CN118169296A - Ellagic acid and metabolite detection method thereof - Google Patents
Ellagic acid and metabolite detection method thereof Download PDFInfo
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- CN118169296A CN118169296A CN202410521338.5A CN202410521338A CN118169296A CN 118169296 A CN118169296 A CN 118169296A CN 202410521338 A CN202410521338 A CN 202410521338A CN 118169296 A CN118169296 A CN 118169296A
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- ellagic acid
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Abstract
The invention discloses a detection method of ellagic acid and a metabolite thereof, and relates to the technical field of detection and analysis. The method optimizes the pretreatment method and the detection technology, adopts the compound extractant N-methyl pyrrolidone-methanol solution to carry out ultrasonic extraction on ellagic acid and urolithin-rich substances, carries out qualitative and quantitative determination on the extracted sample by utilizing a high performance liquid chromatography tandem ultra-high resolution mass spectrometer, and develops a simple high performance liquid chromatography determination method according to experimental results. The method overcomes the defects of difficult extraction of ellagic acid and urolithin substances in samples and inaccurate quantitative results in the traditional detection method due to poor solubility of the ellagic acid and the urolithin substances, has the advantages of simple processing steps, good separation effect, short detection time, high sensitivity, accurate nature, simple operation and good specificity, can analyze the change trend of the metabolic pathway of the ellagic acid, and provides a new method for researching the biological metabolic process of the ellagic acid.
Description
Technical Field
The invention relates to the technical field of detection and analysis, in particular to a detection method of ellagic acid and a metabolite thereof.
Background
Ellagic acid is a natural polyphenol dilactone widely existing in fruits or nuts such as pomegranate, blueberry, hickory, chestnut and the like, has proved to have potential preventive or therapeutic effects on cancers, diabetes, hypertension, atherosclerosis, neuropathy and the like, and has good biological safety. However, ellagic acid has poor water solubility and permeability, resulting in poor absorption in the gastrointestinal tract of mammals, low bioavailability, and often insufficient concentration to reach the target tissue through the intestinal wall via blood circulation, and part of the unabsorbed ellagic acid is converted into more readily absorbable uroliths by the intestinal flora. Urolithin is produced by the loss of a lactone ring from ellagic acid and the gradual dehydroxylation. Because of different dehydroxylation sites and sequences, the variety of urolithin is more, the metabolic process of ellagic acid also has different rules in different individuals, and the biological activity of different urolithin also has corresponding differences due to different hydroxyl numbers and sites. Therefore, qualitative and quantitative detection of ellagic acid and urolithin is a research basis for judging the type of ellagic acid metabolism and analyzing the bioactive mechanism of ellagic acid.
The high performance liquid chromatography is a modern chromatographic separation means, has the advantages of high analysis speed, strong separation effect, high selectivity, strong column effect, wide application range and the like, is often combined with various detectors, determines substances contained in a sample by comparing different peak signals detected by the detectors, and can also realize the purpose of separating a mixture. At present, ellagic acid is quantitatively detected by high performance liquid chromatography after being dissolved by organic reagents such as methanol, but the ellagic acid is poor in water solubility and low in methanol and ethanol, so that the ellagic acid has poor peak shape, high detection limit and strong interference caused by similar components in the detection process. The metabolite urolithin of ellagic acid has more isomers, has similar retention behavior, and cannot be accurately and qualitatively detected by high-performance chromatograph alone.
Therefore, there is a need to optimize the extraction, separation and identification methods of samples rich in ellagic acid and urolithin substances so as to overcome the defects of poor peak shape, difficult separation of isomers, difficult accurate determination of the content of the to-be-detected substances in the samples, high detection cost and the like existing in the existing detection methods.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a detection method of ellagic acid and a metabolite thereof, which aims to quantitatively detect ellagic acid and urolithin substances in a sample at the same time and improve the detection stability.
The invention is realized in the following way:
in a first aspect, the present invention provides a method for detecting ellagic acid and metabolites thereof, comprising:
Extracting a sample to be detected by using a mixed extractant formed by methanol and N-methyl pyrrolidone to prepare a liquid to be detected; in the mixed extractant, the volume fraction of N-methyl pyrrolidone is 5% -10%, and the volume ratio of the sample to be detected to the mixed extractant is 1: (2-6);
preparing a standard sample liquid by using an ellagic acid standard substance, a plurality of urolithin standard substances and a mixed extractant;
Quantitative analysis is carried out by high performance liquid chromatography or liquid chromatography-mass spectrometry.
In an alternative embodiment, the quantitative analysis using high performance liquid chromatography comprises: eluting and separating the standard sample liquid by utilizing high performance liquid chromatography, drawing a standard curve graph according to the known standard substance concentration and peak area, and obtaining a quantitative correction equation; and testing the liquid to be tested by adopting the same method, substituting the peak areas of ellagic acid and urolithin detected by the ultraviolet detector into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolithin in the sample to be tested.
In an alternative embodiment, the instrument conditions for high performance liquid chromatography are as follows: the instrument model is ThemoScientific U & lt 3000 & gt, the chromatographic column is ThemoScientific Acclaim TM & lt 18 & gt, the inner diameter of a filling of the chromatographic column is 3-7 mu m, the column temperature is 25-45 ℃, the flow rate is 0.5-2.0 mL/min, the sample injection amount is 2-10 mu L, and the ultraviolet detection wavelength is 250-260 nm;
In an alternative embodiment, the quantitative analysis using the liquid chromatography-mass spectrometry method comprises: carrying out chromatographic elution and separation on ellagic acid and urolith in a standard sample liquid by utilizing high performance liquid chromatography, detecting by ultra-high resolution mass spectrum, calculating according to a signal-to-noise ratio to obtain a quantitative detection limit and an identification detection limit, drawing a standard curve graph according to the known standard concentration and peak area, and obtaining a quantitative correction equation;
and (3) adopting the same method to sample and test the liquid to be tested, substituting the peak areas of ellagic acid and urolithin detected by the mass spectrum into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolithin in the sample to be tested.
In an alternative embodiment, the instrument conditions for high performance liquid chromatography tandem ultra-high resolution mass spectrometry employed by the lc method are as follows: the instrument model is ThemoScientific Q Exactive, the chromatographic column is C18, the inner diameter of the filling of the chromatographic column is 1.5-2.0 mu m, the column temperature is 25-45 ℃, the flow rate is 0.2-0.4 mL/min, the sample injection amount is 2-5 mu L, the scanning mode is an electrospray ionization negative mode, the spray voltage is-3 Kv to-2 kV, the sheath gas flow rate is 25-35 arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320-340 ℃.
In an alternative embodiment, ultra-high resolution mass spectrometry employs 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, and secondary fragmentation with HCD, with a secondary resolution of 17000-18000, collision energies in collision energy normalization mode of: 20/40/60eV, xcalibur software was selected in combination with Compound Discover software for metabolite identification.
In an alternative embodiment, when quantitative analysis is performed by high performance liquid chromatography or liquid chromatography-mass spectrometry, the mobile phase used includes mobile phase a and mobile phase B, wherein mobile phase a is an aqueous formic acid solution with a volume fraction of 0.05% -0.15%, and mobile phase B is acetonitrile.
In an alternative embodiment, gradient elution is performed under the following conditions:
0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;
5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;
6-10min, the volume fraction of the mobile phase A is kept 65%;
10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;
14-16min, and the volume fraction of the mobile phase A is kept 100%;
16-18min, the volume fraction of mobile phase A increases from 0% to 88%;
18-20min, the volume fraction of the mobile phase A is kept 88%;
wherein, in the gradient elution process, the sum of the volume fractions of the mobile phase A and the mobile phase B is 100 percent.
In an alternative embodiment, the preparation process of the liquid to be tested includes: mixing a sample to be detected with the mixed extractant, performing vortex and ultrasonic extraction, performing centrifugal treatment after dilution, and filtering the supernatant;
preferably, the vortex time is 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is 20min-40min;
preferably, the centrifugation temperature is 0-4 ℃, the centrifugation time is 5-10 min, and the centrifugal force is 10000-12000 g.
In an alternative embodiment, the standard sample fluid is prepared by a process comprising: mixing ellagic acid standard, multiple urolithin standard and mixed extractant, diluting to different concentrations with mixed extractant, and filtering.
The invention has the following beneficial effects: the mixed extractant formed by methanol and N-methyl pyrrolidone is adopted to extract a sample to be detected, and the concentration of the N-methyl pyrrolidone in the mixed extractant and the dosage 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 target substances in the sample is reduced, and the detection accuracy is remarkably improved; the chromatographic and mass spectrometry method for optimizing high performance liquid chromatography tandem ultra-high resolution mass spectrometry detection can obviously separate ellagic acid and urolithin from other impurities in a sample, and has good peak shape, short detection time, high sensitivity and accurate quality; the simple high performance liquid chromatography determination method is developed, the operation is simple, the detection cost is reduced, and the detection accuracy and stability are ensured.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a structural formula of a common urolithin;
FIG. 2 is a diagram of the identification result of the method according to the embodiment of the present invention;
FIG. 3 is a diagram showing the identification result of the conventional detection method;
FIG. 4 is a graph showing the content of ellagic acid and urolithin in an in vitro glycolysis system for 0-15 days, wherein (A) shows ellagic acid, and (B) - (H) are urolithin M5, urolithin M6, urolithin C, urolithin M7, isourolithin A, urolithin A, and urolithin B, respectively;
FIG. 5 is a graph comparing dissolution behavior of ellagic acid in methanol and N-methylpyrrolidone, respectively;
FIG. 6 is a chromatogram of ellagic acid after optimization of extraction conditions;
FIG. 7 is a graph of detection results of other chromatographic methods;
FIG. 8 is a graph showing the separation effect of other chromatographic methods;
Fig. 9 is a diagram showing the separation effect of the method according to the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The structure of ellagic acid is as follows:
the metabolite of ellagic acid is named urolithin, and its specific structure is shown in figure 1, and comprises urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isourolithin A, and urolithin B, and nine kinds of urolithin are shown in figure 1, wherein Uro represents urolithin.
The embodiment of the invention provides a detection method of ellagic acid and a metabolite thereof, which comprises the following steps:
S1, preparing a liquid to be tested
Extracting a sample to be detected by using a mixed extractant formed by methanol and N-methyl pyrrolidone to prepare a liquid to be detected; in the mixed extractant, the volume fraction of N-methyl pyrrolidone is 5% -10%, and the volume ratio of the sample to be detected to the mixed extractant is 1: (2-6). Through optimizing the composition of the extractant, ellagic acid and metabolites thereof can be better dissolved, the peak shape after detection is good, and the detection data is more stable.
Specifically, methanol and N-methyl pyrrolidone are mixed to obtain a mixed extractant, wherein the volume fraction of the N-methyl pyrrolidone in the mixed extractant can be 5%, 6%, 7%, 8%, 9%, 10% and the like, and the volume ratio of a sample to be detected to the mixed extractant can be 1:2, 1:3, 1:4, 1:5, 1:6 and the like.
In some embodiments, the preparation process of the liquid to be tested includes: mixing a sample to be detected with the mixed extractant, performing vortex and ultrasonic extraction, performing centrifugal treatment after dilution, and filtering supernatant to obtain a solution for loading analysis. The vortex time is controlled to be 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is controlled to be 20min-40min, so that ellagic acid and urolithin are fully extracted. After extraction is completed, dilution is performed, the diluted concentration is not limited, and the concentration can be accurately calculated through a standard curve. And (3) carrying out centrifugal treatment after dilution, controlling the centrifugal temperature to be 0-4 ℃, controlling the centrifugal time to be 5-10 min, and controlling the centrifugal force to be 10000-12000 g, and filtering the supernatant after centrifugation to ensure that the obtained liquid to be tested meets the requirement of a machine.
Specifically, the vortex time may be 30s, 40s, 50s, 60s, 70s, 80s, 90s, etc., the ultrasonic power may be 400W, 500W, 600W, etc., and the ultrasonic time may be 20min, 25min, 30min, 40min, etc. The centrifugation temperature may be 0 ℃,1 ℃,2 ℃,3 ℃,4 ℃ and the like, the centrifugation time may be 5min, 8min, 10min and the like, and the centrifugal force may be 10000g, 11000g, 12000g and the like. The filtration mode is not limited, and filtration can be performed by using a PTFE membrane of 0.22. Mu.m.
S2, preparing standard sample liquid
The standard sample liquid is prepared from ellagic acid standard substances, various urolithin standard substances and a mixed extractant, and can be mixed standard liquid or standard liquid with single component. The mixed standard solution is prepared by mixing ellagic acid standard substance, 9 urolithin standard substances and mixed extractant. The standard solution of single component is prepared by respectively mixing ellagic acid standard substance and 9 urolithin standard substances with mixed extractant to prepare standard sample solutions with different concentrations. For the sake of simple method, the embodiment of the invention adopts mixed standard solution, but in consideration of the requirement of further improving the detection accuracy, single-component standard solution can be prepared.
In the actual operation process, the preparation process of the standard sample liquid comprises the following steps: mixing ellagic acid standard substance, multiple urolithin standard substances and mixed extractant, performing ultrasonic treatment to fully dissolve the ellagic acid standard substance, the multiple urolithin standard substances and the mixed extractant, diluting the ellagic acid standard substance to different concentrations by using the mixed extractant, and filtering to obtain standard sample liquid meeting the requirements of the previous machine. The filtration mode is not limited, and filtration can be performed by using a PTFE membrane of 0.22. Mu.m.
Specifically, the composition of the mixed extractant is described with reference to step S1.
S3, quantitative analysis
Quantitative analysis is carried out by high performance liquid chromatography or liquid chromatography-mass spectrometry, and two methods are respectively described below:
(1) High performance liquid chromatography
The quantitative analysis process by high performance liquid chromatography comprises: eluting and separating the standard sample liquid by utilizing high performance liquid chromatography, drawing a standard curve graph according to the known standard substance concentration and peak area, and obtaining a quantitative correction equation; and testing the liquid to be tested by adopting the same method, substituting the peak areas of ellagic acid and urolithin detected by the ultraviolet detector into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolithin in the sample to be tested. The content of ellagic acid and various uroliths can be accurately detected by adopting a common standard curve method.
In some embodiments, the instrument conditions for high performance liquid chromatography are as follows: the instrument model is ThemoScientific U3000,3000, the chromatographic column is ThemoScientific Acclaim TM C18, the inner diameter of the filling of the chromatographic column is 3-7 μm, the column temperature is 25-45 ℃, the flow rate is 0.5-2.0 mL/min, the sample injection amount is 2-10 μL, and the ultraviolet detection wavelength is 250-260 nm. Through optimizing the chromatographic column and the operation parameters, each component can be better separated, and the peak-out effect is improved.
Specifically, the packing inside diameter of the column may be 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, etc., and the column ThemoScientific Acclaim TM 120C18,5 μm. The column temperature can be 25 ℃,30 ℃, 35 ℃, 40 ℃, 45 ℃, and the like, the flow rate can be 0.5mL/min, 1.0mL/min, 1.5mL/min, 2.0mL/min, and the like, the sample injection amount can be 2 mu L, 3 mu L, 5 mu L, 8 mu L, 10 mu L, and the like, and the ultraviolet detection wavelength can be 250nm, 252nm, 255nm, 258nm, 260nm, and the like.
Further, when quantitative analysis is performed by using high performance liquid chromatography, the mobile phase adopted comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is formic acid aqueous solution with the volume fraction of 0.05% -0.15%, and the mobile phase B is acetonitrile. The mobile phase is optimized to improve the eluting effect, so that the peak-out effect is better.
Specifically, the mobile phase a is an aqueous solution of formic acid, and the volume fraction of formic acid may be 0.05%, 0.08%, 0.10%, 0.12%, 0.15%, etc.
In some embodiments, gradient elution is used, in which the sum of the volume fractions of mobile phase a and mobile phase B is 100%, and the conditions for the whole gradient elution are as follows:
0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;
5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;
6-10min, the volume fraction of the mobile phase A is kept 65%;
10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;
14-16min, and the volume fraction of the mobile phase A is kept 100%;
16-18min, the volume fraction of mobile phase A increases from 0% to 88%;
The volume fraction of mobile phase A was kept 88% for 18-20 min.
The gradient elution conditions are precisely controlled to further improve the elution effect, so that each component is better separated, and the peak-yielding effect is improved.
(2) Liquid chromatography-mass spectrometry
The quantitative analysis process by adopting the liquid chromatography-mass spectrometry comprises the following steps: carrying out chromatographic elution and separation on ellagic acid and urolith in a standard sample liquid by utilizing high performance liquid chromatography, detecting by ultra-high resolution mass spectrum, calculating according to a signal-to-noise ratio to obtain a quantitative detection limit and an identification detection limit, drawing a standard curve graph according to the known standard concentration and peak area, and obtaining a quantitative correction equation; and (3) adopting the same method to sample and test the liquid to be tested, substituting the peak areas of ellagic acid and urolithin detected by the mass spectrum into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolithin in the sample to be tested. The detection principle is consistent with the conventional liquid chromatography-mass spectrometry method, and will not be described in more detail herein.
In an alternative embodiment, the instrument conditions for high performance liquid chromatography tandem ultra-high resolution mass spectrometry employed by the lc method are as follows: the instrument model is ThemoScientific Q Exactive, and the chromatographic column is C18 such as Waters acquisitionBEH C18, the inner diameter of the filling of the chromatographic column is 1.5-2.0 mu m (1.7 mu m), the column temperature is 25-45 ℃, the flow rate is 0.2-0.4 mL/min, the sample injection amount is 2-5 mu L, the scanning mode is an electrospray ionization (ESI) negative mode, the spraying voltage is-3 Kv to-2 kV, the sheath gas flow rate is 25-35 arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320-340 ℃. By optimizing the chromatographic column and the operation parameters, each component can be better separated, and the detection accuracy is improved.
In particular, the chromatographic column may be a Waters acquisitionBEH C18,1.7 μm. The column temperature may be 25deg.C, 30deg.C, 35deg.C, 40deg.C, 45deg.C, etc., the flow rate may be 0.2mL/min, 0.3mL/min, 0.4mL/min, etc., and the sample injection amount may be 2 μL, 3 μL, 4 μL, 5 μL, etc. The spray voltage may be-3.0 Kv, -2.5Kv, -2.0Kv, etc., the sheath gas flow rate may be 25arb, 28arb, 30arb, 32arb, 35arb, etc., the assist gas flow rate may be 8arb, 9arb, 10arb, 11arb, 12arb, 13arb, etc., and the capillary temperature may be 320 ℃, 325 ℃, 330 ℃, 335 ℃, 340 ℃, etc.
Further, the ultra-high resolution mass spectrum adopts typical data dependent mass spectrum acquisition (DDA), the primary full scanning resolution is 68000-72000, the primary ion scanning range is 50m/z-750m/z, the secondary fragmentation is carried out by adopting HCD, the secondary resolution is 17000-18000, and the collision energy is respectively as follows in a collision energy normalization mode: 20/40/60eV, xcalibur software was selected in combination with Compound Discover software for metabolite identification. The resolution ratio of the primary mass spectrum and the secondary mass spectrum is controlled, and proper NCE mode and analysis software are selected, so that the peak-out effect is improved, and the detection accuracy is further improved.
Specifically, the first-order full scan resolution may be 68000, 70000, 72000, etc., the dd-MS 2 resolution may be 17000, 17500, 18000, etc., and the second-order resolution may be 17000, 17200, 17500, 17800, 18000, etc.
Further, when quantitative analysis is performed by using a liquid chromatography-mass spectrometry method, the adopted mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution with the volume fraction of 0.05% -0.15%, and the mobile phase B is acetonitrile. The mobile phase is optimized to improve the eluting effect, so that the peak-out effect is better.
Specifically, the mobile phase a is an aqueous solution of formic acid, and the volume fraction of formic acid may be 0.05%, 0.08%, 0.10%, 0.12%, 0.15%, etc.
In some embodiments, a gradient elution is used, in which the sum of the volume fractions of mobile phase a and mobile phase B is 100%, and the whole gradient elution is performed as in step S2, which is not repeated here.
The invention adopts the high-efficiency chromatograph-mass spectrometer to measure ellagic acid and the metabolites thereof, and the liquid chromatography-mass spectrometer technology can accurately identify and quantify trace compounds in complex sample matrixes such as cell and tissue lysate, blood, plasma, urine, oral liquid and the like, has high accuracy, high resolution and high sensitivity, but has high requirements on samples and high detection finished products.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The embodiment provides a detection method of ellagic acid and metabolites thereof, comprising the following steps:
The instrument used is as follows: high performance liquid chromatography-mass spectrometry (Thermo Scientific, Q Exactive); ultrasonic cleaners (KQ-700 VDE, kunshan ultrasonic instruments Co., ltd.); electronic balance (METTER TOLEDO, XSE-205 DU); water purifier (Merck, MILLI-Q IQ 7015).
The reagent used is as follows: ellagic acid standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 96%), urolithin A standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 98%), urolithin B standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 98%), urolithin C standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 95%), urolithin M5 standard (Shanghai Yuan Ye Biotechnology Co., purity: 95%), urolithin M6 standard (Sichuan Vickers Biotechnology Co., ltd., purity: 98%), isouricuryl A standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin D standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin E standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin M7 standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), N-methylpyrrolidone (Shanghai Michelin biotechnology Co., ltd.), methanol (Simer's flight, mass spectrum purity), acetonitrile (Simer's flight mass spectrum purity), formic acid (national pharmaceutical Co., ltd.).
Sample to be measured: an ellagic acid and urolithin mixed solution of unknown concentration.
High performance liquid chromatography tandem ultra-high resolution mass spectrometry qualitative and quantitative detection instrument conditions: instrument model: themoScientific Q Exactive; column temperature: 30 ℃; flow rate: 0.3mL/min; sample injection amount: 3 μL; mobile phase a:0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column and elution mode: waters AcquityBEH C18,1.7 μm, gradient elution; ultraviolet detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;6-10min, the volume fraction of the mobile phase A is kept 65%;10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;14-16min, and the volume fraction of the mobile phase A is kept 100%;16-18min, the volume fraction of mobile phase A increases from 0% to 88%; the volume fraction of mobile phase A was kept 88% for 18-20 min. 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 is an electrospray ionization (ESI) negative mode, the spraying voltage is-2.5 kV, the sheath gas flow rate is 30arb, the auxiliary gas flow rate is 10arb, the capillary temperature is 325 ℃, the primary full-scanning resolution is 70000, the primary ion scanning range is 50m/z-750m/z, the HCD is adopted for secondary pyrolysis, the secondary resolution is 17500, and the collision energy is respectively: 20/40/60, xcalibur software was selected in combination with Compound Discover software for metabolite identification.
Drawing standard curves of ellagic acid and urolithin:
Ellagic acid and urolithin standard are accurately weighed (ten standard substances in total, equivalent weight) respectively, a mother liquor of 0.1mg/mL is prepared by using 5% (v/v) of N-methylpyrrolidone-methanol solution, and further, a series of concentration gradients are formed by diluting the mother liquor with 5% (v/v) of N-methylpyrrolidone-methanol solution. The final concentration of the standard substance for the qualitative and quantitative detection of the high performance liquid chromatography-mass spectrometry is 10,20,30,40,50,60,70 mug/mL (based on the total concentration of the standard substance). The detection was performed according to the conditions in this example, and a standard curve was drawn. The standard curves of ellagic acid and urolithin are shown in table 1, and figure 2 is a mass spectrum of the standard.
TABLE 1 Standard Curve of ellagic acid and urolithin (LC-MS)
Taking a sample to be detected which is rich in ellagic acid and urolithin, taking 5% (v/v) of N-methyl pyrrolidone-methanol solution as a mixed extractant, mixing the sample to be detected with the mixed extractant, and carrying out vortex and ultrasonic extraction. And after the ultrasonic treatment is finished, diluting a sample to be detected by using 5% (v/v) of N-methylpyrrolidone-methanol solution for 10 times, centrifuging, and taking a supernatant of the sample to pass through a 0.22 mu m PTFE film for standby to obtain a liquid to be detected. 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 ℃, the centrifugal time is 10min, and the centrifugal force is 11000g.
And (3) detecting the liquid to be detected on a machine, eluting and separating the standard sample liquid by utilizing high performance liquid chromatography, testing the liquid to be detected by adopting a method identical to that of a standard sample, substituting the peak areas of ellagic acid and urolith detected by an ultraviolet detector into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolith in the sample to be detected.
(1) Precision test
The sample solution to be tested is prepared by utilizing the sample to be tested, and is continuously injected for 5 times under the chromatographic condition, the chromatographic analysis condition is the same as that of the embodiment 1, the chromatogram is recorded, and the system precision is inspected. The results are shown in Table 2. Sample solution to be tested is prepared into a sample to be tested and is continuously injected for 5 times under the chromatographic condition, the chromatographic analysis condition is the same as that of the embodiment 2, a chromatogram is recorded, and the precision of the system is inspected. As shown in Table 2, it can be seen that the sample solutions were sampled 5 times in succession, and the RSD values of ellagic acid and urine Dan Sufeng areas were less than 2%, indicating that the method of the present invention has good system precision.
TABLE 2 precision results
(2) Repeatability test
5 Parts of test solutions were prepared in parallel, and the chromatographic conditions were the same as in example 1, and the chromatograms were recorded to examine the reproducibility. The results are shown in Table 3. From the results, the results of the content of ellagic acid, urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isourolithin A and urolithin B are not obviously different, which indicates that the method of the invention has good repeatability.
TABLE 3 repeatability results (nM)
Example 2
The embodiment provides a detection method of ellagic acid and metabolites thereof, comprising the following steps:
The instrument used is as follows: high performance liquid chromatograph (Thermo Scientific, U3000); ultrasonic cleaners (KQ-700 VDE, kunshan ultrasonic instruments Co., ltd.); electronic balance (METTER TOLEDO, XSE-205 DU); water purifier (Merck, MILLI-Q IQ 7015).
The reagent used is as follows: ellagic acid standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 96%), urolithin A standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 98%), urolithin B standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 98%), urolithin C standard (Shanghai Yuan Ye Biotechnology Co., ltd., purity: 95%), urolithin M5 standard (Shanghai Yuan Ye Biotechnology Co., purity: 95%), urolithin M6 standard (Sichuan Vickers Biotechnology Co., ltd., purity: 98%), isouricuryl A standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin D standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin E standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), urolithin M7 standard (Sichuan-Vickers biotechnology Co., ltd., purity > 98%), N-methylpyrrolidone (Shanghai Michelin biotechnology Co., ltd.), methanol (Simerremia, chromatographic purity), acetonitrile (Simerremia chromatography purity), formic acid (national pharmaceutical Chemicals Co., ltd.).
Sample to be measured: an ellagic acid and urolithin mixed solution of unknown concentration.
Quantitative detection instrument conditions of high performance liquid chromatography: instrument model: themoScientific U3000,3000; column temperature: 30 ℃; flow rate: 1.0mL/min; sample injection amount: 5. Mu.L; mobile phase a:0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column and elution mode: themoScientific Acclaim TM 120C18,5 μm, gradient elution; ultraviolet detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;6-10min, the volume fraction of the mobile phase A is kept 65%;10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;14-16min, and the volume fraction of the mobile phase A is kept 100%;16-18min, the volume fraction of mobile phase A increases from 0% to 88%; the volume fraction of mobile phase A was kept 88% for 18-20 min. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.
Drawing standard curves of ellagic acid and urolithin:
Ellagic acid and urolithin standard are accurately weighed respectively (ten standard substances are weighed in equal quantity), 5% (v/v) of N-methylpyrrolidone-methanol solution is used for preparing a mother solution of 0.1mg/mL, and further N-methylpyrrolidone-methanol solution is used for diluting into a series of concentration gradients. The final concentrations of the final standard substances for high performance liquid quantitative detection are respectively 0.01,0.02,0.04,0.06,0.08 and 0.1mg/mL (based on the total concentration of the standard substances). The detection was performed according to the conditions in this example, and a standard curve was drawn. The standard curves of ellagic acid and urolithin are shown in Table 4, and FIG. 2 is a high performance liquid chromatogram of the standard.
TABLE 4 Standard Curve of ellagic acid and urolithin (high Performance liquid chromatography)
Taking a sample to be detected which is rich in ellagic acid and urolithin, taking 5% (v/v) of N-methyl pyrrolidone-methanol solution as a mixed extractant, mixing the sample to be detected with the mixed extractant, and carrying out vortex and ultrasonic extraction. And diluting the sample to be detected by using an N-methyl pyrrolidone-methanol solution for 10 times after the ultrasonic treatment is finished, centrifuging, and taking a supernatant of the sample to pass through a 0.22 mu m PTFE film for standby to obtain the liquid to be detected. 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 ℃, the centrifugal time is 10min, and the centrifugal force is 11000g.
And (3) detecting the liquid to be detected on a machine, eluting and separating the standard sample liquid by utilizing high performance liquid chromatography, testing the liquid to be detected by adopting a method identical to that of a standard sample, substituting the peak areas of ellagic acid and urolith detected by an ultraviolet detector into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolith in the sample to be detected.
(1) Precision test
Sample solution to be tested is prepared into a sample to be tested and is continuously injected for 5 times under the chromatographic condition, the chromatographic analysis condition is the same as that of the embodiment 2, a chromatogram is recorded, and the precision of the system is inspected. As shown in Table 5, it can be seen that the sample solutions were sampled 5 times in succession, and the RSD values of ellagic acid and urine Dan Sufeng areas were less than 2%, indicating that the method of the present invention has good system precision.
TABLE 5 precision results
| 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 |
| Isouracillin 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 |
(2) Repeatability test
5 Parts of test solutions were prepared in parallel, and the chromatographic conditions were the same as in example 2, and the chromatograms were recorded to examine the reproducibility. The results are shown in Table 6. From the results, the results of the content of ellagic acid, urolithin M5, urolithin M6, urolithin D, urolithin E, urolithin M7, urolithin C, urolithin A, isourolithin A and urolithin B are not obviously different, which indicates that the method of the invention has good repeatability.
TABLE 6 repeatability results (mg/mL)
Example 3
The detection of ellagic acid and urolithin content in rat feces comprises the following specific steps:
(1) Source of sample to be measured
Urolithin is a metabolite of ellagic acid that undergoes a series of reactions under the action of intestinal microorganisms. Typically, after ellagic acid or ellagic acid-rich substances (e.g. punica granatum juice, etc.) are administered, urolithin is produced in part of the organism. The sample to be tested in this example was stool collected 90min after fasting lavage of ellagic acid (lavage dose 100 mg/mL) in normal rats.
(2) Pretreatment of sample to be tested
A proper amount of feces is taken, smashed by a glass rod, added with N-methyl pyrrolidone-methanol solution (the volume fraction of N-methyl pyrrolidone is 5%) according to the ratio of 1:5 (w/v), vortexed for 60s, and extracted by 400W ultrasonic for 30 min. After the ultrasonic treatment is finished, adding a methanol solution with the volume of 4 times into the system for dilution, and after vortex mixing, centrifuging at 12000rpm and 4 ℃ for 10min, wherein the centrifugal force is 11000g, and taking a sample supernatant to pass through a PTFE film with the thickness of 0.22 mu m for later use for loading analysis.
(3) Preparation of standard substance
1.0Mg of ellagic acid/urolithin standard is accurately weighed, a mother liquor of 0.1mg/mL is prepared by using 5% (v/v) of N-methyl pyrrolidone-methanol solution, and the mother liquor is further diluted into a series of concentration gradients, and the final concentrations are 10,20,30,40,50,60 and 70 mug/mL respectively.
(4) Detection of high performance liquid chromatograph-mass spectrometer
Instrument model: themoScientific Q Exactive; column temperature: 30 ℃; flow rate: 0.3mL/min; sample injection amount: 2. Mu.L; mobile phase a:0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column Waters acquisitionBEH C18,1.7 μm, gradient elution; scan Mode: ESI NEGATIVE Mode; scanning range: 50-750m/z. Gradient elution conditions: 0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;6-10min, the volume fraction of the mobile phase A is kept 65%;10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;14-16min, and the volume fraction of the mobile phase A is kept 100%;16-18min, the volume fraction of mobile phase A increases from 0% to 88%; the volume fraction of mobile phase A was kept 88% for 18-20 min. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%. The mass spectrum is detected by adopting a DDA and Full MS method, the Full MS resolution is 70000, the dd-MS2 resolution is 17500, and the NCE mode is selected to be 20,40 and 60eV; metabolite identification was performed by Xcalibur software in combination with Compound Discover software.
Note that: the parameters not mentioned in this example can be referred to in example 1.
(5) Identification result
Production of ellagic acid EA, urolithin M5, urolithin C, urolithin E and urolithin a was detected in the mouse feces collected over different time periods. The change in the content of the metabolite measured by the method of this example is shown in FIG. 2, and the change in the content of the metabolite measured by the conventional method is shown in FIG. 3. As can be seen from the results, the content measured by the method of the example is higher than that of the traditional method, the urolithin E which cannot be detected by the traditional method can be detected, the detection types are more, and the measured value is more accurate.
Example 4
The detection of urolithin produced by in vitro glycolysis of ellagic acid comprises the following specific steps:
(1) Source of sample to be measured
Intestinal flora samples were taken from 10 healthy volunteers of different ages (5 men and 5 women), respectively. Volunteers were on a regular diet prior to intestinal flora sample collection and had not taken antibiotics for 3 months. The collected fresh fecal sample is placed in a centrifuge tube, sealed and stored at-80 ℃. Before use, the fecal sample was thawed in a 37 ℃ water bath and following the fecal sample: sterile phosphate buffer solution=1:9 (W: V) fecal suspension was prepared, vortexed and mixed well, and then two layers of gauze were filtered to obtain intestinal flora samples for in vitro glycolytic ellagic acid. When in use, 10 percent of the inoculum size is added into brain heart infusion broth culture medium (BHI) containing ellagic acid, the final concentration of the ellagic acid is 1 percent (w/v), and the culture is put into a constant-temperature anaerobic workstation at 37 ℃ for shaking culture (120 r/min), and the concentration change rule analysis of ellagic acid and urolithin substances in a glycolysis system is sampled and detected every 24 hours.
(2) Pretreatment of sample to be tested
And (3) taking a proper amount of glycolysis liquid, adding an N-methyl pyrrolidone-methanol solution (the volume fraction of the N-methyl pyrrolidone is 5%) into the glycolysis liquid according to a ratio of 1:3 (w/v), and performing vortex 60s and 400W ultrasonic extraction for 30 min. After the ultrasonic treatment is finished, 5 times of N-methyl pyrrolidone-methanol solution (the volume fraction of the N-methyl pyrrolidone is 5%) is added into the system for dilution, after vortex mixing, the mixture is centrifuged for 10min at 12000rpm and 4 ℃, the centrifugal force is 11000g, and the supernatant of the sample is taken and is filtered through a 0.22 mu m PTFE film for later use for loading analysis.
(3) Preparation of standard substance
1.0Mg of ellagic acid/urolithin standard is accurately weighed, a mother liquor of 0.1mg/mL is prepared by using 5% (v/v) of N-methyl pyrrolidone-methanol solution, and the mother liquor is further diluted into a series of concentration gradients, and the final concentrations are respectively 0.01,0.02,0.04,0.06,0.08 and 0.1mg/mL.
(4) Qualitative and quantitative detection of high performance liquid chromatography
Instrument model: themoScientific U3000,3000; column temperature: 25-45 ℃; flow rate: 0.5-2mL/min; sample injection amount: 2-10 mu L; mobile phase a:0.1% formic acid water; mobile phase B: acetonitrile; chromatographic column and elution mode: themoScientific AcclaimTM 120C18,5 μm, gradient elution; ultraviolet detection: 254nm. Gradient elution conditions: 0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;6-10min, the volume fraction of the mobile phase A is kept 65%;10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;14-16min, and the volume fraction of the mobile phase A is kept 100%;16-18min, the volume fraction of mobile phase A increases from 0% to 88%; the volume fraction of mobile phase A was kept 88% for 18-20 min. In gradient elution, the sum of the volume fractions of mobile phase A and mobile phase B is 100%.
(5) Identification result
In the in vitro glycolysis model, glycolysis liquid is taken every 24 hours to detect the content of ellagic acid and urolithin substances, and the glycolysis days are 15 days.
As can be seen from the results of FIG. 4, ellagic acid content gradually decreases with fermentation time, and urolithin tends to increase with both type and content of fermentation time. Wherein, the content of urolithin M6, urolithin C and isourolithin A is obviously increased within 10 days of glycolysis; urolithin M7 and urolithin a start to be produced after day 4 of glycolysis.
Example 5
The extraction conditions and detection conditions of the invention are optimized:
(1) Extraction conditions
The experiment selects methanol, ethanol, ethyl acetate and N-methyl pyrrolidone as sample extractant for extracting low water-solubility and fat-solubility phenolic compounds such as ellagic acid. The results show that ellagic acid has a significant difference in dissolution behavior in different solvents. The saturated solubility of ellagic acid in the above solvents was further determined and as shown in table 7, the solubility of ellagic acid in N-methylpyrrolidone was significantly higher than in other solvents. The ellagic acid was dissolved in a certain amount with methanol and N-methylpyrrolidone, respectively, as shown in FIG. 5, and it was seen that methanol did not completely dissolve ellagic acid, and there was a large amount of precipitate at the bottom of the sample.
TABLE 7 saturation solubility of ellagic acid in different solvents
| Methanol | Ethanol | Acetic acid ethyl ester | N-methylpyrrolidone | |
| Ellagic acid solubility (mg/mL) | 2.20±0.20 | 2.61±0.08 | 0.14±0.00 | 11.59±0.27 |
Further, the difference between the two different extraction methods of methanol extraction and N-methylpyrrolidone extraction was examined for the ellagic acid content measurement of the same sample. Dissolving excessive ellagic acid in deionized water, centrifuging at 12000rpm for 10min, collecting supernatant, extracting ellagic acid in supernatant with methanol and N-methylpyrrolidone respectively, performing high performance liquid chromatography under the same chromatographic condition for content measurement, dissolving ellagic acid standard with methanol and N-methylpyrrolidone respectively to obtain standard curves of different extraction methods, measuring saturated solubility of ellagic acid in water by methanol extraction method to 0.041+ -0.002 mg/mL, and measuring saturated solubility of ellagic acid in water by N-methylpyrrolidone extraction method to 0.063+ -0.001 mg/mL. The results show that the simple methanol extraction can lead to low ellagic acid measurement value, and the measurement work can not be completed efficiently and accurately. The N-methylpyrrolidone extraction can reflect the content of ellagic acid in the sample more accurately, and the data is stable. However, because the polarity of the N-methyl pyrrolidone is stronger, and the reagent with too high concentration has certain damage to chromatographic columns, chromatographs, mass spectrometers and the like, the N-methyl pyrrolidone-methanol extraction is adopted to combine the two, so that the content of a sample can be accurately reflected, and the peak shape is good. And the damage to the chromatographic column and the instrument is less, and the chromatogram detected by the method is shown in figure 6.
(2) Chromatographic conditions
The experiment shows that the peak shape and ionization effect can be effectively improved by adding a small amount of acid into the mobile phase as a result of gradient elution of methanol-water, methanol-0.1% formic acid water and acetonitrile-0.1% formic acid water solution in the mobile phase. Acetonitrile can improve peak shape better than methanol. Thus acetonitrile-0.1% formic acid water was finally selected as mobile phase.
The gradient elution conditions were optimized in this experiment. The chromatograms before and after the optimization are shown in fig. 7, 8 and 9. Other chromatographic methods in FIG. 7, the urolithin A and the isourolithin A could not be separated (the peak with a retention time of 9.192min is the peak for urolithin A and isourolithin A); other chromatographic methods in fig. 8: the peak shape of urolithin M5 is strange, urolithin D and ellagic acid can not be separated, and the separation effect of urolithin A and isourolithin is poor; FIG. 9 shows that ellagic acid and urolithin are both significantly separated and the peak shape is good in the method provided by the embodiment of the invention. It can be seen that the optimized elution condition has good peak shape, can separate the urolithin isomers with similar retention behaviors, and reduces the interference of other compounds on the target compound.
(3) Mass spectrometry conditions
In order to more systematically and comprehensively complete identification and authentication of ellagic acid/urolithin substances, positive and negative ion scanning modes are adopted for detection in the experiment, and collision energy and mass spectrum resolution are optimized. As a result, it was found that the peak shape of ellagic acid/urolithin-like substances in the negative ion scanning mode was good and the detection limit was low, so that the negative ion mode was selected for detection. When the collision energy is 20eV, 40eV and 60eV, the first-stage ion information of chemical components can be reserved, and the richer second-stage fragment ion information can be obtained. Resolution refers to the degree of separation of two adjacent ion peaks on a mass spectrum, the higher the resolution, the higher the mass accuracy, but the corresponding sensitivity decreases. After the experiment is optimized, the quality precision and the sensitivity are high when the primary resolution is 68000-72000 and the secondary resolution is 17000-18000.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for detecting ellagic acid and metabolites thereof, comprising:
Extracting a sample to be detected by using a mixed extractant formed by methanol and N-methyl pyrrolidone to prepare a liquid to be detected; in the mixed extractant, the volume fraction of N-methyl pyrrolidone is 5% -10%, and the volume ratio of the sample to be detected to the mixed extractant is 1: (2-6);
Preparing a standard sample liquid by using an ellagic acid standard substance, a plurality of urolithin standard substances and the mixed extractant;
Quantitative analysis is carried out by high performance liquid chromatography or liquid chromatography-mass spectrometry.
2. The method according to claim 1, wherein the quantitative analysis by the high performance liquid chromatography comprises: eluting and separating the standard sample liquid by utilizing high performance liquid chromatography, drawing a standard curve graph according to the known standard substance concentration and peak area, and obtaining a quantitative correction equation; and testing the liquid to be tested by adopting the same method, substituting the peak areas of ellagic acid and urolith detected by the ultraviolet detector into a quantitative correction equation, and calculating to obtain the content of ellagic acid and urolith in the sample to be tested.
3. The method according to claim 2, wherein the instrument conditions of the high performance liquid chromatography are as follows: the instrument model is ThemoScientific U3000,3000, the chromatographic column is ThemoScientific Acclaim TM C18, the inner diameter of the filling of the chromatographic column is 3-7 μm, the column temperature is 25-45 ℃, the flow rate is 0.5-2.0 mL/min, the sample injection amount is 2-10 μL, and the ultraviolet detection wavelength is 250-260 nm.
4. The method of claim 1, wherein the step of performing quantitative analysis using the liquid chromatography-mass spectrometry comprises: carrying out chromatographic elution and separation on ellagic acid and urolith in a standard sample liquid by utilizing high performance liquid chromatography, detecting by ultra-high resolution mass spectrum, calculating according to a signal-to-noise ratio to obtain a quantitative detection limit and an identification detection limit, drawing a standard curve graph according to the known standard concentration and peak area, and obtaining a quantitative correction equation;
And (3) adopting the same method to sample the liquid to be tested, substituting the peak areas of ellagic acid and urolithin detected by mass spectrum into a quantitative correction equation, and calculating to obtain the content of the ellagic acid and the urolithin in the sample to be tested.
5. The method according to claim 4, wherein the conditions of the high performance liquid chromatography tandem ultra-high resolution mass spectrometry adopted by the liquid chromatography are as follows: the instrument model is ThemoScientific Q Exactive, the chromatographic column is C18, the inner diameter of the filling of the chromatographic column is 1.5-2.0 mu m, the column temperature is 25-45 ℃, the flow rate is 0.2-0.4 mL/min, the sample injection amount is 2-5 mu L, the scanning mode is an electrospray ionization negative mode, the spray voltage is-3 Kv to-2 kV, the sheath gas flow rate is 25-35 arb, the auxiliary gas flow rate is 8arb-13arb, and the capillary temperature is 320-340 ℃.
6. The method according to claim 5, wherein ultra-high resolution mass spectrometry is performed by typical data-dependent mass spectrometry, the primary full-scan resolution is 68000-72000, the primary ion scan range is 50m/z-750m/z, HCD is used for secondary fragmentation, the secondary resolution is 17000-18000, and collision energies in collision energy normalization mode are respectively: 20/40/60eV, xcalibur software was selected in combination with Compound Discover software for metabolite identification.
7. The method according to claim 2 or 4, wherein the mobile phase used in the quantitative analysis by high performance liquid chromatography or liquid chromatography comprises mobile phase A and mobile phase B, wherein mobile phase A is formic acid aqueous solution with volume fraction of 0.05% -0.15%, and mobile phase B is acetonitrile.
8. The method according to claim 7, wherein the gradient elution is performed under the following conditions:
0-5min, the volume fraction of the mobile phase A is reduced from 88% to 70%;
5-6min, the volume fraction of the mobile phase A is reduced from 70% to 65%;
6-10min, the volume fraction of the mobile phase A is kept 65%;
10-14min, the volume fraction of the mobile phase A is reduced from 65% to 0%;
14-16min, and the volume fraction of the mobile phase A is kept 100%;
16-18min, the volume fraction of mobile phase A increases from 0% to 88%;
18-20min, the volume fraction of the mobile phase A is kept 88%;
wherein, in the gradient elution process, the sum of the volume fractions of the mobile phase A and the mobile phase B is 100 percent.
9. The method according to claim 1, wherein the preparation process of the liquid to be tested comprises: mixing the sample to be detected with the mixed extractant, performing vortex and ultrasonic extraction, performing centrifugal treatment after dilution, and filtering the supernatant;
preferably, the vortex time is 30s-90s, the ultrasonic power is 400W-600W, and the ultrasonic time is 20min-40min;
preferably, the centrifugation temperature is 0-4 ℃, the centrifugation time is 5-10 min, and the centrifugal force is 10000-12000 g.
10. The method according to claim 1, wherein the preparation process of the standard sample solution comprises: mixing the ellagic acid standard, the plurality of urolithin standards and the mixed extractant, diluting to different concentrations by using the mixed extractant, and filtering.
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