2, 3-difluoro-4-(trans-4-propylcyclohexyl) butoxybenzene exposure biomarker and application thereof in monitoring load in human body

Abstract

The invention discloses a 2, 3-difluoro-4-(trans-4-propylcyclohexyl) butoxybenzene exposure biomarker and application of the 2, 3-difluoro-4-(trans-4-propylcyclohexyl) butoxybenzene exposure biomarker in human body load monitoring, and the exposure biomarker is more than one of compounds shown in a table 4. According to the invention, based on the I / II phase metabolic transformation rule of BDPRB, the full-spectrum screening of the exposed biomarker is realized by combining high-resolution mass spectrum data and a fragment ion search system; meanwhile, through metabolite peak area semi-quantitative analysis, the high-sensitivity exposure marker for monitoring the load in the human body is determined for the first time. According to the method, a potential target is locked through metabolic site energy simulation, and accurate molecular structure analysis of the BDPRB exposed biomarker can be realized by utilizing a fragment ion spectrum intelligent matching algorithm.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollutant detection, specifically relating to 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers and their application in monitoring human body load. Background Technology

[0002] Organic pollutant exposure biomarkers refer to the quantitatively detectable original pollutants, metabolic transformation products, and adducts formed with endogenous substances in the tissues, body fluids, or excrement of organisms. These biomarkers serve as a bridge between environmental pollution exposure and health effects, providing key technical support for population exposure assessment and environmental health risk early warning systems.

[0003] With the rapid development of the liquid crystal display (LCD) industry, the global volume of waste LCDs is growing exponentially. Liquid crystal monomers (LCMs), the core component of LCDs, fill the gaps between polarizers through physical adsorption, causing them to be continuously released into the environment throughout the entire life cycle of the equipment. Due to the low cost-effectiveness of existing recycling technologies (>80% of waste LCDs are disposed of in landfills), the environmental pollution problem caused by LCMs is becoming increasingly serious.

[0004] Toxicological studies have shown that exposure to LCMs can induce toxic effects such as endocrine disruption, metabolic dysfunction, and abnormal liver / placental development. Among them, 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene (BDPrB, CAS: 208709-55-1), as the most produced and widely exposed fluorinated LCM, still has significant gaps in the study of its metabolic transformation mechanism: on the one hand, the phase I metabolism of BDPrB in vivo (cytochrome P450-mediated oxidation) may generate cytotoxic active intermediates, which can cause damage by forming adducts with proteins / nucleic acids, or be excreted as water-soluble conjugates through phase II metabolism; on the other hand, existing exposure assessment methods mainly rely on the determination of serum parent compound concentrations, but if BDPrB exhibits rapid metabolic characteristics (metabolite concentration >> parent compound), this traditional method will lead to a serious underestimation of exposure levels. Therefore, elucidating the metabolic pathway of BDPrB and identifying its specific exposure biomarkers (including stable metabolites and characteristic adducts) has become an urgent need to improve LCD waste management strategies and health risk early warning systems. Summary of the Invention

[0005] Current research often uses maternal BDPrB in serum to assess its internal load, neglecting the metabolic transformation process of BDPrB in vivo, resulting in high uncertainty in exposure assessment. Therefore, this invention aims to provide 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers and their application in human load monitoring, clarify the metabolic transformation process of BDPrB in vivo, and rapidly provide exposure biomarkers for human load monitoring.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] Application of 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers in human load monitoring; The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol (BDPrB_M1), 2,3-difluoro-4-(4-(1-hydroxypropyl)cyclohexyl)phenol (BDPrB_M2), and 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol (BDPrB_M2). 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol, BDPrB_M3, 3,4-difluoro-5-(4-(2-hydroxypropyl)cyclohexyl)benzene-1,2-diol, BDPrB_M4, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propane-1,2-diol, BDPrB_M4 (exyl)propane-1,2-diol, BDPrB_M5), 4-(2,3-difluoro-4-(4-propylcyclohexyl)phenoxy)-2-hydroxybutanal, BDPrB_M6), 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, BDPrB_M7), 1-(4-(2,3-difluoro-4- ...propane-1,2-diol, BDPrB_M5), 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, BDPrB_M7), 1-(4-(2,3-difluoro-4-propane-1,2-diol, BDPrB_M5), 4-(2,3-difluoro-4-propane-1,2-diol, BDPrB_M5), 4-(4-propylcyclohexyl)phenoxy)-2-hydroxybutanal, BDPrB_M6), 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, BDPrB_M7), 1-(4-(2,3-difluoro-4-propane-1,2-diol, BDPrB_M5), 4-(4-propylcyclohexyl)phenoxy)-2-hydroxybutanal, BDPrB_M6), 4-(4-allyl-3- 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxypropan-1-one (BDPrB_M8), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propylacetate (BDPrB_M9), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-1-hydroxypropan-2-ylacetate (BDPrB_M9), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-1-hydroxypropan-2-ylacetate (BDPrB_M9),One or more of the following: 3-difluoro-4-hydroxyphenyl)cyclohexyl)-1-hydroxypropan-2-yl acetate, BDPrB_M10, or 3,4-difluoro-2-hydroxy-5-(4-(2-hydroxypropyl)cyclohexyl)phenyl hydrogen sulfate, BDPrB_M11; The structures of the exposure biomarkers are shown in Table 4: Table 4: Molecular structural formulas of BDPrB parent compound (BDPrB_M0) and its 11 in vivo exposure biomarkers

[0008] Preferably, the 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one (BDPrB_M8) and 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate (BDPrB_M9). The relative proportion of the normalized peak area of ​​these two biomarkers exceeds 18%, and their combined proportion is close to 40%, which can be used as sensitive biomarkers for indicating BDPrB exposure. Preferably, the 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one (BDPrB_M8), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate (BDPrB_M9), and 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol (BDPrB_M7), and the combined normalized peak area of ​​these three biomarkers accounts for more than 50%. Preferably, the 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one (BDPrB_M8), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate (BDPrB_M9), 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol (BDPrB_M7), and 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol (BDPrB_M3), and the combined normalized peak area of ​​these four biomarkers accounts for more than 65%. Preferably, the 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one (BDPrB_M8), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate (BDPrB_M9), 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol (BDPrB_M7), 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol (BDPrB_M3), and 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol (BDPrB_M1), and the combined normalized peak area of ​​these five biomarkers accounts for more than 80%. More preferably, the 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarker is 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one (BDPrB_M8), 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate (BDPrB_M9), or 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol (BDPrB_M9). The combined normalized peak areas of these six markers—BDPrB_M7, 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol (BDPrB_M3), 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol (BDPrB_M1), and 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propane-1,2-diol (BDPrB_M5)—account for more than 95% of the total.

[0009] A screening method for biomarkers of 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene (BDPrB) exposure includes the following steps: (1) Exposure and sample preparation: Experimental animals were given the drug by gavage for at least 15 consecutive days, and then samples were collected for further analysis. The experimental animals mentioned include rodents or non-rodents; The preferred rodents are rats and mice; When rats are used as experimental animals, the gavage dose of BDPrB shall not be less than 1 mg / kg / day; The preferred non-rodent animals are rabbits, dogs, monkeys, or others; The sample may be blood or urine; When the sample is blood, serum is further separated for analysis. The serum was further purified; The purification process includes the following steps: take serum, add 3 times the volume of methanol pre-cooled to -80°C, vortex mix, let stand at -80°C for at least 60 minutes, and then centrifuge to collect the supernatant. (2) Mass spectrometry data acquisition: fragment spectra were acquired using an ultra-high performance liquid chromatography-tandem quadrupole / orbit trap high-resolution mass spectrometer; The acquisition includes full mass spectrometry scan, dynamic exclusion, target mass exclusion, peak detection, and fragment ion scanning; the full scan resolution is 120,000 (m / z 100-800), and the fragment ion scanning settings are: isolation window 2 m / z, HCD collision energy step 20%-80% (step size 20%), resolution 15,000, and fragment spectra are acquired simultaneously. (3) Screening of suspected exposure biomarkers: Using BioTransformer and Compound Discoverer software, a theoretical exposure biomarker molecular formula library was constructed based on the phase I / II metabolic transformation law of BDPrB; the fragment spectrum collected in step (2) was screened by Compound Discoverer software, and the theoretical exposure biomarker molecular formula library was matched with the measured mass spectrometry data. The set of biomarkers with mass error <5 ppm was used as the potential BDPrB exposure biomarker molecular formula candidate set. The screening includes one or more of the following operations: peak extraction, peak alignment, screening of desired compounds, combination of desired compounds, compound identification and annotation, and fragment ion search scoring; (4) Structural analysis and validation of exposure biomarkers: The activation energy thresholds of each reaction site of BDPrB molecule were evaluated using a metabolic simulation platform to locate highly propensity metabolically active sites. Based on the principle of metabolic energy optimization, the preliminary structural formulas of candidate biomarkers were derived from the molecular formula candidate set of potential BDPrB exposure biomarkers. The preliminary structure is imported into Compound Discoverer software, and the fragment ion intelligent matching engine is enabled. The system compares the characteristic ion clusters between the measured mass spectrometry fragments (HCD multi-level spectrum) of the analyte and the fragments of the preliminary structure, and completes the structure confirmation based on the spectrum similarity threshold. By integrating metabolic site prediction and mass spectrometry validation data, a multi-pathway metabolic transformation network diagram of BDPrB was constructed. Through characteristic peak area normalization analysis, the top 20% of high-sensitivity exposure biomarkers with the highest peak intensities were screened as BDPrB exposure biomarkers.

[0010] The preferred metabolic simulation platform is ADMET Predictor™ and / or BioTransformer; The aforementioned structural confirmation based on spectral similarity thresholds refers to selecting potential exposure markers with the highest theoretical and actual fragment ion matching degree.

[0011] The present invention has the following advantages and effects compared with the prior art: 1. Based on the phase I / II metabolic transformation law of BDPrB, this invention combines high-resolution mass spectrometry data and fragment ion search system to achieve full-spectrum screening of exposure biomarkers; at the same time, through semi-quantitative analysis of metabolite peak area, it establishes for the first time highly sensitive exposure biomarkers that can be used for monitoring human body load.

[0012] 2. This invention identifies potential targets through metabolic site energy simulation and utilizes a fragment ion spectrum intelligent matching algorithm (theoretical / experimental similarity calculation) to achieve precise molecular structure analysis of BDPrB exposure biomarkers. Attached Figure Description

[0013] Figure 1 The in vivo metabolic transformation pathways of 11 BDPrB exposure biomarkers in rats were identified.

[0014] Figure 2 The spectra of the parent ion (MS1) and fragment ion (MS2) of BDPrB_M3 are shown.

[0015] Figure 3 The spectra of the parent ion (MS1) and fragment ion (MS2) of BDPrB_M5 are shown.

[0016] Figure 4 The spectra of the parent ion (MS1) and fragment ion (MS2) of BDPrB_M10 are shown.

[0017] Figure 5 The spectra of the parent ion (MS1) and fragment ion (MS2) of BDPrB_M11 are shown. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0019] Example The screening method for BDPrB exposure biomarkers includes the following steps: (1) Grouping and exposure regimen: Based on the main human exposure route of BDPrB (dietary intake), 8-week-old SD rats (18 males and 18 females, female / male = 1:1) were selected and randomly divided into a control group (gavage with corn oil) and an exposure group (also known as the exposure group, gavage with 1 mg / kg-day BDPrB corn oil solution). The administration was carried out by gavage once a day for 15 consecutive days.

[0020] (2) Serum sample preparation: After fasting for 12 hours following the last exposure, 15 rats from the control group and 15 rats from the exposed group were randomly selected (the remaining 3 rats from each group were used as the validation set). The rats were anesthetized with isoflurane inhalation, fixed in a supine position, and underwent abdominal incision to expose the abdominal aorta and collect blood. 2 mL of whole blood was collected and immediately centrifuged at 3500 r / min for 5 minutes to separate the supernatant serum, which was then aliquoted and stored at -80℃ for later testing.

[0021] (3) Serum purification and enrichment: Accurately measure 400 μL of serum, add 1.2 mL of methanol pre-cooled to -80℃, vortex mix for 30 seconds, and let stand at -80℃ for 60 minutes. After centrifugation at 14,000 rpm for 15 minutes (4℃), take 800 μL of supernatant and vacuum dry it. Redissolve the residue with 200 μL of 1% formic acid methanol solution, centrifuge again at 14,000 rpm for 15 minutes (4℃), and finally take 120 μL of supernatant and transfer it to a sample vial for testing.

[0022] (4) Mass spectrometry data acquisition: An ultra-high performance liquid chromatography-tandem quadrupole / orbitrap high-resolution mass spectrometer (UPLC-Orbitrap Exploris 240) was used. Chromatographic separation was performed using a Hypersil GOLD AQ C18 column (150×2.1 mm, 1.9 μm). The mobile phase was methanol (organic phase B) and deionized water (aqueous phase A). The column temperature was 35℃, the flow rate was 0.3 mL / min, and the injection volume was 2 μL. Gradient elution program: 0-1 min 2% B; 1-8 min linearly up to 98% B; 8-16 min maintain 98% B; 16.01-19.5 min equilibrate to 2% B. Mass spectrometry detection was performed in ESI negative ion mode (spray voltage –2.5 kV). Ion source parameters: sheath gas 45 Arb, auxiliary gas 8 Arb, purge gas 1 Arb, transfer tube 320℃, vaporizer 350℃. The data acquisition process includes full mass spectrometry scan, dynamic exclusion, target mass exclusion, peak detection, and fragment ion scanning. The full scan resolution is 120,000 m / z (100-800 m / z). The fragment ion scanning settings are: isolation window 2 m / z, HCD collision energy step 20%-80% (step size 20%), resolution 15,000 m / z, and 5 fragment spectra are acquired simultaneously.

[0023] (5) Screening for suspected exposure biomarkers: Using BioTransformer 3.0 and Compound Discoverer software, a theoretical exposure biomarker molecular formula library was constructed based on the phase I / II metabolic transformation rules of BDPrB (Table 1), covering 7 types of phase I reactions such as oxidation / reduction / hydrolysis and 17 types of phase II reactions such as acetylation / methylation / glucuronidation (total metabolic number ≤ 5, maximum single-phase reaction 4 times). An automated screening process was established using Compound Discoverer software (covering modules such as peak extraction, peak alignment, screening of expected compounds, combination of expected compounds, compound identification and annotation, fragment ion search scoring and statistical difference analysis between groups) to screen differential metabolites for further processing. The theoretical exposure biomarker molecular formula library was precisely matched with the measured high-resolution mass spectrometry data (mass error < 5 ppm) to screen a candidate set of potential BDPrB exposure biomarker molecular formulas (Tables 2 and 3).

[0024] Table 1: Library of Theoretical BDPrB Exposure Biomarkers

[0025] Table 2: Candidate molecular formulas of potential BDPrB exposure biomarkers screened from the molecular formula library generated by BioTransformer

[0026] Table 3: Candidate molecular formulas of potential BDPrB exposure biomarkers screened from the molecular formula library generated by Compound Discoverer

[0027] (6) Structural analysis and validation of exposure biomarkers: Using the ADMET Predictor™ and BioTransformer 3.0 metabolic simulation platform, the activation energy thresholds of each reaction site in the BDPrB molecule were evaluated through quantum chemical calculations (density functional theory model), and highly propensity metabolic active sites such as hydroxylation / defluorination were accurately located. Based on the principle of metabolic energy optimization, the preliminary structural formulas of candidate biomarkers were derived from the molecular formula candidate set of potential BDPrB exposure biomarkers.

[0028] The preliminary structural formula is imported into the compound annotation editor module of Compound Discoverer 3.3 SP2 software. The fragment ion intelligent matching engine is enabled. The system compares the characteristic ion clusters between the measured mass spectrometry fragments (HCD multi-level spectrum) of the analyte and the fragments of the preliminary structural formula. The structure is confirmed based on the spectrum similarity threshold (m / z deviation < 5 ppm).

[0029] By integrating metabolic site prediction and mass spectrometry validation data, a multi-pathway metabolic transformation network diagram of BDPrB was constructed. Through characteristic peak area normalization analysis, the top 20% of highly sensitive exposure biomarkers with peak intensity were screened as core targets for biomonitoring.

[0030] Through the above steps, the present invention identified a total of 11 BDPrB exposure markers (Table 4). The structural confidence of most of the exposure markers reached the L2 level, and at least two fragment ions were matched in the database with a mass deviation of <5 ppm (Table 5).

[0031] (7) Take 3 rats from each of the two validation sets and repeat steps (2), (3) and (4) to analyze the concentration of 11 BDPrB exposure markers in the serum of validation set rats.

[0032] These 11 BDPrB exposure biomarkers were not detected in the control group (including the validation set of the control group) (Table 6). In the exposure group, except for BDPrB_M6 and BDPrB_M7, the detection rate of the other biomarkers was 100% (including the validation set of the exposure group), indicating that these biomarkers have high specificity and stability.

[0033] The most extensive metabolic transformation of BDPrB in vivo involves dealkylation, followed by oxidation, desaturation, acetylation, and sulfation. Based on the metabolic transformation reaction patterns, the in vivo metabolic transformation pathways of 11 BDPrB exposure biomarkers were further mapped to characterize their biotransformation specificity. Figure 1Furthermore, semi-quantitative analysis of 11 BDPrB exposure biomarkers based on normalized peak area revealed that BDPrB_M8 had the highest abundance, accounting for 19.8%, followed by BDPrB_M9 (18.6%), BDPrB_M7 (15.7%), BDPrB_M3 (13.9%), BDPrB_M1 (13.7%), and BDPrB_M5 (13.6%). Therefore, BDPrB_M8 and BDPrB_M9 can serve as sensitive biomarkers indicating BDPrB exposure.

[0034] Table 5: Eleven BDPrB exposure biomarkers identified in serum

[0035] Table 6: Detection levels of 11 BDPrB exposure biomarkers in the serum of rats in the control and exposed groups.

[0036] Furthermore, the precursor ion isotope matching of the 11 BDPrB exposure biomarkers was 100%, and the precursor ion isotope distribution spectra (MS1) and fragment ion (MS2) of typical compounds were as follows: Figures 2-5 As shown. BDPrB_M1, BDPrB_M2, and BDPrB_M3 are the dealkylation and oxidation products of BDPrB; BDPrB_M4 and BDPrB_M5 are the oxidation products of BDPrB_M2; BDPrB_M11 is the sulfation product of BDPrB_M4; BDPrB_M9 is the acetylation product of BDPrB_M2; BDPrB_M10 is the acetylation product of BDPrB_M5; BDPrB_M7 is the desaturation product of BDPrB_M3; BDPrB_M8 is the dealkylation, oxidation, oxidation, and desaturation product of BDPrB_M0; and BDPrB_M6 is the oxidation, oxidation, and desaturation product of BDPrB_M0.

[0037] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

The application of 1,2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers in human load monitoring, characterized by: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol, 2,3-difluoro-4-(4-(1-hydroxypropyl)cyclohexyl)phenol, 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol, 3,4-difluoro-5-(4-(2-hydroxypropyl)cyclohexyl)phenyl-1,2-diol, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propane-1,2-diol, 4-(2,3-difluoro- One or more of the following: 4-(4-propylcyclohexyl)phenoxy)-2-hydroxybutanal, 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxypropyl-1-one, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-1-hydroxypropyl-2-yl acetate, or 3,4-difluoro-2-hydroxy-5-(4-(2-hydroxypropyl)cyclohexyl)phenyl hydrogen sulfate.

2. The application according to claim 1, characterized in that: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one and 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate.

3. The application according to claim 1, characterized in that: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate, and 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol.

4. The application according to claim 1, characterized in that: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate, 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, and 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol.

5. The application according to claim 1, characterized in that: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate, 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol, and 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol.

6. The application according to claim 1, characterized in that: The 2,3-difluoro-4-(trans-4-propylcyclohexyl)butoxybenzene exposure biomarkers are 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)-3-hydroxyprop-1-one, 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propyl acetate, 4-(4-allyl-3-hydroxycyclohexyl)-2,3-difluorophenol, 2,3-difluoro-4-(3-hydroxy-4-propylcyclohexyl)phenol, 2,3-difluoro-4-(4-(2-hydroxypropyl)cyclohexyl)phenol, and 1-(4-(2,3-difluoro-4-hydroxyphenyl)cyclohexyl)propane-1,2-diol.

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