阅读说明：本技术 食蟹猴血浆中covid-19潜在治疗药物的检测方法 (Method for detecting COVID-19 potential therapeutic drug in cynomolgus monkey plasma ) 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 陈淑敏 谢一 于 2020-05-13 设计创作，主要内容包括：本发明提供了一种食蟹猴血浆中COVID-19潜在治疗药物的检测方法,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潜在治疗药物进行分析检测；其中,所述的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。本发明能够快速、简便地测定食蟹猴血浆中的COVID-19潜在治疗药物。(The invention provides a method for detecting a COVID-19 potential therapeutic drug in cynomolgus monkey plasma, which comprises the following steps: analyzing and detecting the COVID-19 potential therapeutic drug in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein, the COVID-19 potential therapeutic drug 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) propionic acid-2-ethylbutyl ester. The invention can quickly, simply and conveniently determine the COVID-19 potential therapeutic drug in the blood plasma of the cynomolgus monkey.)

1. A method for detecting COVID-19 potential therapeutic drugs in cynomolgus monkey plasma is characterized by comprising the following steps:

analyzing and detecting the COVID-19 potential therapeutic drug in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein 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;

the treated cynomolgus monkey plasma is the cynomolgus monkey plasma treated by a protein precipitation method;

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.

2. The method of claim 1, wherein the gradient elution procedure comprises: in an initial state, the volume percentage of the mobile phase A is 90-99%, and the volume percentage of the mobile phase B is 1-10%; the volume percentage of the mobile phase A gradually decreases to 1-10% and the volume percentage of the mobile phase B gradually increases to 90-99% in a time period from an initial state to 1.50 +/-0.50 min, and the volume percentage of the mobile phase A is maintained at 1-10% and the volume percentage of the mobile phase B is maintained at 90-99% in a time period from 1.50 +/-0.50 min to 2.50 +/-0.50 min; gradually increasing the volume percentage of the mobile phase A to 90-99% and gradually decreasing the volume percentage of the mobile phase B to 1-10% in a time period from 2.50 +/-0.50 min to 2.51 +/-0.50 min; the volume percentage of the mobile phase A is maintained between 90 and 99 percent and the volume percentage of the mobile phase B is maintained between 1 and 10 percent in a time period of between 2.51 plus or minus 0.50min and 3.00 plus or minus 0.50 min.

3. The method of claim 1, wherein the gradient elution procedure comprises: 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 1.50min, 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 1.50min to 2.50 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 2.50min 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 time period of 2.51min to 3.00 min.

4. The method of claim 1, wherein the precipitating agent in the protein precipitation method is acetonitrile.

5. The method for detecting COVID-19 potential therapeutic drugs in the plasma of the cynomolgus monkey according to claim 1, wherein the sample size in the liquid chromatography-mass spectrometry is 1-10 μ L, preferably 2 μ L;

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

6. The method of claim 1, wherein the total flow rate of mobile phase a and mobile phase B is 0.5 mL/min.

7. The method of claim 1, wherein the mobile phase a is 0.1% formic acid solution by volume.

8. The method of claim 1, wherein the detection of the covi-19 potential therapeutic agent in the cynomolgus monkey plasma is performed by a liquid chromatography-mass spectrometry combination method, wherein the detection is performed on the treated standard curve sample, the treated quality control sample and the treated 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 of 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 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 standard 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.

10. The method of claim 9, wherein the diluent in steps 1, a and I is DMSO, and the diluent in steps 2, b and II is a methanol aqueous solution with a volume concentration of 30% to 70%; the diluent in the steps 2, b and II is preferably a methanol aqueous solution with the volume concentration of 50%;

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

and/or the concentration gradient of the quality control working solution is 240ng/mL,3000ng/mL and 60000ng/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 4ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL and 4000 ng/mL;

and/or, the concentration of the quality control sample is 12ng/mL, 150ng/mL and 3000 ng/mL.

Technical Field

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

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.

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.

The currently relevant potential therapeutic drugs against the new coronaviruses are mainly small molecule drugs. The metabolism of small molecule drugs (molecular weight 200-700) is measured by chromatography, and the method is mainly used for screening used solvents, reagents, matrixes and preparation and processing steps in the development process, so that the method for accurately measuring the metabolism of the small molecule drugs is formed. Therefore, the development of a preclinical safety evaluation method of the potential therapeutic drugs of COVID-19 is of great importance.

Disclosure of Invention

The invention provides a method for detecting a COVID-19 potential therapeutic drug in cynomolgus monkey plasma, which can quickly and simply determine the COVID-19 potential therapeutic drug in the cynomolgus monkey plasma.

The invention provides a method for detecting a COVID-19 potential therapeutic drug in cynomolgus monkey plasma, which comprises the following steps:

analyzing and detecting the COVID-19 potential therapeutic drug in the treated cynomolgus monkey plasma by adopting a liquid chromatography-mass spectrometry combined method; wherein 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;

the treated cynomolgus monkey plasma is the cynomolgus monkey plasma treated by a protein precipitation method;

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.

Preferably, the procedure of gradient elution is as follows: in the initial state, the volume percentage of the mobile phase A is 90-99%, and the volume percentage of the mobile phase B is 1-10%; the volume percentage of the mobile phase A is gradually reduced to 1-10% and the volume percentage of the mobile phase B is gradually increased to 90-99% in a time period from an initial state to 1.50 +/-0.50 min, and the volume percentage of the mobile phase A is maintained at 1-10% and the volume percentage of the mobile phase B is maintained at 90-99% in the time period from 1.50 +/-0.50 min to 2.50 +/-0.50 min; in the time period from 2.50 +/-0.50 min to 2.51 +/-0.50 min, the volume percentage of the mobile phase A is gradually increased to 90-99 percent, and the volume percentage of the mobile phase B is gradually decreased to 1-10 percent; the volume percentage of the mobile phase A is maintained between 90 and 99 percent and the volume percentage of the mobile phase B is maintained between 1 and 10 percent in a time period from 2.51 +/-0.50 min to 3.00 +/-0.50 min. (the volume percentages of the mobile phases A and B are based on the total volume of the mobile phases A and B)

In one specific example, the gradient elution procedure is: 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 1.50min, 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 1.50min to 2.50 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 2.50min 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 the mobile phases A and B are based on the total volume of the mobile phases A and B)

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

Preferably, the precipitating agent in the protein precipitation method is acetonitrile.

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

In a specific example, the mobile phase a is a 0.1% strength by volume aqueous formic acid solution.

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 acetonitrile.

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 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 standard 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 80ng/mL,200ng/mL,1000ng/mL,2000ng/mL,5000ng/mL,10000ng/mL,20000ng/mL and 80000ng/mL in sequence.

More preferably, the concentration gradient of the quality control working solution is 240ng/mL,3000ng/mL and 60000ng/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 4ng/mL, 10ng/mL, 50ng/mL, 100ng/mL, 250ng/mL, 500ng/mL, 1000ng/mL, and 4000 ng/mL.

More preferably, the concentration of the quality control sample is 12ng/mL, 150ng/mL and 3000 ng/mL.

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

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.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention develops a method for detecting the COVID-19 potential therapeutic drug in a cynomolgus monkey plasma sample, and the method can quickly and simply determine the concentration of the COVID-19 potential therapeutic drug in the cynomolgus monkey plasma, thereby being used for analyzing the pharmacokinetics/toxicity kinetics of the COVID-19 potential therapeutic drug in the cynomolgus monkey.

2. The pretreatment 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 consumption and easy operation; the method adopts the liquid chromatography-tandem mass spectrometry, utilizes a proper mobile phase and a gradient elution program, improves the proportion of methanol in the middle part of the operation time, ensures that the peak pattern is good, makes a standard curve linear and accurate, ensures the accuracy and reliability of detection, and simultaneously has small sample amount and short required analysis time; 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 lower limit of quantitation (4ng/mL) of the standard curve (COVID-19 potential therapeutic drug retention time 1.86 min);

FIG. 2 is a standard curve quantitative lower limit spectrum (tolbutamide retention time 1.77 min);

FIG. 3 is a graph of the upper limit of quantitation (4000ng/mL) of the standard curve (COVID-19 potential therapeutic drug retention time 1.86 min);

FIG. 4 is a standard curve quantitation upper limit map (tolbutamide retention time 1.77 min);

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 codv-19 potential therapeutic agent described in the examples was 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 1:

a method for detecting COVID-19 potential therapeutic drugs in cynomolgus monkey plasma comprises the following steps:

1. preparation of reagents

1.1 Mobile Phase A (MPA): formic Acid (FA) in water at a concentration of 0.1% by volume.

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:

the following stock solutions and working solutions were stored in an ultra-low temperature freezer (-70 to-90 ℃).

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 volume of DMSO is calculated from the concentration of the standard stock and the mass of the reduced COVID-19 potential therapeutic drug metabolite, wherein the mass of the reduced COVID-19 potential therapeutic drug metabolite is obtained by multiplying the weighed mass of the COVID-19 potential therapeutic drug metabolite by its corresponding mass reduction factor of 0.992).

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 curve & SST working solutions) having a concentration gradient were diluted with a second diluent according to table 1 below using a standard stock solution;

table 1: preparation of standard curve of COVID-19 potential therapeutic drug standard and 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 standard curve & SST sample

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 standard substance into a transparent sample bottle, adding a proper amount of DMSO, dissolving, shaking up, and preparing into a quality control stock solution with the concentration of 1.000 mg/mL. (the volume of DMSO is calculated from the concentration of the quality control stock and the mass of the reduced COVID-19 potential therapeutic drug metabolite, wherein the mass of the reduced COVID-19 potential therapeutic drug metabolite is obtained by multiplying the mass of the weighed COVID-19 potential therapeutic drug metabolite by its corresponding mass conversion factor of 0.992).

2.5 preparation of quality control working solution (Diluent: MeOH: H2O ═ 1:1, v/v)

Using a quality control stock solution, prepared according to table 3 below, diluted with a second diluent to a plurality of quality control working solutions having a concentration gradient:

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

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 quality control sample of COVID-19 potential therapeutic drug standard

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. (the volume of DMSO is calculated from the concentration of the internal standard stock solution and the mass of the converted tolbutamide, wherein the mass of the converted tolbutamide is obtained by multiplying the weighed mass of tolbutamide by its corresponding mass conversion factor of 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: preparation of tolbutamide internal standard working solution

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 sample, QC quality control sample, DB, Carryover and Blank control;

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 treating by adopting a protein precipitation method: after vortex mixing, 160 μ L of protein precipitant ACN was added 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 drugs 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, elution gradient program as in table 6:

table 6: elution gradient procedure

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 1.50min, 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 1.50min to 2.50 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 2.50min 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 the mobile phases A and B are based on the total volume of the mobile phases A and B)

Needle washing solvent: strong 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;

measuring the line purge amount of 100 μ 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

The instrument parameters are shown in table 8:

table 8 instrument parameters:

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 in SST sample is abscissa and takes standard curve&The peak area ratio of the COVID-19 potential therapeutic drug 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 tested₂Comparison of analyte (i.e. COVID-19 potential therapeutic drug) peak area in plasma sample with internal standard peak area for theoretical concentration of analyte in standard curveAnd calculating the measured concentration of the analyte in the sample according to 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 assay batch acceptance criteria

1. The calculated values for the concentrations of each of the treated standard curve samples were calculated based on the ratio of the peak area of the analyte (i.e., the COVID-19 potential therapeutic drug) to the peak area of the internal standard in each of the treated standard curve samples in combination with the standard curve described above, and the deviation between the calculated values and the labeled values for each of the treated standard curve sample concentrations should be within ± 15.0% (within ± 20.0% of 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., codv-19 potential therapeutic drug) 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. 5, where the standard curve regression equation is: y 0.000718x +0.000224(r 0.9969, r)²Greater than 0.98), the result of the back calculation deviation of the labeled value of the labeled sample and the labeled value of the quality control sample is shown in table 9 and table 10, and the standard of the analysis batch acceptance is met. In conclusion, the method can be used for determining the cynomolgus monkey EDTA-K₂Concentration of COVID-19 potential therapeutic drug in plasma.

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

Table 10: data table for back calculation of deviation between marked music and marked value

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.