Method for detecting nano black carbon particles in animal biological sample

Abstract

The invention discloses a method for detecting nano black carbon particles in an animal biological sample, and relates to the field of atmospheric particulate pollutant research. The method is applied to detection of black carbon particles including human body samples. The method comprises the following steps: after freeze-drying an animal biological sample, digesting with concentrated nitric acid, adjusting the pH value, adding diatomite, uniformly mixing and settling, carrying out high-speed suction filtration to capture nano black carbon particles on a quartz fiber filter membrane pre-paved with diatomite, and washing the filter membrane with an oxidizing agent and an organic solvent in sequence to remove residual organic matters. Transferring the filter membrane into a reaction kettle, oxidizing the filter membrane with concentrated nitric acid at high temperature and high pressure, and oxidizing the black carbon particles into corresponding molecular marker benzene polycarboxylic acid; and carrying out integrated separation, enrichment, purification and derivation treatment, and carrying out quantitative analysis on the benzene polycarboxylic acid by utilizing GC-MS / MS (Gas Chromatography-Mass Spectrometry). And converting the concentration of the benzene polycarboxylic acid to obtain the concentration of the black carbon in the biological sample. The benzene polycarboxylic acid molecular marker method is successfully applied to detection of ultra-trace nano black carbon particles in animal biological samples, and accurate quantitative analysis of the black carbon particles in the biological samples is realized.

Description

Technical Field

[0001] The invention relates to the research field of atmospheric particle pollutants, and in particular to a method for detecting nanometer black carbon particles in animal biological samples. Background Art

[0002] Since the advent of the industrial age, human use of fossil fuels has increased exponentially, and the resulting environmental pollution problems have become increasingly serious. In the process of promoting modernization, my country's economic level and comprehensive national strength have rapidly improved, but it is also facing environmental pollution problems brought about by rapid development, and air pollution is one of the prominent problems. As the main source of air pollution, PM2.5 (particles with an aerodynamic diameter of less than 2.5 microns) that can enter the lungs has always posed a great threat to the lives and health of residents. Its main emission sources include industry, transportation, and biomass combustion. Black carbon is a key component of PM2.5, so as an important particulate air pollutant, the health hazards it brings need to be clarified.

[0003] Existing epidemiological studies have shown that there is a significant association between exposure to black carbon and adverse health effects on the respiratory, cardiovascular and nervous systems, especially for women and children. For example, black carbon exposure can increase oxidative stress levels and induce respiratory inflammation ([1] 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); long-term black carbon exposure is associated with decreased lung function in young urban women ([2] Suglia SF, 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 ([3] 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); black carbon exposure may cause myocardial ischemia ([4] Chuang KJ, et al. Particulate air pollution as a risk factor for ST-segment depression in patients with coronary artery disease. Circulation, 2008, 118: 1314–1320), arrhythmia ([5] Zanobetti A, et al. Associations between arrhythmia episodes and temporally and spatially resolved black carbon and particulate matter in elderly patients.OccupEnviron 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. EnvironPollut, 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. Some studies have found that black carbon exposure is associated with decreased intelligence, memory and learning ability in children ([7] Suglia SF, 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 the 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-pollutionand brain tumors. Environ Int, 2020,144: 106046, among which black carbon may be the main influencing factor; the latest research uses femtosecond pulse 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), which is the first time that black carbon breaks through the placental barrier. Provide direct evidence that black carbon exposure can cause reduced birth weight (

[10] Rokoff LB, 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 premature birth (

[11] Riddell CA, 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). Black carbon exposure during pregnancy may also increase the mother's risk of gestational diabetes (

[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 exposure during pregnancy can enter the fetal circulatory system and penetrate into the fetal liver, lungs, brain and other tissues and organs. The concentration of black carbon particles in the placenta and umbilical cord blood is positively correlated with the black carbon concentration in the mother's residence during pregnancy (

[13] Bongaerts E, et al. Maternal exposure to ambientblack 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 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 based on long-term exposure estimated by models or short-term exposure based on individual substitution. Due to the high spatiotemporal variability of black carbon, its concentration varies greatly in different microenvironments, so the assessment of its exposure level is not accurate enough, which greatly weakens the reliability of epidemiological evidence and the strength of the correlation. Therefore, accurate measurement of black carbon exposure in organisms is more convincing and more accurate. In recent years, foreign teams have developed femtosecond pulse laser technology based on the absorption characteristics of black carbon and conducted experiments in different human biological samples, but the quantitative concentration it can provide is not comprehensive in analysis. It is a semi-quantitative analysis method and lacks molecular-level material structure information; in addition, the method and instruments are expensive, the professional threshold for computer-assisted particle counting is high, and the promotion is poor. Summary of the invention

[0005] The purpose of the present invention is to provide a method for detecting black carbon particles in animal biological samples with low cost, high universality, simple process and wide application in order to quantitatively detect the distribution of black carbon particles in different types of organs or tissues in order to solve the above technical problems in the prior art. The present invention collects animal biological samples, oxidizes the black carbon particles in the samples into small molecular benzene polycarboxylic acids under high temperature and high pressure conditions, detects the concentration of benzene polycarboxylic acids using a gas chromatography-mass spectrometry platform, and finally converts the concentration of benzene polycarboxylic acids into the concentration of black carbon particles.

[0006] The present invention provides a method for detecting nano black carbon particles in an animal biological sample, comprising the following steps:

[0007] 1) Sample homogenization: Take an appropriate amount of fresh animal samples, freeze them at -80°C, put them into clean glass bottles for freeze drying, and then crush the samples with a clean glass rod and stir them to homogenize;

[0008] 2) Sample digestion: Take an appropriate amount of biological sample dry powder in a 15mL centrifuge tube, add concentrated nitric acid until the sample is completely digested, and then neutralize it. At this time, 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 burn it at high temperature to remove organic residues;

[0010] 4) Washing of diatomite: Put some diatomite in a round-bottom flask, add sulfuric acid of a certain concentration, mix well and incubate in a water bath; then collect the diatomite in the flask by filtering with a filter membrane, and wash with water, dilute nitric acid and water in turn; finally, transfer the diatomite on the filter membrane into a crucible, dry it in an oven, and then calcine it in a muffle furnace. At this time, the diatomite has been washed;

[0011] 5) Sample filtration: Weigh diatomaceous earth, add alkali and vortex mix, drip the suspension drop by drop into a 15mL centrifuge tube, mix and settle the digested sample and incubate it, which is set as liquid A; prepare liquid B at the same time, by adding diatomaceous earth to the alkali solution and vortex mix; filter 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 drip liquid B evenly on the quartz fiber filter membrane drop by drop, and then drip liquid A evenly on the quartz fiber filter membrane drop by drop in the same way to achieve particle sample enrichment;

[0012] 6) Sample purification: Wash the filter membrane and diatomaceous earth drop by drop with oxidant and organic solvent, 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 diatomaceous earth into the reactor liner, add concentrated nitric acid and tighten it, put it into the oven for reaction, turn off the power after the oven cools down, and cool it down; take the supernatant and treat it for later use;

[0014] 8) Enrichment of molecular markers: Solid phase extraction is used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation, and HLB columns are selected for solid phase extraction operations; after four steps of activation, equilibration, loading, and elution, the benzene polycarboxylic acid molecular markers in the concentrated acid matrix have been dissolved in the organic solvent, and the enrichment of the entire molecular marker has been completed;

[0015] 9) Derivatization of molecular markers: blow the eluent to dryness with nitrogen blower, then re-dissolve it with methanol, add dichloromethane and internal standard, vortex mix, then add derivatization agent, vortex mix, and derivatize; after derivatization, quench, blow to dryness with nitrogen, then re-dissolve it with organic solvent, and then transfer it to the injection bottle for instrument detection and analysis;

[0016] 10) Instrumental analysis: The derivative product in step 9) is analyzed by GC-MS / MS. The instrument parameters should be optimized for the detection and analysis.

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

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

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

[0020] 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, a diameter of 50 mm, a diatomaceous earth particle size of 7 μm, and the specific experimental conditions for diatomaceous earth acid boiling are to take 10 to 20 g of diatomaceous earth with a particle size of 7 μm in a round-bottom flask, add deionized water and concentrated sulfuric acid in a certain proportion to prepare a sulfuric acid solution with a final concentration of 4 to 5 mol / L, and the solid-liquid ratio should be between 1:3 and 1:4. After mixing, boil in a water bath at 99°C for 3 to 4 hours; the specific steps of washing are to rinse with 200 to 300 mL of deionized water 2 to 3 times, 700 to 800 mL of 2% nitric acid once, and 200 to 300 mL of nitric acid. Rinse with deionized water for 3 to 5 times, then transfer the diatomaceous earth on the filter membrane into a ceramic dry pot and dry it in an oven at 100 to 120°C for 2 to 3 hours, and then calcine it in a muffle furnace at 550 to 600°C for 3 to 4 hours.

[0021] In step 5), the specific method of mixing and sedimentation is to weigh 50-100 mg of diatomaceous earth, add 1.5-2 mL of 10% potassium hydroxide and vortex mix, then drip the suspension drop by drop into a 15 mL centrifuge tube, mix and sediment the digested sample, and incubate at 37-38° C. for 3-5 hours; the configuration method of liquid B is to weigh 50-100 mg of diatomaceous earth, add 2-3 mL of 10% potassium hydroxide and vortex mix, and stand at room temperature for 3-5 hours; the pore size of the high-purity quartz fiber filter is 0.3 μm, and the diameter is 25 mm or 50 mm.

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

[0023] In step 7), the concentrated nitric acid used for oxidation is electronic grade concentrated nitric acid. The specific operation of the oxidation process is to add 1-2.5 mL of concentrated nitric acid to the lining of the reactor and tighten it, put it in an oven and react at 170-180° C. for 8-9 hours, turn off the power when the oven is cooled to 70° C. or below, open the oven door and cool it naturally to room temperature. At this time, the oxidation is completed. The method for treating the supernatant of the oxidation product is to slowly open the reactor cooled to room temperature in a fume hood, take 1200-1600 μL of the supernatant, centrifuge at 8000-12000 rpm for 15-25 minutes, and rinse the reactor liner with deionized water while centrifuging. After the centrifugation, take 800-1200 μL of the supernatant into the original reactor liner, and ventilate it in a fume hood overnight.

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

[0025] In step 9), the specific experimental condition parameters of the derivatization are: blowing the eluent to dryness with nitrogen at 65-70°C with a nitrogen blower, re-dissolving with 400 μL of methanol, adding 1000 μL of dichloromethane, 100 μL of internal standard (concentration 200ppm, 2'2-biphenyldicarboxylic acid), vortexing and then adding 400 μL of derivatizing agent (2 mol / L trimethylsilylated diazomethane), vortexing and sealing, and derivatizing at room temperature for more than 12 hours; the specific method of quenching is to add 40 μL of quenching agent (50% formic acid / methanol) after the derivatization, blowing to dryness with nitrogen at about 40°C, re-dissolving with dichloromethane, vortexing and transferring to an injection bottle, and waiting for instrument detection and analysis;

[0026] In step 10), the optimal parameter conditions for the instrument analysis are that the chromatographic column can be 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 present invention provides a detection method for evaluating black carbon particles in biological samples of ordinary people or animals. The key points of the present invention are: 1. using concentrated nitric acid to completely digest organic matter at room temperature and pressure without destroying black carbon particles; 2. establishing a "mixing sedimentation-high-speed impact" system and determining parameters of some key links, such as the selection of filter membrane and diatomaceous earth sizes, the acid washing process of diatomaceous earth, the optimal acid-base conditions for filtration, the incubation time of diatomaceous earth before filtration, and the filtration method; 3. using an oxidant and an organic solvent to wash successively to remove residual organic matter on the filter membrane, which greatly improves the matrix interference of the present method; 4. using a solid phase extraction method to extract the benzoic acid molecules. The markers were enriched, and the commercial HLB column selected was simple to operate, had a high recovery rate, and was environmentally friendly; ⑤ The derivatization conditions of benzene polycarboxylic acid were optimized, and the relevant factors affecting the derivatization efficiency were systematically investigated; Unlike other previous methods, this method is the first to successfully apply the benzene polycarboxylic acid molecular marker method originally applied to environmental samples with high black carbon content to the analysis of extremely low black carbon content in biological samples after a lot of improvements, which makes this method have great application prospects; At the same time, the benzene polycarboxylic acid molecular marker method also has great limitations when applied to environmental samples rich in organic matter. The successful application of this method on biological samples also solves this key problem in the environmental field. This method will provide relevant internal exposure doses of air pollution by accurately quantifying the black carbon content in different organs of the human body, thereby providing more reliable evidence for related epidemiological studies, thereby revealing the impact of environmental pollution on human life and health; The core of the present invention is to provide a black carbon particle detection method that is quantitatively accurate, highly sensitive, simple and efficient, and has strong universality and can be applied to different types of biological samples.

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

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

[0030] 2. The present invention develops a particle enrichment system based on "mixing sedimentation-high-speed impact", combined with a gas chromatography-mass spectrometry device to achieve accurate quantification of extremely low-content black carbon particles in biological samples. This method has high sensitivity and strong scalability.

[0031] 3. The present invention provides black carbon particle exposure data in the human body for the environmental health field, making epidemiological research more reliable; at the same time, it provides a solution to the inaccurate determination of black carbon particle content in organic-rich samples by the benzene polycarboxylic acid method for the environmental field.

[0032] 4. The present invention will provide methodological support for studying the migration characteristics of black carbon particles between different organs and tissues of the same organism, as well as the changes in local biochemical conditions caused by black carbon and the resulting health effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The GC-MS / MS quantitative ion extraction chromatogram of a mixture of six benzene polycarboxylic acid standards.

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

[0035] Figure 3 This is the spike recovery rate of biomass black carbon particles at different spike levels in this method.

[0036] Figure 4 This is the removal effect of biological matrix interference achieved by this method through a specific washing process.

[0037] Figure 5 This is the GC-MS / MS quantitative ion extraction chromatogram of black carbon particles in the lungs of normally fed cattle that were not exposed to high doses of human factors. DETAILED DESCRIPTION

[0038] The present invention is described in detail below in conjunction with specific examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0039] The embodiment of the present invention includes the following steps:

[0040] (1) Sample homogenization: Take an appropriate amount of fresh animal samples, freeze them at -80°C, and then place them in clean glass bottles for freeze drying. The cold trap temperature is -40 to -50°C, and the freeze drying time is more than 48 hours. Then, crush the samples with a clean glass rod and stir to homogenize.

[0041] (2) Sample digestion: Take an appropriate amount of biological sample dry powder in a 15 mL centrifuge tube, add 1 mL of 68% concentrated nitric acid, and place it at room temperature for more than 24 h until the sample is completely digested. Then add 2 mL of water and 7 mL of 30% potassium hydroxide to neutralize. At this time, the sample has been completely digested;

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

[0043] (4) Washing of diatomite: Take 10-20g of diatomite with a particle size of 7μm 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 4-5mol / L. The solid-liquid ratio should be between 1:3 and 1:4. After mixing, place in a water bath at 99°C for 3-4h. Then, filter and collect the diatomite in the flask with a 0.3μm high-purity quartz fiber filter membrane, and rinse it with 200-300mL deionized water 2-3 times, 700-800mL 2% nitric acid once, and 200-300mL deionized water 3-5 times. Then, transfer the diatomite on the filter membrane into a ceramic dry pot, and dry it in an oven at 100-120°C for 2-3h, then calcine it in a muffle furnace at 550-600°C for 3-4h, then naturally cool it to room temperature and set it aside. At this time, the diatomite has been washed;

[0044] (5) Sample filtration: Weigh 50-100 mg of diatomaceous earth, add 1.5-2 mL of 10% potassium hydroxide and vortex mix, then drip the suspension drop by drop into a 15 mL centrifuge tube, mix and settle the digested sample, and incubate it at 37-38°C for 3-5 h to obtain liquid A. At the same time, weigh 50-100 mg of diatomaceous earth, add 2-3 mL of 10% potassium hydroxide and vortex mix, then let it stand at room temperature for 3-5 h to obtain liquid B. Then filter using a vacuum filtration device with a sand core filter head, 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 drip liquid B evenly on the quartz fiber filter membrane drop by drop, then drip liquid A evenly on the quartz fiber filter membrane drop by drop in the same way to achieve particulate sample enrichment.

[0045] (6) Sample purification: Wash the filter membrane and diatomaceous earth drop by drop with 2-3 mL 30% hydrogen peroxide, then wash drop by drop with 2-6 mL acetone, 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 diatomaceous earth into the inner tank of the reactor, add 1-2.5 mL of concentrated nitric acid and tighten it, place it in an oven at 170-180°C for 8-9 hours, turn off the power when the oven temperature drops to 70°C or below, open the oven door and let it cool naturally to room temperature; slowly open the reactor cooled to room temperature in a fume hood, take 1200-1600 μL of the supernatant, centrifuge at 8000-12000 rpm for 15-25 minutes, and rinse the reactor tank with deionized water while centrifuging. After the centrifugation, take 800-1200 μL of the supernatant into the original reactor tank and ventilate it in a fume hood overnight;

[0047] (8) Molecular marker extraction: HLB column was used to separate, enrich and purify the benzene polycarboxylic acid molecular markers produced by oxidation, and NanoChrom SelectCore HLB SPE Cartridge solid phase extraction column was selected for solid phase extraction operation. The activation process is as follows: ① 4-5 beds of methanol activation; ② 5-6 beds of 2% nitric acid balance; ③ The oxidation product after overnight was diluted to about 11mL with deionized water and mixed, and added to the solid phase extraction column for natural filtration; ④ The liquid on the extraction column was drained under negative pressure, and a 15mL centrifuge tube was prepared at the same time, and 100μL of 37% concentrated hydrochloric acid was added to it as the eluent collection tube; ⑤ 4 beds of methanol solution were used for elution; At this point, the enrichment of the entire molecular marker has been completed;

[0048] (9) Derivatization of molecular markers: The eluent was blown dry with nitrogen at 65-70°C, then reconstituted with 400 μL of methanol, 1000 μL of dichloromethane and 100 μL of internal standard (concentration 200 ppm, 2'2-biphenyldicarboxylic acid) were added, vortexed and then 400 μL of derivatization agent (2 mol / L trimethylsilylated diazomethane) was added, vortexed and sealed, and derivatized at room temperature for more than 12 h;

[0049] (10) Instrumental analysis: After derivatization, add 40 μL of quencher (50% formic acid / methanol), blow dry with nitrogen at about 40 °C, re-dissolve with dichloromethane, vortex mix, and transfer to a sample injection bottle for instrument detection and analysis.

[0050] Example 1

[0051] This embodiment includes the following steps:

[0052] (1) Sample homogenization: Take 3g of fresh animal sample, freeze it at -80℃ for 48h, and then put it into a clean glass bottle for freeze drying. The cold trap temperature is set to -50℃, and the freeze drying time lasts for more than 48h. After completion, crush the sample with a clean glass rod and stir it until it is homogenous.

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

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

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

[0056] (5) Sample filtration: Weigh 50 mg of diatomaceous earth, add 1.5 mL of 10% potassium hydroxide and vortex to mix. Pour the suspension drop by drop into a 15 mL centrifuge tube, mix with the digested sample and mix and settle, incubate at 37 °C for 3 h. At the same time, prepare liquid B (weigh 50 mg of diatomaceous earth, add 2 mL of 10% potassium hydroxide and vortex to mix, and let stand at room temperature for 3 h). Use a vacuum filtration device with a sand core filter head for filtration, 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, and then add liquid A drop by drop to achieve the enrichment of the particulate sample.

[0057] (6) Sample purification: Use 2 mL of 30% hydrogen peroxide and 2 mL of acetone as washing liquid to wash the residue on the filter membrane and diatomaceous earth drop by drop, and continue the filtration operation until the filter membrane is completely dry.

[0058] (7) Sample oxidation: Carefully transfer the entire upper filter membrane and diatomaceous earth to the inner tank of the reactor, add 1 mL of electronic grade concentrated nitric acid and tighten the lid of the reactor. Place the reactor in an oven and react at 170°C for 8 hours. After the reaction is completed, turn off the power and open the oven door when the oven temperature drops below 70°C to cool naturally to room temperature. Slowly open the lid of the reactor in a fume hood, take about 1200 μL of the supernatant, and centrifuge at 8000 r / min for 15 minutes. After centrifugation, transfer 800 μL of the supernatant to the original reactor liner and ventilate overnight to further remove residues.

[0059] (8) Enrichment of molecular markers: Solid phase extraction technology was used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. NanoChrom SelectCore HLB SPE Cartridge (model: HLB060-030250-1, 250 mg / 3 mL, 60 μm) was selected as the solid phase extraction column. The operation was carried out in four steps: activation, equilibration, loading, and elution.

[0060] (9) Derivatization of molecular markers: Use a nitrogen blower to blow the eluent to dryness at 65°C, and add 400 μL of methanol to reconstitute. Then add 1000 μL of dichloromethane and 100 μL of internal standard (2'2-biphenyldicarboxylic acid at a concentration of 200 ppm), vortex to mix, then add 400 μL of derivatization agent (2 mol / L trimethylsilylated diazomethane), vortex to mix again and seal with a lid, and derivatize at room temperature for more than 12 hours. After the derivatization is completed, add 40 μL of quencher (50% formic acid / methanol mixture), blow nitrogen to dryness at about 40°C, then reconstitute with dichloromethane, vortex to mix, and transfer to the injection bottle for instrument detection and analysis.

[0061] (10) Instrumental analysis: GC-MS / MS was used to analyze and detect the derivative products. The chromatographic column used was an Agilent HP-5MS weakly polar gas chromatograph column; the temperature program was set to 60°C for 1 min, then to 200°C at a rate of 30°C / min, then to 240°C at a rate of 3°C / min, then to 290°C at a rate of 20°C / min, and finally to 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.

[0062] Example 2

[0063] (1) Sample homogenization: Take 3g of fresh animal sample, freeze it at -80℃ for 48h, and then put it into a clean glass bottle for freeze drying. The cold trap temperature is set to -50℃, and the freeze drying time lasts for more than 48h. After completion, crush the sample with a clean glass rod and stir it until it is homogenous.

[0064] (2) Sample digestion: Take 0.3 g of biological sample dry powder and place it in a 15 mL centrifuge tube. Add 1 mL of 68% concentrated nitric acid and place it at room temperature for 24 hours until the solution is clear and transparent. Then add 2 mL of water and 7 mL of 30% potassium hydroxide to neutralize and adjust the solution alkaline concentration to about 10%.

[0065] (3) Quartz fiber filter pretreatment: A high-purity quartz fiber filter with a pore size of 0.3 μm and a diameter of 50 mm was placed in a crucible and burned at 600 °C for 3 h to remove organic residues.

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

[0067] (5) Sample filtration: Weigh 50 mg of diatomaceous earth, add 1.5 mL of 10% potassium hydroxide and vortex to mix. Pour the suspension drop by drop into a 15 mL centrifuge tube, mix with the digested sample and mix and settle, incubate at 37 °C for 3 h. At the same time, prepare liquid B (weigh 50 mg of diatomaceous earth, add 2 mL of 10% potassium hydroxide and vortex to mix, and let stand at room temperature for 3 h). Use a vacuum filtration device with a sand core filter head for filtration, 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, and then add liquid A drop by drop evenly to achieve the enrichment of the particulate sample.

[0068] (6) Sample purification: Use 2 mL of 30% hydrogen peroxide and 2 mL of acetone as washing liquid to wash the residue on the filter membrane and diatomaceous earth drop by drop, and continue the filtration operation until the filter membrane is completely dry.

[0069] (7) Sample oxidation: Carefully transfer the entire upper filter membrane and diatomaceous earth to the inner tank of the reactor, add 1 mL of electronic grade concentrated nitric acid and tighten the lid of the reactor. Place the reactor in an oven and react at 170°C for 8 hours. After the reaction is completed, turn off the power and open the oven door when the oven temperature drops below 70°C to cool naturally to room temperature. Slowly open the lid of the reactor in a fume hood, take about 1200 μL of the supernatant, and centrifuge at 8000 r / min for 15 minutes. After centrifugation, transfer 800 μL of the supernatant to the original reactor liner and ventilate overnight to further remove residues.

[0070] (8) Enrichment of molecular markers: Solid phase extraction technology was used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. NanoChrom SelectCore HLB SPE Cartridge (model: HLB060-030250-1, 250 mg / 3 mL, 60 μm) was selected as the solid phase extraction column. The operation was carried out in four steps: activation, equilibration, loading, and elution.

[0071] (9) Derivatization of molecular markers: Use a nitrogen blower to blow the eluent to dryness at 65°C, and add 400 μL of methanol to reconstitute. Then add 1000 μL of dichloromethane and 100 μL of internal standard (2'2-biphenyldicarboxylic acid at a concentration of 200 ppm), vortex to mix, then add 400 μL of derivatization agent (2 mol / L trimethylsilylated diazomethane), vortex to mix again and seal with a lid, and derivatize at room temperature for more than 12 hours. After the derivatization is completed, add 40 μL of quencher (50% formic acid / methanol mixture), blow nitrogen to dryness at about 40°C, then reconstitute with dichloromethane, vortex to mix, and transfer to the injection bottle for instrument detection and analysis.

[0072] (10) Instrumental analysis: GC-MS / MS was used to analyze and detect the derivative products. The chromatographic column used was an Agilent HP-5MS weakly polar gas chromatograph column; the temperature program was set to 60°C for 1 min, then to 200°C at a rate of 30°C / min, then to 240°C at a rate of 3°C / min, then to 290°C at a rate of 20°C / min, and finally to 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.

[0073] Example 3

[0074] (1) Sample homogenization: Take 3g of fresh animal sample, freeze it at -80℃ for 48h, and then put it into a clean glass bottle for freeze drying. The cold trap temperature is set to -50℃, and the freeze drying time lasts for more than 48h. After completion, crush the sample with a clean glass rod and stir it until it is homogenous.

[0075] (2) Sample digestion: Take 0.3 g of biological sample dry powder and place it in a 15 mL centrifuge tube. Add 1 mL of 68% concentrated nitric acid and place it at room temperature for 24 hours until the solution is clear and transparent. Then add 2 mL of water and 7 mL of 30% potassium hydroxide to neutralize and adjust the solution alkaline concentration to about 10%.

[0076] (3) Quartz fiber filter pretreatment: A high-purity quartz fiber filter with a pore size of 0.3 μm and a diameter of 50 mm was placed in a crucible and burned at 600 °C for 3 h to remove organic residues.

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

[0078] (5) Sample filtration: Weigh 50 mg of diatomaceous earth, add 1.5 mL of 10% potassium hydroxide and vortex to mix. Pour the suspension drop by drop into a 15 mL centrifuge tube, mix with the digested sample and mix and settle, incubate at 37 °C for 3 h. At the same time, prepare liquid B (weigh 50 mg of diatomaceous earth, add 2 mL of 10% potassium hydroxide and vortex to mix, and let stand at room temperature for 3 h). Use a vacuum filtration device with a sand core filter head for filtration, 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, and then add liquid A drop by drop evenly to achieve the enrichment of the particulate sample.

[0079] (6) Sample purification: Use 2 mL of 30% hydrogen peroxide and 2 mL of acetone as washing liquid to wash the residue on the filter membrane and diatomaceous earth drop by drop, and continue the filtration operation until the filter membrane is completely dry.

[0080] (7) Sample oxidation: Carefully transfer the entire upper filter membrane and diatomaceous earth to the inner tank of the reactor, add 1 mL of electronic grade concentrated nitric acid and tighten the lid of the reactor. Place the reactor in an oven and react at 170°C for 8 hours. After the reaction is completed, turn off the power and open the oven door when the oven temperature drops below 70°C to cool naturally to room temperature. Slowly open the lid of the reactor in a fume hood, take about 1200 μL of the supernatant, and centrifuge at 8000 r / min for 15 minutes. After centrifugation, transfer 800 μL of the supernatant to the original reactor liner and ventilate overnight to further remove residues.

[0081] (8) Enrichment of molecular markers: Solid phase extraction technology was used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. NanoChrom SelectCore HLB SPE Cartridge (model: HLB060-030250-1, 250 mg / 3 mL, 60 μm) was selected as the solid phase extraction column. The operation was carried out in four steps: activation, equilibration, loading, and elution.

[0082] (9) Derivatization of molecular markers: Use a nitrogen blower to blow the eluent to dryness at 65°C, and add 400 μL of methanol to reconstitute. Then add 1000 μL of dichloromethane and 100 μL of internal standard (2'2-biphenyldicarboxylic acid at a concentration of 200 ppm), vortex to mix, then add 400 μL of derivatization agent (2 mol / L trimethylsilylated diazomethane), vortex to mix again and seal with a lid, and derivatize at room temperature for more than 12 hours. After the derivatization is completed, add 40 μL of quencher (50% formic acid / methanol mixture), blow nitrogen to dryness at about 40°C, then reconstitute with dichloromethane, vortex to mix, and transfer to the injection bottle for instrument detection and analysis.

[0083] (10) Instrumental analysis: GC-MS / MS was used to analyze and detect the derivative products. The chromatographic column used was an Agilent HP-5MS weakly polar gas chromatograph column; the temperature program was set to 60°C for 1 min, then to 200°C at a rate of 30°C / min, then to 240°C at a rate of 3°C / min, then to 290°C at a rate of 20°C / min, and finally to 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: Take 3g of fresh animal sample, freeze it at -80℃ for 48h, and then put it into a clean glass bottle for freeze drying. The cold trap temperature is set to -50℃, and the freeze drying time lasts for more than 48h. After completion, crush the sample with a clean glass rod and stir it until it is homogenous.

[0086] (2) Sample digestion: Take 0.3 g of biological sample dry powder and place it in a 15 mL centrifuge tube. Add 1 mL of 68% concentrated nitric acid and place it at room temperature for 24 hours until the solution is clear and transparent. Then add 2 mL of water and 7 mL of 30% potassium hydroxide to neutralize and adjust the solution alkaline concentration to about 10%.

[0087] (3) Quartz fiber filter pretreatment: A high-purity quartz fiber filter with a pore size of 0.3 μm and a diameter of 50 mm was placed in a crucible and burned at 600 °C for 3 h to remove organic residues.

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

[0089] (5) Sample filtration: Weigh 50 mg of diatomaceous earth, add 1.5 mL of 10% potassium hydroxide and vortex to mix. Pour the suspension drop by drop into a 15 mL centrifuge tube, mix with the digested sample and mix and settle, incubate at 37 °C for 3 h. At the same time, prepare liquid B (weigh 50 mg of diatomaceous earth, add 2 mL of 10% potassium hydroxide and vortex to mix, and let stand at room temperature for 3 h). Use a vacuum filtration device with a sand core filter head for filtration, 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, and then add liquid A drop by drop evenly to achieve the enrichment of the particulate sample.

[0090] (6) Sample purification: Use 2 mL of 30% hydrogen peroxide and 2 mL of acetone as washing liquid to wash the residue on the filter membrane and diatomaceous earth drop by drop, and continue the filtration operation until the filter membrane is completely dry.

[0091] (7) Sample oxidation: Carefully transfer the entire upper filter membrane and diatomaceous earth to the inner tank of the reactor, add 1 mL of electronic grade concentrated nitric acid and tighten the lid of the reactor. Place the reactor in an oven and react at 170°C for 8 hours. After the reaction is completed, turn off the power and open the oven door when the oven temperature drops below 70°C to cool naturally to room temperature. Slowly open the lid of the reactor in a fume hood, take about 1200 μL of the supernatant, and centrifuge at 8000 r / min for 15 minutes. After centrifugation, transfer 800 μL of the supernatant to the original reactor liner and ventilate overnight to further remove residues.

[0092] (8) Enrichment of molecular markers: Solid phase extraction technology was used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation. NanoChrom SelectCore HLB SPE Cartridge (model: HLB060-030250-1, 250 mg / 3 mL, 60 μm) was selected as the solid phase extraction column. The operation was carried out in four steps: activation, equilibration, loading, and elution.

[0093] (9) Derivatization of molecular markers: Use a nitrogen blower to blow the eluent to dryness at 65°C, and add 400 μL of methanol to reconstitute. Then add 1000 μL of dichloromethane and 100 μL of internal standard (2'2-biphenyldicarboxylic acid at a concentration of 200 ppm), vortex to mix, then add 400 μL of derivatization agent (2 mol / L trimethylsilylated diazomethane), vortex to mix again and seal with a lid, and derivatize at room temperature for more than 12 hours. After the derivatization is completed, add 40 μL of quencher (50% formic acid / methanol mixture), blow nitrogen to dryness at about 40°C, then reconstitute with dichloromethane, vortex to mix, and transfer to the injection bottle for instrument detection and analysis.

[0094] (10) Instrumental analysis: GC-MS / MS was used to analyze and detect the derivative products. The chromatographic column used was an Agilent HP-5MS weakly polar gas chromatograph column; the temperature program was set to 60°C for 1 min, then to 200°C at a rate of 30°C / min, then to 240°C at a rate of 3°C / min, then to 290°C at a rate of 20°C / min, and finally to 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.

[0095] Example 5

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

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

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

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

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

[0101] Example 6

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

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

[0104] In step (2), the mixture is placed at room temperature for 48 hours.

[0105] In step (3), the burning time is 4 hours.

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

[0107] In step (5), 75 mg of diatomaceous earth and 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, the difference is that:

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

[0112] In step (2), the mixture is placed at room temperature for 36 hours.

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

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

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

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

[0117] In step (8), 5 beds of methanol were used for activation and 6 beds of 2% nitric acid were used for balance.

[0118] Example 8

[0119] Similar to Example 1, the difference is that:

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

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

[0122] In step (3), the burning time is 3.5 h.

[0123] In step (4), the mass of diatomaceous earth is 20 g.

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

[0125] In step (9), the nitrogen blowing temperature is 70°C and the derivatization time is 16 hours.

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

[0127] Example 9

[0128] Similar to Example 1, the difference is that:

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

[0130] In step (2), the mixture is placed at room temperature for 30 h.

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

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

[0133] In step (5), 80 mg of diatomaceous earth 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 is 175°C.

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

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

[0138] A specific application example is given below.

[0139] This example is based on normally raised cattle without human intervention exposure, so it can better represent the living environment of the general population. This example detects the concentration of black carbon particles in their lungs. The specific implementation steps are as follows:

[0140] 1. Sample collection

[0141] Organ samples of freshly slaughtered cattle are collected at the slaughterhouse. After arriving at the laboratory, the lungs are dissected, and the visceral pleura on the surface is opened with a clean scalpel and tweezers, and the internal lung tissue that has not been exposed to the outside is taken for testing.

[0142] 2. Sample Homogenization

[0143] Take an appropriate amount of bovine lung sample, freeze it at -80℃, and then put it into a clean glass bottle for freeze-drying. The cold trap temperature is -50℃ and the freeze-drying time is more than 48h. Then crush the sample with a clean glass rod and stir it homogenously.

[0144] 3. Sample digestion

[0145] Take an appropriate amount of freeze-dried powder of lung tissue and place it in a 15mL centrifuge tube. Add 1mL of 68% concentrated nitric acid and place it at room temperature for 48h. Then add 2mL of water and 7mL of 30% potassium hydroxide for neutralization.

[0146] 4. Diatomaceous earth washing

[0147] Take 20g of diatomaceous earth with a particle size of 7μm in a round-bottom flask, then add 47mL of water, then add 17mL of 98% concentrated sulfuric acid, mix well and place in a water bath at 99℃ for 4h, then filter and collect the diatomaceous earth in the flask with a 0.3μm high-purity quartz fiber filter membrane, and rinse it with 250mL of deionized water twice, 750mL of 2% nitric acid once, and 250mL of deionized water four times in sequence, then transfer the diatomaceous earth on the filter membrane into a ceramic dry pot, and dry it in an oven at 110℃ for 2h, then calcine it at 600℃ for 3h in a muffle furnace, then naturally cool to room temperature for use.

[0148] 5. Sample Filtration

[0149] Weigh 100 mg of diatomaceous earth, add 1.5 mL of 10% potassium hydroxide and vortex mix, then drip the suspension drop by drop into a 15 mL centrifuge tube, mix and settle the digested lung sample, and incubate at 37°C for 5 hours to obtain liquid A; at the same time, weigh 100 mg of diatomaceous earth, add 3 mL of 10% potassium hydroxide and vortex mix, and let stand at room temperature for 5 hours to obtain liquid B; then filter using a vacuum filtration device with a sand core filter head, place two layers of high-purity quartz fiber filter membranes with a pore size of 0.3 μm on the filter head, and evenly drip liquid B drop by drop on the quartz fiber filter membrane, and then evenly drip liquid A drop by drop on the quartz fiber filter membrane in the same way to achieve particle sample enrichment in the lung sample.

[0150] 6. Sample purification

[0151] Wash the filter membrane and diatomaceous earth dropwise with 2 mL of 30% hydrogen peroxide, then dropwise with 6 mL of acetone, and continue to filter until the filter membrane is completely dry to complete the sample purification.

[0152] 7. Sample oxidation

[0153] Transfer the entire upper filter membrane and diatomaceous earth into the reactor liner, add 2.5 mL of concentrated nitric acid and tighten it, put it in an oven and react at 170°C for 8 hours; turn off the power when the oven temperature drops to 70°C or below, open the oven door and cool naturally to room temperature. Slowly open the reactor cooled to room temperature in a fume hood, take 1600 μL of the supernatant, centrifuge at 12000 rpm for 25 minutes, and rinse the reactor liner with deionized water while centrifuging. After the centrifugation, take 1200 μL of the supernatant in the original reactor liner and ventilate overnight in a fume hood.

[0154] 8. Molecular marker extraction

[0155] Solid phase extraction was used to separate, enrich and purify the benzene polycarboxylic acid molecular markers produced by oxidation, and NanoChrom SelectCore HLB SPE Cartridge solid phase extraction column was selected for solid phase extraction operation. The activation process is: ① 4 beds of methanol activation; ② 5 beds of 2% nitric acid balance; ③ The oxidation product after overnight was diluted to 11mL with deionized water and mixed, and added to the solid phase extraction column for natural filtration; ④ The liquid on the extraction column was drained under negative pressure conditions, and a 15mL centrifuge tube was prepared at the same time, and 100μL of concentrated hydrochloric acid with a mass fraction of 37% was added to it as an eluent collection tube; ⑤ Elution was performed with 4 beds of methanol solution; At this point, the enrichment of the entire molecular marker has been completed;

[0156] IX. Derivation of Molecular Markers

[0157] The eluate was blown dry with nitrogen at 65°C, then reconstituted with 400 μL of methanol, 1000 μL of dichloromethane and 100 μL of internal standard (200 ppm, 2'2-biphenyldicarboxylic acid) were added, vortexed and then 400 μL of derivatizing agent (2 mol / L trimethylsilylated diazomethane) was added, vortexed and sealed, and derivatized at room temperature for 12 h;

[0158] 10. Instrumental Analysis

[0159] After derivatization, 40 μL of quencher (50% formic acid / methanol) was added, and then nitrogen was blown to dryness at 40°C, and then re-dissolved with dichloromethane, vortexed and mixed, and transferred to the injection bottle, and tested on the machine. The qualitative and quantitative analysis of the compounds was performed using the instrument's own software. The standard curve used for quantification was the working standard curve, which was achieved by spiking fetal bovine serum matrix without black carbon particles. At the same time, process blank samples and biological matrix spiked samples were added during the experiment as blank controls and quality controls for the entire experimental process.

[0160] The GC-MS / MS quantitative ion extraction chromatogram of the mixture of the polycarboxylic acid standards of the present invention is shown in Figure 1 ,from Figure 1 (Chromatogram of the mixture of benzene polycarboxylic acid standards) It can be seen that the chromatographic peaks of each standard can be clearly separated from the baseline, and the peak shape is symmetrical without tailing, and there is no overlap between the chromatographic peaks of the compounds, which meets the needs of quantitative analysis.

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

[0162] The recoveries of biomass black carbon particles at different spike levels are shown in Figure 3 ,from Figure 3 (Recovery of black carbon particles spiked in biomass at different spiking levels) It can be seen that this method achieves good recovery of nano black carbon particles regardless of the matrix spiking 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 Figure 4 ,from Figure 4 (The removal effect of a specific washing process on biological matrix interference) It can be seen that after washing with an oxidant and an organic solvent, benzene pentacarboxylic acid, an indicator of organic residues, showed a significant decrease, indicating that the washing method used has a strong ability to remove organic interference. In the case of organic matter that will cause substrate interference, this can greatly improve the sensitivity of the method. Figure 3 It can be seen that this washing method will not affect the recovery rate of black carbon particles.

[0164] The GC-MS / MS quantitative ion extraction chromatogram of black carbon particles in the lungs of normally fed cattle not exposed to high doses of human factors is shown in Figure 5 .from Figure 5 (Chromatogram of black carbon particles in the lungs of normally raised cattle that were not exposed to high doses by humans) It can be seen that a significant response signal of benzene polycarboxylic acid was found in the cattle lungs, which means that black carbon particles that can generate benzene polycarboxylic acid are present in the cattle lungs, and the concentration has reached a level that can be accurately quantified. This not only verifies the feasibility of this method, but also reveals the great potential of this method in daily biological monitoring. It also means that black carbon particles can enter the lungs of organisms through the respiratory tract and are likely to bring corresponding health effects.

[0165] The present invention uses concentrated nitric acid to digest the sample, so that the sample digestion is cleaner; uses the "mixing sedimentation-high-speed impact" particle enrichment system to enrich nano black carbon particles, and obtains a good recovery rate; combines the washing method of oxidant plus organic solvent to well reduce the interference of biological substrate; uses HLB small column to enrich benzene polycarboxylic acid, which simplifies the process and protects the environment compared with the traditional method, and has a good recovery rate; finally uses GC-MS / MS system for detection and analysis, so that the method has high sensitivity and accuracy. Thus, a set of detection and analysis methods with simple operation, wide applicability, high sensitivity, and can be used as daily biological monitoring is established. The present invention successfully applies the benzene polycarboxylic acid molecular marker method to the detection of ultra-trace (note: 1 ng detection is achieved when the solid to be tested is >100 mg (constant), or 0.1-1 ng detection is achieved when the liquid to be tested is 1-10 mL (semi-trace).) nano black carbon particles in animal biological samples for the first time, and realizes accurate quantitative analysis of black carbon particles in biological samples.

[0166] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for detecting nano black carbon particles in animal biological samples, characterized in that The steps include: 1) Sample homogenization: Take an appropriate amount of fresh animal samples, freeze them at -80°C, put them into clean glass bottles for freeze drying, and then crush the samples with a clean glass rod and stir them to homogenize; 2) Sample digestion: Take an appropriate amount of biological sample dry powder in a 15mL centrifuge tube, add concentrated nitric acid until the sample is completely digested, and then neutralize it. At this time, the sample has been completely digested; 3) Quartz fiber filter membrane pretreatment: Place the high-purity quartz fiber filter membrane in a crucible and burn it at high temperature to remove organic residues; 4) Washing of diatomite: Put some diatomite in a round-bottom flask, add sulfuric acid of a certain concentration, mix well and incubate in a water bath; then collect the diatomite in the flask by filtering with a filter membrane, and wash with water, dilute nitric acid and water in turn; finally, transfer the diatomite on the filter membrane into a crucible, dry it in an oven, and then calcine it in a muffle furnace. At this time, the diatomite has been washed; 5) Sample filtration: Weigh diatomaceous earth, add alkali and vortex mix, drip the suspension drop by drop into a 15mL centrifuge tube, mix and settle the digested sample and incubate it, which is set as liquid A; prepare liquid B at the same time, by adding diatomaceous earth to the alkali solution and vortex mix; filter 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 drip liquid B evenly on the quartz fiber filter membrane drop by drop, and then drip liquid A evenly on the quartz fiber filter membrane drop by drop in the same way to achieve particle sample enrichment; 6) Sample purification: Wash the filter membrane and diatomaceous earth drop by drop with oxidant and organic solvent, and continue to filter 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 reactor liner, add concentrated nitric acid and tighten it, put it into the oven for reaction, turn off the power after the oven cools down, and cool it down; Take the supernatant and treat it for later use; 8) Enrichment of molecular markers: Solid phase extraction is used to enrich the benzene polycarboxylic acid molecular markers produced by oxidation, and HLB columns are selected for solid phase extraction operations; after four steps of activation, equilibration, loading, and elution, the benzene polycarboxylic acid molecular markers in the concentrated acid matrix have been dissolved in the organic solvent, and the enrichment of the entire molecular marker has been completed; 9) Molecular marker derivatization: The eluent was blown dry with nitrogen blower, then re-dissolved with methanol, dichloromethane and internal standard were added, vortexed to mix, and then the derivatization agent was added, vortexed to mix, and derivatized; After the derivatization, the product is quenched, blown to dryness with nitrogen, and then re-dissolved with an organic solvent, and then transferred to a sample injection bottle for instrument detection and analysis; 10) Instrumental analysis: The derivative product in step 9) is analyzed by GC-MS / MS. The instrument parameters should be optimized for the detection and analysis.

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

3. The method for detecting nano black carbon particles in animal biological samples according to claim 1, characterized in that In step 2), the specific method of acid digestion is to add 1 mL of 68% concentrated nitric acid and place it at room temperature for more than 24 hours until the solution becomes clear and transparent; The neutralization method is to add 2 mL of water and 7 mL of 30% potassium hydroxide, that is, to adjust the final solution alkali concentration to about 10%.

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

5. The method for detecting nano black carbon particles in animal biological samples according to 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; the diatomaceous earth particle size is 7 μm; The specific experimental conditions of the diatomite acid boiling are as follows: 10-20g of diatomite with a particle size of 7μm is taken into a round-bottom flask, and then a sulfuric acid solution with a concentration of 4-5mol / L is added, and the solid-liquid ratio should be between 1:3 and 1:

4. After mixing, the mixture is boiled in a water bath at 99°C for 3-4h; The specific steps of washing are: washing with 200-300 mL of deionized water for 2-3 times, washing with 700-800 mL of 2% nitric acid for 1 time, and washing with 200-300 mL of deionized water for 3-5 times, then transferring the diatomaceous earth on the filter membrane into a ceramic dry pot, drying it in an oven at 100-120° C. for 2-3 hours, and then calcining it in a muffle furnace at 550-600° C. for 3-4 hours; The specific method of mixing and sedimentation is to weigh 50-100 mg of diatomaceous earth, add 1.5-2 mL of 10% potassium hydroxide and vortex mix, then drip the suspension dropwise into a 15 mL centrifuge tube, mix and sediment the digested sample, and incubate at 37-38° C. for 3-5 hours.

6. The method for detecting nano black carbon particles in animal biological samples according to 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 standing at room temperature for 3-5 hours; The pore size of the high-purity quartz fiber filter is 0.3 μm and the diameter is 25 mm or 50 mm; In step 6), the oxidant and the organic solvent may be 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 according to claim 1, characterized in that In step 7), the concentrated nitric acid with a mass fraction of 68% for oxidation is electronic grade concentrated nitric acid; The specific operation of the oxidation process is to add 1-2.5 mL of concentrated nitric acid to the lining of the reactor and tighten it, put it in an oven and react at 170-180° C. for 8-9 hours, turn off the power when the oven temperature drops to 70° C. or below, open the oven door and cool it naturally to room temperature, at which time the oxidation is completed; The method for treating the supernatant of the oxidation product is to slowly open the reactor cooled to room temperature in a fume hood, take 1200-1600 μL of the supernatant, centrifuge at 8000-12000 rpm for 15-25 minmin, rinse the reactor liner with deionized water while centrifuging, take 800-1200 μL of the supernatant after centrifugation into the original reactor liner, and ventilate in a fume hood overnight.

8. The method for detecting nano black carbon particles in animal biological samples according to claim 1, characterized in that In step 8), the HLB column used is NanoChrom SelectCore HLB SPE Cartridge, HLB060-030250-1, 250 mg 3 mL (60 μm) solid phase extraction column; In step 8), the solid phase extraction process is: ① 4 to 5 beds of methanol activation; ② 5 to 6 beds of 2% nitric acid balance; ③ the oxidation product after overnight is diluted to about 11 mL with deionized water and mixed, and added to the solid phase extraction column for natural filtration; ④ under negative pressure conditions, the liquid on the extraction column is drained, and at the same time, a 15mL centrifuge tube is prepared, and 100 μL of concentrated hydrochloric acid with a mass fraction of 37% is added thereto as an eluent collection tube; ⑤ 4 beds of methanol solution are used for elution.

9. The method for detecting nano black carbon particles in animal biological samples according to claim 1, characterized in that In step 9), the specific experimental condition parameters of the derivatization are: blowing the eluent to dryness with nitrogen at 65-70° C. with a nitrogen blower, then re-dissolving with 400 μL of methanol, adding 1000 μL of dichloromethane, 100 μL of internal standard (concentration 200 ppm, 2'2-biphenyldicarboxylic acid), vortexing, then adding 400 μL of derivatizing agent (2 mol / L trimethylsilylated diazomethane), vortexing, sealing, and derivatizing at room temperature for more than 12 hours; The specific method of quenching is to add 40 μL of quenching agent (50% formic acid / methanol) after the derivatization, blow it to dryness with nitrogen at about 40° C., re-dissolve it with dichloromethane, vortex mix it, and then transfer it to the injection bottle for instrument detection and analysis.

10. The method for detecting nano black carbon particles in animal biological samples according to claim 1, characterized in that In step 10), the optimal parameter conditions for the instrument analysis are: the chromatographic column uses 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.

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