CN111323511A - Rapid detection kit and method for inactivating new coronavirus - Google Patents
Rapid detection kit and method for inactivating new coronavirus Download PDFInfo
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Abstract
The invention discloses a rapid detection kit for inactivating a new coronavirus, which comprises the following components: (1) an inactivation reaction test tube containing trypsin freeze-dried powder, (2) an enzymolysis reaction solution, and (3) rapidly detecting standard polypeptide; the amino acid sequence of the rapid detection standard polypeptide is as follows: ITFGGPTDSTDNNQNGGR are provided. On the premise of ensuring the sensitivity and specificity of detection, the invention can rapidly detect the inactivated virus, obtain an accurate detection result and reduce the infection risk of medical care personnel and detection personnel to the maximum extent.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a rapid detection kit and a method for inactivating a new coronavirus.
Background
Viruses are a class of non-cellular microorganisms. Mature intact virus particles become virions, composed of nucleic acids and proteins. The virus is characterized by small volume and can pass through a sterilizing filter; simple structure, only contains one type of nucleic acid (DNA or RNA); lacking genes encoding mitochondria and ribosomes, it is necessary to parasitize living cells and reproduce progeny in a replicative manner.
Coronaviruses belong phylogenetically to the order of the nested viruses (Nidovirales) the family of Coronaviridae (Coronaviridae) the genus coronaviruses (Coronavirus). The coronavirus is an RNA virus with an envelope (envelope) and a linear single-strand positive strand genome, is a large virus widely existing in nature, has a diameter of about 80-120 nm, has a methylated cap structure at the 5 'end of a genome, has a poly (A) tail at the 3' end, has a genome full length of about 27-32kb, is the virus with the largest genome in the existing known RNA viruses, and only infects vertebrates such as human, mice, pigs, cats, dogs, wolfs, chickens, cattle and birds. The coronavirus is firstly separated from the chicken in 1937, and the virus particles have the diameter of 60-200 nm, the average diameter of 100nm, are spherical or elliptical and have polymorphism. The virus has an envelope, spinous processes exist on the envelope, the whole virus is like coronas, and the spinous processes of different coronaviruses have obvious difference. Tubular inclusions are sometimes visible in coronavirus infected cells. 2019 the novel coronavirus (2019-nCoV, causing novel coronavirus pneumonia COVID-19) is the 7 th coronavirus which is known at present and can infect people, people infected with the coronavirus can have symptoms with different degrees, some people can have fever or slight cough, some people can develop pneumonia, and some people can be more serious or even die. The main transmission route of the novel coronavirus is respiratory aerial transmission and contact transmission.
In virus clinical testing methods, gene (DNA or RNA) based methods (e.g., PCR or sequencing) and protein fingerprint based methods (e.g., time-of-flight mass spectrometry) are commonly used. They all need to collect fresh samples (pharyngeal swab, sputum, lung perfusate or blood) of patients for detection, which can cause great infection risk to medical staff and detection staff who collect the samples due to the very strong infectivity of the novel coronavirus. The collected sample is immediately inactivated, so that the infection risk of medical staff and detection staff can be effectively reduced, but after the sample is inactivated (an autoclave is usually adopted), although the virus loses the original pathogenicity, detection markers (DNA, RNA and protein with a spatial structure) of the virus are easy to damage and cannot be detected. Therefore, how to effectively reduce the infection risk of medical staff and detection staff, and meanwhile, the detection is not influenced, which becomes a problem to be solved urgently.
Disclosure of Invention
The invention aims to provide a rapid detection kit for inactivating a new coronavirus, which can rapidly detect the inactivated virus on the premise of ensuring the sensitivity and specificity of detection, obtain an accurate detection result and reduce the infection risk of medical staff and detection staff to the maximum extent.
The invention also provides a rapid detection method for the new coronavirus capable of reducing the infection risk of medical personnel, which can rapidly carry out on-site rapid enzymolysis and inactivation treatment on the collected virus, complete the enzymolysis process by utilizing the time of returning the sample transportation process to a laboratory, immediately detect the virus after the sample transportation process reaches the laboratory, and effectively reduce the infection risk of the medical personnel and the detection personnel.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a rapid detection kit for inactivating a new coronavirus is characterized by comprising the following components: (1) an inactivation reaction test tube containing trypsin freeze-dried powder, (2) an enzymolysis reaction solution, and (3) rapidly detecting standard polypeptide; the amino acid sequence of the rapid detection standard polypeptide is as follows: ITFGGPTDSTDNNQNGGR are provided.
The scheme abandons the traditional and easily-destroyed detection markers (DNA, RNA and protein with a spatial structure), and selects the virus protein shell zymolyte with stable chemical property as the marker. The virus protein coat enzymolysis product is usually polypeptide, has an amino acid sequence, and can ensure the specificity of detection. And the detection of 1 sample can be completed in 1-2 minutes on the premise of ensuring higher sensitivity and accuracy by matching with mass spectrum detection. After enzymatic hydrolysis, the viral protein coat loses its activity and therefore its risk of infection is greatly reduced. The chemical structure of the amino acid sequence is stable and can bear higher temperature. Therefore, the sample can be heated before the incubator is opened, potential infection sources (mainly from viruses which are not completely enzymolyzed) are thoroughly killed, medical workers and detection workers are protected from the risk of microbial infection to the greatest extent, and meanwhile, laboratories with lower biological safety levels can also accept detection work.
The liquid chromatogram tandem mass spectrum is a device for identifying the molecular weight of a detected object, and has higher sensitivity and accuracy. The amino acid sequence is one of the materials which embody the genetic code in organisms, and the specific amino acid sequence can be selected to ensure the specificity of detection. Compared with the spatial structure of DNA, RNA and protein, the amino acid sequence has higher chemical stability. Compared with the traditional classical method, the invention greatly shortens the detection time on the premise of ensuring the sensitivity and specificity of detection, and can detect the inactivated virus without influencing the detection result.
The enzymolysis reaction solution comprises the following components: ammonium bicarbonate with the concentration of 500mmol/L, mercaptoethanol with the concentration of 50mmol/L and isotope labeling long polypeptide with the concentration of 100 ng/mL.
The ammonium bicarbonate has the main function of controlling the pH value of a reaction system and ensuring the enzymolysis speed and efficiency. Mercaptoethanol, main role: the spatial structure of the protein is largely maintained by disulfide bonds between cysteines. Under the condition that the spatial structure of the protein is compact, trypsin is not easy to contact with enzymolysis sites, so that the enzymolysis efficiency is poor, and the enzymolysis time is prolonged. The mercaptoethanol is added, so that the disulfide bonds of the protein can be destroyed, on one hand, the protein enzymolysis efficiency can be improved, the enzymolysis time can be shortened, and on the other hand, the virus can be inactivated in a mode of destroying the spatial structure of the protein.
The amino acid sequence of the isotope labeled long polypeptide is as follows: PQSNQRSAPRI × TFGGPTDSTDNNQNGGRNGARPKQRRP, whichWherein I is [ alpha ]13C6,15N]-isoleucine.
The trypsin is alkaline trypsin. The sequence was extended 10 amino acids in length from the C-and N-termini of the coronary virus based on the sequence of the N protein, using ITFGGPTDSTDNNQNGGR as a template. The long polypeptide is artificially synthesized by adopting isotope labeled isoleucine, and the false negative detection result is mainly verified.
In the actual detection process, various factors (such as the existence of trypsin inhibitor in a sample and the failure of a kit) can cause the reduction of the enzymolysis efficiency, so that a false negative result is caused. PQSNQRSAPRI x TFGGPTDSTDNNQNGGRNGARPKQRRP produces I x TFGGPTDSTDNNQNGGR under the action of trypsin. When the polypeptide is measured, the enzymolysis process of the sample is considered to be normal, and the result is accurate and reliable. When the polypeptide cannot be detected, the enzymolysis process is judged to have problems, and the problem is eliminated and then the detection is carried out again.
I × TFGGPTDSTDNNQNGGR has exactly the same physicochemical properties as ITFGGPTDSTDNNQNGGR. However, the former has isotope labeling, and does not generate cross reaction with the latter during mass spectrum detection, so that the detection can be carried out simultaneously.
The inactivation reaction tube is a 2mL tube, and contains 100 mug of trypsin freeze-dried powder.
The concentration of the rapid detection standard polypeptide is 100 ng/ml.
A non-medical new coronavirus rapid detection method for reducing the infection risk of medical personnel comprises the following steps: (1) collecting a sample on site and directly putting the sample into an inactivation reaction test tube added with an enzymolysis reaction solution; when a sample is collected on site, in the laboratory, the rapid detection standard polypeptide is used as a standard substance, the liquid chromatography tandem mass spectrometry is used as a detection instrument, sample introduction analysis is carried out, and a standard map is established;
(2) transporting the inactivated reaction test tube back to a laboratory, placing the inactivated reaction test tube into a portable incubator, incubating for 30-60 minutes at 37 ℃, and then raising the temperature of the incubator to 100 ℃ for 30 minutes to stop the enzymolysis reaction;
(3) the sample returned to the laboratory was filtered through a 0.22 micron filter and then detected by liquid chromatography tandem mass spectrometry.
The field sample collection is directly put into the inactivation reaction test tube added with the enzymolysis reaction liquid, so that inspectors do not need to open the cover for the second time to carry out sample pretreatment operation, and the infection risk is reduced.
Incubating for 30-60 minutes at 37 ℃, on one hand, performing enzymolysis on the virus protein shell to obtain an amino acid sequence required by detection, and on the other hand, achieving the purpose of reducing the infection risk by destroying the virus protein shell. The temperature of the incubator is raised to 100 ℃ and kept for 30 minutes, so that the enzymolysis reaction is stopped on one hand, and possible residual viruses are inactivated on the other hand, and the infection risk is further reduced. The enzymolysis inactivation time is short, and the method is suitable for rapid detection.
Usually, the time of collecting the sample to be sent to a laboratory is more than 30 minutes, the method effectively utilizes the sample transportation time to carry out enzymolysis, so that the sample can be detected immediately when arriving at the laboratory, the sample turnover period is shortened, and viruses after enzymolysis are inactivated, so that the infection risk of detection personnel is reduced.
The purpose of the scheme is not to judge the health condition of the detected object, and the scheme is only to improve the detection program so as to reduce the infection risk of medical staff and detection personnel as far as possible and prevent the medical staff and the detection personnel from being infected by viruses due to the detected object.
The liquid phase detection conditions of the liquid chromatography tandem mass spectrum are as follows:
chromatographic column Waters BEH 300C18, specification 100mm × 2.1.1 mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 2% to 45% in 1 minute;
flow rate: 0.3 mL/min;
sample introduction volume: 10 μ L.
The mass spectrum detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
ESI + ion source, capillary voltage: 3.5kv, cone voltage: 25V, desolventizing temperature:at 400 ℃, desolventizing agent airflow is 650L/min, cone hole blowback airflow is 20L/hr, and collision chamber pressure is 3.0 × 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 120 ℃, extractor voltage: 3.0V, the monitoring mode adopts a multi-reaction detection mode MRM, and the concentration of parent ions: 925.9, daughter ion: 1062.4, 645.3.
The invention has the beneficial effects that:
1. and (3) fast: the enzymolysis work can be finished by utilizing the sample transportation time, so that the sample turnover time is reduced; the detection speed of each sample is only 1-2 minutes, and the flux is high.
2. Low biosafety risk: the virus is inactivated by two means of proteolysis and high temperature, so that the biological safety risk is greatly reduced, the medical staff and the detection workers are protected from the risk of virus infection to the maximum extent, and meanwhile, laboratories with lower biological safety levels (such as primary hospitals, customs and the like) can also accept the detection work, thereby achieving the purpose of graded diagnosis and treatment and reducing the pressure of the third hospital.
3. On the premise of ensuring the sensitivity and specificity of detection, the inactivated virus can be quickly detected to obtain an accurate detection result.
Drawings
FIG. 1 shows the relationship between different polypeptides and the time of enzymatic digestion.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples.
In the present invention, the raw materials and equipment used are commercially available or commonly used in the art, unless otherwise specified. The methods in the following examples are conventional in the art unless otherwise specified.
Example 1:
a rapid detection kit for inactivating a new coronavirus comprises the following components:
(1) an inactivation reaction test tube (2mL test tube) contains 100 mu g of alkaline trypsin freeze-dried powder,
(2) enzymolysis reaction liquid: ammonium bicarbonate with the concentration of 500mmol/L, mercaptoethanol with the concentration of 50mmol/L and isotope labeling long polypeptide with the concentration of 100 ng/mL; the amino acid sequence of the isotope-labeled long polypeptide is as follows: PQSNQRSAPRI or TFGGPTDSTDNNQNGGRNGARPKQRRP (SEQ ID No.10), wherein I is [ alpha ]13C6,15N]-isoleucine.
(3) Rapidly detecting a standard polypeptide; the amino acid sequence of the rapid detection standard polypeptide is as follows: ITFGGPTDSTDNNQNGGR (SEQ ID No. 1). The concentration of the rapid detection standard polypeptide is 100ng/ml (aqueous solution).
The present invention adopts novel coronavirus (SARS-CoV-2, positive sample is collected by hospital) to make detection.
Example 2:
a non-medical new coronavirus rapid detection method for reducing the infection risk of medical personnel comprises the following steps:
(1) opening the kit, adding 1mL of enzymolysis reaction solution into 2mL of inactivation reaction test tube containing 100 ug of alkaline trypsin freeze-dried powder, shaking up, collecting throat swab of patient on site by medical staff, and rapidly placing into the inactivation reaction test tube; meanwhile, in a laboratory, rapid detection standard polypeptide is used as a standard substance, liquid chromatography tandem mass spectrometry is used as a detection instrument, sample injection analysis is carried out, and a standard map is established;
(2) transporting the inactivated reaction test tube back to a laboratory, putting the inactivated reaction test tube into a portable incubator (sold in the market), incubating for 30-60 minutes at 37 ℃ (the incubation time is longer when the inactivated reaction test tube is farther away from the laboratory according to the distance from the laboratory), raising the temperature of the incubator to 100 ℃ before uncapping operation, and keeping for 30 minutes to stop the enzymolysis reaction;
(3) returning to the laboratory, filtering the enzymolysis product by a 0.22 micron filter membrane, and detecting by a liquid chromatography tandem mass spectrum.
The liquid phase detection conditions of the liquid chromatography tandem mass spectrum are as follows:
chromatographic column Waters BEH 300C18, specification 100mm × 2.1.1 mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 2% to 45% in 1 minute;
flow rate: 0.3 mL/min;
sample introduction volume: 10 μ L.
The mass spectrum detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
ESI + ion source, capillary voltage 3.5kv, taper hole voltage 25V, desolventizing temperature 400 deg.C, desolventizing gas flow 650L/min, taper hole back blowing gas flow 20L/hr, collision chamber pressure 3.0 × 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 120 ℃, extractor voltage: 3.0V, the monitoring mode adopts a multi-reaction detection mode MRM and the monitoring mode adopts a multi-reaction detection mode MRM.
MRM parameters are as follows
Identification of specificity
To verify the specificity of the assay of the present invention, pharyngeal swabs from patients with influenza A, influenza B and novel coronavirus were selected as specimens (two replicates per specimen) and assayed according to the method of example 2.
| Sample numbering | To confirm the diagnosis of the disease | Using the results of the test of example 2 |
| 1 | Influenza A | Negative of |
| 2 | Influenza A | Negative of |
| 3 | Influenza A | Negative of |
| 4 | Influenza B | Negative of |
| 5 | Influenza B | Negative of |
| 6 | Influenza B | Negative of |
| 7 | Novel coronavirus | Positive for |
| 8 | Novel coronavirus | Positive for |
The detection result shows that the method has specificity and can be effectively distinguished from other viruses.
Screening specific polypeptide according to enzymolysis time
Generally, a protein is subjected to enzymolysis to obtain several polypeptides, and each polypeptide has different physicochemical properties, including sensitivity, stability, specificity, and the like. Therefore, the selection of proper polypeptide is the key of the invention, and the enzymolysis time is the main concern factor of the invention, therefore, the invention firstly screens the polypeptide according to the enzymolysis time to achieve the purpose of rapid detection.
The research aims at the novel coronavirus N protein, and 31 theoretical enzymolysis polypeptides are provided. After N proteolysis, 23 polypeptides were identified. Because the sensitivity of partial polypeptide is low and the detection requirement is not met, the MRM detection method of 9 polypeptides is established. The polypeptide sequences are shown in the following table.
Taking a positive sample for enzymolysis according to the method of the embodiment 2, setting the enzymolysis time to be 0, 10, 20, 30, 40, 50 and 60 minutes, selecting a sample for enzymolysis overnight, and detecting an enzymolysis product by adopting mass spectrometry. The recovery of the polypeptide at other digestion times was calculated with the recovery of the overnight digested sample set at 100%, and the results are shown in fig. 1. As shown in figure 1, the polypeptide with the sequence of ITFGGPTDSTDNNQNGGR, EGIVWVATEGALNTPK, QLQNSMSGASADSTQA can be completely enzymolyzed within 20 minutes, and the requirement of the invention on enzymolysis time is met. Thus, all three polypeptides may serve as candidate markers. And adopting thermal stability as a judgment condition in further investigation for further screening.
Stability testing of candidate markers
The amino acid composition of different polypeptides varies, as does their thermostability. Therefore, the present invention performed a thermostability test on the above three candidate marker polypeptides.
And (3) placing the enzymolysis product in a portable incubator, adjusting the temperature to 100 ℃, and respectively keeping the temperature for 15-90 minutes for detection.
As shown, ITFGGPTDSTDNNQNGGR, EGIVWVATEGALNTPK was stable for 45 minutes. QLQNSMSGASADSTQA, the stability is poor, and the stability can be maintained for only 15 minutes in an environment of 100 ℃. Therefore, ITFGGPTDSTDNNQNGGR with the best stability is selected as the detection marker in the invention.
Two positive samples are respectively detected by adopting the normal kit reagent and the kit reagent after the accelerated destruction test (namely, the reagent is treated for 72 hours at the temperature of 70 ℃), and the detection results are as follows:
| ITFGGPTDSTDNNQNGGR | I*TFGGPTDSTDNNQNGGR | |
| normal reagent | Detect out | Detect out |
| Reagent after accelerated destruction test | Not detected out | Not detected out |
The result shows that the normal reagent can simultaneously detect the detected polypeptide and the isotope labeled detected polypeptide, and the result is accurate and reliable. After the reagent is treated by the accelerated destruction test, the polypeptide to be detected cannot be hydrolyzed out of the virus, and a false negative detection result is generated. The isotope-labeled polypeptide to be detected can not be detected at the same time, so that the reagent state is well indicated, the false negative detection result can not be generated, and the accuracy and reliability of the result can be ensured.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
SEQUENCE LISTING
<110> Zhejiang university medical college affiliated with the fourth hospital (Zhejiang Uygur Hospital, Zhejiang university medical college affiliated with the fourth hospital)
<120> quick detection kit and method for inactivating new coronavirus
<130>2020.03
<160>10
<170>PatentIn version 3.3
<210>1
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<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>1
Ile Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly
1 5 10 15
Gly Arg
<210>2
<211>20
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>2
Asn Pro Asn Asn Asn Ala Ala Thr Val Leu Gln Leu Pro Gln Gly Thr
1 5 10 15
Thr Leu Pro Lys
20
<210>3
<211>19
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>3
Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly
1 5 10 15
Ala Ile Lys
<210>4
<211>17
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>4
Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp Met Asp Asp Phe Ser
1 5 10 15
Arg
<210>5
<211>16
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>5
Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Asp Leu Ile Arg
1 5 10 15
<210>6
<211>17
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>6
Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu Leu Leu Leu Asp
1 5 10 15
Arg
<210>7
<211>16
<212>PRT
<213> novel coronavirus (SARS-CoV-2)
<400>7
Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys
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35
Claims (8)
1. A rapid detection kit for inactivating a new coronavirus is characterized by comprising the following components: (1) an inactivation reaction test tube containing trypsin freeze-dried powder, (2) an enzymolysis reaction solution, and (3) rapidly detecting standard polypeptide; the amino acid sequence of the rapid detection standard polypeptide is as follows: ITFGGPTDSTDNNQNGGR are provided.
2. The rapid detection kit for the inactivation of a new coronavirus according to claim 1, wherein: the enzymolysis reaction solution comprises the following components: ammonium bicarbonate with the concentration of 500mmol/L, mercaptoethanol with the concentration of 50mmol/L and isotope labeling long polypeptide with the concentration of 100 ng/mL.
3. The rapid detection kit for the inactivation of a new coronavirus according to claim 2, wherein: the amino acid sequence of the isotope labeled long polypeptide is as follows: PQSNQRSAPRI or TFGGPTDSTDNNQNGGRNGARPKQRRP wherein I is [ alpha ], [13C6,15N]-isoleucine.
4. The rapid detection kit for the inactivation of a new coronavirus according to claim 1, wherein: the trypsin is alkaline trypsin.
5. The rapid detection kit for the inactivation of a new coronavirus according to claim 1, wherein: the concentration of the rapid detection standard polypeptide is 100 ng/ml.
6. A non-medical new coronavirus rapid detection method for reducing the infection risk of medical personnel is characterized by comprising the following steps:
(1) directly putting a field collected sample into an inactivation reaction test tube added with an enzymolysis reaction solution; when a sample is collected on site, in a laboratory, rapid detection standard polypeptide is used as a standard substance, liquid chromatography tandem mass spectrometry is used as a detection instrument, sample introduction analysis is carried out, and a standard map is established;
(2) transporting the inactivated reaction test tube back to a laboratory, placing the inactivated reaction test tube into a portable incubator, incubating for 30-60 minutes at 37 ℃, and then raising the temperature of the incubator to 100 ℃ for 30 minutes to stop the enzymolysis reaction;
(3) the sample returned to the laboratory was filtered through a 0.22 micron filter and then detected by liquid chromatography tandem mass spectrometry.
7. The method of claim 6, wherein: the liquid phase detection conditions of the liquid chromatography tandem mass spectrum are as follows:
chromatographic column Waters BEH 300C18, specification 100mm × 2.1.1 mm, particle size 1.7 μm;
column temperature: 40 ℃; sample temperature: room temperature;
mobile phase: mobile phase A: an aqueous solution containing 0.1% formic acid; mobile phase B: acetonitrile solution containing 0.1% formic acid;
chromatographic separation gradient conditions: the mobile phase B content was increased from 2% to 45% in 1 minute;
flow rate: 0.3 mL/min;
sample introduction volume: 10 μ L.
8. The method of claim 6, wherein: the mass spectrum detection conditions of the liquid chromatogram tandem mass spectrum are as follows:
ESI + ion source, capillary voltage: 3.5kv, cone voltage: 25V, desolventizing temperature: 400 deg.CThe desolventizing gas flow rate is 650L/min, the cone hole back blowing gas flow rate is 20L/hr, and the pressure of the collision chamber is 3.0 × 10-3mbar; low-end resolution 1: 2.5V, high-end resolution 1: 15.0V, ion energy 1: 0.5 eV; low-end resolution 2: 2.8V, high-end resolution 2: 15.0V, ion energy 2: 1.0 eV; ion source temperature: 120 ℃, extractor voltage: 3.0V, the monitoring mode adopts a multi-reaction detection mode MRM, and the concentration of parent ions: 925.9, daughter ion: 1062.4, 645.3.
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