Method for separating intestinal flora related metabolites in serum and application

Abstract

The invention belongs to the technical field of serum analysis, and particularly relates to a method for separating intestinal flora related metabolites in serum and application. The method comprises the following steps: carrying out gradient elution separation on a to-be-detected sample by using liquid chromatography; mobile phases of the liquid chromatography comprise a mobile phase A and a mobile phase B, the mobile phase A is a formic acid aqueous solution with the mass percent of 0.05%-0.15%, and the mobile phase B is a formic acid acetonitrile solution with the mass percent of 0.05%-0.15%. According to the detection method provided by the invention, at most 22 metabolites can be simultaneously separated and quantitatively detected within 6 minutes, the accuracy range of each concentration in a standard curve is 85-115%, and the lower limit of quantification is 80-120%; in addition, within-batch and inter-batch precision and recovery rate experiments of the 22 metabolites meet the requirements of the guide; after internal standard correction, most analytes have no obvious matrix effect, and the background interference and the pollution carrying rate also meet the requirements.

Description

Technical Field

[0001] The present invention belongs to the technical field of serum analysis, and particularly relates to a method for separating gut microbiota-related metabolites in serum and its application. Background Art

[0002] Colorectal cancer has become an increasingly severe global challenge, and early diagnosis is crucial for improving the survival rate of colorectal cancer patients. There are various methods for colorectal cancer detection, including non-invasive techniques such as fecal occult blood test (FOBT) and carcinoembryonic antigen (CEA) test, and invasive surgeries such as colonoscopy. However, the widespread use of these methods is hindered by the low accuracy of non-invasive detection and the potential harm of invasive detection. Therefore, a non-invasive and accurate method for detecting colorectal cancer is needed.

[0003] Existing studies have determined stage-specific characteristics of the gut microbiome and metabolites in colorectal adenomas and early / late colorectal cancers through metabolomic and metagenomic analyses of fecal samples. Existing studies have found that several metabolites, including carnitine and bile acids, are associated with the gut microbiome of colorectal cancer patients and show significant changes.

[0004] Human bile acids (BAs) are C24 molecules with a steroid nucleus and a carboxylic acid side chain, and are extensively enterohepatic circulation metabolites affected by host and gut microbiota metabolism. Carnitine compounds play a key role in carnitine metabolism, providing energy and metabolic intermediates for cell growth by transporting fatty acids into mitochondria for β-oxidation. 2-Octenoyl carnitine has been reported as a non-targeted metabolomic bile biomarker for cholangiocarcinoma.

[0005] Liquid chromatography-mass spectrometry (LC-MS) is the mainstream method for analyzing BAs, and there is currently no quantitative method for gut microbiota-related metabolites in serum. In addition, no study has used LC-MS / MS to simultaneously quantify BAs and other gut microbiota-related metabolites in human serum. Summary of the Invention

[0006] The present invention has developed a method for separating and detecting 22 gut microbiota-related metabolites in serum based on liquid chromatography-mass spectrometry (LC-MS).

[0007] To achieve the above object, the present invention can adopt the following technical solutions:

[0008] On the one hand, the present invention provides a method for separating 22 gut microbiota-related metabolites in serum, comprising: performing gradient elution separation on a sample to be detected using liquid chromatography; the mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, mobile phase A is an aqueous solution of formic acid with a mass percentage of 0.05%-0.15%, and mobile phase B is an acetonitrile solution of formic acid with a mass percentage of 0.05%-0.15%; the gut microbiota-related metabolites are one or more of GLCA, GCA, DCA, CA, TUDCA, UDCA, TCA, TDCA, GCDCA, TCDCA, GDCA, LCA, CDCA, GUDCA, TLCA, BN01, BN02, BN03, BP01, BP02, BP03 or BP04; the names of the above gut microbiota-related metabolites are as follows:

[0009]

[0010] Preferably, the gradient elution program in the above method is: 0-0.2 min, 25% B phase by volume, 0.2 min - 0.8 min, 25%-40% B phase by volume, 0.8 min - 2 min, 40%-45% B phase by volume, 2 min - 2.5 min, 45%-60% B phase by volume, 2.5 min - 3.6 min, 60%-70% B phase by volume, 3.6 min - 5 min, 70%-98% B phase by volume, 5 min - 5.1 min, 98%-25% B phase by volume.

[0011] Preferably, the chromatographic column of the liquid chromatography in the above method is a Shim-pack velox column, C18, 2.7 μm, 50 mm x 2.1 mm.

[0012] Preferably, the flow rate in the above method is 0.075 mL / min.

[0013] Preferably, the preparation method of the sample to be detected in the above method comprises: mixing the serum to be detected with an IS mixture, then adding a mixed solvent of acetonitrile and isopropanol, vortexing, adding an ammonium formate solution, vortexing, centrifuging, and adding water to the supernatant to obtain the sample to be detected.

[0014] Preferably, in the mixed solvent of acetonitrile and isopropanol in the above method, the volume ratio of acetonitrile to isopropanol is 4:1.

[0015] On the other hand, the present invention provides a method for separating 22 gut microbiota-related metabolites in serum, comprising: separating using the method for separating 22 gut microbiota-related metabolites in serum of the present invention, and then detecting using a mass spectrometer.

[0016] Preferably, the mass spectrometer detection conditions are as follows:

[0017]

[0018]

[0019] The beneficial effects of the present invention at least include:

[0020] The detection method provided by the present invention can simultaneously separate and quantitatively detect up to 22 metabolites within 6 minutes, and the accuracy range of each concentration in its standard curve is within 85%-115%, and the lower limit of quantification is within 80%-120%; in addition, the intra-batch, inter-batch precision and recovery experiments of 22 metabolites all meet the requirements of the guidelines; after internal standard correction, most analytes have no obvious matrix effect, and the background interference and carry-over contamination rate also meet the requirements. Description of the Drawings

[0021] Figure 1 It is a representative chromatogram of 22 compounds and their corresponding ISs in serum samples. Detailed Embodiments

[0022] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation solutions based on the above-mentioned invention content still fall within the protection scope of the present invention.

[0023] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having significantly different meanings in the context, the singular forms of expressions include the plural forms of expressions. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or their combinations may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0024] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited only to the examples below.

[0025] In the following examples, a total of 220 serum samples were collected from the Cancer Hospital of the Chinese Academy of Medical Sciences, including healthy individuals undergoing cancer screening, patients with colorectal adenomas, and patients with colorectal adenocarcinomas. All individual serum samples were obtained before treatment and centrifuged at 1500 g for 15 minutes at 4 - 8 °C. Subsequently, the serum supernatant was transferred to a new centrifuge tube and stored at -80 °C for later use. All procedures of this study were approved by the Independent Ethics Committee of the National Cancer Center / Cancer Hospital of the Chinese Academy of Medical Sciences and Peking Union Medical College.

[0026] In the following examples, the compounds used included: HPLC grade acetonitrile (ACN), methanol (MeOH), isopropanol (IPA), ammonium formate, and formic acid (FA) were purchased from Thermo Fisher Scientific. Deionized water (>18.2 MΩ·cm) was purchased from Watson. Glycocholic acid (GLCA), glycocholic acid (GCA), deoxycholic acid (DCA), cholic acid (CA), taurodeoxycholic acid (TUDCA), ursodeoxycholic acid (UDCA), taurochenodeoxycholic acid (TCA), taurochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (GCDCA), glycochenodeoxycholic acid monohydrate (GDCA), lithocholic acid (LCA), chenodeoxycholic acid (CDCA), glycoursodeoxycholic acid (GUDCA), (±)15-HETE, 12,13-dihomo, pinellic acid, carnitine, trans-2-octanoyl-l-carnitine, trans-2-hexadecenoyl-l-carnitine, and (±)-hexenoyl chloride were obtained from Shanghai Yuanye Bio-Technology, Alfa Aesar, National Institutes for Food and Drug Control, zzstandard, BePuro, ISOREAG, MCE, sigma, and TRC, respectively. Glycocholic acid-d4, glycocholic acid-d4, [2,2,4,4-2H4]-deoxycholic acid, [2H4]-cholic acid, taurolithocholic acid-d4, ursodeoxycholic acid-d4, taurolithocholic acid-d4, [2H4]-chenodeoxycholic acid, taurodeoxycholic acid-d4, glycochenodeoxycholic acid-d4, lithocholic acid-d4, chenodeoxycholic acid-d4, glycodeoxycholic acid-d4, [pyridine-2H4]-(±)-cotinine, [2H4]-(±)12(13)-dihomo, [14,14,14-2H3]-l-carnitine (chloride) and [2H3]-hexanolyl-l-carnitine HCL were purchased from ISOREAG and BePure.

[0027] In the following examples, external and internal standards (Internal standard, IS) were weighed using an analytical balance accurate to one ten-thousandth, and then high-concentration stock solutions were prepared by diluting to a fixed volume with water or isopropanol as the solvent, stored in amber glass bottles at a storage temperature of -80 °C. To further prepare working solutions and quality control solutions, the stock solution of each compound was diluted to different concentrations with 50% ACN (Table 1), and then stored in glass bottles at -20 °C. The IS mixture was freshly prepared each time samples were prepared.

[0028] Table 1 Concentrations of each analyte and internal standard in different working solutions

[0029]

[0030]

[0031] Continued from Table 1

[0032]

[0033] I. Detection method construction

[0034] (I) Sample preparation

[0035] During metabolite extraction, 10 μL of the IS solution (internal standard solution) was added to 80 μL of serum (or 70 μL of blank matrix plasma and 10 μL of the standard solution), then 150 μL of acetonitrile:isopropanol (volume ratio 4:1) was added and vortexed for 2 min, then 50 μL of ammonium formate (0.5 g / mL) was added and vortexed for 2 min, centrifuged at 17949 g for 10 min, 60 μL of the supernatant was aspirated, and then 150 μL of HPLC-grade water was added and reserved for injection.

[0036] (II) LC-MS / MS procedure

[0037] LC-MS / MS used AB SCIEX Triple Quad TM4500 system; the injection volume for each mode is 10 μL; the metabolite standard solution (the solution prepared in Table 1 above) is eluted from a Shim-pack velox column (C18, 2.7 μm, 50 x 2.1 mm) at a flow rate of 0.075 mL / min; mobile phase A (water containing 0.1% formic acid) and mobile phase B (acetonitrile containing 0.1% formic acid) are gradient eluted (the gradient elution program is shown in Table 2 below); by infusing the corresponding standards, the mass spectrometer parameters for each metabolite are optimized by electrospray ionization (ESI) in positive and negative ion modes, and the optimized parameters are shown in Table 3 below; the metabolite peaks are integrated using Sciex Analyst 1.6.3 software and multiQuant 3.0.2 software.

[0038] Table 2 Gradient Elution Program

[0039] 0 - 0.2 min 25% phase B 0.2 - 0.8 min 25% - 40% phase B 0.8 - 2 min 40% - 45% phase B 2 - 2.5 min 45% - 60% phase B 2.5 - 3.6 min 60% - 70% phase B 3.6 - 5 min 70% - 98% phase B 5 - 5.1 min 98% - 25% phase B

[0040] Table 3 Mass Spectrometer Parameters

[0041]

[0042]

[0043] Representative chromatograms of 22 compounds and their corresponding IS solutions (internal standard solutions) in the serum samples after extraction are shown as Figure 1 shown; in addition, the total analysis time of the LC-MS / MS method is 6.0 min.

[0044] II. Detection Method Validation

[0045] In the following validation, the linearity, precision, recovery and interference, carry-over rate, lower limit of quantification (LLOQ), matrix effect and stability of the method are verified according to the CLSI C62A guideline.

[0046] In the following validation, statistical calculations are performed using excel or GraphPad Prism software. All data are expressed as the mean ± SD of at least three replicate experiments. Unless otherwise specified, the differences between two groups are analyzed by Student's t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, no statistical significance.

[0047] (I) Linearity and Lower Limit of Quantification

[0048] The linear range (6 concentrations, 6 replicates) was calculated with a weighting factor of 1 / (x^2); the acceptance criteria for each concentration in the linear range were that the relative error (%RE) of the measured concentration of the calibration standard compared to the theoretical concentration was within ±15%, and the linear correlation coefficient (r2) was greater than or equal to 0.99. To determine the lower limit of quantitation, the following concentration ranges (6 replicates) were measured for each metabolite; at the LLOQ concentration, the %RE was ±20% and the relative standard deviation (%RSD) had to be less than 20%.

[0049] A wide range of concentrations was observed among the 22 compounds; the 22 compounds showed a linear relationship over a wide concentration range, with a correlation coefficient (r2) greater than 0.99 and an accuracy between 85% and 115% (Table 2); the LLOQs of the 22 compounds were determined (Table 4) and the accuracy was acceptable (80% - 120%).

[0050] Table 4 LLOQs of 22 Compounds

[0051]

[0052]

[0053] (II) Intra - batch and inter - batch precision

[0054] The intra - batch precision (3 concentrations, 18 single analyses) and inter - batch precision (3 concentrations, 18 replicates over 3 days) of blank matrix plasma were determined; the blank matrix plasma was analyzed with three different concentrations of working solutions including all metabolites. The three concentrations observed were based on low, medium, and high levels covering the linear range; the %RSD of intra - batch precision and inter - batch precision for each concentration was set at ±15%. The results are shown in Table 5 below.

[0055] Table 5 Intra - batch and inter - batch precision at different concentrations

[0056]

[0057]

[0058] (III) Recovery experiment

[0059] Evaluate the accuracy of the recovery rate evaluation method. Add standard substances at three different concentrations to blank matrix plasma. All metabolites are detected 6 times at each level, and the ratio of the measured concentration to the expected concentration is calculated as the recovery rate. In terms of the recovery rate, the recovery rates at each concentration are 85% - 115%, and the recovery rates at the lowest concentration are 80% - 120%. Except for BN002, BN019, UDCA, and TCA, the recovery rates of all compounds at the LQC concentration are 85% - 115% (Table 5). Except for BN002, BP007, UDCA, and TCDCA, the recovery rates of all compounds at the MQC concentration are 85% - 115% (Table 5). Except for BN002 and BN019, the recovery rates of all compounds at the HQC concentration are 85% - 115% (Table 6).

[0060] Table 6 Results of recovery rate evaluation

[0061]

[0062]

[0063] (IV) Matrix effect

[0064] To determine the matrix effect, the present invention collects clinical samples from six different sources, mixes them and divides them into aliquots; subsequently, standard samples at at least two different concentration levels of high and low are prepared using blank matrix (substitute matrix), and the standard samples and patient matrix samples from 6 different sources are mixed at a ratio of 1:1 to obtain mixed samples.

[0065] The difference between the response value of the mixed sample (analyte / internal standard) and the average response of the clinical sample and the standard solution should be less than 20%, indicating no relative matrix effect. Except for the matrix effects of BN002, BP010, DCA, CDCA, and UDCA being 139.1%, 151.2%, 118.9%, 117.4%, and 124.7% respectively (Table 6), the matrix effects of the remaining compounds at low concentrations are all between 85% - 115% (Table 6). At high concentrations, except for the matrix effects of BN002, BP012, and GDCA being 143.9%, 115.6%, and 115.1% respectively, the matrix effects of the remaining compounds are all between 85% - 115% (Table 7).

[0066] Table 7 Matrix effect situation

[0067]

[0068] (V) Carryover contamination rate and interference experiment

[0069] The blank matrix samples (without analyte and internal standard), blank solvent samples (water without analyte and internal standard), internal standard blank samples (containing the analyte at the upper limit of quantification but without internal standard), and analyte blank samples (containing internal standard but without analyte) were repeated 3 times, and the injection order of the 4 samples could be interchanged. When there was background interference, the peak area of the analyte interference did not exceed 20% of the peak area of the analyte in the sample at the limit of quantification. At the same time, the peak area of the internal standard interference did not exceed 5% of the peak area of the internal standard interference in the sample at the limit of quantification. For the internal standard interference of the analyte, the peak area of the analyte interference did not exceed 20% of the peak area of the analyte in the sample at the limit of quantification. For the interference of the analyte on the internal standard, the peak area of the internal standard interference did not exceed 5% of the peak area of the internal standard in the sample at the limit of quantification.

[0070] To determine the carryover rate, three samples each of the upper limit of quantification (ULOQ) sample and the blank sample (double blank) were processed and analyzed by alternating injection. The peak area of the analyte in the last two blank samples after the ULOQ sample should not exceed 20% of the peak area of the analyte in the qualified LLOQ sample, and the peak area of the internal standard should not exceed 5% of the peak area of the internal standard in the LLOQ sample.

[0071] Except for BN011, BN019, LCA, GDCA, and GCDCA, the carryover rates of the internal standards for all compounds were below 20% and all were below 5% (Table 8). The background interferences for all compounds were acceptable, with the background interference ratio below 20%, the background IS interference ratio below 5%, and the interference ratio between IS and analyte below 20% (Table 8). However, the background interferences of BN002, BN011, BN019, LCA, GDCA, and GCDCA were unacceptable, and the interference percentages are shown in Table 8.

[0072] Table 8 Carryover Rate and Interference Experiment Test Results

[0073]

[0074]

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A method for separating intestinal flora-related metabolites in serum, characterized in that: include: Liquid chromatography is used to perform gradient elution separation on the sample to be tested; the mobile phase of the liquid chromatography includes mobile phase A and mobile phase B, mobile phase A is a 0.05%-0.15% by mass formic acid aqueous solution, and mobile phase B is a 0.05%-0.15% by mass formic acid acetonitrile solution; the intestinal flora-related metabolites are one or more of GLCA, GCA, DCA, CA, TUDCA, UDCA, TCA, TDCA, GCDCA, TCDCA, GDCA, LCA, CDCA, GUDCA, TLCA, BN01, BN02, BN03, BP01, BP02, BP03 or BP04; the names of the above-mentioned intestinal flora-related metabolites are as follows:

2. The method according to claim 1, characterized in that The gradient elution program was: 0-0.2min, volume fraction 25% B phase, 0.2min-0.8min, volume fraction 25%-40% B phase, 0.8min-2min, volume fraction 40%-45% B phase, 2min-2.5min, volume fraction 45%-60% B phase, 2.5min-3.6min, volume fraction 60%-70% B phase, 3.6min-5min, volume fraction 70%-98% B phase, 5min-5.1min, volume fraction 98%-25% phase B.

3. The method according to claim 1 or 2, characterized in that: The chromatographic column for liquid chromatography was a Shim-pack velox column, C18, 2.7 μm, 50 mm x 2.1 mm.

4. The method according to claim 1 or 2, characterized in that: The flow rate of the liquid chromatography was 0.075 mL / min.

5. The method according to claim 1 or 2, characterized in that: The preparation method of the sample to be tested includes: mixing the serum to be tested with the IS mixed solution, then adding a mixed solvent of acetonitrile and isopropanol, vortexing, adding an ammonium formate solution, vortexing, centrifuging, and adding water to the supernatant to obtain the sample to be tested.

6. The method according to claim 5, characterized in that In the mixed solvent of acetonitrile and isopropanol, the volume ratio of acetonitrile to isopropanol is 4:

1.

7. A method for separating 22 intestinal flora-related metabolites in serum, characterized in that: include: The separation is carried out using the scheme described in any one of claims 1 to 6, and then the detection is carried out using a mass spectrometer.

8. The method according to claim 7, characterized in that The mass spectrometer detection conditions are as follows:

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