阅读说明：本技术 Covid-19潜在治疗药物代谢物的检测方法 (Detection method of COVID-19 potential therapeutic drug metabolite ) 是由 缪峰 刘洁 季金风 邢溪溪 陶庭磊 周叶兰 耿家豪 季中秋 郭文静 谢一帆 于 2020-05-13 设计创作，主要内容包括：本发明提供了一种COVID-19潜在治疗药物代谢物的检测方法,其包括下列步骤：采用液相色谱-质谱联用法,对经处理的食蟹猴血浆中的COVID-19潜在治疗药物代谢物进行分析检测；其中,所述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；所述的经处理的食蟹猴血浆为经蛋白沉淀法处理的食蟹猴血浆；所述的蛋白沉淀法中的蛋白沉淀剂为甲醇。本发明能够快速、简便地检测食蟹猴血浆中COVID-19潜在治疗药物代谢物。(The invention provides a detection method of a COVID-19 potential therapeutic drug metabolite, which comprises the following steps: analyzing and detecting COVID-19 potential therapeutic drug metabolites in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein, the COVID-19 potential therapeutic drug metabolite 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 cynomolgus monkey plasma is the cynomolgus monkey plasma treated by a protein precipitation method; the protein precipitator in the protein precipitation method is methanol. The invention can quickly, simply and conveniently detect the COVID-19 potential therapeutic drug metabolite in the cynomolgus monkey plasma.)

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

analyzing and detecting COVID-19 potential therapeutic drug metabolites in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein, the COVID-19 potential therapeutic drug metabolite 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 cynomolgus monkey plasma is the cynomolgus monkey plasma treated by a protein precipitation method; the protein precipitator in the protein precipitation method is methanol.

2. The method for detecting a COVID-19 potential therapeutic drug metabolite according to claim 1, wherein the sample size in the liquid chromatography-mass spectrometry is 1-10 μ L, preferably 2 μ L.

3. The method of detecting a COVID-19 potential therapeutic drug metabolite according to claim 1, wherein the column temperature in the liquid chromatography-mass spectrometry is 20 ℃ to 30 ℃, preferably 25 ℃.

4. The method for detecting a metabolite of a COVID-19 potential therapeutic drug according to claim 1, wherein the liquid chromatography-mass spectrometry is a gradient elution method, wherein the gradient elution method comprises a step of eluting a mobile phase A with 0.01-0.5% by volume of formic acid in water, and a step of eluting a mobile phase B with methanol; preferably, the mobile phase A is a formic acid aqueous solution with the concentration of 0.1% by volume.

5. The method of detecting a COVID-19 potential therapeutic drug metabolite according to claim 4, wherein the volume ratio of mobile phase A to mobile phase B at the time of gradient elution is 95: 5-5: 95.

6. the method of detecting a COVID-19 potential therapeutic drug metabolite according to claim 4, wherein the gradient elution procedure is: in an initial state, the volume percentage of the mobile phase A is 95%, and the volume percentage of the mobile phase B is 5%; gradually decreasing the volume percentage of the mobile phase A to 5% and gradually increasing the volume percentage of the mobile phase B to 95% in a time period from an initial state to 1.5min, and maintaining the volume percentage of the mobile phase A at 5% and the volume percentage of the mobile phase B at 95% in a time 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 the mobile phase a is maintained at 95% and the volume percentage of the mobile phase B is maintained at 5% in a period of 2.51min to 3 min.

7. The method for detecting a COVID-19 potential therapeutic drug metabolite according to claim 4, wherein the total flow rate of mobile phase A and mobile phase B is 0.5 mL/min.

8. The method of claim 1, wherein the detection of the COVID-19 potential therapeutic drug metabolite is performed by a liquid chromatography-mass spectrometry method, wherein the detection is performed on the processed standard curve sample, the processed quality control sample, and the processed cynomolgus monkey plasma sample; wherein, the processed standard curve sample is processed by a protein precipitation method; the processed quality control sample is a quality control sample processed by a protein precipitation method; and internal standard working solution is added into the standard curve sample, the quality control sample and the cynomolgus monkey plasma sample.

9. The method for detecting a COVID-19 potential therapeutic drug metabolite according to claim 8, wherein the standard curve sample is formulated by a method comprising the steps of:

step 1: mixing the COVID-19 potential therapeutic drug metabolite standard with the diluent to obtain a standard stock solution;

step 2: mixing the standard substance stock solution with the diluent to obtain a plurality of standard substance working solutions with concentration gradients;

and step 3: mixing the standard substance working solution with the blank plasma to obtain a standard curve sample;

and/or, the preparation method of the quality control sample comprises the following steps:

step a: mixing the COVID-19 potential therapeutic drug metabolite standard substance with the diluent to obtain a quality control stock solution;

step b: mixing the quality control stock solution with the diluent to obtain a plurality of quality control working solutions with concentration gradients;

step c: mixing the quality control working solution with blank plasma to obtain a quality control sample;

and/or, the preparation method of the internal standard working solution comprises the following steps:

step I: mixing the tolbutamide standard substance with the diluent to obtain an internal standard stock solution;

step II: mixing the internal standard stock solution with the diluent to obtain an internal standard working solution;

more preferably, the diluent in the steps 1, a and I is DMSO, and the diluent in the steps 2, b and II is a methanol aqueous solution with the volume concentration of 30-70%; further, the diluent in the steps 2, b and II is preferably a 50% methanol aqueous solution by volume concentration.

10. The method for detecting a metabolite of a COVID-19 potential therapeutic drug of claim 9, wherein the concentration gradient of the standard working solution is sequentially 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, 2000 μ g/mL;

and/or the concentration gradient of the quality control working solution is 75 mug/mL, 1500 mug/mL and 3000 mug/mL in sequence;

and/or the concentration of the internal standard working solution is 1000 ng/mL;

and/or, the concentration of the standard curve sample is 100ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL, 6250ng/mL, 12500ng/mL, 25000ng/mL, 100000 ng/mL;

and/or, the concentration of the quality control sample is 3750ng/mL, 75000ng/mL and 150000 ng/mL.

Technical Field

The invention belongs to the technical field of drug analysis, and particularly relates to a detection method of a potential therapeutic drug metabolite of COVID-19.

Background

By the end of 2019, a new coronavirus pneumonia (covi-19) outbreak caused by 2019 infection with a new coronavirus (2019-nCoV). The virus is spread rapidly, has strong infectivity, is universally and easily felt by people, and quickly becomes the focus of global attention. However, there is no approved treatment for COVID-19 on a global scale, and the development of anti-new coronavirus drugs is imminent.

Ruidexivir (RDV, GS5734) is an antiviral drug developed by Jilide scientific Inc. of America, 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 action 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 detection method of a potential therapeutic drug metabolite of COVID-19. The method can quickly, simply and conveniently detect the COVID-19 potential therapeutic drug metabolite in the cynomolgus monkey plasma.

The invention provides a detection method of a COVID-19 potential therapeutic drug metabolite, which comprises the following steps:

analyzing and detecting COVID-19 potential therapeutic drug metabolites in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein, the COVID-19 potential therapeutic drug metabolite 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 cynomolgus monkey plasma is the cynomolgus monkey plasma treated by a protein precipitation method; the protein precipitator in the protein precipitation method is methanol.

In the invention, the cynomolgus monkey plasma refers to untreated cynomolgus monkey plasma; the treated cynomolgus plasma becomes treated cynomolgus plasma.

Preferably, the cynomolgus monkey plasma is cynomolgus monkey EDTA-K₂Plasma.

Preferably, the liquid chromatography-mass spectrometry combined method is an LC-MS/MS method.

Preferably, the sample amount in the liquid chromatography-mass spectrometry combined method is 1-10 μ L, and preferably 2 μ L.

Preferably, the column temperature in the liquid chromatography-mass spectrometry is 20-30 ℃, preferably 25 ℃.

Preferably, the liquid chromatography-mass spectrometry combined method adopts gradient elution, wherein a mobile phase A in the gradient elution is a formic acid aqueous solution with the volume concentration of 0.01-0.5%, and a mobile phase B is methanol. In a specific example, the mobile phase a is a 0.1% strength by volume aqueous formic acid solution.

More preferably, the volume ratio of the mobile phase a to the mobile phase B at the time of gradient elution may be 95: 5-5: 95.

more preferably, the gradient elution procedure is as follows: in the initial state, the volume percentage of the mobile phase A is 95 percent, and the volume percentage of the mobile phase B is 5 percent; the volume percentage of the mobile phase A is gradually reduced to 5% and the volume percentage of the mobile phase B is gradually increased to 95% in a time period from the initial state to 1.5min, and the volume percentage of the mobile phase A is maintained at 5% and the volume percentage of the mobile phase B is maintained at 95% in a time 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 min. (the volume percentages of the mobile phases A and B are based on the total volume of the mobile phases A and B)

More preferably, the total flow rate of the mobile phase A and the mobile phase B is 0.5 mL/min.

Preferably, in the liquid chromatography-mass spectrometry combined method, the processed standard curve sample, the processed quality control sample and the processed cynomolgus monkey plasma sample are respectively detected; wherein, the processed standard curve sample is processed by a protein precipitation method; the processed quality control sample is a quality control sample processed by a protein precipitation method; and internal standard working solution is added into the standard curve sample, the quality control sample and the cynomolgus monkey plasma sample.

In the invention, the standard curve sample refers to an untreated standard curve sample; the treated standard curve sample becomes a treated standard curve sample. The quality control sample refers to an unprocessed quality control sample; the processed quality control sample becomes a processed quality control sample.

More preferably, the protein precipitating agent in the protein precipitation method is methanol.

More preferably, the protein precipitation method comprises mixing a standard curve sample, a quality control sample or a cynomolgus monkey plasma sample with a protein precipitant, centrifuging, and taking a supernatant.

More preferably, the preparation method of the standard curve sample comprises the following steps:

step 1: mixing the COVID-19 potential therapeutic drug metabolite standard with the diluent to obtain a standard stock solution;

step 2: mixing the standard substance stock solution with the diluent to obtain a plurality of standard substance working solutions with concentration gradients;

and step 3: mixing the standard substance working solution with the blank plasma to obtain a standard curve sample;

and/or, the preparation method of the quality control sample comprises the following steps:

step a: mixing the COVID-19 potential therapeutic drug metabolite standard substance with the diluent to obtain a quality control stock solution;

step b: mixing the quality control stock solution with the diluent to obtain a plurality of quality control working solutions with concentration gradients;

step c: mixing the quality control working solution with blank plasma to obtain a quality control sample;

and/or, the preparation method of the internal standard working solution comprises the following steps:

step I: mixing the tolbutamide standard substance with the diluent to obtain an internal standard stock solution;

step II: and mixing the internal standard stock solution with the diluent to obtain the internal standard working solution.

More preferably, the diluent in the steps 1, a and I is DMSO, and the diluent in the steps 2, b and II is a methanol aqueous solution with the volume concentration of 30-70%. Further, the diluent in the steps 2, b and II is preferably a 50% methanol aqueous solution by volume concentration.

More preferably, the concentration gradient of the standard working solution is 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 sequence.

More preferably, the concentration gradient of the quality control working solution is 75 mug/mL, 1500 mug/mL and 3000 mug/mL in sequence.

More preferably, the concentration of the internal standard working solution is 1000 ng/mL.

More preferably, the concentration of the standard curve sample is 100ng/mL, 250ng/mL, 1250ng/mL, 2500ng/mL, 6250ng/mL, 12500ng/mL, 25000ng/mL, 100000 ng/mL.

More preferably, the concentration of the quality control sample is 3750ng/mL, 75000ng/mL and 150000 ng/mL.

In the present invention, blank plasma refers to plasma that does not contain COVID-19 potential therapeutic drug metabolites and internal standard working fluids.

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 invention develops a method for measuring the COVID-19 potential therapeutic drug metabolites in the cynomolgus monkey plasma, which can quickly and simply detect the COVID-19 potential therapeutic drug metabolites in the cynomolgus monkey plasma, thereby analyzing the pharmacokinetics/toxicity kinetics of the COVID-19 potential therapeutic drug metabolites in the cynomolgus monkey body.

2. The pretreatment method adopted by the invention is a protein precipitation method, and compared with the common extraction method, the method has simpler flow, short time consumption and easy operation; meanwhile, the linear range of the standard curve is good, so that the response value of the lower limit of the standard curve is good, and the sample injection amount of a required sample is small; the analysis time required by LC-MS/MS detection is short;

3. the pretreatment and LC-MS/MS detection method adopted by the invention enables the standard curve to be linear and accurate, and ensures the accuracy and reliability of detection; therefore, the invention can ensure that more accurate, high-precision and high-sensitivity detection data can be obtained in shorter analysis time, and lays a foundation for tamping for the analysis of non-clinical test biological samples.

Drawings

FIG. 1 is a graph of the COVID-19 potential therapeutic drug metabolite profile in the lower limit of quantitation sample of the standard curve (COVID-19 potential therapeutic drug metabolite retention time 1.01 min);

FIG. 2 is a graph of tolbutamide in a quantitative lower limit sample of a standard curve (tolbutamide retention time 1.77 min);

FIG. 3 is a graph of the spectrum of COVID-19 potential therapeutic drug metabolites in a sample at the upper limit of quantitation of the standard curve (COVID-19 potential therapeutic drug metabolite retention time 1.01 min);

FIG. 4 is a graph of tolbutamide in the upper limit of quantitation sample of the standard curve (tolbutamide retention time 1.77 min);

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 without specifying specific conditions in the following examples were selected according to the 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.

Example 1:

a method for determining COVID-19 potential therapeutic drug metabolites in cynomolgus monkey plasma, comprising:

1. preparation of reagents

1.1 Mobile Phase A (MPA): formic Acid (FA) aqueous solution with a volume concentration of 0.1%

Take 1000mL of H₂And adding 1mL of FA into the O solution in a solvent bottle, and mixing uniformly. Storing at room temperature for 1 month.

1.2 Mobile Phase B (MPB): MeOH (methanol)

1000mL MeOH was taken in a solvent bottle. Storing at room temperature for 3 months.

1.3 Strong Wash Solution (SNW): MeOH, ACN, IPA, DMSO, 1:1:1:1, v/v/v/v.

500mL MeOH, 500mL ACN (acetonitrile), 500mL IPA (isopropanol), 500mL DMSO (dimethyl sulfoxide) were mixed well. Storing at room temperature for 3 months.

1.4 Weak Wash solution (WNW): MeOH H₂O＝1:1，v/v。

1000mL of this solution were taken and 1000mL of H₂And O, and mixing uniformly. Storing at room temperature for 3 months.

1.5 precipitant & internal standard diluent: MeOH.

1000 mM LEOH were taken. Storing at room temperature for 1 month.

1.6 second dilution: MeOH H₂O＝1:1，v/v。

500mL of NaOH solution was taken and 500mL of H solution was added₂And O, and mixing uniformly. Storing at room temperature for 3 months.

1.7 first dilution: DMSO.

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

2. Preparing a standard curve & SST sample, a quality control sample and an internal standard working solution:

each stock solution and working solution prepared as follows was stored in an ultra-low temperature refrigerator (-70 to-90 ℃).

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. (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 by the concentration of the standard stock solution and the mass of the reduced COVID-19 potential therapeutic drug metabolite calculated by multiplying the mass of the weighed COVID-19 potential therapeutic drug metabolite by the mass reduction factor 0.988 of the COVID-19 potential therapeutic drug metabolite).

2.2 Standard music&System adaptability sample working solution (standard curve)&SST working solution) (diluent: MeOH H₂O＝1:1，v/v)

A plurality of standard working solutions (standard & SST working solutions) with concentration gradients were diluted with a second diluent according to table 1 below using a standard stock solution:

table 1: preparation of COVID-19 potential therapeutic drug metabolite koji & SST working solution

2.3 preparation of Standard Curve & SST samples

Standard curve & SST samples (i.e. standard curve samples) were obtained using standard curve & SST working fluids mixed with blank plasma according to table 2 below:

table 2: preparation of COVID-19 potential therapeutic drug metabolite standard curve & SST samples

Standard curve & SST samples were stored in an ultra-low temperature freezer (-70 to-90 ℃).

2.4 preparation of quality control stock solution

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 to dissolve, shaking up, and preparing into a quality control stock solution with the concentration of 5.000 mg/mL. (since the standard of COVID-19 potential therapeutic drug metabolites is not itself 100% pure or with water or salts, the volume of DMSO is calculated by the concentration of the quality control stock and the mass of the reduced COVID-19 potential therapeutic drug metabolite calculated by multiplying the mass of the measured COVID-19 potential therapeutic drug metabolite by the mass reduction factor of the COVID-19 potential therapeutic drug metabolite 0.988.)

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

Using the quality control stock solution, a plurality of quality control working solutions with concentration gradients were diluted with a second diluent according to table 3 below:

table 3: preparation of COVID-19 potential therapeutic drug metabolite quality control working solution

2.6 preparation of quality control samples

Quality control samples were obtained by mixing the blank plasma with the quality control working solution according to table 4 below:

table 4: preparation of COVID-19 potential therapeutic drug metabolite quality control sample

The quality control samples were stored in an ultra-low temperature freezer (-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. (since tolbutamide itself is not 100% pure or with water or salt, the volume of DMSO is calculated from the concentration of the internal standard stock solution and the converted tolbutamide mass, calculated by multiplying the measured tolbutamide mass by the tolbutamide mass conversion factor 0.999.)

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

Using an internal standard stock solution, preparing according to the following table 5, and diluting with a second diluent to obtain an internal standard working solution;

TABLE 5 Tribenesulfonylurea internal standard working solution preparation

3. Sample processing step

3.1 vortex the sample (if the sample needs to be thawed, re-vortex after thawing at room temperature).

3.2 suck 20. mu.L of STD, QC, DB, Carryover, Blank and cynomolgus monkey EDTA-K to be tested respectively₂Transferring the plasma sample to a 96-well plate or a polypropylene centrifugal tube; wherein STD is a standard curve&SST samples, QC quality control samples, DB, Carryover and Blank controls.

3.3 adding 20 μ L of internal standard working fluid (ISWS1) to the STD, QC, Blank and the cynomolgus monkey plasma samples to be tested;

3.4 pretreatment by adopting a protein precipitation method: vortex and mix well, add 160 μ L protein precipitant MeOH to all samples;

3.5 vortex and mix. Centrifugation was carried out at 4000rpm for 10min at 4 ℃.

3.6 taking 100 mu L of centrifuged supernatant to a 96-well plate or a polypropylene centrifuge tube, sealing the membrane, and uniformly mixing the supernatant with vortex at 1000rpm for 10min to obtain the treated STD, QC, DB, Carryover, Blank and cynomolgus monkey EDTA-K to be detected₂Plasma samples were analyzed by injection.

4. Subjecting the treated STD, QC, DB, Carryover, Blank and cynomolgus monkey EDTA-K to be tested to liquid chromatography-mass spectrometry (LC-MS/MS)₂Analytical detection of COVID-19 potential therapeutic drug metabolites in plasma samples:

wherein, the liquid chromatogram-mass spectrum combination condition is 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, and the total flow rate of the mobile phase A and the mobile phase B is 0.5 mL/min;

operating time: 3.0 min;

gradient elution was used, with elution gradients as in table 6:

TABLE 6 gradient of mobile phase

Time (minutes)	Flow rate (mL/min)	A(vol.％)	B(vol.％)
				Initial state	0.5	95	5
1.5	0.5	5	95
				2.5	0.5	5	95
2.51	0.5	95	5
				3	0.5	95	5

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

Needle washing solvent: a vigorous wash (MeOH: ACN: IPA: DMSO ═ 1:1:1:1, v/v/v/v);

weak wash (MeOH: H)₂O＝1:1,v/v)。

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;

the line purge amount was measured at 100. mu.L.

The type of a mass spectrometer: AB SCIEX TRIPLE QUAD^TM 4500；

An ion source: ESI;

ionization mode: a positive ion;

the MRM ion pairs are shown in table 7:

table 7: MRM ion pair

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

The instrument parameters are shown in table 8:

table 8: parameters of the instrument

Parameter(s)	COVID-19 potential therapeutic drug metabolites	Tolbutamide
			Ionization voltage (v)	5500	5500
Temperature (. degree.C.)	500	500
			Air for collision	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 MRM chromatogram, fitting standard curve, and collecting standard curve&Concentration of COVID-19 potential therapeutic drug metabolites in SST samples is represented by a standard curve with the abscissa&The peak area ratio of the COVID-19 potential therapeutic drug metabolite to the internal standard in the SST sample is the ordinate, the weight is set to be 1/x²Neglecting the origin, a linear standard curve is fitted. The corresponding spectra of the STD1 sample are shown in fig. 1 and 2; the corresponding spectra for the STD8 sample are shown in fig. 3 and 4.

With cynomolgus monkey EDTA-K to be tested₂The peak area of the analyte (i.e., COVID-19 potential therapeutic drug metabolite) in the plasma sample is compared to the peak area of the internal standard and a linear least squares regression calculation is performed on the theoretical concentration of the analyte in the standard curve to obtain a regression equation to calculate the measured concentration of the analyte in the sample.

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 assay batch acceptance criteria

1) The recalculated value for each concentration of the treated standard curve sample is calculated based on the ratio of the peak area of the analyte (i.e., the COVID-19 potential therapeutic drug metabolite) to the peak area of the internal standard in each treated standard curve sample in conjunction with the standard curve described above, and the deviation between the recalculated value and the labeled value for each concentration of the treated standard curve sample should be within ± 15.0% (within ± 20.0% at the lower limit of quantitation).

2) At least 75% of the treated 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 concentration of each treated control sample was calculated from the ratio of the peak area of the analyte (i.e., COVID-19 potential therapeutic drug metabolite) to the peak area of the internal standard in each treated control sample in conjunction with the standard curve described above, and the assay lot was considered acceptable when the concentration calculations for at least 67% of the treated control samples (at least 50% of the samples at each concentration point) were within ± 15.0% of their corresponding indices.

The results are shown in FIG. 3, where the standard curve regression equation is: y 0.000139x +0.000864(r 0.9985, r)²Greater than 0.98), the results of the standard curve and the back calculation deviation of the quality control and the labeled value are shown in tables 9 and 10, and the analysis batch acceptance standard is met. In conclusion, the method can be used for determining the concentration of COVID-19 potential therapeutic drug metabolites in the cynomolgus monkey EDTA-K2 plasma.

Table 9: quality control and marking value back calculation deviation data table

TABLE 10 notes and notes back-calculated deviation data sheet

While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that this is by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.