A High-Sensitivity, High-Throughput Scanning Analysis Method for Halogenated Organic Compounds

Abstract

This invention relates to a highly sensitive, high-throughput scanning analysis method for halogenated organic compounds. The method includes the following steps: extracting halogenated organic compounds from a sample to obtain a halogenated organic compound extract; using Ph4PCl as a mobile phase additive, performing high-resolution mass spectrometry (HMS) detection on the halogenated organic compound extract; and using the HMS detection results to identify unknown halogenated organic compounds and analyze the content of known halogenated organic compounds. This invention's highly sensitive, high-throughput, non-targeted scanning method for the determination of halogenated organic compounds using Ph4PCl as a mobile phase additive is original. This method has advantages such as low sample consumption, simple and convenient pretreatment, accurate determination, high specificity, and low interference, providing a new approach and method for the non-targeted detection of halogenated organic compounds in biological and environmental samples.

Description

Technical Field

[0001] This invention relates to the fields of chemical analysis technology and biological and environmental detection technology, specifically to a highly sensitive, high-throughput, and rapid detection method for halogenated organic compounds. Background Technology

[0002] Humans are continuously exposed to a wide variety of industrial chemicals, synthetic byproducts, and transformation products in the environment. Among them, a large class of halogenated organic compounds, including organochlorine pesticides, chlorinated paraffins, decaclones, and brominated flame retardants, are receiving increasing attention due to their persistence, bioaccumulation, and toxicity, and have been or are being included in the inventory of persistent organic pollutants (POPs). Despite global regulatory bans or production restrictions, halogenated organic compounds and their biotransformation products are widely present in various environmental media such as water, air, and soil, and accumulate in organisms, posing ecological and health hazards. Therefore, high-throughput scanning analysis of halogenated organic compounds is crucial for assessing the exposure risk of these substances in organisms. Currently, halogenated organic compounds are enriched and purified using similar sample pretreatment methods, and different chromatographic separation and mass spectrometry methods are used for different substances, resulting in a significant workload and analysis time. Overall, current scanning methods for halogenated organic compounds are mainly limited by the sensitivity, resolution, and scanning throughput of the methods.

[0003] Therefore, it is necessary to find a reagent that can produce characteristic, consistent, and highly responsive ionization of different types of halogenated organic compounds, and to develop a highly sensitive and high-throughput scanning method for halogenated organic compounds. Summary of the Invention

[0004] This invention is the first to discover that tetraphenylphosphine chloride (Ph4PCl) significantly enhances the ionization efficiency of various halogenated organic compounds (including organochlorine pesticides, chlorinated paraffins, decaclones, and brominated flame retardants) in liquid chromatography-mass spectrometry analysis. These halogenated organic compounds all contain the structural features of haloalkanes, generating a single, specific chloride-enhanced ionization signal ([M+Cl)) at the ESI ion source. - This method significantly improves analytical sensitivity, with mass spectrometry response 50-7000 times higher than existing liquid chromatography-mass spectrometry (LC-MS) methods. Based on this, and combining the characteristic isotope peak shapes of chlorine-enhanced ionization products of halogenated organic compounds, a highly sensitive and high-throughput scanning method for halogenated organic compounds using Ph4PCl chlorine-enhanced ionization combined with LC-MS / MS was established. This provides a new technical means for the detection and non-targeted screening of halogenated organic compounds in biological and environmental samples.

[0005] Specifically, the technical solution adopted in this invention is as follows:

[0006] A high-throughput non-targeted identification and highly sensitive detection method for halogenated organic compounds, comprising the following steps:

[0007] Halogenated organic compounds are extracted from the sample to be tested (which may be a biological sample or an environmental sample) to obtain a halogenated organic compound extract.

[0008] Ph4PCl was used as a mobile phase additive to perform high-resolution mass spectrometry detection on the extract of halogenated organic compounds.

[0009] High-resolution mass spectrometry was used to identify unknown halogenated organic compounds and to analyze their content.

[0010] Furthermore, the specific method for extracting halogenated organic compounds from the sample to be tested is as follows:

[0011] Add 0.2 ng of internal standard to 100-2000 μL of blood sample, 0.1-100 mg of biological tissue sample, or 10-1000 mg of environmental sample, and add 200-2000 μL of frozen acetone. Sonicate and homogenize the sample. Extract with 500-2000 μL of n-hexane, centrifuging at at least 5000 g for 3-10 minutes. Collect the n-hexane in a new bottle and repeat three times. Inject the remaining liquid into a glass column containing anhydrous sodium sulfate and elute with an appropriate amount of dichloromethane. Mix the dichloromethane eluent with the n-hexane extract to obtain the halogenated organic compound extract. Dry the extract under weak nitrogen, dilute to volume with 20-100 μL of acetonitrile, and analyze using UPLC-High Resolution Mass Spectrometer (UPLC-HRMS).

[0012] Preferably, the internal standard is 13 C 10 -1,5,5,6,6,10-hexachlorodecan、 3 C 10 -anti-DP and 13 C 12 -4,4'-DDT (internal standard types can also be added depending on the known target object).

[0013] Furthermore, high-resolution mass spectrometry methods for detecting halogenated organic compounds include:

[0014] High-resolution mass spectrometry equipped with an electrospray ionization source was used, and halogenated organic compounds were separated by reverse-phase chromatography. The mobile phase consisted of methanol and water, with tetraphenylphosphine chloride (Ph4PCl) added to the methanol. The halogenated organic compounds were scanned using negative ion-Full-MS mode. The concentration of halogenated organic compounds in the sample was determined by the peak area corrected by the internal standard method.

[0015] More preferably, the preferred concentration of Ph4PCl added to the methanol mobile phase is 10 μM, and the reverse chromatographic column is a C8 column.

[0016] Furthermore, methods for detecting the content of halogenated organic compounds include:

[0017] For organochlorine pesticides, declone and brominated flame retardants, quantification is performed using the internal standard method: a standard curve solution of such halogenated organic compounds is prepared, and the concentration is determined by UPLC-HRMS. Based on the internal standard method, the concentration of such halogenated organic compounds in the sample is calculated by linear regression analysis of the peak area corrected by the internal standard.

[0018] For the quantification of chlorinated paraffins, the linear relationship between the response factor and the chlorine content is used: UPLC-HRMS is applied to analyze various short-chain, medium-chain, and long-chain chlorinated paraffin standards with different chlorine contents, calculate the actual chlorine content and response factor of the standards, obtain the regression equation through linear regression analysis, then measure the chlorine content of chlorinated paraffins in the test sample, and calculate the concentration of short-chain, medium-chain, and long-chain chlorinated paraffins in the test sample based on the regression equation.

[0019] Furthermore, non-targeted identification methods for unknown halogenated organic compounds include:

[0020] First, peak extraction was performed on the mass spectrometry data to obtain the mass-to-charge ratio, retention time, and intensity of the chemical signals. Then, non-targeted screening was conducted based on the isotopic peaks characteristic of halogenated organic compounds. The extracted mass spectrometry signals were then mass-aligned, and the mass spectrometry signals of halogenated organic compound clusters were screened and paired based on the precise mass numbers and peak height ratios of the chlorine and bromine isotope peaks. Finally, elemental composition analysis was performed on the screened halogenated organic compound clusters. Simultaneously, isotopic peak theoretical simulations were performed on the determined chemical formulas, and the results were compared with actual mass spectrometry signals to verify the accuracy of the elemental composition analysis.

[0021] Preferably, the peak extraction parameters are set as follows: mass-to-charge ratio tolerance of 3 ppm; signal-to-noise ratio threshold of 3; peak width of 5-10 s; number of pre-screening attempts of 3; and pre-screening level of 1000. The mass spectrometry signal screening and pairing parameters are: retention time tolerance of 5 s; mass-to-charge ratio tolerance of 0.5 mDa. The elemental composition analysis parameters are: upper limit of the number of atoms for each element in the chemical formula of 60C, 150H, 60O, 60N, 60P, 60S, 150Cl, 150Br, and 150F; and a mass tolerance of 3 ppm.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention reveals that Ph4PCl, as a mobile phase additive, can significantly improve the mass spectrometry detection sensitivity of halogenated organic compounds, reducing the detection sensitivity of each substance to as low as 30-180 fg, representing a sensitivity increase of 50-7000 times. Furthermore, this invention also provides a high-throughput non-targeted identification method for halogenated organic compounds. In an implementation example based on this method, 214 known halogenated organic pollutants were detected in human blood, and 497 unknown halogenated organic pollutants were successfully identified through non-targeted scanning.

[0024] This invention presents an original method for the high-sensitivity, high-throughput, non-targeted scanning determination of halogenated organic compounds using Ph4PCl as a mobile phase additive. This method offers advantages such as low sample consumption, simple and convenient pretreatment, accurate determination, high specificity, and low interference, providing a new approach and method for the non-targeted detection of halogenated organic compounds in biological and environmental samples. By adding the special additive Ph4PCl to the mobile phase to enhance the ionization of halogenated compounds through chlorination, and combining this with UPLC-HRMS for ultra-sensitive and high-resolution non-targeted detection, this method enables accurate non-targeted detection of halogenated organic compounds with a small sample size. It overcomes the shortcomings of traditional methods, such as low sensitivity, low resolution, low throughput, large sample consumption, complex pretreatment, and long analysis time, providing a new and powerful tool for the detection and non-targeted screening of halogenated organic compounds in biological and environmental samples. Attached Figure Description

[0025] Figure 1 Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of DDT-type substance standard samples;

[0026] Figure 2 Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of chlordane, toxaphene, trichlorfon, hexabromocyclododecane, α / β-TBECH and DPTE standard samples;

[0027] Figure 3 For some of the decaclones (Dec 601, Dec 603, Dec 604A, Dec 604B, and C) 13 H4Cl 10 Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of standard samples;

[0028] Figure 4Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of dieldrin, isodrin, HCHs, endosulfan, and some declolon (cis-declolon, trans-declolon, aCl10DP, axCl11DP, aCl11DP, and axxCl10DP) standards.

[0029] Figure 5 For anticides, heptachlor, decachlor, and some declofenac (Dec 602 and C) 15 H6Cl 12 Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of standard samples;

[0030] Figure 6 Chromatographic and mass spectra of Ph4PCl-enhanced UPLC-ESI-Orbitrap-MS analysis of short-chain, medium-chain, and long-chain chlorinated paraffin standards;

[0031] Figure 7 Linear regression working curves of overall corresponding factors and measured chlorine content for short-chain, medium-chain, and long-chain chlorinated paraffin standard samples;

[0032] Figure 8 H / Cl-scale mass defect plots of known (Fig. a) and newly identified (Fig. b) halogenated organic chemical signals detected by high-sensitivity, high-throughput scanning analysis of blood samples from the general population;

[0033] Figure 9 Chromatograms of known and newly identified halogenated organic compounds in blood samples from the general population;

[0034] Figure 10 Mass spectra for high-sensitivity, high-throughput scanning analysis of blood samples from the general population;

[0035] Figure 11 This represents the concentration levels of known and newly identified halogenated organic compounds in blood samples from the general population. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below.

[0037] The following describes the technical solution of the present invention in more detail by taking the scanning and analysis of halogenated contaminants in the blood of normal people as an example.

[0038] 1. Instruments and reagents:

[0039] Ultra-high performance liquid chromatography-tandem mass spectrometry (Thermo Fisher Scientific), including the UltiMate 3000 ultra-high performance liquid chromatography system, Q Exactive Plus Orbitrap mass spectrometer; high-speed refrigerated centrifuge; rotary evaporator; analytical balance; nitrogen evaporator; vortex mixer.

[0040] Tetraphenylphosphine chloride, methanol, acetonitrile, acetone, dichloromethane, n-hexane (LC / MS grade), Milli-Q water.

[0041] 2. Sample Pretreatment. Accurately weigh 200 μL of the blood sample to be tested, add 0.2 ng of internal standard and 200 μL of frozen acetone, and sonicate to mix the tissue sample. Extract with 500 μL of n-hexane, centrifuge at 5000 g for 3 minutes, and collect the n-hexane extract in a new bottle. Repeat three times. Inject the remaining liquid into a glass column containing anhydrous sodium sulfate and elute with an appropriate amount of dichloromethane. Collect the dichloromethane eluent and mix it with the n-hexane extract to obtain the halogenated organic compound extract. Concentrate the halogenated organic compound extract to approximately 1 mL using a rotary evaporator, transfer and dry with a weak nitrogen atmosphere, make up to volume with 20 μL of acetonitrile, thoroughly rinse the inner wall and vortex, then transfer to an inner liner tube for UPLC-HRMS analysis.

[0042] 3. UPLC-HRMS detection using ultra-high performance liquid chromatography-tandem mass spectrometry:

[0043] Preferably, the UPLC-HRMS detection conditions are as follows:

[0044] (1) Chromatographic separation conditions: The chromatographic column was a Poroshell 120EC-C8 with a length of 100 mm. The mobile phase gradient consisted of water (A) and methanol with 10 μM Ph4PCl (B), with the following gradient changes: 0-1 min, 10% B; 1-1.5 min, 10%-30% B; 1.5-2 min, 30%-60% B; 2-4 min, 60%-100% B; 4-10 min, 100% B; 10-10.5 min, 100%-30% B; 10.5-11.5 min, 30%-10% B; 11.5-13 min, 10% B; the mobile phase flow rate was 0.1 mL / min; the column temperature was 40 ℃; and the injection volume was 5 μL.

[0045] (2) Mass spectrometry detection conditions: the ion source is an ESI source; the data scanning mode is negative ion mode; the resolution is 140000; the AGC target is 5e6; the maximum IT is 250ms; the spray voltage is 2.5kV; the capillary temperature is 200℃; the Aux gas heater temperature is 300℃; the sheath gas flow rate is 35arb; the Aux gas flow rate is 10arb; the purge gas flow rate is 1arb; and the RF value of the cascaded ion guide is 60.

[0046] 4. Standard curve and limit of detection:

[0047] (1) Preparation of working solutions for standard substances

[0048] Prepare 18 organochlorine pesticides (o,p'-DDD,p,p'-DDD,o,p'-DDE,p,p'-DDE,o,p'-DDT,p,p'-DDT,β-HCH,γ-HCH, chlordane, heptachlor, dieldrin, isodrin, α / β-thiodan, methylphenidate, decachlor, trichlorfon, and toxaphene), and 13 decaclones (cis-decaclones, trans-decaclones, Dec601, Dec602, Dec603, Dec604A, Dec604B, aCl10DP, aCl11DP, axCl11DP, axxCl10DP, C...). 15 H6Cl 12 , and C 13 H4Cl 10 A mixed standard solution of acetonitrile and four brominated flame retardants (hexabromocyclododecane, α / β-tetrabromoethylcyclohexane (α / β-TBECH), and 2,3-dibromopropyl-2,4,6-tribromophenylallyl ether (DPTE)) was prepared. The standard concentrations were 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, and 500 ng / mL, respectively, and the internal standard concentration was 10 ng / mL.

[0049] Standard solutions of chlorinated paraffin with different concentrations and chlorine contents were prepared: short-chain (51.5%, 53.5%, 55.5%, 59.25%, and 63.0%), medium-chain (42.0%, 47.0%, 52.0%, 54.5%, and 57.0%), and long-chain (36.0%, 39.25%, 42.5%, 45.75%, and 49.0%) chlorinated paraffins. Acetonitrile was used as the solvent, and the concentrations were 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 2 ng / mL, 10 ng / mL, 50 ng / mL, 200 ng / mL, and 500 ng / mL, respectively.

[0050] (2) Preparation of working curves: 5 μL of standard solution was analyzed by UPLC-HRMS to determine the ionization mode, quantitative ions, and retention time of the substances. See Table 1 for details. Figures 1 to 6 At the same time, a working curve is created.

[0051] Working curves were prepared for organochlorine pesticides, declofenac, and brominated flame retardants. Linear regression analysis was performed on the peak area ratio (Y) and concentration ratio (X) of each standard substance to internal standard to obtain the working curves. The results showed that within the range of 0.01 ng / mL–500 ng / mL, the peak area ratio and concentration ratio of the standard substance to internal standard exhibited a good linear relationship, with a correlation coefficient (γ) of [missing value]. 2 All are greater than 0.99.

[0052] Working curves were prepared for short-chain, medium-chain, and long-chain chlorinated paraffins. First, the measured chlorine content of each standard solution was calculated based on the theoretical chlorine content and peak area ratio of each individual chlorinated paraffin component. Then, the ratio of the peak area of ​​total chlorinated paraffin to the internal standard in the standard solution to the concentration ratio was used to obtain the overall response factor of chlorinated paraffin in each standard solution. Linear regression analysis was performed on the overall response factor (Y) of chlorinated paraffin in the standard solutions and the measured chlorine content (X) to obtain regression curves. The results showed that within the range of 0.01 ng / mL–500 ng / mL, the overall response factor of short-chain, medium-chain, and long-chain chlorinated paraffins exhibited a good linear relationship with the chlorine content, and the correlation coefficient (γ) was relatively high. 2 All are greater than 0.99, see details. Figure 7 .

[0053] Table 1. Ionization mode, quantitative ions, retention time, instrument detection limit, method detection limit, and spiked recovery rate of standard substances.

[0054]

[0055] (3) Instrument detection limit

[0056] Six spiked samples (5 μL each) and nine blank samples were analyzed by UPLC-HRMS to determine the instrument detection limit, method detection limit, and spike recovery rate of each substance, as detailed in Table 1. The instrument detection limit was the lowest concentration of the standard substance that produced a response with a signal-to-noise ratio greater than three times. The method detection limit was determined as three times the standard deviation of the responses in the nine blank samples or calculated based on the instrument detection limit.

[0057] 5. High-sensitivity, high-throughput scanning identification of halogenated organic compounds in human blood samples

[0058] (1) Data processing

[0059] Five μL of pre-processed blood samples were analyzed using UPLC-HRMS. Peak extraction and mass number alignment were performed on the collected mass spectrometry data to obtain information such as retention time, mass number, and intensity of chemical signals. Then, based on the characteristic isotope peak shapes and precise mass numbers of halogenated organic compounds, the chemical signals of halogenated organic compounds were screened. Approximately 5000 chemical signals belonging to halogenated organic compounds were found in each blood sample. Furthermore, the chemical signals of halogenated organic compounds were paired to obtain halogenated organic compound clusters composed of multiple chemical signals.

[0060] (2) Targeted analysis of known halogenated organic compounds

[0061] Furthermore, the retention time and mass number of the halogenated organic cluster peaks detected in the blood sample were compared with those of organochlorine pesticides, declone, brominated flame retardants, and chlorinated paraffins in the standard samples. The results showed that 1939 chemical signals detected in human blood samples corresponded to 192 halogenated organic pollutants, including 33 short-chain chlorinated paraffins, 52 medium-chain chlorinated paraffins, 97 long-chain chlorinated paraffins, 18 organochlorine pesticides (o,p'-DDD,p,p'-DDD,o,p'-DDE,p,p'-DDE,o,p'-DDT,p,p'-DDT,β-HCH,γ-HCH, chlordane, heptachlor, dieldrin, isodrin, α-endothelium, β-endothelium, methylphenidate, decachlorophen, trichlorfon, and toxaphene), and 13 decaclones (cis-decaclones, trans-decaclones, Dec601, Dec602, Dec603, Dec604A, Dec604B, aCl10DP, aCl11DP, axCl11DP, axxCl10DP, C... 15 H6Cl 12 , and C 13 H4Cl 10 ) and one brominated flame retardant (hexabromocyclododecane), see Figure 8 a) diagram, Figure 9 , Figure 10 . Figure 8 Figure a) shows the mass defect map of H / Cl scale, which is obtained by multiplying the measured mass number of the chemical signal by 1.001174 (34 / 33.960128) and using it as the x-axis. The decimal part of the H / Cl scale mass number is the mass defect and is used as the y-axis. Each chemical signal cluster in the figure corresponds to a halogenated organic compound.

[0062] (3) Non-targeted scanning identification of unknown halogenated organic compounds

[0063] Furthermore, elemental composition analysis was performed on the chemical signals of unknown halogenated organic compound clusters that did not match the standard substances. The results showed that 175 chemical signals were identified as 22 ultrashort-chain chlorinated paraffins (carbon chain length less than 10), 243 chemical signals as 57 unsaturated chlorinated paraffins (containing 1-4 degrees of unsaturation), 2470 chemical signals as 308 oxygen-containing chlorinated paraffins, 132 chemical signals as 2 HCH analogs, pentachlorophenol and 2 analogs, tribromophenol, 3 brominated chlorinated paraffins, and 23 nitrogen-containing or nitrogen- and oxygen-containing chlorinated organic compounds. (See details...) Figure 8 Figure b) Figure 9 , Figure 10 Table 2 Figure 8 Figure b) shows the mass defect plot at the H / Cl scale, plotted using the chemical signals of halogenated organic compounds identified by non-targeted scanning.

[0064] Table 2. Newly identified HCH analogues, pentachlorophenol and its analogues, tribromophenol, brominated chlorinated paraffins, and nitrogen- or nitrogen-oxygen-containing chlorinated organic compounds in blood samples from the general population using high-sensitivity, high-throughput scanning.

[0065] mass number Isotope peaks Retention time (min) Molecular formula Ionization Deviation (ppm) Degree of unsaturation (Ω) 290.86471 288.86771,292.86172 7.25 <![CDATA[C6H7Cl5]]> <![CDATA[[M+Cl] - ]]> 1.1 1 306.85963 304.86264,308.85666 7.74 <![CDATA[C6H7Cl5O]]> <![CDATA[[M+Cl] - ]]> 1.0 1 194.91683 196.91385,198.91072 7.96 <![CDATA[C6H3Cl3O]]> <![CDATA[[M-H] - ]]> 1.3 4 230.87501 228.87804,232.87197 8.03 <![CDATA[C6H2Cl4O]]> <![CDATA[[M-H] - ]]> 1.6 4 264.83582 262.83877,266.83285,268.82991 8.01 <![CDATA[C6HCl5O]]> <![CDATA[[M-H] - ]]> 0.5 4 328.76314 330.76103,326.76531,332.75891 8.09 <![CDATA[C6H3O1Br3]]> <![CDATA[[M-H] - ]]> 0.5 4 443.1841 445.18201,447.17902 8.46 <![CDATA[C 21 H 42 ClBr]]> <![CDATA[[M+Cl] - ]]> -0.1 0 626.97269 624.97549,628.96965 8.48 <![CDATA[C 21 H 38 Cl4Br2]]> <![CDATA[[M+Cl] - ]]> -0.1 0 640.9886 638.99154,642.98579 8.51 <![CDATA[C 22 H 40 Cl4Br2]]> <![CDATA[[M+Cl] - ]]> 0.3 0 524.89148 522.89448,526.88849 7.81 <![CDATA[C 13 H 22 Cl8N2]]> <![CDATA[[M+Cl] - ]]> -0.1 0 586.88483 584.88782,588.88185 7.84 <![CDATA[C 15 H 25 Cl9N2]]> <![CDATA[[M+Cl] - ]]> 1.7 0 673.13545 671.13845,675.13249 7.84 <![CDATA[C 26 H 49 Cl7N2]]> <![CDATA[[M+Cl] - ]]> 2.2 0 353.13109 355.12812 8.24 <![CDATA[C 16 H 28 Cl2N2]]> <![CDATA[[M+Cl] - ]]> -0.5 3 395.17846 397.17542,399.17258 8.6 <![CDATA[C 19 H 34 Cl2N2]]> <![CDATA[[M+Cl] - ]]> 0.6 3 397.93384 395.93685,399.93083 8.84 <![CDATA[C8H 16 Cl5N3O2]]> <![CDATA[[M+Cl] - ]]> -0.1 0 392.00435 390.00739,394.00133 8.88 <![CDATA[C 10 H 21 Cl4N3O2]]> <![CDATA[[M+Cl] - ]]> 0.5 0 718.80423 716.8072,720.80121 9.14 <![CDATA[C 16 H 25 Cl 11 N2O3]]> <![CDATA[[M+Cl] - ]]> 1.2 0 594.97866 590.98400,592.98161,596.97571 9.13 <![CDATA[C 17 H 31 Cl7N2O3]]> <![CDATA[[M+Cl] - ]]> 1.4 0 746.83591 742.84098,744.83891,748.83287 9.22 <![CDATA[C 18 H 29 Cl 11 N2O3]]> <![CDATA[[M+Cl] - ]]> 1.7 0 685.00231 683.00529,686.99933,688.99581 10.13 <![CDATA[C 21 H 38 Cl8N2O3]]> <![CDATA[[M+Cl] - ]]> 1.2 0 333.92593 331.92896,335.92296 8.31 <![CDATA[C6H 11 Cl4N3O2]]> [M+Cl]- 0.1 1 395.90745 393.91044,397.90449 8.47 <![CDATA[C9H 14 Cl5NO3]]> <![CDATA[[M+Cl] - ]]> 1.2 1 443.8843 441.88732,445.88126 8.78 <![CDATA[C 10 H 15 Cl6NO3]]> <![CDATA[[M+Cl] - ]]> 1.4 1 357.99537 355.99837,359.99241 8.73 <![CDATA[C 10 H 18 Cl3NO4]]> <![CDATA[[M+Cl] - ]]> -0.4 1 479.84229 477.84529,481.83929 8.76 <![CDATA[C 10 H 14 Cl7NO3]]> <![CDATA[[M+Cl] - ]]> 1.1 1 594.93441 592.93746,596.93139,598.92803 8.91 <![CDATA[C 17 H 28 Cl8N2O]]> <![CDATA[[M+Cl] - ]]> 1.8 1 589.00358 587.00659,591.00055 8.93 <![CDATA[C 19 H 33 Cl7N2O]]> <![CDATA[[M+Cl] - ]]> -0.1 1 520.82407 518.82704,522.82103 8.32 <![CDATA[C 12 H 14 Cl8N2O]]> <![CDATA[[M+Cl] - ]]> 0.4 3 852.87605 848.88209,850.87903,854.87307 8.78 <![CDATA[C 25 H 35 Cl 11 N2O4]]> <![CDATA[[M+Cl] - ]]> -0.6 4 633.87582 631.87881,635.87284 9.28 <![CDATA[C 22 H 21 Cl8NO]]> <![CDATA[[M+Cl] - ]]> 0.7 9 680.18058 678.18357,682.17759 9.34 <![CDATA[C 33 H 45 Cl4NO3]]> <![CDATA[[M+Cl] - ]]> -0.2 10 722.22783 720.23084,724.22482 9.34 <![CDATA[C 36 H 51 Cl4NO3]]> <![CDATA[[M+Cl] - ]]> 0.2 10

[0066] 6. Concentration levels of halogenated organic compounds in the blood

[0067] (1) The quantitative and concentration levels of halogenated organic compounds are known.

[0068] For organochlorine pesticides, declone, and brominated flame retardants, the concentration of these halogenated organic compounds in blood samples is calculated based on the peak area corrected by the internal standard method. For short-chain, medium-chain, and long-chain chlorinated paraffins, the measured chlorine content is calculated based on the peak area ratio of each individual component. Then, the overall corresponding factor of the blood sample is obtained according to the regression equation. The total concentration of chlorinated paraffins is calculated based on the overall peak area corrected by the internal standard. Finally, the concentration of each individual component is calculated based on the peak area ratio of each individual component. The results showed that the concentration levels of various halogenated organic compounds in the blood of 50 ordinary individuals were (mean ± standard deviation) as follows: total concentration of short-chain chlorinated paraffins was 292.7 ± 212.6 ng / mL, total concentration of medium-chain chlorinated paraffins was 142.8 ± 144.6 ng / mL, total concentration of long-chain chlorinated paraffins was 11.7 ± 7.9 ng / mL, total concentration of DDT derivatives was 13.9 ± 8.1 ng / mL, total concentration of decaclones was 0.78 ± 0.67 ng / mL, and concentration of hexabromocyclododecane was 0.11 ± 0.08 ng / mL. The average concentration of organochlorine pesticides ranged from 0.28 ng / mL to 2.9 ng / mL. (See details...) Figure 11 .

[0069] (2) Estimation of concentration levels of newly identified halogenated organic compounds

[0070] The newly identified halogenated organic compounds detected in the blood samples of this case included ultrashort-chain chlorinated paraffins, unsaturated chlorinated paraffins, oxychlorinated paraffins, HCH analogs, pentachlorophenol and two analogs, tribromophenol, three brominated chlorinated paraffins, and 23 nitrogen-containing or nitrogen- and oxygen-containing chlorinated organic compounds. For pentachlorophenol and tribromophenol, standard solutions were used to establish working curves, and quantification was performed using the internal standard method. For the other newly identified halogenated organic compounds, no commercial standard samples were available for quantification; therefore, their concentrations were estimated using the corresponding factors of structurally similar known halogenated organic compound standards. The results showed that the concentrations of newly identified halogenated organic compounds in human blood samples were mainly contributed by long-chain chlorinated hydrogen oxychloride (LCCPO2s), ultra-short-chain chlorinated paraffins (vSCCPs), and long-chain chlorinated trioxychloride (LCCPO3s), with concentrations of 172.9±302.6 ng / mL, 137.6±148.2 ng / mL, and 10.1±5.3 ng / mL, respectively. The average concentrations of the remaining halogenated organic compounds were below 10 ng / mL. (See details...) Figure 11 .

[0071] In summary, this invention successfully detected and accurately quantified 192 known halogenated organic pollutants in blood samples from the general population, and successfully identified 519 unknown halogenated organic pollutants and estimated their concentration levels using non-targeted scanning. This invention provides an effective method for high-sensitivity, high-throughput scanning analysis of halogenated organic compounds in various environmental media.

[0072] Unless otherwise specified in the above embodiments, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. For reagents whose manufacturers are not specified, commercially available standard products can be used. All instruments, equipment, and consumables used can be common commercially available products after simple adjustments.

[0073] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention.

Claims

1. A highly sensitive and high-throughput scanning analytical method for the analysis of halogenated organic compounds using Ph4PCl-enhanced ionization combined with liquid chromatography-high-resolution mass spectrometry, characterized in that, Includes the following steps: Halogenated organic compounds were extracted from the sample to be tested to obtain halogenated organic compound extract; Ph4PCl was used as a mobile phase additive to perform high-resolution mass spectrometry detection on the extract of halogenated organic compounds. By adding Ph4PCl to the mobile phase, the ion source of the halogenated compounds was enhanced by chlorination, thereby improving the mass spectrometry detection sensitivity of the halogenated organic compounds. The ion source for the high-resolution mass spectrometry detection was an ESI source, and the data scanning mode was negative ion mode. High-resolution mass spectrometry was used to identify unknown halogenated organic compounds and to analyze their content. The method of using Ph4PCl as a mobile phase additive to perform high-resolution mass spectrometry detection on the halogenated organic compound extract includes: High-resolution mass spectrometry equipped with an electrospray ionization source was used to separate halogenated organic compounds by reverse chromatography. The mobile phase was methanol and water, with Ph4PCl added to the methanol. The halogenated organic compounds were scanned using negative ion-Full-MS mode. The concentration of halogenated organic compounds in the sample was determined using the internal standard method with the peak area corrected by the internal standard. The concentration of Ph4PCl added to the methanol is 10 μM.

2. The method according to claim 1, characterized in that, The sample to be tested is a biological sample or an environmental sample.

3. The method according to claim 1, characterized in that, The extraction of halogenated organic compounds from the sample to be tested yields a halogenated organic compound extract, comprising: Add 0.2 ng of internal standard to 100-2000 μL of blood sample, 0.1-100 mg of biological tissue sample, or 10-1000 mg of environmental sample, and add 200-2000 μL of frozen acetone. Then, sonicate and homogenize the sample. Then extract with 500-2000 μL of n-hexane, centrifuge at a force of not less than 5000 g for 3-10 minutes, and collect the n-hexane in a new bottle. Repeat three times. The remaining liquid was injected into a glass column containing anhydrous sodium sulfate and eluted with an appropriate amount of dichloromethane. The dichloromethane eluent was then mixed with the n-hexane extract to obtain the halogenated organic extract.

4. The method according to claim 3, characterized in that, The internal standard is 13 C 10 -1,5,5,6,6,10-hexachlorodecan、 3 C 10 -anti-DP and 13 C 12 -4,4'-DDT.

5. The method according to claim 1, characterized in that, The reverse chromatographic column is a C8 column.

6. The method according to claim 1, characterized in that, The content analysis of halogenated organic compounds includes: For organochlorine pesticides, declone and brominated flame retardants, quantification is performed using the internal standard method: standard curve solutions of such halogenated organic compounds are prepared and determined by UPLC-HRMS. Based on the internal standard method, the concentration of such halogenated organic compounds in the sample is calculated by linear regression analysis of the peak area corrected by the internal standard. For the quantification of chlorinated paraffins, the linear relationship between the response factor and the chlorine content is used: UPLC-HRMS is applied to analyze various short-chain, medium-chain, and long-chain chlorinated paraffin standards with different chlorine contents, calculate the actual chlorine content and response factor of the standards, obtain the regression equation through linear regression analysis, then measure the chlorine content of chlorinated paraffins in the test sample, and calculate the concentration of short-chain, medium-chain, and long-chain chlorinated paraffins in the test sample based on the regression equation.

7. The method according to claim 1, characterized in that, The identification of unknown halogenated organic compounds includes: First, peak extraction is performed on the mass spectrometry data to obtain the mass-to-charge ratio, retention time, and intensity of the chemical signal; Then, non-targeted screening was performed based on the isotope peaks characteristic of halogenated organic compounds. The extracted mass spectrometry signals were mass aligned, and the mass spectrometry signals of halogenated organic compound cluster peaks were screened and paired according to the precise mass number and peak height ratio of chlorine and bromine isotope peaks. Finally, the elemental composition of the selected halogenated organic cluster peaks was analyzed, and the isotope peaks of the determined chemical formulas were simulated theoretically and compared with the actual mass spectrometry signals to verify the accuracy of the elemental composition analysis.

8. The method according to claim 7, characterized in that, The peak extraction parameters are set as follows: peak mass-to-charge ratio tolerance is 3 ppm; signal-to-noise ratio threshold is 3; chromatographic peak width is 5-10 s; pre-screening times are 3; pre-screening level is 1000; the parameters for screening and pairing the mass spectrometry signals are: retention time tolerance is 5 s; mass-to-charge ratio tolerance is 0.5 mDa; the parameters for elemental composition analysis are: upper limit of the number of atoms of each element in the chemical formula is 60C, 150H, 60O, 60N, 60P, 60S, 150Cl, 150Br, 150F; The quality tolerance is 3 ppm.

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