Systematic screening method for rubber-related chemicals in plasma sample

Abstract

The invention belongs to the technical field of chemical screening, and particularly relates to a systematic screening method for rubber-related chemicals in a plasma sample, which comprises the following steps: S1, taking the plasma sample, adding a mixed extraction solvent, extracting, centrifugally layering, collecting supernate, repeatedly extracting, merging the supernate, concentrating, and freezing to obtain a rubber-related chemical sample; centrifuging again, adding an isotope internal standard, and carrying out data dependence acquisition in a positive and negative ion analysis mode by adopting liquid chromatography-tandem high-resolution mass spectrometry. According to the invention, the comprehensive coverage type monitoring of 1300-1500 RDCs in the plasma sample is realized. By means of combination of a positive and negative ion double analysis mode and data dependence acquisition, the detection range and the signal response stability of the RDCs with different polarities are effectively considered, and the technical problem that in the prior art, the composite effect generated by coexistence of multiple RDCs is ignored is solved.

Description

Technical Field

[0001] This invention belongs to the field of chemical screening technology, and in particular relates to a systematic screening method for rubber-related chemicals in plasma samples. Background Technology

[0002] As an indispensable resource-based industry globally, the rubber industry has maintained strong growth momentum in recent years, with its products widely used in industrial production, transportation, daily life, and many other fields. Due to the inherent aging characteristics of rubber materials, they are prone to problems such as appearance deterioration, hardness fluctuations, and degradation of physical and mechanical properties during use. Therefore, in the rubber production and application stages, various rubber-derived chemicals (RDCs), such as vulcanizing agents, antioxidants, plasticizers, and adhesives, need to be added to achieve cross-linking of rubber molecules, delay the aging process, improve processing performance, and ultimately enhance the service life and safety of rubber products.

[0003] With the development of environmental monitoring technology, an increasing number of studies have confirmed that reactive carbon deposits (RDCs) can enter various environmental media such as soil, water, and atmosphere through production emissions, product wear and tear, and waste degradation, and are widely present in human living and working environments. Meanwhile, related toxicological studies have shown that some RDCs have various adverse effects on aquatic organisms such as fish and terrestrial organisms, including genotoxicity and endocrine disruption. Their environmental risks and potential harm to human health have attracted widespread attention from researchers worldwide.

[0004] However, current research on human exposure to RDCs still has significant shortcomings: existing technologies mostly focus on the independent detection and evaluation of single or specific types of RDCs, lacking systematic screening methods for various RDCs with different structures and functions in biological samples such as plasma. Because the types of RDCs added to rubber products are numerous and their physicochemical properties vary greatly, and multiple compounds may coexist during human exposure, independent detection of a single compound cannot reflect the combined effects of exposure to multiple RDCs. This makes it difficult to comprehensively and accurately assess the characteristics and health risks of RDC exposure in occupational groups (such as rubber manufacturing workers) and the general population, and also fails to provide scientific data support for the development of targeted protective measures.

[0005] Therefore, developing a plasma sample screening method that can simultaneously cover thousands of RDCs, is easy to operate, has high accuracy and efficiency, and solves the technical defects of existing technologies such as narrow detection range, neglect of compound effects and lack of systematicity has become a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned technical problems by providing a systematic screening method for rubber-related chemicals in plasma samples.

[0007] In view of this, the present invention provides a systematic screening method for rubber-related chemicals in plasma samples, comprising the following steps: S1. Take a plasma sample, add a mixed extraction solvent, extract, centrifuge and separate the layers, collect the supernatant, repeat the extraction and combine the supernatants, concentrate and freeze, centrifuge again and add an isotope internal standard, and use liquid chromatography-tandem high-resolution mass spectrometry in positive and negative ion analysis modes for data-dependent acquisition. S2, Construct a screening list containing rubber industry chemicals and related compounds. The list records the name, CAS number, molecular formula and structural formula of each compound, and is constructed based on the environmental relevance data of the compounds and the feasibility of liquid chromatography-tandem high-resolution mass spectrometry analysis. S3 uses mixed QC samples for screening, extracts features and identifies compounds, removes background interference features, and screens compounds based on quality accuracy, isotope patterns and fragment ion matching to classify potential rubber-related chemicals into confidence levels.

[0008] Furthermore, in step S1, the mixed extraction solvent is a mixture of ethyl acetate and hexane in a volume ratio of 3:2, and the mixed extraction solvent contains 0.1% formic acid; the plasma sample transfer volume is 180~220 μL, and the mixed extraction solvent added volume is 2.5~3.5 mL.

[0009] Furthermore, in step S1, the extraction is an oscillating extraction, the extraction time is 8-12 minutes, and after extraction, it is centrifuged at 2800-3200×g and 23-27℃ for 2-4 minutes. The extraction step is repeated 2-3 times and the supernatant is combined.

[0010] Furthermore, in step S1, the concentration is carried out by nitrogen purging concentration under the conditions of a 28~32 ℃ water bath, and the solution volume is concentrated to approximately 80~120 μL; the freezing treatment is carried out by freezing at -85~-75 ℃ for 10~14 hours; after freezing, the solution is centrifuged at 14000~16000×g and -12~-8 ℃ for 4~6 minutes.

[0011] Furthermore, in step S1, the amount of the isotope internal standard added is 0.8~1.2 ng, and after addition, it is mixed and injected into the sample, with an injection volume of 4~6 μL.

[0012] Furthermore, in step S1, the column temperature of the liquid chromatography-tandem high-resolution mass spectrometry column is 28~32 ℃; the mobile phase in both positive and negative ion modes includes phase A containing ammonium formate solution and phase B containing methanol, and the gradient elution program is as follows: 0~2 min, phase B accounts for 38%~42%, flow rate 0.28~0.32 mL / min; 2~3 min, increases to 64%~68%; 3~12 min, increases to 68%~72%; 12~14 min, increases to 98%~100%; 14~16 min, maintains 98%~100%; 16~16.1 min, decreases to 38%~42%; 16.1~19 min, maintains 38%~42%.

[0013] Furthermore, in step S1, the ion source of the liquid chromatography-tandem high-resolution mass spectrometry is H-ESI, the spray voltage in positive ion mode is 3300~3700 V, and the spray voltage in negative ion mode is 2300~2700 V; the sheath gas flow rate is 38~42 units, the auxiliary gas flow rate is 8~12 units, and the purge gas flow rate is 0.8~1.2 units; the ion transmission tube temperature is 315~335 ℃, and the evaporator temperature is 340~360 ℃.

[0014] Furthermore, in step S1, the Orbitrap resolution of the liquid chromatography-tandem high-resolution mass spectrometry is 110,000~130,000 during the first-stage scan and 14,000~16,000 during the second-stage scan; the RF lens is 68%~72%; the scan range is 90~1100 m / z; the activation type is HCD, the HCD collision energy is 28%~32%, and the ±HCD collision energy is 4%~6%; the isolation window is 1.8~2.2 m / z, and the number of data-dependent scans is 18~22.

[0015] Furthermore, in step S3, the criteria for removing background interference features are: features with an intensity less than 4 to 6 times that of the solvent blank or program blank are marked as background and removed; the screening criteria also include: peak intensity of 80,000 to 120,000 cps; mass error of <4 to 6 ppm; and the number of fragments matched in the tandem mass spectrometry spectrum ≥3.

[0016] Furthermore, in step S3, the confidence levels include level 1, level 2a, level 2b, and level 3, where level 1 is the structure confirmed by commercial standards, level 2a is the possible structure determined by database spectral matching, level 2b is the possible structure determined by diagnostic evidence, and level 3 is a provisional candidate lacking evidence of secondary mass spectrometry structure.

[0017] The beneficial effects of this invention are: Compared to existing technologies that can only independently detect single or class-specific rubber-related chemicals, this invention achieves comprehensive detection of 1300-1500 RDCs in plasma samples. By combining positive and negative ion dual analysis modes with data-dependent mass spectrometry acquisition, it effectively balances the detection range and signal response stability of RDCs with different polarities, solving the technical problem of existing technologies neglecting the combined effects of multiple coexisting RDCs. Furthermore, by introducing a confidence level grading standard, the reliability levels of the detection results are clearly defined, providing comprehensive and accurate technical support for assessing RDC exposure characteristics and analyzing health risks in occupational and general populations. This method is streamlined, simple, and controllable, requiring no complex modifications to existing laboratory equipment, and possesses strong practicality and widespread application value. Attached Figure Description

[0018] Figure 1 This is a flowchart for screening RDCs in blood; Figure 2 This is a classification and functional grading chart for RDCs with a confidence level of 3 or higher identified from plasma samples. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] In the description of this application, it should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. For ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0021] It should be noted that the terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and are not limited in number; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0022] It should be noted that in the description of this application, the directional terms such as "front, back, up, down, left, right", "horizontal, vertical, horizontal" and "top, bottom" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description. Unless otherwise stated, these directional terms do not indicate or imply that the device or element referred to must have a specific orientation or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation on the scope of protection of this application. The directional terms "inner" and "outer" refer to the inner and outer contours relative to the outline of each component itself.

[0023] It should be noted that, in this application, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0024] Reference Figure 1 and Figure 2 A systematic screening method for rubber-related chemicals in plasma samples, comprising the following steps: S1. Take plasma samples, add mixed extraction solvent, extract, centrifuge and collect supernatant, repeat extraction and combine supernatants, concentrate and freeze, centrifuge again and add isotope internal standard, use liquid chromatography-tandem high-resolution mass spectrometry in positive and negative ion analysis mode to acquire data-dependent mass spectrometry data. S2, Construct a screening list containing rubber industry chemicals and related compounds. The list records the name, CAS number, molecular formula and structural formula of each compound, and is constructed based on the environmental relevance data of the compounds and the feasibility of liquid chromatography-tandem high-resolution mass spectrometry analysis. S3 uses mixed QC samples for screening, extracts features and identifies compounds, removes background interference features, and screens compounds based on quality accuracy, isotope patterns and fragment ion matching to classify potential rubber-related chemicals into confidence levels.

[0025] Compared to existing technologies that can only independently detect single or class-specific rubber-related chemicals, this invention achieves comprehensive detection of 1300-1500 RDCs in plasma samples. By combining positive and negative ion dual analysis modes with data-dependent tandem mass spectrometry acquisition, it effectively balances the detection range and signal response stability of RDCs with different polarities, solving the technical problem of existing technologies neglecting the combined effects of multiple coexisting RDCs. Furthermore, by introducing a confidence level grading standard, the reliability levels of the detection results are clearly defined, providing comprehensive and accurate technical support for assessing RDC exposure characteristics and analyzing health risks in occupational and general populations. This method is streamlined, simple, and controllable, requiring no complex modifications to existing laboratory equipment, and possesses strong practicality and widespread application value.

[0026] In the example of this application, in step S1, the mixed extraction solvent is a mixture of ethyl acetate and hexane in a volume ratio of 3:2, and the mixed extraction solvent contains 0.1% formic acid; the plasma sample transfer volume is 200 μL, and the mixed extraction solvent added volume is 3 mL.

[0027] As a preferred example of the present invention, the mixed extraction solvent can specifically enhance the separation selectivity of RDCs and plasma matrix, minimize the co-extraction of impurities such as proteins and lipids, and precisely match the 200μL sample and 3mL solvent volume to the 15mL glass centrifuge tube specification. This ensures sufficient contact and release between the target analyte and the solvent, avoids reagent waste, and significantly improves the extraction recovery rate and purity of the target analyte, laying a high-purity sample foundation for subsequent detection.

[0028] In the example of this application, in step S1, the extraction is an oscillating extraction, the extraction time is 10 minutes, and after extraction, it is centrifuged at 3000×g and 25℃ for 3 minutes. The extraction step is repeated twice and the supernatant is combined.

[0029] As a preferred example of the present invention, the synergistic optimization of 10-minute oscillating extraction and centrifugation parameters of 3000×g and 25℃ can fully release free and bound RDCs in plasma and avoid residual loss of target substances; the two repeated extractions not only ensure sufficient extraction but also do not excessively increase the operation time. Combined with centrifugation and stratification, it can achieve efficient separation of target substances and matrix, further improve sample purity, and ensure the accuracy of subsequent mass spectrometry detection.

[0030] In the example of this application, in step S1, the concentration is carried out by nitrogen purging concentration under the conditions of a 30°C water bath, and the concentration is carried out to a solution volume of approximately 100 μL; the freezing treatment is carried out by freezing at -80°C for 12 hours; after freezing, the solution is centrifuged at 15000×g and -10°C for 5 minutes.

[0031] As a preferred example of the present invention, gentle nitrogen blowing concentration at 30°C can prevent the degradation of thermosensitive RDCs due to high temperature, ensuring the structural stability of the target analyte and the authenticity of the detection; the concentration to a volume of nearly 100 μL can meet the sensitivity requirements of mass spectrometry detection and facilitate subsequent processing; freezing at -80°C for 12 hours can efficiently precipitate residual lipids and other interfering matrices in the plasma, and the sample is further purified by a second centrifugation at 15000×g and -10°C, which significantly reduces the interference of the matrix on the detection signal and improves the purity and signal-to-noise ratio of the target analyte detection signal.

[0032] In the example of this application, in step S1, the amount of the isotope internal standard added is 1 ng, and after addition, it is mixed and injected into the sample, with an injection volume of 5 μL.

[0033] As a preferred example of the present invention, the dosage of 1 ng of isotope internal standard can accurately calibrate the loss of target analytes such as adsorption and volatilization during sample pretreatment and instrument detection, effectively improving the quantitative accuracy and repeatability of the detection results; the 5 μL injection volume is highly compatible with the specifications of the Phenomenex Luna® Omega C18 column (3.0 μm 3*100 mm), which can avoid peak distortion caused by injection overload, ensure the resolution and peak symmetry of different RDCs, reduce human error, and improve the repeatability of the method.

[0034] In the example of this application, in step S1, the column temperature of the liquid chromatography-tandem high-resolution mass spectrometry column is 30°C; the mobile phase in both positive and negative ion modes includes phase A containing ammonium formate solution (pH=4) and phase B containing methanol, and the gradient elution program is as follows: 0~2 min, phase B accounts for 40%, flow rate 0.3 mL / min; 2~3 min, increase to 66%; 3~12 min, increase to 70%; 12~14 min, increase to 100%; 14~16 min, maintain 100%; 16~16.1 min, decrease to 40%; 16.1~19 min, maintain 40%.

[0035] As a preferred example of the present invention, the synergistic effect of a column temperature of 30°C and an ammonium formate solution A at pH=4 can optimize the chromatographic retention behavior of RDCs with different polarities, significantly improve the resolution between target analytes, and avoid qualitative and quantitative errors caused by peak overlap. The optimized gradient elution program can complete the efficient separation and detection of all target analytes within 19 minutes, balancing separation effect and analytical efficiency. Furthermore, the positive and negative ion modes share the same mobile phase system, eliminating the need to change reagents, reducing equipment cleaning time and reagent consumption, and lowering experimental costs.

[0036] In the example of this application, in step S1, the ion source of the liquid chromatography-tandem high-resolution mass spectrometry is H-ESI, the positive ion mode spray voltage is 3500V, the negative ion mode spray voltage is 2500V; the sheath gas flow rate is 40 arbitrary units, the auxiliary gas flow rate is 10 arbitrary units, the purge gas flow rate is 1 arbitrary unit; the ion transfer tube temperature is 325℃, and the evaporator temperature is 350℃.

[0037] As a preferred example of the present invention, the combination of a spray voltage of 3500V (positive ion) and 2500V (negative ion) with optimized gas flow rate and temperature parameters can significantly improve the ionization efficiency of RDCs of different polarities and effectively reduce the occurrence of ion suppression. The synergistic matching of the parameters ensures the stability of the ion source and the ion transmission efficiency, reduces background noise interference, provides a guarantee for the effective detection of low-concentration RDCs, and improves the sensitivity of the detection method.

[0038] In the example of this application, in step S1, the Orbitrap resolution of the liquid chromatography-tandem high-resolution mass spectrometry is 120,000 for the first scan and 15,000 for the second scan; the RF lens is 70%; the scan range is 100-1000 m / z; the activation type is HCD, the HCD collision energy is 30%, and the ±HCD collision energy is 5%; the isolation window is 2 m / z, and the number of data-dependent scans is 20.

[0039] As a preferred example of the present invention, the high-resolution parameters of 120,000 for the first stage and 15,000 for the second stage ensure high accuracy in the determination of target analytes, providing a foundation for precise identification of compounds; the scanning range of 100-1000 m / z can fully cover the molecular weight range of the 1409 RDCs in the screening list, avoiding missed detection of target analytes; the optimized HCD collision energy and number of scans can generate fragment ions with strong characteristics, improve the matching reliability of MS / MS spectra, and avoid unnecessary scanning time, further improving analytical efficiency.

[0040] In the example of this application, in step S3, the criteria for removing background interference features are: features with an intensity less than 5 times that of the solvent blank or program blank are marked as background and removed; the screening criteria also include: peak intensity > 100000cps; mass error < 5ppm; number of fragments matching the secondary mass spectrometry spectrum ≥ 3.

[0041] As a preferred example of this invention, the standard of "marking background with an intensity less than 5 times that of blank" can efficiently eliminate matrix interference signals and significantly reduce the probability of false positive results. The multi-dimensional screening standard of peak intensity > 100,000 cps, quality error < 5 ppm, and fragment number ≥ 3 forms a complementary verification system to ensure the accuracy and rigor of RDC identification, adapt to the precision of conventional laboratory instruments, and enhance the applicability and repeatability of the method without reducing the detection standards, thus helping different laboratories to promote its application.

[0042] In the example of this application, in step S3, the confidence level includes level 1, level 2a, level 2b and level 3, wherein level 1 is the structure confirmed by commercial standard, level 2a is the possible structure determined by database spectrum matching, level 2b is the possible structure determined by diagnostic evidence, and level 3 is a provisional candidate lacking secondary mass spectrometry structure evidence.

[0043] As a preferred example of the present invention, the four-level confidence level classification standard clearly defines the reliability levels of different identification results. Level 1 ensures absolute accuracy through verification with commercial standard products, Levels 2a and 2b meet different precision requirements, and Level 3 provides candidate directions for subsequent research. The standardized classification method provides a clear basis for the interpretation of test results, meets the usage needs of different scenarios such as scientific research, occupational health monitoring, and environmental risk assessment, facilitates cross-laboratory data sharing and comparison, and provides technical specifications and data support for multi-center studies related to exposure within RDCs.

[0044] To further clarify the technical details of this invention and ensure the repeatability, comprehensiveness, and effectiveness of the method, the following supplementary explanations are provided regarding key technical aspects, core supporting conditions, and experimental verification results, making the technical solution of this invention more compliant with the "clear and complete" requirements of patent law: Detailed basis and scope for constructing the screening checklist: The screening list described in step S2 of this invention specifically includes 1409 rubber-related chemicals (RDCs), comprehensively covering typical chemical types used in rubber production and application. The list is constructed to balance authoritativeness and practicality, and the data sources specifically include: The U.S. Environmental Protection Agency (EPA) CompTox database, whose core data is based on information from the Federal Research Action Plan (FRAP) regarding chemicals used to recycle tire debris in sports fields and playgrounds, ensures the relevance of the inventory to environmental exposure scenarios; The Norman database contains specific data on tire-related chemicals in environmental media, supplementing the RDCs that may be involved in environmental migration. The literature review results on potential exposed compounds throughout the entire rubber manufacturing process cover characteristic chemicals in raw materials, production, and processing.

[0045] Each compound in the list has its name, CAS number, molecular formula, and structural formula recorded in full. The construction process is based on the environmental relevance and human exposure risk of the compound, and also fully considers the analytical feasibility of liquid chromatography-tandem high-resolution mass spectrometry (UHPLC-HRMS) (such as molecular weight matching scan range, ionization efficiency, etc.) to ensure the comprehensiveness and practicality of the screening work.

[0046] Software operation and quality control details of the screening process: Specific operations for feature extraction and compound recognition: In step S3 of this invention, feature extraction and compound identification are performed using Compound Discoverer 3.2.1 software (ThermoFisher Scientific, USA). The specific process includes: Peak selection: accurately identify the effective signal peaks in the sample mass spectrum and eliminate baseline noise interference; Retention time (RT) alignment: Corrects retention time discrepancies between different samples to ensure consistent matching of the same compound in different samples; Componentization processing: Isotope peaks and adduct peaks are grouped and classified to avoid duplicate counting and misidentification; Peak Unified Grouping: The characteristic peaks of all samples are standardized and grouped to form a unified feature set, laying the foundation for subsequent screening.

[0047] To ensure the reliability of the test results, this invention incorporates a dual quality control system: Program blank control: While processing plasma samples, three HPLC water samples are processed simultaneously as laboratory program blanks. Background contamination caused by experimental environment, reagent residues, etc. is eliminated by blank subtraction method. QC Sample Calibration: Before analysis, 5 μL is transferred from each plasma sample and mixed to prepare a quality control (QC) sample. The QC sample is used as the benchmark for screening analysis to monitor the stability and repeatability of the entire testing process and ensure the consistency of instrument performance and pretreatment operations.

[0048] Specific specifications of core instruments and consumables: The instruments, consumables, and reagents used in this invention are all clearly defined to ensure the reproducibility and scalability of the method, as detailed below: Core testing instrument: ThermoFisher Scientific Vanquish UHPLC coupled with ThermoFisher Orbitrap 240 Mass Spectrometer; Analytical column: Phenomenex Luna® Omega C18 100Å column (3.0μm 3*100mm); Auxiliary equipment: Oganomation 12 N-Evap™ nitrogen evaporator, Hunan Xiangyi centrifuge; Reagent specifications: Formic acid, ethyl acetate, n-hexane, methanol, acetonitrile, and ammonium formate are all Optima grade (purchased from Fisher Scientific) to avoid interference from reagent impurities on the test results; Sample storage conditions: Plasma samples should be sealed and stored in polypropylene centrifuge tubes at −80°C until they are removed before analysis to ensure the stability of RDCs in the samples.

[0049] Functional classification system of RDCs: The RDCs in the screening list of this invention can be clearly divided into five categories according to their functional system. The core functions and specific sub-categories of each category are as follows, providing a classification basis for subsequent exposure risk analysis: Vulcanizing aids: Their core function is to crosslink chain rubber molecules to form a three-dimensional network structure, thereby improving the hardness and elasticity of rubber. They include vulcanizing agents (crosslinking agents), vulcanization accelerators, vulcanization activators, and anti-reversion agents. Protective additives: mainly used to delay rubber aging and extend the storage and service life of products, including antioxidants, anti-ozone agents, anti-flexural cracking agents, light stabilizers, ultraviolet absorbers, harmful metal inhibitors, physical antioxidants, and mildew inhibitors; Processing aids: These enable the rubber to bond firmly to the chemical fiber cord or copper-plated steel wire cord (skeleton material), forming chemical bonds between dissimilar materials at the bonding interface. They include white adhesive systems and cobalt salt bonding accelerators. Adhesive additives: improve the processing performance and operating conditions of rubber compounds, enhance production efficiency and the internal and external quality of products, including anti-scorching agents, plastic solvents, plasticizers, homogenizers, dispersants, tackifiers, lubricants, flow aids, release agents, mold release agents, etc. Other functional additives: These impart specific functional properties to rubber products, including coupling agents, colorants, foaming agents, flame retardants, antistatic agents, and fragrances.

[0050] Experimental validation results of the method's effectiveness: To verify the practical application effect of the technical solution of this invention, the optimal technical parameters described above were used to screen RDCs in plasma samples from 408 rubber manufacturing workers, obtaining clear and reliable experimental results, which fully demonstrate the practicality and advancement of this method (as shown in the table below): Name CAS Levels Molecular formula m / z RT (min) Use classification Caprolactam 105-60-2 Level 1 <![CDATA[C 6 H 11 NO]]> 114.0913 2.00 Adhesive additives 3,5-Di-tert-butyl-4-hydroxybenzaldehyde 1620-98-0 Level 1 <![CDATA[C 15 H 22 O2]]> 235.1693 16.56 Protective additives p-Phenylenediamine 106-50-3 Level 1 <![CDATA[C6H 8 N2]]> 109.0765 2.23 Protective additives Cyclohexanone oxime 100-64-1 Level 1 <![CDATA[C6H 11 NO]]> 114.0918 1.94 Protective additives Isophorone 78-59-1 Level 1 <![CDATA[C9H 14 O]]> 139.1117 7.37 Others N-Cyclohexylformamide 766-93-8 Level 1 <![CDATA[C7H 13 NO]]> 128.1069 2.31 Others Erucamide 112-84-5 Level 1 <![CDATA[C 22 H 43 NO]]> 338.3423 18.39 Others Benzyl-2-ethylhexyl adipate 58394-64-2 Level 2a <![CDATA[C 21 H 32 O4]]> 349.2376 16.97 Adhesive additives Dicyclohexylamine 101-83-7 Level 2a <![CDATA[C 12 H 23 N]]> 182.1903 3.60 Adhesive additives Phthalic anhydride 85-44-9 Level 2a <![CDATA[C8H4O3]]> 149.0233 18.02 Adhesive additives Tributylamine 102-82-9 Level 2a <![CDATA[C 12 H 27 N]]> 186.2216 3.65 Adhesive additives 2,6-Di-tert-butyl-p-benzoquinone 719-22-2 Level 2a <![CDATA[C 14 H 20 O2]]> 221.1535 12.29 Protective additives 3-Phenylprop-2-enal 14371-10-9 Level 2a <![CDATA[C9H8O]]> 133.0648 15.32 Others 1,3-dicyclohexylurea 2387-23-7 Level 2a <![CDATA[C 13 H 24 N2O]]> 225.1961 16.10 Others N-Nitrosopiperidine 100-75-4 Level 2a <![CDATA[C5H 10 N2O]]> 115.0865 3.15 Others 2,2,4-Trimethyl-1,2-dihydroquinoline 26780-96-1 Level 2b <![CDATA[C 12 H 15 N]]> 174.1282 3.38 Adhesive additives 2,5-Cyclohexadiene-1,4-dione, 2-methyl-5-(1-methylethyl)- 490-91-5 Level 2b <![CDATA[C 10 H 12 O2]]> 165.0915 12.42 Adhesive additives 2-Cyanobenzoic acid 3839-22-3 Level 2b <![CDATA[C8H5N O2]]> 148.0398 3.38 Adhesive additives Dihexyl phthalate 84-75-3 Level 2b <![CDATA[C 20 H 30 O4]]> 335.2218 15.99 Adhesive additives Di(2-ethylhexyl) phthalate 117-81-7 Level 2b <![CDATA[C 24 H 38 O4]]> 391.2843 18.02 Adhesive additives Dibutyl sebacate 109-43-3 Level 2b <![CDATA[C 18 H 34 O4]]> 315.253 17.88 Adhesive additives 2-Ethylhexanoic acid 149-57-5 Level 2b <![CDATA[C8H 16 O2]]> 145.1223 5.65 Adhesive additives Diethylphthalate 84-66-2 Level 2b <![CDATA[C 12 H 14 O4]]> 223.0964 17.00 Adhesiveadditives 4-(1,1-Dimethylethyl)phenol 98-54-4 Level 2b <![CDATA[C 10 H 14 O]]> 151.1117 16.56 Adhesiveadditives Dibutyl1,2-benzenedicarboxylate 84-74-2 Level 2b <![CDATA[C 16 H 22 O4]]> 279.159 17.02 Adhesiveadditives 2,4,6-tris(2-methyl-2-propanyl)phenol 732-26-3 Level 2b <![CDATA[C 18 H 30 O]]> 263.2369 17.09 Adhesiveadditives Ethyleneglycoldimethacrylate 97-90-5 Level 2b <![CDATA[C 10 H 14 O4]]> 199.0965 5.75 Adhesiveadditives N-Cyclohexylacetamide 1124-53-4 Level 2b <![CDATA[C8H 15 BI]]> 142.1232 5.32 Processingaids 1,1'-Disulfanediyldiazepan-2-one 23847-08-7 Level 2b <![CDATA[C 12 H20N2O2S2]]> 306.1296 2.72 Processingaids 3-tert-Butyl-4-methoxyphenol 88-32-4 Level 2b <![CDATA[C 11 H 16 O2]]> 181.1228 6.94 Protectiveadditives N,N-Dicyclohexyl-2-benzothiazolsulfeneamide 4979-32-2 Level 2b <![CDATA[C 19 H 26 N2S2]]> 347.1603 6.48 Protectiveadditives 2,4-bis(1,1-dimethylethyl)phenol 96-76-4 Level 2b <![CDATA[C 14 H 22 O]]> 207.1743 16.47 Protectiveadditives 4,4'-Di-tert-butyldiphenylamine 4627-22-9 Level 2b <![CDATA[C 20 H 27 N]]> 282.2217 7.28 Protectiveadditives 4'-Methylformanilide 3085-54-9 Level 2b <![CDATA[C8H9NO]]> 136.0762 4.37 Vulcanizingagent Hexamethylenediisocyanate 822-06-0 Level 2b <![CDATA[C8H 12 N2O2]]> 169.0971 1.91 Vulcanizingagent 1,4-Bis(allyloxy)butane 1471-16-5 Level 2b <![CDATA[C 10 H 18 O2]]> 171.1379 8.48 Vulcanizingagent 2,5-Bis(tert-butylperoxy)-2,5-dimethyl-3-hexyne 1068-27-5 Level 2b <![CDATA[C 16 H 30 O4]]> 287.2218 16.43 Vulcanizingagent 2-(Methylsulfinyl)benzothiazole 3507-54-8 Level 2b <![CDATA[C8H7NOS2]]> 198.004 5.77 Vulcanizingagent 2,2,6,6-Tetramethyl-piperidin-4-ol 2403-88-5 Level 2b <![CDATA[C9H 19 NO]]> 158.154 1.50 Others 1,6-Bis(allyloxy)hexane 81866-56-0 Level 2b <![CDATA[C 12 H 22 O2]]> 199.1693 16.43 Others 2,4-Dihydroxybenzophenone 131-56-6 Level 2b <![CDATA[C 13 H 10 O3]]> 215.0703 16.26 Others 2-Phenyl-2-propanol 617-94-7 Level 2b <![CDATA[C9H 12 O]]> 137.0961 12.29 Others N-Nitrosomorpholine 59-89-2 Level 2b <![CDATA[C4H8N2O2]]> 117.0658 1.48 Others 4'-Hydroxybutyranilide 101-91-7 Level 2b <![CDATA[C 10 H 13 NO2]]> 180.1019 15.86 Others 4-Hydroxybenzeneethanol 501-94-0 Level 2b <![CDATA[C8H 10 O2]]> 139.0754 18.72 Others Dehydroabieticacid 1740-19-8 Level 2b <![CDATA[C 20 H 28 O2]]> 301.2163 15.86 Others 5-Aminosalicylicacid 89-57-6 Level 2b <![CDATA[C7H7NO3]]> 154.0498 5.67 Others tert-Butylperoxybenzoate 614-45-9 Level 2b <![CDATA[C11H 14 O3]]> 195.1016 10.86 Others N-Nitroso-N-methylaniline 614-00-6 Level 2b <![CDATA[C7H8N2O]]> 137.0709 8.01 Others N-[2-(2,3,7,8-Tetrahydrofuro[2,3-g]indol-1-yl)ethyl]acetamide 170729-12-1 Level 2b <![CDATA[C 14 H 18 N2O2]]> 247.1442 2.32 Others N-[4-(phenylamino)phenyl]acetamide 38674-90-7 Level 2b <![CDATA[C 14 H 14 N2O]]> 227.1179 7.29 Others 9-Anthracenethiol 17534-14-4 Level 2b <![CDATA[C 14 H 10 S]]> 211.0578 6.49 Others Ethylacrylate 140-88-5 Level 2b <![CDATA[C5H8O2]]> 101.0597 6.21 Others 1-cyclohexyl-3-phenylurea 886-59-9 Level 2b <![CDATA[C 13 H 18 N2O]]> 219.1491 8.24 Others 2-(2-Butoxyethoxy)ethanol 112-34-5 Level 2b <![CDATA[C8H 18 O3]]> 163.1328 16.02 Others Methylidynetri-p-phenylenetriisocyanate 2422-91-5 Level 2b <![CDATA[C 22 H 13 N3O3]]> 368.1042 6.89 Others 1,2-Diphenyl-3-cyclohexylguanidine 4833-44-7 Level 2b <![CDATA[C 19 H23N3]]> 294.1965 6.31 Others tert-Butylperoxy2-methylbenzoate 22313-62-8 Level 2b <![CDATA[C 12 H 16 O3]]> 209.1172 13.18 Others Phthalic acid 88-99-3 Level 2b <![CDATA[C8H6O4]]> 167.0338 16.74 Others 1-Nitroso-2-naphthol 131-91-9 Level 3 <![CDATA[C 10 H7NO2]]> 174.0546 5.81 Adhesiveadditives Triphenylarsenite 1529-86-8 Level 3 <![CDATA[C 18 H 15 AsO3]]> 355.032 1.52 Adhesiveadditives 1,2,3-Triphenylguanidine 101-01-9 Level 3 <![CDATA[C 19 H17N3]]> 288.1495 5.57 Adhesiveadditives 2-mercaptobenzothiazole 149-30-4 Level 3 <![CDATA[C7H5NS2]]> 167.9936 7.20 Adhesiveadditives 2,2'-Methylenebis(4,6dimethylphenol) 6538-35-8 Level 3 <![CDATA[C 17 H 20 O3]]> 273.1486 16.86 Adhesiveadditives 3-(Trimethoxysilyl)propane-1-thiol 4420-74-0 Level 3 <![CDATA[C6H 16 O3SSi]]> 197.0648 1.58 Processingaids Butylatedhydroxyanisole 25013-16-5 Level 3 <![CDATA[C 11 H 16 O2]]> 181.1223 6.94 Protectiveadditives N,N'-diacetyl-p-phenylenediamine 140-50-1 Level 3 <![CDATA[C 10 H 12 N2O2]]> 193.0972 1.88 Protectiveadditives Rosamox 3650-09-7 Level 3 <![CDATA[C 20 H 28 O4]]> 333.2062 16.81 Protectiveadditives Pterostilbene 537-42-8 Level 3 <![CDATA[C 16 H 16 O3]]> 257.1173 17.88 Protectiveadditives Ethylideneaniline 6052-11-5 Level 3 <![CDATA[C8H9N]]> 120.0807 2.04 Vulcanizingagent N-Phenylheptan-1-imine 4275-05-2 Level 3 <![CDATA[C 13 H 19 N]]> 190.159 16.72 Vulcanizingagent Phthalimide 85-41-6 Level 3 <![CDATA[C8H5NO2]]> 148.0393 3.44 Vulcanizingagent Phenylisocyanate 103-71-9 Level 3 <![CDATA[C7H5NO]]> 120.0444 10.18 Vulcanizingagent Diethyleneglycoldimethacrylate 2358-84-1 Level 3 <![CDATA[C 12 H 18 O5]]> 243.1227 16.73 Vulcanizingagent N,N-Diisopropylbenzothiazole-2-sulfenamide 95-29-4 Level 3 <![CDATA[C 13 H 18 N2S2]]> 267.0977 1.57 Vulcanizingagent 2-Methyl-6-(1-methylcyclohexyl)-p-cresol NA Level 3 <![CDATA[C 15 H 22 O]]> 219.1744 16.56 Others Dipentyldithiocarbamate 15337-18-5 Level 3 <![CDATA[C 11 H 23 NS2]]> 234.1337 6.24 Others Isocyanatocyclohexane 3173-53-3 Level 3 <![CDATA[C7H 11 NO]]> 126.0914 2.03 Others N-isopropyl-N'a-phenyl-1,4-phenylenediaminequinone NA Level 3 <![CDATA[C 15 H 16 N2O2]]> 257.1286 6.66 Others Pentaethyleneglycoldimethylether NA Level 3 <![CDATA[C 12 H 26 O6]]> 267.1803 3.01 Others Azobenzene 103-33-3 Level 3 <![CDATA[C 12 H 10 N2]]> 183.0917 3.24 Others 2-Bromomethylnaphthalene 939-26-4 Level 3 <![CDATA[C 11 H9Br]]> 220.9968 1.59 Others 1-(2-Butoxyethoxy)ethanol 54446-78-5 Level 3 <![CDATA[C8H 18 O3]]> 163.1328 5.64 Others Dibutyldithiocarbamicacidmethylester 38351-44-9 Level 3 <![CDATA[C 10 H 21 NS2]]> 220.118 5.83 Others Diethyleneglycol 111-46-6 Level 3 <![CDATA[C4H 10 O3]]> 107.0702 8.56 Others 1-[4-(2-hydroxypropan-2-yl)phenyl]ethanone 54549-72-3 Level 3 <![CDATA[C 11 H 14 O2]]> 179.1066 7.32 Others Feature matching and screening results: After program blank subtraction and software feature extraction, a total of 161 features matching the screening list were obtained; combined with database comparison, software simulation prediction and manual review, 86 candidate compounds were finally identified (confidence levels covering Level 1 to Level 3). Confidence level distribution: 7 compounds were verified by retention time and MS / MS spectra of commercial standards and are classified as Level 1 (structure confirmed); 8 compounds were classified as Level 2a (possible structure) by database spectral matching; 44 compounds were identified by diagnostic evidence and are classified as Level 2b (possible structure); 27 compounds are tentatively classified as Level 3 (provisional candidates) due to lack of secondary mass spectrometry structural evidence. Functional classification coverage: Among the compounds with confidence levels of Level 3 and above, 48 have clear functional classification uses, comprehensively covering five major categories: vulcanizing aids, protective aids, processing aids, adhesive aids and other functional aids, proving that this method can achieve comprehensive screening of RDCs of different functional types; Application value: The experimental results systematically revealed for the first time the internal exposure characteristics of rubber manufacturing workers' RDCs, providing accurate data support for occupational health risk assessment and the formulation of targeted protective measures, and further verifying the practical application value of the method of this invention.

[0051] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. A systematic screening method for rubber-related chemicals in plasma samples, characterized in that, Includes the following steps: S1. Take a plasma sample, add a mixed extraction solvent, extract, centrifuge and separate the layers, collect the supernatant, repeat the extraction and combine the supernatants, concentrate and freeze, centrifuge again and add an isotope internal standard, and use liquid chromatography-tandem high-resolution mass spectrometry in positive and negative ion analysis modes for data-dependent acquisition. S2, Construct a screening list containing rubber industry chemicals and related compounds. The list records the name, CAS number, molecular formula and structural formula of each compound, and is constructed based on the environmental relevance data of the compounds and the feasibility of liquid chromatography-tandem high-resolution mass spectrometry analysis. S3 uses mixed QC samples for screening, extracts features and identifies compounds, removes background interference features, and screens compounds based on quality accuracy, isotope patterns and fragment ion matching to classify potential rubber-related chemicals into confidence levels.

2. The systematic screening method for rubber-related chemicals in plasma samples according to claim 1, characterized in that, In step S1, the mixed extraction solvent is a mixture of ethyl acetate and hexane in a volume ratio of 3:2, and the mixed extraction solvent contains 0.1% formic acid; the plasma sample transfer volume is 180~220 μL, and the mixed extraction solvent added volume is 2.5~3.5 mL.

3. The systematic screening method for rubber-related chemicals in plasma samples according to claim 2, characterized in that, In step S1, the extraction is an oscillating extraction, the extraction time is 8-12 minutes, and after extraction, it is centrifuged at 2800-3200×g and 23-27℃ for 2-4 minutes. The extraction step is repeated 2-3 times and the supernatants are combined.

4. The systematic screening method for rubber-related chemicals in plasma samples according to claim 3, characterized in that, In step S1, the concentration is carried out by nitrogen purging and concentration in a water bath at 28~32 ℃ until the solution volume is approximately 80~120 μL; the freezing treatment is carried out by freezing at -85~-75 ℃ for 10~14 hours; after freezing, the solution is centrifuged at 14000~16000×g at -12~-8 ℃ for 4~6 minutes.

5. The systematic screening method for rubber-related chemicals in plasma samples according to claim 4, characterized in that, In step S1, the amount of the isotope internal standard added is 0.8~1.2 ng, and after addition, it is mixed and injected into the sample, with an injection volume of 4~6 μL.

6. The systematic screening method for rubber-related chemicals in plasma samples according to claim 5, characterized in that, In step S1, the column temperature of the liquid chromatography-tandem high-resolution mass spectrometry column is 28-32 °C; the mobile phase in both positive and negative ion modes includes phase A containing ammonium formate solution and phase B containing methanol, and the gradient elution program is as follows: 0-2 min, phase B accounts for 38%-42%, flow rate 0.28-0.32 mL / min; 2-3 min, increases to 64%-68%; 3-12 min, increases to 68%-72%; 12-14 min, increases to 98%-100%; 14-16 min, maintains 98%-100%; 16-16.1 min, decreases to 38%-42%; 16.1-19 min, maintains 38%-42%.

7. The systematic screening method for rubber-related chemicals in plasma samples according to claim 6, characterized in that, In step S1, the ion source for the liquid chromatography-tandem high-resolution mass spectrometry is H-ESI, the spray voltage for positive ion mode is 3300~3700 V, and the spray voltage for negative ion mode is 2300~2700 V; the sheath gas flow rate is 38~42 units, the auxiliary gas flow rate is 8~12 units, and the purge gas flow rate is 0.8~1.2 units; the ion transfer tube temperature is 315~335 ℃, and the evaporator temperature is 340~360 ℃.

8. The systematic screening method for rubber-related chemicals in plasma samples according to claim 7, characterized in that, In step S1, the Orbitrap resolution of the liquid chromatography-tandem high-resolution mass spectrometry is 110,000~130,000 for the first-stage scan and 14,000~16,000 for the second-stage scan; the RF lens is 68%~72%; the scan range is 90~1100 m / z; the activation type is HCD, the HCD collision energy is 28%~32%, and the ±HCD collision energy is 4%~6%; the isolation window is 1.8~2.2 m / z; and the number of data-dependent scans is 18~22.

9. A systematic screening method for rubber-related chemicals in plasma samples according to claim 8, characterized in that, In step S3, the standard for removing background interference features is: features with an intensity less than 4 to 6 times that of the solvent blank or program blank are marked as background and removed; The screening criteria also include: peak intensity of 80,000~120,000 cps; mass error of <4~6 ppm; and the number of fragments matched in the tandem mass spectrometry spectrum of ≥3.

10. A systematic screening method for rubber-related chemicals in plasma samples according to claim 9, characterized in that, In step S3, the confidence levels include level 1, level 2a, level 2b, and level 3, where level 1 is the structure confirmed by commercial standards, level 2a is the possible structure determined by database spectral matching, level 2b is the possible structure determined by diagnostic evidence, and level 3 is a provisional candidate lacking evidence of secondary mass spectrometry structure.

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