Method for detecting cigarette smoke exposure biomarker based on nanofiber solid phase extraction

Abstract

The invention relates to the technical field of analysis and detection, and discloses a method for detecting cigarette smoke exposure biomarkers based on nanofiber solid-phase extraction, which comprises the following steps of: treating biological samples such as liver, lung, blood and urine samples by using a polystyrene / polypyrrole nanofiber material as a solid-phase extraction material; and simultaneously detecting a plurality of cigarette smoke biomarkers and an oxidative stress marker 8-isoprostaglandin in different biological samples of the rat after smoke exposure by using a liquid chromatography-mass spectrometer. According to the method, the influence of toxic and harmful substances in cigarette smoke on the health of organisms is reflected by detecting smoke exposure biomarkers and oxidative stress markers in biological samples, and evaluation of the safety performance of tobacco products is facilitated. According to the method, the limitation of small target object coverage of the existing smoke exposure biomarker detection method is solved, and the problems of large use amount of an organic solvent and tedious and time-consuming pretreatment in the existing sample pretreatment are also solved.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction. Background Art

[0002] Traditional tobacco products have been a consumer product for hundreds of years. However, the health disadvantages of traditional tobacco products are becoming increasingly prominent, and the impact of smoking and secondhand smoke on human health has always attracted much attention. Tobacco products react chemically through the burning of tobacco, releasing various chemical substances including nicotine, nitrosamines, polycyclic aromatic hydrocarbons, crotonaldehyde, 1,3-butadiene, benzene and other toxic and harmful compounds. At the same time, they also release high concentrations of nitrogen oxides, free radicals and other substances that induce oxidative stress reactions in organisms. In order to evaluate the safety of tobacco products, some national regulatory agencies have begun to use the method of detecting biomarkers to conduct health and safety risk assessments on tobacco products. Biomarkers in cigarette smoke can reflect the actual exposure or in vivo dose of tobacco products to human bodies, and measuring their concentration levels can more accurately evaluate the safety performance of tobacco products.

[0003] However, the current analysis methods of tobacco-related biomarkers are mainly limited to a single or a few compounds, and there is no method that can simultaneously monitor multiple biomarkers. Biomarkers in biological samples are still a hot topic and difficult to study because of their complex composition and usually low concentration.

[0004] Currently, the common methods used for separation, enrichment and purification of metabolites in biological samples mainly include liquid-liquid extraction (LLE), solid phase extraction (SPE), solid phase microextraction (SPME), etc. Among them, LLE consumes a large amount of organic solvents, SPE consumes a large amount of extraction fillers and organic solvents, and solid phase microextraction is expensive and not suitable for the detection of large quantities of samples.

[0005] In recent years, nanofiber-based solid phase extraction devices and apparatuses, such as Chinese patents 200210123148.5, 200910033559.3, and 20102050026.X, have overcome the shortcomings of the existing SPE technology, such as expensive devices and complicated operations. In addition, they have low cost, simple structure, easy installation and use, save manpower, and can process samples in batches.

[0006] However, the above-mentioned existing technologies still have some defects: whether they can realize the simultaneous and rapid detection of multiple cigarette smoke exposure biomarkers in different biological samples, and the existing smoke exposure biomarker detection methods have the limitation of small target coverage, and the existing sample pretreatment uses a large amount of organic solvents and the pretreatment is cumbersome and time-consuming.

[0007] Based on this, the present invention provides a method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction to solve the above technical problems. Summary of the invention

[0008] The object of the present invention is to provide a method for simultaneously detecting smoke exposure biomarkers in animal organ tissues and body fluid samples by liquid chromatography-mass spectrometry, using polystyrene / polypyrrole nanofiber material to pretreat samples. Biological samples include liver, lung, blood and urine, and detection targets include cotinine, N-acetyl-S-(2-aminoformylethyl)-L-cysteine ​​(AAMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine ​​(MHBMA), 4-(methylnitrosamino)-1-(3-pyridyl)butanol (NNAL), N-acetyl-S-(3-hydroxypropyl)-L-cysteine ​​(3-HPMA), N-acetyl-S-(3-carboxy-1-methylpropyl)-L-cysteine ​​(3-HMPMA), phenylmercaptouric acid (SPMA), 8-isomerized prostaglandins, α-OH naphthalene, β-OH naphthalene, 1-OH phenanthrene, 3-OH phenanthrene, 2-OH fluorene, 9-OH fluorene, 1-OH pyrene and other biomarkers of cigarette smoke exposure. This method first uses a polystyrene / polypyrrole nanofiber solid phase extraction column to enrich and concentrate the target substances in the liver, lungs, blood and urine of experimental animals, and then uses liquid chromatography-mass spectrometry to detect multiple target substances. This method achieves simultaneous and rapid detection of multiple cigarette smoke exposure biomarkers in different biological samples, solving the limitation of the small target coverage of existing smoke exposure biomarker detection methods, and also solves the problem of large amount of organic solvents used in existing sample pretreatment and cumbersome and time-consuming pretreatment.

[0009] The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction provided by the present invention comprises:

[0010] Step 1, dissect the experimental rats to obtain various organs, take the lung or liver, add buffer in a ratio of weight (g): volume (mL) = 1:9, homogenize with a handheld homogenizer, centrifuge at 12000rpm for 10min, remove the supernatant, take 200μL of the homogenate, add 120μL of 5% perchloric acid, vortex for 1min, and centrifuge at 12000r / min for 5min, take the supernatant, add 10μL of cortisol D-4 as an internal standard and make its concentration in the sample 100ng / mL, and the above mixed solution is used as the liver or lung sample test solution;

[0011] Step 2, using a negative pressure venous blood collection tube containing EDTA to collect blood from rats, taking 200 μL of upper plasma after centrifugation, adding 120 μL of 5% perchloric acid, vortexing for 1 min, and centrifuging at 12000 r / min for 5 min, taking the supernatant, adding 10 μL of cortisol D-4 as an internal standard and making its concentration in the sample 100 ng / mL, and the above mixed solution is used as the plasma sample test solution;

[0012] Step 3, the rat's urine was collected by the restraint tube of the smoking machine, 1 mL of urine was added with 10 μL of β-glucuronidase / arylsulfatase and incubated at 37°C for 4 hours, 500 μL of the above solution was added with 10 μL of cortisol D-4 as an internal standard and the concentration in the sample was 100 ng / mL, and the above mixed solution was used as the urine sample test solution;

[0013] Step 4, enriching and concentrating the target substance using a solid phase extraction column filled with polystyrene / polypyrrole composite nanofibers in the above tissue sample solutions containing the internal standard;

[0014] Step 5, using an organic solvent to elute the target substance enriched on the nanofiber solid phase extraction column to obtain a test solution;

[0015] Step 6, analyzing the test solution by liquid chromatography-mass spectrometry, and quantitatively calculating the concentration of the target substance in urine by using the internal standard method.

[0016] Preferably, the reagents used in steps 1-3 are all analytically pure reagents.

[0017] Preferably, the polystyrene nanofibers in step 4 are prepared by dissolving 1 g of polystyrene particles in an organic solvent having a volume ratio of N,N-dimethylformamide to tetrahydrofuran of 4:6 to form a spinning solution, and using electrospinning technology to make the spinning solution into polystyrene nanofibers; the prepared polystyrene nanofibers are immersed in an aqueous solution containing a certain amount of pyrrole, and a certain amount of FeCl3 is added and stirred sufficiently for reaction, and then in situ reduction is carried out on the surface of the polystyrene nanofibers to prepare polystyrene / polypyrrole composite nanofibers.

[0018] Preferably, the solid phase extraction column filled with polystyrene / polypyrrole composite nanofibers in step 4 needs to be activated with methanol and water before use.

[0019] Preferably, the organic solvent in step 5 is an eluent composed of one or more organic substances such as methanol, ethanol and acetonitrile.

[0020] Preferably, in step 6, the conditions of liquid chromatography-mass spectrometry are as follows:

[0021] Chromatographic column: C18 (3.5 μm, 150 mm × 2.1 mm); mobile phase: acetonitrile as B: water as A (0.1% formic acid), gradient elution: 0-0.1 min: 5% B, 0.1-20 min: 5% B-100% B, 20-30 min: 100% B, 30-35 min: 100% B-5% B, 35-40 min: 5% B; 0.45 μm microporous membrane filtration followed by ultrasonic degassing; flow rate: 0.3 m L / min; injection volume: 10μL; column temperature: 30℃; mass spectrometry conditions: electrospray ion source (ESI), negative ion scanning and positive ion scanning modes, detection method is multiple reaction monitoring mode (MRM); spray voltage 3500V; nebulizer gas pressure 50 polystyrene i; curtain gas pressure 30psi; auxiliary nebulizer gas pressure 45psi; ion source temperature gradient 500℃; inlet voltage is 10v; outlet voltage is 10v; dwell time is 200ms.

[0022] Preferably, the cigarette smoke biomarkers and oxidative stress markers to be tested include cotinine, N-acetyl-S-(2-aminoformylethyl)-L-cysteine ​​(AAMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine ​​(MHBMA), 4-(methylnitrosamino)-1-(3-pyridyl)butanol (NNAL), N-acetyl-S-(3-hydroxypropyl)-L-cysteine ​​(3-HPMA), N-acetyl-S-(3-carboxy-1-methylpropyl)-L-cysteine ​​(3-HMPMA), phenylmercaptouric acid (SPMA), α-OH naphthalene, β-OH naphthalene, 1-OH phenanthrene, 3-OH phenanthrene, 2-OH fluorene, 9-OH fluorene, 1-OH pyrene and 8-isomeric prostaglandins.

[0023] Preferably, the polystyrene / polypyrrole nanofibers are prepared by firstly using electrospinning technology to prepare polystyrene nanofibers, which are then used as substrates to perform in-situ polymerization of polypyrrole on the surface of the prepared polystyrene nanofibers, thereby obtaining the polystyrene / polypyrrole composite nanofibers.

[0024] Compared with the related art, the method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction provided by the present invention has the following beneficial effects:

[0025] The present invention uses a polystyrene / polypyrrole composite nanofiber solid phase extraction column to enrich and concentrate target objects in the liver, lungs, blood and urine of experimental animals, and then uses liquid chromatography-mass spectrometry to detect 15 target objects; this method can achieve simultaneous, efficient and rapid detection of multiple cigarette smoke exposure biomarkers in different biological samples, solving the limitation problem of small target coverage of conventional smoke exposure biomarker detection methods.

[0026] This method can simultaneously detect biomarkers of substances in smoke including nicotine, nitrosamines, polycyclic aromatic hydrocarbons, crotonaldehyde, 1,3-butadiene, benzene, and other substances, as well as biomarkers of oxidative stress, thereby achieving rapid and efficient monitoring of biomarkers of exposure to toxic and harmful substances in tobacco smoke. The test results can reflect the exposure of organisms to toxic and harmful substances in cigarette smoke and provide a basis for the safety assessment of tobacco products.

[0027] In addition, compared with traditional sample pretreatment methods, the use of nanofiber solid phase extraction technology to enrich and purify biomarkers in different biological samples simplifies the sample pretreatment operation steps and reduces the amount of samples and organic reagents used. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The electron microscopy and transmission electron microscopy characterization results of polystyrene / polypyrrole composite nanofiber solid phase extraction materials: (A) is the SEM image of polystyrene; (B) is the SEM image of polystyrene / polypyrrole; (C) is the TEM image of polystyrene; (D) is the TEM image of polystyrene / polypyrrole.

[0029] Figure 2 These are the infrared (FTIR) characterization results of polystyrene / polypyrrole composite nanofiber solid phase extraction materials.

[0030] Figure 3 The chromatograms of 15 biomarkers of smoke exposure at a concentration of 100 ng / mL and the internal standard cortisol-D4 spiked in blank samples are as follows: (A, A') are chromatograms of the target in lung tissue detected in positive and negative ion scanning modes; (B, B') are chromatograms of the target in liver tissue detected in positive and negative ion scanning modes; (C, C') are chromatograms of the target in blood samples detected in positive and negative ion scanning modes; (D, D') are chromatograms of the target in urine detected in positive and negative ion scanning modes.

[0031] Figure 4The concentrations of the target substances in different tissues are as follows: (A) the concentrations of each compound in the lung tissue of different dose groups and the control group; (B) the concentrations of each compound in the liver tissue of different dose groups and the control group; (C) the concentrations of each compound in the plasma of different dose groups and the control group; (D) the concentrations of each compound in the urine of different dose groups and the control group. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and implementation modes.

[0033] The present invention uses electrospinning technology to prepare polystyrene nanofibers, and then in-situ polymerizes polypyrrole on the surface of the spun polyacrylonitrile nanofibers to prepare polystyrene / polypyrrole nanofibers, and uses the nanofibers in combination with polystyrene nanofibers as adsorption materials to prepare solid phase extraction columns. Organ tissues, blood, urine and other samples are added with internal standards and then homogenized or centrifuged. The solution is pre-treated with the prepared nanofiber solid phase extraction column, and then a variety of smoke exposure biomarkers are detected simultaneously using liquid chromatography-mass spectrometry, wherein the chromatography-mass spectrometry conditions are as follows:

[0034] Chromatographic column: C18 (3.5 μm, 150 mm × 2.1 mm); mobile phase: acetonitrile as B: water as A (0.1% formic acid), gradient elution: 0-0.1 min: 5% B, 0.1-20 min: 5% B-100% B, 20-30 min: 100% B, 30-35 min: 100% B-5% B, 35-40 min: 5% B. Ultrasonic degassing after 0.45 μm microporous membrane filtration; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 30°C. Mass spectrometry conditions: electrospray ion source (ESI), negative ion scanning and positive ion scanning modes, detection mode is multiple reaction monitoring mode (MRM); spray voltage 3500V; nebulizer gas pressure 50psi; curtain gas pressure 30psi; auxiliary nebulizer gas pressure 45psi; ion source temperature gradient 500℃; inlet voltage 10V; outlet voltage 10V; dwell time 200ms.

[0035] The retention times and mass spectrometry conditions of various smoke exposure markers are as follows:

[0036] Table 1 Retention time and mass spectrometry conditions of the target compounds

[0037]

[0038] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example 1

[0040] Preparation of nanofibers: The preparation process of polystyrene nanofibers is as follows: First, accurately weigh 1.0g of polystyrene particles and dissolve them in a mixed solvent composed of tetrahydrofuran and N,N-dimethylformamide (V:V=4:6), add a magnet, and stir the solution at 25° using a magnetic stirrer for 12 hours as a spinning solution. Quickly load the electrospinning solution into a glass syringe with a stainless steel needle, install a No. 6 stainless steel needle with a diameter of 0.6mm, and keep the needle horizontal during the spinning process. Use a wire to connect the stainless steel needle to the positive pole of the high-voltage power supply, and place a collecting screen connected to the negative pole of the power supply 22cm away from the stainless steel needle. Adjust the voltage to 23kV and the liquid delivery rate to 1.5mL / h. The prepared nanofibers fall on the aluminum collecting screen to form a mesh. The preparation process of polystyrene / polypyrrole nanofibers is as follows: First, a piece of polystyrene nanofiber is immersed in a 0.04 mol / L concentration of pyrrole solution, and FeCl3 solution (0.084 mol / L) is added to the solution to initiate the polymerization of pyrrole into polypyrrole and deposit on the surface of polystyrene. Then, the mixture is vigorously stirred for 2 hours until the appearance of the polystyrene nanofiber changes from white to black, which is a symbol of the end point of the reaction, indicating that the polystyrene / polypyrrole nanofiber is successfully prepared. After washing several times with water, the polystyrene / polypyrrole nanofiber is dried in a vacuum drying oven before use.

[0041] Filling and activation of nanofiber solid phase extraction column: Weigh 5 mg of polystyrene / polypyrrole composite nanofibers and fill them in the solid phase extraction column. Before use, take 200 μL of methanol and 200 μL of distilled water to activate the nanofibers. The solid phase extraction column can use the extractor shell disclosed in Chinese patent 2005101231485 or 201020500026X.

[0042] Nanofiber characterization: The nanofiber solid phase extraction material polystyrene / polypyrrole was characterized by SEM and TEM methods. Figure 1 As shown in the figure, the particle size of polystyrene / polypyrrole nanofibers is relatively uniform and the surface is smooth. Compared with polystyrene nanofibers, the diameter of nanofibers after being composited with polypyrrole is increased. The structural characteristics of polystyrene and polystyrene / polypyrrole composite nanofibers were studied by FTIR. In the spectrum of polystyrene / polypyrrole fibers, some absorption bands of polystyrene overlap with those of polypyrrole, and other characteristic peaks of polystyrene are weakened (as shown in the red box). 1454cm -1 , 3025cm -1 The characteristic peaks at 1548 cm are attributed to the CH and C=C stretching vibrations of the benzene ring in the spectra of polystyrene and polystyrene / polypyrrole. -1 and 1475cm -1The characteristic peaks observed at 1182 cm-1 are related to the fundamental vibration of the pyrrole ring in the polystyrene / polypyrrole spectrum, while they are not observed in the polystyrene spectrum, indicating that the polypyrrole nanofibers were successfully coated on the surface of polystyrene. In addition, the peaks of CN stretching vibration and CH in-plane vibration are 1182 cm-1, -1 and 1042cm -1 , while the CC deformation vibration corresponds to 916cm -1 , which further proves that the polystyrene / polypyrrole composite nanofibers were successfully prepared.

[0043] Example 2

[0044] Experimental rats that have not been exposed to smoke were used as sampling objects, and their liver, lungs, blood, urine and other tissues were collected as blank samples. The lungs and liver were added with Sorensen buffer (buffer composed of Na2HPO4 and KH2PO4, pH 6.8) at a ratio of weight (g): volume (mL) = 1:9, and then homogenized with a handheld homogenizer after adding different concentrations of target standard solutions. 200μL of the organ homogenate was added with 120μL of 5% perchloric acid solution and vortexed for 5 minutes, and then 10μL of cortisol-D4 was added as an internal standard to make its concentration in the sample 100ng / mL, and then centrifuged at 12000rpm for 10min to take the supernatant as the test solution. Take 200 μL of plasma sample and add different concentrations of target standard solution, then add 120 μL of 5% perchloric acid solution and vortex for 5 minutes, then centrifuge at 12000rpm for 10 minutes to take the supernatant, then add 10 μL of cortisol-D4 as an internal standard to make its concentration in the sample 100 ng / mL, and use the above mixed solution as the plasma sample test solution. Take 1 mL of urine and add 10 μL of β-glucuronidase / arylsulfatase to incubate at 37°C for 4 hours, take 500 μL of the above solution and add different concentrations of target, then add 10 μL of cortisol-D4 as an internal standard to make its concentration in the sample 100 ng / mL, and use the above mixed solution as the urine sample test solution. Pass the sample solution with different concentrations of target through a polystyrene / polypyrrole solid phase extraction column and elute with 50 μL of methanol, and the obtained eluate is detected by liquid chromatography-mass spectrometry. This method uses the internal standard quantitative method to draw the working curve. The detection limit and quantification limit of the detection method are calculated by the signal-to-noise ratio (S / N). The detection limit (LOD) is S / N ≥ 3, and the quantification limit (LOQ) is S / N ≥ 10. The specific data are shown in Table 1.

[0045] Table 1-1 Linear range, correlation coefficient, detection limit and quantification limit of each target in the liver

[0046]

[0047] Table 1-2 Linear range, correlation coefficient, detection limit and quantification limit of each target in the lung

[0048]

[0049]

[0050] Table 1-3 Linear range, correlation coefficient, detection limit and quantification limit of each target in plasma

[0051]

[0052] Table 1-4 Linear range, correlation coefficient, detection limit and quantification limit of each target in urine

[0053]

[0054]

[0055] As can be seen from the table above, the targets in liver tissue have a good linear relationship within the detection range, and the correlation coefficients are all greater than 0.99. The LOD of the target is 0.04-2.0ng / mL, and the LOQ is 0.15-6.6ng / mL. The targets in lung tissue have a good linear relationship within the detection range, and the correlation coefficients are all greater than 0.99. The LOD of the target is 0.04-2.9ng / mL, and the LOQ is 0.13-9.8ng / mL. The targets in plasma have a good linear relationship within the detection range, and the correlation coefficients are all greater than 0.99. The LOD of the target is 0.04-2.9ng / mL, and the LOQ is 0.13-9.8ng / mL. There is a good linear relationship within the detection range, with correlation coefficients greater than 0.99, the LOD of the target is 0.15-3.2ng / mL, and the LOQ is 0.5-10.8ng / mL; each target in urine has a good linear relationship within the detection range, with correlation coefficients greater than 0.99, the LOD of the target is 0.02-2.7ng / mL, and the LOQ is 0.06-9.0ng / mL. This method can meet the detection needs of various cigarette smoke markers in various tissue samples.

[0056] Example 3

[0057] Mixed standard solutions were added to various biological blank samples to make the target concentrations 20.0 ng / ml, 100 ng / ml and 400 ng / ml, respectively. Five samples were prepared in parallel for each addition level, and the samples were processed according to Example 2 and then measured on the machine. The recovery rate and relative standard deviation (RSD) were calculated based on the solvent working curve. The recovery rate and precision test results are shown in Table 2.

[0058] Table 2-1 Recovery rate and relative standard deviation of target substances in liver (n=5)

[0059]

[0060]

[0061] Table 2-2 Recovery rate and relative standard deviation of target substances in lungs (n=5)

[0062]

[0063] Table 2-3 Recovery rate and relative standard deviation of target substances in plasma (n=5)

[0064]

[0065]

[0066] Table 2-4 Recovery rate and relative standard deviation of target substances in urine (n=5)

[0067]

[0068] It can be seen from Table 2-1 to Table 2-4 that the recoveries of the target substances at three different added concentrations in liver tissue were 97.7%-117.6%, and the relative standard deviation (RSD) was 2.5%-8.7%; the recoveries of the target substances at three different added concentrations in lung tissue were 85.7%-111.8%, and the relative standard deviation (RSD) was 1.5%-9.5%; the recoveries of the target substances at three different added concentrations in plasma were 85.4%-116.4%, and the relative standard deviation (RSD) was 3.4%-7.2%; the recoveries of the target substances at three different added concentrations in urine were 86.2%-115.0%, and the relative standard deviation (RSD) was 3.3%-7.6%. The recovery rate and stability of this method can meet the detection requirements of cigarette smoke markers in various tissue samples.

[0069] Example 4

[0070] Twelve SPF-grade 4-6-week-old male rats were randomly divided into a control group and low-, medium-, and high-dose experimental groups. The experimental group was exposed to cigarette smoke in the form of nasal inhalation using an LM4E smoking machine. The low exposure dose was smoking 9 heat-not-burn new cigarettes within 2 hours, the medium dose group was smoking 18 heat-not-burn new cigarettes within 2 hours, and the high dose was smoking 36 heat-not-burn new cigarettes within 2 hours. After taking out the tissue samples of the experimental rats, the samples were pre-treated according to the method of Example 2 and tested on the machine. The concentrations of different cigarette smoke biomarkers in the control group and each tissue of the experimental group calculated by the working curve are shown in the results. Figure 4 .

[0071] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction, characterized in that: The following steps are involved: Step 1, dissect the experimental rats to obtain various organs, take the lung or liver, add buffer in a ratio of weight (g): volume (mL) = 1:9, homogenize with a handheld homogenizer, centrifuge at 12000rpm for 10min, remove the supernatant, take 200μL of the homogenate, add 120μL of 5% perchloric acid, vortex for 1min, and centrifuge at 12000r / min for 5min, take the supernatant, add 10μL of cortisol D-4 as an internal standard and make its concentration in the sample 100ng / mL, and the above mixed solution is used as the liver or lung sample test solution; Step 2, using a negative pressure venous blood collection tube containing EDTA to collect blood from rats, taking 200 μL of upper plasma after centrifugation, adding 120 μL of 5% perchloric acid, vortexing for 1 min, and centrifuging at 12000 r / min for 5 min, taking the supernatant, adding 10 μL of cortisol D-4 as an internal standard and making its concentration in the sample 100 ng / mL, and the above mixed solution is used as the plasma sample test solution; Step 3, the rat's urine was collected by the restraint tube of the smoking machine, 1 mL of urine was added with 10 μL of β-glucuronidase / arylsulfatase and incubated at 37°C for 4 hours, 500 μL of the above solution was added with 10 μL of cortisol D-4 as an internal standard and the concentration in the sample was 100 ng / mL, and the above mixed solution was used as the urine sample test solution; Step 4, enriching and concentrating the target substance using a solid phase extraction column filled with polystyrene / polypyrrole composite nanofibers in the above tissue sample solutions containing the internal standard; Step 5, using an organic solvent to elute the target substance enriched on the nanofiber solid phase extraction column to obtain a test solution; Step 6, analyzing the test solution by liquid chromatography-mass spectrometry, and quantitatively calculating the concentration of the target substance in urine by using the internal standard method.

2. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: The reagents used in steps 1-3 are all analytically pure reagents.

3. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: The polystyrene nanofibers described in step 4 are prepared by dissolving 1 g of polystyrene particles in an organic solvent having a volume ratio of N,N-dimethylformamide to tetrahydrofuran of 4:6 to form a spinning solution, and using electrospinning technology to make the spinning solution into polystyrene nanofibers; the prepared polystyrene nanofibers are immersed in an aqueous solution containing a certain amount of pyrrole, and a certain amount of FeCl3 is added to the solution and stirred for reaction, and then the polystyrene nanofibers are in situ reduced on the surface of the polystyrene nanofibers to prepare polystyrene / polypyrrole composite nanofibers.

4. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: In step 4, the solid phase extraction column filled with polystyrene / polypyrrole composite nanofibers needs to be activated with methanol and water before use.

5. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: The organic solvent in step 5 is an eluent composed of one or more organic substances such as methanol, ethanol and acetonitrile.

6. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: In step 6, the conditions of liquid chromatography-mass spectrometry are as follows: Chromatographic column: C18 (3.5 μm, 150 mm×2.1 mm); mobile phase: acetonitrile as B: water as A (0.1% formic acid), gradient elution: 0-0.1 min: 5% B, 0.1-20 min: 5% B-100% B, 20-30 min: 100% B, 30-35 min: 100% B-5% B, 35-40 min: 5% B; Ultrasonic degassing after filtration through a 0.45 μm microporous filter membrane; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 30°C; mass spectrometry conditions: electrospray ion source (ESI), negative ion scanning and positive ion scanning modes, and multiple reaction monitoring mode (MRM); spray voltage 3500 V; nebulizer gas pressure 50 polystyrene i; curtain gas pressure 30 psi; auxiliary nebulizer gas pressure 45 psi; ion source temperature gradient 500°C; inlet voltage 10 V; outlet voltage 10 V; dwell time 200 ms.

7. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: The cigarette smoke biomarkers and oxidative stress markers to be tested include cotinine, N-acetyl-S-(2-aminoformylethyl)-L-cysteine ​​(AAMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine ​​(MHBMA), 4-(methylnitrosamino)-1-(3-pyridyl)butanol (NNAL), N-acetyl-S-(3-hydroxypropyl)-L-cysteine ​​(3-HPMA), N-acetyl-S-(3-carboxy-1-methylpropyl)-L-cysteine ​​(3-HMPMA), phenylmercaptopuric acid (SPMA), α-OH naphthalene, β-OH naphthalene, 1-OH phenanthrene, 3-OH phenanthrene, 2-OH fluorene, 9-OH fluorene, 1-OH pyrene and 8-isoprostaglandins.

8. The method for detecting cigarette smoke exposure biomarkers based on nanofiber solid phase extraction according to claim 1, characterized in that: The polystyrene / polypyrrole nanofibers are prepared by firstly using an electrostatic spinning technique to prepare polystyrene nanofibers, which are then used as substrates to perform in-situ polymerization of polypyrrole on the surface of the prepared polystyrene nanofibers, thereby obtaining the polystyrene / polypyrrole composite nanofibers.

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