A kind of intestinal flora polyamine metabolic activity detection system based on liquid chromatography mass spectrometry

Abstract

The present application relates to the technical field of polyamine detection, and particularly relates to a kind of intestinal flora polyamine metabolic activity detection system based on liquid chromatography mass spectrometry, detection includes the following steps: collecting intestinal microorganism in the sample to be measured, incubated with 13 C-inulin, collect culture medium layer and cell layer, extract metabolite respectively, carry out derivatization treatment, analysis is carried out using liquid chromatography mass spectrometry.The intestinal flora polyamine metabolic activity detection system disclosed in the present application includes intestinal microorganism anaerobic culture medium, derivatization reagent, 13 C-inulin solution, buffer solution, termination liquid, extraction solvent, liquid chromatography device and mass spectrometry device.Using the detection system provided by the present application to detect intestinal flora polyamine, only one sample processing is needed, qualitative and quantitative results of intestinal flora polyamine synthesis function can be obtained simultaneously, detection time is short, sensitivity is high, operation difficulty is low, and it is suitable for widely used in large-scale experiment and analysis.

Description

Technical Field

[0001] This invention relates to the field of polyamine detection technology, and in particular to a detection system for polyamine metabolic activity of gut microbiota based on liquid chromatography-mass spectrometry. Background Technology

[0002] Polyamines (including putrescine PUT, spermine SPM, spermidine SPD and their acetylated metabolites, etc.) are important substances for maintaining the growth and development of the body. Therefore, analyzing changes in the level of polyamines in the intestine is helpful in evaluating disease and health status.

[0003] Existing technologies typically employ high-throughput sequencing to characterize the gut microbiota composition in depth. However, due to the high polarity of polyamines, high-throughput sequencing can only limit research to the gut microbiota level and cannot evaluate the metabolic function of polyamine production by the gut microbiota. Therefore, many methods for quantitative analysis of polyamines in blood or urine have emerged. Xiong et al. disclosed a method for detecting the concentration of derivatized polyamines in human plasma samples using high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) (Chromatographia, 2016). This method evaluates the polyamine status in the plasma of cancer patients after derivatization. CN106381326A discloses an in vitro detection kit and method for detecting acetylpolyamine, utilizing acetylpolyamine oxidase to catalyze natural substrates and detect the content of acetylpolyamine in samples. CN110887910A discloses a method for detecting polyamines and their synthetic pathway substances, using dansyl chloride or benzoyl chloride as derivatization reagents, and establishing a method for the extraction and accurate quantitative detection of nine biogenic amines in citrus roots using LC-MS / MS technology.

[0004] The aforementioned techniques use blood or urine as sample sources and perform derivatization or acetylation treatments to quantify the static concentration of polyamines. However, gut microbiota continuously engage in metabolic activities within the human body, and these methods cannot dynamically evaluate the metabolic levels of gut polyamines. Furthermore, while plasma polyamine concentrations can reflect the polyamine levels in systemic organs and tissues, they cannot directly reflect intestinal polyamine levels. In addition, a single sample processing session only yields a single result for polyamine detection, failing to provide a comprehensive evaluation of the gut microbiota's polyamine synthesis function, including both the types and concentrations of polyamines. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to develop a method for detecting the polyamine metabolic activity of gut microbiota and quantifying polyamines in the gut. By detecting polyamine levels in human feces, the method evaluates polyamines in the gut. The detection system provided by this invention simplifies the polyamine processing of test samples, enabling large-scale detection of polyamines in metabolites in a single step. It divides the study of gut microbiota into cellular and culture media, revealing differences in metabolites inside and outside cells. Combining isotope-traced metabolomics with liquid chromatography and mass spectrometry, the sample is eluted and traced sequentially. Qualitative and quantitative analytical results of gut microbiota polyamines can be obtained with a single sample processing step.

[0006] The first objective of this invention is to provide a detection system for polyamine metabolic activity of gut microbiota based on liquid chromatography-mass spectrometry, the detection system comprising an anaerobic culture medium of gut microbiota, 13 C-inulin solution, derivatization reagent, buffer solution, stop solution, extraction solvent, liquid chromatography apparatus, and mass spectrometry apparatus.

[0007] Furthermore, the detection system targets the feces of the person being tested.

[0008] Furthermore, the above-mentioned detection system employs the following steps for detection:

[0009] Step S1: Anaerobic culture of gut microbiota in the sample to be tested, incubation with 13C-inulin, collection of supernatant and precipitate as culture medium layer and cell layer respectively, and quenching treatment of cell layer;

[0010] Step S2: Extract metabolites from the culture medium layer and cell layer to obtain test solutions containing metabolites from the culture medium layer and cell layer, respectively, and perform derivatization treatment. The volume ratio of derivatization reagent to test solution is 2-5:1.

[0011] Step S3: Terminate the derivatization reaction, use the extraction solvent to perform liquid-liquid extraction to obtain the derivatized product, the volume ratio of the extraction solvent to the solution to be extracted is 2-10:1, and analyze it using liquid chromatography-mass spectrometry.

[0012] Furthermore, the anaerobic culture medium for gut microbiota includes an oxygen indicator, cysteine ​​hydrochloride, yeast extract, tryptone, potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, diaminosulfuric acid, magnesium sulfate, and calcium dichloride.

[0013] Preferably, the oxygen indicator is resazurite.

[0014] Preferably, the intestinal microbial culture medium consists of 300-303 mg of cysteine ​​hydrochloride, 250-260 mg of yeast extract, 500-550 mg of tryptone, 120-125 mg of potassium dihydrogen phosphate, 157-160 mg of potassium hydrogen phosphate, 241-243.2 mg of sodium chloride, 242-244 mg of diaminosulfuric acid, 25-26 mg of magnesium sulfate, and 25-27 mg of calcium dichloride.

[0015] Furthermore, the derivatizing reagent is fluorenemethyloxycarbonylsuccinimide (Fmoc-osu). The derivatized product obtained using this reagent is stable, can be detected at room temperature, and does not degrade even after prolonged storage.

[0016] Preferably, during the derivatization process, Fmoc-osu is dissolved in acetonitrile.

[0017] Preferably, the buffer solution consists of sodium bicarbonate, sodium carbonate, and EDTA, with the pH adjusted to 10.2.

[0018] Furthermore, the stationary phase of the liquid chromatography is a C18 column, the mobile phase A is formic acid-water solution, and the mobile phase B is acetonitrile.

[0019] Preferably, the mass ratio of formic acid to water is 1:1000.

[0020] Furthermore, the gradient elution conditions for liquid chromatography are as follows:

[0021] Within 0-10 minutes, the gradient of mobile phase B increases from 10% to 90%.

[0022] The gradient of mobile phase B is maintained at 90% for 10-14 minutes;

[0023] Over 14-14.5 minutes, the gradient of mobile phase B decreases from 90% to 10%.

[0024] For 14.5–20 minutes, the gradient of mobile phase B is maintained at 10%.

[0025] Furthermore, the high-resolution mass spectrometry device employs positive ion scanning, with the ion source parameters set as follows: capillary temperature maintained at 300°C, HESI probe temperature maintained at 325°C, sheath gas flow rate at 35 units, spray voltage set at 4000V, and mass-to-charge ratio collection range at 75-1000.

[0026] Preferably, in step S1, the cell layer is quenched using an acetonitrile-formic acid mixture.

[0027] Preferably, in step S2, the volume ratio of the reagent used for derivatization to the test solution is 2:1.

[0028] Preferably, in step S3, formic acid is used to terminate the derivatization reaction.

[0029] Preferably, the volume ratio of formic acid to the test solution is 2:5.

[0030] Preferably, in step S3, ethyl acetate is used for liquid-liquid extraction.

[0031] Furthermore, in step S3, the intestinal polyamines in the sample to be tested are first separated by liquid chromatography, and then the intestinal polyamines to be tested are quantified by high-resolution mass spectrometry.

[0032] A second objective of this invention is to provide the application of the above-described detection system in the preparation of colorectal cancer diagnostic products or products for detecting intestinal polyamine metabolism levels.

[0033] The technical solution of the present invention has the following advantages compared with the prior art:

[0034] (1) This invention simplifies the treatment of polyamines in fecal samples. After removing food residues, the intestinal microorganisms are incubated separately, and the concentration of polyamines in the intestinal microbiome in feces is detected. The interference of exogenous polyamines produced by dietary metabolism is eliminated, thereby evaluating the polyamine status in the patient's body and evaluating the metabolic level of intestinal polyamines in a dynamic form.

[0035] (2) This invention divides the study of gut microbiota into cellular and culture medium layers to reveal differences in metabolites between intracellular and extracellular environments. Using the detection system provided by this invention, it is possible not only to evaluate known polyamine metabolic pathways (putrescine pathways) but also to evaluate the contribution of new metabolic pathways of gut microbiota to polyamine synthesis (non-putrescine pathways);

[0036] (3) This invention couples liquid chromatography and mass spectrometry, with 13 Combining C-inulin-labeled stable isotope tracing with metabolomics, this invention enables rapid and direct detection of polyamine synthesis function in gut microbiota. The detection system of this invention is used to analyze the polyamine biosynthesis process and metabolites in gut microbiota. 13 The C-labeling mode allows for the simultaneous qualitative and quantitative analysis of the intestinal flora's polyamine synthesis function (types of polyamines and metabolites) through a single sample processing. 13 (C isotope enrichment ratio) results. Attached Figure Description

[0037] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0038] Figure 1 This is the metabolic pathway of putrescine;

[0039] Figure 2 It is a derivative of ornithine, spermidine, and putrescine. 13 C enrichment status;

[0040] Figure 3 This is the metabolic pathway of non-corrosive amines;

[0041] Figure 4 It is a derivative of arginine and guanidine. 13 C enrichment status;

[0042] Figure 5 It is 5'-methionine, acetylated spermidine, and acetylated putrescine. 13 C enrichment status;

[0043] Figure 6 It is the standard curve of putrescine derivatives;

[0044] Figure 7 It is the standard curve of spermidine derivatization products;

[0045] Figure 8 It is the standard curve of spermine derivatives;

[0046] Figure 9 The results are liquid chromatographic results of putrescine, spermine and spermidine derivatives with the internal standard tolbutamide. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0048] Intestinal microbial culture medium: 303 mg cysteine ​​hydrochloride, 250 mg yeast extract, and 500 mg tryptone were dissolved in a solvent consisting of 120.6 mg potassium dihydrogen phosphate, 157.8 mg potassium hydrogen phosphate, 241.7 mg sodium chloride, 242.8 mg diaminosulfate, 25.1 mg magnesium sulfate, and 25.3 mg calcium dichloride (using 500 mL of deionized water). The pH was adjusted to 6.5 with sodium hydroxide, and then 0.1% / L resazurin was added. The medium was sterilized, and carbon dioxide was bubbled through until it became clear, removing any oxygen.

[0049] Polyamine derivatization reagent: 1.05 g sodium bicarbonate, 1.325 g sodium carbonate, and 0.042 g EDTA were dissolved in 25 mL of ultrapure water, and the pH was adjusted to 10.2 using 5 M sodium hydroxide as a buffer solution. 10.12 mg of Fmoc-Osu (fluorenemethyloxycarbonyl succinimide) was dissolved in 6 mL of acetonitrile as a derivatization reagent.

[0050] Example 1: Fecal sample treatment

[0051] 1. Collection of human fecal samples.

[0052] Approximately 50 mg of fresh human fecal sample was collected in a sterile microcentrifuge tube and transferred to an anaerobic bag within 30 minutes to preserve the viability of gut microbiota.

[0053] 2. Isolation and... 13 Incubation of C-inulin.

[0054] Using a syringe, transfer 2 mL of intestinal microbial culture medium to a microcentrifuge tube containing a fecal sample. After crushing the sample with a clean glass rod, transfer it to anaerobic tube 1. Add 700 μL of intestinal microbial culture medium to the microcentrifuge tube for washing, and then transfer the mixture to anaerobic tube 1. Centrifuge anaerobic tube 1 at low speed (600 rpm, 10 min) to remove undigested, larger food particles from the lower precipitate. Using a syringe, transfer the supernatant to anaerobic tube 2 and centrifuge at 3000 rpm for 10 min to collect the intestinal microorganisms. Discard the supernatant, and using a syringe, add 5.4 mL of intestinal microbial culture medium to the intestinal microorganisms. Vortex to mix, and then transfer the mixture to anaerobic tubes 3 and 4, each containing 1.8 mL of intestinal microbial suspension. Add... 13 0.2 mL of C-inulin solvent was added to each tube. After incubation at 37°C for 24 hours, the three anaerobic tubes were centrifuged (3000 rpm, 10 min). The supernatant collected was the culture medium layer. The lower precipitate was washed with 1 mL of intestinal microbial culture medium and then centrifuged again at 3000 rpm, 5 min. The precipitate collected was the cell layer.

[0055] Example 2: Fecal Sample Analysis

[0056] 1. Extracting metabolites from culture media for non-targeted, stable isotope-traced metabolomics analysis.

[0057] The culture medium was aliquoted into microcentrifuge tubes, 1 mL per tube, and then lyophilized to remove water. The lyophilized powder was dissolved in acetonitrile (containing 0.2% formic acid), and the supernatant was collected by centrifugation. The supernatant was derivatized with a derivatizing reagent (twice the volume of the supernatant), and the derivatization reaction was terminated with formic acid (0.4 times the volume of the supernatant). Liquid-liquid extraction was performed with ethyl acetate (10 times the volume of the supernatant). Finally, the ethyl acetate was dried under nitrogen, reconstituted with acetonitrile and water, and then subjected to high-resolution mass spectrometry analysis.

[0058] 2. Extracting metabolites from cell layers for non-targeted stable isotope-traced metabolomics analysis.

[0059] Acetonitrile (containing 0.2% formic acid) was added to the obtained cell extract to quench it. After a brief homogenization by vortexing, the mixture was centrifuged at 5000 rpm for 10 min, and the supernatant was collected. Derivatization, nitrogen blowing, and analysis were performed in the same manner as described above.

[0060] 3. Stable isotope-traced metabolomics analysis using liquid chromatography-mass spectrometry.

[0061] HPLC-MS analysis was performed using a Thermo Fisher Scientific SII HPLC system and a Thermo Fisher Scientific Q Exactive Orbitrap mass spectrometer with a heated electrospray ionization source (including a VF-D11-A UV detector, a VF-P10-A autosampler, and a VH-C10-A column chamber) interface. The mass spectrometer was operated in positive ion mode, collecting complete scans at a resolution of 70,000. Ion source parameters were set as follows: heated capillary maintained at 330°C, HESI probe maintained at 325°C, sheath gas flow rate set to 35 units, auxiliary gas flow rate set to 12 units, standby gas flow rate set to 3 units, and spray voltage set to 4000V. Mass spectrometric data acquisition was performed within a mass-to-charge ratio range of 75–1000. Chromatographic separations were achieved using an Ajer Venusil XBP C18 column (2.1 × 50 mm, 5 μm) and a guard column (SecurityGuard Cartridges C18 4 × 2.0 mm). Buffer phase A was 0.1% formic acid aqueous solution; buffer phase B was acetonitrile. The chromatographic gradient was run at a flow rate of 0.200 mL / min as follows: 0–10 min, linear gradient from 10% to 90%, phase B; 10–14 min, maintained at 90%, phase B; 14–14.5 min, linear gradient to 10%, phase B; 14.5–20 min, equilibration at 10% phase B; 32 min, stop the run. The injection volume was 5 μL.

[0062] 4. Verification using LC-MS / MS

[0063] The liquid chromatography system used was a Shimadzu HPLC system from Japan, including a CBM-20A controller, a Sil-20A autosampler, an LC-20AC binary pump, and a DUG-20A3 degasser. The tandem mass spectrometer was an AB SCIEX4000 QTrap, equipped with an electrospray ionization source. The column oven, CTO-20A, maintained the temperature at 40℃. The chromatographic column used was an Agilent Venusil XBP C18 column (2.1 × 50 mm, 5 μm). The mobile phase for liquid chromatography separation was 0.1% formic acid in water (phase A) and pure acetonitrile (phase B). The mobile phase gradient was: 0.5 min, 10% phase B; 1 min, 90% phase B; 3.0 min, 90% phase B; 3.1 min, 10% phase B; 5 min, stop. The flow rate was 0.4 mL / min, and the injected sample volume was 10 μL. Analyst was used for LC-MS / MC collection and analysis. The tandem mass spectrometers used electrospray ionization (ESI) mode, scanning in positive ion mode, with a total scan time of 1.2601 sec. Specific mass spectrometry parameters were as follows: Curtain Gas 10 psi, GS1 30 psi, GS2 30 psi, IonSpray Voltage 4500 V, ion source temperature (TEM) 450 °C, CXP value 13, and EP value 10.

[0064] Example 3: 13 C-marking results

[0065] The method of the present invention was used to label and evaluate the metabolic activity of intestinal bacteria on polyamines in known pathways. Figure 1 The effects of derivatized Ornithine (ornithine), SPD (spermidine), and PUT (putrescine) on the derivatization were evaluated. 13 C enrichment status ( Figure 2 [Fmoc] represents the functional group that binds to the target compound after the derivatization reaction, and SPD and PUT are abbreviations for Spermidine and Putrescine, respectively. According to the image, ornithine and putrescine... 13 The enrichment patterns of C are quite similar: ornithine is present at M+3, 4, and 5, with a high enrichment level at M+5; putrescine is present at M+2, 3, and 4, with a high enrichment level at M+4, which is consistent with... Figure 1 Ornithine in the sample is a precursor compound of putrescine. Spermine exhibits M+1, 2, 4, 6, and 7, with a high enrichment at M+2, which differs from the enrichment pattern of putrescine. Since putrescine is a precursor compound of spermidine, this does not conform to the criteria. Figure 1 The metabolic pathways in which gut microbiota metabolize polyamines were investigated. Therefore, by using the method of this invention, we evaluated the activity of gut microbiota in polyamine metabolism along known pathways.

[0066] The detection system of this invention was used to evaluate the metabolic activity of gut bacteria on polyamines in a newly discovered pathway. Figure 3 This invention evaluates the derivatized Arginine and Agmatine guanidine. 13 C enrichment status ( Figure 4 According to the image, both arginine and guanidine M+2 are present and 13 The enrichment level of C is relatively high compared to other markers, and it is similar to that of SPD spermidine. 13 The enrichment of C is similar, and guanidine in the F pathway is a precursor compound of putrescine. Therefore, the activity of gut bacteria in polyamine metabolism in the novel pathway was discovered and evaluated using the method of the present invention.

[0067] In the above metabolic pathways Figure 1 and Figure 3 In this study, the activity of other compounds produced by gut microbial metabolism was analyzed using the method of this invention, and the related properties of these other compounds were also analyzed. 13 C isotope enrichment is as follows: Figure 5 As shown. It mainly includes MTA: 5'-methionine; derivatized SPD_Acyl: acetylated spermidine; and derivatized Acetyl Putrescine: acetylated putrescine. Using the method of this invention, the polyamine acetylated products can be detected in human fecal samples, and all of them possess… 13 The C-inulin labeling, wherein MTA has a full labeling range from M+2 to M+8. MTA, polyamines, and polyamine acetylated compounds all have significant effects on tumorigenesis; therefore, the method of this invention for detecting the activity of these metabolites produced by gut microbiota has potential in the treatment of cancer patients.

[0068] Example 4: Quantitative Results

[0069] (1) Standard curve and lower limit of quantitation

[0070] Using the detection system of this invention, a quantitative curve is obtained by analyzing the ratio of the peak area of ​​polyamine standards at different concentrations to that of the internal standard relative to the polyamine. The ratio of polyamine concentration to internal standard concentration is plotted on the x-axis, and the ratio of the polyamine peak area to the internal standard compound peak area is plotted on the y-axis, using a weighted average (W = 1 / X). 2 The least squares regression method was used to obtain the linear equation, which is the standard curve. During method validation, three standard curves were prepared using derivatization reagents. The regression coefficient r of each linear regression equation was greater than 0.9926. The regression coefficient r is shown in the table below. The linearity was good, and the average accuracy was within ±15%. The quantitative lower limit of [Fmoc]2-PUT was 5 ng / mL, and the quantitative lower limit of [Fmoc]2-SPD and [Fmoc]3-SPM was 1 ng / mL. The results are shown below.

[0071] Table 1 Standard curves for three derivatives

[0072]

[0073] (2) Accuracy and Precision

[0074] Intra-day precision and accuracy were verified using quality control (QC) samples at four concentration gradients: LLOQ, LQC, MQC, and HQC. The average errors in precision and accuracy were all within 15%, meeting the relevant requirements. Specifically, the QC sample concentrations for [Fmoc]2-PUT were 5, 10, 50, and 400 ng / mL, and the QC sample concentrations for [Fmoc]2-SPD and [Fmoc]3-SPM were 1, 3, 10, and 80 ng / mL, respectively. After treatment with the derivatization reagents of this invention, analysis was performed using LC-MS / MS. Accuracy and precision were calculated based on the corresponding standard curves. The QC samples prepared during the method validation period are shown in the table below.

[0075] Table 2. Accuracy and precision of the three derivatized products

[0076]

[0077] (3) Stability

[0078] The stability of LQC and HQC after extraction of three compound samples was investigated using the derivatization reagent of this invention under different conditions, including short-term stability after 2 hours at room temperature, storage stability after 24 hours at 4°C, and overnight stability after 24 hours on the injection tray. The results showed that the stability under different conditions met the requirements, as shown in the table below:

[0079] Table 3. Stability of the three derivatives

[0080]

[0081] (4) Matrix effect

[0082] Three different LQC and HQC methods were analyzed to assess matrix effects. The accuracy of [Fmoc]2-PUT was 88.83%, and its precision was 4.59%; the accuracy of [Fmoc]2-SPD was 97.28%, and its precision was 7.81%; and the accuracy of [Fmoc]3-SPM was 99.77%, and its precision was 12.47%. All three methods achieved accuracies within 100 ± 15% and precisions less than 15%, meeting the relevant standards.

[0083] (5) Residual effect

[0084] After analyzing the samples at the upper limit of quantification, blank samples were analyzed. The residues of [Fmoc]2-PUT samples were 19.79% and internal standard residues were 0.26%; the residues of [Fmoc]2-SPD samples were 10.91% and internal standard residues were 0.22%; and the residues of [Fmoc]3-SPM samples were 25.74% and internal standard residues were 0.22%. The residues of internal standards were all below 5%, meeting the relevant requirements, and the residues of analytes were all below 20%, meeting the relevant requirements.

[0085] (6) Selectivity

[0086] The three polyamine derivatized products [Fmoc]2-Put, [Fmoc]2-Spd, and [Fmoc]3-Spm generated using the derivatization reagent of the present invention exhibit good peak shapes and stable signals with the internal standard tolbutamide, with no interference between them. Furthermore, the blank fecal matrix treated with activated carbon does not interfere with the peaks of the products and the internal standard.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A detection system for polyamine metabolic activity of intestinal flora based on liquid chromatography-mass spectrometry, characterized in that, The detection system includes an anaerobic culture medium for intestinal microorganisms. 13 C-inulin solution, derivatization reagent, buffer solution, stop solution, extraction solvent, liquid chromatography apparatus and high-resolution mass spectrometry apparatus; The following steps are used for testing: Step S1: Anaerobic culture of gut microbiota in the sample to be tested, adding... 13 C-inulin incubation was performed, and the supernatant and precipitate were collected separately to form a culture medium layer and a cell layer. The culture medium layer was freeze-dried and the cell layer was quenched. Step S2: Extract metabolites from the culture medium layer and cell layer to obtain test solutions containing metabolites from the culture medium layer and cell layer, respectively, and perform derivatization treatment. The volume ratio of derivatization reagent to test solution is 2-5:

1. Step S3: Terminate the derivatization reaction, use the extraction solvent to perform liquid-liquid extraction to obtain the derivatized product, the volume ratio of the extraction solvent to the solution to be extracted is 2-10:1, and use liquid chromatography-mass spectrometry for analysis; The derivatizing agent is fluorenemethyloxycarbonyl succinimide Fmoc-osu; The detection system detects the feces of the person being tested. The substances detected by the detection system are ornithine, spermidine, putrescine, spermine, arginine, and guanidine.

2. The intestinal flora polyamine metabolic activity detection system according to claim 1, characterized in that: The intestinal microbial anaerobic culture medium includes an oxygen indicator, cysteine ​​hydrochloride, yeast extract, tryptone, potassium dihydrogen phosphate, potassium hydrogen phosphate, sodium chloride, diaminosulfuric acid, magnesium sulfate, and calcium dichloride.

3. The intestinal flora polyamine metabolic activity detection system according to claim 1, characterized in that: The stationary phase of the liquid chromatography is a C18 column, the mobile phase A is formic acid-water solution, and the mobile phase B is acetonitrile.

4. The intestinal flora polyamine metabolic activity detection system according to claim 1, characterized in that, The gradient elution conditions for the liquid chromatography are as follows: Within 0-10 minutes, the gradient of mobile phase B increases from 10% to 90%. The gradient of mobile phase B is maintained at 90% for 10-14 minutes; Over 14-14.5 minutes, the gradient of mobile phase B decreases from 90% to 10%. For 14.5-20 minutes, the gradient of mobile phase B is maintained at 10%.

5. The intestinal flora polyamine metabolic activity detection system according to claim 1, characterized in that: The high-resolution mass spectrometer uses positive ion scanning, with the ion source parameters set as follows: capillary temperature maintained at 300-350°C, HESI probe temperature maintained at 300-350°C, sheath gas flow rate at 20-35 units, and spray voltage set at 3500-4500V.

6. The intestinal flora polyamine metabolic activity detection system according to claim 1, characterized in that: The high-resolution mass spectrometer has a mass-to-charge ratio collection range of 75-1000.

7. The application of the intestinal flora polyamine metabolic activity detection system as described in any one of claims 1-6 in the preparation of colorectal cancer diagnostic products or intestinal polyamine metabolism level detection products.

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