CN113777209B

CN113777209B - Synchronous detection and application of exposure and effect markers of volatile pollutants in urine

Info

Publication number: CN113777209B
Application number: CN202111310594.2A
Authority: CN
Inventors: 郑晶; 梅航; 唐斌; 汤送雄; 秦瑞欣; 蔡凤珊; 于云江
Original assignee: South China Institute of Environmental Science of Ministry of Ecology and Environment
Current assignee: South China Institute of Environmental Science of Ministry of Ecology and Environment
Priority date: 2021-11-08
Filing date: 2021-11-08
Publication date: 2022-02-08
Anticipated expiration: 2041-11-08
Also published as: CN113777209A

Abstract

The invention discloses a method for synchronously detecting exposure of volatile pollutants and effect markers in urine and application thereof, wherein the method selects OH-PAHs, mVOCs and 8-OHdG which possibly have correlation in the same urine sample as target detection objects to synchronously extract, detect and analyze, and comprises the following steps: pretreating a sample; enriching and separating a sample; concentration and detection of samples: and (3) respectively carrying out on-machine detection on OH-PAHs, mVOCs and 8-OHdG by adopting HPLC-MS/MS, correspondingly analyzing to obtain content data of various target detection substances in the urine, and analyzing the correlation among the three. The invention applies the relation between the content data of the analysis target object to the evaluation of the human body exposure characteristics of the two pollutants. According to the invention, through an optimization scheme, various target detection objects can be synchronously detected and analyzed, the detection steps are simplified, the detection cost is reduced, and the application range of the health exposure risk assessment result is expanded.

Description

Synchronous detection and application of exposure and effect markers of volatile pollutants in urine

Technical Field

The invention belongs to the technical field of pollutant detection, health exposure risk assessment and medical treatment, and particularly relates to synchronous detection and application of exposure of volatile pollutants in urine and an effect marker.

Background

Polycyclic Aromatic Hydrocarbons (PAHs) are semi-volatile organic pollutants (SVOCs) generated by incomplete combustion of organic matters such as coal, petroleum and tobacco. Researches show that PAHs exposure can cause stimulation effects on digestive tracts and respiratory tracts, and long-term exposure can cause damages to the circulatory system and the nervous system of a human body to produce teratogenesis, carcinogenesis, mutagenesis and other damages. Volatile Organic Compounds (VOCs) are ubiquitous pollutants in the atmosphere, are widely available, and are produced in industrial production, motor vehicle exhaust, cigarette smoke, high temperature cooking foods, and the like. Chronic exposure of certain VOCs may increase the risk of developing cancer, birth defects, and neurocognitive disorders. Exposure levels of PAHs, VOCs are generally assessed by two methods, external exposure and internal exposure: 1) the external exposure method comprises the steps of measuring PAHs and VOCs prototype compounds in the environment; 2) the internal exposure method is to measure PAHs and VOCs in human blood or to measure metabolites of PAHs and VOCs in urine. Currently, aiming at the human body exposure of environmental chemical pollutants such as PAHs, VOCs and the like, the concentrations of the environmental chemical pollutants in air, water, soil and the like are mainly analyzed through an external exposure method, and the concentrations are indirectly evaluated by combining an exposure model. The method is simple in sampling and easy to develop, and is developed more mature at present. However, the ways of exposing PAHs and VOCs to human body are complex and various, and the detection result of the environmental sample can not be extrapolated to the actual exposure dose in human body, so that it is difficult to accurately reflect the internal exposure load level of human body. Second, detection of PAHs and VOCs in blood can also be used to assess the exposure characteristics in humans. The method has the disadvantages that the blood collection belongs to invasive sampling, and the requirement on the sampling process is high and the difficulty is high; in addition, the biological half-life of VOCs in blood is short, volatility is high, target substances are easily lost, and accurate detection is difficult. Compare in measuring blood VOCs, VOCs metabolite (mVOCs) has more stable advantage because of its longer biological half-life and difficult volatility in the urine to the urine sample is because of not having the wound sampling process, and is convenient more to obtain.

Internal exposure is typically reflected by assessing the level of exposure markers in organisms, particularly humans. PAHs in the environment mainly enter the human body through air inhalation, water and food intake, and the like. Subsequently, under the action of various enzymes, PAHs are metabolized into hydroxylated metabolites (OH-PAHs), which are further combined with glucuronic acid or sulfate and excreted out of the body through urine. Therefore, the concentration of OH-PAHs in urine can reflect the exposure condition of PAHs in human body, and is widely used for evaluating the exposure level in PAHs in human body. After entering human body, VOCs can be hydrolyzed into alcohols (such as chlorophenol), and the alcohols are discharged out of the body through urine; the alcohol can also be further oxidized to carboxylic acid (e.g., trans-muconic acid) and then excreted; in addition, part of the major primary metabolites of VOCs can also be eliminated via urine via the formation of N-acetylated mercaptoacids via the thiol metabolic pathway. Therefore, the metabolites of VOCs in urine can also effectively reflect the actual exposure characteristics of VOCs in human bodies. In addition, relevant researches show that PAHs and VOCs exposure can also induce oxidative stress effect, 8-hydroxy-2 ' -deoxyguanosine (8-OHdG) in urine is used as a biomarker for evaluating oxidative stress and DNA oxidative damage caused by human carcinogen exposure, is widely applied to experimental research and clinical diagnosis, participates in index detection and mechanism analysis of oxidative stress and pathological changes, and has great application value in clinical diagnosis and treatment of cardiovascular diseases, diabetes, neurodegenerative diseases (Parkinson's disease, Alzheimer's disease) and cancers. Currently, 8-hydroxydeoxyguanosine (8-OHdG) which is the mainstream in the market is detected by using a special quantitative kit.

Because the dose of PAHs in vivo is influenced by various factors such as individual difference, living habits, diet and the like, one biomarker hardly reflects the actual exposure level of the PAHs, particularly the level of carcinogenic PAHs such as benzopyrene in vivo, more and more scholars comprehensively reflect the exposure level of the PAHs by the joint detection of various PAHs metabolites. At present, the PAHs exposure biomarkers in human bodies mainly comprise: metabolites of PAHs in urine, thioethers in urine, mutagenic substances in urine, PAH-DNA adducts, and PAH-protein adducts. However, most of the current detection methods are to hydrolyze the glucuronide and sulfuric acid combined product of monohydroxy PAHs into free OH-PAHs by using beta-glucuronidase or sulfatase, and then to perform the processes of extraction, concentration and enrichment and the like. At present, an analysis method for synchronously measuring various environmental chemical pollutants such as OH-PAHs, mVOCs and the like is lacked. For example, chinese patent application No. cn201610911596.x discloses a rapid detection method for urine hydroxyl polycyclic aromatic hydrocarbon, comprising the following steps: centrifuging the urine sample and carrying out enzymolysis treatment on the supernatant to free OH-PAHs so as to obtain a pretreated urine sample; adding evenly mixed dispersing agent acetone and extracting agent n-undecanol into the pretreated urine sample, evenly mixing, layering, and suspending the n-undecanol layer on the surface; the sample was cooled until the n-undecanol layer solidified, and the solidified n-undecanol layer was taken out and dissolved in an organic solvent for chromatography. The Chinese patent application No. 202010728067.2 discloses a solid phase extraction-liquid chromatography triple quadrupole mass spectrometry isotope dilution method for on-line determination of hydroxyl polycyclic aromatic hydrocarbon in urine, which comprises the steps of adding an isotope internal standard into urine, then adding beta-glucuronidase-arylsulfatase, carrying out enzymolysis on a constant temperature shaking table, adding an organic solvent after enzymolysis to precipitate protein, freezing and centrifuging, transferring centrifuged supernatant, carrying out on-line solid phase extraction through automatic valve switching, and then carrying out quantitative determination through liquid chromatography triple quadrupole mass spectrometry. And (4) obtaining the content of the hydroxyl polycyclic aromatic hydrocarbon in the urine through data processing and quantitative calculation. The Chinese invention application number: CN201710918513.4 discloses a screening technology for urine specific VOCs related to urinary system diseases by using methylene dichloride induced protonation reaction mass spectrometry, which is a novel liquid-phase VOCs detection means and can be used for directly and rapidly screening specific VOCs related to urinary system diseases from human urine. Firstly, adjusting a dichloromethane sampling system to ensure that the pressure in an ionizer is stabilized at 200 Pa; then extracting 50 microliters of pathological urine by using a disposable microsyringe and quickly injecting the pathological urine into a heat pipe, then carrying the gasified VOCs into an ionizer by carrier gas, and immediately carrying out protonation ionization by dichloromethane protonation and vacuum ultraviolet light; the ions enter a V-shaped mass spectrum through the migration cavity to be detected. And finally, comparing and analyzing the detection result with the normal values of the 16 VOCs in the urine, thereby determining the concentration range of the VOCs in the urine of the patient with the urinary system disease. The invention has important significance in reducing the lethality rate of bladder cancer and prostatic cancer. In addition, chinese patent document CN103175920A provides a method for detecting 8 OH-PAHs in urine by liquid-liquid extraction-gas chromatography-mass spectrometry; chinese patent document CN108872415A also establishes a method for simultaneously measuring 10 OH-PAHs in human urine by combining solid phase extraction-liquid chromatography-tandem mass spectrometry; patent CN112198239A discloses a method for rapidly detecting 8-OHdG in urine by combining an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technology. In the gas chromatography-mass spectrometry combined method adopted by the prior art, target compounds need to be derived in the sample pretreatment process, and the detection process is complicated; more importantly, the three methods can only detect a single metabolite or a target detection object, and if a plurality of metabolites and target detection objects need to be detected, sample pretreatment and detection need to be carried out for a plurality of times.

Therefore, the methods for detecting and analyzing the biological monitoring and internal exposure levels of PAHs and VOCs in human bodies, which are disclosed at present, can not complete synchronous detection of multiple detection objects of PAHs, VOCs and 8-OHdG in one detection process at present and obtain accurate and stable detection results because the detectable metabolites in a pretreated sample are less in variety or the target detection object is single in variety, or the detection methods, instruments and the like are matched with each other, so that the detection of a single marker of PAHs, VOCs and 8-OHdG is respectively carried out; meanwhile, data obtained by three times of detection is influenced by sample changes, various detection condition changes and other reasons, and has large errors, so that the data is difficult to be used for accurately analyzing the correlation among PAHs, VOCs and 8-OHdG.

In the prior art, the quantitative detection data of PAHs, VOCs and 8-OHdG can be respectively obtained by analyzing the samples for multiple times, the processing process of the quantitative detection data has long time, large solvent amount, high processing cost and analysis time, the detection and analysis of a large number of samples in epidemiology can not be carried out, and meanwhile, the quantitative detection data is not beneficial to environmental protection and is not beneficial to protecting the health of detection and analysis operators.

Disclosure of Invention

Aiming at the defects in the prior art, in particular to the problem that when the existing detection method is adopted to detect OH-PAHs, mVOCs and 8-OHdG, the adopted sample pretreatment, detection instruments and analysis methods are different and the detection can be completed in three times, a novel synchronous detection method for the exposure of volatile pollutants in urine and effect markers is provided, the three target detection objects are brought into one detection process of one sample to be completed by improving the sample pretreatment, detection and analysis schemes, and the method is convenient, quick and solvent-saving; meanwhile, three target detection object data synchronously obtained by one-time detection of one sample are adopted, and because the sample and the detection condition of the three target detection object data are not changed, the obtained data have no error, and the correlation among PAHs, VOCs and 8-OHdG can be further accurately analyzed.

The invention also aims at the defect that the current 8-OHdG data required by clinical diagnosis needs to be detected by using a kit or preparing a sample independently, and the detection is completed in the same detection process of the same sample with PAHs and VOCs, so that the detection data of three target detection objects are obtained by using one sample, a mathematical model is constructed to analyze the relationship among the detection data of the three target detection objects, the relationship among the group 8-OHdG, PAHs and VOCs exposure is researched, and the relationship is further used for revealing the rising contribution value, the main influence factors and other data of the 8-OHdG together, thereby providing support for the analysis and prediction of health exposure risk of related individual personnel, and the clinical diagnosis, treatment and epidemiological analysis and prevention of diseases.

The technical scheme provided by the invention for solving the problems is as follows:

a synchronous detection method for exposure of volatile pollutants and effect markers in urine is characterized in that multiple related monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs), volatile organic metabolites (mVOCs) and exposure effect markers (8-hydroxy-2-deoxyguanosine 8-OHdG) in the same urine sample are selected as target detection objects to be synchronously extracted, detected and analyzed, and the method comprises the following steps:

s1: pretreatment of the sample: unfreezing collected individual urine samples at 4 ℃ respectively, then uniformly mixing, putting 1 mL of the individual urine samples into a 15-mL PP centrifugal tube, and adding an internal standard indicator with a known amount into the centrifugal tube; subsequently, 1 mL of 1 mol/L sodium acetate-acetic acid buffer with pH =4.5 was added to adjust to pH = 5 or so, and then 10 μ L of 30U/mL β -glucuronidase/arylsulfatase was added; vortex, mixing uniformly, and performing enzymolysis for 6 h in water bath at 37 ℃; cooling to room temperature, adding 50 μ L of 1% formic acid to adjust pH, and mixing by vortex for 5 s;

s2: enrichment and separation of samples: transferring the solution after enzymolysis to a 500mg and 6 mL Poly-Sery HLB Pro solid phase extraction column activated by 6 mL dichloromethane, 6 mL methanol and 12 mL 0.1% formic acid aqueous solution in sequence for enrichment; then, first, 6 mL of formic acid 2: methanol 49: acetonitrile 49, and 8 mL of methanol 1: eluting with dichloromethane 1 solvent, and collecting the effluent solution into 15-mL glass centrifuge tube to obtain eluate containing two metabolites, namely OH-PAHs, mVOCs and exposure effect marker 8-OHdG;

s3: concentration and detection of samples: the eluate containing the above two metabolites and exposure effect marker is near-dried by blowing and concentrating, redissolved with 180 μ L methanol, added with 20 μ L of 1ppm back marker, i.e. dissolved to 200 μ L at-20%^oStanding in a refrigerator for one night, taking supernatant, centrifuging for 5 min at 12000 r/min, and taking supernatant again; performing on-board detection of OH-PAHs, mVOCs and 8-OHdG by HPLC-MS/MS respectively, and analyzing correspondingly to obtain content data of target detection objects OH-PAHs, mVOCs and 8-OHdG in urine;

s4: repeating the steps S1-S3, respectively completing the detection of OH-PAHs, mVOCs and 8-OHdG in a plurality of individual urine samples of the same batch to obtain the detection data of a plurality of urine samples of the batch, then summarizing and classifying the detection data, and carrying out the health exposure characteristic analysis of the OH-PAHs population: taking exposure and non-exposure as independent variables, and dividing factors of age, sex, post and operation time into continuous variables and classification variables, wherein the sex and the post belong to the classification variables, the age and the operation time belong to the continuous variables, and the content data of OH-PAHs, mVOCs and 8-OHdG are used as dependent variables; adopting SPSS analysis software to perform difference analysis on the classified variables and OH-PAHs, mVOCs and 8-OHdG detection data, and performing correlation analysis on the continuous variables and OH-PAHs, mVOCs and 8-OHdG detection data to obtain the correlation among three target detection objects;

s5: on the basis of the detection data of three target detection objects and the correlation among the detection data, a mathematical model for the individual damage prediction and evaluation analysis of the monohydroxy polycyclic aromatic hydrocarbon OH-PAHs is constructed, the detection data of the target detection objects of a single individual sample is substituted into the mathematical model, and the contribution value causing the increase of the individual DNA damage marker 8-OHdG is calculated, so that the method is further applied to the prediction and evaluation of the OH-PAHs health exposure risk of a single individual.

The application of the synchronous detection method for the exposure of the volatile pollutants in the urine and the effect markers is characterized in that the content data of target objects OH-PAHs, mVOCs and 8-OHdG in a detected urine sample are analyzed to be related to each other, and the method is applied to the evaluation of the exposure characteristics or clinical diagnosis of two types of pollutants ubiquitous in the environment in crowds, and specifically comprises the following steps:

(1) collecting sampling object information: collecting basic information of a sampling object, including age, gender, post and operation time;

(2) data classification: dividing factors of age, gender, post and operation time into continuous variables and classification variables, wherein the gender and the post belong to the classification variables, the age and the operation time belong to the continuous variables, and OH-PAHs, mVOCs and 8-OHdG content data are used as dependent variables and are classified correspondingly;

(3) and (3) correlation analysis: performing difference analysis on the classified variables and OH-PAHs, mVOCs and 8-OHdG detection data by adopting SPSS analysis software; carrying out correlation analysis on the continuous variable, OH-PAHs, mVOCs and 8-OHdG detection data to obtain a P value;

(4) and (4) judging a result: when P < 0.05, the exposure characteristics are significant; p < 0.01, indicating that the exposure characteristics are very significant; p > 0.05, no difference in exposure characteristics is indicated.

The application of the synchronous detection method is characterized in that the content data of target objects OH-PAHs, mVOCs and 8-OHdG in a plurality of individual urine samples in the same batch obtained by detection are analyzed to be mutually related, and the content data are applied to the evaluation of the group health exposure risk or the epidemiological analysis of the same batch of people together, and the method specifically comprises the following steps:

(1) data acquisition: acquiring data of hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs) of various pollutants, mVOCs of metabolites of various volatile organic compounds and an exposure effect marker 8-OHdG in urine samples of a plurality of individual persons, and then summarizing and unifying units, mg/L;

(2) statistical analysis: with not less than 20 pollutants and metabolites as independent variables and 8-OHdG as dependent variable, the mathematical model for calculating the contribution y to the increase in the dependent variable 8-OHdG concentration is:

y=aX₁+bX₂+…+nX_n(formula 1)

Wherein a and b … n are coefficients, X₁、X₂… Xn is an independent variable;

then using logistic regression analysis to obtain a P value;

(3) and (4) judging a result:

when P < 0.05, significance is present, indicating that the independent variable and the dependent variable are related, and the weight number of the variable is included in formula 1, and when P > 0.05, significance is absent, indicating that the independent variable and the dependent variable are unrelated, and no calculation is included.

The application of the synchronous detection method is characterized in that the content data of target objects OH-PAHs, mVOCs and 8-OHdG in the urine sample obtained by detection and the further analysis of the mutual relation are applied to the analysis or clinical diagnosis of the individual injury influence level of individual personnel together, and the method specifically comprises the following steps:

(1) data acquisition: acquiring data of various hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), various volatile organic compounds (mVOCs) metabolites and an exposure effect marker 8-OHdG in a urine sample of an individual to be detected, and unifying units (mg/L);

(2) influence analysis: and (3) respectively substituting the detection data of each target detection object in the sample into the formula 1, and calculating to obtain a contribution value y causing the increase of the individual DNA damage marker 8-OHdG so as to further judge the influence factors and the influence level.

Compared with the prior art, the application of the method for synchronously detecting the exposure of the volatile pollutants in the urine and the effect markers has the beneficial effects that:

1. the invention provides a synchronous detection method for multiple volatile pollutant exposure and effect markers in urine, which overcomes the defects of the prior detection technology, particularly solves the problems of long detection time, high cost, large data error and the like caused by the adoption of the prior detection method and the adoption of different sample pretreatment, detection instruments and analysis methods which need to finish detection of PAHs, VOCs and 8-OHdG in three times, selects multiple related monohydroxy polycyclic aromatic hydrocarbons OH-PAHs, volatile organic matter metabolites mVOCs and exposure effect markers 8-hydroxy-2-deoxyguanosine 8-OHdG in the same urine sample as target detection objects according to the requirements of specific application, and brings the three target detection objects into one detection process of one sample by improving the schemes of sample pretreatment, detection and analysis, the method is convenient and quick, saves solvent, adopts three target detection object data synchronously obtained by one-time detection of a sample, has no error in the obtained data because the sample and the detection condition are not changed, and can further carry out accurate analysis on the correlation among PAHs, VOCs and 8-OHdG.

2. According to the synchronous detection method for the exposure of the volatile pollutants and the effect markers in the urine, provided by the invention, 12 kinds of hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), 22 kinds of volatile organic metabolites (mVOCs) and the exposure effect marker 8-hydroxyl-2-deoxyguanosine (8-OHdG) in the urine can be synchronously extracted at one time through synchronously improving a target detection object and a sample pretreatment, detection and analysis scheme, and the separation and detection of the two kinds of metabolites and 8-OHGD can be respectively realized by adopting the same liquid chromatography column and HPLC-MS/MS, so that the efficiency of sample pretreatment and analysis and detection is greatly improved.

3. According to the synchronous detection method for the exposure of the volatile pollutants in the urine and the effect markers, the same solid-phase extraction column is used, so that the synchronous enrichment and separation of two types of target objects with obvious polarity difference are realized, meanwhile, the effect biomarkers 8-OHdG of the exposure of the pollutants are brought into the analysis range, time is saved, convenience is realized, the final recovery rate of all the target compounds is between 80% and 120%, and the accuracy of quantitative analysis is greatly improved. According to the invention, a Poly-Sery HLB Pro commodity column is adopted in the steps of synchronously enriching and separating the target substances of the sample, so that the solvent is greatly saved, and the chromatogram of the fraction can also keep a higher separation effect.

4. According to the synchronous detection method for the exposure of the volatile pollutants in the urine and the effect markers, provided by the invention, OH-PAHs, VOCs metabolites (mVOCs) and 8-hydroxy-2' -deoxyguanosine (8-OHdG) in the same sample are synchronously processed and analyzed, so that the error caused by multiple pretreatment can be reduced, and the accuracy of a detection result and an evaluation result is improved.

5. The application of the synchronous detection method provided by the invention further aims at the defect that the 8-OHdG data required by the current clinical diagnosis needs to be detected by using a kit or preparing a sample independently, and the detection is completed in the same detection process of the same sample with PAHs and VOCs, so that the detection data of three target detection objects are obtained by using one sample, the detection data of the three target detection objects and the correlation among the detection data are further applied to the research of the relationship among the exposure of the 8-OHdG, the PAHs and the VOCs together, the data such as the rising contribution value and the main influence factors of the 8-OHdG are further disclosed, and the support is provided for the analysis and prediction of the health exposure risk of related individuals, and the clinical diagnosis, treatment and epidemiological analysis and prevention of diseases.

6. The application of the synchronous detection method provided by the invention can more accurately judge and evaluate the exposure level and the damage condition of the human body by synchronously monitoring the exposure markers and the human body damage markers in various volatile pollutants in the same sample.

7. The application of the synchronous detection method provided by the invention obtains various pollution-free or exposure marker data by optimizing the special selection of the target detection object and the design of the detection scheme, overcomes the defect that the accuracy of the evaluation result is not strong because the health exposure risk of the PAHs (environmental pollutants) of the crowd and the (individual) human body is evaluated by adopting a single pollutant component at present, obtains various pollutants and exposure marker data synchronously reflected by the same sample and the correlation among the pollutants and exposure marker data by adopting the synchronous detection of the exposure and effect markers of various volatile pollutants in urine, and can greatly improve the accuracy of the evaluation result when being applied to the exposure condition evaluation of the PAHs and mVOCs.

8. The application of the synchronous detection method provided by the invention further aims at the defect that the 8-OHdG data required by the current clinical diagnosis needs to be detected by using a kit or preparing a sample independently, and the detection is completed in the same detection process with the same sample of PAHs and VOCs, so that the detection data of three target detection objects are obtained by using one sample, and the detection data of the three target detection objects and the correlation (difference and correlation) thereof are further applied to the research of the relationship among the 8-OHdG, PAHs and VOCs exposure together, so as to reveal the source of the 8-OHdG, and provide support for the clinical diagnosis, treatment, epidemiological analysis and prevention of related diseases.

9. The synchronous detection and application of the exposure of the volatile pollutants in the urine and the effect markers are based on the characteristics of metabolites of PAHs and VOCs and damage markers in the urine, not only are PAHs metabolites (OH-PAHs) and VOCs metabolites (mVOCs) in the urine brought into a detection range simultaneously, but also 8-OHdG is used as the effect markers of the exposure of the pollutants (indexes of various pollutants participating in oxidative stress and pathological changes) for synchronous analysis and detection, and the synchronous detection and application of the exposure of the volatile pollutants and the effect markers in the urine have important significance for evaluating the exposure characteristics of two types of pollutants commonly existing in the environment in crowds and the influence of the two types of pollutants on the health of human bodies.

10. The synchronous detection and application of the exposure of the volatile pollutants in the urine and the effect marker, provided by the invention, can be used for detecting the detection method of the effect marker 8-OHdG, analyzing a model (difference and correlation) of data and correlation, can also be used for clinically screening and diagnosing diseases such as colorectal cancer and the like of a single individual, can be further used for analyzing an induction factor of the disease, and can be used for carrying out epidemiological analysis on population and individual induction influence factors of specific diseases (particularly occupational diseases) induced by the pollutants and the like, so that the synchronous detection and application of the detection data and the health exposure risk assessment result have wide medical application prospects, and the application range of the detection data and the health exposure risk assessment result is expanded.

Drawings

FIG. 1 is a schematic flow chart of a method for simultaneous detection of exposure of volatile contaminants and effector markers in urine according to an embodiment of the present invention.

FIG. 2 is a chromatogram of HPLC-MS/MS analysis of OH-PAHs in an example of the present invention.

FIG. 3 is a chromatogram of HPLC-MS/MS analysis of mVOCs and 8-OHdG in an example of the present invention.

FIG. 4 is a bar graph of the internal standard recovery of target compounds in urine matrix low (LL), Medium (ML) and High (HL) spiked samples in accordance with an example of the present invention.

Detailed Description

The invention is explained in detail below with reference to the figures and examples:

example 1:

referring to fig. 1-4, the synchronous detection and application of exposure of volatile contaminants and effect markers in urine provided in this embodiment is a method for detecting a group urine sample and an application thereof, and specifically, the synchronous detection and application of exposure of volatile contaminants and effect markers in urine of the present invention are specifically described by taking urine samples of 38 male workers (21 office workers and 17 workshop workers) in south china, which are aged 20 to 50 years in a certain petrochemical enterprise.

The synchronous detection method for the exposure of the volatile pollutants and the effect marker in the urine and the application thereof provided by the embodiment are characterized in that 38 parts of samples are collected firstly, then the samples are sequentially and respectively detected in sequence, and multiple related monohydroxy polycyclic aromatic hydrocarbons OH-PAHs, volatile organic metabolites mVOCs and exposure effect marker 8-hydroxy-2-deoxyguanosine 8-OHdG existing in the same (each) part of urine sample are selected as target detection objects to be synchronously extracted, detected and analyzed, and the synchronous detection method comprises the following steps:

(1) sampling object basic information acquisition

Exposure influencing factors of 38 professional workers, including basic information such as sex, age, post position, time of employment and the like, are collected.

(2) Sample collection

The middle morning urine of 38 workers was collected, stored frozen at-80 ℃ in a pre-treated 70 mL polyethylene urine cup, and the specific gravity of the urine was measured over 12 hours.

(3) Pretreatment of the sample (i.e., step S1:): referring to fig. 1, 38 individual urine samples were collected, one at a time: unfreezing collected individual urine samples at 4 ℃ respectively, then uniformly mixing, putting 1 mL of the individual urine samples into a 15 mL PP centrifugal tube, and adding an internal standard indicator with a known amount into the PP centrifugal tube; subsequently, 1 mL of 1 mol/L sodium acetate-acetic acid buffer with pH =4.5 was added to adjust to pH = 5 or so, and then 10 μ L of 30U/mL β -glucuronidase/arylsulfatase was added; vortex, mixing uniformly, and performing enzymolysis for 6 h in water bath at 37 ℃; cooling to room temperature, adding 50 μ L of 1% formic acid to adjust pH, and mixing by vortex for 5 s; the internal standard indicator comprises: 20 ng 2-OH-Nap-d₇, 20 ng 2-OH-Flu-d₉, 20 ng 3-OH-Phe-¹³C₆, 20 ng AAMA-d₄, 20 ng PGA-d₅, 20 ng MA-d₅,20 ng BPMA-d₇, 20 ng PMA-d₅, 20 ng TCVMA-d₃, 20 ng 8-OHdG-¹³C,¹⁵N₂. Wherein, 1 mL of 1 mol/L, pH =4.5 sodium acetate-acetic acid buffer solution is used for adjusting the pH of urine, an optimal pH condition is created for the subsequently added beta-glucuronidase/arylsulfatase, and the beta-glucuronidase/arylsulfatase performs enzymolysis in a water bath at 37 ℃, so that the beta-glucuronidase/arylsulfatase can exert the optimal enzymolysis effect, and the combined state metabolite and 8-OHdG in the urine are fully released; wherein after cooling to room temperature, adding 50 μ L of 1% formic acid to adjust pH before solid phase extraction, which is favorable for retaining target metabolite on HLB Pro solid phase extraction columnHigher recovery rate is obtained.

(4) Enrichment and isolation of the sample (i.e., step S2): transferring the solution after enzymolysis to a 500mg and 6 mL Poly-Sery HLB Pro solid phase extraction column activated by 6 mL dichloromethane, 6 mL methanol and 12 mL 0.1% formic acid aqueous solution in sequence for enrichment; then, first, 6 mL of formic acid 2: methanol 49: acetonitrile 49, and 8 mL of methanol 1: eluting with dichloromethane 1 solvent, and collecting the effluent solution into 15-mL glass centrifuge tube to obtain eluate containing two metabolites, namely OH-PAHs, mVOCs and exposure effect marker 8-OHdG; the solid phase extraction column adopts a Poly-Sery HLB Pro column with the specification of 500mg and 6 ml, and nonpolar and polar target substances with wide range of properties are well reserved through the arrangement, so that the conditions of enrichment, separation and quantitative research are met, and the use of a solvent is reduced;

(5) concentration and detection of sample (i.e., step S3): the eluate containing the above two metabolites and exposure effect marker is near-dried by blowing and concentrating, redissolved with 180 μ L methanol, added with 20 μ L of 1ppm back marker, i.e. dissolved to 200 μ L at-20%^oStanding in a refrigerator for one night, taking supernatant, centrifuging for 5 min at 12000 r/min, and taking supernatant again; performing on-board detection of OH-PAHs, mVOCs and 8-OHdG by HPLC-MS/MS respectively, and analyzing correspondingly to obtain content data of target detection objects OH-PAHs, mVOCs and 8-OHdG in urine; wherein the label returning indicators are as follows: 20 ng of 1-OH-Pyr-d9 and 20 ng of HA-d 5; the HPLC-MS/MS is a combination of high performance liquid chromatography and mass spectrum, the liquid chromatography is used as a separation system, and the mass spectrum is used as a detection system; wherein, HPLC is an initial compound, primary MS is used for carrying out mass spectrum analysis on each peak in an HPLC spectrogram, and secondary MS is used for carrying out further dissociation analysis on the peak in the primary MS so as to determine the peak structure of the primary MS.

(6) Repeating the steps (3) to (5) (namely step S4), respectively completing the detection of OH-PAHs, mVOCs and 8-OHdG in the same batch of 38 individual urine samples, obtaining the detection data of the 38 urine samples in the batch, then summarizing and classifying, and performing the health exposure characteristic analysis of the OH-PAHs population: taking exposure and non-exposure as independent variables, and dividing factors of age, sex, post and operation time into continuous variables and classification variables, wherein the sex and the post belong to the classification variables, the age and the operation time belong to the continuous variables, and the content data of OH-PAHs, mVOCs and 8-OHdG are used as dependent variables; adopting SPSS analysis software to perform difference analysis on the classified variables and OH-PAHs, mVOCs and 8-OHdG detection data, and performing correlation analysis on the continuous variables and OH-PAHs, mVOCs and 8-OHdG detection data to obtain the correlation among three target detection objects;

(7) exposure feature analysis (i.e., step S5): on the basis of detection data of three target detection objects and correlation among the detection data, a mathematical model for individual damage prediction and evaluation analysis of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) and mVOCs is constructed, the detection data of the target detection objects of a single individual sample is substituted into the mathematical model (formula 1), and a contribution value causing the increase of an individual DNA damage marker 8-OHdG is calculated, so that the method is further applied to prediction and evaluation of the healthy exposure risk of the OH-PAHs and the mVOCs of the individual and judgment of influence factors and influence levels.

The urine detection data of 38 occupational workers are sorted, summarized and classified, workshop workers are used as an exposure group, office workers are used as a comparison group, SPSS analysis software is adopted to analyze the differences and the correlations of mVOC and OHPAH one by one, and a P value is obtained, and is shown in tables 5 and 6.

In the foregoing steps (1) - (7), the present embodiment provides more detailed steps as follows:

1. material preparation

1.1 instruments and materials: agilent 1260 liquid chromatograph, AB SCIEX API 4000 triple quadrupole mass spectrometer (applied biosystems, usa), Agilent infinity lab poroshell 120 EC-C18 (4.6 × 50mm, 2.7 μm) liquid chromatography column, water bath (manufactured by sincery analytical instruments, shanghai), nitrogen blower (berlin hesy, usa), Milli-Q ultrapure water system (merck, germany), vortex shaker (tranner, usa), 15 mL PP centrifuge tube, 15 mL glass screw cap centrifuge tube (sekko, japan), short glass pasteur pipette (shanghai spectrum experiment, china), Poly-series HLB Pro (500 mg, 6 mL) (shanghai spectrum experiment, china), weighing paper (shanghai bo bio science, china).

1.2 reagent and standard: chromatographically pure ammonium acetate, acetic acid, sodium acetate, methanol, acetonitrile (shanghai' an spectral experimental science, china), chromatographic grade dichloromethane (merck, germany), 12 OH-PAHs standards: 1-OH-Nap, 1-OH-Pyr, 6-OH-Chr (Accustandard Inc, New Haven, USA), 2-OH-Nap, 2-OH-Flu (Chiron AS, Trondheim, Norway), 3-OH-Flu, 3-OH-BaP (Toronto Research Chemicals, Toronto, Canada), 1-OH-Phe, 2-OH-Phe, 3-OH-Phe, 4-OH-Phe, 9-OH-Phe (Dr. Ehrenstontorfer, Augsburg, Germany), and 4 isotopic labels thereof: 2-OH-Nap-d₇, 2-OH-Flu-d₉, 1-OH-Pyr-d₉(Toronto Research Chemicals, Toronto, Canada), 3-OH-Phe-¹³C₆(Cambridge Isotrope Laboratories, Andover, MA, USA); 22 volatile organic metabolite standards: AAMA, CYMA, BPMA, DHBMA, MHBMA3, PMA, AMCC, IPMA3, PHEMA, TCVMA, BMA, 2,2-DCVMA (Toronto Research Chemicals, Toronto, Canada), MU, TTCA, PGA (Sigma-Aldrich, Bormem, Belgium), 1,2-DB (Accustandard Inc., New Haven, Chemical), 4-CCT, TGA (Dr. Ehrenstorfer, Augsburg, Germany), PPA, MA (Tokyo Chemical Industry Co., Tokyo, Japan), 2MHA (alfer Aesar, Shanghai, China), HA (Addin Co., Shanghai, Shanna); and 7 isotopic marker standards: AAMA-d₄, PGA-d₅, MA-d₅, BPMA-d₇, PMA-d₅, TCVMA-d₃, HA-d₅(Torto Research Chemicals, Torto, Canada), 8-OHdG (allele Aesar, Shanghai, China) and isotopic marker 8-OHdG-¹³C,¹⁵N₂(Toronto Research Chemicals, Toronto, Canada); isotope standard 2-OH-Nap-d₇, 2-OH-Flu-d₉, 3-OH-Phe-¹³C₆Used as an internal standard for OH-PAHs quantification and an isotope standard AAMA-d₄, PGA-d₅, MA-d₅, BPMA-d₇, PMA-d₅, TCVMA-d₃8-OhdG-ion as an internal standard for quantitation of mVOCs¹³C,¹⁵N₂Used in 8-OhdG quantitationAnd (4) marking. Isotope standard product 1-OH-Pyr-d₉Used as OH-PAHs retro-label, HA-d₅Used as a retro-standard for mVOCs. The basic information and instrumental analysis parameters for each target analyte are detailed in table 1.

TABLE 1 basic information of target analytes and instrumental analysis parameters

2. Analytical method

2.1 sample information

38 male workers between 20 and 50 years of age were recruited from a petrochemical enterprise in south China. The collected urine samples were mid-morning urine, frozen at-80 ℃ in a pre-treated 70 mL polyethylene urine cup, and the specific gravity of the urine was measured within 12 hours.

2.2 pretreatment of samples

(1) Enzymolysis of the sample: thawing collected urine samples at 4 ℃ and then uniformly mixing, putting 1 mL of the urine samples into a 15 mL PP centrifugal tube, adding an internal standard indicator with a known amount (20 ng) into the PP centrifugal tube, then adding 1 mL of 1 mol/L sodium acetate-acetic acid buffer (pH = 4.5) to adjust the pH, adding 10 mu L of beta-glucuronidase/arylsulfatase (30U/mL), vortex uniformly mixing, carrying out water bath enzymolysis at 37 ℃ for 6 h, cooling to room temperature, adding 50 mu L of 1% formic acid to adjust the pH, vortex 5 s and uniformly mixing;

(2) enrichment and separation of samples: the solution after the above enzymatic hydrolysis was loaded on a Poly-Sery HLB Pro (500 mg, 6 mL) solid phase extraction cartridge activated sequentially with 6 mL of dichloromethane, 6 mL of methanol, and 12 mL of 0.1% formic acid aqueous solution, for enrichment, and then, 6 mL of formic acid: methanol: acetonitrile (2: 49:49, v/v/v), and then 8 mL of methanol: eluting with dichloromethane (1: 1, v/v), and collecting the effluent solution into a 15 mL glass centrifuge tube to obtain an eluent containing two metabolites and 8-OHdG;

(3) concentration and detection of samples: the eluate containing the above two metabolites and 8-OhdG is concentrated by blowing to near dryness, redissolved with 180 μ L methanol, added with 20 μ L of 1ppm back standard indicator (i.e. dissolved to 200 μ L) and dried to obtain a solution of-20%^oAnd C, standing the mixture in a refrigerator overnight, taking supernatant, centrifuging the mixture at 12000 r/min for 5 min, taking the supernatant again, respectively carrying out on-machine detection on OH-PAHs, mVOCs and 8-OHdG by adopting HPLC-MS/MS, and analyzing the content of the target substance. The complete pretreatment process is shown in detail in FIG. 1.

2.3 HPLC-MS/MS instrumental analysis method

OH-PAHs, mVOCs and 8-OHdG are all analyzed by adopting a high performance liquid chromatography-triple quadrupole mass spectrometer (HPLC-MS/MS), the Agilent Infinity Lab poroshell 120 EC-C18 (4.6 multiplied by 50mm, 2.7 mu m) chromatographic column is adopted to realize the chromatographic separation of the target, the temperature of a column box is set to be 40 ℃, the sample injection volume is 10 mu L, the mass spectrum ion source is ESI, and the mass spectrum is in an MRM scanning mode. Other instrument parameters were as follows: the temperature of the drying gas is 550^oC, gas flow rate of 10 mL/min^-1The input voltage is-10V, the output voltage of the collision cell is-15V, the ion spray voltage is-4500V, and the ion source mode of negative electric spray ionization is adopted.

(1) Chromatographic separation of OH-PAHs: methanol (phase A) and ultrapure water (phase D) were used as mobile phases, the flow rates were set to 0.4 mL/min, the chromatograms of 12 OH-PAHs and 4 isotopic markers thereof and the mobile phase elution program were as shown in FIG. 2 and Table 2, respectively, and the total analysis time was 17.1 min.

TABLE 2.12 mobile phase elution procedure for OH-PAHs

(2) Chromatographic separation of mVOCs and 8-OHdG: acetonitrile (phase A) and 0.1% formic acid aqueous solution (phase B) were used as mobile phases with a flow rate of 0.4 mL/min, 22 mVOCs and 6 internal isotope standards thereof, 8-OHdG and 8-OHdG-¹³C,¹⁵N₂The chromatogram and the mobile phase elution program of (1) are respectively shown in FIG. 3 and Table 3, and the whole analysis process is 14.1 min.

TABLE 3.22 procedures for mobile phase elution of mVOCs and 8-OHdG

2.4 detection results and applications

After the detection of OH-PAHs, mVOCs and 8-OHdG in 38 individual urine samples in the same batch is completed, the correlation between the OH-PAHs, mVOCs and 8-OHdG detection data in a plurality of urine samples in the same batch is analyzed, and the correlation is used as the basis for evaluating the healthy exposure risk of the monohydroxy polycyclic aromatic hydrocarbon OH-PAHs and mVOCs groups, so that the accuracy of the healthy exposure risk evaluation result of the groups OH-PAHs and mVOCs is improved.

Summarizing and classifying the obtained detection data of the 38 urine of the batch, and performing healthy exposure characteristic analysis of the population OH-PAHs: taking exposure and non-exposure as independent variables, and dividing factors of age, sex, post and operation time into continuous variables and classification variables, wherein the sex and the post belong to the classification variables, the age and the operation time belong to the continuous variables, and the content data of OH-PAHs, mVOCs and 8-OHdG are used as dependent variables; adopting SPSS analysis software to perform difference analysis on the classified variables and OH-PAHs, mVOCs and 8-OHdG detection data, and performing correlation analysis on the continuous variables and OH-PAHs, mVOCs and 8-OHdG detection data to obtain the correlation among three target detection objects;

the steps of predicting and assessing the health exposure of an individual are: on the basis of detection data of three target detection objects and correlation among the detection data, a mathematical model for individual damage prediction and evaluation analysis of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) and mVOCs is constructed, the detection data of the target detection objects of an individual sample to be detected is substituted into the mathematical model, and a contribution value causing the increase of the DNA damage marker 8-OHdG of the individual is calculated, so that the method is further applied to prediction and evaluation of the healthy exposure risk of the OH-PAHs and the mVOCs of the individual, and judgment of influence factors and influence levels is carried out.

2.4.1 method validation

Due to the lack of standard reference substances suitable for urine analysis, the present invention verifies the applicability of the method by means of a labeling matrix (mixed urine sample) and blank experiments. To ensure a low background value of the target analyte in the urine matrix sample, a middle-morning urine sample of general population in the low exposure area was collected, mixed well and divided into 12 aliquots (1 mL per aliquot); of these, 3 were spiked with low concentration (LL, 2 ng) of target compound, 3 with medium concentration (ML, 10 ng), and 3 with high concentration (HL, 40 ng); the remaining 3 replicate samples were used as non-spiked controls. Setting three blank samples to monitor background pollution; procedure blank and average concentration of target compound in non-spiked urine samples were used for calibration of assay results for spiked samples. The reliability and applicability of the developed method was evaluated by the linearity of the calibration curve, the limit of detection of the target compound (LOD), the limit of quantitation (LOQ), and the accuracy and precision of each analyte, as detailed below.

(1) Linearity

Two calibration curves were prepared based on the target compound species analyzed: 12 OH-PAHs standard mixtures (1-OH-Nap, 2-OH-Nap, 2-OH-Flu, 3-OH-Flu, 1-OH-Phe, 2-OH-Phe, 3-OH-Phe, 4-OH-Phe, 9-OH-Phe, 1-OH-Pyr, 6-OH-Chr, 3-OH-BaP); 22 mVOCs and 8-OHdG standard mixtures (AAMA, CYMA, BPMA, DHBMA, MHBMA3, PMA, AMCC, IPMA3, PHEMA, TCVMA, BMA, 2,2-DCVMA, MU, TTCA, PGA, 1,2-DB, 4-CCT, TGA, PPA, MA, 2MHA, HA, 8-OHdG). The concentration range of the calibration curve sets 9 data points, respectively, according to the expected concentration in the urine sample. The calibration curves are all linear models, passing through the linear correlation coefficient (R)²) And evaluated. In the present invention, all target analytes have good correlationProperty, R²Not less than 0.996. See table 4 for details.

TABLE 4 verification results of the established urinalysis method

(2) Limit of quantitation (LOD) and limit of detection (LOQ)

LOD was calculated from the 3-fold signal-to-noise ratio (S/N = 3) of the corresponding compound at the lowest concentration standard curve point. LOQ is set as the mean of the target compounds detected in the program blank plus three times the standard deviation. For target compounds not detected in the program blank, LOQ was set to 10 signal-to-noise ratio (S/N = 10) of the target compound at the lowest concentration standard curve point. In the study, LOQ of OH-PAHs, mVOCs and 8-OHdG is 0.010-7.567 ng/mL, 0.036-4.733 ng/mL and 0.204 ng/mL respectively, which are detailed in Table 4.

(3) Accuracy and precision

Accuracy was assessed by recovery of each target compound in the urine matrix spiked sample, i.e., the percentage of the detected value of each analyte in the spiked sample (corrected by the procedure and the amount in the matrix blank sample) to the actual spiked amount. The precision of the analytical method (also referred to as the reproducibility of the method) is the Relative Standard Deviation (RSD) of three replicate samples under reproducible conditions.

Overall, validation results show that the analytes in the LL, ML and HL groups all have good accuracy and precision. The average accuracy range of OH-PAHs, mVOCs and 8-OHdG, the accuracy of OH-PAHs, mVOCs and 8-OHdG in the low-labeling (LL) sample is 81-118% (RSD < 12%), 81-117% (RSD < 15%) and 108% (RSD < 4%); the corresponding accuracies in the medium-labeled (HL) samples are 81-113% (RSD < 18%), 76-114% (RSD < 11%), and 101% (RSD < 6%); the corresponding accuracies in the highly spiked (HL) samples were 81-117% (RSD < 11%), 77-120% (RSD < 10%), 91% (RSD < 3%) (see Table 4), respectively. The accuracy and precision of the detection results of all the targets in the embodiment are equivalent to or better than those of the published documents. Recovery of the respective Internal Standard (IS) in the spiked matrix samples of OH-PAHs, mVOCs and 8-OHdG ranged from 73. + -.2% to 101. + -.2%, 82. + -.5% to 120. + -.2%, and 86. + -.8% to 118. + -.3%, respectively (see FIG. 4).

TABLE 5 mVOC exposure analysis Table

TABLE 6 analysis table of exposure of OHPAHs

The application of the method for synchronously detecting exposure of volatile pollutants and effect markers in urine provided in this embodiment is to analyze content data of target objects OH-PAHs, mVOCs and 8-OHdG in a plurality of (population) urine samples obtained by detection and a relationship therebetween on the basis of the detection data, and apply the data and the relationship together to evaluate the exposure characteristics or clinical diagnosis of two types of pollutants ubiquitous in the environment in a population, and specifically includes the following steps:

(1) collecting sampling object information: collecting basic information of a sampling object, including collecting basic information of exposure influence factors of 38 professional workers, including age, gender, post, operation time (working time) and the like;

(2) data classification: dividing factors such as age, gender, post, operation time and the like into continuous variables and classification variables, wherein the gender and the post belong to the classification variables, the age and the operation time belong to the continuous variables, and OH-PAHs, mVOCs and 8-OHdG content data are used as dependent variables and are classified correspondingly;

(4) and (4) judging a result: when P < 0.05, the difference of the exposure characteristics is significant; p > 0.05, no statistical difference in exposure profile is indicated.

And (5) carrying out an exposure characteristic judgment result: when the condition "P < 0.05, it is significant, and when P > 0.05, there is no difference" the exposure was judged, and it is understood from table 5 that the P value of the mfcos such as TGA, MU, 1,2-DB, IPMA3, HA, 2MHA, BPMA, 2-DCVMA, 4-CCT, TCVMA, PPA, etc. of the exposed group (plant worker) was less than 0.05, and the difference between the exposed group and the non-exposed group was significant, that is, GA, MU, 1,2-DB, IPMA3, HA, 2MHA, BPMA, 2-DCVMA, 4-CCT, TCVMA, PPA in urine of the plant worker was higher than that of office workers.

Similarly, it can be judged from Table 6 that the urine of workers in the workshop contains higher amounts of 1-OH-Nap, 2-OH-Flu, 3-OH-Phe, 1-OH-Pyr and 6-OH-Chr than office workers.

Another application of the synchronous detection method of the embodiment of the invention is further obtained by analyzing and detecting content data and mutual relations of target objects, namely, OH-PAHs, mVOCs and 8-OHdG, in a plurality of individual urine samples in the same batch, and is applied to evaluating group health exposure risk or epidemiological analysis of the same batch of people together, and the method specifically comprises the following steps:

(1) data acquisition: acquiring data of various PAHs metabolites, namely hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), various volatile organic metabolites (mVOCs) and an exposure effect marker (8-OHdG) in urine samples of 38 individuals, and summarizing and unifying units (mg/L);

(2) statistical analysis: with 32 pollutants and metabolites as independent variables and 8-OHdG as dependent variable, the mathematical model for calculating the contribution y to the rise in the dependent variable 8-OHdG concentration is:

y=aX₁+bX₂+…+nX_n(formula 1)

then using logistic regression analysis to obtain a P value;

(3) and (4) judging a result:

when P is less than 0.05, significance exists, which indicates that the independent variable is a protection or danger factor of the dependent variable and has statistical significance, the weight number of the variable is calculated in the formula 1, and when P is more than 0.05, significance does not exist, which indicates that the influence of the independent variable on the dependent variable has no statistical significance and is not included in the calculation.

Concretely, 12 kinds of hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), 22 kinds of volatile organic metabolites (mVOCs) and 8-OHdG data are firstly arranged, a unit is unified, mg/L or ug/L is unified, 32 kinds of pollutant metabolites are used as independent variables, 8-OHdG is used as dependent variables, and y = aX₁+bX₂+…+nX_nWherein a and b … n are coefficients, X₁、X₂… Xn are independent variables and were analyzed by logistic regression to give P values. When P is less than 0.05, significance exists, which indicates that the independent variable is a protection or danger factor of the dependent variable, statistical significance exists, the weight number of the variable is included in the calculation, and when P is more than 0.05, significance is absent, which indicates that the influence of the independent variable on the dependent variable has no statistical significance and is not included in the calculation. The results are shown in Table 7.

TABLE 7 weight coefficients and significance of mVOC, OHPAHS and 8-OHdG

As can be seen from Table 7, the P values of 4-OH-Phe, MU and TCVMA in the pollutants are less than 0.05, and the 4-OH-Phe, MU and TCVMA are related to 8-OHdG, i.e., the 4-OH-Phe, MU and TCVMA have certain harm to human health and are one of the important reasons for increasing the concentration of 8-OHdG. The contribution y y of the above factors to the increase in concentration of 8-OHdG =0.26 × X₁+0.84*X₂+0.72*X₃The higher the value of y, the more damage to the human body caused by the relevant factors.

Example 2:

the synchronous detection and application of exposure of volatile pollutants and effect markers in urine provided by this embodiment are basically the same as those in embodiment 1, except that the data and the correlation are further applied to analysis or clinical diagnosis of individual injury influence levels of individual individuals on the basis of the detection method, data and population application of the group urine samples obtained by analyzing 38 samples in embodiment 1. Namely, the content data and the relationship among the content data of the target substances, namely, OH-PAHs, mVOCs and 8-OHdG in the urine sample obtained by detecting and analyzing the 38 samples obtained in the embodiment 1 are applied to the analysis or clinical diagnosis of the individual injury influence level of single individual personnel, and the method specifically comprises the following steps:

(1) data acquisition: acquiring data of multiple hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs), multiple volatile organic compounds (mVOCs) metabolites and exposure effect markers (8-OHdG) in urine samples (1 in 38 samples) of a single individual to be detected, unifying units and mg/L;

(2) influence analysis: the detection data of each target detection object of the sample is respectively substituted into the formula 1: y = aX₁+bX₂+…+nX_nThe contribution y that leads to an increase in the individual's DNA damage marker 8-OHdG is calculated. The higher the value of y, the more the human body is damaged by the relevant factors.

In this embodiment, urine sample detection data of zhang san among 38 persons is randomly extracted, individual injury prediction and evaluation are performed, and the detection data and analysis results are specifically substituted into the mathematical model y = 0.26X₁+0.84*X₂+0.72*X₃Analyzing the level of the effect of the injury in the subject. The concentration of 4-OH-Phe in the Zhang Sanxue urine sample data is 0.04 ng/L, the MU concentration is 2.78 ng/L, TCVMA is 15.3 ng/L, and the above factors can result in the contribution value of the 8-OHdG concentration increase: y =0.26 × 0.04+0.84 × 2.78+0.72 × 15.3=13.36 ng/L. The calculation result of the contribution value shows that the body is damaged after the human body is exposed by MU, 4-OH-Phe and TCVMA, wherein the damage contribution of TCVMA is the largest.

It should be noted that, in other embodiments of the present invention, different schemes obtained by specifically selecting steps, components, ratios, process parameters and conditions within the scope of the steps, components, ratios, process parameters and conditions described in the present invention can achieve the technical effects described in the present invention, and therefore, the present invention is not listed one by one.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Those skilled in the art can make numerous possible variations and modifications to the present invention, or modify equivalent embodiments, using the methods and techniques disclosed above, without departing from the scope of the present invention. All equivalent changes in the components, proportions and processes according to the present invention are intended to be covered by the scope of the present invention.

Claims

1. A synchronous detection method for exposure of volatile pollutants and effect markers in urine is characterized in that multiple related monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs), volatile organic metabolites (mVOCs) and exposure effect markers (8-hydroxy-2-deoxyguanosine 8-OHdG) in the same urine sample are selected as target detection objects to be synchronously extracted, detected and analyzed, and the method comprises the following steps:

s1: pretreatment of the sample: unfreezing collected individual urine samples at 4 ℃ respectively, then uniformly mixing, putting 1 mL of the individual urine samples into a 15-mL PP centrifugal tube, and adding an internal standard indicator with a known amount into the centrifugal tube; subsequently, 1 mL of 1 mol/L sodium acetate-acetic acid buffer pH =4.5 was added to adjust to pH = 5, followed by 10 μ L, 30U/mL β -glucuronidase/arylsulfatase; vortex, mixing uniformly, and performing enzymolysis for 6 h in water bath at 37 ℃; cooling to room temperature, adding 50 μ L of 1% formic acid to adjust pH, and mixing by vortex for 5 s;

s3: concentration and detection of samples: blowing, concentrating and nearly drying the elution liquid nitrogen containing the two metabolites and the exposure effect marker, re-dissolving the elution liquid nitrogen with 180 mu L of methanol, adding 20 mu L of 1ppm label returning indicator, namely, dissolving the mixture to 200 mu L, standing the mixture for one night in a refrigerator at the temperature of 20 ℃ below zero, taking supernatant, centrifuging the supernatant for 5 min at 12000 r/min, and taking the supernatant again; performing on-board detection of OH-PAHs, mVOCs and 8-OHdG by HPLC-MS/MS respectively, and analyzing correspondingly to obtain content data of target detection objects OH-PAHs, mVOCs and 8-OHdG in urine;

s4: repeating the steps S1-S3, respectively completing the detection of OH-PAHs, mVOCs and 8-OHdG in a plurality of individual urine samples of the same batch to obtain the detection data of a plurality of urine samples of the batch, then summarizing and classifying, and performing the health exposure characteristic analysis of the groups OH-PAHs and mVOCs: dividing factors of age, gender, post and operation time into continuous variables and classification variables, wherein the gender and the post belong to the classification variables, the age and the operation time belong to the continuous variables, OH-PAHs and mVOCs content data are used as independent variables, and 8-OHdG content data are used as dependent variables; and (3) adopting SPSS analysis software to perform differential analysis on the classified variables and the OH-PAHs, mVOCs and 8-OHdG detection data, and performing correlation analysis on the continuous variables and the OH-PAHs, mVOCs and 8-OHdG detection data to obtain the correlation among the three target detection objects.

2. The method for the simultaneous detection of exposure of volatile contaminants and effector markers in urine according to claim 1, further comprising the steps of:

s5: based on the detection data of three target detection objects and the correlation among the detection data, a mathematical model for individual damage prediction and evaluation analysis of monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) and mVOCs is constructed, the detection data of the target detection objects of a single individual sample is substituted into the mathematical model, and the contribution value causing the increase of the individual DNA damage marker 8-OHdG is calculated.

3. The method for the simultaneous detection of exposure of volatile contaminants and effector markers in urine according to claim 1, further comprising the steps of:

the internal standard indicator in step S1 includes: 20 ng 2-OH-Nap-d₇, 20 ng 2-OH-Flu-d₉, 20 ng 3-OH-Phe-¹³C₆, 20 ng AAMA-d₄, 20 ng PGA-d₅, 20 ng MA-d₅, 20 ng BPMA-d₇, 20 ng PMA-d₅, 20 ng TCVMA-d₃, 20 ng 8-OHdG-¹³C,¹⁵N₂。

4. The method for synchronously detecting the exposure of the volatile pollutants and the effect markers in the urine as claimed in claim 1, wherein in step S1, 1 mL of 1 mol/L, pH =4.5 sodium acetate-acetic acid buffer is used for adjusting the pH of the urine, so as to create an optimal pH condition for the subsequently added β -glucuronidase/arylsulfatase, and perform enzymolysis in a water bath at 37 ℃ to enable the β -glucuronidase/arylsulfatase to exert an optimal enzymolysis effect, thereby fully releasing the combined metabolites and 8-OHdG in the urine; after cooling to room temperature in step S1, 50 μ L of 1% formic acid was added to adjust the pH before solid phase extraction, in order to facilitate retention of the target metabolite on the HLB Pro solid phase extraction cartridge, resulting in higher recovery.

5. The method for synchronously detecting the exposure of the volatile pollutants and the effect markers in the urine as claimed in claim 1, wherein the solid phase extraction column in step S2 adopts a Poly-Sery HLB Pro column with the specification of 500mg and 6 ml, and by the arrangement, both nonpolar and polar targets with wide range of properties can be well retained, so as to meet the conditions of enrichment, separation and quantitative research, and simultaneously reduce the use of solvents.

6. The method for synchronously detecting the exposure of the volatile pollutants in the urine and the effect markers according to claim 1, wherein the back-marking indicators in the step S3 are as follows: 20 ng of 1-OH-Pyr-d9 and 20 ng of HA-d 5.

7. The method for synchronously detecting the exposure of the volatile pollutants and the effect markers in the urine according to claim 1, wherein the HPLC-MS/MS in the step S3 is a combination of high performance liquid chromatography and mass spectrometry, the liquid chromatography is used as a separation system, and the mass spectrometry is used as a detection system; wherein, HPLC is an initial compound, primary MS is used for carrying out mass spectrum analysis on each peak in an HPLC spectrogram, and secondary MS is used for carrying out further dissociation analysis on the peak in the primary MS so as to determine the peak structure of the primary MS.

8. The application of the method for synchronously detecting the exposure of the volatile pollutants and the effect markers in the urine as claimed in any one of claims 1 to 7, wherein the content data of the target substances, namely OH-PAHs, mVOCs and 8-OHdG, in the detected urine sample are further analyzed to obtain the relationship among the target substances, and the relationship is jointly applied to the evaluation of the exposure characteristics of the two types of pollutants commonly existing in the environment in the crowd, and the method specifically comprises the following steps:

(2) data classification: dividing factors of age, gender, post and operation time into continuous variables and classification variables, wherein the gender and the post belong to the classification variables, the age and the operation time belong to the continuous variables, OH-PAHs and mVOCs content data are used as independent variables, and 8-OHdG content data are used as dependent variables and are classified correspondingly;

9. The use of the synchronous detection method according to claim 8, wherein the content data of the target objects OH-PAHs, mVOCs and 8-OHdG in the urine samples of the same batch of individuals obtained by the detection and the further analysis of the relationship therebetween are used together to evaluate the exposure risk of the group health of the same batch of people, which comprises the following steps:

(1) data acquisition: acquiring data of a plurality of PAHs metabolites namely monohydroxy polycyclic aromatic hydrocarbons OH-PAHs, a plurality of volatile organic metabolites mVOCs and an exposure effect marker 8-OHdG in urine samples of a plurality of individual persons, and then summarizing and unifying units (mg/L);

(2) statistical analysis: with metabolites of not less than 20 pollutants as independent variables and 8-OHdG as dependent variable, the mathematical model for calculating the contribution y to the rise in the dependent variable 8-OHdG concentration is:

y=aX₁+bX₂+…+nX_nequation 1

then using logistic regression analysis to obtain a P value;

(3) and (4) judging a result:

when P is less than 0.05, significance exists, which means that the independent variable and the dependent variable have a relationship, the weight number of the variable is calculated in the formula 1, and when P is more than 0.05, significance does not exist, which means that the independent variable and the dependent variable have no relationship, and are not calculated.

10. The use of the synchronous detection method according to claim 9, wherein the content data of the target objects OH-PAHs, mVOCs and 8-OHdG in the urine sample obtained by detection and the further analysis of the relationship therebetween are applied together to analyze the individual injury influence level of the individual person, which specifically comprises the following steps:

(1) data acquisition: acquiring detection data of various monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs), various volatile organic compounds (mVOCs) metabolites and exposure effect marker 8-OHdG in a urine sample of a single individual to be detected, and unifying units (mg/L);

(2) influence analysis: respectively substituting the detection data of each target detection object of the single individual sample into the formula 1 to calculate a contribution value y causing the increase of the individual DNA damage marker 8-OHdG; the higher the value of y, the more damage the human body is subjected to by the relevant factors.