Method and kit for detecting intestinal microbial metabolites in biological sample

Abstract

The invention provides a method and a kit for detecting intestinal microbial metabolites in a biological sample, and the intestinal microbial metabolites in the method for detecting the intestinal microbial metabolites in the biological sample comprise at least one of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate and indol sulfate. According to the method for detecting the intestinal microbial metabolites in the biological sample, multiple intestinal microbial metabolites can be detected at the same time, pretreatment is simple, the total analysis time needed by detection is short, and the method is beneficial to being popularized to practical application.

Description

Technical Field

[0001] The invention belongs to the field of detection, and specifically relates to a method and a kit for detecting intestinal microbial metabolites in biological samples. Background Art

[0002] Fecal microbiota transplantation (FMT) is a treatment method that transplants the intestinal flora from the feces of healthy people into the patient's intestines to rebuild a new intestinal flora and achieve the treatment of intestinal and extraintestinal diseases. The selection of donors is crucial to the safety and effectiveness of FMT. According to the "Chinese Expert Consensus on the Screening and Management of Fecal Microbiota Transplantation Donors (2022 Edition)", donor screening needs to consider multiple aspects, including six dimensions: physiology, psychology, personal history, stability, continuity, and tolerance to food restriction.

[0003] During the donor screening process, a series of health checks are required, including routine physical examinations, stool tests, serological tests, etc., to rule out infectious diseases and chronic diseases. In addition, the donor's mental health, medication history, family genetic disease history, and lifestyle habits need to be assessed to ensure that the donor's intestinal flora is in good condition.

[0004] Small molecule metabolites produced by intestinal flora metabolism, such as choline, trimethylamine, trimethylamine oxide, betaine, L-carnitine, etc., are closely related to the occurrence and development of cardiovascular diseases. Indole sulfate, a metabolite of tryptophan, and p-cresol sulfate, a metabolite of tyrosine, can affect the progression of chronic kidney disease. Therefore, detecting the levels of these bacterial metabolites that affect physical condition is of great significance for evaluating the status of FMT donors.

[0005] In the "Chinese Expert Consensus on the Screening and Management of Intestinal Microbiota Transplantation Donors (2022 Edition)", microbiota metabolomics testing is listed as an evaluation indicator for donor screening. This means that in the donor screening process, in addition to traditional health checks, the donor's intestinal microbiota metabolites need to be tested to ensure that the donor's intestinal microbiota metabolite levels are within a safe range, thereby improving the safety and effectiveness of FMT.

[0006] People in the industry often use liquid chromatography tandem mass spectrometry (LC-MS) detection methods to detect some intestinal flora metabolites. Liquid chromatography tandem mass spectrometry technology has the advantages of high sensitivity, high specificity, and the ability to detect multiple compounds at the same time, and is widely used in many fields such as medicine, food, environment, forensics, and clinical. However, at present, there are still some defects in the LC-MS detection method for the detection of intestinal flora metabolites that have not been overcome. For example, most LC-MS detection methods can detect fewer types of intestinal flora metabolites and cannot meet the detection needs; or they require more complex pre-treatment of biological samples, involving more operational processes, which are difficult to directly promote to practical applications; or they require a longer analysis time, which is not conducive to the detection of a large number of samples. Summary of the invention

[0007] The object of the present invention is to provide a method and a kit for detecting intestinal microbial metabolites in biological samples. The method for detecting intestinal microbial metabolites in biological samples can simultaneously detect trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate, and the pretreatment is simple, the total analysis time required for the detection is short, and it is conducive to promotion to practical applications.

[0008] According to one aspect of the present invention, a method for detecting intestinal microbial metabolites in a biological sample is provided, wherein the intestinal microbial metabolites include at least one of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate; the method for detecting intestinal microbial metabolites includes the following operations: S1. using methanol and aqueous solution to prepare standard storage solutions containing gradient concentrations of intestinal microbial metabolites, and using the standard storage solutions to obtain standard curve equations of intestinal microbial metabolites; S2. adding an extracting solution to the biological sample containing intestinal microbial metabolites for centrifugal extraction, the extracting solution including acetonitrile and water to obtain a supernatant; S3. taking the supernatant for ultra-performance liquid chromatography-tandem mass spectrometry analysis to obtain the peak area of ​​the intestinal microbial metabolites ; Ultra-high performance liquid chromatography includes gradient elution using mobile phase A and mobile phase B, mobile phase A includes formic acid, water and ammonium acetate, mobile phase B includes acetonitrile, water, formic acid and ammonium acetate, and the gradient elution program is from 0 minutes to 1.5 minutes, mobile phase B is maintained at 100%; from 1.5 minutes to 3 minutes, mobile phase B is changed from 100% to 45-55%, and maintained for 1 minute; from 4 minutes to 4.5 minutes, mobile phase B is changed from 45-55% to 40-50%, and maintained for 0.5 minutes; from 5 minutes to 5.5 minutes, mobile phase B is changed from 40-50% to 90-100%, and maintained for 2 minutes; S4. The concentration of intestinal microbial metabolites in biological samples is obtained by using the peak area of ​​intestinal microbial metabolites through the standard curve equation.

[0009] The method for detecting intestinal microbial metabolites in biological samples provided by this scheme adopts the above-mentioned mobile phase A and mobile phase B, and analyzes the biological samples to be detected according to the above-mentioned gradient elution procedure, and can simultaneously detect trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate, and there are many types of detectable intestinal microbial metabolites. This detection method can shorten the total elution time on the basis of reducing the loss of the chromatographic column and optimizing the peak shape presented by each intestinal microbial metabolite, and achieve the effect of completing one detection in 7.5 minutes, and the detection results of the above-mentioned 10 intestinal microbial metabolites are highly accurate and sensitive. At the same time, the method for detecting intestinal microbial metabolites in biological samples uses the above-mentioned specific extraction liquid to pre-treat the biological sample, so that the protein in the biological sample is precipitated, which can not only ensure the extraction effect of intestinal microbial metabolites, but also reduce the interference of impurities, thereby ensuring that the sample analysis is completed in a relatively short analysis time, and the pre-treated supernatant can be directly subjected to ultra-high performance liquid chromatography tandem mass spectrometry analysis, which is simple to operate and suitable for large-scale promotion and application. In addition, it should be noted that the method provided in this application is not intended for the diagnosis and treatment of diseases.

[0010] Preferably, mobile phase A includes 2-8 mmol / L ammonium acetate and 0.1-0.2 v / v% formic acid; mobile phase B includes 2-8 mmol / L ammonium acetate, 0.1-0.2 v / v% formic acid and 85-95 v / v% acetonitrile. By regulating the proportion of mobile phase A and mobile phase B, and maintaining the ammonium acetate content and formic acid content in mobile phase A and mobile phase B within the above range, it is helpful to obtain a better peak shape in ultra-high performance liquid chromatography tandem mass spectrometry analysis, which is characterized by good symmetry, flat peak bottom and clear peak height, which helps to improve the separation between multiple intestinal microbial metabolites, avoid peak overlap, thereby reducing errors and obtaining more accurate analysis results. Since the quality of the peak shape will directly affect the accuracy and reliability of the analysis results.

[0011] Preferably, mobile phase A comprises 5 mmol / L ammonium acetate and 0.1-0.2 v / v% formic acid.

[0012] Preferably, mobile phase A comprises 5 mmol / L ammonium acetate and 0.15 v / v% formic acid.

[0013] Preferably, mobile phase B comprises 5 mmol / L ammonium acetate, 0.1-0.2 v / v% formic acid and 85-95 v / v% acetonitrile.

[0014] Preferably, mobile phase B comprises 5 mmol / L ammonium acetate, 0.15 v / v% formic acid and 85-95 v / v% acetonitrile.

[0015] Preferably, mobile phase B comprises 5 mmol / L ammonium acetate, 0.15 v / v% formic acid and 90 v / v% acetonitrile.

[0016] Preferably, the extract comprises 2-8 mmol / L ammonium acetate and 85-95 v / v% acetonitrile. The extract is close to the composition of mobile phase B as the initial mobile phase, so that the pre-treated biological sample can be dispersed and transmitted more evenly in the mobile phase when it enters the chromatographic column later, and the supernatant obtained by the treatment of the biological sample can be better mixed with the mobile phase, reducing the local concentration difference caused by the sudden change of the solvent composition, thereby avoiding the adverse effects of the solvent effect, and is conducive to forming a sharp and symmetrical peak shape, avoiding the broadening and tailing of the peak, reducing the peak width, and completing the analysis and detection in a shorter time.

[0017] Preferably, the extract comprises 5 mmol / L ammonium acetate and 85-95 / v% acetonitrile.

[0018] Preferably, the extract is 90 v / v% acetonitrile containing 5 mmol / L ammonium acetate.

[0019] Preferably, the volume ratio of the biological sample to the extract is 1:3.5-4.5.

[0020] Preferably, the volume ratio of the biological sample to the extract is 1:4.

[0021] Preferably, the program of gradient elution is as follows: from 0 minutes to 1.5 minutes, mobile phase B is maintained at 100%; from 1.5 minutes to 3 minutes, mobile phase B is changed from 100% to 50% and maintained for 1 minute; from 4 minutes to 4.5 minutes, mobile phase B is changed from 50% to 45% and maintained for 0.5 minutes; from 5 minutes to 5.5 minutes, mobile phase B is changed from 45% to 100% and maintained for 2 minutes.

[0022] Preferably, during the ultra-high performance liquid chromatography process, the temperature of the chromatographic column is 35-45° C., and the chromatographic column is ACQUITY UPLC BEH Amide.

[0023] Preferably, the conditions for mass spectrometry analysis are: in electrospray ionization mode, multiple reaction monitoring is used for positive ion mode scanning; the curtain gas pressure is 28-32 psi, the ion source temperature is 520-580°C, the ion spray voltage is 5200-5800V, the nebulizer gas pressure is 50-60 psi, and the auxiliary heating gas pressure is 50-60 psi.

[0024] Preferably, the declustering voltage of trimethylamine is 65-70V, and the collision energy is 25-30V; and / or, the declustering voltage of trimethylamine oxide is 28-33V, and the collision energy is 13-18V; and / or, the declustering voltage of choline is 28-32V, and the collision energy is 20-25V; and / or, the declustering voltage of L-carnitine is 81-87V, and the collision energy is 23-28V; and / or, the declustering voltage of betaine is 18-22V, and the collision energy is 35-40V; and / or, The declustering voltage of creatinine is 60 to 65 V, and the collision energy is 21 to 26 V; and / or, the declustering voltage of tryptophan is 57 to 63 V, and the collision energy is 57 to 63 V; and / or, the declustering voltage of tyrosine is 55 to 60 V, and the collision energy is 17 to 23 V; and / or, the declustering voltage of p-cresol sulfate is -50 to -55 V, and the collision energy is -35 to -30 V; and / or, the declustering voltage of indoxyl sulfate is -93 to -87 V, and the collision energy is -38 to -32 V.

[0025] Preferably, the mass / charge ratio of the parent ion of trimethylamine is 60.2, and the mass / charge ratio of the daughter ion of trimethylamine is 44.1.

[0026] Preferably, the mass / charge ratio of the parent ion of trimethylamine oxide is 76.1, and the mass / charge ratio of the daughter ion of trimethylamine oxide is 59.3.

[0027] Preferably, the mass / charge ratio of the parent ion of choline is 104.1, and the mass / charge ratio of the daughter ion of choline is 60.1.

[0028] Preferably, the mass / charge ratio of the parent ion of L-carnitine is 162.1, and the mass / charge ratio of the daughter ion of L-carnitine is 103.1.

[0029] Preferably, the mass / charge ratio of the parent ion of betaine is 118.4, and the mass / charge ratio of the daughter ion of betaine is 58.1.

[0030] Preferably, the mass / charge ratio of the parent ion of creatinine is 114, and the mass / charge ratio of the daughter ion of creatinine is 44.2.

[0031] Preferably, the mass / charge ratio of the parent ion of tryptophan is 205, and the mass / charge ratio of the daughter ion of tryptophan is 115.

[0032] Preferably, the mass / charge ratio of the parent ion of tyrosine is 182, and the mass / charge ratio of the daughter ion of tyrosine is 136.

[0033] Preferably, the mass / charge ratio of the parent ion of p-cresol sulfate is 187, and the mass / charge ratio of the daughter ion of p-cresol sulfate is 80.

[0034] Preferably, the mass / charge ratio of the parent ion of indoxyl sulfate is 211.9, and the mass / charge ratio of the daughter ion of indoxyl sulfate is 80.

[0035] Preferably, the biological sample includes at least one of serum, blood and plasma.

[0036] Preferably, the biological sample is serum.

[0037] According to another aspect of the present invention, a detection kit for detecting intestinal microbial metabolites in biological samples is provided, wherein the intestinal microbial metabolites include at least one of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate; the detection kit includes an extract, a mobile phase A and a mobile phase B, the extract includes acetonitrile and water, the mobile phase A includes formic acid, water and ammonium acetate, and the mobile phase B includes acetonitrile, water, formic acid and ammonium acetate.

[0038] Preferably, the mobile phase A is a 0.1-0.2% formic acid aqueous solution containing 2-8 mmol / L ammonium acetate.

[0039] Preferably, mobile phase B is an 85-95% acetonitrile aqueous solution containing 2-8 mmol / L ammonium acetate and 0.1-0.2% formic acid.

[0040] Preferably, mobile phase A is a 0.1-0.2% formic acid aqueous solution containing 5 mmol / L ammonium acetate.

[0041] Preferably, mobile phase A is a 0.15% formic acid aqueous solution containing 5 mmol / L ammonium acetate.

[0042] Preferably, mobile phase B is an 85-95% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate and 0.15% formic acid.

[0043] Preferably, mobile phase B is a 90% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate and 0.15% formic acid.

[0044] Preferably, the extract is a 85-95% acetonitrile aqueous solution containing 2-8 mmol / L ammonium acetate.

[0045] Preferably, the extract is an 85-95% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate.

[0046] Preferably, the extract is a 90% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 is the total chromatogram of each intestinal microbial metabolite;

[0048] Figure 2 1 is a chromatogram of trimethylamine, trimethylamine oxide, and choline, wherein from top to bottom are chromatograms of trimethylamine, trimethylamine oxide, and choline;

[0049] Figure 3 The chromatograms of L-carnitine, betaine and creatinine are shown in FIG. 1 , wherein from top to bottom are the chromatograms of L-carnitine, betaine and creatinine;

[0050] Figure 4 The chromatograms are tryptophan, tyrosine, and p-cresol sulfate, wherein from top to bottom are the chromatograms of tryptophan, tyrosine, and p-cresol sulfate;

[0051] Figure 5 This is the chromatogram of indoxyl sulfate. DETAILED DESCRIPTION

[0052] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the drawings in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0053] Example 1

[0054] S1. Mixed standard curve solution: Accurately weigh appropriate amounts of standard substances of trimethylamine (TMA), trimethylamine oxide (TMAO), choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresyl sulfate, and indoxyl sulfate, mix the above intestinal microbial metabolites, and dissolve the mixture of the above intestinal microbial metabolites with 50% methanol aqueous solution to prepare a standard storage solution. Then, the standard storage solution is diluted with 50% methanol aqueous solution in sequence to obtain a standard storage solution of gradient concentrations to obtain a standard curve of each intestinal microbial metabolite.

[0055] The gradient concentration of the standard storage solution of trimethylamine is 28.4-14545.5 ng / mL; the gradient concentration of the standard storage solution of trimethylamine oxide is 1.77-909.1 ng / mL; the gradient concentration of the standard storage solution of choline is 17.7-9091 ng / mL; the gradient concentration of the standard storage solution of L-carnitine is 4.63-2363.6 ng / mL; the gradient concentration of the standard storage solution of betaine is 4.63-2363.6 ng / mL. L; the gradient concentration of the standard stock solution of creatinine is 73.3~37545.5ng / mL; the gradient concentration of the standard stock solution of tryptophan is 0.106~54.5μg / mL; the gradient concentration of the standard stock solution of tyrosine is 0.199~101.8μg / mL; the gradient concentration of the standard stock solution of p-cresol sulfate is 1.77~909.1ng / mL; the gradient concentration of the standard stock solution of indoxyl sulfate is 3.99~2045ng / mL.

[0056] S2-1. Prepare quality control products: Quality control products are biological samples containing intestinal microbial metabolites.

[0057] Accurately weigh trimethylamine, trimethylamine oxide, L-carnitine, choline, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate, mix the above intestinal microbial metabolites, and dissolve the mixture of the above intestinal microbial metabolites with 50% methanol aqueous solution, prepare the quality control storage solution, dilute the quality control storage solution with 50% methanol aqueous solution, and prepare low-value, medium-value, and high-value quality control working solutions. Then take 20 serum samples and mix them in equal volumes to obtain mixed serum. Add no more than 10% of the total volume of low-value, medium-value, and high-value quality control working solutions to the mixed serum to prepare low-value quality control products, medium-value quality control products, and high-value quality control products. The concentrations of intestinal microbial metabolites in the low-value, medium-value, and high-value quality control products are shown in the following table:

[0058] Table 1. Concentrations of intestinal microbial metabolites in low, medium, and high value quality control products

[0059]

[0060] S2-2 Sample pretreatment: 50 μL of standard, 50 μL of mixed serum, and 50 μL of quality control were respectively pipetted into 1.5 mL Eppendorf tubes. 200 μL of extraction solution was added to the Eppendorf tubes and shaken thoroughly for 5 minutes. The extraction solution was a 90% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate. The extraction was centrifuged at 14,000 rpm at 4°C for 10 minutes and the supernatant was taken.

[0061] S3. Take the supernatant and perform ultra-high performance liquid chromatography tandem mass spectrometry analysis to obtain the peak area of ​​intestinal microbial metabolites. Specifically, the total chromatogram of each intestinal microbial metabolite is as follows: Figure 1As shown, the chromatograms of trimethylamine, trimethylamine oxide, and choline are as follows Figure 2 As shown; the chromatograms of L-carnitine, betaine and creatinine are shown Figure 3 As shown; the chromatograms of tryptophan, tyrosine, and p-cresol sulfate are as shown Figure 4 As shown; the chromatogram of indoxyl sulfate is as shown Figure 5 shown.

[0062] Ultra-high performance liquid chromatography includes gradient elution using mobile phase A and mobile phase B. The chromatographic column is ACQUITY UPLC BEH Amide, the chromatographic column temperature is 40°C, and the injection volume is 5 μL. Mobile phase A is a 0.15% formic acid aqueous solution containing 5 mmol / L ammonium acetate; mobile phase B is a 90% acetonitrile aqueous solution containing 5 mmol / L ammonium acetate and 0.15% formic acid.

[0063] The gradient elution procedure is shown in the following table:

[0064] Table 2. Gradient elution program

[0065] Time (min) Flow rate (mL / min) A％ B％ 0 0.15 0 100 1.5 0.4 0 100 3 0.4 50 50 4 0.4 50 50 4.5 0.4 55 45 5 0.4 55 45 5.5 0.4 0 100 7.5 0.4 0 100

[0066] Note: "A%" shown in the above table represents the percentage of the volume of mobile phase A in the sum of the volumes of mobile phase A and mobile phase B, and "B%" represents the percentage of the volume of mobile phase B in the sum of the volumes of mobile phase A and mobile phase B.

[0067] The conditions for mass spectrometry analysis are shown in the following table:

[0068] Table 3. Mass spectrometry analysis parameters

[0069] Parameter Type Parameter size Curtain Gas Pressure 30psi Ion source temperature (Temperatμre) 550℃ Ion Spray Voltage (IS) 5500V Nebulizer gas pressure (Ion Source Gas1) 55psi Auxiliary heating gas pressure (Ion Source Gas2) 55psi Ion mode Electrospray ionization mode (ESI) Scan type Multiple Reaction Monitoring (MRM)

[0070] The multiple reaction monitoring parameters for the above mass spectrometry analysis are shown in the following table:

[0071] Table 4. Multiplex reaction monitoring parameters

[0072]

[0073] After the intestinal microbial metabolites flow out of the analytical column with the mobile phase, they enter the mass spectrometer ion source under the action of pressure, and the sample channel entering the ion source is controlled by a six-way valve. In the ion source, the intestinal microbial metabolites are vaporized and ionized into charged molecules. Under the action of voltage and vacuum, the charged molecules enter Q1, Q2 and Q3, where Q1 and Q3 are mass filters that only allow parent ions and daughter ions selected according to the mass-to-charge ratio of the above-mentioned intestinal microbial metabolites to pass through, and Q2 is a collision unit where parent ions collide with inert gas atoms to produce specific fragment ions. The first quadrupole (Q1) of the mass spectrometer selects parent ions with specific mass-to-charge ratios m / z of intestinal microbial metabolites, and parent ions with these mass-to-charge ratios are allowed to enter Q2. The fragment ions generated by Q2 enter Q3, where the fragment ions (daughter ions) of the analyte and its internal standard are selected to pass through, while other ions are removed. In positive ion mode, DP was optimized between 0-300 volts and CE was optimized between 5-180 volts. In negative ion mode, DP was optimized between -300 and 0 volts and CE was optimized between -180 and -5 volts. As shown in the table above, the highest target peak intensity was selected as the final method parameter.

[0074] S4. The concentration of the intestinal microbial metabolites in the biological sample is obtained by using the peak area of ​​the intestinal microbial metabolites through the standard curve equation.

[0075] Example 2

[0076] The methodological performance of Example 1 was verified, and the verification results are shown below.

[0077] (1) Linear relationship, detection limit and quantification limit of each intestinal microbial metabolite:

[0078] The mixed standard working solution prepared above was centrifugally extracted and then injected. According to the determination conditions provided in Example 1, the concentration was determined from low to high, and the quantitative chromatographic peak area-concentration was plotted to obtain a standard curve. The results showed that the linear range, detection limit and quantitative limit of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, indoxyl sulfate and p-cresol sulfate were as follows:

[0079] Table 5. Linear range, detection limit and quantification limit of each intestinal microbial metabolite

[0080]

[0081] (2) Precision and recovery of this method

[0082] The precision experiment was carried out using three quality control products of low, medium and high levels. The products were processed and analyzed according to the method provided in Example 1. The test was repeated three times. The precision and recovery rate of the method for each intestinal microbial metabolite were calculated. The precision is shown in Table 6, and the recovery rate is shown in Table 7.

[0083] Table 6. Precision of the method

[0084]

[0085] Table 7. Recovery of the method

[0086]

[0087] In summary, the above embodiments show that the detection limit, recovery rate, precision and other technical indicators of the method for detecting intestinal microbial metabolites in biological samples meet the requirements, and the method can simultaneously detect the contents of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, indoxyl sulfate and p-cresol sulfate in serum. In addition, the method for detecting intestinal microbial metabolites in biological samples has a short total elution time, high accuracy of the detection results and high sensitivity.

[0088] The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting intestinal microbial metabolites in a biological sample, characterized in that: The intestinal microbial metabolites include at least one of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate; The method for detecting intestinal microbial metabolites comprises the following operations: S1. using methanol-water solution to prepare standard stock solutions containing gradient concentrations of the intestinal microbial metabolites, and using the standard stock solutions to obtain standard curve equations of the intestinal microbial metabolites; S2. adding an extract to the biological sample containing the intestinal microbial metabolites for centrifugal extraction, wherein the extract comprises acetonitrile and water to obtain a supernatant; S3. The supernatant is subjected to ultra-high performance liquid chromatography tandem mass spectrometry analysis to obtain the peak area of ​​intestinal microbial metabolites; the ultra-high performance liquid chromatography comprises gradient elution using mobile phase A and mobile phase B, wherein the mobile phase A comprises formic acid, ammonium acetate and water, and the mobile phase B comprises acetonitrile, formic acid, ammonium acetate and water, The gradient elution procedure is as follows: from 0 minutes to 1.5 minutes, the mobile phase B is maintained at 100%; from 1.5 minutes to 3 minutes, the mobile phase B is changed from 100% to 45-55% and maintained for 1 minute; from 4 minutes to 4.5 minutes, the mobile phase B is changed from 45-55% to 40-50% and maintained for 0.5 minutes; from 5 minutes to 5.5 minutes, the mobile phase B is changed from 40-50% to 90-100% and maintained for 2 minutes; S4. Using the standard curve equation and the peak area of ​​the intestinal microbial metabolite, the concentration of the intestinal microbial metabolite in the biological sample is obtained.

2. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The mobile phase A comprises 2-8 mmol / L ammonium acetate and 0.1-0.2 v / v% formic acid; The mobile phase B includes 2-8 mmol / L ammonium acetate, 0.1-0.2 v / v% formic acid and 85-95 v / v% acetonitrile.

3. The method for detecting intestinal microbial metabolites in a biological sample as claimed in claim 2, characterized in that: The extract comprises 2-8 mmol / L ammonium acetate and 85-95 v / v % acetonitrile.

4. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The volume ratio of the biological sample to the extract is 1:3.5-4.

5.

5. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The program of the gradient elution is as follows: from 0 minutes to 1.5 minutes, the mobile phase B is maintained at 100%; from 1.5 minutes to 3 minutes, the mobile phase B is changed from 100% to 50% and maintained for 1 minute; from 4 minutes to 4.5 minutes, the mobile phase B is changed from 50% to 45% and maintained for 0.5 minutes; from 5 minutes to 5.5 minutes, the mobile phase B is changed from 45% to 100% and maintained for 2 minutes.

6. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: During the ultra-high performance liquid chromatography process, the temperature of the chromatographic column is 35-45° C., and the chromatographic column is ACQUITY UPLC BEH Amide.

7. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The mass spectrometry analysis conditions are: in electrospray ionization mode, multiple reaction monitoring is used for positive ion mode scanning; the curtain gas pressure is 28-32 psi, the ion source temperature is 520-580°C, the ion spray voltage is 5200-5800V, the nebulizer gas pressure is 50-60 psi, and the auxiliary heating gas pressure is 50-60 psi.

8. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The declustering voltage of trimethylamine is 65-70V, and the collision energy is 25-30V; And / or, the declustering voltage of trimethylamine oxide is 28 to 33 V, and the collision energy is 13 to 18 V; And / or, the betaine has a declustering voltage of 18 to 22 V and a collision energy of 35 to 40 V; And / or, the declustering voltage of the L-carnitine is 81 to 87 V, and the collision energy is 23 to 28 V; and / or, the tryptophan has a declustering voltage of 57 to 63 V and a collision energy of 57 to 63 V; and / or, the creatinine has a declustering voltage of 60 to 65 V and a collision energy of 21 to 26 V; and / or, the declustering voltage of choline is 28 to 32 V, and the collision energy is 20 to 25 V; and / or, the tyrosine declustering voltage is 55 to 60 V and the collision energy is 17 to 23 V; And / or, the declustering voltage of p-cresol sulfate is -50 to -55 V, and the collision energy is -35 to -30 V; And / or, the declustering voltage of the indoxyl sulfate is -93 to -87V, and the collision energy is -38 to -32V.

9. The method for detecting intestinal microbial metabolites in a biological sample according to claim 1, characterized in that: The biological sample includes at least one of serum, blood and plasma.

10. A detection kit for detecting intestinal microbial metabolites in biological samples, characterized in that: The intestinal microbial metabolites include at least one of trimethylamine, trimethylamine oxide, choline, L-carnitine, betaine, creatinine, tryptophan, tyrosine, p-cresol sulfate, and indoxyl sulfate; The detection kit comprises an extracting solution, a mobile phase A and a mobile phase B, wherein the extracting solution comprises acetonitrile and water, the mobile phase A comprises formic acid, water and ammonium acetate, and the mobile phase B comprises acetonitrile, water, formic acid and ammonium acetate.

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