Identification and detection method of whole grain wheat urine intake marker

Abstract

The invention relates to an identification and detection method of a whole grain wheat urine intake marker, and the whole grain wheat urine intake marker comprises a glycine conjugate of 3, 5-dihydroxybenzoic acid, a sulfuric acid conjugate of ortho-aminophenol, a sulfuric acid conjugate of 2-acetaminophen, a glycine conjugate of 3, 5-dihydroxybenzoic acid, a sulfuric acid conjugate of 2-acetaminophen, a glycine conjugate of 3, 5-dihydroxybenzoic acid and a sulfuric acid conjugate of 2-acetaminophen. The invention also relates to a sulfuric acid conjugate of 2, 5-dihydroxyphenyl valeric acid, a glucuronic acid conjugate of ortho-aminophenol or a glucuronic acid conjugate of 2-acetaminophen. The invention further relates to a high-throughput screening method of the whole-grain wheat urine intake marker, a whole-grain wheat diet intervention experiment is designed, and the urine metabolism marker of the whole-grain wheat is screened out by utilizing a non-targeted metabonomics technology through contrastive analysis of a human urine metabolism spectrum. And the method is provided for accurately evaluating the intake of the whole-grain wheat.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarkers, and in particular relates to an identification and detection method for urine markers of whole grain wheat intake. Background Art

[0002] In 1999, the American Association of Cereal Chemists (AACC) first clearly defined whole grains as consisting of intact, crushed, or flaked caryopsis, with the starchy endosperm, germ, and bran in the same proportions as in intact caryopsis. This definition covers cereals such as wheat, oats, and barley. This definition has been adopted by many countries, but regional variations exist in the specific wording and types of grains used.

[0003] As research into the health effects of whole grains deepens, recall bias, a key issue with traditional dietary assessment methods (such as the 24-hour dietary recall), which rely on subjective reporting, has become increasingly prominent. This severely limits causal inferences between whole grain intake and health outcomes. Against this backdrop, metabolomics-based biomarkers of food intake (BFIs) have emerged. This technology objectively reflects dietary intake levels by detecting characteristic metabolites in biological samples. Its advantages include correcting self-reported dietary survey data, improving the objectivity and accuracy of dietary assessments, and helping to elucidate the relationship between diet and disease.

[0004] Urine has become an ideal matrix for BFIs research due to its characteristics such as non-invasive sampling, dynamic monitoring of metabolic characteristics, and a wide range of metabolite concentrations. Compared with blood, food-related metabolites are more easily enriched in urine, and its low protein characteristics simplify the pre-treatment process, which is conducive to large-scale nutritional epidemiological studies. However, urine currently still faces three major challenges in the field of whole grains: first, metabolite identification relies on standards, with a high false positive rate and poor reproducibility; second, there is a lack of unified standards for sample processing and analysis procedures, resulting in insufficient comparability of cross-study results; third, existing studies are mostly based on Nordic whole-grain rye consumers. However, in China, whole-grain wheat is the main type of whole-grain consumption. Due to differences in genetic background and lifestyle habits, individuals have significant differences in dietary choices and metabolic responses, which makes the applicability and effectiveness of whole-grain wheat urine markers in the Chinese population questionable. Summary of the Invention

[0005] To solve the above problems, the present application designs a dietary intervention experiment based on the dietary characteristics of whole grain wheat in Chinese population, compares and analyzes the urine metabolome by non-targeted metabolomics technology, screens potential markers, introduces human liver microsomal / cytoplasmic liquid in vitro metabolism model for high-confidence identification, and constructs an LC-MS / MS targeted detection method. Ultimately, 6 specific urine markers of whole grain wheat are successfully screened, and the verification results show that these markers have high specificity, stability and repeatability, and have a dose-effect relationship with whole wheat intake, which can accurately identify whole grain wheat consumers and realize intake level stratification. In addition, the present application develops a supporting high-throughput and high-sensitivity targeted analysis method, which provides technical support for the standardized application of whole grain wheat intake markers in Chinese nutritional epidemiological research.

[0006] In a first aspect, the present application provides a urine marker of whole grain wheat intake, which includes glycine-bound 3,5-dihydroxybenzoic acid (3,5-DHBA), sulfate-bound 2-aminophenol (AP), sulfate-bound 2-acetamidophenol (HPAA), sulfate-bound 3,5-dihydroxyphenyl propanoic acid (DHPPTA), glucuronide-bound 2-aminophenol (AP) or glucuronide-bound 2-acetamidophenol (HPAA).

[0007] Further, the urine marker of whole grain wheat intake includes one or more of the above-mentioned substances, preferably, the urine marker of whole grain wheat intake includes glycine-bound DHBA and sulfate-bound AP.

[0008] More preferably, the urine marker of whole grain wheat intake includes the above-mentioned 6 substances.

[0009] Further, the urine marker of whole grain wheat intake is obtained by screening and identifying the urine sample of the intervention experiment subject through chromatography-mass spectrometry detection.

[0010] In a second aspect, the present application provides a high-throughput screening method of a urine marker of whole grain wheat intake, which includes the following steps: (1) Two-stage cross-over dietary intervention experiment: Set up intervention group whole wheat flour group and control group refined wheat flour group, carry out two-stage dietary intervention, and exchange intervention food between the two groups within two stages.

[0011] The intervention experiment sets up an intervention group, a whole wheat flour group, and a control group, a refined wheat flour group, and the number of subjects in each group is the same.

[0012] The intervention experiment includes an introduction period, a first-stage intervention period, a washout period, and a second-stage intervention period, a total of 4 stages, and a total of 15 days.

[0013] The introduction period is 5 days: the subjects in both groups avoid consuming whole grain wheat and processed foods for the first 3 days; and avoid consuming pasta foods for the 4th-5th days.

[0014] The first-stage intervention period is 2 days, and the whole wheat flour group consumes 50g of food (steamed buns) made of whole wheat flour for breakfast on the intervention day, and the refined wheat flour group consumes 50g of food (steamed buns) made of refined wheat flour for breakfast on the intervention day, and the rest of the day The intake of cereal food is rice, and the total amount of cereal food intake per day in both groups is equal.

[0015] The washout period is 5 days, and the dietary restrictions during the washout period are the same as during the introduction period. After the washout period, the second-stage intervention period begins, which is 2 days, and the intervention food is exchanged between the two groups.

[0016] During the intervention period, the intake of whole wheat steamed buns in breakfast needs to be completed under the supervision of researchers to ensure that the whole grain wheat reaches the pre-set intake amount of the experiment. Dinner and lunch are designed by researchers based on the principle of balanced diet, and the meal only limits the intake of pasta (mainly rice), and meets the daily nutrient and energy needs of men and women on this basis.

[0017] In the marker screening stage, in order to maximize the identification of metabolic differences directly related to the intake of whole grain wheat, potential confounding factors are controlled. The inclusion criteria for subjects are: age 18-40 years old, BMI 18.5-23.9 kg / m 2 The exclusion criteria are: suffering from chronic diseases, having a history of smoking, using nutritional supplements within 3 months, using antibiotics within 1 month, changing body weight by more than 3kg within 3 months, being in pregnancy, lactation or menstrual period, being a vegetarian or having special dietary habits, being allergic to wheat, and being unable to follow the relevant dietary requirements during the experiment.

[0018] During the intervention experiment, only the food uniformly distributed by the researchers can be eaten. Food and beverages other than the distributed meals cannot be consumed at will. Three meals a day are scheduled at 8:00 a.m., 12:00 p.m., and 18:00 p.m. (must be eaten within 20 minutes). No food is consumed after dinner every day, and the uniformly distributed plain water can be drunk.

[0019] (2) Urine collection by time period: Baseline urine was collected at 0 h before the intervention, and urine was collected at multiple preset time periods from 0 (not included) to 48 h after the intervention meal.

[0020] Before the intervention breakfast, 50 mL of midstream urine sample (0 h) was collected from the subjects as a baseline control (i.e., 0 h urine was used as the baseline urine sample).

[0021] After the intervention meal, urine was collected in different time periods: 0 (not included) to 2 hours, 2 (not included) to 4 hours, 4 (not included) to 6 hours, 6 (not included) to 9 hours, 9 (not included) to 12 hours, 12 (not included) to 24 hours, 24 (not included) to 36 hours, and 36 (not included) to 48 hours. The urine volume of each period was accurately recorded for future use.

[0022] (3) Sample mixing and detection: The urine samples from different time periods were mixed according to the volume ratio to obtain a mixed urine sample, and metabolites were detected by high performance liquid chromatography-mass spectrometry.

[0023] 3.1 Mixing and pretreatment: Mix, centrifuge, and dilute the urine; The urine mixing is to mix urine from different time periods according to volume ratio to form a urine mixed sample: Among them, P-12h mixed urine: mixed urine from 0 to 12 hours (including 0 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 9 hours, and 9 to 12 hours); P-24h mixed urine: mixed urine from 0 to 24h (including 0 to 2h, 2 to 4h, 4 to 6h, 6 to 9h, 9 to 12h, and 12 to 24h); P-24~48h mixed urine: mixed urine from 24 (not including) to 48h (including 24~36h and 36~48h); Further, centrifugation: 7000-10000×g for 10-30 min at 0-4°C; Furthermore, the diluent is 10-20% methanol-water; the amount of the diluent added is the same as the sample volume.

[0024] The present invention mixes urine of subjects from different time periods together, and the mixed urine sample can represent the overall state after eating complete grain wheat and is more representative.

[0025] Since the amount of urine samples collected at each time period is large and inconvenient to store, the method of equal proportion mixing is adopted: the volume of urine from each time period is reduced according to the original proportion and then mixed to obtain mixed urine samples representing different time periods.

[0026] In one embodiment of the present invention, the mixed urine is heated at 9000× g (Relative centrifugal force units) Centrifuge for 15 minutes to remove particulate matter and sediment, then accurately transfer 500 μL of urine sample with a pipette and add 500 μL of 20% methanol-water (1:1) dilution for testing.

[0027] 3.2 High performance chromatography-mass spectrometry detection: Chromatographic conditions: Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm), column temperature 30–40°C, injection volume 1–2 μL. Mobile phase A consisted of methanol containing 0.01–0.05% acetic acid, and mobile phase B consisted of ultrapure water containing 0.01–0.05% acetic acid. The HPLC flow rate was 0.2–0.4 mL / min, with a linear gradient elution program: starting with 2% A, then increasing linearly to 100% A over 15 minutes, holding for 5 minutes, returning to the starting mobile phase ratio, equilibration for 3 minutes, and waiting for the next injection.

[0028] Mass spectrometry conditions: High-throughput screening of urine biomarkers was performed using an Orbitrap high-resolution mass spectrometer. Spray voltages were 3.5–3.8 kV (+) and 3.0–3.5 kV (-); heating temperature was 300–350°C; and capillary temperature was 300–320°C. Scan mode: FullMS-ddMS2 (Top 8); scan range: 100–1000 m / z; secondary fragmentation mode: Higher energy collision induced dissociation (HCD) with collision energies set at 15 eV, 30 eV, and 50 eV. The primary full scan resolution was 60,000; the secondary scan resolution was 15,000.

[0029] 3.3 Data collection and preliminary identification: The collected data were subjected to peak extraction, peak alignment, background subtraction, and data filtering; and the compound types were preliminarily identified using a self-built library.

[0030] Xcalibur v.4.0 software was used for data acquisition, and Compound Discoverer v.3.2 software was used for peak extraction, peak alignment and background subtraction of raw data.

[0031] The data filtering conditions include accurate mass deviation (<5ppm), signal-to-noise ratio (S / N>10), peak intensity (Peakarea>100000) and retention time shift (RT<0.2 min).

[0032] The compound types were preliminarily identified using databases; the databases included self-built Mass List database, MZcloud, Chemspider, HMDB, FOODB, PhytoHub and other databases.

[0033] (4) Screening for urine markers of whole grain wheat intake 4.1 Preliminary Screening: Whole grain wheat urine BFIs were screened using the t-test (normally distributed data) and the Mann-Whitney rank sum test (non-normally distributed data). Given the special requirements of BFIs for specificity, sensitivity, and reproducibility, the preliminary screening of whole grain wheat urine markers must meet the following requirements: (i) Comparison between the intervention group and the control group: In each stage, the differences in metabolite concentrations in urine samples of the intervention group and the control group at the corresponding time points were compared, and the following conditions must be met: P-12h, P-24h mixed urine samples: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine sample: P value < 0.05 and fold difference log2 FC > 1; That is, in each stage, between the intervention group and the control group, and between the corresponding P-12h mixed urine samples, P value < 0.001 and the difference fold log2 FC> 2; between the corresponding P-24h mixed urine samples, P The value was <0.001 and the difference multiple log2 FC>2; the corresponding P-24h~48h mixed urine samples, P value < 0.05 and the fold difference log2 FC > 1.

[0034] (ii) Comparison within intervention groups: In each phase, the metabolite concentration differences between the post-meal urine samples and the pre-meal baseline samples at 0 h within the intervention group were compared, and the following conditions must be met: Compared with the 0h sample, the mixed urine samples of P-12h and P-24h were: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine samples compared with 0h samples: P value < 0.05 and the fold difference log2 FC > 1.

[0035] That is, in each stage, the mixed urine samples of the intervention group at postprandial time P-12h were compared with those at P-0h. P The value was <0.001 and the difference fold log2 FC>2; compared with P-0h, the mixed urine samples at P-24h were P The value was <0.001 and the difference fold log2 FC>2; compared with P-0h, the mixed urine samples of P-24~48h were P value < 0.05 and the fold difference log2 FC > 1.

[0036] Using log2FC value and -log 10 P Volcano plots were drawn to screen the metabolite data of mixed urine at different time periods.

[0037] 4.2 Marker identification: Through peak detection, isotope cluster analysis, characteristic MS / MS fragment identification and mass spectral library matching, exogenous specific differential metabolites that were reproduced in both phases of the cross-intervention experiment were screened as urinary markers of whole grain wheat intake.

[0038] In one embodiment of the present invention, after whole grain wheat intake, 20 and 14 differential metabolites were screened in the 12-hour mixed urine (P-12h) and 24-hour mixed urine (P-24h), respectively, the latter being a subset of the former. Only one significantly differential metabolite was found in the mixed urine 24 (excluding) to 48 hours after the intervention meal (P-24~48h). m / z 188.0022.

[0039] The information of each mass spectrometry fragment was analyzed in detail to complete the identification and specificity evaluation of the candidate markers. A total of 6 urine markers specific to whole grain wheat were found, with mass-to-charge ratios of m / z 210.0406, m / z 188.0022, m / z 230.0127, m / z 289.0386, m / z 284.0775 and m / z 326.0879.

[0040] Among them, through standard comparison, m / z 210.0406 was identified as 3,5-DHBA-glycine, a glycine conjugate of 3,5-dihydroxybenzoic acid (3,5-DHBA), a secondary metabolite of the whole grain-specific phytochemical alkylresorcinol. m / z 188.0022, m / z 230.0127 are AP-sulfate and HPAA-sulfate, the sulfate conjugates of secondary metabolites of whole grain phytochemicals, 2-aminophenol (AP) and 2-acetamidophenol (HPAA), respectively.

[0041] m / z 289.0386 is DHPPTA-sulfate, a sulfate conjugate of 3,5-dihydroxyphenyl propanoic acid (DHPPTA), a secondary metabolite of the whole grain wheat phytochemical alkylresorcinol.

[0042] m / z 284.0775, m / z 326.0879 are AP-glucuronide and HPAA-glucuronide, the glucuronic acid conjugates of AP and HPAA, the secondary metabolites of benzoxazine phytochemicals in whole grain wheat, respectively.

[0043] A third aspect of the present invention provides a method for identifying whole-grain wheat urine biomarkers using an in vitro metabolic model. Because there are no commercially available standards for o-aminophenol sulfate conjugate AP-sulfate, 2-acetaminophen sulfate conjugate HPAA-sulfate, o-aminophenol glucuronic acid conjugate HPAA-glucuronide, and 3,5-dihydroxyphenylvaleric acid sulfate conjugate DHPPTA-sulfate, biomarker analysis relies solely on high-resolution mass spectrometry. To address these issues, the present invention utilizes a hybrid human liver microsome / human liver cytosol in vitro metabolic model to further identify potential biomarkers with high confidence.

[0044] This invention utilizes a mixed human liver microsome / human liver cytosol in vitro metabolic model to identify potential biomarkers with high confidence. The identification method involves incubating known precursors (o-aminophenol (AP), 2-acetaminophen (HPAA), and 3,5-dihydroxyphenylvaleric acid (DHPPTA) standards) with a mixed system of human liver microsomes and / or human liver cytosol. The mixed system contains essential cofactors such as uridine diphosphate glucose (UDPGA) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS).

[0045] Then, the retention times, fragment ions, and ion abundance ratios of the in vitro metabolites were compared with those of the urine sample metabolites.

[0046] In one embodiment of the present invention, in vitro metabolic assay conditions are as follows: uridine diphosphate glucose (UDPGA) / 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and human liver microsomes / human liver cytosol were removed from -80°C and thawed on ice. To an Eppendorf tube, 475µL of 100mM Tris-buffer (pH 7.4), 10µL of mixed human liver microsomes (20mg / mL) / mixed human liver cytosol (10mg / mL), and 10µL of 100mM / 10mM UDPGA / PAPS were added. This mixed solution was pre-incubated in a 37°C water bath for 5 minutes before the reaction was initiated by adding 5.0µL of the phytochemical or target compound to be identified (2.0mg / mL), maintaining the total reaction volume at 500µL. After initiating the reaction, incubate in a 37°C shaking water bath. After 12 hours, 100 µL of the total reaction volume was removed and added to an Eppendorf tube containing 200 µL of acetonitrile to terminate the reaction. After centrifugation, the supernatant was obtained and diluted 10-fold with 50% methanol in water. Metabolic products were identified using a high-resolution LC-Orbitrap MS system.

[0047] The results showed that the retention times, fragment ions, and ion abundance ratios of four metabolite markers (AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate) in the urine of the intervention group subjects within 24 hours after consuming whole grain wheat were completely consistent with the relevant data of metabolites obtained by in vitro incubation, indicating that the identification of metabolite markers was accurate and reliable.

[0048] This invention is the first to use the human liver microsome / human liver cytoplasmic fluid in vitro metabolic model for the identification of whole grain wheat urine markers, significantly improving the identification confidence of suspicious metabolites and laying the foundation for the biological interpretation of markers and the development of detection methods.

[0049] A fourth aspect of the present invention provides an LC-MS / MS targeted detection method for whole grain wheat intake urine markers, wherein the whole grain wheat intake urine markers include the six markers described in the first aspect.

[0050] The targeted detection method comprises the following steps: S1. Mass spectrometry conditions: Based on the mass spectrometry identification results of the first aspect, targeted optimization of the MS / MS mass spectrometry parameters of each marker, including retention time, qualitative ions, and quantitative ions.

[0051] Target compounds were detected using an AB Sciex 6500 triple quadrupole mass spectrometer. Specific mass spectrometry conditions included an ESI ion source in negative ion mode and multiple reaction monitoring (MRM) acquisition; high-purity nitrogen for the nebulizer, curtain, auxiliary heater, and collision gases; electrospray voltage: 4500 V; ion source temperature: 500°C; nebulizer gas pressure: 55 psi; and curtain gas pressure: 35 psi. Data were acquired and analyzed using AB Analyst software.

[0052] S2. Chromatographic Conditions: Chromatographic separation was performed using a Waters ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.7 μm), column temperature 30–40°C, injection volume 1–2 μL; mobile phase A consisted of methanol containing 0.01–0.05% acetic acid, and mobile phase B consisted of ultrapure water containing 0.01–0.05% acetic acid, with a flow rate of 0.1–0.3 mL / min. A linear gradient elution was used, with the following elution program: initial mobile phase concentration of 5% A, linearly increasing to 100% A over 8.5 min, maintaining the concentration for 2.0 min, and returning to the initial mobile phase for equilibrium before the next injection.

[0053] Based on the high-throughput screening of whole grain wheat intake urine markers in the second aspect of the present invention, given that the target marker has been identified, optimizing the gradient elution parameters of the mobile phase is beneficial to the targeted detection process.

[0054] Specifically, in the second aspect of target biomarker screening, high-resolution mass spectrometry (such as Orbitrap-MS) is used for non-targeted analysis. Its advantage lies in its high throughput, making it suitable for large-scale screening of unknowns. However, its quantitative accuracy is relatively limited. Given that six candidate target biomarkers have been successfully identified through this non-targeted screening, targeted mass spectrometry detection (LC-MS / MS) is required for subsequent verification and analysis. This method is designed for precise quantification and enables highly sensitive and precise quantitative detection of target biomarkers.

[0055] Given that there are no commercial standards for the sulfate conjugate of o-aminophenol, AP-sulfate; the sulfate conjugate of 2-acetaminophen, HPAA-sulfate; the glucuronic acid conjugate of 2-acetaminophen, HPAA-glucuronide; and the sulfate conjugate of 3,5-dihydroxyphenylvaleric acid, DHPPTA-sulfate, the present invention selects isomers or structural analogs of the above markers to prepare standard curves for quantification of the target substances: 4-acetaminophen sulfate potassium salt (CAS: 32113-41-0) is used for quantitative analysis of AP-sulfate and HPAA-sulfate; and 3-(4-methoxy-3-(sulfoxy)phenyl)propionic acid, dihydro isoferulic acid 3-O-sulfate (CAS: 1258842-21-5) is used for quantitative analysis of the sulfated metabolic marker, DHPPTA-sulfate.

[0056] In one embodiment of the present invention, spike recovery experiments were conducted by spiking reference standards at low, medium, and high concentrations of 2, 10, and 50 ng / mL, respectively, into preprandial baseline urine samples of subjects. The experimental results showed that the average recoveries of whole-grain wheat urine markers in the urine matrix ranged from 87.6% to 107.8%, with precisions ranging from 2.3% to 13.8%, demonstrating the excellent precision and recovery of the LC-MS / MS targeted detection method. Using matrix spike concentrations of 3x and 10x the signal-to-noise ratio (S / N) as the limits of detection (LOD) and quantification (LOQ), respectively, the LOD and LOQ ranges for whole-grain markers in urine were 0.3-0.7 ng / mL and 1.0-2.0 ng / mL, respectively, demonstrating the excellent sensitivity of this method. Using this method, the present invention achieved, for the first time, the highly sensitive simultaneous analysis and detection of six whole-grain wheat urine markers in urine by LC-MS / MS.

[0057] Beneficial effects of the present invention: 1. Based on the dietary characteristics of the Chinese population, this invention screened and identified six whole-grain wheat urine markers in healthy Chinese adults through a two-stage crossover intervention experiment for the first time, providing a new method for the accurate assessment of whole-grain intake.

[0058] 2. This invention is the first to use the human liver microsome / human liver cytoplasmic fluid in vitro metabolic model for the identification of whole grain wheat urine markers, verifying the biotransformation pathway of the markers, significantly improving the confidence level in the identification of suspected metabolites, laying the foundation for the biological interpretation of the markers, and providing a reference for the in vitro enzymatic synthesis of future marker reference standards.

[0059] 3. This invention establishes for the first time a high-sensitivity LC-MS / MS targeted synchronous detection method for six markers, which has high sensitivity and good reproducibility. It can be used as an economical and efficient routine laboratory detection method to promote the nutritional epidemiological application of whole grain markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 This is a schematic diagram of the randomized, controlled, crossover design whole grain wheat urine dietary intervention experiment in Example 1; Figure 2 For Example 1, log2FC values ​​and -log 10 P drew a volcano plot to screen urine markers for whole grain wheat intake in mixed urine between groups, where (a) was the screening of differential metabolites in mixed urine P-12h 12h after a meal; (b) was the screening of differential metabolites in mixed urine P-24h 24h after a meal; (c) was the screening of differential metabolites in mixed urine P-24~48h 24h (excluding) to 48h after a meal; Figure 3 For Example 1, a Venn diagram was used to screen for overlapping biomarkers in two phases of a randomized crossover dietary intervention experiment; Figure 4 This is the qualitative deconstruction spectrum of the whole grain wheat urine marker DHBA-glycine by high-resolution mass spectrometry in Example 1; Figure 5 This is the qualitative deconstruction spectrum of the high-resolution mass spectrometry of the whole grain wheat urine marker AP-sulfate in Example 1; Figure 6 This is the qualitative deconstruction spectrum of the HPAA-sulfate high-resolution mass spectrometry marker in whole grain wheat urine in Example 1; Figure 7 This is the qualitative deconstruction spectrum of the whole grain wheat urine marker DHPPTA-sulfate by high-resolution mass spectrometry in Example 1; Figure 8 This is the qualitative deconstruction spectrum of the whole grain wheat urine marker AP-glucuronide by high-resolution mass spectrometry in Example 1; Figure 9 This is the qualitative deconstruction spectrum of the HPAA-glucuronide high-resolution mass spectrometry marker in whole grain wheat urine in Example 1; Figure 10 Schematic diagram of the human liver microsome / human liver cytoplasmic fluid in vitro metabolic model assisting in the identification of four markers in Example 2; Figure 11 For Example 2, the retention time and fragment ion identification of four urine markers without standard substances were performed using an in vitro metabolism experiment; Figure 12This is a representative chromatogram of the targeted detection of six whole grain wheat urine markers by LC-MS / MS in Example 3; Figure 13 This is a schematic diagram of the dose-effect relationship experiment of whole grain wheat urine markers in Example 4; Figure 14 This is the linear regression analysis of the dose-effect relationship of the six whole grain wheat urine markers in Example 4. DETAILED DESCRIPTION

[0061] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0062] Example 1 A method for identifying urine markers of whole grain wheat intake comprises the following steps: (1) Two-stage crossover dietary intervention experiment: The two-stage crossover dietary intervention trial is a randomized, controlled, crossover design acute dietary intervention study. The trial lasted 15 days and included four phases: the introduction phase, the first phase intervention phase, the washout phase, and the second phase intervention phase. The specific experimental design process is shown in Figure 1 .

[0063] This study was approved by the Biomedical Ethics Committee of Peking University (approval number: IRB00001052-23***), and the research protocol was also registered with the U.S. Clinical Trial Registry (ClinicalTriab.Gov) (NCT05837***). Before the start of the experiment, the research team posted a recruitment advertisement to recruit subjects. Subsequently, eligible participants contacted the research team and were screened through interviews. After questionnaires, physical examinations, and blood biochemical index screening, a total of 22 subjects (12 males and 10 females) completed the study. The average age of the subjects was 24.6±3.2 years old, and the BMI was 21.2±1.5 kg / m 2 .

[0064] The introduction period was 5 days. During the first 3 days, both groups of subjects avoided consuming whole grain wheat and processed foods; and on the 4th and 5th days, both groups avoided consuming pasta foods.

[0065] During the run-in period, participants were required to strictly avoid foods containing whole-grain wheat, including but not limited to whole-wheat bread, whole-wheat steamed buns, whole-wheat crackers, instant cereals, and pre-packaged foods that listed whole-wheat flour in the ingredient list. Otherwise, participants were free to eat whatever they wanted. To minimize the impact of dietary habits on experimental results, participants were asked to avoid pasta for two days before the formal dietary intervention.

[0066] The subjects entered the standardized diet phase starting from dinner on the day before the intervention experiment. During the experiment, they could only eat the food uniformly distributed by the researchers and could not eat any food or drink other than the distributed meals at will. Three meals a day were scheduled at 8:00 a.m., 12:00 noon, and 18:00 p.m. (required to be eaten within 20 minutes). No food was consumed after dinner every day, and they could drink the uniformly distributed plain water.

[0067] This experiment set up an intervention group - the "whole wheat flour group" and a control group - the "refined wheat flour" group. Referring to the daily recommended intake standard for whole grains set by the "Dietary Guidelines for Chinese Residents (2022)" (50~150g, including miscellaneous beans), this study used 50g of whole wheat flour as the intervention dose. Steamed buns, a pasta consumed daily by the Chinese population, were used as intervention foods. 100% whole wheat flour obtained by grinding whole wheat grains was used as the raw material for making intervention foods. The process of making the intervention food is as follows: 50g of refined wheat flour / whole wheat flour, 30g of water and 0.5g of yeast were mixed, and then fermented, shaped and steamed. The final weight of the steamed buns was 82.9±4.4g ( n =10). Before the formal intervention, the subjects were divided into two groups based on gender, and then randomly divided into two groups A and B using a computer.

[0068] During the first two-day intervention period, Group A consumed 50g of steamed bread made with whole-wheat flour for breakfast on the intervention day, while Group B consumed 50g of steamed bread made with refined wheat flour for breakfast on the intervention day. All other cereals consumed on the same day were rice, and both groups consumed the same total daily cereal intake. This was followed by a five-day washout period, during which the dietary restrictions remained the same as during the run-in period. Following the washout period, the second two-day intervention period began, during which the two groups exchanged their intervention foods. Breakfast intake of whole-wheat steamed bread was supervised by the researchers to ensure that the pre-determined intake of whole-grain wheat was achieved. Dinner and lunch were designed by the researchers based on balanced diets, restricting only pasta (rice being the staple food) to meet the daily nutrient and energy requirements of both men and women.

[0069] (2) Urine collection by time period: Before the intervention breakfast, participants emptied their bladders and collected a 50 mL mid-stream urine sample (at 0 hours) as a baseline blank control. Urine samples were collected after the intervention meal at the following time periods: 0 (not included) to 2 hours, 2 (not included) to 4 hours, 4 (not included) to 6 hours, 6 (not included) to 9 hours, 9 (not included) to 12 hours, 12 (not included) to 24 hours, 24 (not included) to 36 hours, and 36 (not included) to 48 hours. During each scheduled time period, participants urinated and collected all urine samples for that period using one or more 500 mL sterile sampling bottles. The collected samples were temporarily stored on ice and delivered to the research staff promptly before the next group meal. Fresh sterile sampling bottles were then used for subsequent collection. Upon receipt of the urine samples, the research staff measured and accurately recorded the urine volume for each time period. The urine samples were then aliquoted into 1 mL aliquots and stored at -80°C for future use.

[0070] (3) Sample mixing and testing: 3.1 Mixing and pretreatment: Before sample analysis, place the urine sample frozen at -80℃ in a 4℃ environment to thaw overnight. Vortex the thawed urine sample thoroughly to mix it evenly. Mix the urine samples at each time point according to the volume ratio to form a mixed urine sample: P-12h mixed urine: mixed urine from 0 to 12h (including 0 to 2h, 2 to 4h, 4 to 6h, 6 to 9h, and 9 to 12h); P-24h mixed urine: mixed urine from 0 to 24h (including 0 to 2h, 2 to 4h, 4 to 6h, 6 to 9h, 9 to 12h, and 12 to 24h); P-24~48h mixed urine: mixed urine from 24 (not included) to 48h (including 24~36h and 36~48h).

[0071] The mixed urine samples were centrifuged at 9000 × g for 15 minutes at 4°C to remove particulate matter and precipitate. Then, 500 μL of urine sample was accurately pipetted and diluted with 500 μL of 20% methanol-water (1:1) for analysis. Additionally, 100 μL of each urine sample was aspirated and mixed again to prepare quality control (QC) samples for analysis.

[0072] The "mixing by volume ratio" described in the present invention is specifically explained as follows: Taking a subject as an example, the original urine volumes collected in the five time periods of 0-2h, 2-4h, 4-6h, 6-9h, and 9-12h are 200mL, 300mL, 400mL, 200mL, and 300mL, respectively. If the actual limitations are not taken into account, in theory, the urine of all time periods (a total of 1.4L) can be directly mixed to obtain a complete 0-12h mixed urine sample (P-12h). However, actual research often involves multiple subjects, and the complete collection and freezing of a large number of original urine samples from all time periods will face huge freezing space pressure. To solve this problem, the present invention adopts the following method: after collecting and recording the urine volume of each time period, 1mL is immediately taken from it as a packaged sample for that time period for freezing. To later prepare a pooled urine sample representing the 0-12 hour period (P-12h), precisely measured volumes of each 1 mL aliquot from each frozen time period are mixed, strictly proportional to the original urine volume: 200 µL for the 0-2 hour sample, 300 µL for the 2-4 hour sample, 400 µL for the 4-6 hour sample, 200 µL for the 6-9 hour sample, and 300 µL for the 9-12 hour sample. These five proportionally measured volumes are combined (a total of 1.4 mL). This yields a significantly smaller P-12h sample (1.4 mL vs. 1.4 L), yet the proportions of urine from each time period in the pooled sample are identical to those of the original urine. This method significantly conserves cryopreservation space while ensuring the representativeness of the pooled sample.

[0073] 3.2 High-performance chromatography-mass spectrometry detection A total of 176 urine samples (22 subjects × 4 time periods (0h, P-12h, P-24h, P-24~48h) × two intervention phases) were subjected to non-targeted metabolomics analysis in four batches in both positive and negative ionization modes. All urine samples collected during the same intervention phase and in the same ionization mode were grouped into the same batch, and samples from different subjects were tested in a random order. Before and after each batch of sample testing, five QC samples were added for instrument testing to ensure that the injection system was fully balanced and to evaluate system stability. At the same time, one blank sample (100% methanol) and one QC sample (Quality Control Sample) were inserted every 10 samples analyzed.

[0074] LC-Orbitrap high-resolution mass spectrometry was performed in both positive and negative ion modes. Liquid chromatography was performed using an HSST3 Acquity column (2.1 mm × 100 mm; 1.8 µm, Waters, USA) for metabolite separation. The column temperature was 40°C, and the injection volume was 2 µL. Mobile phase A consisted of methanol containing 0.05% acetic acid, and mobile phase B consisted of ultrapure water containing 0.05% acetic acid. The elution rate was 0.35 mL / min, and a linear gradient elution program was used: starting with 2% acetic acid, then increasing linearly to 100% acetic acid over 15 minutes, holding for 5 minutes, returning to the starting mobile phase ratio, equilibrating for 3 minutes, and waiting for the next injection.

[0075] Mass spectrometry conditions for high-throughput screening of urine biomarkers were as follows: Orbitrap high-resolution mass spectrometry with spray voltages of 3.8 kV (+) and 3.0 kV (-); heating temperature of 350°C; and capillary temperature of 320°C. Scanning mode: Full MS-ddMS2 (Top8); scan range: 100–1000 m / z; secondary fragmentation mode: higher energy collision-induced dissociation (HCD) with collision energies of 15 eV, 30 eV, and 50 eV. The primary full scan resolution was 60,000; the data-dependent secondary scan resolution was 15,000.

[0076] 3.3 Data collection and preliminary identification: Data acquisition was performed using Xcalibur v.4.0 software, and raw data were subjected to peak extraction, peak alignment, and background subtraction using Compound Discoverer v.3.2 software. Data filtering criteria included accurate mass deviation (<5 ppm), signal-to-noise ratio (S / N>10), peak intensity (peak area>100,000), and retention time shift (RT<0.2 min). Compounds were initially identified using databases such as the self-built Mass List database, MZcloud, ChemSpider, HMDB, FOODB, and PhytoHub.

[0077] (4) Screening for urine markers of whole grain wheat intake 4.1 Preliminary Screening: Whole grain wheat urine BFIs were screened using the t-test (normally distributed data) and the Mann-Whitney rank sum test (non-normally distributed data). Given the special requirements of BFIs for specificity, sensitivity, and reproducibility, the preliminary screening of whole grain wheat urine markers must meet the following requirements: (i) Comparison between the intervention group and the control group: In each stage, the differences in metabolite concentrations in urine samples of the intervention group and the control group at the corresponding time points were compared, and the following conditions must be met: P-12h, P-24h mixed urine samples: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine sample: P value < 0.05 and fold difference log2 FC > 1; And, (ii) comparison within intervention group: In each phase, the metabolite concentration differences between postprandial urine samples and preprandial baseline samples in the intervention group were compared, and the following conditions must be met: Compared with the 0h sample, the mixed urine samples of P-12h and P-24h were: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine samples compared with 0h samples: P value < 0.05 and the fold difference log2 FC > 1.

[0078] That is, the inter-group comparison (whole wheat flour group vs refined wheat flour group) and intra-group comparison (whole wheat flour group post-meal vs pre-meal baseline 0h) of the two-stage urine metabolites met P Value < 0.001 (P-12h, P-24h) or P The value was <0.05 (P-24~48h); the difference folds of urine metabolites between the two-stage groups (whole wheat flour group vs refined wheat flour group) and within the group (whole wheat flour group post-meal vs pre-meal baseline 0h) met log2FC>2 (P-12h, P-24h) or log2FC>1 (P-24~48h).

[0079] Using log2FC value and -log 10 P draws a volcano diagram to screen the urine metabolite data of each period ( Figure 2 ).

[0080] 4.2 Marker identification: Through peak inspection, isotope cluster analysis, characteristic MS / MS fragment identification and mass spectral library matching, differential metabolites reproduced in both phases of the cross-intervention experiment were screened as urinary markers of whole grain wheat intake.

[0081] Finally, 20 differential metabolites that could be reproduced in both phases of the cross-intervention experiment were screened out (cross-combination). Figure 3As shown in the results, after whole grain wheat intake, 20 and 14 differential metabolites were screened in the 12h mixed urine (P-12h) and 24h mixed urine (P-24h), respectively, and the latter is a subset of the former. The results showed that the number of observable differential metabolites decreased significantly over time, indicating that the difference in urine metabolic profiles between the intervention group and the control group gradually narrowed. Only one significantly differential metabolite was found in the mixed urine 24 to 48 hours after the intervention meal (P-24~48h). m / z 188.0022 ( Figure 3 ).

[0082] The information of each mass spectrometry fragment was analyzed in detail to complete the identification and specificity evaluation of candidate markers. A total of 6 urine markers specific to whole grain wheat were found ( Figure 3 ). By comparing with standard products, m / z 210.0406 was identified as 3,5-DHBA-glycine, a glycine conjugate of 3,5-dihydroxybenzoic acid (3,5-DHBA), a secondary metabolite of the whole grain-specific phytochemical alkylresorcinol ( Figure 4 ). In addition, the differential metabolic characteristics m / z 188.0022, m / z 230.0127, m / z Consistent mass spectrometric behavior was observed in the secondary fragments of 289.0386, specifically, they all contained characteristic fragments SO3 - ( m / z 79.9564, corresponding to sulfate ion) and parent ion loss HSO3 - Therefore, it can be considered that the above types of differential metabolites are sulfate conjugates. Through manual mass spectrometry analysis and literature search, m / z 188.0022, m / z 230.0127 are the sulfate conjugates of o-aminophenol (2-aminophenol, AP) and 2-acetamidophenol (2-acetamidophenol, HPAA), which are secondary metabolites of whole grain phytochemicals. Figure 5~Figure 6 ).and m / z 289.0386 was identified as DHPPTA-sulfate, a sulfate conjugate of 3,5-dihydroxyphenyl propanoic acid (DHPPTA), a secondary metabolite of the whole grain wheat phytochemical alkylresorcinol. Figure 7 ). Similarly, in m / z 284.0775, m / z 326.0879 in the secondary spectrum of the marker ion C6H7O6 - ( m / z 175.0246, corresponding to a glucuronide fragment ion), suggesting that the above metabolites are glucuronide conjugates of AP and HPAA. Since the above target fragment ions contain the same skeleton fragments as AP and HPAA, such as m / z 108.0445、 m / z 150.0560, it can be concluded that they are glucuronide conjugates of AP and HPAA, AP-glucuronide ( Figure 8 ) and HPAA-glucuronide ( Figure 9 ), respectively, of the secondary metabolites of whole grain wheat benzoxazine phytochemicals AP and HPAA. Among them, AP-glucuronide has been confirmed by standard comparison. The structures of the 6 markers are shown in Table 1.

[0083] Table 1 Information of 6 markers in urine of whole grain wheat .

[0084] Example 2 A method for identifying urine markers of whole grain wheat assisted by an in vitro metabolism model: First, the known precursors (AP, HPAA and DHPPTA standards) were incubated with a human liver microsomal / human liver cytosol mixed system under the condition of containing necessary cofactors such as uridine diphosphate glucose (UDPGA) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Then, the accurate mass number, MS / MS spectrum and chromatographic retention time of the target metabolites obtained in the in vitro metabolism experiment were compared and verified with the potential metabolites detected in the urine samples (within 24 hours after meals).

[0085] The specific in vitro metabolism experiment conditions are as follows (such as Figure 10(As shown): Uridine diphosphate glucose (UDPGA) / 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and human liver microsomes / human liver cytosol were removed from -80°C and thawed on ice. To an Eppendorf tube, 475µL of 100mM Tris-buffer (pH 7.4), 10µL of mixed human liver microsomes (20mg / mL) / mixed human liver cytosol (10mg / mL), and 10µL of 100mM / 10mM UDPGA / PAPS were added. This mixture was pre-incubated in a 37°C water bath for 5 minutes before the reaction was initiated by adding 5.0µL of the target compound (2.0mg / mL) to maintain a total reaction volume of 500µL. After incubation, the reaction was incubated in a 37°C shaking water bath. After 12 hours, 100µL of the reaction mixture was removed and added to Eppendorf tubes containing 200µL of acetonitrile to terminate the reaction. After centrifugation, the supernatant was obtained and diluted 10-fold with 50% methanol-water. The conversion type of the target compound in the phase II metabolic reaction was identified by LC-Orbitrap MS.

[0086] The results showed that the retention times, fragment ions, and ion abundance ratios of the four metabolite markers, AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate, in urine after whole-grain wheat ingestion were completely consistent with the relevant data of metabolites obtained by in vitro incubation ( Figure 11 ), indicating that the identification of metabolic markers is accurate and reliable.

[0087] Example 3 A method for targeted detection of whole grain wheat intake urine markers, wherein the whole grain wheat intake urine markers include the six markers mentioned above, specifically comprising the following steps: S1. Mass Spectrometry Parameter Optimization: Based on the mass spectrometry identification results of Example 1, targeted optimization of MS / MS parameters for each marker was performed, including retention time, qualifier ions, and quantifier ions. Specific parameters, such as mass spectrometry ion pair information and collision energy, are shown in Table 2.

[0088] Table 2 Mass spectrometry parameters for the LC-MS / MS targeted detection method of six whole grain wheat urine markers ; Note: CE: collision energy, unit: electron volt (eV).

[0089] S2. Chromatographic conditions: Three chromatographic columns, BEH C18, BEH Shield RP18 and BEH HSS T3, were selected to carry out chromatographic separation experiments on the target substances. Since biomarkers in urine are usually low-molecular-weight terminal metabolites with high hydrophilicity, the peak elution time of the target substances in the first two chromatographic columns is relatively early, overlapping with the solvent peak, thus affecting the detection sensitivity and specificity. Compared with the former, the BEH HSST3 chromatographic column has 100% aqueous phase compatibility, which not only significantly improves the chromatographic retention ability of polar compounds, but also maintains good tolerance under high aqueous phase conditions. Therefore, it was finally selected as the chromatographic column for urine metabolic marker analysis. In addition, since the metabolic markers contain a variety of glucuronidation, sulfation and amino acid conjugates, most of these target metabolites have weak acidic characteristics.

[0090] Chromatographic separations are susceptible to changes in urine pH, leading to peak broadening and retention time fluctuations, which in turn affect the reproducibility of the analytical method. Adding 0.05% acetic acid to the mobile phase, making it a weakly acidic system, can inhibit the ionization of weakly acidic compounds, enhance the chromatographic retention of target compounds, and thus improve the stability of analytical results.

[0091] The instrument parameters of the optimized method for targeted detection of whole grain urine markers are as follows: Chromatographic separation uses a Waters ACQUITY UPLC HSS T3 column (100mm × 2.1mm, 1.7μm), column temperature: 40°C, injection volume 2µL; mobile phase A is methanol containing 0.05% acetic acid, mobile phase B is ultrapure water containing 0.05% acetic acid, flow rate is 0.3mL / min, and the chromatographic mobile phase uses linear gradient elution. The elution program is: maintain the initial mobile phase 5% A, linearly increase to 100% A within 8.5min, maintain for 2.0min, return to the initial mobile phase equilibrium before the next injection. The target substances were detected using an AB Sciex 6500 triple quadrupole mass spectrometer. Finally, through the optimization of chromatographic gradient elution conditions, the simultaneous separation and detection of 6 whole grain wheat urine markers were achieved within 10min ( Figure 12 ).

[0092] Because commercial standards for AP-sulfate, HPAA-sulfate, HPAA-glucuronide, and DHPPTA-sulfate are unavailable, standard curves were prepared using isomers or structural analogs of these markers for quantification (Table 2). For example, 4-acetaminophen sulfate (CAS: 32113-41-0) was used for quantification of AP-sulfate and HPAA-sulfate, while dihydro isoferulic acid 3-O-sulfate (CAS: 1258842-21-5) was used for quantification of the sulfated metabolite marker DHPPTA-sulfate. To overcome variability in urine matrices, matrix-matched standard curves were prepared using mixed blank urine over a concentration range of 0.1–200 ng / mL. The results showed that all standard curves had correlation coefficients greater than 0.99, meeting practical analytical requirements.

[0093] Recovery experiments were performed using a reference standard spiked into preprandial baseline urine (P-0h) at low, medium, and high concentrations of 2, 10, and 50 ng / mL. The results showed that the average recoveries of whole grain wheat urinary markers in urine matrix ranged from 87.6% to 107.8%, with precisions ranging from 2.3% to 13.8%, demonstrating the excellent precision and recovery of the targeted LC-MS / MS detection method (Table 3). Using matrix spike concentrations at 3x and 10x the signal-to-noise ratio (S / N) as the limits of detection (LOD) and quantification (LOQ), respectively, the LOD and LOQ ranges for whole grain markers in urine were 0.3–0.7 ng / mL and 1.0–2.0 ng / mL (Table 3), demonstrating the excellent sensitivity of this method. This method, for the first time, achieved the highly sensitive simultaneous analysis and detection of six whole grain wheat urinary markers in urine by LC-MS / MS.

[0094] Table 3 Sensitivity, accuracy, and precision of whole grain urine markers assays evaluated by standard addition (n=6) .

[0095] Example 4: Validation of Whole Grain Wheat Urine Marker Application (Validation of Marker Dose-Effect Relationship) In order to further verify the dose-effect relationship between whole grain wheat intake and markers, a 6-day experiment was conducted. The validation trial lasted for 6 days, including a 5-day lead-in period and a 1-day concentrated dietary intervention period ( Figure 13 During the run-in period, participants were required to avoid whole-grain wheat, including pre-packaged foods that listed whole-wheat flour as an ingredient. Otherwise, participants were free to choose other foods.

[0096] After the run-in period, participants began a centralized dietary intervention period, which consisted of four standardized meals: dinner at the end of the run-in period (dinner on the fifth day of the run-in period), and breakfast, lunch, and dinner on the first day of the intervention period. Participants consumed 0g, 25g, 50g, or 100g of whole-wheat flour steamed buns for breakfast, depending on their randomized group. Other foods included rice porridge, eggs, and milk. This breakfast was conducted under the supervision of research staff to ensure that the whole-grain wheat content met the study design requirements. Dinner and lunch were designed by research staff based on balanced diet principles, restricting only pasta (rice was the staple food) to meet the daily nutrient and energy requirements of both men and women. During the centralized meal period, participants ate at the designated location and consumed only food distributed by the researchers. They were not allowed to consume any food or beverages other than those distributed. Meals were scheduled at 8:00, 12:00, and 18:00 (consumed within 20 minutes). After dinner, no food was consumed, but plain water was provided.

[0097] The inclusion and exclusion criteria for subjects were the same as those in Example 1. This validation study was approved by the Biomedical Ethics Committee of Peking University (approval number: IRB00001052-23***), and the study protocol was registered with ClinicalTrials.gov (NCT06358***). Subjects who met the inclusion criteria were informed of the specific trial procedures and subject management system by the researchers before participating in the formal trial and signed an informed consent form.

[0098] After questionnaires, physical examinations, and screening with blood biochemical parameters, a total of 40 subjects (20 males and 20 females) entered the study. During the intervention period, subjects were randomly divided into four subgroups: A, B, C, and D, with 10 subjects in each subgroup, ensuring a 50 / 50 split between males and females. During the trial, one subject each in subgroup A (0 g whole-wheat flour group) and subgroup C (50 g whole-wheat flour group) withdrew, resulting in a total of 38 subjects completing the dietary intervention and biological sample collection.

[0099] On the day of the experiment, participants consumed breakfast at 8:00 AM (consumed within 20 minutes). Urine samples were collected from 0 to 2 hours, 2 to 4 hours, 4 to 6 hours, 6 to 9 hours, 9 to 12 hours, and 12 to 24 hours after breakfast. After eating at a designated location, participants were free to move around with their urine collection containers. According to experimental requirements, urine samples were collected at different time points and temporarily stored in a cooler containing an ice pack. Participants returned to the designated location at 12:00 PM and 6:00 PM for lunch and dinner, respectively, and handed their collected urine samples to researchers. After each time point, researchers recorded the urine volume, aliquoted 1 mL of urine, and stored frozen at -80°C. Urine samples from each time point were combined according to volume ratio to create a 24-hour pooled urine (P-24h). Quantitative analysis of whole grain urinary markers was performed using an established LC-MS / MS targeted detection method.

[0100] The diagnostic performance of whole-grain wheat markers in urine for whole-grain intake was assessed by plotting receiver operating characteristic (ROC) curves. The ROC curve plots the true positive rate (sensitivity) against the false positive rate (1-specificity) on the horizontal axis. The diagnostic value of the marker was assessed by calculating the area under the ROC curve (AUC). AUC results were interpreted as follows: 0.9–1.0 indicates excellent; 0.8–0.9 indicates good; 0.7–0.8 indicates fair; 0.6–0.7 indicates poor; and 0.5–0.6 indicates model failure.

[0101] Receiver operating characteristic (ROC) analysis was used to evaluate the performance of whole-grain wheat urinary markers in identifying whole-grain wheat consumers and their consumption levels in the validation trial (Table 4). Validation results showed that, among the six urinary markers in 24-hour urine (P-24h), AP-sulfate demonstrated excellent discriminatory power (AUC = 1.0) in distinguishing whole-grain wheat flour consumers from non-whole-grain wheat flour consumers. Furthermore, five urinary markers—AP-sulfate, AP-glucuronide, HPAA-sulfate, DHPPTA-sulfate, and DHBA-glycine—also performed well in determining whether whole-grain wheat intake reached 50g, as well as in distinguishing between moderate (50g) and high (100g) whole-grain wheat intake levels (AUC > 0.9).

[0102] The 24-hour urine (P-24h) marker concentrations increased in a dose-dependent manner with increasing whole grain wheat intake, demonstrating that urinary markers can be used as continuous variables to objectively reflect whole grain wheat intake. Figure 14 Overall, AP-sulfate and DHBA-glycine had higher concentration levels in urine after whole grain wheat intake (both 24h urine concentrations exceeded 100 ng / mL after 100 g whole wheat flour intake), and had the strongest linear relationship with whole wheat flour intake ( R 2 = 0.98~0.99), and had the least individual heterogeneity, which were the best performing urinary markers in the validation study.

[0103] Table 4 ROC analysis of whole grain wheat urinary markers to identify whole grain wheat consumers and consumption levels .

[0104] The above-described embodiments are merely specific implementations of the present application, which are used to illustrate the technical solutions of the present application, but not to limit the same. The protection scope of the present application is not limited thereto. Although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those skilled in the art that any person skilled in the art can make modifications or easily think of changes to the technical solutions recorded in the foregoing embodiments, or make equivalent replacements to some of the technical features, without departing from the technical scope disclosed by the present application. Such modifications, changes or replacements do not cause the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A whole grain wheat intake urine marker, characterized in that The markers include glycine conjugates of 3,5-dihydroxybenzoic acid, sulfate conjugates of o-aminophenol, sulfate conjugates of 2-acetaminophenol, sulfate conjugates of 3,5-dihydroxyphenylvaleric acid, glucuronic acid conjugates of o-aminophenol or glucuronic acid conjugates of 2-acetaminophenol.

2. A high-throughput screening method for urine markers of whole grain wheat intake, characterized in that: The steps include: (1) Two-stage crossover dietary intervention experiment: a whole wheat flour intervention group and a refined wheat flour control group were set up for two-stage dietary intervention. The two groups exchanged intervention foods during the two-stage intervention period. (2) Time-based urine collection: baseline urine was collected at 0 h before the intervention, and urine was collected at multiple preset time periods from 0 (excluding) to 48 h after the intervention meal; (3) Sample mixing and detection: The urine samples collected at different time periods were mixed according to the volume ratio to obtain a mixed urine sample, and metabolites were detected by high performance liquid chromatography-mass spectrometry; (4) Screening for urine markers of whole grain wheat intake: Based on the results of the cross-intervention experiment, the differences in metabolite concentrations between the groups and within the groups after meals and at baseline were compared to screen for specific urine markers of whole grain wheat intake.

3. The screening method according to claim 2, characterized in that The intervention experiment in step (1) includes four phases: the introduction phase, the first phase intervention phase, the washout phase, and the second phase intervention phase; The lead-in period was 5 days: for the first 3 days, both groups of subjects avoided consuming whole grain wheat and processed foods; on the 4th and 5th days, both groups avoided consuming pasta foods; The first phase intervention period was 2 days. The whole wheat flour group consumed whole wheat flour foods for breakfast on the intervention day, while the refined wheat flour group consumed the same amount of refined wheat flour foods for breakfast on the intervention day. The rest of the cereal food consumed on that day was rice. The total daily cereal food intake of the two groups was equal. The washout period is 5 days, and the dietary restrictions are the same as those during the introduction period; The second phase intervention period lasted for 2 days, and the two groups exchanged intervention foods.

4. The screening method according to claim 2, wherein Step (2) Collect urine at different time periods after the intervention meal: collect urine from 0 (not included) to 2 hours, 2 (not included) to 4 hours, 4 (not included) to 6 hours, 6 (not included) to 9 hours, 9 (not included) to 12 hours, 12 (not included) to 24 hours, 24 (not included) to 36 hours, and 36 (not included) to 48 hours, and record the urine volume in each time period; Step (3) includes: 3.1 Mixing and pretreatment: Mix, centrifuge, and dilute the urine; The urine mixing is to mix urine from different time periods according to the volume ratio to form a mixed urine sample: Among them, P-12h mixed urine: mixed urine from 0 to 12 hours; P-24h mixed urine: mixed urine from 0 to 24h; P-24~48h mixed urine: mixed urine from 24 (not including) to 48h; The centrifugation: 0-4°C, 7000-10000×g for 10-30 min; The dilution: using 10-20% methanol-water dilution solution; 3.2 High performance chromatography-mass spectrometry detection; and, 3.3 Data acquisition and preliminary identification: Data acquisition includes peak extraction, peak alignment, background subtraction, and data filtering, and preliminary identification of compound types using a self-built library.

5. The screening method according to claim 2, characterized in that Step (4) includes: 4.1 Preliminary screening: (i) Comparison between the intervention group and the control group: In each stage, the differences in metabolite concentrations in urine samples of the intervention group and the control group at the corresponding time points were compared, and the following conditions must be met: P-12h, P-24h mixed urine samples: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine sample: P value < 0.05 and fold difference log2 FC > 1; And, (ii) comparison within intervention group: In each phase, the metabolite concentration differences between postprandial urine samples and preprandial baseline samples in the intervention group were compared, and the following conditions must be met: Compared with the 0h sample, the mixed urine samples of P-12h and P-24h were: P value < 0.001 and fold difference log2 FC > 2; P-24~48h mixed urine samples compared with 0h samples: P value < 0.05 and fold difference log2 FC > 1; 4.2 Marker identification: Through peak detection, isotope cluster analysis, characteristic MS / MS fragment identification and mass spectral library matching, exogenous specific differential metabolites that were reproduced in both phases of the cross-intervention experiment were screened as urinary markers of whole grain wheat intake.

6. The method according to claim 4, characterized in that In step 3.2, the chromatographic conditions were: column temperature 30–40°C, injection volume 1–2 µL; mobile phase A was methanol containing 0.01–0.05% acetic acid, and mobile phase B was ultrapure water containing 0.01–0.05% acetic acid; the HPLC flow rate was 0.2–0.4 mL / min, with a linear gradient elution. Mass spectrometry conditions: spray voltage 3.5–3.8 kV (+), 3.0–3.5 kV (–); heating temperature 300–350 °C; capillary temperature 300–320 °C; scan range 100–1000 m / z; collision energies 15 eV, 30 eV, and 50 eV.

7. The method according to claim 4, characterized in that In step 3.3, Xcalibur software was used for data acquisition, and Compound Discoverer software was used for peak extraction, peak alignment, and background subtraction; Data filtering conditions included accurate mass deviation <5 ppm, signal-to-noise ratio (S / N) > 10, peak area > 100,000, and retention time offset (RT) < 0.2 min.

8. The method according to claim 2, characterized in that Six urine markers specific to whole grain wheat were screened out, with mass-to-charge ratios of m / z 210.0406, m / z 188.0022, m / z 230.0127, m / z 289.0386, m / z 284.0775 and m / z 326.0879; in, m / z 210.0406 is a glycine conjugate of 3,5-dihydroxybenzoic acid. m / z 188.0022 is a sulfuric acid conjugate of o-aminophenol, m / z 230.0127 is a sulfuric acid conjugate of 2-acetaminophen, m / z 289.0386 is the sulfuric acid conjugate of 3,5-dihydroxyphenylvaleric acid, m / z 284.0775 is a glucuronic acid conjugate of o-aminophenol, m / z 326.0879 is a glucuronic acid conjugate of 2-acetaminophen.

9. A method for identifying urine markers of whole grain wheat intake using an in vitro metabolic model, characterized in that: First, precursor o-aminophenol, 2-acetaminophen and 3,5-dihydroxyphenylvaleric acid standards are incubated with a mixed system of human liver microsomes and / or human liver cytosol; then, the retention times, fragment ions and ion abundance ratios of the in vitro metabolites and the metabolites in urine samples are compared; wherein the mixed system includes cofactors.

10. A targeted detection method for urine markers of whole grain wheat intake as claimed in claim 1, characterized in that: The steps include: S1. Mass spectrometry parameters: Targeted optimization of MS / MS parameters for each marker, including retention time, qualifier ions, and quantifier ions; Chromatographic conditions: A Waters ACQUITY UPLC HSS T3 column was used, with a column temperature of 30–40°C and an injection volume of 1–2 µL. Mobile phase A consisted of methanol containing 0.01–0.05% acetic acid, and mobile phase B consisted of ultrapure water containing 0.01–0.05% acetic acid. The flow rate was 0.1–0.3 mL / min.

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