WO2022021886A1 - Method for separation and structural identification of relevant metabolites of lianhua qingwen drugs - Google Patents

Abstract

The present invention provides a method for separation and structural identification of relevant metabolites of Lianhua Qingwen drugs. The method comprises: separating, by using the following chromatographic conditions, relevant metabolites in body fluids of individuals administrated Lianhua Qingwen drugs: a chromatographic column: C18 chromatographic column; mobile phases: A: 0.1 % aqueous formic acid solution, and B: acetonitrile, the volume ratio of A to B being (98-5):(2-95); flow velocity: 0.3 mL/min; and column temperature: 35°C.

Description

A method for separation and structure identification of Lianhuaqingwen drug-related metabolites technical field

The invention relates to the field of medicine, in particular to a method for separation and structure identification of Lianhuaqingwen drug-related metabolites.

Background technique

At the end of 2019, a new type of coronavirus (SARS-CoV-2) that had never been discovered before suddenly broke out, and large-scale human-to-human transmission of the virus occurred worldwide, with more than 5 million confirmed infections. people. The high morbidity rate caused by the rapid spread of the new coronavirus and the current lack of effective therapeutic drugs make it urgent to research effective drugs to prevent or treat the new coronavirus. In China's anti-epidemic work, traditional Chinese medicine has played an irreplaceable role. Traditional Chinese medicine can effectively relieve symptoms, reduce the development of mild and common types to severe types, improve the cure rate, reduce the mortality rate, and promote the recovery of the convalescent population. The application of traditional Chinese medicine has provided an important guarantee for China to effectively control the development of new coronary pneumonia. Among them, Lianhua Qingwen Capsule has been confirmed to have clear clinical therapeutic effect after clinical research and observation. In February 2020, Lianhua Qingwen Capsules (granules) were included in the "New Coronavirus Pneumonia Diagnosis and Treatment Program (Trial Version 6)" by the National Health Commission of China. In April 2020, the "Approval for Supplementary Drug Application" issued by the State Food and Drug Administration showed that Lianhua Qingwen Capsules (granules) were approved to be used for fever, cough, and fatigue caused by mild and common types of new coronavirus pneumonia. The course of treatment is: 7 to 10 days.

Lianhua Qingwen Capsule is a Chinese patent medicine produced by Shijiazhuang Yiling Pharmaceutical Co., Ltd., and is included in the Chinese Pharmacopoeia. Lianhua Qingwen Prescription draws on the essence of medicines used by physicians in the past dynasties to treat epidemic syndromes, and uses Maxing Shigan Decoction, which was specially used to treat epidemic diseases in Zhang Zhongjing's "Treatise on Febrile Diseases" in the Han Dynasty, and Wu Jutong's "Diagnosis of Warm Diseases" in the Qing Dynasty. Yinqiao Powder is mainly based on the experience of using rhubarb to treat epidemic syndrome in Wu Youke's "The Treatise on Epidemic Diseases" in Ming Dynasty. The formula is formulated according to the theory of traditional Chinese medicine (monarch, minister, assistant, and envoy), and is made by modern technology. Baked ephedra, gypsum, and fried bitter almonds are the three herbs for ministers, which can help the monarch to clear the lung fire, and can also clear the lungs and relieve asthma; Banlangen, Mianma Guanzhong, Houttuynia cordata, menthol, patchouli , Rhubarb, Rhodiola and seven adjuvants are used together, which not only helps the monarch and ministers to clear the lungs and detoxify, disperse the lungs and relieve heat, but also dispel dampness and turbidity and regulate Qi and neutralize. Licorice, as a medicine, has the functions of clearing away heat and detoxifying, and reconciling various medicines. The prescription is based on traditional Chinese medicine theory and consists of 13 traditional Chinese medicines. It has the effects of clearing temperature and detoxification, dispelling heat in the lungs and expelling heat. It can be seen from the composition of the prescription that Lianhua Qingwen Capsule is complex in composition, and exploring its material basis and mechanism of action will play an important role in the clinical application of Lianhua Qingwen Capsule and the development of new anti-epidemic Chinese medicines based on TCM theory.

In the research strategy of traditional Chinese medicine active ingredients, phytochemical separation and analysis are often used as the guide, and the in vitro activity is gradually transitioned to the in vivo activity research. Regardless of Chinese medicine or Western medicine, the understanding of the material basis for the treatment of diseases is unified, and they all believe that the components and their metabolites entering the body are the material guarantee for the efficacy of the drug. In this way, through the study of substances in the body, we can more intuitively observe the pharmacodynamic material basis of traditional Chinese medicine as a whole, and can better reflect the overall view of traditional Chinese medicine. Different from new chemical drugs, the clinical safety of traditional Chinese medicine has been guaranteed by thousands of years of clinical practice experience. Traditional Chinese medicine is a discipline developed from clinical practice. The research of traditional Chinese medicine should start with clinical research, and directly carry out human exposure research under safe doses, which will be able to more intuitively reflect the actual material basis of traditional Chinese medicine. This study preliminarily analyzed the composition of Lianhua Qingwen Capsules in human body.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a separation method of Lianhuaqingwen drug-related metabolites;

Another object of the present invention is to provide a structure identification method for Lianhuaqingwen drug-related metabolites. This will not only help to dig out the main medicinal components of Lianhua Qingwen Capsules, but also reveal the metabolic transformation process and excretion pathways of Lianhua Qingwen Capsules in the body; it can also be helpful for the toxicological research of Lianhua Qingwen Capsules and subsequent medicines. In addition, it can also provide theoretical guidance for clinical treatment, thereby promoting its clinical application.

In order to achieve the above object, on the one hand, the present invention provides a method for separating related metabolites of Lianhuaqingwen medicine, wherein the method comprises using the following chromatographic conditions to separate the relevant metabolites in the body fluids of individuals who are administered Lianhuaqingwen medicine. Metabolites are separated:

Chromatographic column: C18 chromatographic column;

Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile, the volume ratio of A and B is (98-5): (2-95);

Flow rate: 0.2-0.3mL/min;

Column temperature: 30-40°C.

According to some specific embodiments of the present invention, wherein the flow rate in the chromatographic conditions is 0.3 mL/min.

According to some specific embodiments of the present invention, the column temperature in the chromatographic conditions is 35°C.

According to some specific embodiments of the present invention, wherein, the method further comprises elution with the following mobile phase gradient:

0-3min: 2%-2%B, 3-4min: 2%-5%B, 4-6min: 5%-5%B, 6-9min: 5%-10%B, 9-14min: 10% -15%B, 14-18min: 15%-20%B, 18-25min: 20%-30%B, 25-28min: 30%-95%, 28-32min: 95%-95%, 32min: 95 %-5%B, 32-37min: 5%-5%B.

It can be understood that the "0-3min: 2%-2%B" refers to that the concentration of B remains unchanged during the 0-3min period; the "3-4min: 2%-5%B" ”, which means that the concentration of B increases from 2% to 5% within a time period of 3-4min; and so on for other time periods.

It can also be understood that the above-mentioned endpoints of each time period, especially the end point of the previous period and the start point of the next period, refer to the starting point of the endpoint time, for example, "0-3min: 2%- 2%B, 3-4min: 2%-5%B" means that the concentration starts to increase from 2% at the beginning of the 3rd minute; however, there are acceptable errors in the art at this time point (for example, 1 second before and after, or even 2%). error in seconds).

The "28-32min: 95%-95%, 32min: 95%-5%B, 32-37min: 5%-5%B" means that the concentration changes from 95% to 5% at the beginning of 32min, and then The concentration was maintained at 5% to the end.

According to some specific embodiments of the present invention, wherein, the method further comprises elution with the following mobile phase gradient:

0-3min: 2%-2%B, 3-4min: 2%-5%B, 4-6min: 5%-5%B, 6-9min: 5%-10%B, 9-14min: 10% -15%B, 14-18min: 15%-20%B, 18-25min: 20%-30%B, 25-28min: 30%-95%, 28-32min: 95%-95%, 32.01min: 95%-5%B, 32.01-37min: 5%-5%B.

According to some specific embodiments of the present invention, wherein, the method further comprises elution with the following mobile phase gradient:

0-3min: 2%-2%B, 3-4min: 2%-5%B, 4-6min: 5%-5%B, 6-9min: 5%-10%B, 9-14min: 10% -15%B, 14-18min: 15%-20%B, 18-25min: 20%-30%B, 25-28min: 30%-95%, 28-32min: 95%-95%, 32.01min: 95%-5%B, 32.01-37min: 5%-5%B; in each time period when the B concentration increased, the B concentration increased at a constant speed.

Wherein, in each time period when the B concentration increases, the B concentration increases at a uniform speed, which means that the B concentration increases at a constant speed during the 3-4min time period such as "3-4min: 2%-5%B". From 2% to 5%.

According to some specific embodiments of the present invention, wherein, the chromatographic column is an ACQUITY UPLC CSH C18 chromatographic column.

According to some specific embodiments of the present invention, wherein, the chromatographic conditions further include that the injection volume is 3 μL.

According to some specific embodiments of the present invention, wherein, the method comprises using chromatography-mass spectrometry combined to separate the body fluids of individuals administered Lianhua Qingwen medicine, wherein the mass spectrometry conditions include: positive and negative ion detection mode; Full MS full scan resolution : 70000; Scan mass range: 100-1200Da; Full ms/ddMS2 collision energy: 30eV; Full MS resolution: 35000; ddMS2 resolution: 17500.

According to some specific embodiments of the present invention, the Lianhua Qingwen medicine is Lianhua Qingwen Capsule or Lianhua Qingwen Granule.

According to some specific embodiments of the present invention, wherein the individual is a mammalian individual.

According to some specific embodiments of the present invention, wherein the individual is a human.

According to some specific embodiments of the present invention, wherein the body fluid is blood and/or urine.

According to some specific embodiments of the present invention, wherein, the method comprises enriching and purifying the body fluid by column chromatography, and then separating.

According to some specific embodiments of the present invention, wherein, the method comprises first enriching and purifying the body fluid with an extraction column, and then performing separation.

According to some specific embodiments of the present invention, wherein, when the body fluid is blood, the extraction column is

HLB 3cc (60mg) Extraction Cartridges (Batch No: 164A39127A) column; when the body fluid is urine, the extraction column is an Oasis HLB SPE (60mg/3cc) column.

According to some specific embodiments of the present invention, the method includes first enriching and purifying the body fluid by column chromatography, eluting with methanol and drying, redissolving the obtained dried product with methanol, and centrifuging the supernatant for separation.

According to some specific embodiments of the present invention, wherein, the drying is drying with nitrogen.

According to some specific embodiments of the present invention, after the obtained dried product is reconstituted with methanol, it is sonicated and vortexed to mix, and the supernatant is collected by centrifugation for separation.

According to some specific embodiments of the present invention, wherein the centrifugation comprises centrifugation at 8000rpm/min-12000rpm/min for 10-5min.

According to some specific embodiments of the present invention, the rotation speed of the centrifugation is 12000 rpm/min.

According to some specific embodiments of the present invention, wherein, the time of centrifugation is 5min.

According to some specific embodiments of the present invention, wherein the body fluid is from an individual on day 8 to day 10 after administration.

According to some specific embodiments of the present invention, wherein, when the body fluid is blood, the body fluid is from 10min, 30min, 1h, 3h, 6h, 9h, 12h, 16h, 25h and 8th day after administration. 48h mixing of the body fluids of individuals; when the body fluid is urine, the body fluids are the mixing of body fluids from the following time periods on day 8 after dosing: 0-4h, 4-10h, 10- 24h, 24-32h and 32-48h.

On the other hand, the present invention also provides a method for identifying the structure of Lianhuaqingwen drug-related metabolites, wherein the method comprises using the separation method of the present invention to separate the body fluids of individuals who are administered Lianhuaqingwen drug. The related metabolites were separated, and then the structure was elucidated by high-resolution mass spectrometry.

According to some specific embodiments of the present invention, wherein, the method comprises using the following mass spectrometry conditions for structure elucidation: ESI ion source; positive and negative ion detection mode; Full MS full scan resolution: 70000, scan mass range: 100-1200 Da; Full ms /ddMS2 collision energy: 30eV; Full MS resolution: 35000; ddMS2 resolution: 17500.

According to some specific embodiments of the present invention, the mass spectrometry conditions further include: spray voltage (Spray Voltage): 3.8kV; capillary voltage (Capillary Voltage): 40V; capillary tube temperature (Ion Transfer Tube Temperature): 300°C; Temperature (Vaporizer Temperature): 350℃; Sheath Cas Flow Rate: 35arb; Auxiliary Gas Flow Rate: 10arb; Sweep Gas Flow Rate: 5arb; RF Lens (%) : 50; AGC Target: 3.0e6.

According to some specific embodiments of the present invention, wherein the sheath gas is N ₂ .

According to some specific embodiments of the present invention, the auxiliary gas is N ₂ .

According to some specific embodiments of the present invention, the scavenging gas is N ₂ .

According to some specific embodiments of the present invention, wherein, the mass spectrometry conditions further include: MS2 uses Data Dependent Scan to set Top 10 ions to perform collision-induced dissociation, isolation window (Isolation Window (m/z)): 2.0; (N) CE/stepped(N) CE: 35; ddMS scanning resolution: 17500; Dynamic Exclusion: 10s.

According to some specific embodiments of the present invention, wherein, the high-resolution mass spectrometer is a Thermo Fisher Q Exactive mass spectrometer (liquid mass spectrometer).

According to some specific embodiments of the present invention, wherein the method comprises using the aforementioned mass spectrometry conditions to obtain high-resolution mass spectrometry data (including retention time, HRMS and MS/MS data sets) of in vivo metabolites of Lianhua Qingwen Capsules, The structure of Lianhuaqingwen drug-related metabolites was identified by the following methods:

1) For the compound with the reference substance, deduce the structure with reference to the cracking rule of the reference substance;

2) For compounds without reference substances, structure characterization based on high-resolution mass spectrometry data;

3) For compounds whose structures have not been deduced by the above two methods, use Compound Discoverer software to assist in the analysis.

According to some specific embodiments of the present invention, wherein, for a compound with a reference substance, deriving the structure with reference to the cleavage rule of the reference substance includes deducing the cleavage rule of the compound according to the collected high-resolution HRMS and MS/MS data. Comparison to identify compound structures.

According to some specific embodiments of the present invention, wherein, for a compound without a reference substance, performing structural characterization according to high-resolution mass spectrometry data includes: first, inferring the molecular formula of the compound according to the acquired accurate HRMS data; then, according to MS/MS characteristic fragment ions The information deduces the fragmentation rule, and compares it with the mass spectrometry information (such as retention time, molecular formula, mass spectrometry fragmentation rule, etc.) of the compounds whose structures have been determined above, as well as literature reports, and deduces the structure of the compound.

According to some specific embodiments of the present invention, wherein, for compounds whose structures have not been deduced by the above two methods, the auxiliary analysis with the help of Compound Discoverer software includes: importing the high-resolution HRMS/MS mass spectrometry data of Lianhua Qingwen capsule body fluid samples into Compound Discoverer Software, set the search parameters, link to Chemspider, Pubmed, m/z Cloud and other local natural product databases built by Thermo for searching, and use the matching score to score; finally, synthesize the matching score results of each database to infer the structure of the compound.

In conclusion, the present invention provides a method for separation and structure identification of Lianhuaqingwen drug-related metabolites. The method of the present invention has the following advantages:

1) The method of the present invention can rapidly and effectively separate metabolites related to Lianhuaqingwen drug in body fluids;

2) The present invention adopts a high-resolution mass spectrometer to comprehensively collect accurate mass spectrometry data of Lianhuaqingwen drug-related metabolites, calculate a reasonable molecular formula, and complete structural identification.

Description of drawings

Fig. 1 is the high-resolution extraction particle flow spectrum of Lianhua Qingwen Capsule-related components in the human plasma sample of Example 2 (A) negative ion high-resolution extraction particle flow spectrum (B) positive ion high-resolution extraction particle flow spectrum;

Fig. 2 is the high-resolution extraction particle flow spectrum of Lianhua Qingwen Capsule-related components in the human urine sample of Example 2 (A) negative ion high-resolution extraction particle flow spectrum (B) positive ion high-resolution extraction particle flow spectrum.

detailed description

The technical solutions of the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but the protection scope of the present invention includes but is not limited thereto.

Example 1

The present embodiment provides a method for separating Lianhuaqingwen drug-related metabolites, including:

1. Clinical Sample Collection

A single-center, randomized, open-label, multiple-dose trial design was used, and a total of 14 healthy subjects or appendectomy patients were enrolled. Each subject has been confirmed by physical examination, laboratory examination, electrocardiogram, etc. to have no heart, liver and kidney diseases; no history of allergies, and has not taken Lianhua Qingwen Capsules or other drugs that may affect drug absorption within one month before the test. , distribution, metabolism, excretion of drugs or food. The study protocol was approved by the Ethics Committee of Hebei Yiling Hospital, and all subjects signed a written informed consent before entering the study. The test was carried out in the form of continuous administration. The continuous administration was administered on the 1st to 8th day (except the 2nd day) (the administration dose was 4.2g (12 Lianhua Qingwen capsules)), sampling (blood and urine) ) Sampling (blood and urine) on day 1 to 48h after administration, blood sampling on day 3-7 before administration, sampling on day 8 (blood and urine); the test period is 11 days. Start to eat breakfast 1 hour before taking the medicine, and finish the meal within 30 minutes, and take the test drug on time 1 hour after the start of the meal. Take with 240mL warm water. The collected plasma and urine samples were stored in a refrigerator at about -80°C for testing.

2. Biological Sample Preparation

Take 6 subjects (subject number 1, 3, 6, 8, 9, 12) 10min, 30min, 1h, 3h, 6h, 9h, 12h, 16h, 25h and 48h on the eighth day after administration Plasma samples were pooled, and each subject took 50 microliters of plasma at each time point to obtain 3 mL of pooled plasma samples. After three-fold dilution with ultrapure water,

The HLB 3cc (60mg) Extraction Cartridges (Batch No: 164A39127A) column was used for enrichment and purification, and the methanol eluent was collected and dried with nitrogen; the residues were redissolved in 100 μL of 70% methanol, dissolved by ultrasonic for 10s, vortexed, and 12000rpm. /min centrifuged for 5 min, and the supernatant was taken for analysis. The blank plasma samples before administration were prepared by the same method.

Urine samples (including 0-4h, 4-10h, 10-24h, 24h) were taken on the eighth day after administration of 6 subjects ( subject number 1, 3, 6, 8, 9, 12). -32h, 32-48h and other time periods), combined after freezing and thawing, each subject took 100 microliters of urine samples at each time point to obtain 3 mL of mixed urine samples; Oasis HLB SPE (60mg/3cc) column enriched After collection and purification, the methanol eluate was collected, dried with nitrogen at 37°C, and the residues were redissolved in 100 μL of 70% methanol respectively, dissolved by ultrasound for 10 s, vortexed, and centrifuged at 12,000 rpm/min for 5 min, and the supernatant was taken for analysis. Blank urine samples were prepared in the same way.

3. UPLC-HRMS analysis conditions

equipment

Thermo Fisher Q Exactive LC/MS instrument, product of Thermo Company, USA; Xcalibur 3.0.63 (Thermo Fisher) ChemStation data processing system.

Chromatographic conditions

Chromatographic column: ACQUITY UPLC CSH C18 (2.1*50mm, 1.7μm);

Mobile phase composition: A: aqueous solution containing 0.1% formic acid; B: acetonitrile

Mobile phase gradient: 0-3min (2%-2%B); 3-4min (2%-5%B); 4-6min (5%-5%B); 6-9min (5%-10%B) ); 9-14min (10%-15%B); 14-18min (15%-20%B); 18-25min (20%-30%B); 25-28min (30%-95%), 28 -32min(95%-95%), 32.01min(95%-5%B); 32.01-37min(5%-5%B).

Flow rate: 0.3 mL/min; injection volume: 3 μL; column temperature: 35°C.

Example 2

The present embodiment provides a structure identification method for Lianhuaqingwen drug-related metabolites, including:

The in vivo metabolites of Lianhua Qingwen Capsules are separated according to the steps of Example 1, and then the structure analysis is carried out with Thermo Fisher Q Exactive LC/MS, wherein the mass spectrometry conditions include:

ESI ion source, positive and negative ion detection mode; Full MS full scan resolution: 70000, scan mass range: 100–1200Da; Full ms/ddMS2 collision energy: 30eV; Full MS resolution: 35000; ddMS2 resolution: 17500; spray voltage : 3.8kV; capillary voltage: 40V; capillary temperature: 300°C; gasification temperature: 350°C; sheath gas (N ₂ ) flow rate: 35arb; auxiliary gas (N ₂ ) flow rate: 10arb _; 5arb; RF Lens: 50; AGC Target: 3.0e6. MS2 uses Data Dependent Scan to set Top 10 ions for collision-induced dissociation, Isolation Window (m/z): 2.0; (N) CE/stepped (N) CE: 35; ddMS scan resolution: 17500; Dynamic Exclusion: 10s.

And follow the steps below for structural identification:

After comprehensively collecting the high-resolution mass spectrometry data (including retention time, HRMS and MS/MS data sets) of the metabolites of Lianhua Qingwen Capsules in vivo according to the above conditions, analyze the reference substance of Lianhua Qingwen Capsules under the same conditions to obtain accurate Retention time, HRMS and MS/MS mass spectral data information, and mass spectral fragmentation patterns. Then the compound structure identification is carried out. The specific structure identification process is mainly divided into three categories:

1) For compounds with reference substances, the structures are deduced with reference to the cracking rules of reference substances. Firstly, according to the collected high-resolution HRMS and MS/MS data, the compound cleavage rule was inferred, and the compound structure was identified by comparing with the cleavage rule of the reference substance.

2) For compounds without reference substance, perform structural characterization based on high-resolution mass spectrometry data. Firstly, according to the obtained accurate HRMS data, the molecular formula of the compound is deduced; then, the fragmentation rule is deduced according to the MS/MS characteristic fragment ion information, and the mass spectrometry information of the compound whose structure has been determined, such as retention time, molecular formula, mass spectrometry fragmentation rule, etc., Compared with literature reports, the structure of the compound was deduced.

3) For compounds whose structures have not been deduced by the above two methods, use Compound Discoverer software to assist in the analysis. Import the high-resolution HRMS/MS mass spectrometry data of Lianhua Qingwen capsule body fluid samples into Compound Discoverer software, set search parameters, and link to Chemspider, Pubmed, m/z Cloud and other local natural product databases built by Thermo Company for searching. Score the matching degree; finally, synthesize the matching score results of each database to infer the structure of the compound. Chinese traditional medicine (TCM) has complex and diverse components, usually containing hundreds of original components of drugs, and each component will change with environmental factors and processing conditions. After entering the human body or animal body, through various biological metabolic transformation pathways More complex and diverse compounds will be produced. There are many difficulties to carry out research on it, especially the research on metabolic process in vivo. A total of 126 compounds were screened in plasma and urine samples; 101 were detected in plasma and 111 in urine. The specific compound-related data information is shown in Table 1 (Table 1 lists the total number of compounds detected in Lianhua Qingwen Capsule, plasma and urine three matrices). Figure 1 and Figure 2 show the high-resolution extraction particle flow patterns of Lianhua Qingwen capsule-related components in human plasma and urine samples.

Claims (17)

1. A method for separating related metabolites of Lianhuaqingwen medicine, wherein the method comprises using the following chromatographic conditions to separate relevant metabolites in the body fluids of individuals who are administered Lianhuaqingwen medicine:

   Chromatographic column: C18 chromatographic column;

   Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile, the volume ratio of A and B is (98-5): (2-95);

   Flow rate: 0.2-0.3mL/min;

   Column temperature: 30-40°C.
2. The method of claim 1, wherein the method further comprises eluting with a mobile phase gradient as follows:

   0-3min: 2%-2%B, 3-4min: 2%-5%B, 4-6min: 5%-5%B, 6-9min: 5%-10%B, 9-14min: 10% -15%B, 14-18min: 15%-20%B, 18-25min: 20%-30%B, 25-28min: 30%-95%B, 28-32min: 95%-95%B, 32min : 95%-5%B, 32-37min: 5%-5%B.
3. The method according to claim 1 or 2, wherein the method further comprises elution with the following mobile phase gradient:

   0-3min: 2%-2%B, 3-4min: 2%-5%B, 4-6min: 5%-5%B, 6-9min: 5%-10%B, 9-14min: 10% -15%B, 14-18min: 15%-20%B, 18-25min: 20%-30%B, 25-28min: 30%-95%, 28-32min: 95%-95%, 32.01min: 95%-5%B, 32.01-37min: 5%-5%B; in each time period when the B concentration increased, the B concentration increased at a constant speed.
4. The method according to any one of claims 1 to 3, wherein the chromatographic column is an ACQUITY UPLC CSH C18 chromatographic column.
5. The method according to any one of claims 1 to 4, wherein the chromatographic conditions further include that the injection volume is 3 μL.
6. The method according to any one of claims 1 to 5, wherein the method comprises using chromatography-mass spectrometry combined to separate the body fluids of individuals administered Lianhua Qingwen medicine, wherein the mass spectrometry conditions include: positive and negative ion detection modes; Full MS full scan resolution: 70000; scan mass range: 100-1200Da; Full ms/ddMS2 collision energy: 30eV; Full MS resolution: 35000; ddMS2 resolution: 17500.
7. The method according to any one of claims 1 to 6, wherein the Lianhua Qingwen medicine is Lianhua Qingwen Capsule or Lianhua Qingwen Granule.
8. The method according to any one of claims 1 to 7, wherein the individual is a mammalian individual, preferably a human.
9. The method according to any one of claims 1 to 8, wherein the body fluid is blood and/or urine.
10. The method according to any one of claims 1 to 9, wherein the method comprises enriching and purifying the body fluid by column chromatography, and then separating.
11. The method according to any one of claims 1 to 10, wherein the method comprises enriching and purifying the body fluid with an extraction column, and then separating.
12. The method according to any one of claims 1 to 10, wherein the method comprises first enriching and purifying the body fluid with column chromatography, eluting with methanol and drying, and redissolving the obtained dried product with methanol, and taking the The clear liquid is separated.
13. The method of any one of claims 1 to 12, wherein the body fluid is from an individual on day 8 to day 10 after administration.
14. The method according to claim 13, wherein when the body fluid is blood, the body fluid is from 10min, 30min, 1h, 3h, 6h, 9h, 12h, 16h, 25h on the 8th day after administration and 48h of the individual's body fluids; when the body fluid is urine, the body fluids are the mixture of body fluids from the following time periods on the 8th day after dosing: 0-4h, 4-10h, 10 -24h, 24-32h and 32-48h.
15. A method for structural identification of related metabolites of Lianhuaqingwen drug, wherein the method comprises using the separation method described in claims 1 to 14 to separate the relevant metabolites in the body fluid of an individual who is administered Lianhuaqingwen drug Then, high-resolution mass spectrometry was used for structural analysis.
16. The identification method according to claim 15, wherein the method comprises using the following mass spectrometry conditions for structure analysis: ESI ion source; positive and negative ion detection mode; Full MS full scan resolution: 70000, scan mass range: 100-1200 Da; Full ms/ddMS2 collision energy: 30eV; Full MS resolution: 35000; ddMS2 resolution: 17500.
17. The identification method according to claim 16, wherein the mass spectrometry conditions further comprise: ESI ion source, spray voltage: 3.8kV; capillary voltage: 40V; capillary temperature: 300°C; vaporization temperature: 350°C; sheath gas flow rate : 35arb; auxiliary gas flow rate: 10arb; sweep gas flow rate: 5arb; RF Lens: 50; AGC Target: 3.0e6.

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