A hypercrosslinked polymer, its preparation method and application in urine exposome analysis

Abstract

The present invention provides a hypercrosslinked polymer, a preparation method thereof, and an application thereof in urine exposome analysis. The present invention provides a preparation method of a hypercrosslinked polymer, comprising the following steps: mixing a halogenated benzene, an acetal, an organic solvent, and a catalyst, and performing a Friedel-Crafts reaction to obtain the hypercrosslinked polymer. The present invention uses a halogenated benzene as a monomer and an acetal as a crosslinking agent, and synthesizes the hypercrosslinked polymer by a simple and efficient one-step Friedel-Crafts reaction. Among them, the halogenated benzene has high reactivity, so the synthesized polymer has a high specific surface area, a high porosity, and a uniform pore structure; due to the presence of halogen groups, the hypercrosslinked polymer has a high adsorption capacity, especially for small molecule substances involved in exposome analysis, thereby achieving the simultaneous and efficient capture of exogenous and endogenous molecules in the treatment of a single sample and realizing ultra-high-throughput exposure characterization.

Description

Technical Field

[0001] The present invention belongs to the technical field of hypercrosslinked polymers, and particularly relates to a hypercrosslinked polymer, a preparation method thereof, and an application thereof in urine exposome analysis. Background Art

[0002] Non-invasive and convenient sampling makes urine analysis very important in biomonitoring or epidemiological studies. The analysis of exogenous and endogenous residues in urine has always been considered an effective method for characterizing human exogenous and endogenous exposure, and is also an important part of human exposure assessment for overall environmental drivers of health and disease and subsequent associations with adverse health effects. Although various methods have been developed to quantitatively determine different types of urine residues, there is still a lack of a comprehensive method to meet the high-throughput determination requirements of exogenous and endogenous substances in urine exposure assessment. Therefore, developing a material that can efficiently capture exogenous and endogenous molecules simultaneously in a single sample treatment and achieve ultra-high-throughput exposure characterization has become a technical problem to be solved urgently in this field. Summary of the Invention

[0003] The purpose of the present invention is to provide a hypercrosslinked polymer, a preparation method thereof, and an application thereof in urine exposome analysis. In an enzyme-treated urine sample, a simple adsorption-desorption process is carried out using the hypercrosslinked polymer prepared by the preparation method provided by the present invention, and then targeted mass spectrometry detection is carried out, realizing the simultaneous and efficient capture of exogenous and endogenous molecules in a single sample treatment and achieving ultra-high-throughput exposure characterization.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a preparation method of a hypercrosslinked polymer, comprising the following steps:

[0006] Mix a halogenated benzene, an acetal, an organic solvent, and a catalyst, and carry out a Friedel-Crafts reaction to obtain a hypercrosslinked polymer.

[0007] Preferably, the halogenated benzene is chlorobenzene.

[0008] Preferably, the acetal is dimethoxymethane.

[0009] Preferably, the organic solvent is a halogenated alkane.

[0010] Preferably, the catalyst is ferric chloride or iron(III) oxide.

[0011] Preferably, the molar ratio of the halogenated benzene to the acetal is 1:(0.4 - 10).

[0012] Preferably, the Friedel-Crafts reaction is carried out at 300-350K for 1-3h first, and then at 350-400K for 10-15h.

[0013] The present invention also provides a hypercrosslinked polymer prepared by the preparation method described in the above technical solution.

[0014] The present invention also provides the application of the hypercrosslinked polymer described in the above technical solution in the analysis of urine exposure groups.

[0015] Preferably, the application includes the following steps:

[0016] (1) Mix urine, a recovery indicator, a buffer solution and β-glucuronidase, and incubate overnight to obtain a urine sample;

[0017] (2) Mix the urine sample obtained in step (1) with the hypercrosslinked polymer, perform adsorption, and separate to obtain a hypercrosslinked polymer adsorbed with endogenous and exogenous substances;

[0018] (3) Soak the hypercrosslinked polymer adsorbed with endogenous and exogenous substances obtained in step (2) in methanol and ethyl acetate in sequence for desorption to obtain endogenous and exogenous substances;

[0019] (4) Quantitatively analyze the endogenous and exogenous substances obtained in step (3) by the internal standard method.

[0020] The present invention provides a method for preparing a hypercrosslinked polymer, comprising the following steps: mixing a halogenated benzene, an acetal, an organic solvent and a catalyst, and carrying out a Friedel-Crafts reaction to obtain the hypercrosslinked polymer. In the present invention, a halogenated benzene is used as a monomer and an acetal is used as a crosslinking agent, and the hypercrosslinked polymer is synthesized by a simple and efficient one-step Friedel-Crafts reaction. Among them, the halogenated benzene has a high reaction activity, so the synthesized polymer has a high specific surface area, a high porosity and a uniform pore structure; due to the presence of halogen groups, the hypercrosslinked polymer has a high adsorption capacity, especially for small molecule substances involved in exposome analysis, thereby realizing the simultaneous and efficient capture of exogenous and endogenous molecules in the treatment of a single sample and achieving ultra-high-throughput exposure characterization. Experimental results show that after the hypercrosslinked polymer prepared by the present invention is applied to exposome analysis, a total of 77 exogenous chemicals are detected in 13 mixed urine samples. Among them, the detection frequency of 64 chemicals in urine samples is 50%, indicating that the study population is exposed to complex environmental chemicals; among participants from 13 cities, the total concentration of detectable exogenous chemicals ranges from 182 to 340 ng / mL, and the main chemicals are monophthalate (32%), hydroxylated polycyclic aromatic hydrocarbons (28%), personal care products (13%) and non-phthalate plasticizers (6%); 97 endogenous molecules are detected by targeted analysis in urine, including alkaloids, amides, amino acids, carnitine, bile acids, carbohydrates, DNA damage markers, neurotransmitters, hormones, organic acids and vitamins. Description of the Drawings

[0021] Figure 1 It is the concentration level of exogenous compounds in 13 cities in Application Example 1;

[0022] Figure 2 It is the SEM image of the hypercrosslinked polymer prepared in Example 1;

[0023] Figure 3 It is the SEM image of the hypercrosslinked polymer after desorption in Application Example 1;

[0024] Figure 4 It is of the hypercrosslinked polymer prepared in Example 1 13 13C nuclear magnetic resonance spectrum;

[0025] Figure 5 It is the FTIR spectra of the hypercrosslinked polymer prepared in Example 1 before and after application;

[0026] Figure 6 It is the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope-labeled compounds in Comparative Application Examples 1 to 3;

[0027] Figure 7To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 4 to 7;

[0028] Figure 8 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 8 to 11;

[0029] Figure 9 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 12 to 16;

[0030] Figure 10 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 17 to 20;

[0031] Figure 11 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 21 to 23;

[0032] Figure 12 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 24 to 27;

[0033] Figure 13 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Application Examples 1, 28 to 29. Detailed implementation mode

[0034] The present invention provides a method for preparing a hypercrosslinked polymer, comprising the following steps:

[0035] Mix a halogenated benzene, an acetal, an organic solvent and a catalyst, and carry out a Friedel-Crafts reaction to obtain a hypercrosslinked polymer.

[0036] Unless otherwise specified, the present invention has no special limitation on the sources of the various raw materials, and commercially available products well-known to those skilled in the art can be used.

[0037] In the present invention, the halogenated benzene is preferably chlorobenzene. In the present invention, the chlorobenzene monomer has higher reaction activity, lower cost, and the synthesized polymer has a higher specific surface area, a higher porosity and a uniform pore structure.

[0038] In the present invention, the acetal is preferably dimethyl formal. In the present invention, the acetal is a crosslinking agent.

[0039] In the present invention, the molar ratio of the halogenated benzene to the acetal is preferably 1:(0.4 to 10), more preferably 1:(1 to 5), and still more preferably 1:3.

[0040] In the present invention, the organic solvent is preferably a halogenated alkane, more preferably 1,2-dichloroethane or carbon tetrachloride. There is no special limitation on the amount of the organic solvent used in the present invention, as long as the raw materials can be dissolved.

[0041] In the present invention, the catalyst is preferably ferric chloride or iron(III) oxide.

[0042] In the present invention, the molar ratio of the halogenated benzene to the catalyst is preferably 1:(2 - 5), more preferably 1:3.

[0043] In the present invention, the mixing of the halogenated benzene, acetal, organic solvent and catalyst is preferably as follows: the halogenated benzene, acetal and organic solvent are mixed, and then the catalyst is added.

[0044] In the present invention, the mixing of the halogenated benzene, acetal and organic solvent is preferably carried out under stirring conditions; the stirring time is preferably 1 - 3 min. There is no special limitation on the stirring rate in the present invention, and the raw materials can be mixed evenly by using the operations well-known to those skilled in the art.

[0045] There is no special limitation on the operation of adding the catalyst in the present invention, and the technical solutions for preparing the mixed materials well-known to those skilled in the art can be adopted.

[0046] In the present invention, the Friedel-Crafts reaction is preferably carried out at 300 - 350 K for 1 - 3 h first, and then at 350 - 400 K for 10 - 15 h, more preferably at 318 - 330 K with stirring for 2 h first, and then at 353 - 360 K for 12 h. Reacting at 300 - 350 K for 1 - 3 h in the present invention can form a network structure, and reacting at 350 - 400 K for 10 - 15 h can generate a microporous polymer, thus obtaining a hypercrosslinked microporous polymer.

[0047] After the Friedel-Crafts reaction is completed, the present invention preferably washes and dries the obtained product in sequence to obtain a hypercrosslinked polymer.

[0048] In the present invention, the washing is preferably carried out by washing with methanol 3 - 5 times first, and then washing with methanol in a Soxhlet apparatus for 10 - 15 h.

[0049] In the present invention, the drying temperature is preferably 350 - 400 K; the drying time is preferably overnight.

[0050] The present invention uses halogenated benzene as a monomer and acetal as a crosslinking agent to synthesize a hypercrosslinked polymer by a simple and efficient one-step Friedel-Crafts reaction. Among them, the halogenated benzene has high reactivity, so the synthesized polymer has a high specific surface area, a high porosity and a uniform pore structure; due to the presence of halogen groups, this hypercrosslinked polymer has a high adsorption capacity, especially for small molecule substances involved in the analysis of the exposome, thus achieving the simultaneous and efficient capture of exogenous and endogenous molecules in the processing of a single sample and realizing ultra-high-throughput exposure characterization.

[0051] The hypercrosslinked polymer prepared by the preparation method provided by the present invention has a large specific surface area and a high inherent porosity, and has good thermal stability and chemical stability, and has great potential in realizing urine exposure characterization and identifying potential exposure markers or biomarkers, providing a new solution for large-scale biological monitoring and disease research.

[0052] The present invention also provides a hypercrosslinked polymer prepared by the preparation method described in the above technical solution.

[0053] The present invention also provides an application of the hypercrosslinked polymer described in the above technical solution in urine exposome analysis.

[0054] In the present invention, the application of the hypercrosslinked polymer in urine exposome analysis preferably includes the following steps:

[0055] (1) Mix urine, a recovery indicator, a buffer solution and β-glucuronidase, and incubate overnight to obtain a urine sample;

[0056] (2) Mix the urine sample obtained in step (1) with the hypercrosslinked polymer, perform adsorption, and separate to obtain a hypercrosslinked polymer adsorbed with endogenous and exogenous substances;

[0057] (3) Soak the hypercrosslinked polymer adsorbed with endogenous and exogenous substances obtained in step (2) in methanol and ethyl acetate in sequence for desorption to obtain endogenous and exogenous substances;

[0058] (4) Quantitatively analyze the endogenous and exogenous substances obtained in step (3) by the internal standard method.

[0059] The application method provided by the present invention is applicable to the exposome analysis of urine obtained by any method.

[0060] The present invention preferably mixes urine, a recovery indicator, a buffer solution and β-glucuronidase, and incubates overnight to obtain a urine sample.

[0061] The present invention has no special limitation on the types of the recovery rate indicators, and the corresponding isotope markers of endogenous and exogenous compounds can be selected according to common sense. In the present invention, the spiked concentration of the recovery rate indicator in urine is preferably 2-40 ng / mL.

[0062] In the present invention, the buffer solution is preferably an ammonium acetate solution; the concentration of the ammonium acetate solution is preferably 1 mol / L. The present invention has no special limitation on the dosage of the buffer solution, and it is only necessary to adjust the pH of the solution to the range of 4-5. The present invention can further improve the spiked recovery rate by controlling the pH of the solution.

[0063] In the present invention, the concentration of the β-glucuronidase is preferably 20,000-30,000 units / mL. The present invention has no special limitation on the dosage of the β-glucuronidase, and it can be judged according to common sense.

[0064] The present invention has no special limitation on the operation of mixing the urine, the recovery rate indicator, the buffer solution and the β-glucuronidase, and the technical solutions for preparing the mixed material well-known to those skilled in the art can be adopted.

[0065] In the present invention, the temperature for overnight incubation is preferably 37 °C.

[0066] After obtaining the urine sample, the present invention preferably mixes the urine sample with a hypercrosslinked polymer for adsorption, and after separation, a hypercrosslinked polymer adsorbed with endogenous and exogenous substances is obtained.

[0067] In the present invention, the mass ratio of the hypercrosslinked polymer to the volume of urine is preferably (8.5-12) mg: 1 mL, and more preferably (9-10) mg: 1 mL.

[0068] The present invention has no special limitation on the operation of mixing the urine sample with the hypercrosslinked polymer, and the technical solutions for preparing the mixed material well-known to those skilled in the art can be adopted.

[0069] In the present invention, the adsorption is preferably carried out under oscillating conditions; the oscillation time is preferably 4-6 min, and more preferably 5 min. The present invention has no special limitation on other operations of the oscillation, and the operations well-known to those skilled in the art can be adopted. The present invention can further improve the spiked recovery rate by controlling the oscillation time.

[0070] In the present invention, the separation is preferably centrifugal separation. The present invention has no special limitation on the operation of the separation, and the operations well-known to those skilled in the art can be adopted. The present invention removes the supernatant by separation to obtain a hypercrosslinked polymer adsorbed with endogenous and exogenous substances.

[0071] After obtaining the hypercrosslinked polymer adsorbed with endogenous and exogenous substances, the present invention preferably immerses the hypercrosslinked polymer adsorbed with endogenous and exogenous substances in methanol and ethyl acetate in sequence for desorption to obtain the endogenous and exogenous substances.

[0072] In the present invention, the mass ratio of the volume of the methanol to the mass of the hypercrosslinked polymer is preferably (0.25 - 0.35) mL: 1 mg, more preferably 0.3 mL: 1 mg; the mass ratio of the volume of the ethyl acetate to the mass of the hypercrosslinked polymer is preferably (0.25 - 0.35) mL: 1 mg, more preferably 0.3 mL: 1 mg. By controlling the ratios of methanol to hypercrosslinked polymer and ethyl acetate to hypercrosslinked polymer, the present invention can further improve the spike recovery rate.

[0073] In the present invention, the desorption carried out by immersing in methanol is preferably shaking desorption; the time of the shaking desorption is preferably 4 - 6 min, more preferably 5 min. By controlling the time of the shaking desorption, the present invention can further improve the spike recovery rate.

[0074] In the present invention, the desorption carried out by immersing in ethyl acetate is preferably shaking desorption; the time of the shaking desorption is preferably 4 - 6 min, more preferably 5 min. By controlling the time of the shaking desorption, the present invention can further improve the spike recovery rate.

[0075] After the desorption is completed, the present invention preferably separates the product obtained by desorption to obtain a supernatant, and then concentrates the supernatant to obtain the endogenous and exogenous substances.

[0076] The present invention has no special limitation on the operations of the separation and concentration, and the operations well-known to those skilled in the art can be adopted.

[0077] After obtaining the endogenous and exogenous substances, the present invention preferably uses the internal standard method to quantitatively analyze the endogenous and exogenous substances.

[0078] The present invention has no special limitation on the operation of the internal standard method, and the internal standard method in the well-known targeted quantification in the art can be adopted for the operation.

[0079] The present invention synthesizes HCPs by using halogenated benzene as a monomer and acetal as a crosslinking agent through a simple and efficient one-step Friedel-Crafts reaction; the chlorobenzene monomer has high reactivity and low cost, so the synthesized polymer material has a high specific surface area, a high porosity and a uniform pore structure; due to the presence of halogen groups, the HCPs have a high adsorption capacity, especially for small molecule substances involved in exposure group analysis; a simple adsorption-desorption process is carried out using HCPs in enzyme-treated urine samples, and then targeted mass spectrometry detection is carried out, avoiding the complex processes of extraction and cleaning required by traditional methods, achieving simultaneous and efficient capture of exogenous and endogenous molecules in single sample processing, realizing ultra-high-throughput exposure characterization, and inhibiting the entry of macromolecules to reduce matrix effects; the method provided by the present invention has incomparable advantages in terms of low time and low cost, and high compatibility with the range of analytes makes it a powerful method for characterizing chemical exposure in large-scale biomonitoring and health research.

[0080] The present invention provides a urine exposure assessment method based on HCPs, demonstrating the great potential of HCPs in realizing urine exposure characterization and identifying potential exposure markers or biomarkers, and providing a new solution for large-scale biomonitoring and disease research.

[0081] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without creative efforts shall fall within the protection scope of the present invention.

[0082] Example 1

[0083] A preparation method of a hypercrosslinked polymer consists of the following steps:

[0084] 0.02 mol of chlorobenzene and 0.06 mol of dimethylol formaldehyde are added to 20 mL of 1,2-dichloroethane, stirred and mixed for 2 min, then 0.06 mol of ferric chloride is added to obtain a mixture, then the mixture is first stirred and reacted at 318 K for 2 h, then heated to 353 K and reacted for 12 h, after that, the obtained HCP powder is washed with methanol 3 times, then washed with methanol in a Soxhlet apparatus for 12 h, and finally vacuum dried at 353 K overnight to obtain the hypercrosslinked polymer HCP.

[0085] Application Example 1

[0086] Characterization of exogenous and endogenous metabolites of 2990 people in China

[0087] 1. Reagents and materials

[0088] There are 296 exogenous substances and 171 endogenous substances as reference standards. According to their molecular structures or application purposes, they can be classified into 12 and 11 categories respectively.

[0089] Exogenous substances include synthetic antioxidants (Antioxidants, n = 25), bisphenol analogues (Bisphenolanalogues, n = 14), brominated flame retardants (Brominatedflame retardants, n = 4), organophosphate esters (Organophosphate esters, n = 45), phenolic polycyclic aromatic hydrocarbons (Phenolicpolycyclic aromatichydrocarbons, n = 15), personal care products (Personal care products, n = 8), pesticides (Pesticides, n = 83), photoinitiators (Photoinitiators, n = 15), mono - phthalates esters (mono-Phthalates esters, n = 21), non - phthalate plasticizers (Plasticizers, n = 25), retarders (Retarders, n = 4) and UV stabilizers (UV stabilizers, n = 37).

[0090] Endogenous substances include alkaloids (Alkaloids, n = 1), amides (Amides, n = 2), amino acids (Aminoacids, n = 24), carnitines (Carnitines, n = 10), bile acids (Bile acids, n = 18), carbohydrates (Carbohydrates, n = 25), DNA damage indicators (DNAdamage indicators, n = 1), hormones (Hormones, n = 56), neurotransmitters (Neurotransmitters, n = 5), organic acids (Organic acids, n = 19), and vitamins (Vitamines, n = 10).

[0091] Table 1 Details of target analytes and isotopically labeled chemical substances

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113] In addition, there are 82 kinds of isotope-labeled chemical substances as internal standards. All the standard samples are purchased from 24 companies such as AccuStandard in the United States, Sigma-Aldrich in the United States, Wellington Laboratories in Canada, and Toronto Research Chemicals in Canada.

[0114] The liquid chromatography-mass spectrometry (5500Q Trap triple quadrupole-linear ion trap mass spectrometer) was purchased from AB Sciex in Toronto, Canada; the liquid chromatography-mass spectrometry (Sciex 7500 Triple quadrupole mass spectrometer equipped with an OptiFlow Pro Ion Source) was purchased from AB Sciex in Toronto, Canada; the nitrogen evaporator (12N-EvapTM) was purchased from Oganomation; the centrifuge was purchased from Hunan Xiangyi Company. The formic acid, ammonium acetate, ammonium formate, water, methanol, ethyl acetate, and acetonitrile used in the experiment were of Optima grade and were all purchased from Fisher Scientific in the United States;

[0115] 2. Sample collection

[0116] A total of 2,990 participants from China were recruited between 2017 and 2019. Participants at each study site were recruited from communities within 25 kilometers of the national air center, with 184 - 260 participants at each site and a gender ratio close to 1:1. From each site, a pooled urine sample was prepared by combining 20 μL aliquots from each participant, for a total of 13 samples, for the determination of exogenous and endogenous molecules. This study was approved by the Ethics Committee of the Institute of Environmental Health, Chinese Center for Disease Control and Prevention.

[0117] 3. Sample pretreatment and instrumental analysis

[0118] Add 0.5 mL of urine to the recovery indicator and 100 μL of 1 M ammonium acetate (pH adjusted to 5.0), buffer with 10 μL of β-glucuronidase (25,000 units / mL), and incubate overnight at 37 °C to obtain a urine sample; then add 5 mg of the hypercrosslinked polymer HCP prepared in Example 1 and shake for 5 min. After centrifugation and removal of the supernatant, first soak in 1.5 mL of methanol and shake for 5 min, then soak in 1.5 mL of ethyl acetate and shake for 5 min for desorption. Concentrate the desorbed supernatant to approximately 100 μL, add the internal standard, and then perform instrumental analysis; all target compounds were analyzed by LC-MS / MS, which was divided into 5 methods. The specific information of the methods is shown in Table 2, and the instrument parameters are shown in Table 3.

[0119] Table 2 Method information for exogenous and endogenous compounds in quantitative detection

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135] Table 3 Instrument Method Parameters

[0136]

[0137]

[0138] 4. Quality Assurance and Quality Control

[0139] Recovery rate evaluation: A mixture of exogenous and endogenous chemical substances with high detection rates in the sample was added to the mixed sample at two different levels (2 ng and 10 ng), and 10 parallel samples were set up. The recovery rate range obtained from the analysis was: 60% - 135%.

[0140] Matrix effect test: 10 parallel samples of the mixed sample were treated with HCP, and each extract was divided into two sub-samples of equal volume. 100 μL of the standard analyte mixture was added to sub-sample A, and 100 μL of methanol was added to sub-sample B; by comparing the response differences of the analytes and external standards in sub-samples A and B, the matrix effect range was calculated to be: 69% - 137%.

[0141] Reusability test: The reusability of HCP was tested in 10 parallel samples. For each repetition, the same HCP particles were used to continuously extract the mixed samples added to the analysis mixture five times. After each extraction, the HCP particles were soaked in methanol for at least 48 h before the next extraction to remove potential residues from the previous extraction. The average recovery rates for 5 repetitions ranged from 53% to 132%.

[0142] Three laboratory blank samples were set in 13 real mixed samples. Each type of compound was quantified by a standard curve with more than 7 concentration gradients, and the R of the standard curve 2 was above 99 in both cases. The method limit of quantification (LOQ) was defined as the compound concentration corresponding to a response 10 times the standard deviation of the noise. If the LOQ was lower than the lowest concentration of the quantitative standard curve or the background contamination value in the blank, the higher of the latter was used. The ranges of LOQs for all exogenous compounds were 0.001 - 2.874 ng / mL, and the ranges of LOQs for exogenous compounds were 0.0001 - 0.266 μmol / L.

[0143] 5. Analysis results

[0144] The concentration levels of exogenous compounds in 13 cities are as Figure 1 shown.

[0145] From Figure 1 it can be seen that a total of 77 exogenous chemicals were detected in 13 mixed urine samples. Among them, the detection frequencies of 64 chemicals in urine samples were 50%, indicating that the study population was exposed to complex environmental chemicals; among the participants from 13 cities, the total concentration of detectable exogenous chemicals was between 182 - 340 ng / mL, and the main chemicals were phthalic acid monoesters (32%), hydroxy polycyclic aromatic hydrocarbons (28%), personal care products (13%), and non-phthalate plasticizers (6%).

[0146] Targeted analysis detected 97 endogenous molecules in urine, including alkaloids, amides, amino acids, carnitine, bile acids, carbohydrates, DNA damage markers, neurotransmitters, hormones, organic acids, and vitamins.

[0147] The SEM images of the hypercrosslinked polymer prepared in Example 1 are as Figure 2 shown, and the SEM images of the hypercrosslinked polymer after desorption in Application Example 1 are as Figure 3 shown; the 13 C nuclear magnetic resonance spectrum of the hypercrosslinked polymer prepared in Example 1 is as Figure 4 shown, where the asterisks indicate spinning sidebands; the FTIR spectra of the hypercrosslinked polymer before and after application prepared in Example 1 are as Figure 5 shown.

[0148] From Figure 2 It can be seen that the prepared hypercrosslinked polymer HCP has a relatively small nanostructure; from Figure 4 It can be seen that the hypercrosslinked polymer HCP is crosslinked by chlorobenzene and dimethylol formaldehyde; from Figure 5 It can be seen that the chemical structure of the hypercrosslinked polymer HCP is similar before and after use, indicating its good chemical stability.

[0149] Comparative Application Example 1

[0150] Based on Application Example 1, 11 exogenous compounds and 6 endogenous isotope markers were selected. The desorption solvent was changed to methanol, the desorption solvent volume was changed to 0.5 mL, and the desorption times were changed to one time. Under the same other conditions as Application Example 1, a series of spike recovery tests were carried out on the samples.

[0151] Comparative Application Example 2

[0152] Based on Comparative Application Example 1, the mass of the hypercrosslinked polymer was modified to 10 mg, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0153] Comparative Application Example 3

[0154] Based on Comparative Application Example 1, the mass of the hypercrosslinked polymer was modified to 15 mg, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0155] Figure 6 To compare the spike recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Comparative Application Examples 1 to 3. From Figure 6 It can be seen that the spike recovery is the best when the mass of the hypercrosslinked polymer is 5 mg.

[0156] Comparative Application Example 4

[0157] Based on Comparative Application Example 1, the adsorption time was changed to 7 min, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0158] Comparative Application Example 5

[0159] Based on Comparative Application Example 1, the adsorption time was changed to 1 min, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0160] Comparative Application Example 6

[0161] Based on Comparative Application Example 1, the adsorption time was changed to 3 min, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0162] Comparative Application Example 7

[0163] On the basis of Comparative Application Example 1, the adsorption time was changed to 9 min, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0164] Figure 7 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope-labeled compounds in Comparative Application Examples 1, 4 to 7. From Figure 7 it can be seen that the optimal adsorption time is 5 min.

[0165] Comparative Application Example 8

[0166] On the basis of Comparative Application Example 1, the desorption time was changed to 7 min, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0167] Comparative Application Example 9

[0168] On the basis of Comparative Application Example 1, the desorption time was changed to 1 min, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0169] Comparative Application Example 10

[0170] On the basis of Comparative Application Example 1, the desorption time was changed to 3 min, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0171] Comparative Application Example 11

[0172] On the basis of Comparative Application Example 1, the desorption time was changed to 9 min, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0173] Figure 8 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope-labeled compounds in Comparative Application Examples 1, 8 to 11. From Figure 8 it can be seen that the optimal desorption time is 5 min.

[0174] Comparative Application Example 12

[0175] On the basis of Comparative Application Example 1, the pH adjusted with ammonium acetate was changed to 1, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0176] Comparative Application Example 13

[0177] On the basis of Comparative Application Example 1, the pH adjusted with ammonium acetate was changed to 3, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0178] Comparative Application Example 14

[0179] On the basis of Comparative Application Example 1, the pH adjusted with ammonium acetate was changed to 7, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0180] Comparative Application Example 15

[0181] On the basis of Comparative Application Example 1, the pH adjusted with ammonium acetate was changed to 9, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0182] Comparative Application Example 16

[0183] On the basis of Comparative Application Example 1, the pH adjusted with ammonium acetate was changed to 11, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0184] Figure 9 For the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope-labeled compounds in Comparative Application Examples 1, 12 - 16. From Figure 9 it can be seen that the optimal pH is 5.

[0185] Comparative Application Example 17

[0186] On the basis of Comparative Application Example 1, the desorption solvent was changed to acetone, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0187] Comparative Application Example 18

[0188] On the basis of Comparative Application Example 1, the desorption solvent was changed to acetonitrile, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0189] Comparative Application Example 19

[0190] On the basis of Comparative Application Example 1, the desorption solvent was changed to ethanol, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0191] Comparative Application Example 20

[0192] On the basis of Comparative Application Example 1, the desorption solvent was changed to ethyl acetate, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0193] Figure 10 For the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope-labeled compounds in Comparative Application Examples 1, 17 - 20. From Figure 10 it can be seen that the optimal desorption solvents are methanol and ethyl acetate.

[0194] Comparative Application Example 21

[0195] On the basis of Comparative Application Example 1, the volume of the desorption solvent methanol was changed to 1.0 mL, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0196] Comparative Application Example 22

[0197] On the basis of Comparative Application Example 1, the volume of the desorption solvent methanol was changed to 1.5 mL, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0198] Comparative Application Example 23

[0199] On the basis of Comparative Application Example 1, the volume of the desorption solvent methanol was changed to 2 mL, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0200] Figure 11 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Comparative Application Examples 1, 21 - 23. From Figure 11 it can be seen that the optimal desorption solvent volume is 1.5 mL.

[0201] Comparative Application Example 24

[0202] On the basis of Comparative Application Example 1, sodium chloride was added before adding the hypercrosslinked polymer to the urine sample. The concentration of sodium chloride in the urine sample was 1 w / w%, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0203] Comparative Application Example 25

[0204] On the basis of Comparative Application Example 24, the concentration of sodium chloride was replaced with 5 w / w%, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0205] Comparative Application Example 26

[0206] On the basis of Comparative Application Example 24, the concentration of sodium chloride was replaced with 10 w / w%, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0207] Comparative Application Example 27

[0208] On the basis of Comparative Application Example 24, the concentration of sodium chloride was replaced with 20 w / w%, and other conditions remained unchanged. A series of spiked recovery tests were carried out on the samples.

[0209] Figure 12 To compare the spiked recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Comparative Application Examples 1, 24 - 27. From Figure 12 it can be seen that the optimal salt concentration is 0, that is, the best effect is achieved without adding sodium chloride.

[0210] Comparative Application Example 28

[0211] On the basis of Comparative Application Example 1, the number of desorption times was changed to 2 times, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0212] Comparative Application Example 29

[0213] On the basis of Comparative Application Example 1, the number of desorption times was changed to 3 times, and other conditions remained unchanged. A series of spike recovery tests were carried out on the samples.

[0214] Figure 13 To compare the spike recoveries of 11 exogenous compounds and 6 endogenous isotope markers in Comparative Application Examples 1, 28 to 29. From Figure 13 it can be seen that the optimal number of desorption times is 2 times.

[0215] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of a hyper-crosslinked polymer in urine exposome analysis, wherein the application is for non-diagnostic purposes and comprises the following steps: (1) mixing urine, a recovery rate indicator, a buffer solution and β-glucuronidase, and incubating overnight to obtain a urine sample; the buffer solution is an ammonium acetate solution, and the pH of the solution is adjusted to 5; (2) mixing the urine sample obtained in step (1) with a hyper-cross-linked polymer, adsorbing the sample, and separating the hyper-cross-linked polymer adsorbing the endogenous and exogenous substances; (3) soaking the hyper-crosslinked polymer adsorbed with endogenous and exogenous substances obtained in step (2) in methanol and ethyl acetate in turn for desorption, separating the desorbed product to obtain a supernatant, and then concentrating the supernatant to obtain the endogenous and exogenous substances; (4) using the internal standard method to quantitatively analyze the endogenous and exogenous substances obtained in step (3); The method for preparing the hyper-crosslinked polymer comprises the following steps: Chlorobenzene, acetal, an organic solvent and a catalyst are mixed to carry out a Friedel-Crafts reaction to obtain a hyper-crosslinked polymer; The acetal is dimethanol formal; The catalyst is ferric chloride or ferric oxide.

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