Method for detecting nano black carbon particles in an animal biological sample

Abstract

The application relates to a method for detecting nano black carbon particles in animal biological samples, and relates to the field of atmospheric particulate pollution research. The method is applied to black carbon particle detection of human samples. After the animal biological sample is freeze-dried, concentrated nitric acid is used for digestion, the pH value is adjusted, diatomite is added, mixed and settled, high-speed filtration is carried out, nano black carbon particles are captured on a quartz fiber filter membrane pre-paved with diatomite, and the filter membrane is washed with an oxidant and an organic solvent to remove residual organic matter. The filter membrane is moved into a reaction kettle, concentrated nitric acid is used for oxidation under high temperature and high pressure, black carbon particles are oxidized into corresponding molecular markers benzene polycarboxylic acid, integrated separation, enrichment, purification and derivative treatment are carried out, and GC-MS / MS is used for quantitative analysis of the benzene polycarboxylic acid. The concentration of the benzene polycarboxylic acid is obtained after conversion, and the concentration of black carbon in the biological sample is obtained. The benzene polycarboxylic acid molecular marker method is successfully applied to the detection of ultra-trace nano black carbon particles in animal biological samples, and accurate quantitative analysis of black carbon particles in biological samples is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of atmospheric particulate pollutants, in particular to a method for detecting nano black carbon particles in animal biological samples. BACKGROUND

[0002] Since the industrial era, the use of fossil fuels by humans has increased exponentially, and the resulting environmental pollution problems have become increasingly serious. In the process of promoting modernization, China's economic level and overall national strength have rapidly improved, but it also faces the problem of environmental pollution brought about by rapid development, with air pollution being a major problem. As a major source of air pollution, PM2.5 (particles with an aerodynamic diameter of less than 2.5 microns) has always posed a great threat to the health of residents, and its main sources of emission include industry, transportation and biomass burning, etc. Black carbon is a key component of PM2.5, so the health hazards caused by black carbon as an important particulate air pollutant need to be clarified.

[0003] Epidemiological studies have shown that exposure to black carbon is significantly associated with adverse health effects on the respiratory, cardiovascular and nervous systems, especially on the health of women and children. For example, exposure to black carbon can increase oxidative stress and trigger respiratory inflammation (Chen X, et al. Respiratory inflammation and short-term ambient air pollution exposures in adult Beijing residents with and without prediabetes: A panel study. Environ Health Perspect, 2020, 128:067004), and long-term exposure to black carbon is associated with decreased lung function in urban women (Suglia S F, et al. Association between traffic-related black carbon exposure and lung function among urban women. Environ Health Perspect, 2008, 116: 1333–1337) and elderly men (Lepeule J, et al. Long-term effects of traffic particles on lung function decline in the elderly. Am J Respir Crit Care Med, 2014, 190: 542–548); exposure to black carbon may cause myocardial ischemia (Chuang K J, et al. Particulate air pollution as a risk factor for ST-segment depression in patients with coronary artery disease. Circulation, 2008, 118: 1314–1320) and arrhythmia (Zanobetti A, et al. Associations between arrhythmia episodes and temporally and spatially resolved black carbon and particulate matter in elderly patients.Occup Environ Med, 2014, 71: 201-207), coronary heart disease, ischemic heart disease ([6] Zhu X, et al. Short and long-term association of exposure to ambient black carbon with all-cause and cause-specific mortality: A systematic review and meta-analysis. Environ Pollut, 2023, 324: 121086) and other adverse cardiovascular outcomes; black carbon can also be transmitted to the brain along the olfactory neurons through the olfactory bulb, causing adverse effects on the nervous system. Studies have found that black carbon exposure is associated with decreased intelligence, memory, and learning ability in children ([7] Suglia S F, et al. Association of black carbon with cognition among children in a prospective birth cohort study. Am J Epidemiol, 2008, 167: 280-286), and case-control studies have revealed an association between brain tumors and the sum of organic carbon and black carbon components ([8] Harbo Poulsen A, et al. Components of particulate matter air-pollution and brain tumors. Environ Int, 2020, 144: 106046), with black carbon possibly being the main influencing factor. Recent studies have used femtosecond pulsed laser technology to find black carbon particles on the fetal side of the placenta ([9] Bové H, et al. Ambient black carbon particles reach the fetal side of human placenta. Nat Commun, 2019, 10: 3866), providing direct evidence that black carbon can cross the placental barrier. Black carbon exposure has been linked to reduced birth weight (

[10] Rokoff L B, et al. Cumulative exposure to environmental pollutants during early pregnancy and reduced fetal growth: the Project Viva cohort.Environ Health, 2018, 17: 1) and increased risk of preterm birth (

[11] Riddell C A, et al. Hyper-localized measures of air pollution and risk of preterm birth in Oakland and San Jose, California. Int J Epidemiol, 2021, 50: 1875-1885), and gestational diabetes mellitus (

[12] Yu G, et al. Fine particular matter and its constituents in air pollution and gestational diabetes mellitus. Environ Int, 2020, 142: 105880); studies have shown that black carbon exposed during pregnancy can enter the fetal circulation and penetrate into fetal liver, lung, brain and other tissues and organs, and the concentration of black carbon particles in the placenta and umbilical cord blood is positively correlated with the concentration of black carbon at the maternal residence during pregnancy (

[13] Bongaerts E, et al. Maternal exposure to ambient black carbon particles and their presence in maternal and fetal circulation and organs: An analysis of two independent population-based observational studies. Lancet Planet Health, 2022, 6: e804-e811), indicating that maternal prenatal exposure to black carbon particles may migrate to the fetus, leading to corresponding health problems in the respiratory, cardiovascular and nervous systems of the offspring.

[0004] In epidemiological studies, exposure levels to black carbon are mostly estimated based on long-term exposure levels using models or short-term exposure levels based on individual substitution. Due to the high spatiotemporal variability of black carbon, its concentration varies greatly across different microenvironments, making the assessment of exposure levels inaccurate. This significantly weakens the reliability of epidemiological evidence and the strength of correlations. Therefore, accurate measurement of black carbon exposure in organisms is more convincing and precise. In recent years, some teams abroad have developed femtosecond pulsed laser technology based on the light absorption characteristics of black carbon and conducted experiments on different human biological samples. However, the concentration data provided is not comprehensive and is considered a semi-quantitative analytical method, lacking molecular-level information on material structure. Furthermore, the methods and equipment are expensive, and computer-aided particle counting has a high professional threshold, limiting its widespread adoption. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned technical problems in existing technologies by providing a low-cost, universally applicable, simple, and widely applicable method for detecting black carbon particles in animal biological samples, in order to quantitatively detect the distribution of black carbon particles in different types of organs or tissues. This invention involves collecting animal biological samples, oxidizing the black carbon particles in the samples into small-molecule benzene polycarboxylic acids under high temperature and high pressure conditions, detecting the concentration of benzene polycarboxylic acids using a gas chromatography-mass spectrometry platform, and finally converting the benzene polycarboxylic acid concentration into the concentration of black carbon particles.

[0006] This invention provides a method for detecting nano-black carbon particles in animal biological samples, comprising the following steps:

[0007] 1) Sample homogenization: Take an appropriate amount of fresh animal sample, freeze it at -80℃, then put it in a clean glass bottle for freeze drying, then crush the sample with a clean glass rod and stir to homogenize it;

[0008] 2) Sample digestion: Take an appropriate amount of biological sample powder into a 15mL centrifuge tube, add concentrated nitric acid until the sample is completely digested, and then neutralize it. At this point, the sample has been completely digested.

[0009] 3) Quartz fiber filter membrane pretreatment: Place the high-purity quartz fiber filter membrane in a crucible and calcine it at high temperature to remove organic residues;

[0010] 4) Diatomaceous earth washing: Take a portion of diatomaceous earth into a round-bottom flask, add a certain concentration of sulfuric acid, mix well and incubate in a water bath; then filter the diatomaceous earth in the flask through a filter membrane to collect it, and wash it with water, dilute nitric acid and water in sequence; finally transfer the diatomaceous earth on the filter membrane into a crucible, dry it in an oven, and then calcine it in a muffle furnace. At this point, the diatomaceous earth has been washed.

[0011] 5) Sample filtration: weigh the diatomite, add the alkali and vortex to mix, drop the suspension into a 15 mL centrifuge tube, mix and settle the digested sample and incubate, set as liquid A; meanwhile, prepare liquid B by adding diatomite to the alkali and vortexing to mix; use a vacuum filtration device with a sand core filter head to filter, place two layers of high-purity quartz fiber filter membranes on the filter head, and drop liquid B evenly on the quartz fiber filter membranes, then drop liquid A evenly on the quartz fiber filter membranes in the same way, to realize enrichment of the particulate matter sample;

[0012] 6) Sample purification: wash the filter membrane and diatomite with an oxidizing agent and an organic solvent drop by drop, and continue to filter until the filter membrane is completely dry, to complete the sample purification;

[0013] 7) Sample oxidation: transfer the entire upper filter membrane and diatomite into the inner tank of the reaction kettle, add concentrated nitric acid and tighten, put into the oven for reaction, turn off the power after the oven cools down, and cool; take the supernatant for treatment after treatment;

[0014] 8) Molecular marker enrichment: use solid phase extraction to enrich the benzene polycarboxylic acid molecular markers generated by oxidation, and select HLB column for solid phase extraction operation; after four steps of activation, balance, sample loading and elution, the benzene polycarboxylic acid molecular markers in the concentrated acid matrix have been dissolved in the organic solvent, and the entire molecular marker enrichment work has been completed;

[0015] 9) Molecular marker derivation: nitrogen blow the eluent with a nitrogen blowing instrument until dry, then redissolve with methanol, add dichloromethane, internal standard, vortex to mix, then add derivatizing agent, vortex to mix, and derive; after derivation, quench, nitrogen blow to dry, redissolve with organic solvent, then transfer to the sample bottle, and wait for instrument detection and analysis;

[0016] 10) Instrument analysis: analyze the derivative product in step 9) with GC-MS / MS, and the instrument parameters should be detected and analyzed under the optimal parameter conditions.

[0017] In step 1), the specific conditions of freeze-drying are cold trap temperature -50°C, and freeze-drying time of 48h or more.

[0018] In step 2), the specific method of acid digestion is to add 1mL of 68% concentrated nitric acid, and place at room temperature for 24h or more until the solution is clear and transparent; the neutralization method is to add 2mL of water and 7mL of 30% potassium hydroxide, so that the final solution alkali concentration can be adjusted to about 10%.

[0019] In step 3), the specific conditions of high-temperature calcination are calcination at 600-800°C for 3-4h.

[0020] In step 4), the filter membrane used for the filtration has a pore size of 0.3 μm, a diameter of 50 mm, and diatomite particles with a particle size of 7 μm. The acid boiling of the diatomite is performed as follows: 10-20 g of diatomite with a particle size of 7 μm is placed in a round-bottom flask, and deionized water and concentrated sulfuric acid are added in a certain proportion to prepare a sulfuric acid solution with a final concentration of 4-5 mol / L. The solid-liquid ratio should be between 1:3 and 1:4. After mixing, the acid boiling is performed in a water bath at 99°C for 3-4 h. The washing steps are as follows: the filter membrane is sequentially washed with 200-300 mL of deionized water for 2-3 times, 700-800 mL of 2% nitric acid for 1 time, and 200-300 mL of deionized water for 3-5 times. Then, the diatomite on the filter membrane is transferred into a ceramic dry pan and dried in an oven at 100-120°C for 2-3 h. Finally, the diatomite is calcined in a muffle furnace at a temperature of 550-600°C for 3-4 h.

[0021] In step 5), the mixing and settling method is as follows: 50-100 mg of diatomite is weighed, 1.5-2 mL of 10% potassium hydroxide is added, and the mixture is vortexed. Then, the suspension is added dropwise into a 15 mL centrifuge tube. The mixed and settled sample is incubated at 37-38°C for 3-5 h. Liquid B is prepared by weighing 50-100 mg of diatomite, adding 2-3 mL of 10% potassium hydroxide, and vortexing. The mixture is then left to stand at room temperature for 3-5 h. The high-purity quartz fiber filter membrane has a pore size of 0.3 μm and a diameter of 25 mm or 50 mm.

[0022] In step 6), the oxidizing agent and organic solvent are 2 mL-3 mL of 30% hydrogen peroxide and 2-6 mL of acetone, respectively.

[0023] In step 7), the concentrated nitric acid used for oxidation is an electronic-grade concentrated nitric acid. The oxidation process is performed as follows: 1-2.5 mL of concentrated nitric acid is added to the inner liner of the reaction kettle and tightened. The reaction kettle is placed in an oven and reacted at 170-180°C for 8-9 h. When the oven temperature drops to 70°C or below, the power is turned off, and the oven door is opened to cool naturally to room temperature. At this time, the oxidation is completed. The treatment method for the supernatant of the oxidation product is as follows: the cooled reaction kettle is slowly opened in a fume hood, and 1200-1600 μL of the supernatant is taken. The reaction kettle is washed with deionized water. After centrifugation at 8000-12000 rpm for 15-25 min, 800-1200 μL of the supernatant is taken in the original reaction kettle liner, and the kettle is left to ventilate overnight in a fume hood.

[0024] In step 8), the HLB column used is NanoChrom Select Core HLB SPE Cartridge, HLB060-030250-1, 250mg 3mL (60μm) solid phase extraction column; the solid phase extraction process is: ① 4-5 bed methanol activation; ② 5-6 bed 2% nitric acid balance; ③ the overnight oxidation product is diluted to about 11 mL with deionized water and mixed, and then added to the solid phase extraction column for natural filtration; ④ under negative pressure, the liquid on the extraction column is dried, and a 15 mL centrifuge tube is prepared, 100 μL of 37% concentrated hydrochloric acid is added as an eluent collection tube; ⑤ elution is performed with 4 bed methanol solution;

[0025] In step 9), the specific experimental condition parameters of the derivative are that the eluent is dried by nitrogen blowing at 65-70°C, 400 μL of methanol is added for re-dissolution, 1000 μL of dichloromethane is added, 100 μL of an internal standard (concentration 200 ppm, 2'2-diphenyl dicarboxylic acid) is added, vortexed and mixed, then 400 μL of a derivatizing agent (2 mol / L trimethylsilyl diazomethane) is added, vortexed and mixed, sealed and covered, and derivatized at room temperature for 12 hours or more; the specific method of quenching is that after the derivation is completed, 40 μL of a quenching agent (50% formic acid / methanol) is added, dried by nitrogen blowing at about 40°C, re-dissolved with dichloromethane, vortexed and mixed, then transferred to a sample bottle, and then instrument detection and analysis is performed;

[0026] In step 10), the optimal parameter conditions for instrument analysis are that the chromatographic column can use an Agilent HP-5MS weak polar gas chromatographic column; the temperature program is 60°C for 1 min, 30°C / min to 200°C, 3°C / min to 240°C, 20°C / min to 290°C, and 290°C for 2 min; carrier gas type: He; carrier gas flow rate: 1 mL / min; split ratio: no split;

[0027] The application provides a detection method for evaluating black carbon particles in common people or animal biological samples, and the key points of the application are as follows: 1, organic matter is digested clean by concentrated nitric acid at normal temperature and pressure, and meanwhile, the black carbon particles are not damaged; 2, a “mixing and settling-high speed impact” system is established, and parameters of some key links are determined, such as the selection of filter membrane and diatomite size, the acid washing process of diatomite, the most suitable acid and alkali conditions for suction filtration, the incubation time of diatomite before suction filtration and the suction filtration mode; 3, the residual organic matter on the filter membrane is removed clean by using an oxidizing agent and an organic solvent in sequence, which greatly improves the matrix interference of the method; 4, the benzene polycarboxylic acid molecular marker is enriched by using a solid phase extraction method, and the selected commercial HLB column is simple to operate, has high recovery rate and is environment-friendly; 5, the benzene polycarboxylic acid derivative conditions are optimized, and the related factors influencing the derivative efficiency are systematically investigated; different from other previous methods, the method is first applied to the benzene polycarboxylic acid molecular marker method originally used in the environment with high black carbon content, and is successfully applied to the analysis of biological samples with extremely low black carbon content after a large number of improvements, so that the method has great application prospect; meanwhile, the benzene polycarboxylic acid molecular marker method has great limitations when applied to the environment samples rich in organic matter, and the successful application of the method to biological samples also solves this key problem in the environmental field. The method can accurately quantify the black carbon content in different organs of the human body, provide the internal exposure dose of air pollution, and thus provide more reliable evidence for relevant epidemiological research, so as to reveal the influence of environmental pollution on the health of the population; the core of the application is to provide a black carbon particle detection method which is accurate in quantification, high in sensitivity, simple and efficient, strong in universality and can be applied to different types of biological samples.

[0028] Therefore, the application has the following outstanding technical effects:

[0029] 1, the application solves the problem of organic matrix interference of the benzene polycarboxylic acid molecular marker method applied to black carbon analysis, so that it can be applied to the analysis of biological samples.

[0030] 2, the application develops a particle enrichment system based on “mixing and settling-high speed impact”, and realizes accurate quantification of extremely low content black carbon particles in biological samples by combining a gas chromatography mass spectrometry device, the method is high in sensitivity and strong in popularization.

[0031] 3, the application provides black carbon particle internal exposure data for the environmental health field, so that the epidemiological research is more reliable; meanwhile, the application provides a solution for solving the inaccurate black carbon particle content in the environment field rich in organic matter.

[0032] 4, the application provides a methodological support for studying the migration characteristics of black carbon particles between different organs of the same organism, and the local biochemical condition changes caused by black carbon and the health effects caused by black carbon. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 GC-MS / MS quantitative ion extraction chromatogram of 6 benzenepolycarboxylic acid standard mixture.

[0034] Figure 2 GC-MS / MS quantitative ion extraction chromatogram of black carbon particles in diesel engine exhaust.

[0035] Figure 3 Bio-matrix black carbon particle recovery rate of the present method at different spiked levels.

[0036] Figure 4 Removal effect of the present method on bio-matrix interference through a specific washing process.

[0037] Figure 5 GC-MS / MS quantitative ion extraction chromatogram of black carbon particles in the lungs of normally fed non-human high-dose exposed cattle. DETAILED DESCRIPTION

[0038] The present application will be described in detail below with specific examples. The following examples will help those skilled in the art to further understand the present application, but do not limit the present application in any form. It should be noted that for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made. These are within the scope of the present application.

[0039] The present application embodiment includes the following steps:

[0040] (1) Sample homogenization: Take an appropriate amount of fresh animal sample, freeze at -80℃, then put into a clean glass bottle for freeze-drying, with a cold trap temperature of -40 to -50℃, freeze-drying time of 48h or more, then crush the sample with a clean glass rod and stir homogenously;

[0041] (2) Sample digestion: Take an appropriate amount of dry biological sample powder in a 15mL centrifuge tube, add 1mL of 68% concentrated nitric acid, and place at room temperature for 24h or more until the sample is digested completely, then add 2mL of water and 7mL of 30% potassium hydroxide for neutralization, at which point the sample has been digested completely;

[0042] (3) Quartz fiber filter membrane pretreatment: Place the high-purity quartz fiber filter membrane in a crucible and calcine at 600-800℃ for 3-4h to remove organic residues;

[0043] (4) Diatomite washing: take 10-20 g of diatomite with a particle size of 7 μm in a round-bottom flask, then add deionized water and concentrated sulfuric acid in a certain proportion to prepare a sulfuric acid solution with a final concentration of 4-5 mol / L, the solid-liquid ratio should be between 1:3-1:4, mix well, then water bath at 99°C for 3-4 h, then filter the diatomite in the flask with a 0.3 μm high-purity quartz fiber filter membrane, and rinse with 200-300 mL of deionized water for 2-3 times, 700-800 mL of 2% nitric acid for 1 time, and 200-300 mL of deionized water for 3-5 times, then transfer the diatomite on the filter membrane into a ceramic baking dish, and dry in an oven at 100-120°C for 2-3 h, then calcine in a muffle furnace at 550-600°C for 3-4 h, then naturally cool to room temperature, and prepare for use, at this time the diatomite has been washed;

[0044] (5) Sample filtration: weigh 50-100 mg of diatomite, add 1.5-2 mL of 10% potassium hydroxide and vortex mix, then drop the suspension into a 15 mL centrifuge tube, mix and settle the digested sample, and incubate at 37-38°C for 3-5 h to obtain liquid A; at the same time, weigh 50-100 mg of diatomite, add 2-3 mL of 10% potassium hydroxide and vortex mix, and stand at room temperature for 3-5 h to obtain liquid B; then use a vacuum filtration device with a sand core filter head to filter, place two layers of high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 25 mm or 50 mm on the filter head, and drop liquid B evenly on the quartz fiber filter membranes, then drop liquid A evenly on the quartz fiber filter membranes in the same way, to realize enrichment of the particulate matter sample.

[0045] (6) Sample purification: wash the filter membrane and diatomite with 2-3 mL of 30% hydrogen peroxide dropwise, then wash with 2-6 mL of acetone dropwise, and continue to filter until the filter membrane is completely dry, to complete the sample purification;

[0046] (7) Sample oxidation: transfer the entire upper filter membrane and diatomite into the inner container of the reaction kettle, add 1-2.5 mL of concentrated nitric acid and tighten, and place in an oven at 170-180°C for 8-9 h, turn off the power when the oven cools to 70°C or below, and naturally cool to room temperature; slowly open the cooled reaction kettle in a fume hood, take 1200-1600 μL of supernatant, centrifuge at 8000-12000 rpm for 15-25 min, rinse the inner container of the reaction kettle with deionized water while centrifuging, and take 800-1200 μL of supernatant in the original inner container of the reaction kettle, and ventilate in a fume hood overnight;

[0047] (8) Molecular marker extraction: The HLB column is used to separate, enrich and purify the molecular markers of benzene polycarboxylic acid produced by oxidation. The NanoChrom Select Core HLB SPE Cartridge solid-phase extraction column is selected for solid-phase extraction operation. The activation process is as follows: ① 4-5 bed methanol activation; ② 5-6 bed 2% nitric acid balance; ③ After overnight, the oxidation product is diluted to about 11 mL with deionized water and mixed well, and then added to the solid-phase extraction column for natural filtration; ④ Under negative pressure, the liquid on the extraction column is drained, and a 15 mL centrifuge tube is prepared, 100 μL of 37% concentrated hydrochloric acid is added as an eluent collection tube; ⑤ Elution with 4 bed methanol solution; the enrichment of the molecular marker has been completed;

[0048] (9) Molecular marker derivation: The eluent is dried by nitrogen blowing at 65-70°C, then redissolved with 400 μL of methanol, added with 1000 μL of dichloromethane, 100 μL of internal standard (concentration 200 ppm, 2'2-biphenyldicarboxylic acid), vortexed and mixed, then added with 400 μL of derivatization agent (2 mol / L trimethylsilyl diazomethane), vortexed and mixed, sealed and covered, and derivatized at room temperature for 12 hours or more;

[0049] (10) Instrument analysis: After derivation, 40 μL of quenching agent (50% formic acid / methanol) is added, then dried by nitrogen blowing at about 40°C, then redissolved with dichloromethane, vortexed and mixed, then transferred to a sample bottle, and then instrument detection and analysis.

[0050] Example 1

[0051] This example includes the following steps:

[0052] (1) Sample homogenization: Take 3g of fresh animal sample, freeze at -80°C for 48h, then put it in a clean glass bottle for freeze-drying, set the cold trap temperature to -50°C, and freeze-dry for 48h or more. After completion, crush the sample with a clean glass rod and stir until homogeneous.

[0053] (2) Sample digestion: Take 0.3g of dry biological sample powder and place it in a 15mL centrifuge tube, add 1mL of 68% concentrated nitric acid, and let it stand at room temperature for 24h until the solution is clear and transparent. Then add 2mL of water and 7mL of 30% potassium hydroxide for neutralization, and adjust the alkali concentration of the solution to about 10%.

[0054] (3) Quartz fiber filter membrane pretreatment: Place the high-purity quartz fiber filter membrane with a pore size of 0.3 μm and a diameter of 50mm in a crucible and calcine at 600°C for 3h to remove organic matter residues.

[0055] (4) Diatomite washing: Take 10 g diatomite with particle size of 7 μm in a round bottom flask, add deionized water and concentrated sulfuric acid to prepare a sulfuric acid solution with a final concentration of 4 mol / L, and the solid-liquid ratio is 1:3. After mixing, acid cook at 99°C for 3h in water bath. The washing steps include rinsing with 200 mL deionized water twice, 700 mL 2% nitric acid once, and 200 mL deionized water three times. Finally, transfer the diatomite on the filter membrane to a ceramic casserole and dry in an oven at 100°C for 2h, then calcine in a muffle furnace at 550°C for 3h.

[0056] (5) Sample filtration: weigh 50 mg diatomite, add 1.5 mL 10% potassium hydroxide and vortex mix. Drop the suspension into a 15 mL centrifuge tube, mix with the digested sample and settle, and incubate at 37°C for 3h. Meanwhile, prepare liquid B (weigh 50 mg diatomite, add 2 mL 10% potassium hydroxide and vortex mix, and stand at room temperature for 3h). Use a vacuum filtration device with a sand core filter head, and place two layers of high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 50 mm on the filter head. First, add liquid B to pre-wet the filter membrane, then add liquid A drop by drop to achieve enrichment of the particulate matter sample.

[0057] (6) Sample purification: use 2 mL 30% hydrogen peroxide and 2 mL acetone as washing liquid, dropwise wash the residues on the filter membrane and diatomite, and continue to perform suction filtration operation until the filter membrane is completely dry.

[0058] (7) Sample oxidation: carefully transfer the entire upper filter membrane and diatomite to the inner container of the reaction kettle, add 1 mL electronic grade concentrated nitric acid, and tighten the kettle cover. Put the reaction kettle into the oven and react at 170°C for 8h. After the reaction is completed, turn off the power when the oven temperature drops below 70°C and open the oven door to cool to room temperature naturally. Slowly open the kettle cover in the fume hood, take about 1200 μL of supernatant, and centrifuge at 8000 r / min for 15 min. After centrifugation, transfer 800 μL of supernatant to the original reaction kettle inner container and ventilate overnight to further remove residues.

[0059] (8) Molecular marker enrichment: use solid phase extraction technology to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. Select NanoChrom Select Core HLB SPE Cartridge (model HLB060-030250-1, 250 mg / 3 mL, 60 microns) as the solid phase extraction column. Follow the four steps of activation, equilibration, sampling and elution.

[0060] (9) Molecular marker derivation: The eluent was blown dry with a nitrogen blower at 65°C, 400 μL of methanol was added for re-dissolution. Then 1000 μL of dichloromethane and 100 μL of internal standard (2',2-biphenyldicarboxylic acid with a concentration of 200 ppm) were added, vortexed and mixed, 400 μL of derivatization agent (2 mol / L of trimethylsilyl diazomethane) was added, vortexed and mixed again, sealed and covered, and derivatized at room temperature for 12 hours or more. After the derivation was completed, 40 μL of quenching agent (50% formic acid / methanol mixture) was added, blown dry at about 40°C, re-dissolved with dichloromethane and vortexed and mixed, and then transferred to a sample injection bottle for instrument detection and analysis.

[0061] (10) Instrument analysis: GC-MS / MS was used to analyze and detect the derivatized product. The chromatographic column used was an Agilent HP-5MS weak polarity gas chromatographic column; the temperature program was set to 60°C for 1 min, then increased to 200°C at a rate of 30°C / min, then increased to 240°C at a rate of 3°C / min, then increased to 290°C at a rate of 20°C / min, and finally held at 290°C for 2 min; the carrier gas type was helium (He), and the carrier gas flow rate was set to 1 mL / min; the split ratio was set to no split mode for detection and analysis.

[0062] Example 2

[0063] (1) Sample homogenization: 3 g of fresh animal sample was frozen at -80°C for 48 hours, then placed in a clean glass bottle for freeze-drying, with the cold trap temperature set to -50°C, and the freeze-drying time lasting for 48 hours or more. After completion, the sample was crushed and stirred to a homogeneous state with a clean glass rod.

[0064] (2) Sample digestion: 0.3 g of dry biological sample powder was placed in a 15 mL centrifuge tube, 1 mL of 68% concentrated nitric acid was added, and the solution was left to stand at room temperature for 24 hours until it was clear and transparent. Then 2 mL of water and 7 mL of 30% potassium hydroxide were added for neutralization, and the alkali concentration of the solution was adjusted to about 10%.

[0065] (3) Quartz fiber filter membrane pretreatment: high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 50 mm were placed in a crucible and calcined at 600°C for 3 hours to remove organic matter residues.

[0066] (4) Diatomite washing: Take 10 g diatomite with particle size of 7 μm in a round bottom flask, add deionized water and concentrated sulfuric acid to prepare a sulfuric acid solution with a final concentration of 4 mol / L, and the solid-liquid ratio is 1:3. After mixing, acid cook at 99°C for 3h in water bath. The washing steps include rinsing with 200 mL deionized water twice, 700 mL 2% nitric acid once, and 200 mL deionized water three times. Finally, transfer the diatomite on the filter membrane to a ceramic casserole and dry in an oven at 100°C for 2h, then calcine in a muffle furnace at 550°C for 3h.

[0067] (5) Sample filtration: weigh 50 mg diatomite, add 1.5 mL 10% potassium hydroxide and vortex mix. Drop the suspension into a 15 mL centrifuge tube, mix with the digested sample and settle, and incubate at 37°C for 3h. Meanwhile, prepare liquid B (weigh 50 mg diatomite, add 2 mL 10% potassium hydroxide and vortex mix, and stand at room temperature for 3h). Use a vacuum filtration device with a sand core filter head, and place two layers of high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 50 mm on the filter head. First, add liquid B to pre-wet the filter membrane, then add liquid A drop by drop to achieve enrichment of the particulate matter sample.

[0068] (6) Sample purification: use 2 mL 30% hydrogen peroxide and 2 mL acetone as washing liquid, dropwise wash the residues on the filter membrane and diatomite, and continue to perform suction filtration operation until the filter membrane is completely dry.

[0069] (7) Sample oxidation: carefully transfer the entire upper filter membrane and diatomite to the inner container of the reaction kettle, add 1 mL electronic grade concentrated nitric acid, and tighten the kettle cover. Put the reaction kettle into the oven and react at 170°C for 8h. After the reaction is completed, turn off the power when the oven temperature drops below 70°C and open the oven door to cool to room temperature naturally. Slowly open the kettle cover in the fume hood, take about 1200 μL of supernatant, and centrifuge at 8000 r / min for 15 min. After centrifugation, transfer 800 μL of supernatant to the original reaction kettle inner container and ventilate overnight to further remove residues.

[0070] (8) Molecular marker enrichment: use solid phase extraction technology to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. Select NanoChrom Select Core HLB SPE Cartridge (model HLB060-030250-1, 250 mg / 3 mL, 60 microns) as the solid phase extraction column. Follow the four steps of activation, equilibration, sampling and elution.

[0071] (9) Molecular marker derivation: The eluent was blown dry with a nitrogen blower at 65°C, 400 μL of methanol was added for re-dissolution. 1000 μL of dichloromethane and 100 μL of internal standard (2',2-biphenyldicarboxylic acid with a concentration of 200 ppm) were added, vortexed and mixed, 400 μL of derivatization agent (2 mol / L of trimethylsilyl diazomethane) was added, vortexed and mixed again, sealed and covered, and derivatized at room temperature for 12 hours or more. After the derivation was completed, 40 μL of quenching agent (50% formic acid / methanol mixture) was added, blown dry at about 40°C, re-dissolved with dichloromethane and vortexed, and then transferred to a sample injection bottle for instrument detection and analysis.

[0072] (10) Instrument analysis: GC-MS / MS was used to analyze and detect the derivatized product. The chromatographic column used was an Agilent HP-5MS weak polarity gas chromatographic column; the temperature program was set to 60°C for 1 min, then increased to 200°C at a rate of 30°C / min, then increased to 240°C at a rate of 3°C / min, then increased to 290°C at a rate of 20°C / min, and finally held at 290°C for 2 min; the carrier gas type was helium (He), and the carrier gas flow rate was set to 1 mL / min; the split ratio was set to no split mode for detection and analysis.

[0073] Example 3

[0074] (1) Sample homogenization: 3 g of fresh animal sample was frozen at -80°C for 48 hours, then placed in a clean glass bottle for freeze-drying, with the cold trap temperature set to -50°C, and the freeze-drying time lasting for 48 hours or more. After completion, the sample was crushed and stirred to a homogeneous state with a clean glass rod.

[0075] (2) Sample digestion: 0.3 g of dry biological sample powder was placed in a 15 mL centrifuge tube, 1 mL of 68% concentrated nitric acid was added, and the solution was left to stand at room temperature for 24 hours until it was clear and transparent. Then 2 mL of water and 7 mL of 30% potassium hydroxide were added for neutralization, and the alkali concentration of the solution was adjusted to about 10%.

[0076] (3) Quartz fiber filter membrane pretreatment: high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 50 mm were placed in a crucible and calcined at 600°C for 3 hours to remove organic matter residues.

[0077] (4) Diatomite washing: Take 10 g diatomite with a particle size of 7 μm in a round-bottom flask, add deionized water and concentrated sulfuric acid to prepare a sulfuric acid solution with a final concentration of 4 mol / L, and the solid-liquid ratio is 1:3. After mixing, acid cook at 99°C for 3h in water bath. The washing steps include rinsing with 200 mL deionized water twice, 700 mL 2% nitric acid once, and 200 mL deionized water three times. Finally, transfer the diatomite on the filter membrane to a ceramic casserole and dry in a 100°C oven for 2h, then calcine in a 550°C muffle furnace for 3h.

[0078] (5) Sample filtration: weigh 50 mg diatomite, add 1.5 mL 10% potassium hydroxide and vortex mix. Drop the suspension into a 15 mL centrifuge tube, mix with the digested sample and settle, and incubate at 37°C for 3h. At the same time, prepare liquid B (weigh 50 mg diatomite, add 2 mL 10% potassium hydroxide and vortex mix, and stand at room temperature for 3h). Use a vacuum filtration device with a sand core filter head, place two layers of high-purity quartz fiber filter membrane with a pore size of 0.3 μm and a diameter of 50 mm on the filter head. First, add liquid B to pre-wet the filter membrane, then add liquid A drop by drop to achieve enrichment of the particulate matter sample.

[0079] (6) Sample purification: use 2 mL 30% hydrogen peroxide and 2 mL acetone as washing liquid, dropwise wash the residues on the filter membrane and diatomite, and continue to perform suction filtration operation until the filter membrane is completely dry.

[0080] (7) Sample oxidation: carefully transfer the entire upper filter membrane and diatomite to the inner container of the reaction kettle, add 1 mL electronic grade concentrated nitric acid, and tighten the kettle cover. Put the reaction kettle into the oven and react at 170°C for 8h. After the reaction is completed, turn off the power when the oven temperature drops below 70°C and open the oven door to cool to room temperature naturally. Slowly open the kettle cover in the fume hood, take about 1200 μL of supernatant, and centrifuge at 8000 r / min for 15 min. After centrifugation, transfer 800 μL of supernatant to the original reaction kettle inner container and ventilate overnight to further remove residues.

[0081] (8) Molecular marker enrichment: use solid phase extraction technology to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. Select NanoChrom Select Core HLB SPE Cartridge (model HLB060-030250-1, 250 mg / 3 mL, 60 microns) as the solid phase extraction column. Follow the four steps of activation, equilibration, sampling and elution.

[0082] (9) Molecular marker derivation: The eluent was dried by nitrogen blowing at 65°C, 400 μL of methanol was added for re-dissolution. Then 1000 μL of dichloromethane and 100 μL of internal standard (2',2-biphenyldicarboxylic acid with a concentration of 200 ppm) were added. After vortex mixing, 400 μL of derivatization agent (2 mol / L of trimethylsilyl diazomethane) was added. The mixture was vortex mixed again and sealed, and then derivatized at room temperature for 12 hours or more. After the derivation was completed, 40 μL of quenching agent (50% formic acid / methanol mixture) was added, and the mixture was dried by nitrogen blowing at about 40°C. Then the mixture was re-dissolved in dichloromethane and vortex mixed, and then transferred to a sample injection bottle for instrument detection and analysis.

[0083] (10) Instrument analysis: The derivatized product was analyzed and detected by GC-MS / MS. The chromatographic column used was an Agilent HP-5MS weak polarity gas chromatographic column. The temperature program was set as follows: 60°C for 1 min, then 30°C / min to 200°C, then 3°C / min to 240°C, then 20°C / min to 290°C, and finally 290°C for 2 min. The carrier gas type was helium (He), and the carrier gas flow rate was set to 1 mL / min. The split ratio was set to the non-split mode for detection and analysis.

[0084] Example 4

[0085] (1) Sample homogenization: 3 g of fresh animal sample was frozen at -80°C for 48 hours, and then placed in a clean glass bottle for freeze-drying. The cold trap temperature was set to -50°C, and the freeze-drying time lasted for 48 hours or more. After completion, the sample was crushed and stirred to a homogeneous state with a clean glass rod.

[0086] (2) Sample digestion: 0.3 g of dry powder of the biological sample was placed in a 15 mL centrifuge tube, 1 mL of concentrated nitric acid with a mass fraction of 68% was added, and the solution was placed at room temperature for 24 hours until it was clear and transparent. Then 2 mL of water and 7 mL of 30% potassium hydroxide were added for neutralization, and the alkali concentration of the solution was adjusted to about 10%.

[0087] (3) Quartz fiber filter membrane pretreatment: high-purity quartz fiber filter membranes with a pore size of 0.3 μm and a diameter of 50 mm were placed in a crucible and calcined at 600°C for 3 hours to remove organic matter residues.

[0088] (4) Diatomite washing: Take 10 g diatomite with a particle size of 7 μm in a round bottom flask, add deionized water and concentrated sulfuric acid to prepare a sulfuric acid solution with a final concentration of 4 mol / L, and the solid-liquid ratio is 1:3. After mixing, acid cook at 99℃ for 3h in water bath. The washing steps include rinsing with 200 mL deionized water twice, 700 mL 2% nitric acid once, and 200 mL deionized water three times. Finally, the diatomite on the filter membrane is transferred to a ceramic casserole and dried in an oven at 100℃ for 2h, and then calcined in a muffle furnace at 550℃ for 3h.

[0089] (5) Sample filtration: weigh 50 mg diatomite, add 1.5 mL 10% potassium hydroxide and vortex mix. Drop the suspension into a 15 mL centrifuge tube, mix with the digested sample and settle, and incubate at 37℃ for 3h. At the same time, prepare liquid B (weigh 50 mg diatomite, add 2 mL 10% potassium hydroxide and vortex mix, and stand at room temperature for 3h). Use a vacuum filtration device with a sand core filter head, and place two layers of high-purity quartz fiber filter membrane with a pore size of 0.3 μm and a diameter of 50 mm on the filter head. First, add liquid B to pre-wet the filter membrane, then add liquid A drop by drop to achieve enrichment of the particulate matter sample.

[0090] (6) Sample purification: use 2 mL 30% hydrogen peroxide and 2 mL acetone as washing liquid, dropwise wash the residues on the filter membrane and diatomite, and continue to perform suction filtration operation until the filter membrane is completely dry.

[0091] (7) Sample oxidation: carefully transfer the entire upper filter membrane and diatomite to the inner container of the reaction kettle, add 1 mL electronic grade concentrated nitric acid, and tighten the kettle cover. Put the reaction kettle into the oven and react at 170℃ for 8h. After the reaction is completed, turn off the power when the oven temperature drops below 70℃, and open the oven door to cool to room temperature naturally. Slowly open the kettle cover in the fume hood, take about 1200 μL of supernatant, and centrifuge at 8000 r / min for 15 min. After centrifugation, transfer 800 μL of supernatant to the original reaction kettle inner container, and ventilate overnight to further remove residues.

[0092] (8) Molecular marker enrichment: use solid phase extraction technology to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. Select NanoChrom Select Core HLB SPE Cartridge (model HLB060-030250-1, 250 mg / 3 mL, 60 microns) as the solid phase extraction column. Follow the four steps of activation, equilibration, sampling, and elution.

[0093] (9) Molecular marker derivation: The eluent was blown dry with a nitrogen blowing instrument at 65°C, 400 μL of methanol was added for re-dissolution. 1000 μL of dichloromethane and 100 μL of internal standard (2',2-biphenyldicarboxylic acid with a concentration of 200 ppm) were added, vortexed and mixed, then 400 μL of derivatization agent (2 mol / L of trimethylsilyl diazomethane) was added, vortexed and mixed again, sealed, and derivatized at room temperature for 12 h or more. After the derivation was completed, 40 μL of quenching agent (50% formic acid / methanol mixture) was added, blown dry at about 40°C, re-dissolved with dichloromethane and vortexed, then transferred to a sample bottle for instrument detection and analysis.

[0094] (10) Instrument analysis: GC-MS / MS was used to analyze and detect the derivatized product. The chromatographic column used was an Agilent HP-5MS weak polar gas chromatographic column; the temperature program was set to 60°C for 1 min, then increased to 200°C at a rate of 30°C / min, then increased to 240°C at a rate of 3°C / min, then increased to 290°C at a rate of 20°C / min, and finally held at 290°C for 2 min; the carrier gas type was helium (He), and the carrier gas flow rate was set to 1 mL / min; the split ratio was set to no split mode for detection and analysis.

[0095] Example 5

[0096] Similar to Example 1, the difference is that:

[0097] In step (1), the freeze-drying time was 72 h.

[0098] In step (2), 1.5 mL of 68% concentrated nitric acid was used.

[0099] In step (3), the calcination temperature was 700°C.

[0100] In step (4), the diatomaceous earth mass was 15 g, the sulfuric acid solution concentration was 4.5 mol / L, the solid-liquid ratio was 1:3.5, and the oven temperature was 110°C.

[0101] Example 6

[0102] Similar to Example 1, the difference is that:

[0103] In step (1), the cold trap temperature was -50°C.

[0104] In step (2), it was placed at room temperature for 48 h.

[0105] In step (3), the calcination time was 4 h.

[0106] In step (4), the oven temperature was 120°C.

[0107] In step (5), 75 mg of celite, 2 mL of 10% potassium hydroxide were used and the incubation temperature was 38 °C.

[0108] In step (6), 3 mL of 30% hydrogen peroxide and 4 mL of acetone were used.

[0109] Example 7

[0110] Similar to Example 1, except that:

[0111] In step (1), the freeze-drying time was 60 h.

[0112] In step (2), the room temperature was maintained for 36 h.

[0113] In step (3), the calcination temperature was 800 °C.

[0114] In step (4), the sulfuric acid solution concentration was 5 mol / L and the solid-to-liquid ratio was 1:4.

[0115] In step (5), the incubation time was 4 h.

[0116] In step (7), the concentrated nitric acid amount was 2.5 mL and the reaction time was 9 h.

[0117] In step (8), 5 bed methanol activation and 6 bed 2% nitric acid equilibration were used.

[0118] Example 8

[0119] Similar to Example 1, except that:

[0120] In step (1), the cold trap temperature was -45 °C.

[0121] In step (2), 0.5 mL of 68% concentrated nitric acid was used.

[0122] In step (3), the calcination time was 3.5 h.

[0123] In step (4), the celite mass was 20 g.

[0124] In step (5), 100 mg of celite, 2.5 mL of 10% potassium hydroxide were used.

[0125] In step (9), the nitrogen blow-down temperature was 70 °C and the derivatization time was 16 h.

[0126] The remaining step parameters were the same as in Example 1.

[0127] Example 9

[0128] Similar to Example 1, except that:

[0129] In step (1), the freeze-drying time was 54 h.

[0130] In step (2), the sample was left at room temperature for 30 h.

[0131] In step (3), the calcination temperature was 650 °C.

[0132] In step (4), the oven temperature was 115 °C.

[0133] In step (5), 80 mg of celite and 1.8 mL of 10% potassium hydroxide were used.

[0134] In step (6), 5 mL of acetone was used.

[0135] In step (7), the oven temperature was 175 °C.

[0136] In step (8), 4.5 bed volumes of methanol were used for activation and 5.5 bed volumes of 2% nitric acid were used for equilibration.

[0137] In step (9), the derivatization time was 14 h.

[0138] A specific application example is given below.

[0139] This example is based on normally fed cattle, exposed without human intervention, so it is more representative of the living environment of the general population. This example detects the concentration of black carbon particles in the lungs, and the specific implementation steps are as follows:

[0140] I. Sample collection

[0141] Freshly slaughtered cattle organ samples were collected at the slaughterhouse. After being taken to the laboratory, the lungs were dissected, the surface of the dirty pleura was removed with a clean scalpel and forceps, and the internal lung tissue that had not been in contact with the outside was taken for testing.

[0142] II. Sample homogenization

[0143] An appropriate amount of cattle lung sample was frozen at -80 °C, then placed in a clean glass bottle for freeze-drying, with a cold trap temperature of -50 °C and a freeze-drying time of more than 48 h. The sample was then crushed and homogenized with a clean glass rod.

[0144] III. Sample digestion

[0145] An appropriate amount of freeze-dried powder of lung tissue was placed in a 15 mL centrifuge tube, 1 mL of 68% concentrated nitric acid was added, and it was left at room temperature for 48 h. Then 2 mL of water and 7 mL of 30% potassium hydroxide were added for neutralization.

[0146] IV. Celite washing

[0147] Take 20 g of diatomite with a particle size of 7 μm in a round-bottom flask, then add 47 mL of water, and then add 17 mL of concentrated sulfuric acid with a mass fraction of 98%. After mixing, place the flask in a water bath at 99°C for 4 h. Then filter the diatomite in the flask with a 0.3-μm high-purity quartz fiber filter membrane, and sequentially rinse the diatomite with 250 mL of deionized water twice, 750 mL of 2% nitric acid once, and 250 mL of deionized water four times. Then transfer the diatomite on the filter membrane into a ceramic baking dish, and bake in an oven at 110°C for 2 h. Then calcine in a muffle furnace at 600°C for 3 h, and then naturally cool to room temperature. The diatomite is ready for use.

[0148] V. Sample filtration

[0149] Weigh 100 mg of diatomite, add 1.5 mL of 10% potassium hydroxide, and vortex to mix. Then drop the suspension into a 15-mL centrifuge tube to mix and settle the digested lung sample, and incubate at 37°C for 5 h to obtain liquid A. At the same time, weigh 100 mg of diatomite, add 3 mL of 10% potassium hydroxide, and vortex to mix. Then let stand at room temperature for 5 h to obtain liquid B. Then use a vacuum filtration device with a sand core filter head to filter. Place two layers of high-purity quartz fiber filter membranes with a pore size of 0.3 μm on the filter head, and drop liquid B evenly onto the quartz fiber filter membranes. Then drop liquid A evenly onto the quartz fiber filter membranes in the same way to enrich the particulate matter sample in the lung sample.

[0150] VI. Sample purification

[0151] Wash the filter membrane and diatomite with 2 mL of 30% hydrogen peroxide drop by drop, and then wash with 6 mL of acetone drop by drop. Continue to filter until the filter membrane is completely dry to complete the sample purification.

[0152] VII. Sample oxidation

[0153] Transfer the entire upper filter membrane and diatomite into the inner container of the reaction kettle, add 2.5 mL of concentrated nitric acid, and tighten the lid. Place the reaction kettle in an oven at 170°C for 8 h. When the oven temperature drops to 70°C or below, turn off the power and open the oven door to naturally cool to room temperature. Slowly open the cooled reaction kettle in a fume hood, and take 1600 μL of supernatant. Centrifuge at 12000 rpm for 25 min. At the same time, rinse the inner container of the reaction kettle with deionized water. After centrifugation, take 1200 μL of supernatant into the original inner container of the reaction kettle, and ventilate in a fume hood overnight.

[0154] VIII. Molecular marker extraction

[0155] The benzene polycarboxylic acid molecular markers generated by oxidation are separated, enriched and purified by solid phase extraction, and a NanoChrom SelectCore HLB SPE Cartridge solid phase extraction column is selected for solid phase extraction operation. The activation process is: ① 4-bed methanol activation; ② 5-bed 2% nitric acid balance; ③ The overnight oxidation product is diluted to 11 mL with deionized water and mixed, and then added to the solid phase extraction column and naturally filtered; ④ Under negative pressure, the liquid on the extraction column is dried, and a 15 mL centrifuge tube is prepared, 100 μL of 37% concentrated hydrochloric acid is added as an eluent collection tube; ⑤ Elution is performed with 4-bed methanol solution; the enrichment of the entire molecular marker has been completed;

[0156] Nine, molecular marker derivation

[0157] The eluent is dried by nitrogen blowing at 65°C, then redissolved with 400 μL of methanol, 1000 μL of dichloromethane, 100 μL of internal standard (concentration 200 ppm, 2'2-biphenyl dicarboxylic acid), vortex mixed, then 400 μL of derivatization agent (2 mol / L trimethylsilyl diazomethane) is added, vortex mixed, sealed and covered, and derivatized at room temperature for 12 hours;

[0158] Ten, instrument analysis

[0159] After the derivation is completed, 40 μL of quenching agent (50% formic acid / methanol) is added, then dried by nitrogen blowing at 40°C, then redissolved with dichloromethane, vortex mixed, then transferred to a sample bottle, and detected by instrument, and compound qualitative and quantitative analysis is performed by using instrument software, the standard curve used for quantification is a working standard curve, which is realized by adding a standard sample of fetal bovine serum matrix without black carbon particles. At the same time, process blank samples and biological matrix standard samples are added during the experiment as blank controls and quality controls during the entire experimental process.

[0160] The GC-MS / MS quantitative ion extraction chromatogram of the benzene polycarboxylic acid standard mixture of the embodiment of the present application is shown in Figure 1 , from Figure 1 It can be seen from the chromatogram of the benzene polycarboxylic acid standard mixture that the chromatographic peaks of each standard can be obviously separated from the baseline, and the peak shape is symmetrical, without tailing, and the chromatographic peaks between the compounds are not overlapped, meeting the needs of quantitative analysis.

[0161] The GC-MS / MS quantitative ion extraction chromatogram of the black carbon particles in the exhaust gas of the diesel engine is shown in Figure 2 , from Figure 2(Chromatogram of black carbon particles in diesel engine exhaust) It can be seen that after black carbon particles in diesel engine exhaust are oxidized by concentrated nitric acid under high temperature and high pressure conditions, they generate corresponding benzene polycarboxylic acid molecular markers. Moreover, 1 μg of black carbon particles also obtained a high response signal, which fully demonstrates the representativeness of benzene polycarboxylic acid to black carbon particles and its potential as a highly sensitive molecular marker.

[0162] The recovery rates of biomass black carbon particles at different spiking levels are shown in the figure. Figure 3 ,from Figure 3 (Recovery of black carbon particles in bio-matrix at different spiking levels) It can be seen that this method achieves good recovery of nano black carbon particles regardless of whether the matrix is ​​spiked at low, medium or high levels, proving the robustness of this method.

[0163] The removal effect of this method on biological matrix interference through a specific washing process is shown in the figure. Figure 4 ,from Figure 4 (Removal effect of specific washing process on biological matrix interference) It can be seen that after washing with oxidant and organic solvent, phenylpentacarboxylic acid, an indicator of organic residue, showed a significant decrease, indicating that the washing method has a strong ability to remove organic interference. In the presence of organic matter that causes matrix interference, this can greatly improve the sensitivity of the method. Meanwhile, due to… Figure 3 It is evident that this washing method does not affect the recovery rate of black carbon particles.

[0164] GC-MS / MS quantitative ion extraction chromatogram of black carbon particles in the lungs of normally fed cattle with non-human high-dose exposure is shown below. Figure 5 .from Figure 5 (Chromatogram of black carbon particles in the lungs of cattle under normal feeding conditions without human-induced high-dose exposure) As can be seen, a significant benzene polycarboxylic acid response signal was found in the cattle lungs, which means that black carbon particles that can generate benzene polycarboxylic acids exist in the cattle lungs and the concentration has reached a level that can be accurately quantified. This verifies the feasibility of this method and reveals its great potential for application in routine biological monitoring. It also means that black carbon particles can enter the lungs of organisms through the respiratory tract and may bring corresponding health effects.

[0165] The application uses concentrated nitric acid to digest samples, so that the sample digestion is cleaner; uses a "mixing and settling-high speed impact" particle enrichment system to enrich nano black carbon particles, and good recovery is obtained; combines the washing method of oxidizing agent and organic solvent, which well reduces the interference of biological matrix; uses HLB column to enrich benzene polycarboxylic acid, compared with the traditional method, the process is simplified, the environment is protected, and good recovery is obtained; finally, a GC-MS / MS system is used for detection and analysis, so that the method has high sensitivity and accuracy. Thus, a detection and analysis method which is simple to operate, widely applicable, high in sensitivity and can be used for daily biological monitoring is established. The application successfully applies the benzene polycarboxylic acid molecular marker method to the detection of ultra-trace nano black carbon particles in animal biological samples for the first time, realizes the accurate quantitative analysis of black carbon particles in biological samples.

[0166] The specific embodiments of the application are described above. It should be understood that the application is not limited to the specific embodiments described above, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which does not affect the essential content of the application.

Claims

1. A method for detecting nano-black carbon particles in animal biological samples, characterized in that... Includes the following steps: 1) Sample homogenization: Take an appropriate amount of fresh animal sample, freeze it at -80℃, then put it in a clean glass bottle for freeze drying, then crush the sample with a clean glass rod and stir to homogenize it; 2) Sample digestion: Take an appropriate amount of biological sample powder into a 15mL centrifuge tube, add concentrated nitric acid until the sample is completely digested, and then neutralize it. At this point, the sample has been completely digested. 3) Quartz fiber filter membrane pretreatment: Place the high-purity quartz fiber filter membrane in a crucible and calcine it at high temperature to remove organic residues; 4) Diatomaceous earth washing: Take a portion of diatomaceous earth into a round-bottom flask, add a certain concentration of sulfuric acid, mix well and incubate in a water bath; then filter the diatomaceous earth in the flask through a filter membrane to collect it, and wash it with water, dilute nitric acid and water in sequence; finally transfer the diatomaceous earth on the filter membrane into a crucible, dry it in an oven, and then calcine it in a muffle furnace. At this point, the diatomaceous earth has been washed. 5) Sample filtration: Weigh diatomaceous earth, add alkali and vortex mix. Drop the suspension dropwise into a 15mL centrifuge tube. Mix the digested sample, allow it to settle and incubate. This is designated as liquid A. Simultaneously, prepare liquid B by adding diatomaceous earth to the alkali solution and vortex mix. Filter the sample using a vacuum filtration device with a sand core filter head. Place two layers of high-purity quartz fiber filter membrane on the filter head and dropwise evenly onto the quartz fiber filter membrane. Then, dropwise evenly onto the quartz fiber filter membrane in the same manner to achieve particulate matter enrichment. 6) Sample purification: Wash the filter membrane and diatomaceous earth dropwise with oxidant and organic solvent successively, and continue filtration until the filter membrane is completely dry to complete the sample purification. 7) Sample oxidation: Transfer the entire upper filter membrane and diatomaceous earth into the inner liner of the reactor, add concentrated nitric acid and tighten, place in an oven for reaction, turn off the power after the oven cools down, and allow to cool. Take the supernatant, process it, and then proceed with further processing; 8) Enrichment of molecular markers: Solid-phase extraction was used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. An HLB column was selected for solid-phase extraction. After four steps of activation, equilibration, loading and elution, the benzene polycarboxylic acid molecular markers in the concentrated acid matrix were dissolved in the organic solvent. At this point, the entire molecular marker enrichment work was completed. 9) Derivatization of molecular markers: The eluent was dried by nitrogen blowing, then reconstituted with methanol, dichloromethane and internal standard were added, and the mixture was vortexed and mixed. Then the derivatizing agent was added, and the mixture was vortexed and mixed to derivatize. After derivatization, the sample is quenched, dried with nitrogen, reconstituted with an organic solvent, and then transferred to a sample vial for instrument analysis. 10) Instrumental analysis: Analyze the derivatives from step 9) using GC-MS / MS. The instrument parameters should be under optimal conditions for detection and analysis.

2. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 1), the specific conditions for freeze drying are a cold trap temperature of -40 to -50°C and a freeze drying time of 48 hours or more.

3. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 2), the specific method for acid digestion is to add 1 mL of concentrated nitric acid with a mass fraction of 68% and leave it at room temperature for more than 24 hours until the solution is clear and transparent. The neutralization method involves adding 2 mL of water and 7 mL of 30% potassium hydroxide, adjusting the final solution's alkali concentration to 10%.

4. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 3), the specific conditions for high-temperature burning are burning at 600-800°C for 3-4 hours.

5. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 4), the specific parameters of the filter membrane used for filtration are: a high-purity quartz fiber filter membrane with a pore size of 0.3 μm and a diameter of 25 mm or 50 mm; and the diatomaceous earth with a particle size of 7 μm. The specific experimental conditions for acid boiling of diatomaceous earth are as follows: take 10-20g of diatomaceous earth with a particle size of 7μm into a round-bottom flask, then add sulfuric acid solution with a concentration of 4-5mol / L, the solid-liquid ratio should be between 1:3 and 1:4, mix well, and then boil in a water bath at 99℃ for 3-4 hours. The specific washing steps are as follows: rinse 2-3 times with 200-300 mL of deionized water, rinse once with 700-800 mL of 2% nitric acid, rinse 3-5 times with 200-300 mL of deionized water, then transfer the diatomaceous earth on the filter membrane into a ceramic dry pot, and dry it in an oven at 100-120℃ for 2-3 hours, and then calcine it in a muffle furnace at 550-600℃ for 3-4 hours. The specific method for mixing and sedimentation is as follows: weigh 50-100 mg of diatomaceous earth, add 1.5-2 mL of 10% potassium hydroxide and vortex mix, then drop the suspension dropwise into a 15 mL centrifuge tube, mix and sediment the digested sample, and incubate at 37-38℃ for 3-5 h.

6. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 5), the liquid B is prepared by weighing 50-100 mg of diatomaceous earth, adding 2-3 mL of 10% potassium hydroxide and vortexing to mix, and then letting it stand at room temperature for 3-5 hours. The high-purity quartz fiber filter membrane has a pore size of 0.3 μm and a diameter of 25 mm or 50 mm. In step 6), the oxidant and organic solvent are 2 mL to 3 mL of 30% hydrogen peroxide and 2 to 6 mL of acetone, respectively.

7. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 7), the concentrated nitric acid with a mass fraction of 68% used for oxidation is electronic-grade concentrated nitric acid; The specific operation of the oxidation process is as follows: add 1-2.5 mL of concentrated nitric acid to the lining of the reaction vessel and tighten it. Place it in an oven and react at 170-180°C for 8-9 hours. When the oven temperature drops to 70°C or below, turn off the power and open the oven door to allow it to cool naturally to room temperature. At this point, the oxidation is complete. The method for treating the supernatant of the oxidation product is as follows: slowly open the reactor that has been cooled to room temperature in a fume hood, take 1200-1600 μL of the supernatant, centrifuge at 8000-12000 rpm for 15-25 min, and rinse the inner liner of the reactor with deionized water while centrifuging. After centrifugation, take 800-1200 μL of the supernatant back into the original inner liner of the reactor and ventilate it overnight in a fume hood.

8. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 8), the HLB column used is a NanoChrom SelectCore HLB SPE Cartridge, HLB060-030250-1, 250mg 3mL solid phase extraction column; In step 8), the solid-phase extraction process is as follows: ① Activation with methanol in beds 4-5; ② Equilibration with 2% nitric acid in beds 5-6; ③ The overnight oxidation product is diluted to 11 mL with deionized water and mixed well, then added to the solid-phase extraction column for natural filtration; ④ The liquid on the extraction column is dried under negative pressure, and a 15 mL centrifuge tube is prepared with 100 μL of 37% hydrochloric acid added as the eluent collection tube; ⑤ Elution is performed with methanol solution in bed 4.

9. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 9), the specific experimental conditions for the derivatization are as follows: the eluent is dried by nitrogen blowing at 65-70°C using a nitrogen blower, then redissolved with 400 μL of methanol, and 1000 μL of dichloromethane, 100 μL of internal standard (200 ppm concentration), and 2',2-biphenyldicarboxylic acid are added. After vortexing and mixing, 400 μL of derivatizing agent and 2 mol / L trimethylsilyl diazomethane are added, vortexed and mixed, and the mixture is sealed and derivatized at room temperature for more than 12 hours. The specific quenching method is as follows: after derivatization, add 40 μL of quenching agent and 50% formic acid / methanol, then blow dry with nitrogen at 40°C, then redissolve with dichloromethane, vortex mix, and transfer to a sample vial for instrument detection and analysis.

10. The method for detecting nano-black carbon particles in animal biological samples as described in claim 1, characterized in that... In step 10), the optimal instrument analysis parameters are as follows: the chromatographic column is an Agilent HP-5MS weakly polar gas chromatographic column; the temperature program is 60℃ for 1 min, 30℃ / min to 200℃, 3℃ / min to 240℃, 20℃ / min to 290℃, and 290℃ for 2 min; the carrier gas type is He; the carrier gas flow rate is 1 mL / min; and the split ratio is splitless.

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