A pretreatment method and detection method for synchronously analyzing polycyclic aromatic hydrocarbons and hydroxyl polycyclic aromatic hydrocarbons in a biological source sample

Abstract

The application relates to the technical field of biological detection, and particularly discloses a pretreatment method and a detection method for synchronously analyzing polycyclic aromatic hydrocarbons (PAHs) and hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs) in a biological sample. The pretreatment method comprises the following steps: S100, treating the biological sample by using an alkaline hydrolysis method to obtain supernatant containing PPAHs and lower solution; treating the lower solution by using an acid hydrolysis method to obtain supernatant containing OH-PAHs; S200, treating the supernatant containing PPAHs to obtain a to-be-detected solution containing PPAHs; and S300, treating the supernatant containing OH-PAHs to obtain a to-be-detected solution containing OH-PAHs. The detection method comprises the pretreatment method. The application provides a method which is universal for multiple biological samples, can realize synchronous determination of PPAHs and OH-PAHs, and greatly reduces the sample amount and pretreatment cost.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to a pretreatment method and detection method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxy PAHs in biological samples. Background Technology

[0002] The harmful effects of environmental pollutants on human health have become a major global concern. Polycyclic aromatic hydrocarbons (PAHs) are a very important class of organic pollutants, composed of two or more fused benzene rings or cyclopentadiene rings. They are widely distributed in nature, mainly originating from the pyrolysis or incomplete combustion of various organic matter or mineral fuels, such as coal, coal tar, petroleum, wood, and cigarettes. There are hundreds of parent polycyclic aromatic hydrocarbons (PPAHs) and their derivatives in nature. The U.S. Environmental Protection Agency (EPA) has designated 16 of them as priority pollutants for control based on their toxicity, prevalence in the environment, and health risks from exposure. PPAHs can enter the human body through various routes, including respiration, diet, and skin absorption, and are metabolized into hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) by mixed-function oxidases in the endoplasmic reticulum. Because of individual differences in absorption and metabolism, measuring only the content of one type of substance is insufficient to accurately assess the true exposure level of PAHs. Therefore, simultaneously measuring the content of both types of substances in an organism is an important way to indicate the environmental exposure status of PAHs. PAHs have carcinogenic, teratogenic, and mutagenic effects, which can lead to a variety of adverse health outcomes. Therefore, accurate measurement of PAH exposure levels in the population is of great significance for evaluating the impact of PAH exposure on human health and for formulating pollutant control policies.

[0003] PPAHs are lipophilic, easily cross biological membranes, and can diffuse and widely distribute throughout the body after entering the human body. Previous studies have detected PPAHs and / or OH-PAHs in various human specimens, such as blood, urine, and hair. Blood is an important medium for substance transport and is one of the most commonly used specimens for exposure monitoring. Urine is an important excretion route, and the OH-PAHs in it can represent the body's PAH exposure level over a short period of time. Substances in hair can accumulate over a long period of time, and due to its advantages of non-invasive collection and easy preservation, it has also been used in recent years to determine the concentration levels of PPAHs and OH-PAHs. Therefore, the detection of contaminants in multiple specimens is an effective means of comprehensively assessing PAH exposure levels.

[0004] Biological samples differ from media such as plants and soil, often containing a variety of complex biomolecules, such as fats, proteins, peptides, and pigments. These not only bind to the target substance but also interfere with its accurate quantification, necessitating the dissociation of the target substance from the biomolecules. Furthermore, PPAHs and OH-PAHs differ in properties; the former is mainly found in fats, while the latter primarily binds to glucuronic acid or sulfuric acid. This leads to differences in their dissociation, extraction, purification, and assay methods, making simultaneous extraction and analysis difficult through a single pretreatment. Therefore, current detection methods mostly detect only PPAHs or OH-PAHs in a specific biological sample (such as hair, blood, or urine), failing to simultaneously detect both PPAHs and OH-PAHs, and requiring different pretreatment methods for different biological samples. Currently, inorganic bases (such as sodium hydroxide) are commonly used for hair sample pretreatment, while glucuronidase and sulfatase are used for blood or urine pretreatment. The key step in dissociation is the decoupling process, converting some glucuronidation / sulfation metabolites into hydroxyl compounds. However, glucuronidase and sulfatase have a negative impact on the hydrolysis and recovery of free phenolic metabolites in biological specimens, thus underestimating the level of phenolic metabolites. Therefore, enzymatic hydrolysis is not an ideal strategy for quantifying OH-PAHs.

[0005] Because the levels of PPAHs and OH-PAHs in non-occupational exposure populations are relatively low, the sample volume required for pretreatment of these two substances separately would be quite large. However, due to the preciousness and uniqueness of human specimens, the required sample volume is often insufficient. Furthermore, the pretreatment process for biological specimens constitutes a significant portion of the entire process for determining the concentration of the target substance, while environmental epidemiological studies often involve large sample sizes. This necessitates pretreatment methods that can rapidly handle large batches of samples.

[0006] In summary, there is a need to develop a method that is applicable to a variety of biological specimens, enables simultaneous determination of PPAHs and OH-PAHs, and significantly reduces sample size and pretreatment costs. Summary of the Invention

[0007] This application provides a pretreatment method and detection method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples. This application provides a method applicable to various biological samples, capable of simultaneous analysis and determination of PPAHs and OH-PAHs, and significantly reducing sample volume and pretreatment costs.

[0008] Firstly, this application provides a pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxy PAHs in biological samples, employing the following technical solution:

[0009] A pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples, the pretreatment method comprising the following steps:

[0010] S100. The biological sample is treated with alkaline hydrolysis to hydrolyze the proteins in the biological sample to obtain an alkaline hydrolysate; a demulsifier is first added to the hydrolysate to demulsify, then a first extractant is added and centrifuged to obtain a supernatant and a lower layer solution containing PPAHs.

[0011] The lower layer solution is treated with acid hydrolysis to obtain an acidic hydrolysate; a second extractant is added to the acidic hydrolysate and centrifuged to obtain a supernatant containing OH-PAHs;

[0012] The demulsifier is selected from C1-C3 organic alcohols;

[0013] The first extractant contains n-hexane and methyl tert-butyl ether in a volume ratio of (7-11):1;

[0014] The second extractant comprises cyclohexane and ethyl acetate in a volume ratio of (3-5):1;

[0015] S200: Process the supernatant containing PPAHs to obtain a test solution containing PPAHs:

[0016] S300: Process the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs.

[0017] In some alternative embodiments of this application, the alkaline solution used in the alkaline hydrolysis method is selected from aqueous solutions of alkali metal hydroxides.

[0018] In some alternative embodiments of this application, the aqueous solution of the alkali metal hydroxide is selected from at least one of an aqueous solution of sodium hydroxide and an aqueous solution of potassium hydroxide.

[0019] In some specific embodiments of this application, the aqueous solution of the alkali metal hydroxide is selected from an aqueous solution of potassium hydroxide.

[0020] In some optional embodiments of this application, the concentration of the aqueous solution of the alkali metal hydroxide is 1 to 5 mol / L; such as 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L or 5 mol / L, etc.

[0021] In some alternative embodiments of this application, the biological sample is selected from liquid or solid samples; the liquid sample is selected from at least one of serum, plasma, and urine; and the solid sample is selected from at least one of hair, nail, and tissue samples.

[0022] In some alternative embodiments of this application, in step S100, the recommended usage amount (including range and optimal value) of each biological source sample is shown in Table 1.

[0023] Table 1. Recommended usage amounts (including ranges and optimal values) for each biological source sample.

[0024]

[0025] If the biological source sample is selected from liquid samples, the concentration of the aqueous solution of the alkali metal hydroxide is selected from 3.5 to 4.5 mol / L (such as 3.6 mol / L, 3.7 mol / L, 3.8 mol / L, 3.9 mol / L, 4 mol / L, 4.1 mol / L, 4.2 mol / L, 4.3 mol / L or 4.4 mol / L, etc.), and the volume is selected from 150 to 250 μL (such as 160 μL, 170 μL, 180 μL, 190 μL, 200 μL, 210 μL, 220 μL, 230 μL or 240 μL, etc.).

[0026] If the biological source sample is selected from solid samples, the concentration of the aqueous solution of the alkali metal hydroxide is selected from 1.5 to 2.5 mol / L (e.g., 1.6 mol / L, 1.7 mol / L, 1.8 mol / L, 1.9 mol / L, 2 mol / L, 2.1 mol / L, 2.2 mol / L, 2.3 mol / L, or 2.4 mol / L, etc.), and the volume is selected from 550 to 650 μL (e.g., 560 μL, 570 μL, 580 μL, 590 μL, 600 μL, 610 μL, 620 μL, 630 μL, or 640 μL, etc.).

[0027] In some optional embodiments of this application, the hydrolysis temperature is 55-65°C (e.g., 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, or 64°C) and the time is 50-90 minutes (e.g., 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, or 85 minutes).

[0028] In some alternative embodiments of this application, the first extractant comprises n-hexane and methyl tert-butyl ether in a volume ratio of (8-10):1, such as 8:1, 8.5:1, 9:1, 9.5:1 or 10:1.

[0029] In some specific embodiments of this application, the first extractant comprises n-hexane and methyl tert-butyl ether in a volume ratio of 9:1.

[0030] In some alternative embodiments of this application, the amount of the first extractant is selected from 550 to 650 μL (e.g., 560 μL, 570 μL, 580 μL, 590 μL, 600 μL, 610 μL, 620 μL, 630 μL, or 640 μL, etc.).

[0031] In some alternative embodiments of this application, the C1-C3 organic alcohols are selected from at least one of methanol, ethanol, n-propanol, and isopropanol.

[0032] In some specific embodiments of this application, the C1-C3 organic alcohols are selected from methanol.

[0033] In some alternative embodiments of this application, the amount of the demulsifier is selected from 80 to 120 microliters (e.g., 85 microliters, 90 microliters, 95 microliters, 100 microliters, 105 microliters, 110 microliters, or 115 microliters, etc.).

[0034] In some optional embodiments of this application, the centrifugation is repeated 2 to 4 times (e.g., 3 times), and the amount of the first extractant used in each centrifugation is selected from 550 to 650 μL (e.g., 560 μL, 570 μL, 580 μL, 590 μL, 600 μL, 610 μL, 620 μL, 630 μL, or 640 μL).

[0035] In some optional embodiments of this application, the acidic solution used in the acid hydrolysis method comprises concentrated hydrochloric acid in a volume ratio of (1 to 1.5):1 and sodium acetate buffer (e.g., pH = 5.2) with a pH of 5.0 to 5.5. The concentrated hydrochloric acid has a weight percentage concentration selected from 36% to 38%.

[0036] In some alternative embodiments of this application, the second extractant ring comprises cyclohexane and ethyl acetate in a volume ratio of (3.5 to 4.5):1, such as 3.5:1, 3.6:1, 3.7:1, 3.8:1, 3.9:1, 4.0:1, 4.1:1, 4.2:1, 4.3:1, 4.4:1, 4.5:1, etc.

[0037] In some specific embodiments of this application, the second extractant ring comprises cyclohexane and ethyl acetate in a volume ratio of 4:1.

[0038] In some optional embodiments of this application, step S200, which processes the supernatant containing PPAHs to obtain a test solution containing PPAHs, includes the following steps:

[0039] S201. Concentrate the supernatant containing PPAHs to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a first redissolved solution;

[0040] S202, The first reconstituted solution is passed through a first solid-phase extraction column and eluted to obtain a first eluent;

[0041] S203. Concentrate the first eluent to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a second redissolved solution;

[0042] S204. The second reconstituted solution is passed through a second solid-phase extraction column and eluted to obtain a second eluent.

[0043] S205. Add a mixed solution of deuterated polycyclic aromatic hydrocarbon internal standards to the second eluent to form a first mixed solution;

[0044] S206. Concentrate the first mixed solution to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain the test solution containing PPAHs.

[0045] In some alternative embodiments of this application, in step S201, the amount of n-hexane used is 400 to 600 microliters (e.g., 500 microliters).

[0046] In some optional embodiments of this application, in step S202, the first solid phase extraction column is selected as a solid phase extraction column specifically for polycyclic aromatic hydrocarbons.

[0047] The specific operation process of step S202 includes: activating and equilibrating the first solid phase extraction column with 3 ml of dichloromethane and 5 ml of n-hexane in sequence;

[0048] The first reconstituted solution is transferred to the first solid-phase extraction column;

[0049] Rinse with 6 ml of n-hexane and discard the eluent;

[0050] The first eluent is obtained by eluting with an eluent containing n-hexane and dichloromethane in a volume ratio of (0.8 to 1.2):1 (e.g., 1:1).

[0051] In step S203, the amount of n-hexane used is 200-300 μL (e.g., 250 μL).

[0052] In some alternative embodiments of this application, in step S204, the second solid-phase extraction column is selected from a neutral alumina solid-phase extraction column. The packing material in the neutral alumina solid-phase extraction column comprises 98% by mass of neutral alumina and 2% by mass of ultrapure water.

[0053] The specific operation process of step S204 includes: inserting a piece of cotton into the bottom of the pipette tip and tamping it down to block the outlet at the bottom of the pipette tip.

[0054] Add 2-5 ml (e.g., 3 ml) of acetone to the pipette tip to clean the inner wall of the pipette tip, and discard the cleaning waste liquid.

[0055] Fill the nozzle with 2% neutral alumina to a height of 2.8–3.2 cm (e.g., 3 cm) and anhydrous sodium sulfate to a height of 0.3–0.7 cm (e.g., 0.5 cm) sequentially from bottom to top;

[0056] Add 3 ml of dichloromethane to the nozzle to clean impurities on the packing material, and discard the cleaning waste liquid;

[0057] When the level of dichloromethane drops to the top of the anhydrous sodium sulfate, add 3 ml of n-hexane;

[0058] When the level of n-hexane drops to the top of anhydrous sodium sulfate, add the second redissolution solution and rinse with 500 ml of n-hexane.

[0059] When the liquid level of n-hexane drops to the upper surface of anhydrous sodium sulfate, add 8 mL of a mixed solution of n-hexane and dichloromethane (volume ratio of n-hexane to dichloromethane is 3:2) to elute and obtain the second eluent.

[0060] Unless otherwise stated, the term "2% neutral alumina" in this application consists of 2% by mass of pure water and 98% by mass of neutral alumina.

[0061] In some alternative embodiments of this application, in step S205, the solute in the deuterated polycyclic aromatic hydrocarbon internal standard mixed solution is selected from at least one of acenaphthene-D10, naphthalene-D8, chrysene-D12, perylene-D12, 1,4-dichlorobenzene-D4, and phenanthrene-D10.

[0062] In some alternative embodiments of this application, in step S205, the solvent in the deuterated polycyclic aromatic hydrocarbon internal standard mixed solution is selected from n-hexane.

[0063] In some optional embodiments of this application, in step S205, the concentration of the deuterated polycyclic aromatic hydrocarbon internal standard mixed solution is 0.35 to 0.45 nanograms per microliter (e.g., 0.4 nanograms per microliter), and the amount used is 45 to 55 microliters (e.g., 50 microliters).

[0064] In some alternative embodiments of this application, in step S206, the amount of n-hexane used is 50 to 150 microliters (e.g., 100 microliters).

[0065] In some optional embodiments of this application, step S300, processing the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs, includes the following steps:

[0066] S301. Concentrate the supernatant containing OH-PAHs to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a third redissolved solution;

[0067] S302, The third reconstituted solution is passed through a third solid-phase extraction column and eluted to obtain a third eluent;

[0068] S303. Add a mixed solution of deuterated hydroxyl polycyclic aromatic hydrocarbon internal standards to the third eluent to form a second mixed solution;

[0069] S304. Concentrate the second mixed solution to near dryness by nitrogen blowing, and redissolve it with acetonitrile to obtain a fourth redissolved solution;

[0070] S305. Add a silanizing agent to the fourth reconstituted solution and react for 20 to 40 minutes under an inert atmosphere and at a temperature of 55 to 65°C to obtain the test solution containing OH-PAHs.

[0071] In some alternative embodiments of this application, in step S305, the solute in the deuterated hydroxyl polycyclic aromatic hydrocarbon internal standard mixed solution is selected from at least one of 1-hydroxy-naphthalene-D7 and 1-hydroxypyrene-D9.

[0072] In some alternative embodiments of this application, in step S305, the solvent in the deuterated hydroxyl polycyclic aromatic hydrocarbon internal standard mixed solution is selected from acetonitrile.

[0073] Secondly, this application provides a method for the simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxy PAHs in biological samples, employing the following technical solution:

[0074] A method for simultaneously analyzing and detecting polycyclic aromatic hydrocarbons (PAHs) and hydroxy PAHs in biological samples, the method comprising the pretreatment method described in the first aspect, detecting the concentration of PAHs in a test solution, and detecting the concentration of hydroxy PAHs in a test solution containing OH-PAHs.

[0075] In some specific embodiments of this application, the concentration of polycyclic aromatic hydrocarbons (PAHs) in the test solution is detected by gas chromatography-mass spectrometry (such as triple quadrupole gas chromatography-mass spectrometry).

[0076] In some specific embodiments of this application, the concentration of hydroxyl polycyclic aromatic hydrocarbons in the test solution containing OH-PAHs is detected by gas chromatography-mass spectrometry (such as triple quadrupole gas chromatography-mass spectrometry).

[0077] In summary, this application has the following beneficial effects:

[0078] First, this application provides a method applicable to a variety of biological specimens, capable of simultaneously analyzing and determining PPAHs and OH-PAHs, and greatly reducing sample volume and pretreatment costs.

[0079] Secondly, the pretreatment method of this application can not only ensure the stability and accuracy of the detection method of this application, but also be applicable to different types of biological samples. Detailed Implementation

[0080] The following embodiments provide a further detailed description of this application.

[0081] Example 1 and Comparative Examples 1-2

[0082] Example 1 and Comparative Examples 1-2 analyzed the same biological source sample, which was human hair. The amount of hair used in Example 1 and Comparative Examples 1-2 was 60 mg dry weight.

[0083] Example 1

[0084] A pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples specifically includes the following steps:

[0085] S100, hair 60 mg The dry weight was added to a brown silanized screw-top vial (labeled vial #1).

[0086] Add to each vial 600 μL A 4 mol / L aqueous solution of potassium hydroxide was prepared. Bottle #1 was placed in a dry, constant-temperature metal bath and heated to 60°C for 1 hour. 100 μL of methanol was added to bottle #1. 600 μL of a hexane / methyl tert-butyl ether mixed solution (hexane to methyl tert-butyl ether volume ratio 9:1) was added to bottle #1. The mixture was vortexed for 5 minutes and centrifuged at 3000 rpm to obtain a supernatant and a lower layer containing PPAHs. The supernatant containing PPAHs was transferred to a new ordinary brown clamp-top bottle (labeled bottle #2). The lower layer in bottle #1 was repeated twice, and the supernatants containing PPAHs obtained from the three repetitions were combined into bottle #2.

[0087] Add to the lower layer of solution in bottle #1 first 150 μL Concentrated hydrochloric acid was added, followed by 100 μL of sodium acetate buffer solution (pH 5.2) to adjust the pH of the lower layer solution to acidic. Then, 0.6 mL of a cyclohexane / ethyl acetate mixture (cyclohexane to ethyl acetate volume ratio 4:1) was added, the mixture was vortexed for 5 minutes, and centrifuged at 3000 rpm for 3 minutes to obtain a supernatant containing OH-PAHs. This supernatant containing OH-PAHs was transferred to a new brown silanized screw-top vial (labeled vial #3) using a Pasteur pipette. The lower layer solution in vial #1 was repeated twice, and the supernatants containing OH-PAHs obtained from all three repetitions were combined into vial #3.

[0088] S200. Process the supernatant containing PPAHs to obtain a test solution containing PPAHs, specifically including the following steps:

[0089] S201. The supernatant containing PPAHs in bottle #2 is concentrated to near dryness by nitrogen blowing, and then redissolved with 50 μL of n-hexane to obtain the first redissolved solution;

[0090] S202. Take a 500mg specification polycyclic aromatic hydrocarbon solid phase extraction column (hereinafter referred to as "MIP column"), and activate and equilibrate the MIP column with 3 ml of dichloromethane and 5 ml of n-hexane in sequence, and discard the waste liquid.

[0091] Using a Pasteur pipette, transfer the first reconstituted solution from bottle #2 to the MIP column, and wash bottle #2 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0092] Rinse with 6 ml of n-hexane and discard the eluent;

[0093] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the eluent in a 15 ml long glass tube (tube #1).

[0094] S203. The first eluent in tube #1 is concentrated to near dryness by nitrogen blowing, and then redissolved with 250 μL of n-hexane to obtain the second redissolved solution;

[0095] S204. Insert a piece of cotton into the bottom of the pipette tip and tamp it down with a Pasteur pipette or glass rod to block the outlet at the bottom of the pipette tip.

[0096] Add 3 ml of acetone to the pipette tip to clean the inner wall of the pipette tip, and discard the cleaning waste liquid.

[0097] Fill the nozzle with approximately 3 cm of 2% neutral alumina and approximately 0.5 cm of anhydrous sodium sulfate sequentially from bottom to top, and tap until stable;

[0098] Add 3 ml of dichloromethane to the nozzle to clean impurities on the packing material, and discard the cleaning waste liquid;

[0099] When the level of dichloromethane drops to the top of the anhydrous sodium sulfate, add 3 ml of n-hexane;

[0100] When the level of n-hexane drops to the top of anhydrous sodium sulfate, quickly add the second redissolution solution and rinse with 500 ml of n-hexane.

[0101] When the liquid level of n-hexane drops to the upper surface of anhydrous sodium sulfate, add 8 mL of a mixed solution of n-hexane and dichloromethane (volume ratio of n-hexane to dichloromethane is 3:2) to elute and obtain a second eluent. Collect the second eluent in a long glass tube (tube #2).

[0102] S205. Add 50 ml of a 0.4 ng / µL internal standard mixed solution of deuterated polycyclic aromatic hydrocarbons to the second eluent in tube #2 to form the first mixed solution;

[0103] S206. Concentrate the first mixed solution to near dryness by nitrogen blowing, redissolve it with n-hexane, and transfer it to a sample vial with a liner using a Pasteur pipette. Then wash tube #2 with 50 μL of n-hexane and transfer the mixture to the liner; to obtain the test solution containing PPAHs.

[0104] The concentration of pPAHs in the test solution containing PPAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0105] S300: Process the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs:

[0106] S301. The supernatant containing OH-PAHs in bottle #3 is concentrated to near dryness by nitrogen blowing, and then redissolved with 500 μL of n-hexane to obtain the third redissolved solution;

[0107] S302. Take a 500mg MIP column and activate and equilibrate it with 3 mL of dichloromethane and 5 mL of n-hexane in sequence, then discard the waste liquid.

[0108] Using a Pasteur pipette, transfer the third reconstituted solution from flask #3 to the MIP column, and wash flask #3 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0109] Rinse with 6 ml of n-hexane and discard the eluent;

[0110] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the third eluent in a 15 ml long glass tube (tube #3);

[0111] S303. Add 50 μL of a 0.4 ng / μL internal standard mixed solution of deuterated hydroxyl polycyclic aromatic hydrocarbons to the third eluent in tube #3 to form a second mixed solution;

[0112] S304. Concentrate the second mixed solution in tube #3 with nitrogen to approximately 200 μL. Transfer the remaining second mixed solution in tube #3 to a new brown screw-top silanized vial (labeled as vial #4) using a long Pasteur pipette. Rinse the long glass tube twice with 100 μL of n-hexane and combine the solutions in vial #4. Blow the liquid in vial #4 with nitrogen until nearly dry, and add 50 μL of acetonitrile to redissolve it, obtaining the fourth redissolved solution.

[0113] S305. Add 100 μL of silanizing reagent (BSTFA:TMCS = 99:1, ≥99%) to the fourth reconstitution solution in bottle #4, purge the air in the bottle with nitrogen, and tighten the cap; place it in a dry constant temperature metal bath and heat at 60°C for 30 minutes to obtain the test solution containing OH-PAHs; after cooling bottle #4 to room temperature, open the cap and transfer all the test solution containing OH-PAHs to a sample vial with a liner, and seal the vial.

[0114] The concentration of OH-PAHs in the test solution containing OH-PAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0115] In Example 1, the experiment was repeated three times, referred to as the first experiment, the second experiment, and the third experiment, and the test results are shown in Table 2.

[0116] Table 2: Detection results of PPAHs and OH-PAHs concentrations in three repeated experiments of Example 1

[0117]

[0118] Comparative Example 1

[0119] The concentration of pPAHs in biological samples was detected using conventional detection methods.

[0120] The specific testing method is as follows:

[0121] Take 60 mg of washed and ground hair sample and add 1.2 ml of acetonitrile. Incubate overnight at 40°C and 100 rpm on a shaker. The next day, sonicate for 30 minutes. Centrifuge at 3000 rpm for 3 minutes and transfer the supernatant to a 1.5 ml centrifuge tube. Add 0.3 ml of acetonitrile to the original tube, repeat the centrifugation, and collect the supernatant, combining it with the above 1.5 ml centrifuge tube. Centrifuge the tube at 20000 g for 5 minutes and collect the supernatant to a silanized transparent sample vial. Blow the liquid nitrogen in the silanized transparent sample vial to near dryness and reconstitute with 0.6 ml of dilute hydrochloric acid (pH=2). Add 0.6 ml of a cyclohexane / ethyl acetate mixture (cyclohexane to ethyl acetate volume ratio of 4:1), vortex for 10 minutes, centrifuge at 3000 rpm for 3 minutes, and let stand for at least 2 minutes. Collect the supernatant to another brown silanized vial. Repeat the extraction three times and combine the supernatants. Add 50 mL of a 0.4 ng / µL internal standard solution of deuterated polycyclic aromatic hydrocarbons (PAHs), pPAHs were purged to near dryness with nitrogen, and then redissolved in 150 µL of n-hexane. The liquid was transferred to a sample vial, and the pPAH concentration was determined using a triple quadrupole gas chromatography-mass spectrometry (GC-MS).

[0122] The detection results of PPAHs concentration in Comparative Example 1 are shown in Table 3.

[0123] Table 3: Detection results of pPAHs concentration in Comparative Example 1 (unit: ng / g hair)

[0124] Chinese name English name English abbreviation Comparative Example 1 Acenaphthylene acenaphthylene ANY 1.2059 Acenaphthene acenaphthene ACE 4.7554 Fluorene fluorene FLU 21.971 Phenanthrene phenanthrene PHE 87.2253 Anthracene anthracene ANT 7.7465 Fluoranthene fluoranthene FLO 24.101 Pyrene pyrene PYR 16.9476 Benzo[a]anthracene benz(a)anthracene BAA 7.7986 Benzo[k]fluoranthene benzo(k)fluoranthene BKF 6.233 Benzo[a]pyrene benzo(a)pyrene BAP 3.3201 Dibenz[a,h]anthracene dibenz(a,h)anthracene DahA 0.1237 Benzo[g,h,i]perylene benzo(g,h,i)perylene BghiP 0.4438

[0125] Comparative Example 2

[0126] The concentration of OH-PAHs in biological samples was detected using conventional detection methods.

[0127] The specific testing method is as follows:

[0128] Take 60 mg of washed and ground hair sample, add 1 mL of 1 mol / L NaOH aqueous solution, incubate at 60 °C for 30 minutes, and centrifuge at 5000 rpm for 10 minutes. Add 1 mL of acetate buffer and 800 μL of 2 mol / L hydrochloric acid to the supernatant to adjust the pH to 5. Add 2 mL of dichloromethane, vortex for 15 minutes, and centrifuge at 5000 rpm for 3 minutes. Aspirate the organic layer, and repeat the extraction once more: add 2 mL of dichloromethane, vortex for 15 minutes, and centrifuge at 5000 rpm for 3 minutes, aspirating the organic layer. Combine the two organic layers, add 50 μL of a 0.4 ng / μL solution of deuterated hydroxyl polycyclic aromatic hydrocarbon internal standard, and purge with nitrogen to near dryness. Redissolved in 50 μL acetonitrile solution, then 100 μL silanizing reagent (N,O-bis(trimethylsilyl)trifluoroacetamide (CAS No. 25561-30-2, abbreviated BSTFA):trimethylchlorosilane (CAS No. 75-77-4, abbreviated TMCS) = 99:1, ≥99%) was added. The air in the vial was purged with nitrogen, the cap was tightened, and the vial was placed in a dry constant-temperature metal bath and heated at 60°C for 30 minutes. After cooling to room temperature, the vial was opened, and the entire contents were transferred to a sample vial with a liner. The concentration of OH-PAHs was detected using a triple quadrupole gas chromatography-mass spectrometry (GC-MS).

[0129] The detection results of OH-PAHs concentration in Comparative Example 2 are shown in Table 4.

[0130] Table 4: Detection results of OH-PAHs concentration in Comparative Example 2 (unit: ng / g hair)

[0131] Chinese name English name English abbreviation Comparative Example 2 1-Naphthol 1-hydroxynaphthalene 1-OH-NAP 20.31 2-Naphthol 2-hydroxynaphthalene 2-OH-NAP 64.7884 2-Hydroxyfluorene 2-hydroxyfluorene 2-OH-FLUO 3.2594 1-Hydroxyphenanthrene 1-hydroxyphenanthrene 1-OH-PHE 4.8357 2-Hydroxyphenanthrene 2-hydroxyphenanthrene 2-OH-PHE 7.2334 3-Hydroxyphenanthrene 3-hydroxyphenanthrene 3-OH-PHE 4.6529 4-Hydroxyphenanthrene 4-hydroxyphenanthrene 4-OH-PHE 1.2897 9-Hydroxyphenanthrene 9-hydroxyphenanthrene 9-OH-PHE 0.6311 1-Hydroxypyrene 1-hydroxypyrene 1-OH-PYR 0.4892

[0132] As can be seen from Tables 2 to 4, this application preprocesses the same biological sample to obtain detection solutions containing pPAHs and OH-PAHs respectively, which can realize the simultaneous determination of PPAHs and OH-PAHs and greatly reduce the sample volume and preprocessing cost.

[0133] The three repeated experiments in Example 1 demonstrate that the pretreatment method of this application can guarantee the stability of the detection method of this application.

[0134] Compared to detecting only the content of pPAHs in biological samples (Comparative Example 1) and only the content of OH-PAHs in biological samples (Comparative Example 2), Example 1 not only enables the simultaneous detection of pPAHs and OH-PAHs in the same biological sample, but also improves the detection values ​​of both pPAHs and OH-PAHs.

[0135] Therefore, the pretreatment method of this application can not only ensure the stability of the detection method of this application, but also improve the detection accuracy of pPAHs content and OH-PAHs content.

[0136] ​

[0137] Example 2 and Comparative Examples 3-4 analyzed the same biological source sample, which was human serum. The amount of serum used in Example 2 and Comparative Examples 3-4 was 400 μL.

[0138] ​

[0139] A pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples specifically includes the following steps:

[0140] S100. Add 400 μL of serum to a brown silanized screw-top vial (labeled as vial #1).

[0141] Add 200 μL of a 4 mol / L potassium hydroxide aqueous solution to each vial; place vial #1 in a dry constant temperature metal bath and incubate at 60°C for 1 hour; add 100 μL of methanol to vial #1; add 600 μL of a hexane / methyl tert-butyl ether mixed solution (hexane to methyl tert-butyl ether volume ratio of 9:1) to vial #1, vortex for 5 minutes, and centrifuge at 3000 rpm to obtain a supernatant and a lower layer containing PPAHs. Transfer the supernatant containing PPAHs to a new ordinary brown clamp bottle (labeled vial #2). Repeat this step twice more for the lower layer in vial #1, and combine the supernatants containing PPAHs obtained from the three steps into vial #2.

[0142] Add to the lower layer of solution in bottle #1 first 100 Add 100 μL of concentrated hydrochloric acid and 100 μL of sodium acetate buffer solution (pH 5.2) to adjust the pH of the lower layer solution to acidic. Then add 0.6 mL of a cyclohexane / ethyl acetate mixture (cyclohexane to ethyl acetate volume ratio 4:1), vortex for 5 minutes, and centrifuge at 3000 rpm for 3 minutes to obtain a supernatant containing OH-PAHs. Transfer the supernatant containing OH-PAHs to a new brown silanized screw-top vial (labeled vial #3) using a Pasteur pipette. Repeat this step twice more with the lower layer solution in vial #1, and combine the supernatants containing OH-PAHs obtained from all three steps into vial #3.

[0143] S200. Process the supernatant containing PPAHs to obtain a test solution containing PPAHs, specifically including the following steps:

[0144] S201. The supernatant containing PPAHs in bottle #2 is concentrated to near dryness by nitrogen blowing, and then redissolved with 50 μL of n-hexane to obtain the first redissolved solution;

[0145] S202. Take a 500mg specification polycyclic aromatic hydrocarbon solid phase extraction column (hereinafter referred to as "MIP column"), and activate and equilibrate the MIP column with 3 ml of dichloromethane and 5 ml of n-hexane in sequence, and discard the waste liquid.

[0146] Using a Pasteur pipette, transfer the first reconstituted solution from bottle #2 to the MIP column, and wash bottle #2 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0147] Rinse with 6 ml of n-hexane and discard the eluent;

[0148] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the eluent in a 15 ml long glass tube (tube #1).

[0149] S203. The first eluent in tube #1 is concentrated to near dryness by nitrogen blowing, and then redissolved with 250 μL of n-hexane to obtain the second redissolved solution;

[0150] S204. Insert a piece of cotton into the bottom of the pipette tip and tamp it down with a Pasteur pipette or glass rod to block the outlet at the bottom of the pipette tip.

[0151] Add 3 ml of acetone to the pipette tip to clean the inner wall of the pipette tip, and discard the cleaning waste liquid.

[0152] Fill the nozzle with approximately 3 cm of 2% neutral alumina and approximately 0.5 cm of anhydrous sodium sulfate sequentially from bottom to top, and tap until stable;

[0153] Add 3 ml of dichloromethane to the nozzle to clean impurities on the packing material, and discard the cleaning waste liquid;

[0154] When the level of dichloromethane drops to the top of the anhydrous sodium sulfate, add 3 ml of n-hexane;

[0155] When the level of n-hexane drops to the top of anhydrous sodium sulfate, quickly add the second redissolution solution and rinse with 500 ml of n-hexane.

[0156] When the liquid level of n-hexane drops to the upper surface of anhydrous sodium sulfate, add 8 mL of a mixed solution of n-hexane and dichloromethane (volume ratio of n-hexane to dichloromethane is 3:2) to elute and obtain a second eluent. Collect the second eluent in a long glass tube (tube #2).

[0157] S205. Add 50 ml of a 0.4 ng / µL internal standard mixed solution of deuterated polycyclic aromatic hydrocarbons to the second eluent in tube #2 to form the first mixed solution;

[0158] S206. Concentrate the first mixed solution to near dryness by nitrogen blowing, redissolve it with n-hexane, and transfer it to a sample vial with a liner using a Pasteur pipette. Then wash tube #2 with 50 μL of n-hexane and transfer the mixture to the liner; to obtain the test solution containing PPAHs.

[0159] The concentration of pPAHs in the test solution containing PPAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0160] S300: Process the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs:

[0161] S301. The supernatant containing OH-PAHs in bottle #3 is concentrated to near dryness by nitrogen blowing, and then redissolved with 500 μL of n-hexane to obtain the third redissolved solution;

[0162] S302. Take a 500mg MIP column and activate and equilibrate it with 3 mL of dichloromethane and 5 mL of n-hexane in sequence, then discard the waste liquid.

[0163] Using a Pasteur pipette, transfer the third reconstituted solution from flask #3 to the MIP column, and wash flask #3 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0164] Rinse with 6 ml of n-hexane and discard the eluent;

[0165] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the third eluent in a 15 ml long glass tube (tube #3);

[0166] S303. Add 50 μL of a 0.4 ng / μL internal standard mixed solution of deuterated hydroxyl polycyclic aromatic hydrocarbons to the third eluent in tube #3 to form a second mixed solution;

[0167] S304. Concentrate the second mixed solution in tube #3 with nitrogen to approximately 200 μL. Transfer the remaining second mixed solution in tube #3 to a new brown screw-top silanized vial (labeled as vial #4) using a long Pasteur pipette. Rinse the long glass tube twice with 100 μL of n-hexane and combine the solutions in vial #4. Blow the liquid in vial #4 with nitrogen until nearly dry, and add 50 μL of acetonitrile to redissolve it, obtaining the fourth redissolved solution.

[0168] S305. Add 100 μL of silanizing reagent (BSTFA:TMCS = 99:1, ≥99%) to the fourth reconstitution solution in bottle #4, purge the air in the bottle with nitrogen, and tighten the cap; place it in a dry constant temperature metal bath and heat at 60°C for 30 minutes to obtain the test solution containing OH-PAHs; after cooling bottle #4 to room temperature, open the cap and transfer all the test solution containing OH-PAHs to a sample vial with a liner, and seal the vial.

[0169] The concentration of OH-PAHs in the test solution containing OH-PAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0170] In Example 2, the experiment was repeated three times, referred to as the first experiment, the second experiment, and the third experiment, and the test results are shown in Table 5.

[0171] Table 5: Detection results of PPAHs and OH-PAHs concentrations in three repeated experiments in Example 2

[0172]

[0173]

[0174] ​

[0175] The concentration of polycyclic aromatic hydrocarbons in biological samples was detected using conventional detection methods.

[0176] The specific testing method is as follows:

[0177] Step 1: Take 400 μL of serum solution, add 20 mL of acetonitrile, and extract using a microwave extraction system for 20 minutes. Specific microwave conditions are: increase the temperature from room temperature to 110°C within 10 minutes and maintain that temperature for 10 minutes.

[0178] Step 2: Filter the above solution using a 37mm (diameter) glass fiber filter to remove lipids. Transfer the filtrate to a 250ml separatory funnel and extract twice with 30ml of n-hexane solution containing 4% sodium sulfate.

[0179] Step 3: Concentrate the above extract (approximately 60 ml of n-hexane) to approximately 1 ml in a vacuum centrifuge and transfer it to a 30 cm × 10 mm purification column. The purification column, from bottom to top, contains a 12 cm high layer of 3% deactivated alumina (3% by mass ultrapure water + 97% by mass neutral alumina powder), a 12 cm high layer of 2% deactivated silica gel (2% by mass ultrapure water + 98% by mass silica gel), and a 1 cm high layer of anhydrous sodium sulfate. The purification column is pre-equilibrated with 10 ml of n-hexane. Elute the purification column with 50 ml of a hexane / dichloromethane mixture (1:1 volume ratio of n-hexane to dichloromethane), concentrate the eluent to approximately 1 ml, and add 50 ml of a 0.4 ng / µL deuterated polycyclic aromatic hydrocarbon internal standard solution.

[0180] Step 4: Concentrate the eluent to 150 μL, transfer it to the sample, and determine the PPAHs concentration using a triple quadrupole gas chromatography-mass spectrometry system.

[0181] The detection results of pPAHs concentration in Comparative Example 3 are shown in Table 6.

[0182] Table 6: Detection results of pPAHs concentration in Comparative Example 3 (unit: ng / g serum)

[0183] ​ ​ ​ ​ ​ ​ ​ 0.0692 ​ ​ ​ 0.8065 ​ ​ ​ 2.047 ​ ​ ​ 1.6044 ​ ​ ​ 0.1042 ​ ​ ​ 0.2721 ​ ​ ​ 0.1891 ​ ​ ​ 0.0839 ​ ​ ​ 0.3821 ​ ​ ​ 0.0586 ​ ​ ​ 0.0873 ​ ​ ​ 0.0984

[0184] ​

[0185] The concentration of hydroxyl polycyclic aromatic hydrocarbons in biological samples was determined using conventional detection methods.

[0186] The specific testing method is as follows:

[0187] Take 400 μL of serum, 3 mL of 5 mol / L acetate-sodium acetate buffer solution (pH=5), and 10 μL of β-glucuronidase / sulfatase and add them to a glass centrifuge tube. Mix thoroughly using a vortex mixer. After capping the centrifuge tube, place it in a constant temperature water bath at 37°C for 12 hours. After 12 hours, remove the tube and prepare it for SPE column processing. Activate and equilibrate the C18 column with 5 mL of methanol and 10 mL of ultrapure water. After the sample passes through the C18 column, rinse the column with 5 mL of ultrapure water. After the eluent passes through the extraction column, maintain a vacuum for 1 minute to thoroughly remove water from the solid-phase extraction column (referred to as the "SPE column"). Then elute the column with 10 mL of methanol, collect the eluent in a test tube, blow it with nitrogen to a volume below 1 mL, and make up to 1 mL with methanol. Transfer the eluent to the sample and determine the concentration of OH-PAHs using a triple quadrupole gas chromatography-mass spectrometry (GC-MS).

[0188] The detection results of OH-PAHs concentration in Comparative Example 4 are shown in Table 7.

[0189] Table 7: Detection results of OH-PAHs concentration in Comparative Example 4 (unit: ng / g serum)

[0190] ​ ​ ​ ​ ​ ​ ​ 0.0277 ​ ​ ​ 0.106 ​ 2-hydroxyfluorene 2-OH-FLUO 0.0023 1-Hydroxyphenanthrene 1-hydroxyphenanthrene 1-OH-PHE 0 2-Hydroxyphenanthrene 2-hydroxyphenanthrene 2-OH-PHE 0 3-Hydroxyphenanthrene 3-hydroxyphenanthrene 3-OH-PHE 0 4-Hydroxyphenanthrene 4-hydroxyphenanthrene 4-OH-PHE 0 9-Hydroxyphenanthrene 9-hydroxyphenanthrene 9-OH-PHE 0 1-Hydroxypyrene 1-hydroxypyrene 1-OH-PYR 0

[0191] As can be seen from Tables 5 to 7, the present application preprocesses the same human serum biological sample to obtain detection solutions containing pPAHs and OH-PAHs respectively, which can realize the simultaneous determination of PPAHs and OH-PAHs and greatly reduce the sample volume and preprocessing cost.

[0192] The three repeated experiments in Example 2 demonstrate that the pretreatment method of this application can guarantee the stability of the detection method of this application.

[0193] Compared to detecting only the content of pPAHs in biological samples (Comparative Example 3) and only the content of OH-PAHs in biological samples (Comparative Example 4), Example 2 not only enables the simultaneous detection of pPAHs and OH-PAHs in the same biological sample, but also improves the detection values ​​of most pPAHs and OH-PAHs.

[0194] Therefore, the pretreatment method of this application can not only ensure the stability of the detection method of this application, but also improve the detection accuracy of pPAHs content and OH-PAHs content.

[0195] Example 3 and Comparative Examples 5-6

[0196] Example 3 and Comparative Examples 5-6 analyzed the same biological source sample, which was mouse urine. In Example 2 and Comparative Examples 3-4, the amount of urine used was 400 microliters.

[0197] Example 3

[0198] A pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxy PAHs in biological samples includes the following steps (same as Example 2, except that serum is replaced with urine):

[0199] S100. Add 400 μL of urine to a brown silanized screw-top vial (labeled as vial #1).

[0200] Add 200 μL of a 4 mol / L potassium hydroxide aqueous solution to each vial; place vial #1 in a dry constant temperature metal bath and incubate at 60°C for 1 hour; add 100 μL of methanol to vial #1; add 600 μL of a hexane / methyl tert-butyl ether mixed solution (hexane to methyl tert-butyl ether volume ratio of 9:1) to vial #1, vortex for 5 minutes, and centrifuge at 3000 rpm to obtain a supernatant and a lower layer containing PPAHs. Transfer the supernatant containing PPAHs to a new ordinary brown clamp bottle (labeled vial #2). Repeat this step twice more for the lower layer in vial #1, and combine the supernatants containing PPAHs obtained from the three steps into vial #2.

[0201] Add to the lower layer of solution in bottle #1 first 100 Add 100 μL of concentrated hydrochloric acid and 100 μL of sodium acetate buffer solution (pH 5.2) to adjust the pH of the lower layer solution to acidic. Then add 0.6 mL of a cyclohexane / ethyl acetate mixture (cyclohexane to ethyl acetate volume ratio 4:1), vortex for 5 minutes, and centrifuge at 3000 rpm for 3 minutes to obtain a supernatant containing OH-PAHs. Transfer the supernatant containing OH-PAHs to a new brown silanized screw-top vial (labeled vial #3) using a Pasteur pipette. Repeat this step twice more with the lower layer solution in vial #1, and combine the supernatants containing OH-PAHs obtained from all three steps into vial #3.

[0202] S200. Process the supernatant containing PPAHs to obtain a test solution containing PPAHs, specifically including the following steps:

[0203] S201. The supernatant containing PPAHs in bottle #2 is concentrated to near dryness by nitrogen blowing, and then redissolved with 50 μL of n-hexane to obtain the first redissolved solution;

[0204] S202. Take a 500mg specification polycyclic aromatic hydrocarbon solid phase extraction column (hereinafter referred to as "MIP column"), and activate and equilibrate the MIP column with 3 ml of dichloromethane and 5 ml of n-hexane in sequence, and discard the waste liquid.

[0205] Using a Pasteur pipette, transfer the first reconstituted solution from bottle #2 to the MIP column, and wash bottle #2 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0206] Rinse with 6 ml of n-hexane and discard the eluent;

[0207] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the eluent in a 15 ml long glass tube (tube #1).

[0208] S203. The first eluent in tube #1 is concentrated to near dryness by nitrogen blowing, and then redissolved with 250 μL of n-hexane to obtain the second redissolved solution;

[0209] S204. Insert a piece of cotton into the bottom of the pipette tip and tamp it down with a Pasteur pipette or glass rod to block the outlet at the bottom of the pipette tip.

[0210] Add 3 ml of acetone to the pipette tip to clean the inner wall of the pipette tip, and discard the cleaning waste liquid.

[0211] Fill the nozzle with approximately 3 cm of 2% neutral alumina and approximately 0.5 cm of anhydrous sodium sulfate sequentially from bottom to top, and tap until stable;

[0212] Add 3 ml of dichloromethane to the nozzle to clean impurities on the packing material, and discard the cleaning waste liquid;

[0213] When the level of dichloromethane drops to the top of the anhydrous sodium sulfate, add 3 ml of n-hexane;

[0214] When the level of n-hexane drops to the top of anhydrous sodium sulfate, quickly add the second redissolution solution and rinse with 500 ml of n-hexane.

[0215] When the liquid level of n-hexane drops to the upper surface of anhydrous sodium sulfate, add 8 mL of a mixed solution of n-hexane and dichloromethane (volume ratio of n-hexane to dichloromethane is 3:2) to elute and obtain a second eluent. Collect the second eluent in a long glass tube (tube #2).

[0216] S205. Add 50 ml of a 0.4 ng / µL internal standard mixed solution of deuterated polycyclic aromatic hydrocarbons to the second eluent in tube #2 to form the first mixed solution;

[0217] S206. Concentrate the first mixed solution to near dryness by nitrogen blowing, redissolve it with n-hexane, and transfer it to a sample vial with a liner using a Pasteur pipette. Then wash tube #2 with 50 μL of n-hexane and transfer the mixture to the liner; to obtain the test solution containing PPAHs.

[0218] The concentration of pPAHs in the test solution containing PPAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0219] S300: Process the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs:

[0220] S301. The supernatant containing OH-PAHs in bottle #3 is concentrated to near dryness by nitrogen blowing, and then redissolved with 500 μL of n-hexane to obtain the third redissolved solution;

[0221] S302. Take a 500mg MIP column and activate and equilibrate it with 3 mL of dichloromethane and 5 mL of n-hexane in sequence, then discard the waste liquid.

[0222] Using a Pasteur pipette, transfer the third reconstituted solution from flask #3 to the MIP column, and wash flask #3 with 500 μL of n-hexane, then transfer the wash solution to the MIP column as well.

[0223] Rinse with 6 ml of n-hexane and discard the eluent;

[0224] Elute with 10 ml of a hexane / dichloromethane mixed solution (hexane and dichloromethane in a volume ratio of 1:1), and collect the third eluent in a 15 ml long glass tube (tube #3);

[0225] S303. Add 50 μL of a 0.4 ng / μL internal standard mixed solution of deuterated hydroxyl polycyclic aromatic hydrocarbons to the third eluent in tube #3 to form a second mixed solution;

[0226] S304. Concentrate the second mixed solution in tube #3 with nitrogen to approximately 200 μL. Transfer the remaining second mixed solution in tube #3 to a new brown screw-top silanized vial (labeled as vial #4) using a long Pasteur pipette. Rinse the long glass tube twice with 100 μL of n-hexane and combine the solutions in vial #4. Blow the liquid in vial #4 with nitrogen until nearly dry, and add 50 μL of acetonitrile to redissolve it, obtaining the fourth redissolved solution.

[0227] S305. Add 100 μL of silanizing reagent (BSTFA:TMCS = 99:1, ≥99%) to the fourth reconstitution solution in bottle #4, purge the air in the bottle with nitrogen, and tighten the cap; place it in a dry constant temperature metal bath and heat at 60°C for 30 minutes to obtain the test solution containing OH-PAHs; after cooling bottle #4 to room temperature, open the cap and transfer all the test solution containing OH-PAHs to a sample vial with a liner, and seal the vial.

[0228] The concentration of OH-PAHs in the test solution containing OH-PAHs was determined using a triple quadrupole gas chromatography-mass spectrometry system.

[0229] In Example 3, the experiment was repeated three times, referred to as the first experiment, the second experiment, and the third experiment, and the test results are shown in Table 8.

[0230] Table 8: Results of PPAHs and OH-PAHs concentrations in 3 repeated experiments (unit: ng / g urine)

[0231]

[0232]

[0233] Comparative Example 5

[0234] The concentration of polycyclic aromatic hydrocarbons (PAHs) in biological samples was detected using conventional detection methods. The specific detection method was (same as Comparative Example 3, except that urine was used instead of serum):

[0235] Step 1: Take 400 μL of urine solution, add 20 mL of acetonitrile, and extract using a microwave extraction system for 20 minutes. Specific microwave conditions are: increase the temperature from room temperature to 110°C within 10 minutes and maintain that temperature for 10 minutes.

[0236] Step 2: Filter the above solution using a 37mm (diameter) glass fiber filter to remove lipids. Transfer the filtrate to a 250ml separatory funnel and extract twice with 30ml of n-hexane solution containing 4% sodium sulfate.

[0237] Step 3: Concentrate the above extract (approximately 60 ml of n-hexane) to approximately 1 ml in a vacuum centrifuge and transfer it to a purification column. This column, from bottom to top, contains 12 cm of 3% deactivated alumina (3% by mass ultrapure water + 97% by mass neutral alumina powder), 12 cm of 2% deactivated silica gel (2% by mass ultrapure water + 98% by mass silica gel), and 1 cm of anhydrous sodium sulfate. The purification column is pre-equilibrated with 10 ml of n-hexane. Elute the purification column with 50 ml of a hexane / dichloromethane mixture (1:1 volume ratio of n-hexane to dichloromethane), concentrate the eluent to approximately 1 ml, and add 50 ml of a 0.4 ng / µL deuterated polycyclic aromatic hydrocarbon internal standard mixture.

[0238] Step 4: Concentrate the eluent to 150 μL, transfer it to the sample, and determine the PPAHs concentration using a triple quadrupole gas chromatography-mass spectrometry system.

[0239] The detection results of pPAHs concentration in Comparative Example 5 are shown in Table 9.

[0240] Table 9: Detection results of pPAHs concentration in Comparative Example 5 (unit: ng / g urine)

[0241]

[0242]

[0243] Comparative Example 6

[0244] The concentration of hydroxyl polycyclic aromatic hydrocarbons in biological samples was determined using conventional detection methods. The specific detection method was (same as Comparative Example 4, only the serum was replaced with urine):

[0245] Take 400 μL of urine, 3 mL of 5 mol / L acetate-sodium acetate buffer solution (pH=5), and 10 μL of β-glucuronidase / sulfatase and add them to a glass centrifuge tube. Mix thoroughly using a vortex mixer. After capping the centrifuge tube, place it in a constant temperature water bath at 37°C for 12 hours. After 12 hours, remove the tube and prepare it for SPE column extraction. Activate and equilibrate the C18 column with 5 mL of methanol and 10 mL of ultrapure water. After the sample passes through the C18 column, rinse the column with 5 mL of ultrapure water. After the eluent passes through the extraction column, maintain a vacuum for 1 minute to thoroughly remove water from the SPE column. Then elute the column with 10 mL of methanol, collect the eluent in a test tube, blow it with nitrogen to a volume below 1 mL, and make up to 1 mL with methanol. Transfer the eluent to the sample and determine the concentration of OH-PAHs using a triple quadrupole gas chromatography-mass spectrometry (GC-MS).

[0246] The detection results of pPAHs concentration in Comparative Example 6 are shown in Table 10.

[0247] Table 10: Detection results of OH-PAHs concentration in Comparative Example 6 (unit: ng / g urine)

[0248] Chinese name English name English abbreviations Comparative Example 6 1-Naphthol 1-hydroxynaphthalene 1-OH-NAP 1.8027 2-Naphthol 2-hydroxynaphthalene 2-OH-NAP 1.8319 2-Hydroxyfluorene 2-hydroxyfluorene 2-OH-FLUO 10.1578 1-Hydroxyphenanthrene 1-hydroxyphenanthrene 1-OH-PHE 31.9993 2-Hydroxyphenanthrene 2-hydroxyphenanthrene 2-OH-PHE 4.0881 3-Hydroxyphenanthrene 3-hydroxyphenanthrene 3-OH-PHE 3.5644 4-Hydroxyphenanthrene 4-hydroxyphenanthrene 4-OH-PHE 3.5146 9-Hydroxyphenanthrene 9-hydroxyphenanthrene 9-OH-PHE 4.1692 1-Hydroxypyrene 1-hydroxypyrene 1-OH-PYR 3.0935

[0249] As can be seen from Tables 5 to 7, the present application preprocesses the same biological sample (mouse urine) to obtain detection solutions containing pPAHs and OH-PAHs respectively, which can realize the simultaneous determination of PPAHs and OH-PAHs and greatly reduce the sample volume and preprocessing cost.

[0250] The three repeated experiments in Example 3 demonstrate that the pretreatment method of this application can guarantee the stability of the detection method of this application.

[0251] Compared to detecting only the content of pPAHs in biological samples (Comparative Example 5) and only the content of OH-PAHs in biological samples (Comparative Example 6), Example 3 not only enables the simultaneous detection of pPAHs and OH-PAHs in the same biological sample, but also improves the detection values ​​of most pPAHs and OH-PAHs.

[0252] Therefore, the pretreatment method of this application can not only ensure the stability of the detection method of this application, but also improve the detection accuracy of pPAHs content and OH-PAHs content.

[0253] In summary, the pretreatment method of this application not only ensures the stability and accuracy of the detection method, but also makes it applicable to different types of biological samples.

[0254] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A pretreatment method for simultaneous analysis of polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples, characterized in that, The preprocessing method includes the following steps: S100. The biological sample is treated with alkaline hydrolysis to hydrolyze the proteins in the biological sample to obtain an alkaline hydrolysate; a demulsifier is first added to the hydrolysate to demulsify, then a first extractant is added and centrifuged to obtain a supernatant and a lower layer solution containing PPAHs. The lower layer solution is treated with acid hydrolysis to obtain an acidic hydrolysate; a second extractant is added to the acidic hydrolysate and centrifuged to obtain a supernatant containing OH-PAHs; The demulsifier is selected from C1-C3 organic alcohols; The first extractant comprises n-hexane and methyl tert-butyl ether in a volume ratio of (7-11):1; The second extractant comprises cyclohexane and ethyl acetate in a volume ratio of (3-5):1; S200: Process the supernatant containing PPAHs to obtain a test solution containing PPAHs; S300: Process the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs.

2. The pretreatment method according to claim 1, characterized in that, The alkaline solution used in the alkaline hydrolysis method is selected from aqueous solutions of alkali metal hydroxides.

3. The pretreatment method according to claim 2, characterized in that, The concentration of the aqueous solution of the alkali metal hydroxide is 1–5 mol / L.

4. The pretreatment method according to claim 2, characterized in that, The aqueous solution of the alkali metal hydroxide is selected from at least one of an aqueous solution of sodium hydroxide and an aqueous solution of potassium hydroxide.

5. The pretreatment method according to claim 2, characterized in that, The biological sample is selected from liquid and / or solid samples; the liquid sample is selected from at least one of serum, plasma and urine; the solid sample is selected from tissue samples.

6. The pretreatment method according to claim 2, characterized in that, The biological sample is selected from liquid and / or solid samples; the liquid sample is selected from at least one of serum, plasma and urine; the solid sample is selected from at least one of hair and nails.

7. The pretreatment method according to claim 5, characterized in that, If the biological source sample is selected from liquid samples, the concentration of the aqueous solution of the alkali metal hydroxide is selected from 3.5 to 4.5 mol / L and the volume is selected from 150 to 250 μL; If the biological source sample is selected from solid samples, the concentration of the aqueous solution of the alkali metal hydroxide is selected from 1.5 to 2.5 mol / L and the amount is selected from 550 to 650 μL.

8. The pretreatment method according to claim 1, characterized in that, The hydrolysis is performed at a temperature of 55–65°C for 50–90 minutes.

9. The pretreatment method according to claim 1, characterized in that, The C1-C3 organic alcohols are selected from at least one of methanol, ethanol, n-propanol, and isopropanol.

10. The pretreatment method according to claim 9, characterized in that, The C1-C3 organic alcohols are selected from methanol.

11. The pretreatment method according to claim 9, characterized in that, The amount of the demulsifier used is selected from 80 to 120 microliters.

12. The pretreatment method according to claim 1, characterized in that, The first extractant contains n-hexane and methyl tert-butyl ether in a volume ratio of (8-10):

1.

13. The pretreatment method according to claim 12, characterized in that, The first extractant comprises n-hexane and methyl tert-butyl ether in a volume ratio of 9:

1.

14. The pretreatment method according to claim 12, characterized in that, The amount of the first extractant is selected from 550 to 650 microliters.

15. The pretreatment method according to claim 12, characterized in that, The centrifugation separation is repeated 2 to 4 times, and the amount of the first extractant used in each centrifugation separation is selected from 550 to 650 microliters.

16. The pretreatment method according to any one of claims 1 to 15, characterized in that, Step S200 involves processing the supernatant containing PPAHs to obtain a test solution containing PPAHs, including the following steps: S201. Concentrate the supernatant containing PPAHs to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a first redissolved solution; S202, The first reconstituted solution is passed through a first solid-phase extraction column and eluted to obtain a first eluent; S203. Concentrate the first eluent to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a second redissolved solution; S204. The second reconstituted solution is passed through a second solid-phase extraction column and eluted to obtain a second eluent. S205. Add a mixed solution of deuterated polycyclic aromatic hydrocarbon internal standards to the second eluent to form a first mixed solution; S206. Concentrate the first mixed solution to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain the test solution containing PPAHs.

17. The pretreatment method according to claim 16, characterized in that, In step S201, the amount of n-hexane used is 400 to 600 microliters.

18. The pretreatment method according to claim 16, characterized in that, In step S202, the first solid phase extraction column is a solid phase extraction column specifically for polycyclic aromatic hydrocarbons.

19. The pretreatment method according to claim 16, characterized in that, In step S202, the eluent used for elution comprises n-hexane and dichloromethane in a volume ratio of 1:

1.

20. The pretreatment method according to claim 16, characterized in that, In step S203, the amount of n-hexane used is 200-300 microliters.

21. The pretreatment method according to claim 16, characterized in that, In step S204, the second solid phase extraction column is selected from a neutral alumina solid phase extraction column.

22. The pretreatment method according to claim 16, characterized in that, In step S204, the eluent used for elution includes n-hexane and dichloromethane in a volume ratio of 3:

2.

23. The pretreatment method according to claim 16, characterized in that... In step S205, the concentration of the deuterated polycyclic aromatic hydrocarbon internal standard mixed solution is 0.35–0.45 ng / µL, and the amount used is 45–55 µL.

24. The pretreatment method according to claim 16, characterized in that, In step S206, the amount of n-hexane used is 50 to 150 microliters.

25. The pretreatment method according to any one of claims 1 to 15, characterized in that, Step S300 involves processing the supernatant containing OH-PAHs to obtain a test solution containing OH-PAHs, including the following steps: S301. Concentrate the supernatant containing OH-PAHs to near dryness by nitrogen blowing, and redissolve it with n-hexane to obtain a third redissolved solution; S302, The third reconstituted solution is passed through a third solid-phase extraction column and eluted to obtain a third eluent; S303. Add a mixed solution of deuterated hydroxyl polycyclic aromatic hydrocarbon internal standards to the third eluent to form a second mixed solution; S304. Concentrate the second mixed solution to near dryness by nitrogen blowing, and redissolve it with acetonitrile to obtain a fourth redissolved solution; S305. Add a silanizing agent to the fourth reconstituted solution and react for 20 to 40 minutes under a nitrogen atmosphere and at a temperature of 55 to 65°C to obtain the test solution containing OH-PAHs.

26. A method for simultaneously analyzing and detecting polycyclic aromatic hydrocarbons (PAHs) and hydroxyl PAHs in biological samples, characterized in that, The detection method includes the pretreatment method according to any one of claims 1 to 25, detecting the concentration of PPAHs in the test solution containing PPAHs, and detecting the concentration of OH-PAHs in the test solution containing OH-PAHs.

27. The detection method according to claim 26, characterized in that, The concentration of PPAHs in the test solution containing PPAHs was determined by gas chromatography-mass spectrometry.

28. The detection method according to claim 26, characterized in that, The concentration of OH-PAHs in the test solution containing OH-PAHs was determined by gas chromatography-mass spectrometry.

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