阅读说明：本技术 大鼠血浆中covid-19潜在治疗药物的检测方法 (Method for detecting COVID-19 potential therapeutic drug in rat plasma ) 是由 季金风 刘洁 邱云良 缪峰 陶庭磊 周叶兰 耿家豪 季中秋 郭文静 陈淑敏 谢一 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潜在治疗药物的检测方法,其包含以下步骤：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；大鼠血浆的稳定剂为敌敌畏；COVID-19潜在治疗药物为(S)-2-(((S)-(((2R,3S,4R,5R)-5-(4-氨基吡咯并[2,1-f][1,2,4]三嗪-7-基)-5-氰基-3,4-二羟基四氢呋喃-2-基)甲氧基)(苯氧基)磷酰基)氨基)丙酸-2-乙基丁基酯。本发明的检测方法,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 in rat plasma, which comprises the following steps: analyzing and detecting the treated rat plasma by adopting a liquid chromatography-mass spectrometry combined method; the stabilizer of the rat plasma is dichlorvos; a potential therapeutic agent for covi-19 is (S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propanoic acid-2-ethylbutyl ester. 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 agent in rat plasma, comprising the steps of:

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

the stabilizer in the rat plasma is dichlorvos;

the COVID-19 potential therapeutic agent is (S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propanoic acid-2-ethylbutyl ester.

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 treated rat plasma is treated with a protein precipitation method.

3. The detection method according to claim 2, wherein the protein precipitation method uses a protein precipitant of methanol or acetonitrile;

and/or, the processing comprises: mixing the rat plasma, the internal standard substance and the protein precipitator, and taking supernatant fluid; preferably, the internal standard is tolbutamide.

4. 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.

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

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.

6. The detection method according to claim 5, 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) -3 95 5

。

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

step (1), respectively preparing standard working solution and quality control working solution of the COVID-19 potential therapeutic drugs with certain concentration gradient, 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 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 in the standard working solution and the internal standard substance in the internal standard working solution and the concentration of the corresponding 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 quality control samples have a deviation of the calculated concentration from its corresponding actual value within ± 15.0%.

8. The detection method according to claim 7, 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 tolbutamide 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.

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

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

10. The detection method according to claim 7, wherein in the step (1), the concentration of the internal standard working solution is 1000 ng/mL;

and/or, in the step (1), the concentration gradient of the standard working solution is 80ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL, 10000ng/mL, 20000ng/mL and 80000ng/mL in sequence;

and/or in the step (1), the concentration gradient of the quality control working solution is 240ng/mL, 3000ng/mL and 60000ng/mL in sequence;

and/or, in the step (2), the concentration of the standard sample solution is 4ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL and 4000ng/mL in sequence;

and/or, in the step (2), the concentration of the quality control sample solution is 12ng/mL, 150ng/mL and 3000ng/mL in sequence.

Technical Field

The invention relates to the field of drug analysis, in particular to a method for detecting 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 imminent.

Drug screening is carried out according to the marketed drugs and related databases, and 30 drugs which have potential therapeutic effects on COVID-19 are rapidly discovered, wherein the drugs comprise drugs such as Remdesivir (remdesivir), Saquinavir (Saquinavir), Lopinavir (Lopinavir), Carfilzomib (Carfilzomib), ritonavir and Chloroquine Phosphate (Chloroquine Phosphonate), most of the drugs can prevent viral RNA synthesis to inhibit virus replication, and related cell level tests show that the drugs can effectively inhibit infection of VID-19 at a cell level, but the effect of the drugs on human bodies is yet to be clinically verified. According to the regulation of the State food and drug administration, the new drug needs corresponding non-clinical test data before clinical verification. Therefore, the non-clinical safety evaluation work for the potential therapeutic drugs against COVID-19 is particularly important, and the accuracy of the drug metabolism and toxicology method in different animal models is an important safety guarantee in clinical trials.

At present, related potential therapeutic drugs for the new coronavirus are mainly micromolecular drugs, the metabolism of the micromolecular drugs (with the molecular weight of 200-700) is measured by chromatography, and solvents, reagents, matrixes and preparation and processing steps used in the development process of the method are mainly screened, so that the method for accurately measuring the metabolism is formed. Therefore, the development of preclinical detection methods for the potential therapeutic drugs COVID-19 is of great importance.

Disclosure of Invention

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

The invention solves the technical problem by the following scheme:

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

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

the stabilizer of the rat plasma is dichlorvos;

the COVID-19 potential therapeutic agent is (S) -2- (((S) - ((((2R, 3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propanoic acid-2-ethylbutyl ester.

In the invention, the rat plasma refers to rat plasma added with a stabilizer only; 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 30 ℃, 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, preferably, the treated rat plasma is treated by a protein precipitation method. The protein precipitating agent adopted by the protein precipitation method is preferably methanol or acetonitrile.

Wherein the processing may include: and mixing the rat plasma, the internal standard substance and the protein precipitator, and taking supernatant fluid.

The internal standard may be tolbutamide.

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 invention, the mobile phase adopted by the liquid chromatography in the liquid chromatography-mass spectrometry combined method can be a polar organic solvent and a formic acid aqueous solution, the formic acid in the formic acid aqueous solution has a volume percentage content of 0.01-0.5%, such as 0.1% formic acid aqueous solution, and the calculation method of the volume percentage content of formic acid in the formic acid aqueous solution is the proportion of the volume of formic acid in the volume of water.

Wherein, the polar organic solvent is preferably methanol or acetonitrile.

Wherein, the liquid chromatogram in the liquid chromatogram-mass spectrum combined 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 in table 1:

TABLE 1

Time (min)	Formic acid aqueous solution (%) (mobile phase A)	Polar organic solvent (%) (mobile phase B)
			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

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 percent of mobile phase a was maintained at 95% and the volume percent 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 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 includes:

step (1), respectively preparing standard working solution and quality control working solution of the COVID-19 potential therapeutic drugs with certain concentration gradient, 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 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 in the standard working solution and the internal standard substance in the internal standard working solution and the concentration of the corresponding 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 quality control samples have a deviation of the calculated concentration (at least 50% of the samples at each concentration level) from its corresponding actual value within ± 15.0%.

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 tolbutamide 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 the step (1), the concentration gradient of the standard working solution can be 80ng/mL, 200ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL, 10000ng/mL, 20000ng/mL and 80000ng/mL in sequence.

In the step (1), the concentration gradient of the quality control working solution can be 240ng/mL, 3000ng/mL and 60000ng/mL in sequence.

In step (2), the concentration of the standard sample solution can be 4ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL and 4000ng/mL in sequence.

In the step (2), the concentration of the quality control sample solution can be 12ng/mL, 150ng/mL and 3000ng/mL in sequence.

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 in the SD rat plasma sample is simple, short in time and easy to operate; thus, the pharmacokinetics of the COVID-19 potential therapeutic drug metabolites in rats were 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.

Drawings

FIG. 1 is a plot of the lower limit of quantitation (i.e., 4ng/mL) of COVID-19 potential therapeutic drugs in a standard curve.

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

FIG. 3 is a plot of the upper limit of quantitation (i.e., 4000ng/mL) of COVID-19 potential therapeutic drugs in a standard curve.

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

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

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 without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The COVID-19 potential therapeutic agent in the following examples is 2-ethylbutyl (S) -2- (((S) - (((2R,3S,4R,5R) -5- (4-aminopyrrolo [2,1-f ] [1,2,4] triazin-7-yl) -5-cyano-3, 4-dihydroxytetrahydrofuran-2-yl) methoxy) (phenoxy) phosphoryl) amino) propionate.

Example specificity of detection method of COVID-19 potential therapeutic drug 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 solution in a solvent bottle, uniformly mixing, and storing at room temperature for 1 month.

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

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. Storing at room temperature 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 standard in a transparent sample bottle, adding a proper amount of DMSO to dissolve, shaking up, and preparing into standard stock solution with the concentration of 1.000 mg/mL. (the mass reduction factor for the COVID-19 potential therapeutic agent is 0.992 (since the COVID-19 potential therapeutic agent standard is not itself 100% pure or with water or salt, the volume of DMSO is calculated from the concentration of the standard stock solution and the mass of the converted COVID-19 potential therapeutic agent calculated by multiplying the mass of the weighed COVID-19 potential therapeutic agent by the mass reduction factor for the COVID-19 potential therapeutic agent, 0.992).

2.2 working solution for Standard substance&Preparation of SST (System adaptive sample) working solutionTo prepare (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 solutions of table 2, prepared according to table 3 below, standard sample solutions were obtained, where plasma was at a volume ratio according to dichlorvos: blank plasma equal to 2: 5 ratio of the mixing station (e.g. 40. mu.l DDV: 100. mu.l blank plasma)

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. Plasma is shown in volume ratio as dichlorvos: blank plasma equal to 2: 5 (e.g., 40 microliters DDV: 100 microliters of blank plasma).

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, dissolving, shaking up, and preparing into an internal standard stock solution with the concentration of 1.000 mg/mL. (the mass conversion factor of tolbutamide is 0.999, since tolbutamide standards are not 100% pure by themselves or with water or salt, the volume of DMSO is calculated from the concentration of the standard stock solution and the converted tolbutamide mass, which is calculated by multiplying the weighed tolbutamide mass by the mass conversion factor of tolbutamide, 0.992).

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 sample pretreatment step

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% 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 drugs 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 percent of mobile phase a was maintained at 95% and the volume percent 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	603.300	402.00	150
Tolbutamide	271.100	154.800	150

TABLE 9 Mass spectrometer Instrument parameters

Parameter(s)	COVID-19 potential therapeutic drug	Tolbutamide
			Ionization voltage (v)	5500	5500
Temperature (. degree.C.)	500	500
			Collision gas (psi)	9	9
Air curtain gas (psi)	20	20
			Spray mist (psi)	50	50
Auxiliary gas (psi)	40	40
			Declustering voltage (v)	93	50
Inlet voltage (v)	10	10
			Collision energy (v)	25	24
Outlet voltage of collision cell (v)	11	10

5. Analytical batch acceptance criteria and standard curve regression method

5.1 regression method

Extracting an MRM chromatogram, fitting a standard curve, setting the weight as 1/x2 by taking the concentration of the standard working solution as an abscissa and the peak area ratio of the standard working solution to the internal standard as an ordinate of the standard curve, neglecting the origin, and fitting a linear standard curve. And (3) performing linear least square regression calculation on the theoretical concentration of the analyte in the standard curve by comparing the peak area of the analyte with the peak area of the internal standard, 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 a section of a standard curveDistance (weight factor 1/x)²)

4.2 analysis 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 nominal values.

The results are shown in FIG. 3 (measured on samples according to Table 3) and the standard curve regression equation is: 0.000958x +0.000926(r 0.9968, r)²> 0.98) and the standard curve is 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 acceptance standards of the analysis batches are met. In conclusion, the method can be used for determining EDTA-K of SD rat₂The concentration of the analyte (i.e., the potential therapeutic agent for COVID-19) in the plasma.

Watch 10

TABLE 11