A method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine

Abstract

The present invention provides a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine, belonging to the field of chemical detection technology. The method involves enzymatic hydrolysis, extraction, purification, nitrogen purging, and derivatization of urine to obtain a test solution, which is then subjected to gas chromatography-mass spectrometry. The detection employs an AEI source and multiple reaction monitoring mode, which increases the yield of ionized products and improves ionization efficiency. This method also enhances the intensity of the response signal, controls collision energy, reduces the detection limit, and improves detection sensitivity, making it more suitable for detecting samples with lower concentrations. It can accurately detect 16 polycyclic aromatic hydrocarbon metabolites simultaneously.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical detection, and in particular relates to a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine. Background Art

[0002] PAHs are mainly derived from the incomplete combustion of organic matter and have mutagenic, teratogenic and carcinogenic effects. When the human body is exposed to an environment containing PAHs, these substances can enter the human body through breathing, skin contact and diet, and the detection of PAH metabolites in urine can well reflect the content of PAHs in the human body. The existing detection method of PAH metabolites in urine is mainly based on low-resolution tandem mass spectrometry of gas chromatography, but the sensitivity of the existing method is relatively low and cannot meet the requirements of simultaneous and accurate detection of multiple metabolites. Therefore, there is an urgent need for a detection method with high sensitivity that can accurately detect multiple PAH metabolites in urine at the same time. Summary of the Invention

[0003] The present invention aims to provide a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine. The detection method provided by the present invention has high sensitivity and can accurately detect 16 polycyclic aromatic hydrocarbon metabolites simultaneously.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine, which comprises the following steps:

[0006] (1) mixing urine with a mixed internal standard solution, an ascorbic acid solution, and an enzyme solution, and performing enzymolysis to obtain an enzymolysis solution;

[0007] (2) mixing the enzymatic hydrolyzate obtained in step (1) with water, toluene and n-pentane, and performing extraction to obtain an extract;

[0008] (3) mixing the extract obtained in step (2) with a silver nitrate solution and purifying the mixture to obtain a purified solution;

[0009] (4) mixing the purified liquid obtained in step (3) with n-dodecane and blowing with nitrogen, and then mixing with toluene, a solution for recovering the internal standard, and a derivatization reagent to obtain a test solution;

[0010] (5) subjecting the test liquid obtained in step (4) to gas chromatography-mass spectrometry to obtain the content of polycyclic aromatic hydrocarbon metabolites in urine; the ion source during the gas chromatography-mass spectrometry detection is an AEI source; the gas chromatography-mass spectrometry detection adopts a multiple reaction monitoring mode; the collision energy of the mass spectrometer during the gas chromatography-mass spectrometry detection is 10 to 35 eV;

[0011] The gas chromatography conditions during the gas chromatography-mass spectrometry detection in step (5) are as follows: an inlet temperature of 250 to 300° C., a carrier gas flow rate of 0.9 to 1 mL / min, a splitless injection method, and an injection volume of 0.9 to 1.1 μL;

[0012] In the step (5), the gas chromatography during the gas chromatography-mass spectrometry detection adopts programmed temperature control of the chromatographic column temperature; the programmed temperature process is as follows: within 0 to 1 minute, the chromatographic column temperature is 90 to 100°C; within 1 to 7.667 minutes, the temperature is increased to 190 to 200°C at a rate of 14 to 16°C / min; within 7.667 to 13.167 minutes, the temperature is increased to 204 to 208°C at a rate of 1 to 3°C / min; within 13.167 to 18.167 minutes, the chromatographic column temperature is 204 to 208°C; within 18.167 to 20.517 minutes, the temperature is increased to 295 to 305°C at a rate of 30 to 50°C / min; within 20.517 to 27.017 minutes, the chromatographic column temperature is 295 to 305°C;

[0013] The mass spectrometry conditions during gas chromatography-mass spectrometry detection in step (5) are: mass spectrometry transmission line temperature is 295-305°C, ion source temperature is 295-305°C, electron energy is 30-40eV, solvent delay time is 4-6min, and collision gas is argon.

[0014] Preferably, the enzyme in the enzyme solution of step (1) comprises β-glucuronidase / arylsulfatase or β-glucuronidase.

[0015] Preferably, the concentration of the enzyme solution in step (1) is 2000-3000 U / mL.

[0016] Preferably, the volume ratio of urine to enzyme solution in step (1) is 1:(0.8-1.2).

[0017] Preferably, the enzymatic hydrolysis temperature in step (1) is 35-38° C., and the enzymatic hydrolysis time is ≥16 h.

[0018] Preferably, the volume ratio of toluene to n-pentane in step (2) is 1:(3-5).

[0019] Preferably, the derivatization reagent in step (4) comprises at least one of N-methyl-N-(trimethylsilyl)trifluoroacetamide, N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide.

[0020] The present invention provides a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine, which comprises the following steps: (1) mixing urine with a mixed internal standard solution, an ascorbic acid solution and an enzyme solution, performing enzymolysis to obtain an enzymolysis solution; (2) mixing the enzymolysis solution obtained in step (1) with water, toluene and n-pentane, performing extraction to obtain an extract; (3) mixing the extract obtained in step (2) with a silver nitrate solution, performing purification to obtain a purified solution; (4) mixing the purified solution obtained in step (3) with n-dodecane, performing nitrogen blowing, and then mixing with toluene, recovering the internal standard solution, and performing nitrogen blowing. The solution and the derivatization reagent are mixed to obtain a test liquid; (5) the test liquid obtained in step (4) is subjected to gas chromatography-mass spectrometry detection to obtain the content of polycyclic aromatic hydrocarbon metabolites in urine; the ion source during the gas chromatography-mass spectrometry detection is an AEI source; the gas chromatography-mass spectrometry detection adopts a multiple reaction monitoring mode; the collision energy of the mass spectrometer during the gas chromatography-mass spectrometry detection is 10 to 35 eV; the gas chromatography conditions during the gas chromatography-mass spectrometry detection in step (5) are: an inlet temperature of 250 to 300 ° C, a carrier gas flow rate of 0.9 to 1 mL / min min, the injection mode is splitless injection, and the injection volume is 0.9-1.1 μL; the gas chromatography during gas chromatography-mass spectrometry detection in step (5) adopts programmed temperature control of the chromatographic column temperature; the programmed temperature process is as follows: within 0-1 min, the chromatographic column temperature is 90-100 ° C; within 1-7.667 min, the temperature is increased to 190-200 ° C at a rate of 14-16 ° C / min; within 7.667-13.167 min, the temperature is increased to 204-208 ° C at a rate of 1-3 ° C / min; within 13.167-18.16 Within 7 minutes, the temperature of the chromatographic column is 204-208°C; within 18.167-20.517 minutes, the temperature is increased to 295-305°C at a rate of 30-50°C / min; within 20.517-27.017 minutes, the temperature of the chromatographic column is 295-305°C; the mass spectrometry conditions during gas chromatography-mass spectrometry detection in step (5) are as follows: the mass spectrometry transmission line temperature is 295-305°C, the ion source temperature is 295-305°C, the electron energy is 30-40 eV, the solvent delay time is 4-6 minutes, and the collision gas is argon. The present invention subjects urine to enzymatic hydrolysis, extraction, purification, nitrogen blowing and derivatization to obtain a test liquid, which is then subjected to gas chromatography-mass spectrometry detection. During detection, an AEI source and a multiple reaction monitoring mode are used, which increases the yield of ionization products and the ionization efficiency. At the same time, the intensity of the response signal is higher, the collision energy is controlled, the detection limit is reduced, and the detection sensitivity is improved. This makes it more suitable for detecting samples with lower concentrations and can accurately detect 16 polycyclic aromatic hydrocarbon metabolites simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing different collision energies and relative abundances of 1-hydroxynaphthalene during mass spectrometry detection in Test Example 1;

[0022] Figure 2 This is a graph showing different collision energies and relative abundances of 2-hydroxynaphthalene during mass spectrometry detection in Test Example 1;

[0023] Figure 3 This is a graph showing different collision energies and relative abundances of 9-hydroxyfluorene during mass spectrometry detection in Test Example 1;

[0024] Figure 4 This is a graph showing different collision energies and relative abundances of 3-hydroxyfluorene during mass spectrometry detection in Test Example 1;

[0025] Figure 5 This is a graph showing different collision energies and relative abundances of 2-hydroxyfluorene during mass spectrometry detection in Test Example 1;

[0026] Figure 6 This is a graph showing different collision energies and relative abundances of 4-hydroxyphenanthrene during mass spectrometry detection in Test Example 1;

[0027] Figure 7 This is a graph showing different collision energies and relative abundances of 9-hydroxyphenanthrene during mass spectrometry detection in Test Example 1;

[0028] Figure 8 This is a graph showing different collision energies and relative abundances of 3-hydroxyphenanthrene during mass spectrometry detection in Test Example 1;

[0029] Figure 9 This is a graph showing different collision energies and relative abundances of 1-hydroxyphenanthrene during mass spectrometry detection in Test Example 1;

[0030] Figure 10 This is a graph showing different collision energies and relative abundances of 2-hydroxyphenanthrene during mass spectrometry detection in Test Example 1;

[0031] Figure 11 This is a graph showing different collision energies and relative abundances of 3-hydroxyfluoranthene during mass spectrometry detection in Test Example 1;

[0032] Figure 12 This is a graph showing different collision energies and relative abundances of 1-hydroxypyrene during mass spectrometry detection in Test Example 1;

[0033] Figure 13 6-hydroxyl group in mass spectrometry detection in Test Example 1 A graph of different collision energies and relative abundances;

[0034] Figure 14 3-hydroxyl group in mass spectrometry detection in Test Example 1 A graph of different collision energies and relative abundances;

[0035] Figure 15 This is a graph showing different collision energies and relative abundances of 3-hydroxybenz[a]anthracene during mass spectrometry detection in Test Example 1;

[0036] Figure 16 This is a graph showing different collision energies and relative abundances of 3-hydroxybenzo[a]pyrene during mass spectrometry detection in Test Example 1;

[0037] Figure 17 is the peak intensity of 1-hydroxynaphthalene and 2-hydroxynaphthalene in Test Example 2 and Comparative Test Example 1;

[0038] Figure 18 is the peak intensity of 9-hydroxyfluorene in Test Example 2 and Comparative Test Example 1;

[0039] Figure 19 is the peak intensity of 3-hydroxyfluorene and 2-hydroxyfluorene in Test Example 2 and Comparative Test Example 1;

[0040] Figure 20 is the peak intensity of 4-hydroxyphenanthrene and 9-hydroxyphenanthrene in Test Example 2 and Comparative Test Example 1;

[0041] Figure 21 is the peak intensity of 3-hydroxyphenanthrene, 1-hydroxyphenanthrene and 2-hydroxyphenanthrene in Test Example 2 and Comparative Test Example 1;

[0042] Figure 22 is the peak intensity of 3-hydroxyfluoranthene and 1-hydroxypyrene in Test Example 2 and Comparative Test Example 1;

[0043] Figure 23 6-hydroxy in Test Example 2 and Comparative Test Example 1 3-Hydroxy and peak intensities of 3-hydroxybenz[a]anthracene;

[0044] Figure 24 is the peak intensity of 3-hydroxybenzo[a]pyrene in Test Example 2 and Comparative Test Example 1. DETAILED DESCRIPTION

[0045] The present invention provides a method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine, which comprises the following steps:

[0046] (1) mixing urine with a mixed internal standard solution, an ascorbic acid solution, and an enzyme solution, and performing enzymolysis to obtain an enzymolysis solution;

[0047] (2) mixing the enzymatic hydrolyzate obtained in step (1) with water, toluene and n-pentane, and performing extraction to obtain an extract;

[0048] (3) mixing the extract obtained in step (2) with a silver nitrate solution and purifying the mixture to obtain a purified solution;

[0049] (4) mixing the purified liquid obtained in step (3) with n-dodecane and blowing with nitrogen, and then mixing with toluene, a solution for recovering the internal standard, and a derivatization reagent to obtain a test solution;

[0050] (5) subjecting the test solution obtained in step (4) to gas chromatography-mass spectrometry to obtain the content of polycyclic aromatic hydrocarbon metabolites in urine;

[0051] The gas chromatography conditions during the gas chromatography-mass spectrometry detection in step (5) are as follows: an inlet temperature of 250 to 300° C., a carrier gas flow rate of 0.9 to 1 mL / min, a splitless injection method, and an injection volume of 0.9 to 1.1 μL;

[0052] In the step (5), the gas chromatography during the gas chromatography-mass spectrometry detection adopts programmed temperature control of the chromatographic column temperature; the programmed temperature process is as follows: within 0 to 1 minute, the chromatographic column temperature is 90 to 100°C; within 1 to 7.667 minutes, the temperature is increased to 190 to 200°C at a rate of 14 to 16°C / min; within 7.667 to 13.167 minutes, the temperature is increased to 204 to 208°C at a rate of 1 to 3°C / min; within 13.167 to 18.167 minutes, the chromatographic column temperature is 204 to 208°C; within 18.167 to 20.517 minutes, the temperature is increased to 295 to 305°C at a rate of 30 to 50°C / min; within 20.517 to 27.017 minutes, the chromatographic column temperature is 295 to 305°C;

[0053] The mass spectrometry conditions during gas chromatography-mass spectrometry detection in step (5) are: mass spectrometry transmission line temperature is 295-305°C, ion source temperature is 295-305°C, electron energy is 30-40eV, solvent delay time is 4-6min, and collision gas is argon.

[0054] Unless otherwise specified, the present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0055] The detection method of the present invention is used to detect polycyclic aromatic hydrocarbon metabolites in urine, including 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxypyrene, 3-Hydroxy 3-Hydroxybenzo[a]anthracene and 3-hydroxybenzo[a]pyrene.

[0056] The invention mixes urine with a mixed internal standard solution, an ascorbic acid solution and an enzyme solution, performs enzymolysis, and obtains an enzymolysis solution.

[0057] In the present invention, when the urine is frozen urine, the present invention preferably thaws the frozen urine. The present invention preferably places the frozen urine at 3-5°C for more than 8 hours and then returns it to room temperature; or thaws the frozen urine at room temperature in the dark or under yellow light.

[0058] In the present invention, the urine is preferably vortex mixed before use. The present invention has no particular limitation on the operation of the vortex mixing, and a technical solution well known to those skilled in the art can be used to ensure that the urine is uniform.

[0059] In the present invention, the internal standard substance in the mixed internal standard solution preferably includes 13 C6-1-hydroxynaphthalene, 13 C6-2-hydroxynaphthalene, 13 C6-9-hydroxyfluorene, 13 C6-3-hydroxyfluorene, 13 C6-2-hydroxyfluorene, 13 C6-4-hydroxyphenanthrene, 13 C6-3-hydroxyphenanthrene, 13 C6-1-hydroxyphenanthrene, D9-2-hydroxyphenanthrene, D9-3-hydroxyfluoranthene, 13 C6-1-hydroxypyrene, D 11 -3-hydroxy and D 11 -3-hydroxybenzo[a]pyrene.

[0060] In the present invention, the concentration of each internal standard substance in the mixed internal standard solution is preferably 20-30 μg / L, more preferably 25 μg / L.

[0061] In the present invention, the volume ratio of the urine to the mixed internal standard solution is preferably (10-15):1, more preferably (12-14):1, and further preferably 12.5:1.

[0062] In the present invention, the concentration of the ascorbic acid solution is preferably 0.2-0.3 mg / L, more preferably 0.25 mg / L. In the present invention, the ascorbic acid solution acts as an antioxidant to prevent oxidative damage to the enzyme, promote the uncoupling of 1-hydroxynaphthalene and 9-hydroxyfluorene, and shorten the uncoupling time.

[0063] In the present invention, the volume ratio of the urine to the ascorbic acid solution is preferably (90-110):1, more preferably 100:1.

[0064] In the present invention, the enzyme in the enzyme solution preferably includes β-glucuronidase / arylsulfatase or β-glucuronidase, more preferably β-glucuronidase / arylsulfatase. The present invention uses β-glucuronidase / arylsulfatase to achieve better enzymatic hydrolysis effect.

[0065] In the present invention, the ratio of the enzyme activities of β-glucuronidase and arylsulfatase in the β-glucuronidase / arylsulfatase is preferably (25-35):1, more preferably 30:1.

[0066] In the present invention, the solvent in the enzyme solution is preferably a sodium acetate buffer solution.

[0067] In the present invention, the concentration of the enzyme solution is preferably 2000-3000 U / mL, more preferably 2500-3000 U / mL; the pH value of the enzyme solution is preferably 5-6, more preferably 5.5.

[0068] In the present invention, the volume ratio of the urine to the enzyme solution is preferably 1:(0.8-1.2), more preferably 1:1.

[0069] In the present invention, the mixing of the urine, the mixed internal standard solution, the ascorbic acid solution and the enzyme solution is preferably performed by shaking. The present invention has no particular limitation on the shaking operation, as long as the raw materials are uniformly mixed.

[0070] In the present invention, the enzymatic hydrolysis temperature is preferably 35-38°C, more preferably 36-37°C; the enzymatic hydrolysis time is preferably ≥16 hours; and the enzymatic hydrolysis is preferably performed in the dark. By controlling the amount of each raw material, the enzymatic hydrolysis temperature, and the time within the above ranges, the present invention can ensure sufficient enzymatic hydrolysis and further improve detection sensitivity.

[0071] After the enzymatic hydrolysis is completed, the enzymatic hydrolysis product is preferably allowed to stand at room temperature to obtain an enzymatic hydrolysis solution.

[0072] After obtaining the enzymatic hydrolysate, the present invention mixes the enzymatic hydrolysate with water, toluene and n-pentane, and performs extraction to obtain an extract.

[0073] In the present invention, the water is preferably pure water.

[0074] In the present invention, the volume ratio of urine to water is preferably 1:(1-3), more preferably 1:2.

[0075] In the present invention, the volume ratio of toluene to n-pentane is preferably 1:(3-5), more preferably 1:4.

[0076] In the present invention, the ratio of the volume of water to the total volume of toluene and n-pentane is preferably 1:(2-3), more preferably 1:2.5.

[0077] In the present invention, the extraction time is preferably 5 to 15 minutes, more preferably 10 minutes; and the extraction is preferably carried out under shaking conditions.

[0078] After the extraction is completed, the present invention preferably centrifuges the extracted product to obtain a first supernatant and a raffinate.

[0079] In the present invention, the centrifugal speed is preferably 3000-4000 rpm, more preferably 3500 rpm; the centrifugal time is preferably 5-15 min, more preferably 10 min.

[0080] After obtaining the raffinate, the present invention preferably mixes the raffinate with toluene and n-pentane, performs extraction, and then centrifuges to obtain a second supernatant.

[0081] In the present invention, the volume ratio of toluene to n-pentane is preferably 1:(3-5), more preferably 1:4.

[0082] In the present invention, the amounts of toluene and n-pentane used are preferably the same as those used in the above extraction.

[0083] In the present invention, the extraction time is preferably 5 to 15 minutes, more preferably 10 minutes; and the extraction is preferably carried out under shaking conditions.

[0084] In the present invention, the centrifugal speed is preferably 3000-4000 rpm, more preferably 3500 rpm; the centrifugal time is preferably 5-15 min, more preferably 10 min.

[0085] The present invention preferably combines the first supernatant and the second supernatant to obtain an extract. The present invention controls various parameters during extraction to allow the substance to be detected to enter the extract more fully, further improving detection sensitivity and accuracy.

[0086] After obtaining the extract, the present invention mixes the extract with a silver nitrate solution and purifies the mixture to obtain a purified solution.

[0087] In the present invention, the concentration of the silver nitrate solution is preferably 0.5 to 1.5 mol / L, more preferably 1 mol / L.

[0088] In the present invention, the volume ratio of the urine to the silver nitrate solution is preferably 1: (0.5-1.5), more preferably 1: 1. The addition of silver nitrate in the present invention reduces the interference of polycyclic aromatic hydrocarbon sulfur metabolites.

[0089] In the present invention, the purification time is preferably 5 to 15 minutes, more preferably 10 minutes; the purification is preferably performed under shaking conditions. The present invention controls the concentration, dosage, and purification time of the silver nitrate solution within the above ranges to achieve more complete purification.

[0090] After purification is completed, the present invention preferably centrifuges the purified product to obtain a purified liquid.

[0091] In the present invention, the centrifugal speed is preferably 3000-4000 rpm, more preferably 3500 rpm; the centrifugal time is preferably 1-10 min, more preferably 5 min.

[0092] After obtaining the purified liquid, the present invention mixes the purified liquid with n-dodecane and blows nitrogen, and then mixes it with toluene, a solution for recovering the internal standard, and a derivatization reagent to obtain a test liquid.

[0093] In the present invention, the volume ratio of urine to n-dodecane is preferably (90-110):1, more preferably 100:1.

[0094] In the present invention, the nitrogen blowing is preferably: blowing at 35-45°C for 5-15 minutes, and then blowing at 55-65°C to near dryness; more preferably blowing at 40°C for 10 minutes, and then blowing at 60°C to near dryness.

[0095] In the present invention, the volume ratio of urine to toluene is preferably (40-60):1, more preferably 50:1.

[0096] In the present invention, the solution for recovering the internal standard is preferably 13 C 12 -PCB105 toluene solution; the concentration of the recovered internal standard solution is preferably 45-55 μg / L, more preferably 50 μg / L.

[0097] In the present invention, the volume ratio of the urine to the solution for recovering the internal standard is preferably (90-110):1, more preferably 100:1.

[0098] In the present invention, the derivatization agent preferably includes at least one of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), and more preferably N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). The use of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) as a derivatization agent in the present invention has better effects than the other two.

[0099] In the present invention, the volume ratio of the urine to the derivatization reagent is preferably (90-110):1, more preferably 100:1.

[0100] In the present invention, the mixing is preferably performed by shaking; and the mixing is preferably performed in the dark.

[0101] After the mixing is completed, the present invention preferably places the mixed product in the dark at 55-65° C. for 40-60 minutes to obtain the test solution; more preferably places the mixed product in the dark at 60° C. for 50 minutes to obtain the test solution.

[0102] After obtaining the test liquid, the present invention performs gas chromatography-mass spectrometry on the test liquid to obtain the content of polycyclic aromatic hydrocarbon metabolites in urine.

[0103] In the present invention, the gas chromatography conditions during the gas chromatography-mass spectrometry detection are: the injection port temperature is 250-300°C, the carrier gas is helium, the carrier gas flow rate is in constant flow mode, 0.9-1 mL / min, the injection method is splitless injection, and the injection volume is 0.9-1.1 μL; preferably: the injection port temperature is 300°C, the carrier gas is helium, the carrier gas flow rate is in constant flow mode, 0.9 mL / min, the injection method is splitless injection, and the injection volume is 1 μL.

[0104] In the present invention, the gas chromatography column preferably has a 95% dimethylpolysiloxane (5% diphenyl) stationary phase filler, a column length of 20 to 40 m, a diameter of 0.2 to 0.3 mm, and a filler film thickness of 0.2 to 0.3 μm; more preferably, a 95% dimethylpolysiloxane (5% diphenyl) stationary phase filler, a column length of 30 m, a diameter of 0.25 mm, and a filler film thickness of 0.25 μm. In the present invention, the preferred model of the chromatographic column is TG-5SilMS.

[0105] In the present invention, the gas chromatography uses programmed temperature to control the temperature of the chromatographic column; the programmed temperature process is: within 0 to 1 minute, the chromatographic column temperature is 90 to 100 ° C; within 1 to 7.667 minutes, the temperature is increased to 190 to 200 ° C at a rate of 14 to 16 ° C / min; within 7.667 to 13.167 minutes, the temperature is increased to 204 to 208 ° C at a rate of 1 to 3 ° C / min; within 13.167 to 18.167 minutes, the chromatographic column temperature is 204 to 208 ° C; within 18.167 to 20.517 minutes, the temperature is increased to 295 to 305 ° C at a rate of 30 to 50 ° C / min; Within 0.517 to 27.017 minutes, the temperature of the chromatographic column is 295 to 305°C; preferably, within 0 to 1 minute, the temperature of the chromatographic column is 95°C; within 1 to 7.667 minutes, the temperature is increased to 195°C at a rate of 15°C / min; within 7.667 to 13.167 minutes, the temperature is increased to 206°C at a rate of 2°C / min; within 13.167 to 18.167 minutes, the temperature of the chromatographic column is 206°C; within 18.167 to 20.517 minutes, the temperature is increased to 300°C at a rate of 40°C / min; within 20.517 to 27.017 minutes, the temperature of the chromatographic column is 300°C.

[0106] In the present invention, the ion source of the mass spectrometer during the gas chromatography-mass spectrometry detection is an AEI source; the mass spectrometer adopts a multiple reaction monitoring mode during the gas chromatography-mass spectrometry detection; and the collision energy of the mass spectrometer during the gas chromatography-mass spectrometry detection is 10 to 35 eV.

[0107] In the present invention, the AEI source is preferably purchased from Thermo Fisher Scientific. The present invention employs an AEI source and multiple reaction monitoring mode to increase the yield of ionized products and ionization efficiency, while also providing a higher intensity response signal, lowering the detection limit and improving detection sensitivity, making it more suitable for detecting samples with lower concentrations.

[0108] In the present invention, the mass spectrometry conditions during gas chromatography-mass spectrometry detection are: the mass spectrometry transmission line temperature is 295-305°C, the ion source temperature is 295-305°C, the electron energy is 30-40eV, the solvent delay time is 4-6min, and the collision gas is argon; preferably: the mass spectrometry transmission line temperature is 300°C, the ion source temperature is 300°C, the electron energy is 35eV, the solvent delay time is 5min, and the collision gas is argon.

[0109] In the present invention, the quantitative parent ion (m / z) of 1-hydroxynaphthalene in the polycyclic aromatic hydrocarbon metabolites during mass spectrometry detection is preferably 216.0, the quantitative daughter ion (m / z) is preferably 185.0, and the collision energy is preferably 20 eV; the qualitative parent ion (m / z) is preferably 216.0, the qualitative daughter ion (m / z) is preferably 201.0, and the collision energy is preferably 10 eV; the retention time is preferably 7.80 min; the quantitative internal standard is preferably 13 The quantitative parent ion (m / z) of C6-1-hydroxynaphthalene and 2-hydroxynaphthalene is preferably 216.0, the quantitative daughter ion (m / z) is preferably 185.0, and the collision energy is preferably 20 eV; the qualitative parent ion (m / z) is preferably 216.0, the qualitative daughter ion (m / z) is preferably 201.0, and the collision energy is preferably 10 eV; the retention time is preferably 8.01 min; the quantitative internal standard is preferably 13 The quantitative parent ion (m / z) of C6-2-hydroxynaphthalene and 9-hydroxyfluorene is preferably 254.0, the quantitative daughter ion (m / z) is preferably 165.0, and the collision energy is preferably 23 eV; the qualitative parent ion (m / z) is preferably 239.0, the qualitative daughter ion (m / z) is preferably 165.0, and the collision energy is preferably 23 eV; the retention time is preferably 10.51 min; the quantitative internal standard is preferably 13C6-9-hydroxyfluorene; the quantitative parent ion (m / z) of 3-hydroxyfluorene is preferably 254.0, the quantitative daughter ion (m / z) is preferably 165.0, and the collision energy is preferably 20 eV; the qualitative parent ion (m / z) is preferably 254.0, the qualitative daughter ion (m / z) is preferably 239.0, and the collision energy is preferably 10 eV; the retention time is preferably 12.76 min; the quantitative internal standard is preferably 13 C6-3-hydroxyfluorene; the quantitative parent ion (m / z) of 2-hydroxyfluorene is preferably 254.0, the quantitative daughter ion (m / z) is preferably 165.0, and the collision energy is preferably 25 eV; the qualitative parent ion (m / z) is preferably 254.0, the qualitative daughter ion (m / z) is preferably 239.0, and the collision energy is preferably 10 eV; the retention time is preferably 13.15 min; the quantitative internal standard is preferably 13 C6-2-hydroxyfluorene; the quantitative parent ion (m / z) of 4-hydroxyphenanthrene is preferably 266.0, the quantitative daughter ion (m / z) is preferably 235.0, and the collision energy is preferably 20 eV; the qualitative parent ion (m / z) is preferably 266.0, the qualitative daughter ion (m / z) is preferably 251.0, and the collision energy is preferably 10 eV; the retention time is preferably 14.99 min; the quantitative internal standard is preferably 13 The quantitative parent ion (m / z) of C6-4-hydroxyphenanthrene and 9-hydroxyphenanthrene is preferably 251.0, the quantitative daughter ion (m / z) is preferably 235.0, and the collision energy is preferably 10 eV; the qualitative parent ion (m / z) is preferably 266.0, the qualitative daughter ion (m / z) is preferably 251.0, and the collision energy is preferably 10 eV; the retention time is preferably 15.96 min; the quantitative internal standard is preferably 13 The quantitative parent ion (m / z) of C6-4-hydroxyphenanthrene; 3-hydroxyphenanthrene is preferably 266.0, the quantitative daughter ion (m / z) is preferably 251.0, and the collision energy is preferably 10 eV; the qualitative parent ion (m / z) is preferably 266.0, the qualitative daughter ion (m / z) is preferably 235.0, and the collision energy is preferably 20 eV; the retention time is preferably 16.73 min; the quantitative internal standard is preferably 13 C6-3-hydroxyphenanthrene; the quantitative parent ion (m / z) of 1-hydroxyphenanthrene is preferably 266.0, the quantitative daughter ion (m / z) is preferably 235.0, and the collision energy is preferably 20 eV; the qualitative parent ion (m / z) is preferably 266.0, the qualitative daughter ion (m / z) is preferably 251.0, and the collision energy is preferably 10 eV; the retention time is preferably 16.89 min; the quantitative internal standard is preferably 13C6-1-hydroxyphenanthrene; the quantitative parent ion (m / z) of 2-hydroxyphenanthrene is preferably 266.0, the quantitative daughter ion (m / z) is preferably 251.0, and the collision energy is preferably 10 eV; the qualitative parent ion (m / z) is preferably 266.0, the qualitative daughter ion (m / z) is preferably 235.0, and the collision energy is preferably 20 eV; the retention time is preferably 17.92 min; the quantitative internal standard is preferably D9-2-hydroxyphenanthrene; the quantitative parent ion (m / z) of 3-hydroxyfluoranthene is preferably 290.1, the quantitative daughter ion (m / z) is preferably 275.2, and the collision energy is preferably 10 eV; the qualitative parent ion The quantification parent ion (m / z) is preferably 275.1, the quantification daughter ion (m / z) is preferably 215.2, and the collision energy is preferably 20 eV; the retention time is preferably 20.80 min; the quantitative internal standard is preferably D9-3-hydroxyfluoranthene; the quantitative parent ion (m / z) of 1-hydroxypyrene is preferably 290.0, the quantification daughter ion (m / z) is preferably 258.9, and the collision energy is preferably 25 eV; the quantification parent ion (m / z) is preferably 290.0, the quantification daughter ion (m / z) is preferably 275.0, and the collision energy is preferably 10 eV; the retention time is preferably 21.06 min; the quantitative internal standard is preferably 13 C6-1-hydroxypyrene; 6-hydroxy The quantitative parent ion (m / z) is preferably 316.0, the quantitative daughter ion (m / z) is preferably 301.0, and the collision energy is preferably 10 eV; the qualitative parent ion (m / z) is preferably 316.0, the qualitative daughter ion (m / z) is preferably 281.0, and the collision energy is preferably 30 eV; the retention time is preferably 22.44 min; the quantitative internal standard is preferably D 11 -3-hydroxy 3-Hydroxy The quantitative parent ion (m / z) is preferably 316.1, the quantitative daughter ion (m / z) is preferably 270.0, and the collision energy is preferably 35 eV; the qualitative parent ion (m / z) is preferably 281.0, the qualitative daughter ion (m / z) is preferably 207.0, and the collision energy is preferably 30 eV; the retention time is preferably 23.05 min; the quantitative internal standard is preferably D 11 -3-hydroxy The quantitative parent ion (m / z) of 3-hydroxybenz[a]anthracene is preferably 301.1, the quantitative daughter ion (m / z) is preferably 226.1, and the collision energy is preferably 34 eV; the qualitative parent ion (m / z) is preferably 316.1, the qualitative daughter ion (m / z) is preferably 301.1, and the collision energy is preferably 10 eV; the retention time is preferably 23.32 min; the quantitative internal standard is preferably D9-3-hydroxyfluoranthene; the quantitative parent ion (m / z) of 3-hydroxybenzo[a]pyrene is preferably 340.0, the quantitative daughter ion (m / z) is preferably 324.9, and the collision energy is preferably 12 eV; the qualitative parent ion (m / z) is preferably 340.0, the qualitative daughter ion (m / z) is preferably 308.9, and the collision energy is preferably 22 eV; the retention time is preferably 26.76 min; the quantitative internal standard is preferably D 11 -3-hydroxybenzo[a]pyrene.

[0110] The present invention preferably utilizes gas chromatography for separation, qualitative analysis based on retention time and mass spectrometric characteristics, and quantitative analysis using isotope labeling. A standard curve is drawn with the ratio of the peak area of ​​the target analyte to the corresponding isotope internal standard as the ordinate and the ratio of the mass concentration of the target analyte to the corresponding isotope internal standard as the abscissa. The content of polycyclic aromatic hydrocarbon metabolites in urine is then calculated based on the standard curve.

[0111] The present invention preferably prepares a series of mixed polycyclic aromatic hydrocarbon metabolite standard solutions, which are then mixed with a mixed internal standard solution, an injection internal standard solution, and a derivatization reagent, respectively, and subjected to gas chromatography-mass spectrometry detection. A standard curve is drawn with the ratio of the peak area of ​​the target analyte to the corresponding isotope internal standard as the ordinate and the ratio of the mass concentration of the target analyte to the corresponding isotope internal standard as the abscissa.

[0112] In the present invention, the polycyclic aromatic hydrocarbon metabolites preferably include 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxy 3-Hydroxy 3-Hydroxybenzo[a]anthracene and 3-hydroxybenzo[a]pyrene.

[0113] In the present invention, the concentrations of 1-hydroxynaphthalene and 2-hydroxynaphthalene in the series of mixed polycyclic aromatic hydrocarbon metabolite standard solutions are preferably 2-3 μg / L, 10-15 μg / L, 20-30 μg / L, 100-150 μg / L, 220-260 μg / L, 1200-1500 μg / L and 2200-2600 μg / L, more preferably 2.5 μg / L. g / L, 12.5 μg / L, 25 μg / L, 125 μg / L, 250 μg / L, 1250 μg / L and 2500 μg / L; 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxyphenanthrene, 1-hydroxypyrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxyphenanthrene, 1-hydroxypyrene, 2-hydroxyphenanthrene, 1-hydroxyfluoranthene ... 3-Hydroxy The concentrations of 3-hydroxybenz[a]anthracene and 3-hydroxybenzo[a]pyrene are preferably 0.3-0.6 μg / L, 2-3 μg / L, 4-6 μg / L, 20-30 μg / L, 40-60 μg / L, 220-260 μg / L and 450-550 μg / L, respectively, from low to high, and more preferably 0.5 μg / L, 2.5 μg / L, 5 μg / L, 25 μg / L, 50 μg / L, 250 μg / L and 500 μg / L.

[0114] In the present invention, the internal standard substance in the mixed internal standard solution preferably includes 13 C6-1-hydroxynaphthalene, 13 C6-2-hydroxynaphthalene, 13 C6-9-hydroxyfluorene, 13 C6-3-hydroxyfluorene, 13 C6-2-hydroxyfluorene, 13 C6-4-hydroxyphenanthrene, 13 C6-3-hydroxyphenanthrene, 13 C6-1-hydroxyphenanthrene, D9-2-hydroxyphenanthrene, D9-3-hydroxyfluoranthene, 13 C6-1-hydroxypyrene, D 11 -3-hydroxy and D 11 -3-hydroxybenzo[a]pyrene.

[0115] In the present invention, the concentration of each internal standard substance in the mixed internal standard solution is preferably 20-30 μg / L, more preferably 25 μg / L.

[0116] In the present invention, the volume ratio of the mixed polycyclic aromatic hydrocarbon metabolite standard solution to the mixed internal standard solution is preferably 1:(0.5-1.5), more preferably 1:1.

[0117] In the present invention, the injection internal standard solution is preferably13 C 12 -Toluene solution of PCB105; the concentration of the injection internal standard solution is preferably 45-55 μg / L, more preferably 50 μg / L.

[0118] In the present invention, the volume ratio of the mixed polycyclic aromatic hydrocarbon metabolite standard solution to the injection internal standard solution is preferably 1:(0.5-1.5), more preferably 1:1.

[0119] In the present invention, the derivatization agent preferably includes at least one of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), more preferably N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA).

[0120] In the present invention, the volume ratio of the mixed polycyclic aromatic hydrocarbon metabolite standard solution to the derivatization reagent is preferably 1:(0.5-1.5), more preferably 1:1.

[0121] In the present invention, the mixing is preferably performed by shaking; and the mixing is preferably performed in the dark.

[0122] After the mixing is completed, the present invention preferably places the mixed product in the dark at 55-65° C. for 40-60 minutes; more preferably places it in the dark at 60° C. for 50 minutes.

[0123] In the present invention, the parameters of the gas chromatography-mass spectrometry detection are preferably the same as the gas chromatography-mass spectrometry parameters during urine detection, and will not be described in detail here.

[0124] The present invention has no special limitation on the operation of drawing the standard curve with the ratio of the peak area of ​​the target analyte to the corresponding isotope internal standard as the ordinate and the ratio of the mass concentration of the target analyte to the corresponding isotope internal standard as the abscissa, and the technical solutions familiar to those skilled in the art can be used.

[0125] The present invention has no particular limitation on the operation of calculating the content of polycyclic aromatic hydrocarbon metabolites in urine according to the standard curve, and a technical solution well known to those skilled in the art can be used.

[0126] The present invention subjects urine to enzymatic hydrolysis, extraction, purification, nitrogen blowing and derivatization to obtain a test liquid, which is then subjected to gas chromatography-mass spectrometry detection. During detection, an AEI source and a multiple reaction monitoring mode are used, and collision energy is controlled, thereby increasing the yield of ionization products and the ionization efficiency. At the same time, the intensity of the response signal is higher, making it more suitable for detecting samples with lower concentrations. The detection method has the advantages of suitable detection limit and quantification limit, high recovery rate, high precision, and small matrix effect, and can accurately detect 16 polycyclic aromatic hydrocarbon metabolites simultaneously.

[0127] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0128] The preparation methods and concentrations of the components of the 16 PAH metabolite mixed standard stock solutions, 16 PAH metabolite mixed standard working solutions, 16 PAH metabolite mixed internal standard stock solutions, and 16 PAH metabolite mixed internal standard working solutions described in each embodiment are as follows:

[0129] Accurately weigh appropriate amounts of 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxy 3-Hydroxy 3-Hydroxybenz[a]anthracene and 3-hydroxybenzo[a]pyrene were dissolved in toluene and diluted to 10 mL, and stored at -20°C in the dark to obtain a mixed standard stock solution of 16 polycyclic aromatic hydrocarbon metabolites. The concentrations of each component in the mixed standard stock solution of 16 polycyclic aromatic hydrocarbon metabolites are shown in Table 1.

[0130] Table 1 Concentration of each component in the mixed standard stock solution of 16 PAH metabolites

[0131]

[0132] Accurately transfer 1.00 mL of the mixed standard stock solution of 16 PAH metabolites to a 10 mL volumetric flask, dilute to volume with toluene, mix thoroughly, and store at -20°C in the dark to obtain the mixed standard working solution of 16 PAH metabolites. The concentrations of the components in the mixed standard working solution of 16 PAH metabolites are shown in Table 2.

[0133] Table 2 Concentration of each component in the mixed standard working solution of 16 PAH metabolites

[0134]

[0135]

[0136] Accurately weigh the appropriate amount 13 C6-1-hydroxynaphthalene, 13 C6-2-hydroxynaphthalene, 13 C6-9-hydroxyfluorene, 13 C6-3-hydroxyfluorene, 13 C6-2-hydroxyfluorene, 13 C6-4-hydroxyphenanthrene, 13 C6-3-hydroxyphenanthrene, 13 C6-1-hydroxyphenanthrene, D9-2-hydroxyphenanthrene, D9-3-hydroxyfluoranthene, 13 C6-1-hydroxypyrene, D 11 -3-hydroxy and D 11 -3-Hydroxybenzo[a]pyrene was placed in a volumetric flask, diluted to the mark with toluene, and stored at -20°C in the dark until use. A mixed internal standard stock solution of 16 polycyclic aromatic hydrocarbon metabolites was obtained. The concentrations of the components in the mixed internal standard stock solution of 16 polycyclic aromatic hydrocarbon metabolites are shown in Table 3.

[0137] Table 3 Concentration of each component in the mixed internal standard stock solution of 16 PAH metabolites

[0138]

[0139] Accurately transfer 2.5 mL of the mixed internal standard stock solution of 16 PAH metabolites to a 100 mL volumetric flask, dilute to volume with toluene, mix thoroughly, and store at 4°C in the dark to obtain the mixed internal standard working solution of 16 PAH metabolites. The concentrations of the components in the mixed internal standard working solution of 16 PAH metabolites are shown in Table 4.

[0140] Table 4 Concentration of each component in the mixed internal standard solution of 16 PAH metabolites

[0141]

[0142] Example 1

[0143] The method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine is as follows: (1) the urine sample to be tested is taken out of a -80°C refrigerator, placed in a 4°C refrigerator to fully thaw, then taken out of the 4°C refrigerator, placed at room temperature, vortexed and mixed, 1 mL of urine is taken, 80 μL of a mixed internal standard solution of 16 polycyclic aromatic hydrocarbon metabolites, 10 μL of ascorbic acid solution (0.25 mg / L) and 1 mL of β-glucuronidase / sulfate arylesterase-sodium acetate buffer solution (3000 U / mL, pH=5.5, β-glucuronidase / sulfate arylesterase enzyme activity ratio of 30:1) are added, the sample is fully shaken to mix, and enzymatically hydrolyzed at 37°C in the dark for 16 hours, taken out and allowed to stand at room temperature to obtain an enzymatic solution;

[0144] (2) Add 2 mL of pure water (the volume ratio of urine to pure water is 1:2, and the ratio of the volume of pure water to the total volume of toluene and n-pentane is 1:2.5) and 5 mL of a mixed solution of toluene-n-pentane (volume ratio 1:4) to the obtained enzymatic hydrolyzate, shake for 10 minutes, centrifuge at 3500 rpm for 10 minutes, take the supernatant and transfer it to another glass centrifuge tube, add 5 mL of a mixed solution of toluene-n-pentane (volume ratio 1:4) to the remaining solution, shake for 10 minutes, centrifuge at 3500 rpm for 10 minutes, take the supernatant, and combine the supernatants of the two extractions to obtain an extract;

[0145] (3) Add 1 mL of silver nitrate solution (1 mol / L, volume ratio of urine to silver nitrate solution is 1:1) to the obtained extract, shake for 10 minutes, and centrifuge at 3500 rpm for 5 minutes to obtain a purified solution;

[0146] (4) The purified solution was poured into a nitrogen-purged test tube, 10 μL of n-dodecane (the volume ratio of urine to n-dodecane was 100:1) was added, and the solution was placed in a nitrogen concentrator. After concentrating at 40°C for 10 minutes, the temperature was raised to 60°C and concentrated to near dryness. 20 μL of toluene (the volume ratio of urine to toluene was 50:1) was added to the nitrogen-purged test tube for redissolution, and then the solution was transferred to a nitrogen-purged test tube to which 10 μL of the recovered internal standard solution ( 13 C 12 -PCB105 toluene solution, 50 μg / L) and 10 μL N-methyl-N-(trimethylsilyl)trifluoroacetamide (the volume ratio of urine to the recovered internal standard solution is 100:1, and the volume ratio of urine to N-methyl-N-(trimethylsilyl)trifluoroacetamide is 100:1) are placed in a brown injection vial, shaken to mix, and placed in the dark at 60°C for 50 min to obtain the test solution;

[0147] (5) The obtained test liquid was subjected to gas chromatography-mass spectrometry detection, and the gas chromatography conditions were as follows: injection port temperature 300°C; carrier gas was helium, constant flow mode, carrier gas flow rate was 0.9 mL / min; injection method was splitless injection, injection volume was 1 μL; chromatographic column specifications: 95% dimethylpolysiloxane (5% diphenyl) stationary phase filler, chromatographic column length 30m, diameter 0.25mm, filler film thickness 0.25μm, model TG-5SilMS; programmed temperature was used to control the chromatographic column temperature, and the programmed temperature process was as follows: within 0 to 1 minute, the chromatographic column temperature was 95°C; within 1 to 7.667 minutes, the temperature was uniformly increased to 195°C at a rate of 15°C / min. From 7.667 to 13.167 min, the temperature was uniformly increased at a rate of 2°C / min to 206°C; from 13.167 to 18.167 min, this temperature was maintained; from 18.167 to 20.517 min, the temperature was uniformly increased at a rate of 40°C / min to 300°C; from 20.517 to 27.017 min, this temperature was maintained; the mass spectrometry conditions were as follows: AEI source, multiple reaction monitoring mode, mass spectrometry transfer line temperature of 300°C, ion source temperature of 300°C, electron energy of 35 eV, solvent delay time of 5 min, collision gas of argon, and multiple reaction monitoring conditions and retention times of 16 PAH metabolites and internal standard substances are shown in Table 5;

[0148] (6) Use toluene to dilute the 16 PAH metabolite mixed standard solution step by step to prepare the 16 PAH metabolite standard series solution. Then, take 10 μL of the 16 PAH metabolite standard series solution and transfer it to the 10 μL internal standard solution and 10 μL injection internal standard solution. 13 C 12 -PCB105 toluene solution, 50 μg / L) and 10 μL MSTFA in a brown injection vial, after shaking and mixing, placed in the dark at 60°C for 50 minutes and then subjected to gas chromatography-mass spectrometry detection. The detection conditions are the same as those in step (5). The ratio of the peak area of ​​the target analyte to the corresponding isotope internal standard is used as the vertical axis, and the ratio of the mass concentration of the target analyte to the corresponding isotope internal standard is used as the horizontal axis. A calibration curve is drawn, and then the content of polycyclic aromatic hydrocarbon metabolites in urine is obtained based on the standard curve and the detection results in step (5). The concentrations of each target analyte and the mixed internal standard solution corresponding to the 16 polycyclic aromatic hydrocarbon metabolites in the standard series of solutions are shown in Table 6.

[0149] Table 5 Multiple reaction monitoring conditions and retention times of 16 PAH metabolites and internal standard substances in step (5) of Example 1

[0150]

[0151]

[0152] Note: * indicates quantitative ion.

[0153] Table 6 Concentrations of the target substances to be measured and the mixed internal standard solutions corresponding to the 16 PAH metabolites in the standard series solutions of the 16 PAH metabolites in step (6) of Example 1

[0154]

[0155]

[0156] The accuracy of the method of Example 1 was evaluated by a spike recovery test. Prepare an artificial urine sample (composition of artificial urine: calcium chloride dihydrate 3.8mmol / L, magnesium chloride hexahydrate 3.0mmol / L, sodium chloride 72.1mmol / L, sodium sulfate 14.5mmol / L, trisodium citrate dihydrate 2.2mmol / L, sodium oxalate 0.15mmol / L, potassium dihydrogen phosphate 18.7mmol / L, potassium chloride 19.3mmol / L, ammonium chloride 17.2mmol / L, urea 41.6mmol / L, total sodium The sample was prepared by adding 108.0 mmol / L of sodium hydroxide, 38.0 mmol / L of potassium, and pH 5.7, and three concentrations of standard substances were added (low concentration: 1 μg / L, medium concentration: 2 μg / L, and high concentration: 4 μg / L for the other 14 target substances except 1-hydroxynaphthalene and 2-hydroxynaphthalene; low concentration: 5 μg / L, medium concentration: 10 μg / L, and high concentration: 20 μg / L for 1-hydroxynaphthalene and 2-hydroxynaphthalene). The same steps were repeated for 6 times and the results are shown in Table 7.

[0157] Table 7: Recovery and precision of 16 target analytes in urine samples (n=6)

[0158]

[0159] As can be seen from Table 7, the recoveries of the 16 target compounds ranged from 72.2% to 122.6%, and the RSDs of the six repeated tests were in the range of 0.4% to 9.2%.

[0160] Test Example 1

[0161] The 16 target analytes and 13 internal standard solutions were diluted with toluene to prepare mixed standard solutions with a mass concentration of 500 μg / L and mixed internal standard solutions with a mass concentration of 100 μg / L, and a recovered internal standard solution with a mass concentration of 100 μg / L was prepared ( 13 C 12-PCB105 toluene solution). Mass spectrometry detection was performed using an AEI source, a mass spectrometry transmission line temperature of 300°C, an ion source temperature of 300°C, an electron energy of 35eV, an argon collision gas, a solvent delay time of 5 minutes, and a mass spectrometry scanning mode of multiple reaction monitoring. Except for the collision energy, other parameters were the same as those in Example 1. The specific process is: first, a full scan of the mixed standard solution and the mixed internal standard solution was performed to determine the retention time of each target and the corresponding isotope internal standard, and ions with a large mass-to-charge ratio and high abundance were selected as parent ions. The daughter ions produced were scanned, and 2 to 4 ions with better responses were selected to obtain alternative ion pair combinations, and the collision voltage of each ion pair was optimized; the obtained alternative ion pair combinations were screened, and two ion pairs with less interference and better response were selected as quantitative ion pairs and qualitative ion pairs, respectively. The relative abundance of each substance at different collision energies during mass spectrometry detection is shown in Tables 8 to 9 and Figures 1 to 16 shown.

[0162] Table 8 Collision energy and relative abundance of different substances during mass spectrometry detection in Test Example 1

[0163]

[0164]

[0165] Table 9 Collision energy and relative abundance of different substances during mass spectrometry detection in Test Example 1

[0166]

[0167]

[0168] From Tables 8 to 9 and Figures 1 to 16 It can be seen that the optimal collision energy of each substance.

[0169] Example 2

[0170] The method of Example 1 was used to analyze 59 urine samples from the general population for the presence of 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxypyrene, 3-Hydroxy Detection of 16 target analytes, including 3-hydroxybenz[a]anthracene and 3-hydroxybenzo[a]pyrene, Except for 3-hydroxybenzo[a]anthracene, the other 14 target analytes were detected. Among them, the median concentration of 1-hydroxynaphthalene was 2.34 ng / mL, and the detection concentration range was 0.14-47.37 ng / mL; the median concentration of 2-hydroxynaphthalene was 3.80 ng / mL, and the detection concentration range was 0.21-62.35 ng / mL; the median concentration of 9-hydroxyfluorene was 0.53 ng / mL, and the detection concentration range was 0.17-4.07 ng / mL; the median concentration of 3-hydroxyfluorene was 0.24 ng / mL, and the detection concentration range was 0.04-5.26 ng / mL; the median concentration of 2-hydroxyfluorene was 0.68 ng / mL, and the detection concentration range was 0.11-7.77 ng / mL; the median concentration of 4-hydroxyphenanthrene was 0.06 ng / mL, and the detection concentration range was 0.02-2.03 ng / mL; The median concentration of 1-hydroxyphenanthrene was 0.22 ng / mL, and the detection range was 0.02-5.45 ng / mL; the median concentration of 3-hydroxyphenanthrene was 0.42 ng / mL, and the detection range was 0.05-3.81 ng / mL; the median concentration of 1-hydroxyphenanthrene was 0.30 ng / mL, and the detection range was 0.05-11.51 ng / mL; the median concentration of 2-hydroxyphenanthrene was 0.28 ng / mL, and the detection range was 0.01-4.40 ng / mL; the median concentration of 3-hydroxyfluoranthene was 0.48 ng / mL, and the detection range was 0.14-3.31 ng / mL; the median concentration of 1-hydroxypyrene was 0.24 ng / mL, and the detection range was 0.07-2.52 ng / mL; The detection concentration of is 0-0.03ng / mL; the median concentration of 3-hydroxybenzo[a]pyrene is 0.41ng / mL, and the detection concentration range is 0.26-0.41ng / mL.

[0171] Example 3

[0172] 40 urine samples were collected from the elderly population and the detection method of Example 1 was used to detect 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxy 3-Hydroxy Detection of 16 target analytes, including 3-hydroxybenz[a]anthracene and 3-hydroxybenzo[a]pyrene, In addition, the other 15 target analytes were detected. Among them, the median concentration of 1-hydroxynaphthalene was 1.33ng / mL, and the detection concentration range was 0.31-7.21ng / mL; the median concentration of 2-hydroxynaphthalene was 1.02ng / mL, and the detection concentration range was 0.26-4.36ng / mL; the median concentration of 9-hydroxyfluorene was 0.33ng / mL, and the detection concentration range was 0.02-1.14ng / mL; the median concentration of 3-hydroxyfluorene was 0.11ng / mL, and the detection concentration range was 0.01-0.42ng / mL; the median concentration of 2-hydroxyfluorene was 0.25ng / mL, and the detection concentration range was 0.03-1.19ng / mL; the median concentration of 4-hydroxyphenanthrene was 0.03ng / mL, and the detection concentration range was 0.01-0.14ng / mL; The median concentration of 1-hydroxyphenanthrene was 0.07 ng / mL, and the detection range was 0.01-0.24 ng / mL; the median concentration of 3-hydroxyphenanthrene was 0.12 ng / mL, and the detection range was 0.01-0.44 ng / mL; the median concentration of 1-hydroxyphenanthrene was 0.18 ng / mL, and the detection range was 0.03-0.55 ng / mL; the median concentration of 2-hydroxyphenanthrene was 0.15 ng / mL, and the detection range was 0.01-0.81 ng / mL; the median concentration of 3-hydroxyfluoranthene was 0.12 ng / mL, and the detection range was 0.05-2.91 ng / mL; the median concentration of 1-hydroxypyrene was 0.15 ng / mL, and the detection range was 0.09-0.27 ng / mL; The detection concentration of benzo[a]anthracene was 0-0.27 ng / mL; the median concentration of 3-hydroxybenzo[a]anthracene was 0.09 ng / mL, and the detection concentration range was 0.03-1.12 ng / mL; the detection concentration of 3-hydroxybenzo[a]pyrene was 0-0.66 ng / mL.

[0173] Test Example 2

[0174] The method of Example 1 was used to detect 16 PAH metabolites, wherein the concentrations of 1-hydroxynaphthalene and 2-hydroxynaphthalene were 100 ng / mL, and the concentrations of the remaining 14 substances were 20 ng / mL.

[0175] Comparative test example 1

[0176] The AEI source used in mass spectrometry detection in Test Example 2 was replaced with an EI source, and 16 PAH metabolites were detected, with the concentrations of 1-hydroxynaphthalene and 2-hydroxynaphthalene being 100 ng / mL, respectively, and the concentrations of the remaining 14 substances being 20 ng / mL, respectively.

[0177] The maximum peak intensities in Test Example 2 and Comparative Test Example 1 are shown in Tables 10 and Figures 17-24 As shown, Figures 17-24The new mass spectrometry mode is test case 2, and the traditional mass spectrometry mode is comparative test case 1.

[0178] Table 10 Peak intensity maximum values ​​in Test Example 2 and Comparative Test Example 1

[0179]

[0180] From Table 10 and Figures 17-24 As can be seen, the response signal intensity of the chromatogram in Test Example 1 is 3 to 17 times lower than that in Test Example 2. For actual urine samples, the concentration is much lower than that in Test Example 2, and some low-concentration substances are difficult to achieve baseline separation. This demonstrates that the present invention uses an AEI source for better detection results.

[0181] Comparative test example 2

[0182] The multiple reaction monitoring mode during mass spectrometry detection in Test Example 2 was replaced with the SIM mode, and the other parameters were the same as those in Test Example 2.

[0183] After the replacement of the comparative test example 2, 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 2-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxy The recoveries of the spiked experiments for 1-hydroxyphenanthrene, 3-hydroxyfluoranthene, and 3-hydroxybenzo[a]anthracene ranged from 25.05% to 147.86%, and the precision ranged from 0.52% to 12.19%. and 3-hydroxybenzo[a]pyrene can only be detected qualitatively but cannot be accurately quantified.

[0184] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting multiple polycyclic aromatic hydrocarbon metabolites in urine, characterized in that: It consists of the following steps: (1) mixing urine with a mixed internal standard solution, an ascorbic acid solution, and an enzyme solution, and performing enzymolysis to obtain an enzymolysis solution; (2) mixing the enzymatic hydrolyzate obtained in step (1) with water, toluene and n-pentane, and performing extraction to obtain an extract; (3) mixing the extract obtained in step (2) with a silver nitrate solution and purifying the mixture to obtain a purified solution; (4) mixing the purified liquid obtained in step (3) with n-dodecane and blowing with nitrogen, and then mixing with toluene, a solution for recovering the internal standard, and a derivatization reagent to obtain a test solution; (5) subjecting the test liquid obtained in step (4) to gas chromatography-mass spectrometry to obtain the content of polycyclic aromatic hydrocarbon metabolites in urine; the ion source during the gas chromatography-mass spectrometry detection is an AEI source; the gas chromatography-mass spectrometry detection adopts a multiple reaction monitoring mode; the collision energy of the mass spectrometer during the gas chromatography-mass spectrometry detection is 10 to 35 eV; The gas chromatography conditions during the gas chromatography-mass spectrometry detection in step (5) are as follows: an injection port temperature of 250-300° C., a carrier gas flow rate of 0.9-1 mL / min, a splitless injection method, and an injection volume of 0.9-1.1 μL; The gas chromatography during gas chromatography-mass spectrometry detection in step (5) adopts programmed temperature control of the chromatographic column temperature; the programmed temperature process is as follows: within 0-1 min, the chromatographic column temperature is 90-100 ° C; within 1-7.667 min, the temperature is increased to 190-200 ° C at a rate of 14-16 ° C / min; within 7.667-13.167 min, the temperature is increased to 204-208 ° C at a rate of 1-3 ° C / min; within 13.167-18.167 min, the temperature is increased to 204-208 ° C at a rate of 1-3 ° C / min; within 13.167-18.167 min, the temperature is increased to 204-208 ° C; within 18.167-19.167 min, the temperature is increased to 204-208 ° C. The column temperature is 204-208°C; the temperature is increased to 295-305°C at a rate of 30-50°C / min within 18.167-20.517 minutes; the column temperature is maintained at 295-305°C within 20.517-27.017 minutes. The gas chromatography column specifications are: 95% dimethylpolysiloxane and 5% diphenyl stationary phase filler, the column length is 20-40m, the diameter is 0.2-0.3mm, and the filler film thickness is 0.2-0.3μm. The mass spectrometry conditions during gas chromatography-mass spectrometry detection in step (5) are as follows: mass spectrometry transmission line temperature is 295-305° C., ion source temperature is 295-305° C., electron energy is 30-40 eV, solvent delay time is 4-6 min, and collision gas is argon; The polycyclic aromatic hydrocarbon metabolites in the urine include 1-hydroxynaphthalene, 2-hydroxynaphthalene, 9-hydroxyfluorene, 3-hydroxyfluorene, 2-hydroxyfluorene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 3-hydroxyphenanthrene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyfluoranthene, 1-hydroxypyrene, 6-hydroxy-3-hydroxy-3-hydroxybenz[a]anthracene, and 3-hydroxybenzo[a]pyrene; The derivatization reagent in step (4) includes at least one of N-methyl-N-(trimethylsilyl)trifluoroacetamide, N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide.

2. The detection method according to claim 1, wherein The enzyme in the enzyme solution of step (1) includes β-glucuronidase / arylsulfatase or β-glucuronidase.

3. The detection method according to claim 1, wherein The concentration of the enzyme solution in step (1) is 2000-3000 U / mL.

4. The detection method according to claim 3, characterized in that The volume ratio of urine to enzyme solution in step (1) is 1:(0.8-1.2).

5. The detection method according to claim 1, wherein The enzymatic hydrolysis temperature in step (1) is 35-38° C., and the enzymatic hydrolysis time is ≥16 h.

6. The detection method according to claim 1, wherein The volume ratio of toluene to n-pentane in step (2) is 1:(3-5).

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