the internal standard working solution and the corresponding concentration of the standard working solution;

step (6), substituting the peak area ratio obtained in the step (4) into the regression equation obtained in the step (5), and calculating to obtain the back calculation concentration;

the specificity of the detection method is good when at least 67% of the calculated concentrations (at least 50% of the samples per concentration level) deviate within ± 15.0% from their corresponding actual values.

In the step (1), the preparation method of the standard working solution may include the following steps: mixing the COVID-19 potential therapeutic drug metabolite with a first diluent to obtain a standard substance stock solution; and mixing the standard substance stock solution with a second diluent to obtain the standard substance working solution with a certain concentration gradient.

In the step (1), the preparation method of the quality control working solution may include the following steps: mixing the COVID-19 potential therapeutic drug metabolite with the first diluent to obtain a quality control stock solution; and mixing the quality control stock solution with the second diluent to obtain the quality control working solution with a certain concentration gradient.

In the step (1), the preparation method of the internal standard working solution may include the following steps: mixing the internal standard substance with the first diluent to obtain an internal standard stock solution; and mixing the internal standard stock solution with the second diluent to obtain the internal standard working solution with a certain concentration.

Wherein the first diluent may be DMSO. The second diluent may be a 30% to 70% aqueous methanol solution.

In the step (1), the concentration of the internal standard working solution can be 1000 ng/mL.

In step (1), the concentration gradient of the standard working solution can be 2 μ g/mL, 5 μ g/mL, 25 μ g/mL, 50 μ g/mL, 125 μ g/mL, 250 μ g/mL, 500 μ g/mL and 2000 μ g/mL in sequence.

In the step (1), the concentration gradient of the quality control working solution can be 75 mug/mL, 1500 mug/mL and 3000 mug/mL in sequence.

In step (2), the concentration of the standard sample solution can be 100ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL, 6250ng/mL, 12500ng/mL, 25000ng/mL and 100000ng/mL in sequence.

In the step (2), the concentration of the quality control sample solution can be 300ng/mL, 3750ng/mL and 75000ng/mL in sequence.

Blank plasma in the present invention generally refers to blank plasma without addition of COVID-19 potential therapeutic drug metabolites, without addition of internal standards, without any treatment.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

1) the detection method of the COVID-19 potential therapeutic drug metabolite in the SD rat plasma sample is simple, short in time and easy to operate, so that the pharmacokinetics of the COVID-19 potential therapeutic drug metabolite in a rat body is analyzed.

2) The method of the invention needs small sample input amount and short analysis time; the processing mode developed by the invention ensures that the standard curve is linear and accurate, and the accuracy and the reliability of detection are ensured.

3) The processing method adopted by the invention is a protein precipitation method, and compared with the common extraction method, the method has the advantages of simpler flow, short time and easy operation.

Drawings

FIG. 1 is a plot of the lower limit of quantitation (i.e., 100ng/mL) of COVID-19 potential therapeutic drug metabolites in a standard curve.

FIG. 2 is a spectrum of tolbutamide in a lower limit of quantitation sample of the standard curve. (i.e., 50ng/mL)

FIG. 3 is a plot of the upper limit of quantitation of a standard curve (i.e., 100000ng/mL) for COVID-19 potential therapeutic drug metabolites in the standard curve.

FIG. 4 is a spectrum of tolbutamide in the upper limit of quantitation sample of the standard curve. (i.e., 50ng/mL)

FIG. 5 is a graph of a COVID-19 standard of potential therapeutic drug metabolites.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods in the following examples, in which specific conditions are not specified, were selected according to conventional methods and conditions, or according to the commercial instructions.

The potential therapeutic drug metabolite of COVID-19 in the following examples is (2R,3R,4S,5R) -2- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -3, 4-dihydroxy-5- (hydroxymethyl) tetrahydrofuran-2-carbonitrile.

The blank plasma in the following examples is rat plasma without any treatment.

EXAMPLE specificity test of the method for detecting COVID-19 potential therapeutic drug metabolites in rat plasma

1. Preparation of reagents

Mobile phase a (mpa), 0.1% aqueous formic acid: take 1000mL of H₂And adding 1mL of Formic Acid (FA) into the O, uniformly mixing, and storing at room temperature for 1 month.

Mobile phase b (mpb), methanol (MeOH): 1000mL of MeOH was taken in a solvent bottle and stored at room temperature for 3 months of validity.

Vigorous wash Solution (SNW), MeOH (acetonitrile) ACN: Isopropanol (IPA), dimethyl sulfoxide (DMSO) ═ 1:1:1:1, v/v/v/v: 500mL MeOH, 500mL ACN, 500mL IPA, 500mL DMSO were mixed well. Storing at room temperature for 3 months.

Weak Wash solution (WNW), MeOH: H₂1:1, v/v: 1000mL of MeOH and 1000mL of H were taken₂And O, and mixing uniformly. The storage at room temperature is effective for 3 months.

Precipitant & internal standard diluent: MeOH.

1000mL of MeOH were taken. Storing at room temperature for 1 month.

A second diluent: MeOH H₂O＝1:1，v/v。

500mL of MeOH and 500mL of H were added₂And O, and mixing uniformly. Storing at room temperature for 3 months.

First diluent: DMSO.

50mL of DMSO was taken. Storing at room temperature for 3 months.

2. Preparation of standard working solution and quality control working solution

The stock solution and the working solution are both stored in an ultra-low temperature refrigerator (70 ℃ below zero to 90 ℃ below zero).

2.1 preparation of stock solutions for standards

Accurately weighing a proper amount of COVID-19 potential therapeutic drug metabolite standard substance into a transparent sample bottle, adding a proper amount of DMSO, dissolving, shaking up, and preparing into standard substance stock solution with the concentration of 5.000 mg/mL. (COVID-19 potential therapeutic drug metabolite mass reduction factor is 0.988, since the COVID-19 potential therapeutic drug metabolite standard is not itself 100% pure or with water or salt, the volume of DMSO is calculated from the concentration of the standard stock and the reduced COVID-19 potential therapeutic drug metabolite mass calculated by multiplying the weighed COVID-19 potential therapeutic drug metabolite mass by the COVID-19 potential therapeutic drug metabolite mass reduction factor of 0.988.)

2.2 working solution for Standard substance&Preparation of SST (System Adaptation sample) working solution (Diluent: MeOH: H)₂O＝1:1，v/v)

Standard working solutions & SST working solutions were obtained using standard stock solutions, formulated according to table 2 below:

TABLE 2

2.3 preparation of Standard sample solution

Using the working liquids of table 2, the standard sample solutions were prepared according to table 3 below.

TABLE 3

The standard sample solution was stored in an ultra-low temperature freezer (-70 to-90 ℃).

2.4 preparation of quality control stock solution

Same as standard stock solutions.

2.5 preparation of quality control working solution (Diluent: MeOH: H)₂O＝1:1，v/v)

The quality control stock solution was used and formulated according to table 4 below to obtain a quality control working solution.

TABLE 4

2.6 preparation of quality control sample solution

The quality control working solution was used and prepared according to table 5 below to obtain a quality control sample solution.

TABLE 5

The quality control sample solution is stored in an ultra-low temperature refrigerator (-70 to-90 ℃).

2.7 preparation of stock solutions for internal standards

Accurately weighing a proper amount of tolbutamide into a transparent sample bottle, adding a proper amount of DMSO (the conversion factor of tolbutamide is 0.999), dissolving and shaking uniformly to prepare an internal standard stock solution with the concentration of 1.000mg/mL, wherein the volume of the DMSO is calculated by the concentration of the internal standard stock solution and the mass of the converted tolbutamide because the purity of the tolbutamide is not 100 percent or water or salt is contained, and the mass of the converted tolbutamide is calculated by multiplying the mass of the weighed tolbutamide by the mass conversion factor of tolbutamide, namely 0.999.)

2.8 preparation of internal standard working solution (Diluent: MeOH: H)₂O＝1:1)

Internal standard working solutions were prepared according to table 6 below using internal standard stock solutions to give internal standard working solutions:

TABLE 6

3. Sample introduction and sample treatment

1) Vortexing the sample (if the sample needs to be thawed, vortexing after thawing at room temperature);

2) pipetting 20. mu.L of sample (including STD, QC, DB, Carryover, Blank) and transferring to 96-well plates or polypropylene centrifuge tubes; wherein, STD corresponds to the table 3 samples; QC corresponds to Table 5 samples; DB: no substance to be detected and no internal standard substance; carryover: no object to be detected is contained; blank: no test substance contained only the internal standard substance.

3) Add 20. mu.L of internal standard working solution (1000.000 ng/mL tolbutamide in 50% aqueous methanol) to all samples;

4) after vortex mixing, 160 mu LMeOH is added into all samples to be used as protein precipitator;

5) mixing by vortex, and centrifuging at 4000rpm at 4 deg.C for 10 min;

6) taking 100 mu L of centrifuged supernatant to a 96-well plate or a polypropylene centrifugal tube, sealing a membrane, and uniformly mixing for 10min by vortex at 1000 rpm; and (6) sample injection analysis.

FIGS. 1 and 2 are measured for STD1 sample injection analysis; FIGS. 3 and 4 are measured for STD8 sample injection analysis; FIGS. 1-4 all show that the concentrations in the standard curve of the present invention can be detected by a combination of chromatography and mass spectrometry.

4. Detection of COVID-19 potential therapeutic drug metabolites in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS):

wherein the liquid phase chromatographic conditions are as follows:

sample introduction amount: 2 mu L of the solution;

a chromatographic column: poroshell 120SB-C18, 2.1X 50mm,2.7 μm, Agilent;

column temperature: 25 ℃;

gradient elution is carried out by adopting a mobile phase A and a mobile phase B;

operating time: 3.0min

TABLE 7

Wherein, the gradient elution procedure is as follows: the volume percentage of mobile phase a was 95% and the volume percentage of mobile phase B was 5% in the period from the initial state to 1.5min, and the volume percentage of mobile phase a was changed to 5% and the volume percentage of mobile phase B was changed to 95% in the period from 1.5min to 2.5 min; gradually increasing the volume percentage of the mobile phase A to 95% and gradually decreasing the volume percentage of the mobile phase B to 5% in a time period of 2.5min to 2.51 min; the volume percentage of mobile phase a was maintained at 95% and the volume percentage of mobile phase B was maintained at 5% over a period of 2.51min to 3.00 min. (the volume percentages of mobile phases A and B are based on the total volume of mobile phases A and B).

Needle washing procedure: the type of flush: only the exterior;

a flushing mode, wherein before and after suction, the immersion time is 2 s;

the pump flushing mode is that the pump is flushed and then stopped for 2 s;

the flushing speed is 35 mu L/s;

the washing volume is 1000 mu L;

measuring the line purge amount of 100 μ L

The mass spectrum conditions are as follows:

the instrument model is as follows: AB SCIEX TRIPLE QUADTM 4500

An ion source: ESI

Ionization mode: positive ion

TABLE 8MRM ion pairs

Analyte	Q1 mass to charge ratio	Q3 mass to charge ratio	Scanning interval (ms)
				COVID-19 potential therapeutic drug metabolites	603.300	402.00	150
Tolbutamide	271.100	154.800	150

TABLE 9 Mass spectrometer Instrument parameters

5. Analytical batch acceptance criteria and standard curve regression method

5.1 regression method

And extracting the MRM chromatogram, fitting a standard curve, comparing the peak area of the analyte with the peak area of an internal standard, performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve, and calculating the actually measured concentration of the analyte in the sample by using the obtained regression equation.

The measured concentration of the analyte in the sample is calculated from the following regression equation:

y＝ax+b

where y is the peak area ratio of analyte to internal standard

a is the slope of the standard curve

x is the analyte concentration (in ng/mL)

b is the intercept of the standard curve (weight factor 1/x)²)

5.2 analysis of batch acceptance criteria

1) The deviation between the recalculated value and the plotted value for each concentration point of the standard curve should be within + -15.0% (deviation at the lower limit of quantitation is within + -20.0%).

2) At least 75% of the standard curve samples, and at least 50% of the samples per concentration point should meet the acceptance criteria.

3) Correlation coefficient (r) of regression equation²) It must be 0.98 or more.

4) The assay lot was considered acceptable when at least 67% of the quality control sample results (at least 50% per concentration) were within ± 15.0% of their corresponding plotted values.

The results are shown in FIG. 3 (based on the data in Table 3), and the standard curve regression equation is: 0.000208x +0.00217(r 0.9992, r)²> 0.98) standard curve as shown in figure 5. The results of the back-calculation deviation of the standard sample solution, the quality control sample solution and the labeled value are shown in tables 10 and 11, and the standard sample solution, the quality control sample solution and the labeled value meet the analysis batch acceptance standard. In conclusion, the method can be used for determining EDTA-K of SD rat₂The concentration of the analyte (i.e., the COVID-19 potential therapeutic drug metabolite) in the plasma.

Watch 10

TABLE 11