A method for detecting ellagic acid and its metabolites

Abstract

The invention discloses a detection method for ellagic acid and its metabolites, and relates to the field of detection and analysis technology. The method of the present invention optimizes the pre-treatment method and detection technology, and adopts a composite extractant N-methylpyrrolidone-methanol solution to ultrasonically extract substances rich in ellagic acid and urolithin, and the extracted samples are qualitatively and quantitatively determined by high performance liquid chromatography tandem ultra-high resolution mass spectrometer, and at the same time, according to the experimental results, a simple high performance liquid chromatography determination method is developed. This method breaks the shortcomings of the traditional detection method that ellagic acid and urolithin substances have poor solubility and are difficult to extract such substances in the sample, and the quantitative results are inaccurate. The method has simple processing steps, good separation effect, short detection time, high sensitivity, qualitative accuracy, simple operation, good specificity, and can analyze the changing trend of the ellagic acid metabolic pathway, providing new ideas and methods for studying its biological metabolic process.

Description

Technical Field

[0001] The present invention relates to the field of detection and analysis technology, and in particular to a method for detecting ellagic acid and its metabolites. Background Art

[0002] Ellagic acid is a natural polyphenol dilactone widely found in fruits or nuts such as pomegranates, blueberries, pecans, chestnuts, etc. It has been proven to have potential preventive or therapeutic effects on cancer, diabetes, hypertension, atherosclerosis, and neuropathies, and has good biosafety. However, the poor water solubility and permeability of ellagic acid lead to its poor absorption in the gastrointestinal tract of mammals, low bioavailability, and the concentration of reaching the target tissue through the intestinal wall through the blood circulation is often insufficient to exert biological activity. Some unabsorbed ellagic acid is converted into more easily absorbed urolithin substances under the action of intestinal flora. Urolithin is generated by ellagic acid losing a lactone ring and gradually dehydroxylating. Due to different dehydroxylation sites and sequences, there are many types of urolithin, and the metabolic process of ellagic acid also has different rules in different individuals. Different urolithin has different numbers and sites of hydroxyl groups, and its biological activity also has corresponding differences. Therefore, the qualitative and quantitative detection of ellagic acid and urolithin is the research basis for judging the metabolic type of ellagic acid and analyzing the biological activity mechanism of ellagic acid.

[0003] High performance liquid chromatography is a modern chromatographic separation method with the advantages of fast analysis speed, strong separation effect, high selectivity, strong column efficiency, and wide application range. It is often used in conjunction with various detectors to determine the substances contained in the sample by comparing the different peak signals detected by the detector, and can also achieve the purpose of separating the mixture. At present, ellagic acid is mostly dissolved in organic reagents such as methanol and then quantitatively detected by high performance liquid chromatography, but ellagic acid is not only poorly water-soluble, but also has low solubility in methanol and ethanol. Therefore, in the detection process, there will be poor peak shape, high detection limit, and similar components will cause strong interference. The metabolites of ellagic acid, urolithins, have many isomers with similar retention behaviors, and accurate qualitative detection cannot be performed using a high performance chromatograph alone.

[0004] Therefore, there is an urgent need to optimize the extraction, separation and identification methods of samples rich in ellagic acid and urolithin-like substances in order to overcome the defects of existing detection methods, such as poor peak shape, difficulty in separating isomers, difficulty in accurately determining the content of the analyte in the sample, and high detection cost.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for detecting ellagic acid and its metabolites, aiming to simultaneously and quantitatively detect ellagic acid and urolithin substances in a sample and improve the stability of detection.

[0007] The present invention is achieved in that:

[0008] In a first aspect, the present invention provides a method for detecting ellagic acid and its metabolites, comprising:

[0009] The sample to be tested is extracted with a mixed extractant formed by methanol and N-methylpyrrolidone to prepare a test solution; wherein the volume fraction of N-methylpyrrolidone in the mixed extractant is 5%-10%, and the volume ratio of the sample to be tested to the mixed extractant is 1:(2-6);

[0010] The standard sample solution was prepared by using ellagic acid standard, various urolithin standards and mixed extractant;

[0011] Quantitative analysis was performed using high performance liquid chromatography or liquid chromatography-mass spectrometry.

[0012] In an optional embodiment, the process of quantitative analysis using high performance liquid chromatography includes: eluting and separating the standard sample solution using high performance liquid chromatography, drawing a standard curve graph based on the known standard concentration and peak area and obtaining a quantitative correction equation; testing the test solution using the same method, substituting the peak areas of ellagic acid and urolithin detected by the ultraviolet detector into the quantitative correction equation, and calculating the content of ellagic acid and urolithin in the test sample.

[0013] In an optional embodiment, the instrument conditions of the high performance liquid chromatography are as follows: the instrument model is ThemoScientific U3000, and the chromatographic column is ThemoScientific Acclaim TM 120C18, the inner diameter of the column filling is 3μm-7μm, the column temperature is 25℃-45℃, the flow rate is 0.5mL / min-2.0mL / min, the injection volume is 2μL-10μL, and the UV detection wavelength is 250nm-260nm;

[0014] In an optional embodiment, the process of using liquid chromatography-mass spectrometry for quantitative analysis includes: using high performance liquid chromatography to perform chromatographic elution separation on ellagic acid and urolithin in a standard sample solution, detecting by ultra-high resolution mass spectrometry, calculating the quantitative detection limit and the identification detection limit according to the signal-to-noise ratio, and drawing a standard curve according to the known standard concentration and peak area to obtain a quantitative correction equation;

[0015] The test solution was sampled and tested in the same manner, and the peak areas of ellagic acid and urolithin detected by mass spectrometry were substituted into the quantitative calibration equation to calculate the contents of ellagic acid and urolithin in the test sample.

[0016] In an optional embodiment, the instrument conditions of the high performance liquid chromatography tandem ultra-high resolution mass spectrometry used in the LC-MS method are as follows: the instrument model is ThemoScientific Q Exactive, the chromatographic column is C18, the inner diameter of the chromatographic column filling is 1.5μm-2.0μm, the column temperature is 25℃-45℃, the flow rate is 0.2mL / min-0.4mL / min, the injection volume is 2μL-5μL, the scanning mode is electrospray ionization negative mode, the spray voltage is -3Kv~-2kV, the sheath gas flow rate is 25arb-35arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320℃-340℃.

[0017] In an optional embodiment, the ultra-high resolution mass spectrometer adopts typical data-dependent mass spectrometry acquisition, with a primary full scan resolution of 68000-72000, a primary ion scan range of 50m / z-750m / z, HCD is used for secondary fragmentation, and the secondary resolution is 17000-18000. In the collision energy normalization mode, the collision energies are 20 / 40 / 60eV, and Xcalibur software is selected in combination with Compound Discover software for metabolite identification.

[0018] In an optional embodiment, when high performance liquid chromatography or liquid chromatography-mass spectrometry is used for quantitative analysis, the mobile phases used include mobile phase A and mobile phase B, mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05%-0.15%, and mobile phase B is acetonitrile.

[0019] In an optional embodiment, a gradient elution method is adopted, and the conditions of the gradient elution are as follows:

[0020] 0-5min, the volume fraction of mobile phase A was reduced from 88% to 70%;

[0021] 5-6min, the volume fraction of mobile phase A was reduced from 70% to 65%;

[0022] 6-10min, the volume fraction of mobile phase A was maintained at 65%;

[0023] 10-14 min, the volume fraction of mobile phase A was reduced from 65% to 0%;

[0024] 14-16 min, the volume fraction of mobile phase A was maintained at 100%;

[0025] At 16-18 min, the volume fraction of mobile phase A increased from 0% to 88%;

[0026] 18-20min, the volume fraction of mobile phase A was maintained at 88%;

[0027] In the gradient elution process, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.

[0028] In an optional embodiment, the preparation process of the test solution includes: mixing the test sample and the mixed extractant, vortexing and ultrasonically extracting, centrifuging after dilution, and filtering the supernatant;

[0029] Preferably, the vortex time is 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is 20min-40min;

[0030] Preferably, the centrifugation temperature is 0°C-4°C, the centrifugation time is 5 min-10 min, and the centrifugal force is 10000g-12000g.

[0031] In an optional embodiment, the preparation process of the standard sample solution includes: mixing ellagic acid standards, multiple urolithin standards and a mixed extractant, diluting the mixed extractant to different concentrations, and filtering.

[0032] The invention has the following beneficial effects: a mixed extractant formed by methanol and N-methylpyrrolidone is used to extract a sample to be tested, and the concentration of N-methylpyrrolidone in the mixed extractant and the amount of the mixed extractant are controlled, so that the dissolution of ellagic acid and urolithin substances in the sample can be improved, the detection limit of the target substance in the sample is reduced, and the detection accuracy is significantly improved; the chromatography and mass spectrometry method of high performance liquid chromatography tandem ultra-high resolution mass spectrometry detection is optimized, so that ellagic acid and urolithin can be significantly separated from other impurities in the sample, the peak shape is good, and the optimized method has a short detection time, high sensitivity and accurate qualitative analysis; a simple high performance liquid chromatography determination method is developed, which is simple to operate, reduces the detection cost, and ensures the accuracy and stability of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 It is the structural formula of common urolithin;

[0035] Figure 2 The identification result diagram of the method provided by the embodiment of the present invention;

[0036] Figure 3 This is the identification result diagram of the traditional detection method;

[0037] Figure 4The content of ellagic acid and urolithin in the in vitro fermentation system from day 0 to day 15, in which (A) represents ellagic acid, and (B)-(H) represent urolithin M5, urolithin M6, urolithin C, urolithin M7, isouroelithin A, urolithin A, and urolithin B, respectively;

[0038] Figure 5 The figure is a comparison of the dissolution behavior of ellagic acid in methanol and N-methylpyrrolidone;

[0039] Figure 6 Chromatogram of ellagic acid after optimization of extraction conditions;

[0040] Figure 7 It is the test result diagram of other chromatographic methods;

[0041] Figure 8 It is the separation effect diagram of other chromatographic methods;

[0042] Fig. 9 A separation effect diagram of a method is provided for an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0044] The structure of ellagic acid is as follows:

[0045]

[0046] The metabolite of ellagic acid is called urolithin, and its specific structure is as follows Figure 1 As shown, there are nine urolithins in total, including urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isourolitoin A, and urolithin B. Figure 1 Uro means urolithin.

[0047] The present invention provides a method for detecting ellagic acid and its metabolites, comprising the following steps:

[0048] S1. Prepare the test solution

[0049] The sample to be tested is extracted with a mixed extractant formed by methanol and N-methylpyrrolidone to prepare a test solution; wherein the volume fraction of N-methylpyrrolidone in the mixed extractant is 5%-10%, and the volume ratio of the sample to be tested to the mixed extractant is 1:(2-6). By optimizing the composition of the extractant, ellagic acid and its metabolites can be better dissolved, the peak shape after detection is good, and the detection data is more stable.

[0050] Specifically, methanol and N-methylpyrrolidone are mixed to obtain a mixed extractant, in which the volume fraction of N-methylpyrrolidone can be 5%, 6%, 7%, 8%, 9%, 10%, etc., and the volume ratio of the sample to be tested to the mixed extractant can be 1:2, 1:3, 1:4, 1:5, 1:6, etc.

[0051] In some embodiments, the preparation process of the test liquid includes: mixing the test sample and the mixed extractant for vortexing and ultrasonic extraction, centrifuging after dilution, taking the supernatant for filtration, and the obtained solution is used for sample analysis. The vortex time is controlled to be 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is 20min-40min, so that ellagic acid and urolithin are fully extracted. After the extraction is completed, dilution is performed, and the concentration of the dilution is not limited, and the concentration can be accurately calculated by the standard curve. Centrifugation is performed after dilution, and the centrifugation temperature is controlled to be 0°C-4°C, the centrifugation time is 5min-10min, and the centrifugal force is 10000g-12000g. After centrifugation, the supernatant is taken and filtered so that the obtained test liquid meets the requirements of the machine.

[0052] Specifically, the vortex time can be 30s, 40s, 50s, 60s, 70s, 80s, 90s, etc., the ultrasonic power can be 400W, 500W, 600W, etc., and the ultrasonic time can be 20min, 25min, 30min, 40min, etc. The centrifugal temperature can be 0°C, 1°C, 2°C, 3°C, 4°C, etc., the centrifugal time can be 5min, 8min, 10min, etc., and the centrifugal force can be 10000g, 11000g, 12000g, etc. The filtering method is not limited, and a 0.22μm PTFE membrane can be used for filtering.

[0053] S2. Preparation of standard sample solution

[0054] The standard sample solution is prepared by utilizing ellagic acid standard, multiple urolithin standard and mixed extractant, and the standard sample solution can be a mixed standard solution or a standard solution of a single component. The mixed standard solution is to mix ellagic acid standard, 9 kinds of urolithin standard and mixed extractant to prepare standard sample solutions of different concentrations. The standard solution of a single component is to mix ellagic acid standard and 9 kinds of urolithin standard with mixed extractant respectively to prepare standard sample solutions of different concentrations. For the sake of method simplicity, the embodiment of the present invention adopts a mixed standard solution, but considering the requirement of further improving the detection accuracy, the standard solution of a single component can also be prepared.

[0055] In actual operation, the preparation process of the standard sample solution includes: mixing ellagic acid standard, multiple urolithin standard and mixed extractant by ultrasonication to fully dissolve them, diluting to different concentrations with the mixed extractant, filtering, and obtaining a standard sample solution that meets the requirements of the machine. The filtering method is not limited, and a 0.22μm PTFE membrane can be used for filtering.

[0056] Specifically, the composition of the mixed extractant is as described in step S1.

[0057] S3. Quantitative analysis

[0058] Quantitative analysis is performed using high performance liquid chromatography or liquid chromatography-mass spectrometry. The following describes the two methods:

[0059] (1) High performance liquid chromatography

[0060] The process of quantitative analysis by high performance liquid chromatography includes: using high performance liquid chromatography to elute and separate the standard sample solution, drawing a standard curve according to the known standard concentration and peak area and obtaining a quantitative correction equation; using the same method to test the test solution, substituting the peak area of ​​ellagic acid and urolithin detected by the ultraviolet detector into the quantitative correction equation, and calculating the content of ellagic acid and urolithin in the test sample. The general standard curve method can accurately detect the content of ellagic acid and various urolithins.

[0061] In some embodiments, the instrument conditions of high performance liquid chromatography are as follows: the instrument model is ThemoScientific U3000, and the chromatographic column is ThemoScientific Acclaim TM 120C18, the inner diameter of the column filling is 3μm-7μm, the column temperature is 25℃-45℃, the flow rate is 0.5mL / min-2.0mL / min, the injection volume is 2μL-10μL, and the UV detection wavelength is 250nm-260nm. By optimizing the chromatographic column and operating parameters, the components can be better separated and the peak effect can be improved.

[0062] Specifically, the inner diameter of the filling material of the chromatographic column can be 3μm, 4μm, 5μm, 6μm, 7μm, etc. The chromatographic column is ThemoScientific Acclaim TM 120C18, 5μm. The column temperature can be 25℃, 30℃, 35℃, 40℃, 45℃, etc. The flow rate can be 0.5mL / min, 1.0mL / min, 1.5mL / min, 2.0mL / min, etc. The injection volume can be 2μL, 3μL, 5μL, 8μL, 10μL, etc. The UV detection wavelength can be 250nm, 252nm, 255nm, 258nm, 260nm, etc.

[0063] Furthermore, when using high performance liquid chromatography for quantitative analysis, the mobile phases used include mobile phase A and mobile phase B, wherein mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05%-0.15%, and mobile phase B is acetonitrile. By optimizing the mobile phase, the elution effect is improved, and the peak effect is better.

[0064] Specifically, the mobile phase A is a formic acid aqueous solution, and the volume fraction of the formic acid can be 0.05%, 0.08%, 0.10%, 0.12%, 0.15%, etc.

[0065] In some embodiments, a gradient elution method is used. During the gradient elution process, the sum of the volume fractions of mobile phase A and mobile phase B is 100%. The conditions of the entire gradient elution process are as follows:

[0066] 0-5min, the volume fraction of mobile phase A was reduced from 88% to 70%;

[0067] 5-6min, the volume fraction of mobile phase A was reduced from 70% to 65%;

[0068] 6-10min, the volume fraction of mobile phase A was maintained at 65%;

[0069] 10-14 min, the volume fraction of mobile phase A was reduced from 65% to 0%;

[0070] 14-16 min, the volume fraction of mobile phase A was maintained at 100%;

[0071] At 16-18 min, the volume fraction of mobile phase A increased from 0% to 88%;

[0072] During 18-20 min, the volume fraction of mobile phase A was maintained at 88%.

[0073] By precisely controlling the conditions of gradient elution, the elution effect can be further improved, the components can be better separated, and the peak elution effect can be improved.

[0074] (2) LC-MS

[0075] The process of quantitative analysis by liquid chromatography-mass spectrometry includes: using high performance liquid chromatography to separate ellagic acid and urolithin in the standard sample solution by chromatographic elution, detecting by ultra-high resolution mass spectrometry, calculating the quantitative detection limit and identification detection limit according to the signal-to-noise ratio, drawing a standard curve according to the known standard concentration and peak area, and obtaining a quantitative correction equation; using the same method to inject and test the test solution, substituting the peak area of ​​ellagic acid and urolithin detected by mass spectrometry into the quantitative correction equation, and calculating the content of ellagic acid and urolithin in the test sample. The detection principle is consistent with the conventional liquid chromatography-mass spectrometry method, and no further detailed description is given here.

[0076] In an optional embodiment, the instrument conditions of the high performance liquid chromatography tandem ultra-high resolution mass spectrometry used in the LC-MS method are as follows: the instrument model is ThemoScientific Q Exactive, and the chromatographic column is C18 such as Waters Acquity BEH C18, the inner diameter of the column filling is 1.5-2.0μm (1.7μm), the column temperature is 25℃-45℃, the flow rate is 0.2mL / min-0.4mL / min, the injection volume is 2μL-5μL, the scanning mode is electrospray ionization (ESI) negative mode, the spray voltage is -3Kv~-2kV, the sheath gas flow rate is 25arb-35arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320℃-340℃. By optimizing the chromatographic column and operating parameters, the components can be better separated and the detection accuracy can be improved.

[0077] Specifically, the chromatographic column can be Waters Acquity BEH C18, 1.7μm. The column temperature can be 25℃, 30℃, 35℃, 40℃, 45℃, etc., the flow rate can be 0.2mL / min, 0.3mL / min, 0.4mL / min, etc., the injection volume can be 2μL, 3μL, 4μL, 5μL, etc. The spray voltage can be -3.0Kv, -2.5Kv, -2.0Kv, etc., the sheath gas flow rate can be 25arb, 28arb, 30arb, 32arb, 35arb, etc., the auxiliary gas flow rate can be 8arb, 9arb, 10arb, 11arb, 12arb, 13arb, etc., and the capillary temperature can be 320℃, 325℃, 330℃, 335℃, 340℃, etc.

[0078] Furthermore, the ultra-high resolution mass spectrometry uses typical data-dependent mass spectrometry acquisition (DDA), with a primary full scan resolution of 68000-72000, a primary ion scan range of 50m / z-750m / z, HCD for secondary fragmentation, a secondary resolution of 17000-18000, and collision energies of 20 / 40 / 60eV in collision energy normalization mode. Xcalibur software is selected in combination with Compound Discover software for metabolite identification. By controlling the resolution of primary and secondary mass spectrometry, and selecting appropriate NCE mode and analysis software, the peak effect is improved, thereby improving the accuracy of detection.

[0079] Specifically, the primary full scan resolution can be 68000, 70000, 72000, etc. 2 The resolution can be 17000, 17500, 18000, etc., and the secondary resolution can be 17000, 17200, 17500, 17800, 18000, etc.

[0080] Furthermore, when using LC-MS for quantitative analysis, the mobile phases used include mobile phase A and mobile phase B, wherein mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05%-0.15%, and mobile phase B is acetonitrile. The mobile phase is optimized to improve the elution effect and achieve a better peak effect.

[0081] Specifically, the mobile phase A is a formic acid aqueous solution, and the volume fraction of the formic acid can be 0.05%, 0.08%, 0.10%, 0.12%, 0.15%, etc.

[0082] In some embodiments, gradient elution is adopted. During the gradient elution process, the sum of the volume fractions of mobile phase A and mobile phase B is 100%. The entire gradient elution process is as in step S2 and will not be repeated here.

[0083] It should be noted that the present invention adopts high performance chromatography coupled to mass spectrometry to determine ellagic acid and its metabolites. Liquid chromatography-mass spectrometry technology can accurately identify and quantify trace compounds in complex sample matrices such as cell and tissue lysates, blood, plasma, urine and oral fluid, with high precision, high resolution and high sensitivity, but it has high requirements on samples and high detection quality.

[0084] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0085] Example 1

[0086] This embodiment provides a method for detecting ellagic acid and its metabolites, comprising the following steps:

[0087] Instruments used: high performance liquid chromatography-mass spectrometry (Thermo Scientific, Q Exactive); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., KQ-700VDE); electronic balance (METTLER TOLEDO, XSE-205DU); water purifier (Merck, MILLI-Q IQ 7015).

[0088] Reagents used: ellagic acid standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 96%), urolithin A standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 98%), urolithin B standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 98%), urolithin C standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 95%), urolithin M5 standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 95%), urolithin M6 standard (Sichuan Weiqiqi Biotechnology Co., Ltd., purity ≥ 98%), isourea Litholithin A standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin D standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin E standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin M7 standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), N-methylpyrrolidone (Shanghai MacLean Biotechnology Co., Ltd.), methanol (Thermo Fisher, mass spectrometry grade), acetonitrile (Thermo Fisher mass spectrometry grade), formic acid (Sinopharm Chemical Reagent Co., Ltd.).

[0089] Test sample: Mixed solution of ellagic acid and urolithin of unknown concentration.

[0090] High performance liquid chromatography tandem ultra-high resolution mass spectrometry qualitative and quantitative detection instrument conditions: instrument model: ThemoScientific Q Exactive; column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 3 μL; mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column and elution method: Waters Acquity BEH C18, 1.7μm, gradient elution; UV detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of mobile phase A is reduced from 88% to 70%; 5-6min, the volume fraction of mobile phase A is reduced from 70% to 65%; 6-10min, the volume fraction of mobile phase A is maintained at 65%; 10-14min, the volume fraction of mobile phase A is reduced from 65% to 0%; 14-16min, the volume fraction of mobile phase A is maintained at 100%; 16-18min, the volume fraction of mobile phase A is increased from 0% to 88%; 18-20min, the volume fraction of mobile phase A is maintained at 88%. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%. Mass spectrometry conditions: the scanning mode was electrospray ionization (ESI) negative mode, the spray voltage was -2.5 kV, the sheath gas flow rate was 30 arb, the auxiliary gas flow rate was 10 arb, the capillary temperature was 325 ° C, the first-level full scan resolution was 70000, the first-level ion scanning range was 50m / z-750m / z, HCD was used for secondary fragmentation, the secondary resolution was 17500, and the collision energies in the collision energy normalization mode were: 20 / 40 / 60, and Xcalibur software combined with Compound Discover software was selected for metabolite identification.

[0091] Drawing of the standard curve of ellagic acid and urolithin:

[0092] Accurately weigh the ellagic acid and urolithin standards (a total of ten standards, weighed in equal amounts), prepare a 0.1 mg / mL mother solution with 5% (v / v) N-methylpyrrolidone-methanol solution, and further dilute it with 5% (v / v) N-methylpyrrolidone-methanol solution to form a series of concentration gradients. The final concentration of the standard used for high performance liquid chromatography-mass spectrometry qualitative and quantitative detection is 10, 20, 30, 40, 50, 60, 70 μg / mL (based on the total concentration of the standard). Perform the test according to the conditions in this example and draw a standard curve. The standard curves of ellagic acid and urolithin are shown in Table 1. Figure 2 is the mass spectrum of the standard.

[0093] Table 1 Standard curves of ellagic acid and urolithin (LC-MS method)

[0094]

[0095] Take a sample rich in ellagic acid and urolithin, use 5% (v / v) N-methylpyrrolidone-methanol solution as a mixed extractant, mix the sample to be tested and the mixed extractant, vortex, and ultrasonic extraction. After the ultrasound is completed, use 5% (v / v) N-methylpyrrolidone-methanol solution to dilute the sample to be tested 10 times and then centrifuge, take the sample supernatant and pass it through a 0.22μm PTFE membrane for standby use to obtain a test solution. The volume ratio of the sample to be tested to the mixed extractant is 1:3, the vortex time is 60s, the ultrasonic power is 500W, the ultrasonic time is 30min, the centrifugal temperature is 4°C, the centrifugal time is 10min, and the centrifugal force is 11000g.

[0096] The test liquid is tested on a machine, and the standard sample liquid is eluted and separated by high performance liquid chromatography. The test liquid is tested in the same way as the standard sample, and the peak areas of ellagic acid and urolithin detected by the ultraviolet detector are substituted into the quantitative correction equation to calculate the contents of ellagic acid and urolithin in the test sample.

[0097] (1) Precision test

[0098] The sample to be tested was used to prepare a test solution, which was continuously injected 5 times under chromatographic conditions, and the chromatographic analysis conditions were the same as in Example 1, and the chromatogram was recorded to examine the precision of the system. The results are shown in Table 2. The sample to be tested was used to prepare a test solution, which was continuously injected 5 times under chromatographic conditions, and the chromatographic analysis conditions were the same as in Example 2, and the chromatogram was recorded to examine the precision of the system. The results are shown in Table 2, and it can be seen that the test solution was injected 5 times in a row, and the RSD values ​​of the peak areas of ellagic acid and urolithin were less than 2%, indicating that the method system of the present invention has good precision.

[0099] Table 2 Precision results

[0100]

[0101] (2) Repeatability test

[0102] Five test solutions were prepared in parallel, and the chromatographic analysis conditions were the same as in Example 1, and the chromatograms were recorded to examine the repeatability. The results are shown in Table 3. From the results, it can be seen that there is no significant difference in the content results of ellagic acid, urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isoureolithin A, and urolithin B, indicating that the method of the present invention has good repeatability.

[0103] Table 3 Repeatability results (nM)

[0104]

[0105] Example 2

[0106] This embodiment provides a method for detecting ellagic acid and its metabolites, comprising the following steps:

[0107] Instruments used: high performance liquid chromatograph (Thermo Scientific, U3000); ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., KQ-700VDE); electronic balance (METTLER TOLEDO, XSE-205DU); water purifier (Merck, MILLI-Q IQ 7015).

[0108] Reagents used: ellagic acid standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 96%), urolithin A standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 98%), urolithin B standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 98%), urolithin C standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 95%), urolithin M5 standard (Shanghai Yuanye Biotechnology Co., Ltd., purity ≥ 95%), urolithin M6 standard (Sichuan Weiqiqi Biotechnology Co., Ltd., purity ≥ 98%), isourea Litholithin A standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin D standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin E standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), urolithin M7 standard (Sichuan Weikeqi Biotechnology Co., Ltd., purity ≥ 98%), N-methylpyrrolidone (Shanghai MacLean Biotechnology Co., Ltd.), methanol (Thermo Fisher, chromatographic grade), acetonitrile (Thermo Fisher chromatographic grade), formic acid (Sinopharm Chemical Reagent Co., Ltd.).

[0109] Test sample: Mixed solution of ellagic acid and urolithin of unknown concentration.

[0110] HPLC quantitative detection instrument conditions: Instrument model: ThemoScientific U3000; Column temperature: 30°C; Flow rate: 1.0 mL / min; Injection volume: 5 μL; Mobile phase A: 0.1% formic acid water; Mobile phase B: acetonitrile; Chromatographic column and elution method: ThemoScientific Acclaim TM 120C18, 5μm, gradient elution; UV detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of mobile phase A is reduced from 88% to 70%; 5-6min, the volume fraction of mobile phase A is reduced from 70% to 65%; 6-10min, the volume fraction of mobile phase A is maintained at 65%; 10-14min, the volume fraction of mobile phase A is reduced from 65% to 0%; 14-16min, the volume fraction of mobile phase A is maintained at 100%; 16-18min, the volume fraction of mobile phase A is increased from 0% to 88%; 18-20min, the volume fraction of mobile phase A is maintained at 88%. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.

[0111] Drawing of the standard curve of ellagic acid and urolithin:

[0112] Accurately weigh the ellagic acid and urolithin standards (a total of ten standards, weighed in equal amounts), prepare a 0.1 mg / mL mother solution with 5% (v / v) N-methylpyrrolidone-methanol solution, and further dilute it with N-methylpyrrolidone-methanol solution to form a series of concentration gradients. The final concentrations of the standards used for high-performance liquid chromatography quantitative detection are 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL (based on the total concentration of the standards). Perform the test according to the conditions in this example and draw a standard curve. The standard curves of ellagic acid and urolithin are shown in Table 4. Figure 2 It is the HPLC chromatogram of the standard product.

[0113] Table 4 Standard curve of ellagic acid and urolithin (HPLC)

[0114]

[0115] Take a sample rich in ellagic acid and urolithin, use 5% (v / v) N-methylpyrrolidone-methanol solution as a mixed extractant, mix the sample to be tested and the mixed extractant, vortex, and ultrasonic extraction. After the ultrasound is completed, the sample to be tested is diluted 10 times with N-methylpyrrolidone-methanol solution and then centrifuged, and the sample supernatant is passed through a 0.22μm PTFE membrane for standby use to obtain a test liquid. The volume ratio of the sample to be tested to the mixed extractant is 1:3, the vortex time is 60s, the ultrasonic power is 500W, the ultrasonic time is 30min, the centrifugal temperature is 4°C, the centrifugal time is 10min, and the centrifugal force is 11000g.

[0116] The test liquid is tested on a machine, and the standard sample liquid is eluted and separated by high performance liquid chromatography. The test liquid is tested in the same way as the standard sample, and the peak areas of ellagic acid and urolithin detected by the ultraviolet detector are substituted into the quantitative correction equation to calculate the contents of ellagic acid and urolithin in the test sample.

[0117] (1) Precision test

[0118] The sample to be tested was prepared into a test solution and injected 5 times continuously under the chromatographic conditions. The chromatographic analysis conditions were the same as those in Example 2. The chromatogram was recorded to examine the system precision. The results are shown in Table 5. It can be seen that the RSD values ​​of the peak areas of ellagic acid and urolithin were less than 2% after 5 consecutive injections of the test solution, indicating that the method of the present invention has good system precision.

[0119] Table 5 Precision results

[0120] 1 2 3 4 5 Average peak area RSD% Ellagic acid 68.37 68.36 68.36 68.23 68.28 68.32 0.08 Urolithin M5 11.87 11.88 11.87 11.85 11.84 11.86 0.12 Urolithin M6 156.51 156.34 156.32 156.05 155.81 156.21 0.14 Urolithin D 12.23 12.27 12.22 12.29 12.23 12.25 0.19 Urolithin E 68.87 68.88 68.86 68.78 68.73 68.82 0.08 Urolithin M7 29.30 29.26 29.24 29.23 29.17 29.24 0.13 Urolithin C 28.22 28.22 28.31 28.26 28.27 28.26 0.11 Urolithin A 225.57 225.43 225.57 225.14 225.28 225.40 0.07 Isourolithin A 23.58 23.46 23.67 23.42 23.63 23.55 0.37 Urolithin B 29.10 29.06 29.05 29.01 28.96 29.04 0.14

[0121] (2) Repeatability test

[0122] Five test solutions were prepared in parallel, and the chromatographic analysis conditions were the same as in Example 2, and the chromatograms were recorded to examine the repeatability. The results are shown in Table 6. From the results, it can be seen that there is no significant difference in the content results of ellagic acid, urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isoureolithin A, and urolithin B, indicating that the method of the present invention has good repeatability.

[0123] Table 6 Repeatability results (mg / mL)

[0124]

[0125]

[0126] Example 3

[0127] The specific steps for detecting the content of ellagic acid and urolithin in rat feces are as follows:

[0128] (1) Source of samples to be tested

[0129] Urolithin is a metabolite of ellagic acid after a series of reactions under the action of intestinal microorganisms. Usually, after taking ellagic acid or substances rich in ellagic acid (such as pomegranate juice, etc.), some organisms will produce urolithin. The sample to be tested in this example is the feces collected within 90 minutes after normal rats were gavaged with ellagic acid (gavage dose 100 mg / mL) on an empty stomach.

[0130] (2) Pretreatment of samples to be tested

[0131] Take an appropriate amount of feces, crush it with a glass rod, add N-methylpyrrolidone-methanol solution (N-methylpyrrolidone volume fraction is 5%) at a ratio of 1:5 (w / v), vortex for 60s, and extract at 400W ultrasound for 30min. After the ultrasound, add 4 times the volume of methanol solution to the system for dilution, vortex mix, centrifuge at 12000rpm, 4℃ for 10min, and the centrifugal force is 11000g. Take the sample supernatant and pass it through a 0.22μm PTFE membrane for standby analysis.

[0132] (3) Preparation of standard products

[0133] Accurately weigh 1.0 mg of ellagic acid / urolithin standard and prepare a 0.1 mg / mL stock solution with 5% (v / v) N-methylpyrrolidone-methanol solution, and further dilute into a series of concentration gradients with final concentrations of 10, 20, 30, 40, 50, 60, and 70 μg / mL, respectively.

[0134] (4) Detection by high performance liquid chromatography-mass spectrometry

[0135] Instrument model: ThemoScientific Q Exactive; column temperature: 30°C; flow rate: 0.3 mL / min; injection volume: 2 μL; mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column: Waters Acquity BEH C18, 1.7μm, gradient elution; Scan Mode: ESI Negative Mode; Scanning range: 50-750m / z. Gradient elution conditions: 0-5min, the volume fraction of mobile phase A is reduced from 88% to 70%; 5-6min, the volume fraction of mobile phase A is reduced from 70% to 65%; 6-10min, the volume fraction of mobile phase A is maintained at 65%; 10-14min, the volume fraction of mobile phase A is reduced from 65% to 0%; 14-16min, the volume fraction of mobile phase A is maintained at 100%; 16-18min, the volume fraction of mobile phase A is increased from 0% to 88%; 18-20min, the volume fraction of mobile phase A is maintained at 88%. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%. The mass spectrometer was detected by DDA and Full MS methods, with a Full MS resolution of 70,000 and a dd-MS2 resolution of 17,500 in NCE mode at 20, 40, and 60 eV. Xcalibur software was combined with Compound Discover software for metabolite identification.

[0136] Note: For parameters not mentioned in this example, please refer to Example 1.

[0137] (5) Identification results

[0138] The production of ellagic acid EA, urolithin M5, urolithin C, urolithin E and urolithin A was detected in the mouse feces collected at different time periods. The changes in the content of metabolites measured by the detection method of this example are as follows Figure 2 As shown in Figure 2, the changes in metabolite content measured by traditional detection methods are as follows Figure 3 As shown in the results, it can be seen that the content measured by this example method is higher than that of the traditional method, and it can detect urolithin E that cannot be detected by the traditional method. It can detect more types and the measured value is more accurate.

[0139] Example 4

[0140] The specific steps for the detection of urolithin produced by in vitro fermentation of ellagic acid are as follows:

[0141] (1) Source of samples to be tested

[0142] Intestinal flora samples were collected from 10 healthy volunteers of different ages (5 males and 5 females). The volunteers had a regular diet before the collection of intestinal flora samples and had not taken antibiotics within 3 months. The collected fresh fecal samples were placed in centrifuge tubes, sealed and stored at -80°C. Before use, the fecal samples were thawed in a 37°C water bath, and a fecal suspension was prepared according to the ratio of fecal sample: sterile phosphate buffer solution = 1:9 (W:V). After vortexing and mixing, two layers of gauze were filtered to obtain intestinal flora samples for in vitro fermentation of ellagic acid. When used, 10% of the inoculum was added to the brain heart infusion broth (BHI) containing ellagic acid, and the final concentration of ellagic acid was 1% (w / v). It was placed in a constant temperature anaerobic workstation at 37°C for shaking culture (120r / min), and samples were taken every 24 hours to detect the concentration changes of ellagic acid and urolithin substances in the fermentation system.

[0143] (2) Pretreatment of samples to be tested

[0144] Take an appropriate amount of fermentation solution, add N-methylpyrrolidone-methanol solution (N-methylpyrrolidone volume fraction is 5%) at a ratio of 1:3 (w / v), vortex for 60s, and extract at 400W ultrasonic for 30min. After the end of ultrasonication, add 5 times the volume of N-methylpyrrolidone-methanol solution (N-methylpyrrolidone volume fraction is 5%) to the system for dilution, vortex mixing, centrifuge at 12000rpm, 4℃ for 10min, centrifugal force is 11000g, take the sample supernatant and pass it through 0.22μm PTFE membrane for standby use for sample analysis.

[0145] (3) Preparation of standard products

[0146] Accurately weigh 1.0 mg of ellagic acid / urolithin standard and prepare a 0.1 mg / mL stock solution with 5% (v / v) N-methylpyrrolidone-methanol solution, and further dilute into a series of concentration gradients with final concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL, respectively.

[0147] (4) Qualitative and quantitative detection by high performance liquid chromatography

[0148] Instrument model: ThemoScientific U3000; column temperature: 25-45℃; flow rate: 0.5-2mL / min; injection volume: 2-10μL; mobile phase A: 0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column and elution method: ThemoScientificAcclaimTM 120C18, 5μm, gradient elution; UV detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of mobile phase A is reduced from 88% to 70%; 5-6min, the volume fraction of mobile phase A is reduced from 70% to 65%; 6-10min, the volume fraction of mobile phase A is maintained at 65%; 10-14min, the volume fraction of mobile phase A is reduced from 65% to 0%; 14-16min, the volume fraction of mobile phase A is maintained at 100%; 16-18min, the volume fraction of mobile phase A is increased from 0% to 88%; 18-20min, the volume fraction of mobile phase A is maintained at 88%. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.

[0149] (5) Identification results

[0150] In the in vitro fermentation model, the fermentation liquid was collected every 24 hours to detect the contents of ellagic acid and urolithin substances, and the fermentation period was 15 days in total.

[0151] from Figure 4 The results showed that the content of ellagic acid gradually decreased with the fermentation time, and the types and contents of urolithins increased with the fermentation time. Among them, the contents of urolithin M6, urolithin C and isourolithin A increased significantly within 10 days of fermentation; urolithin M7 and urolithin A began to be generated after the 4th day of fermentation.

[0152] Example 5

[0153] Optimization of extraction conditions and detection conditions of the present invention:

[0154] (1) Extraction conditions

[0155] In this experiment, methanol, ethanol, ethyl acetate, and N-methylpyrrolidone were selected as sample extractants for the extraction of ellagic acid, a phenolic compound with low water solubility and fat solubility. The results show that the dissolution behavior of ellagic acid in different solvents is significantly different. The saturated solubility of ellagic acid in the above solvents was further determined. The results are shown in Table 7. It can be seen that the solubility of ellagic acid in N-methylpyrrolidone is significantly higher than that in other solvents. A certain amount of ellagic acid was dissolved in methanol and N-methylpyrrolidone, respectively. The dissolution conditions are shown in Table 7. Figure 5 As shown, it can be seen that methanol cannot completely dissolve ellagic acid, and there is a large amount of precipitation at the bottom of the sample.

[0156] Table 7 Saturated solubility of ellagic acid in different solvents

[0157] Methanol Ethanol Ethyl acetate N-Methylpyrrolidone Ellagic acid solubility (mg / mL) 2.20±0.20 2.61±0.08 0.14±0.00 11.59±0.27

[0158] Further, the difference in the determination of ellagic acid content of the same sample by two different extraction methods, methanol extraction and N-methylpyrrolidone extraction, was investigated. Excess ellagic acid was dissolved in deionized water, and the supernatant was taken after centrifugation at 12000rpm for 10 minutes. The ellagic acid in the supernatant was extracted with methanol and N-methylpyrrolidone respectively, and the content was determined by high performance liquid chromatography under the same chromatographic conditions. The standard sample of ellagic acid was dissolved with methanol and N-methylpyrrolidone respectively to obtain the standard curve of different extraction methods. The calculated result was that the saturated solubility of ellagic acid in water detected by methanol extraction was 0.041±0.002mg / mL, and the saturated solubility of ellagic acid in water detected by N-methylpyrrolidone extraction was 0.063±0.001mg / mL. The results show that the use of methanol extraction alone will lead to a low measurement value of ellagic acid, and the determination work cannot be completed efficiently and accurately. N-methylpyrrolidone extraction can more accurately reflect the content of ellagic acid in the sample, and the data is stable. However, due to the strong polarity of N-methylpyrrolidone, excessive concentration of the reagent will cause certain damage to the chromatographic column, chromatograph and mass spectrometer, so we choose to combine the two and use N-methylpyrrolidone-methanol extraction, which can not only accurately reflect the sample content, but also has a good peak shape. It also causes less damage to the chromatographic column and instrument. The chromatogram detected by this method is as follows Figure 6 shown.

[0159] (2) Chromatographic conditions

[0160] In this experiment, methanol-water, methanol-0.1% formic acid water and acetonitrile-0.1% formic acid water were selected as the mobile phase for gradient elution. The results showed that adding a small amount of acid to the mobile phase can effectively improve the peak shape and ionization effect. Acetonitrile can better improve the peak shape than methanol. Therefore, acetonitrile-0.1% formic acid water was finally selected as the mobile phase.

[0161] In this experiment, the gradient elution conditions were optimized. The chromatograms before and after optimization are shown in Figure 2. Figure 7 , Figure 8 and Fig. 9 shown. Figure 7 In other chromatographic methods, urolithin A and isourolitin A could not be separated (the peak with a retention time of 9.192 min was the peak of urolithin A and isourolitin A); Figure 8 Other chromatographic methods: The peak shape of urolithin M5 was strange, urolithin D and ellagic acid could not be separated, and the separation effect of urolithin A and isourolitin was poor; Fig. 9 According to the method provided in the embodiment of the present invention, ellagic acid and urolithin are clearly separated with good peak shape. It can be seen that the peak shape of the optimized elution condition is good, and urolithin isomers with similar retention behaviors can be separated, reducing the interference of other compounds on the target compound.

[0162] (3) Mass spectrometry conditions

[0163] In order to complete the identification and identification of ellagic acid / urolithin substances in a more systematic and comprehensive manner, this experiment used positive and negative ion scanning modes for detection, and optimized the collision energy and mass spectrometry resolution. The results showed that the peak shape of ellagic acid / urolithin substances in the negative ion scanning mode was better and the detection limit was lower, so the negative ion mode was selected for detection. When the collision energy was 20eV, 40eV, and 60eV, the primary ion information of the chemical composition could be retained, and more abundant secondary fragment ion information could be obtained. Resolution refers to the degree of separation of two adjacent ion peaks on the mass spectrum. The higher the resolution, the higher the mass accuracy, but the corresponding sensitivity will decrease. After optimization, this experiment found that when the primary resolution was 68000-72000 and the secondary resolution was 17000-18000, the mass accuracy was high and the sensitivity was high.

[0164] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting ellagic acid and its metabolites, characterized in that: include: The sample to be tested is extracted using a mixed extractant formed by methanol and N-methylpyrrolidone to prepare a test solution; wherein the volume fraction of N-methylpyrrolidone in the mixed extractant is 5%-10%, and the volume ratio of the sample to be tested to the mixed extractant is 1:(2-6); A standard sample solution is prepared by using ellagic acid standard, multiple urolithin standards and the mixed extractant; the multiple urolithin standards are urolithin A, urolithin B, urolithin C, urolithin M5, urolithin M6, isoureolithin A, urolithin D, urolithin E and urolithin M7; Quantitative analysis using high performance liquid chromatography or liquid chromatography-mass spectrometry; When using high performance liquid chromatography for quantitative analysis, the chromatographic column is ThemoScientific AcclaimTM 120C18, and the UV detection wavelength is 250nm-260nm; When using liquid chromatography-mass spectrometry for quantitative analysis, the chromatographic column is C18, the scanning mode is electrospray ionization negative mode, the spray voltage is -3Kv to -2kV, the sheath gas flow rate is 25arb-35arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320°C-340°C; When using high performance liquid chromatography or liquid chromatography-mass spectrometry for quantitative analysis, the mobile phases used include mobile phase A and mobile phase B, wherein the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05%-0.15%, and the mobile phase B is acetonitrile; a gradient elution method is used, and the conditions of the gradient elution are as follows: 0-5min, the volume fraction of mobile phase A was reduced from 88% to 70%; 5-6min, the volume fraction of mobile phase A was reduced from 70% to 65%; 6-10min, the volume fraction of mobile phase A was maintained at 65%; 10-14 min, the volume fraction of mobile phase A was reduced from 65% to 0%; 14-16 min, the volume fraction of mobile phase A was maintained at 0%; At 16-18 min, the volume fraction of mobile phase A increased from 0% to 88%; 18-20min, the volume fraction of mobile phase A was maintained at 88%; In the gradient elution process, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.

2. The detection method according to claim 1, characterized in that: The process of quantitative analysis using the high performance liquid chromatography method includes: eluting and separating the standard sample solution using high performance liquid chromatography, drawing a standard curve according to the known standard concentration and peak area, and obtaining a quantitative correction equation; testing the test solution using the same method, substituting the peak areas of ellagic acid and urolithin detected by the ultraviolet detector into the quantitative correction equation, and calculating the contents of ellagic acid and urolithin in the test sample.

3. The detection method according to claim 2, characterized in that: The instrument conditions of the HPLC are as follows: the instrument model is ThemoScientific U3000, the inner diameter of the filling material of the chromatographic column is 3 μm-7 μm, the column temperature is 25°C-45°C, the flow rate is 0.5 mL / min-2.0 mL / min, and the injection volume is 2 μL-10 μL.

4. The detection method according to claim 1, characterized in that: The process of using the LC-MS method for quantitative analysis includes: using high performance liquid chromatography to perform chromatographic elution separation on ellagic acid and urolithin in a standard sample solution, detecting by ultra-high resolution mass spectrometry, calculating the quantitative detection limit and the identification detection limit according to the signal-to-noise ratio, and drawing a standard curve according to the known standard concentration and peak area to obtain a quantitative correction equation; The test solution was sampled and tested in the same manner, and the peak areas of ellagic acid and urolithin detected by mass spectrometry were substituted into the quantitative calibration equation to calculate the contents of ellagic acid and urolithin in the test sample.

5. The detection method according to claim 4, characterized in that: The instrument conditions of the high performance liquid chromatography tandem ultra-high resolution mass spectrometry used in the LC-MS method are as follows: the instrument model is ThemoScientific Q Exactive, the inner diameter of the chromatographic column filling material is 1.5μm-2.0μm, the column temperature is 25℃-45℃, the flow rate is 0.2mL / min-0.4mL / min, and the injection volume is 2μL-5μL.

6. The detection method according to claim 5, characterized in that: The ultra-high resolution mass spectrometry adopted typical data-dependent mass spectrometry acquisition, with a primary full scan resolution of 68000-72000, a primary ion scan range of 50m / z-750m / z, HCD for secondary fragmentation, a secondary resolution of 17000-18000, and collision energies of 20 / 40 / 60eV in collision energy normalization mode. Xcalibur software was selected in combination with Compound Discover software for metabolite identification.

7. The detection method according to claim 1, characterized in that: The preparation process of the test liquid includes: mixing the test sample and the mixed extractant, performing vortex and ultrasonic extraction, centrifuging after dilution, and filtering the supernatant.

8. The detection method according to claim 7, characterized in that: The vortex time is 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is 20min-40min.

9. The detection method according to claim 7, characterized in that: The centrifugation temperature is 0℃-4℃, the centrifugation time is 5min-10min, and the centrifugal force is 10000g-12000g.

10. The detection method according to claim 1, characterized in that: The preparation process of the standard sample solution includes: mixing the ellagic acid standard, a plurality of urolithin standards and the mixed extractant, diluting the mixed extractant to different concentrations, and filtering.

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