Method for Simultaneously Detecting 16 Kinds of Hydroxylated Polycyclic Aromatic Hydrocarbons in Urine

Abstract

The present invention relates to a method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine, belonging to the technical field of detection. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine includes: a pretreatment of the sample: mixing the sample to be tested with a buffer solution and an enzyme, and then enzymolyzing; b enrichment and purification by a solid-phase extraction column; c concentration; d reconstitution; e determination. The average spiked recovery rate of the 16 hydroxy polycyclic aromatic hydrocarbons in human urine measured by the method of the present invention ranges from 71.25% to 114.07%, and the RSD ranges from 0.53% to 11.90%. It has good repeatability, high sensitivity, good stability, low detection cost, and has extensive economic and social benefits.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine, belonging to the technical field of detection. Background Art

[0002] Polycyclic aromatic hydrocarbons (PAHs) originate from the pyrolysis or incomplete combustion of various fossil fuels (such as coal, petroleum, natural gas, etc.) and organic substances, and are widely present in various environmental media such as air, water, and soil. Especially with the rise of modern industry, the increasing number of various motor vehicles such as airplanes and cars and other anthropogenic emissions have led to the increasingly serious pollution of PAHs. In daily life, people are exposed to PAHs to varying degrees through breathing, diet, drinking water, and even skin contact. Research shows that PAHs exposure can damage the central nervous system of animals, disrupt the micronucleus rate of lymphocytes, liver function, and DNA repair ability, and is a chemical substance with "carcinogenic, teratogenic, and mutagenic" toxicity. In addition, PAHs also have an endocrine disrupting effect, which can affect the function of the reproductive endocrine system and cause genetic damage. PAHs are considered to be one of the environmental carcinogens with the largest number, the widest distribution, the closest relationship with humans, and the greatest threat to human health. The problem of population exposure to polycyclic aromatic hydrocarbons has gradually become an increasingly important topic.

[0003] Initially, people estimated the degree of human exposure to PAHs by analyzing the amount of polycyclic aromatic hydrocarbons in media such as air, food, and water, which is called external exposure assessment. However, due to the constantly changing environment in which people live and work in daily life and the diverse exposure routes to PAHs, the external exposure method cannot accurately estimate the actual exposure dose of individuals, and the workload is large. Since the 1980s, some studies have started to use biomarkers to comprehensively evaluate human exposure to PAHs. Biomarkers can comprehensively reflect the exposure status of humans to PAHs by detecting PAHs or their metabolites in human tissues or body fluids. The PAHs that enter the body are converted into conjugated monohydroxy polycyclic aromatic hydrocarbons (OH-PAHs) in two steps in the liver, and then excreted from the body through urine or feces. The urinary polycyclic aromatic hydrocarbon hydroxy metabolites can more comprehensively reflect the exposure of humans to PAHs. And urine is easy to obtain, easy to preserve, and non-destructive. Scholars generally use the method of enzymatic hydrolysis to hydrolyze the conjugated products into free OH-PAHs, and evaluate the degree of human exposure by measuring the content of OH-PAHs in human urine. Common polycyclic aromatic hydrocarbons and their monohydroxy metabolites include: 1-hydroxynaphthalene, 2-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyfluorene, 9-hydroxyfluorene, 1-hydroxyphenanthrene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, 9-hydroxyphenanthrene, 1-hydroxypyrene, 1-hydroxybenzo[a]anthracene, 2-hydroxybenzo[a]anthracene, 3-hydroxybenzo[a]anthracene, 1-hydroxy 2-hydroxy 3-hydroxy 4-Hydroxy 6-Hydroxy 3-Hydroxybenzo[a]pyrene, 9-Hydroxybenzo[a]pyrene.

[0004] Among them, 1-hydroxypyrene is the biomarker that has been studied the most and used the most widely. This is because compared with other PAHs, the relative content of pyrene in the air is relatively stable, and the content of the metabolite 1-hydroxypyrene generated after in vivo metabolism is sufficient. However, in actual determination, the concentration of 1-hydroxypyrene in the human body is often affected by various factors such as smoking and diet, resulting in a large difference in the content of 1-hydroxypyrene in individuals under the same exposure conditions. To reflect the internal exposure levels of PAHs in various populations and reduce or eliminate the influence brought by individual differences, combining multiple PAHs metabolites as biomarkers can more comprehensively evaluate the human PAHs exposure level. Therefore, establishing a rapid, sensitive, accurate and stable method for simultaneously determining multiple OH-PAHs in urine is of great significance for the exposure assessment of PAHs.

[0005] Generally, HPLC can simultaneously detect 2-8 OH-PAHs in urine. Yue Qiang, Wang Dechao, Yu Zhiqiang, etc. Simultaneous determination of 10 hydroxy polycyclic aromatic hydrocarbons in human urine [J]. Chinese Journal of Public Health, 2009, 25(4): 2. disclosed a method for simultaneously detecting 10 hydroxy polycyclic aromatic hydrocarbons in urine. Zhu Yusui, Yang Hongguo, An Caixiu, etc. Determination of hydroxy polycyclic aromatic hydrocarbons in human urine by aqueous two-phase extraction-liquid chromatography [J]. Chinese Journal of Analysis Laboratory, 2012, 31(10): 5. established an analytical method for simultaneously detecting 10 hydroxy polycyclic aromatic hydrocarbons in human urine by acetonitrile-(NH4)2SO4 aqueous two-phase extraction and liquid chromatography-fluorescence method. However, the number of detected species needs to be further improved.

[0006] CN113777209A discloses a method and application for synchronous detection of exposure and effect markers of volatile pollutants in urine. This method selects OH-PAHs, mVOCs and 8-OHdG that may be correlated in the same urine sample as the target analytes, and performs synchronous extraction, detection and analysis, which includes the following steps: pretreatment of the sample; enrichment and separation of the sample; concentration and detection of the sample: using HPLC-MS / MS to perform the on-machine detection of OH-PAHs, mVOCs and 8-OHdG respectively, corresponding analysis to obtain the content data of various target analytes in urine, and analyzing the correlation among the three. It requires an internal standard, and can only synchronously extract 12 hydroxy polycyclic aromatic hydrocarbons (OH-PAHs) in urine at one time.

[0007] CN114563502A discloses a method for simultaneously and quantitatively determining the concentrations of multiple types of aromatic compounds and metabolites in urine. In this method, an isotope internal standard, a buffer solution, and β-glucuronidase-arylsulfatase are successively added to urine, followed by constant-temperature enzymatic hydrolysis. After enzymatic hydrolysis, an organic solvent is added for extraction, and the supernatant is transferred after centrifugation and concentrated and fixed in a polar solution. Through quantification and data processing, the concentration levels of multiple types of target substances in urine are obtained. It also requires internal standards and can only simultaneously extract 12 hydroxy polycyclic aromatic hydrocarbons (OH-PAHs) in urine at one time.

[0008] Shi Xinyuan, Wang Xiaoju, Liu Baoxian, et al. Determination of 16 polycyclic aromatic hydrocarbons in water quality by solid-phase extraction-high performance liquid chromatography [J]. Modern Instruments & Medical Treatment, 2014, 20(6): 6. Wang Leilei, Zheng Liwen, Kong Xue, et al. Determination of 16 polycyclic aromatic hydrocarbons in oilfield soil by accelerated solvent extraction-GPC purification-gas chromatography-mass spectrometry [J]. Shandong Science, 2015, 28(2): 6. Although 16 polycyclic aromatic hydrocarbons in water or soil can be detected, these literatures all determine the original forms of the 16 polycyclic aromatic hydrocarbons. However, after polycyclic aromatic hydrocarbons enter the human body, they will be metabolized into monohydroxy polycyclic aromatic hydrocarbons, and their content levels are trace or even ultra-trace levels. These methods cannot simultaneously detect 16 hydroxy polycyclic aromatic hydrocarbon substances in urine by the external standard method. Summary of the Invention

[0009] The object of the present invention is to provide a new method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbon substances in urine.

[0010] To achieve the above object, the method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbon substances in urine includes:

[0011] a Pretreatment of the sample: Mix the sample to be measured with a buffer solution and an enzyme and then perform enzymatic hydrolysis;

[0012] b Enrichment and purification by a solid-phase extraction column: Pass the enzymatically hydrolyzed sample through a solid-phase extraction column, and obtain an eluate after rinsing and eluting; the solid-phase extraction column is an HLB column, Prime HLB or Agilent bond; the rinsing is carried out using rinsing program 1 or 2; rinsing program 1 is to successively rinse with water and 30% acetonitrile, and the volume ratio of water to 30% acetonitrile in rinsing program 1 is 1:1, rinsing program 2 is to successively rinse with water, 30% acetonitrile, and 50% acetonitrile, and the volume ratio of water, 30% acetonitrile, and 50% acetonitrile in rinsing program 2 is 2:2:1; the solvent for solid-phase extraction elution is a mixed solution of ethyl acetate and acetonitrile, and the volume ratio of ethyl acetate to acetonitrile is more than 5:1, preferably the volume ratio of ethyl acetate to acetonitrile is 6-6.5:1;

[0013] Concentration: Blow the elution liquid nitrogen to a volume V, and the volume ratio of V to the volume of the sample to be measured in step a is 0.01 - 0.02:1;

[0014] Redissolution: Redissolve the concentrated eluate in step c with a diluent to obtain the sample to be measured and processed. The diluent includes acetonitrile, methanol, 50% methanol, 50% acetonitrile, 20% methanol, 20% acetonitrile, 0.05% ammonia acetonitrile, 0.5% ammonia acetonitrile, 0.5% acetic acid acetonitrile, 0.05% acetic acid acetonitrile, ethyl acetate, V 乙腈 :V 乙酸乙酯 =1:1, V 甲醇 :V 乙酸乙酯 =1:1 or 0.1% Vc acetonitrile; preferably, the diluent is 0.1% Vc acetonitrile;

[0015] Determination: Prepare a standard solution, and detect the sample to be measured and processed and the standard solution respectively by liquid chromatography - tandem mass spectrometry, and compare the detection results of the sample to be measured and processed with the standard solution to obtain the detection result.

[0016] Using an acetonitrile solution containing 0.1% vitamin C can not only significantly improve the mass spectrometry response of 3 - OHBaP with poor response, but also increase the stability of target substances such as 3 - OHBaP, 6 - OHNP, and 6 - OHChr during the pretreatment process (which is prone to degradation due to the influence of light, temperature, oxygen, etc.). Therefore, 0.1% vitamin C acetonitrile is preferably used as the redissolution solvent.

[0017] In a specific embodiment, the 16 hydroxy - polycyclic aromatic hydrocarbon substances are 2 - hydroxynaphthalene, 1 - hydroxynaphthalene, 3 - hydroxyfluorene, 2 - hydroxyfluorene, 3 - hydroxyphenanthrene, 2 - hydroxyphenanthrene, 9 - hydroxyphenanthrene, 1 - hydroxyphenanthrene, 4 - hydroxyphenanthrene, 1 - hydroxypyrene, 6 - hydroxy - 1 - nitropyrene, 3 - hydroxybenzo[a]anthracene, 3 - hydroxy 6 - hydroxy 9 - hydroxybenzo[a]pyrene, 3 - hydroxybenzo[a]pyrene.

[0018] In a specific embodiment, the buffer solution in step a is saturated acetic acid - ammonium acetate, and the enzyme is β - glucuronidase / arylsulfatase; the conditions for enzymatic hydrolysis in step a are enzymatic hydrolysis at 37°C in the dark for 16 h.

[0019] β - glucuronidase / arylsulfatase is a mixture, and the enzyme activity used in the experiment of the present invention is aqueoussolution>100000 units / mL; sulfatase activity<20000 units / mL.

[0020] In a specific embodiment, before the sample in step b passes through the solid-phase extraction column, the solid-phase extraction column is also activated, and the activation is carried out by passing 3 mL of acetonitrile and 3 mL of water through the solid-phase extraction column in sequence.

[0021] In a specific embodiment, the sample in step b passing through the solid-phase extraction column is an HLB column; preferably, elution program 2 in step b is adopted; the volume ratio of ethyl acetate to acetonitrile is preferably 6:1.

[0022] In a specific embodiment, the volume ratio of the sample to be tested, the buffer solution, and the enzyme is 1:1:0.01; the volume ratio of the sample to be tested to the water in the elution program is 5:2; the volume ratio of the sample to be tested to acetonitrile in the solvent for solid-phase extraction elution is 5:1.

[0023] In a specific embodiment, the volume ratio of the sample to be tested in step a to the diluent for reconstitution in step d is 5:1; preferably, it further includes passing the reconstituted sample to be tested through a 0.22 μm organic filter membrane and storing it in the dark and refrigerated for later use; steps a to d are preferably carried out in the dark at a temperature below 20°C.

[0024] In a specific embodiment, the chromatographic conditions in step e are: Agilent Eclipse PAH chromatographic column; the mobile phase is a 0.1% acetic acid-acetonitrile system or a 0.1% formic acid-acetonitrile system, preferably a 0.1% acetic acid-acetonitrile system.

[0025] In a specific embodiment, the elution of the chromatography in step e adopts the gradient elution conditions of elution program 15, the organic phase is a 5% methanol-acetonitrile solution, and the flow rate is 0.18 mL / min to 0.30 mL / min; preferably 0.20 mL / min; the gradient elution conditions of elution program 15 are:

[0026] Table 1 Gradient elution conditions of elution program 15

[0027]

[0028] In a specific embodiment, the conditions of the mass spectrometry in step e are a drying gas temperature of 200 - 240°C, a drying gas flow rate of 5 L / min, a nebulizer pressure of 40 psi, a capillary voltage of 3000 - 5000 V, a sheath gas temperature of 250°C, a sheath gas flow rate of 12 L / min, and a nozzle voltage of 0 - 1000 V;

[0029] Preferably, the conditions of the mass spectrometry are a drying gas temperature of 240°C, a drying gas flow rate of 5 L / min, a nebulizer pressure of 40 psi, a capillary voltage of 3000 V, a sheath gas temperature of 250°C, a sheath gas flow rate of 12 L / min, and a nozzle voltage of 1000 V.

[0030] In a specific embodiment, the MRM information of the 16 monohydroxy polycyclic aromatic hydrocarbons in step e is shown in Table 2:

[0031] Table 2 MRM Information of Monohydroxy Polycyclic Aromatic Hydrocarbons

[0032]

[0033]

[0034] Beneficial effects:

[0035] 1. The method of the present invention for determining monohydroxy polycyclic aromatic hydrocarbon compounds has good sensitivity and can well meet the needs of trace and even ultra-trace determination of 16 hydroxy polycyclic aromatic hydrocarbons in urine.

[0036] 2. The average recovery rate of the method of the present invention for determining 16 hydroxy polycyclic aromatic hydrocarbons in human urine ranges from 71.25% to 114.07%, and the RSD ranges from 0.53% to 11.90%, both meeting the requirements of relevant standards such as GB / T 32465-2015 "Requirements for Verification, Validation and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for the Verification and Validation of Chemical Analysis Methods in Conformity Assessment", indicating that the method of the present invention can meet the accurate determination needs of the content of 16 hydroxy polycyclic aromatic hydrocarbons in human urine.

[0037] 3. The test solutions prepared by the method of the invention have good stability within 24 hours.

[0038] 4. The method of the present invention has good repeatability for determining 16 hydroxy polycyclic aromatic hydrocarbons in human urine.

[0039] 5. The method of the present invention has established a liquid chromatography-tandem mass spectrometry determination standard method for the content of 16 hydroxy polycyclic aromatic hydrocarbons in human urine, filling the relevant blanks, and can effectively guide and standardize the monitoring and analysis work of the content of hydroxy polycyclic aromatic hydrocarbons in human urine by relevant testing institutions, with extensive economic and social benefits and low detection costs.

[0040] 6. By preferably using 0.1% Vc acetonitrile solution as the diluent, the method of the present invention not only significantly improves the mass spectrometry response of 3-OHBaP with poor response, but also can significantly increase the stability of target substances such as 3-OHBaP, 6-OHNP, and 6-OHChr during the pretreatment process, realizing the need for accurate determination.

[0041] 7. The detection method of the present invention does not require an internal standard. Description of the Drawings

[0042] Figure 1 Linear equation and standard curve of 2-OHNap;

[0043] Figure 2 TIC chromatogram of Agilent RRHD SB-C18 column;

[0044] Figure 3 TIC chromatogram of Agilent Eclipse PAH column;

[0045] Figure 2 and Figure 3 The chromatographic peaks numbered 1-16 in are 2-OHNap, 1-OHNap, 3-OHFlu, 2-OHFlu, 3-OHPhe, 2-OHPhe, 9-OHPhe, 1-OHPhe, 4-OHPhe, 1-OHPyr, 6-OHNP, 3-OHBaA, 3-OHChr, 6-OHChr, 9-OHBaP, 3-OHBaP in sequence. Detailed implementation manners

[0046] To achieve the above object, the method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine includes:

[0047] a Pretreatment of the sample: Mix the sample to be measured with a buffer solution and an enzyme, and then carry out enzymatic hydrolysis;

[0048] b Enrichment and purification by solid phase extraction column: Pass the enzymatically hydrolyzed sample through a solid phase extraction column, and after rinsing and eluting, an eluate is obtained; the solid phase extraction column is an HLB column, Prime HLB or Agilent bond; the rinsing is carried out using rinsing program 1 or 2; rinsing program 1 is to sequentially rinse with water and 30% acetonitrile, and the volume ratio of water to 30% acetonitrile in rinsing program 1 is 1:1, rinsing program 2 is to sequentially rinse with water, 30% acetonitrile, and 50% acetonitrile, and the volume ratio of water, 30% acetonitrile, and 50% acetonitrile in rinsing program 2 is 2:2:1; the solvent for solid phase extraction elution is a mixed solution of ethyl acetate and acetonitrile, and the volume ratio of ethyl acetate to acetonitrile is more than 5:1, preferably the volume ratio of ethyl acetate to acetonitrile is 6-6.5:1;

[0049] c Concentration: Blow the eluate to volume V by nitrogen blowing, and the volume ratio of volume V to the volume of the sample to be measured in step a is 0.01-0.02:1;

[0050] d Reconstitution: Reconstitute the eluate concentrated in step c with a diluent to obtain a sample to be measured after treatment, and the diluent includes acetonitrile, methanol, 50% methanol, 50% acetonitrile, 20% methanol, 20% acetonitrile, 0.05% ammonia water acetonitrile, 0.5% ammonia water acetonitrile, 0.5% acetic acid acetonitrile, 0.05% acetic acid acetonitrile, ethyl acetate, V 乙腈 :V 乙酸乙酯 =1:1, V 甲醇 :V乙酸乙酯 = 1:1 or 0.1% Vc acetonitrile; preferably, the diluent is 0.1% Vc acetonitrile;

[0051] e Determination: Prepare a standard solution, and detect the sample to be treated and the standard solution by liquid chromatography-tandem mass spectrometry respectively. Compare the detection results of the sample to be treated and the standard solution to obtain the detection result.

[0052] The experiment verified the enrichment and purification effects of solid-phase extraction cartridges such as Prime HLB (150mg / 3cc), HLB (60mg / 3cc), Agilent bond (200mg / 6cc), and Cleanert ODS C 18 on monohydroxy polycyclic aromatic hydrocarbon compounds. Cleanert ODS C 18 has the worst recovery rate for each compound, and the recovery rate of most compounds is about 30%. Prime HLB (150mg / 3cc), HLB (60mg / 3cc), and Agilent bond (200mg / 6cc) have better effects, but the hydrophilic Prime HLB and Agilent bond have poor retention of compound 2-OHNap, and the HLB cartridge is the best.

[0053] The recovery rates of elution programs 1 and 2 are both good. The recovery rates of 2-OHNap and 1-OHNap in elution program 2 are slightly better, and the solution is clearer.

[0054] In a specific embodiment, the 16 kinds of hydroxy polycyclic aromatic hydrocarbon substances are 2-hydroxy naphthalene, 1-hydroxy naphthalene, 3-hydroxy fluorene, 2-hydroxy fluorene, 3-hydroxy phenanthrene, 2-hydroxy phenanthrene, 9-hydroxy phenanthrene, 1-hydroxy phenanthrene, 4-hydroxy phenanthrene, 1-hydroxy pyrene, 6-hydroxy-1-nitro pyrene, 3-hydroxy benzo[a]anthracene, 3-hydroxy 6-hydroxy 9-hydroxy benzo[a]pyrene, 3-hydroxy benzo[a]pyrene.

[0055] In a specific embodiment, the buffer solution in step a is saturated acetic acid-ammonium acetate, and the enzyme is β-glucuronidase / arylsulfatase; the conditions for enzymatic hydrolysis in step a are enzymatic hydrolysis at 37°C in the dark for 16 h.

[0056] In a specific embodiment, before the sample passes through the solid-phase extraction cartridge in step b, the solid-phase extraction cartridge is also activated, and 3 mL of acetonitrile and 3 mL of water are sequentially passed through the solid-phase extraction cartridge.

[0057] In a specific embodiment, the sample passing through the solid-phase extraction cartridge in step b is an HLB cartridge; preferably, elution program 2 in step b is used; the volume ratio of ethyl acetate to acetonitrile is preferably 6:1.

[0058] In a specific embodiment, the volume ratio of the sample to be tested, buffer and enzyme is 1:1:0.01; the volume ratio of the sample to be tested and water in the elution procedure is 5:2; and the volume ratio of the sample to be tested and acetonitrile in the solvent of solid phase extraction elution is 5:1.

[0059] In a specific embodiment, the volume ratio of the sample to be tested in step a to the diluent for re-dissolving in step d is 5:1; preferably, the re-dissolved treated sample to be tested is passed through a 0.22 μm organic filter membrane and then shielded from light and refrigerated for later use; steps a to d are preferably carried out in a light-shielded environment at a temperature below 20°C.

[0060] Through experiments, it was found that under room temperature and light-proof conditions, the parallelism of two sample compounds prepared under the same conditions was poor, especially for compound 3-OHBaP, with a recovery rate of 18.64% for one sample and 86.87% for the other sample, which may be due to the effect of oxygen in the solvent on the compound. For most compounds, the recovery rate under light-proof conditions is better than that under non-light-proof conditions. Under room temperature and light-proof conditions, the recovery rates of compounds 3-OHBaP, 6-OHNP, and 6-OHChr were significantly reduced. Whether it was a brown injection bottle or a white translucent injection bottle stored in a refrigerated and light-proof environment at 4°C, the recovery rate of the compound was not affected. It is possible that light, temperature, and oxygen in the diluent may cause the degradation of such compounds, especially compound 3-OHBaP, so the entire pretreatment process and standard solution storage process should be kept away from light as much as possible and kept at a low temperature.

[0061] In a specific embodiment, the chromatographic conditions in step e are: Agilent Eclipse PAH chromatographic column; the mobile phase is 0.1% acetic acid-acetonitrile system or 0.1% formic acid-acetonitrile system, preferably 0.1% acetic acid-acetonitrile system.

[0062] Many mobile phases were experimented, such as 0.05% acetic acid water - acetonitrile, 5 mmol / L ammonium acetate water - acetonitrile, 5 mmol / L ammonium acetate water - methanol, 0.05% acetic acid water - methanol, 0.1% formic acid water - acetonitrile, 0.1% formic acid water - methanol, water - methanol, water - acetonitrile, 0.02% acetic acid water - acetonitrile, 0.1% acetic acid water - acetonitrile, 0.02% acetic acid - 2 mmol / L ammonium acetate water - acetonitrile. It was found that the acidity had little effect on the peak emergence time of PAHs compounds, but had an impact on the response of the compounds. In the systems of 0.02% acetic acid, 0.05% acetic acid, and 0.1% acetic acid - acetonitrile, the response of most compounds decreased slightly with the increase of acid concentration. However, the response of 3 - OHBaP was greatly affected by the acid. The response of the 0.1% acetic acid - acetonitrile system was 5 times that of the 0.05% acetic acid - acetonitrile system, and the response of the 0.1% formic acid - acetonitrile system was 1.5 times that of the 0.1% acetic acid - acetonitrile system, but the response of other compounds was low. The addition of salt would reduce the response of the compounds. Compared with the methanol system, the acetonitrile system had a better response for most compounds.

[0063] In a specific embodiment, the elution of the chromatography in step e adopts the gradient elution conditions of elution program 15. The organic phase is a 5% methanol - acetonitrile solution, and the flow rate is 0.18 mL / min to 0.30 mL / min; preferably 0.20 mL / min. The gradient elution conditions of the elution program 15 are as follows:

[0064] Table 1 Gradient elution conditions of elution program 15

[0065]

[0066] Many elution programs were experimented, and it was found that the gradient elution conditions of program 15 were the best. The organic phase was a 5% methanol - acetonitrile solution, which slightly increased the response of some compounds, especially compound 3 - OHBaP, and the isomers could also be well separated.

[0067] When the flow rate was in the range of 0.18 mL / min to 0.30 mL / min, the resolution of each target did not change. When the flow rate gradually decreased, the response of each analyte increased to a certain extent. However, when the flow rate continued to decrease, the retention time of each analyte was delayed, and the peak became wider. Selecting 0.20 mL / min was the best.

[0068] In a specific embodiment, the conditions of the mass spectrometry in step e are as follows: the drying gas temperature is 200 - 240 °C, the drying gas flow rate is 5 L / min, the nebulizer pressure is 40 psi, the capillary voltage is 3000 - 5000 V, the sheath gas temperature is 250 °C, the sheath gas flow rate is 12 L / min, and the nozzle voltage is 0 - 1000 V;

[0069] The preferred conditions for mass spectrometry are as follows: drying gas temperature 240 °C, drying gas flow rate 5 L / min, nebulizer pressure 40 psi, capillary voltage 3000 V, sheath gas temperature 250 °C, sheath gas flow rate 12 L / min, and nozzle voltage 1000 V.

[0070] When the drying gas temperature is 200 °C, the responses of each target peak are relatively high, but for compound 3-OHBaP, the response is slightly better at a higher drying gas temperature. When the drying gas flow rate is 5 L / min, the responses of most target peaks are better than those under other conditions. When the nebulizer pressure is 40 psi, the responses of the remaining target peaks are better than those under other conditions except for compound 6-OHNP. When the capillary voltage is 3000 - 5000 V, the responses of most compounds show little difference. For compound 3-OHBaP, the response is the best when the capillary voltage is 3000 V. The sheath gas temperature has little effect on the responses of each compound. When the sheath gas temperature is 250 °C, the response of compound 3-OHBaP is better than that under other conditions. When the sheath gas flow rate is 12 L / min, the responses of most compounds are better than those under other conditions. When the nozzle voltage is 0 - 1000 V, the responses of each target peak are better than those under other conditions. However, for compound 3-OHBaP, the response of the target peak is better than that under other conditions when the nozzle voltage is 1000 V. In a specific embodiment, the MRM information of the 16 monohydroxy polycyclic aromatic hydrocarbons in step e is shown in Table 2:

[0071] Table 2 MRM information of monohydroxy polycyclic aromatic hydrocarbons

[0072]

[0073]

[0074] The following further describes the specific embodiments of the present invention in conjunction with examples, and the present invention is not limited to the scope of the described examples.

[0075] Example 1

[0076] 1 Experimental materials and instruments

[0077] 1.1 Reagent materials

[0078] Standard of 2-hydroxynaphthalene (2-OHNap): purity ≥ 99%; molecular formula: C 10 H8O; CAS number: 135-19-3.

[0079] Standard of 1-hydroxynaphthalene (1-OHNap): purity ≥ 99.6; molecular formula: C 10 H8O; CAS number: 90-15-3.

[0080] Standard of 3-hydroxyfluorene (3-OHFlu): purity ≥ 99%; molecular formula: C 13H 10 O; CAS No.: 6344-67-8.

[0081] 2-Hydroxyfluorene (2-OHFlu) reference standard: purity ≥ 99%; molecular formula: C 13 H 10 O; CAS No.: 2443-58-5.

[0082] 3-Hydroxyphenanthrene (3-OHPhe) reference standard: purity ≥ 99%; molecular formula: C 14 H 10 O; CAS No.: 605-87-8.

[0083] 2-Hydroxyphenanthrene (2-OHPhe) reference standard: purity ≥ 99%; molecular formula: C 14 H 10 O; CAS No.: 605-55-0.

[0084] 9-Hydroxyphenanthrene (9-OHPhe) reference standard: purity ≥ 99%; molecular formula: C 14 H 10 O; CAS No.: 484-17-3.

[0085] 1-Hydroxyphenanthrene (1-OHPhe) reference standard: purity ≥ 99%; molecular formula: C 14 H 10 O; CAS No.: 2433-56-9.

[0086] 4-Hydroxyphenanthrene (4-OHPhe) reference standard: purity ≥ 99%; molecular formula: C 14 H 10 O; CAS No.: 7651-86-7.

[0087] 1-Hydroxypyrene (1-OHPyr) reference standard: purity ≥ 98%; molecular formula: C 16 H 10 O; CAS No.: 5315-79-7.

[0088] 6-Hydroxy-1-nitropyrene (6-OHNP) reference standard: purity ≥ 99%; molecular formula: C 16 H9NO3; CAS No.: 1767-28-8.

[0089] 3-Hydroxybenzo[a]anthracene (3-OHBaA) reference standard: purity ≥ 99%; molecular formula: C 18 H 12 O; CAS No.: 4834-35-9.

[0090] 3-Hydroxychrysene (3-OHChr) reference standard: purity ≥ 99%; molecular formula: C 18 H12 O; CAS No.: 63019-39-6.

[0091] 6-Hydroxychrysene (6-OHChr) reference standard: purity ≥99%; molecular formula: C 18 H 12 O; CAS No.: 37515-51-8.

[0092] 9-Hydroxybenzo[a]pyrene (9-OHBaP) reference standard: purity ≥99%; molecular formula: C 20 H 12 O; CAS No.: 17573-21-6.

[0093] 3-Hydroxybenzo[a]pyrene (3-OHBaP) reference standard: purity ≥99%; molecular formula: C 20 H 12 O; CAS No.: 13345-21-6.

[0094] Oasis HLB solid phase extraction cartridge (specification: 60 mg, 3 mL); ammonium acetate (C2H7NO2; HPLC grade); glacial acetic acid (C2H4O2; chromatographic grade); methanol (CH3OH; chromatographic grade); acetonitrile (C2H3N; chromatographic grade); L-ascorbic acid (C6H8O6; guaranteed reagent grade); 0.22 μm nylon filter membrane; the experimental water is all grade I water, and the specific requirements are referred to GB / T 6682.

[0095] 1.2 Solution preparation

[0096] Saturated acetic acid-ammonium acetate buffer solution: Weigh 740 g of ammonium acetate, add 500 mL of water, dissolve it by oscillating with an ultrasonic extractor, add 500 mL of glacial acetic acid, mix well, and adjust the pH to 5.5 with glacial acetic acid.

[0097] 30% acetonitrile solution: Pipette 30 mL of acetonitrile, dilute it with water to 100 mL, and shake well.

[0098] 50% acetonitrile solution: Pipette 50 mL of acetonitrile, dilute it with water to 100 mL, and shake well.

[0099] 5% methanol-acetonitrile solution: Pipette 50 mL of methanol, dilute it with acetonitrile to 1000 mL, and mix well.

[0100] 0.1% vitamin C-acetonitrile solution: Weigh 100 mg of vitamin C, add 2 mL of water, vortex to dissolve it, dilute it with acetonitrile to 100 mL, and mix well.

[0101] Single standard stock solution: Weigh approximately 10 mg (accurate to 0.1 mg) of each hydroxy polycyclic aromatic hydrocarbon standard, dissolve them separately with methanol and make up the volume to 10 mL to prepare single standard stock solutions with a mass concentration of 1000 mg / L each, store them at -18 °C, and the validity period is half a year.

[0102] Mixed standard stock solution: Pipette appropriate amounts of each single-hydroxy polycyclic aromatic hydrocarbon standard stock solution into the same 10 mL volumetric flask, dilute and make up the volume with 0.1% vitamin C acetonitrile solution, mix well to prepare a mixed standard stock solution with a mass concentration of 10 mg / L each, store it at -18 °C, and the validity period is one month.

[0103] Mixed standard intermediate solution: Pipette 1.0 mL of the mixed standard stock solution into a 100 mL volumetric flask, dilute and make up the volume with 0.1% vitamin C acetonitrile solution to prepare a mixed standard intermediate solution with a mass concentration of 100 μg / L for each single-hydroxy polycyclic aromatic hydrocarbon, and use it immediately after preparation.

[0104] Series of standard working solutions: Accurately pipette appropriate amounts of the mixed standard intermediate solution separately, dilute and make up the volume with 0.1% vitamin C acetonitrile solution, shake well to prepare a series of standard working solutions with concentrations of 0.1 ng / mL, 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, and 50 ng / mL for single-hydroxy polycyclic aromatic hydrocarbons, and use them immediately after preparation.

[0105] 1.3 Instrument and equipment

[0106] Liquid chromatography-tandem mass spectrometer: Equipped with an electrospray ionization source (ESI source).

[0107] Analytical balance: With sensitivities of 0.1 g, 0.01 mg, and 0.0001 g.

[0108] pH meter: With a precision of 0.01 pH unit and a pH measurement range of 0 - 14.

[0109] Vortex mixer, electric pipette, constant temperature water bath, ultrasonic extractor, parallel concentrator.

[0110] 2 Experimental methods

[0111] 2.1 Sample pretreatment

[0112] Transfer 5 mL of the sample into a 15-mL capped glass centrifuge tube, add 5 mL of saturated acetic acid-ammonium acetate buffer, vortex to mix evenly, then add 50 μL of β-glucuronidase / arylsulfatase, vortex again to mix evenly, and place it in a 37 °C water bath for enzymatic hydrolysis in the dark for 16 h. Take it out and cool it in the dark to room temperature, mix evenly, pass through a pre-activated HLB column (the method for activating the HLB column is to activate it successively with 3 mL of acetonitrile and 3 mL of water), wash the column successively with 2 mL of water, 2 mL of 30% acetonitrile solution, and 1 mL of 50% acetonitrile solution, discard the washing solution, then elute with 1 mL of acetonitrile and 6.5 mL of ethyl acetate, collect the eluate, blow it to about 50 μL in a 30 °C water bath with nitrogen, dissolve the residue with 0.1% vitamin C acetonitrile solution to a volume of 1.0 mL, mix evenly, pass through a 0.22-μm organic filter membrane, and store it in a brown injection vial for testing.

[0113] 2.2 Chromatographic reference conditions

[0114] a) Chromatographic column: PAH (polycyclic aromatic hydrocarbon) chromatographic column, 2.1 mm × 100 mm, particle size 1.8 μm or equivalent;

[0115] b) Mobile phase: A is 0.1% acetic acid aqueous solution, B is 5% methanol-acetonitrile solution, and the gradient elution program is shown in Table 3;

[0116] c) Flow rate: Refer to Table 3;

[0117] d) Column temperature: 40 °C;

[0118] e) Injection volume: 2 μL.

[0119] Table 3 Gradient elution program of Example 1

[0120] Time / min Volume fraction of mobile phase A / % Volume fraction of mobile phase B / % Flow rate mL / min 0.0 69.55 30.45 0.2 3.0 58.00 42.00 0.2 6.0 58.00 42.00 0.2 12.0 42.25 57.75 0.2 15.0 21.25 78.75 0.2 20.0 3.40 96.60 0.2 20.1 3.40 96.60 0.3 25.0 3.40 96.00 0.3 25.1 69.55 30.45 0.3 29.0 69.55 30.45 0.3 29.1 69.55 30.45 0.2 30.0 69.55 30.45 0.2

[0121] 2.3 Mass spectrometry reference conditions

[0122] a) Ion source: Electrospray ionization source (ESI source); b) Scanning mode: Negative ion mode;

[0123] c) Detection mode: Multiple reaction monitoring (MRM); d) Drying gas temperature: 240 °C;

[0124] e) Nebulizing gas pressure: 40 psi (1 psi = 6.895 kPa); f) Drying gas flow rate: 5 L / min;

[0125] g) Capillary voltage: 3000 V; h) Sheath gas temperature: 250 °C; i) Sheath gas flow rate: 12 L / min;

[0126] j) Nozzle voltage: 1000 V; k) Qualitative ion pairs, quantitative ion pairs, collision energy, in-source fragmentation voltage, and internal standard are shown in Table 2.

[0127] 2.4 Qualitative determination

[0128] Under the same experimental conditions, the retention time of the chromatographic peak of the analyte in the sample solution is the same as that of the standard working solution (within the range of ±2.5%), and in the chromatogram of the sample solution after background subtraction, the selected ions all appear. If the relative abundances of the qualitative ions and the relative abundance ratios of the standard product ions do not exceed the range specified in Table 4, it can be judged that the corresponding analyte exists in the sample.

[0129] Table 4 Maximum allowable deviation of relative ion abundances for qualitative confirmation

[0130] Relative ion abundance / % ＞50 ＞20～50 ＞10～20 ≤10 Allowed relative deviation / % ±20 ±25 ±30 ±50

[0131] 2.5 Quantitative determination

[0132] Under the same experimental conditions, a series of standard working solutions are injected for analysis in order of increasing concentration from low to high. The peak area of the analyte is divided by the peak area of the internal standard as the ordinate, and the mass concentration of the analyte solution is used as the abscissa to plot the standard working curve. The sample is quantified using the standard working curve, and the mass concentration of each analyte in the sample solution should be within the mass concentration range of the standard working curve. Samples exceeding the upper limit of the mass concentration of the standard working curve should be re-sampled with a reduced sampling volume and re-prepared for determination.

[0133] 2.6 Result analysis

[0134] The content of the target substance in the test sample is calculated according to formula (1).

[0135]

[0136] Where:

[0137] w—the content of the target substance in the test sample, unit: nanogram per milliliter (ng / mL);

[0138] ρ—the mass concentration of the target substance in the test sample obtained from the standard working curve, unit: nanogram per milliliter (ng / mL);

[0139] V—the constant volume of the test sample, unit: milliliter (mL);

[0140] V 样 —the sampling volume of the test sample, unit: milliliter (mL).

[0141] The calculation result should subtract the blank value and be expressed as the arithmetic mean of two independent determination results obtained under repeatability conditions, and the result is retained to three significant figures.

[0142] 3 In-laboratory verification of the method

[0143] 3.1 Verification of the linear range, detection limit, and quantification limit of the method

[0144] Under the optimal working conditions of the instrument, inject the series of mixed standard working solutions for determination into the liquid chromatography-tandem mass spectrometer respectively. Plot the standard working curve with the peak area of the target quantitative ion pair as the ordinate and the mass concentration of the standard working solution as the abscissa, as shown in Table 5 and some representatives Figure 1 As shown. It can be seen from the table that the linear relationships of all targets are good within the specified range, and the correlation coefficients r are all > 0.99. To investigate the sensitivity of this method, the signal-to-noise ratio method is used to calculate the quantification limit (LOQ) of this method. The concentration with a signal-to-noise ratio S / N ≥ about 3 is the detection limit concentration of this method, and the concentration with a signal-to-noise ratio S / N ≥ about 10 is the quantification limit concentration of this method. The specific results are shown in Table 5. It can be seen from the table that this method has good sensitivity for the determination of monohydroxy polycyclic aromatic hydrocarbon compounds and can well meet the needs of trace and even ultra-trace determination of 16 monohydroxy polycyclic aromatic hydrocarbon compounds in urine

[0145] Table 5 Linear equations, correlation coefficients, detection limits, and quantification limits of the method

[0146]

[0147] 3.2 Verification of the accuracy and repeatability of the method

[0148] Take 15 portions of human urine. Among them, 3 portions are prepared into test solutions according to the sample pretreatment method specified in Example 2.1 of this embodiment. The other 12 portions are respectively added with an appropriate amount of mixed standard intermediate solution to make the final test solution concentrations form spiked samples at three different concentration levels: low, medium, and high. Then, prepare the test solutions according to the sample pretreatment method specified in Example 2.1 of this embodiment to examine the spiked recovery rate and precision of this method. The specific results are shown in Table 6. It can be seen from the table that the average recovery rate range of the 16 monohydroxy polycyclic aromatic hydrocarbon compounds in human urine determined by this method is 71.25% - 114.07%, and the RSD range is 0.53% - 11.90%. They all meet the requirements of relevant standards such as GB / T 32465-2015 "Requirements for Verification, Confirmation, and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for the Confirmation and Verification of Chemical Analysis Methods in Conformity Assessment", indicating that this method can meet the accurate determination requirements of the contents of 16 monohydroxy polycyclic aromatic hydrocarbon compounds in human urine

[0149] Table 6 Results of the spiked recovery experiment for 16 hydroxy polycyclic aromatic hydrocarbons

[0150]

[0151]

[0152] 3.3 Verification of the solution stability

[0153] Absorb 2 μL of the mixed standard working solution and the spiked solution of the concentration in urine respectively, and inject them into the liquid chromatography-tandem mass spectrometer for analysis at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 16 h, 20 h and 24 h according to the optimized best instrument conditions of this method, so as to investigate the stability of each solution to be measured. The specific results are shown in Tables 7-8. It can be seen from the tables that there is no obvious regular decreasing or increasing change rule in the response results of each solution within 24 hours. According to the requirements of relevant standards such as GB / T 32465-2015 "Requirements for Verification, Validation and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for the Validation and Verification of Chemical Analysis Methods in Conformity Assessment", the RSDs of the detection results of each solution to be measured all meet the requirements, indicating that the solutions to be measured prepared by this method have good stability within 24 h.

[0154] Table 7 Test Results of the Stability of the Mixed Standard Working Solution

[0155]

[0156] Table 8 Test Results of the Stability of the Spiked Solution of the Concentration in Urine

[0157]

[0158]

[0159] 3.4 Verification of Method Precision

[0160] Take 6 portions of urine, add 100 μL of the mixed standard solution at 50 ng / mL, and then prepare the solution to be measured according to the sample pretreatment method specified in Example 2.1 of this embodiment. Inject 1 needle for each portion, and calculate the repeatability of each spiked solution. The specific results are shown in Table 9. Under the same experimental conditions, Person B takes 6 portions of urine, adds 100 μL of the mixed standard solution at 50 ng / mL, and then prepares the solution to be measured according to the sample pretreatment method specified in Example 2.1 of this embodiment. Inject 1 needle for each portion, compare with the repeatability data, and calculate the intermediate precision of each spiked solution. The specific results are shown in Table 10. It can be seen from the tables that the precision of measuring 16 hydroxy polycyclic aromatic hydrocarbons in human urine by this method fully meets the requirements of relevant standards such as GB / T 32465-2015 "Requirements for Verification, Validation and Internal Quality Control of Chemical Analysis Methods" and GB / T 27417-2017 "Guidelines for the Validation and Verification of Chemical Analysis Methods in Conformity Assessment" for method repeatability and precision, indicating that this method has good precision in measuring the contents of 16 hydroxy polycyclic aromatic hydrocarbons in urine.

[0161] Table 9 Test Results of the Repeatability of the Spiked Urine (n = 6)

[0162]

[0163]

[0164] Table 10 Results of the precision test of spiked urine (n = 12)

[0165]

[0166] Examples 2 to 15

[0167] In Examples 2 to 15, diluents: acetonitrile, methanol, 50% methanol, 50% acetonitrile, 20% methanol, 20% acetonitrile, 0.05% ammonia water acetonitrile, 0.5% ammonia water acetonitrile, 0.5% acetic acid acetonitrile, 0.05% acetic acid acetonitrile, ethyl acetate, V 乙腈 :V 乙酸乙酯 = 1:1, V 甲醇 :V 乙酸乙酯 = 1:1, 0.1% Vc acetonitrile were used to dilute the standard solution. The effects of different diluents on the response of the compounds are shown in Table 11. The results show that various diluents and diluents added with acids and bases have little effect on the response of polycyclic aromatic hydrocarbon compounds with hydroxyl groups, but 0.1% Vc acetonitrile can increase the response of compound 3-OHBaP by 2.4 times, and the hydroxyl groups on the rings of polycyclic aromatic hydrocarbon compounds with hydroxyl groups are easily oxidized. The addition of antioxidant Vc can also increase their stability. Using 0.1% Vc acetonitrile as the diluent and reconstitution solvent has the best effect.

[0168] Table 11 Effects of different diluents on the response of compounds

[0169]

[0170]

[0171] Examples 16 to 20

[0172] Others are similar to Example 1. 5 equal amounts of mixed standard intermediate solutions were taken and 0, 5 mL of acetonitrile, 5 mL of acetonitrile, 5 mL of methanol, 5 mL of ethyl acetate were added as simulated eluents respectively. The concentration methods were direct dilution for injection (without concentration, as a control), dilution after nitrogen blowing to 50 - 100 μL, reconstitution after nitrogen drying for 30 minutes, dilution after nitrogen blowing to 50 - 100 μL, and reconstitution after drying with 5 ml of ethyl acetate. The experimental results are shown in Table 12. Complete nitrogen drying will affect the recovery rates of some compounds, especially compounds 2-OHNap and 1-OHNap, with a loss of up to 90%. Compounds 3-OHFlu, 2-OHFlu, 9-OHPhe, 1-OHPyr, 6-OHChr, 3-OHBaP will also have varying degrees of loss. Using nitrogen blowing to 50 - 100 μL as the concentration condition has good results.

[0173] Investigation of Sample Concentration Methods for Table 12

[0174]

[0175]

[0176] Examples 21 - 22

[0177] Others are similar to Example 1, except that in Example 21, different types of eluents were used, and in Example 22, 1 mL of acetonitrile was added with different volumes of ethyl acetate eluent to investigate the recovery rate of hydroxy polycyclic aromatic hydrocarbons. The results are shown in Table 13 and Table 14. It can be seen from Table 13 that the best elution effect is achieved by first using 1 mL of acetonitrile and then adding 6.5 mL of ethyl acetate. It can be seen from Table 14 that at least 5 mL of ethyl acetate is required for complete elution, and further increasing the amount of eluent has no effect on the recovery rate of the compound.

[0178] Investigation of Solid Phase Extraction Elution Solvents for Table 13

[0179]

[0180] Investigation of Solid Phase Extraction Elution Solvent Volumes for Table 14

[0181]

[0182]

[0183] Examples 23 - 33

[0184] The chromatographic column model specifications for Examples 23 - 33 are Agilent RRHD SB - C 18 (2.1 mm × 100 mm, 1.8 um), Agilent SB - Phenyl(2.1 mm × 100 mm, 1.8 um), Agilent Eclipse PAH(2.1 mm × 100 mm, 1.8 um), Agilent RRHD Extend - C 18 (2.1 mm × 100 mm, 1.8 um), Agilent Eclipse XDB - C 18 (3.0 mm × 100 mm, 1.8 um), Agilent Eclipse Plus - C 18 (2.1 mm × 100 mm, 1.8 um), AgilentSB - Aq(2.1 mm × 100 mm, 1.8 um), Agilent RRHD SB - C8(2.1 mm × 100 mm, 1.8 um), ACE ExcelSuper - C 18(2.1 mm × 100 mm, 1.8 um), Waters HSS T3 (2.1 mm × 100 mm, 1.8 um), numbered ZHU01 - ZHU10 in sequence. For partial representative diagrams of the total ion chromatogram (TIC diagram) of each hydroxy - polycyclic aromatic hydrocarbon compound, see Figure 2 and 3 , Tables 15 and 16. It can be seen from the figures and tables that: The peak shape of Agilent SB - Phenyl (2.1 mm × 100 mm, 1.8 um) is the best, but except for Agilent Eclipse PAH (2.1 mm × 100 mm, 1.8 um), all columns cannot separate the two pairs of isomers, namely compound 3, 4 and compound 8, 9. Except for the three compounds 1 - OHPhe, 3 - OHChr, and 3 - OHBaP, the responses of other compounds on the Agilent Eclipse PAH (2.1 mm × 100 mm, 1.8 um) column are better or not significantly different compared to other chromatographic columns.

[0185] Table 15 Response values of compounds on various types of chromatographic columns

[0186]

[0187]

[0188] Table 16 Comparison of response values of compounds on various types of chromatographic columns

[0189]

Claims

1. Method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine, characterized in that, The method includes: a Pretreatment of the sample: Mix the sample to be tested with a buffer solution and an enzyme, and then perform enzymatic hydrolysis. b Enrichment and purification using a solid-phase extraction cartridge: Pass the enzymatically hydrolyzed sample through a solid-phase extraction cartridge, and obtain an eluate after rinsing and eluting; the solid-phase extraction cartridge is an HLB cartridge or Prime HLB; the rinsing is performed using rinsing program 1 or 2; rinsing program 1 is to sequentially rinse with water and 30% acetonitrile, and the volume ratio of water to 30% acetonitrile in rinsing program 1 is 1:1, rinsing program 2 is to sequentially rinse with water, 30% acetonitrile, and 50% acetonitrile, and the volume ratio of water, 30% acetonitrile, and 50% acetonitrile in rinsing program 2 is 2:2:1; the solvent for solid-phase extraction elution is a mixed solution of ethyl acetate and acetonitrile, and the volume ratio of ethyl acetate to acetonitrile is more than 5:

1. c Concentration: Blow the eluate to volume V using nitrogen, and the volume ratio of volume V to the volume of the sample to be tested in step a is 0.01 - 0.02:

1. d Reconstitution: Reconstitute the concentrated eluate in step c with a diluent to obtain the sample to be tested after treatment, and the diluent is acetonitrile, methanol, 50% methanol, 50% acetonitrile, 20% methanol, 20% acetonitrile, 0.05% ammonia acetonitrile, 0.5% ammonia acetonitrile, 0.5% acetic acid acetonitrile, 0.05% acetic acid acetonitrile, ethyl acetate, V acetonitrile:V ethyl acetate = 1:1, V methanol:V ethyl acetate = 1:1, or 0.1% vitamin C acetonitrile solution. e Determination: Prepare a standard solution, and separately detect the sample to be tested after treatment and the standard solution using liquid chromatography - tandem mass spectrometry. Compare the detection results of the sample to be tested after treatment and the standard solution to obtain the detection result. The chromatographic conditions in step e are: Agilent Eclipse PAH chromatographic column; the mobile phase is a 0.1% acetic acid - acetonitrile system or a 0.1% formic acid - acetonitrile system; the elution of the chromatography in step e uses gradient elution conditions, the organic phase is a 5% methanol acetonitrile solution, and the flow rate is 0.18 mL / min - 0.30 mL / min; the gradient elution conditions are: Table 1 Gradient elution conditions The 16 kinds of hydroxy polycyclic aromatic hydrocarbons are 2 - hydroxynaphthalene, 1 - hydroxynaphthalene, 3 - hydroxyfluorene, 2 - hydroxyfluorene, 3 - hydroxyphenanthrene, 2 - hydroxyphenanthrene, 9 - hydroxyphenanthrene, 1 - hydroxyphenanthrene, 4 - hydroxyphenanthrene, 1 - hydroxypyrene, 6 - hydroxy - 1 - nitropyrene, 3 - hydroxybenzo[a]anthracene, 3 - hydroxychrysene, 6 - hydroxychrysene, 9 - hydroxybenzo[a]pyrene, 3 - hydroxybenzo[a]pyrene.

2. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The volume ratio of ethyl acetate to acetonitrile is 6 - 6.5:

1.

3. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The diluent is 0.1% vitamin C acetonitrile solution.

4. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The mobile phase is a 0.1% acetic acid - acetonitrile system.

5. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The flow rate is 0.20 mL / min.

6. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The buffer solution in step a is saturated acetic acid - ammonium acetate, and the enzyme is β - glucuronidase / arylsulfatase; the conditions for enzymatic hydrolysis in step a are enzymatic hydrolysis at 37°C in the dark for 16 h.

7. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, Before passing the sample through the solid-phase extraction cartridge in step b, the solid-phase extraction cartridge was also activated, and the activation was sequentially performed by passing 3 mL of acetonitrile and 3 mL of water through the solid-phase extraction cartridge.

8. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, wherein The sample passing through the solid-phase extraction cartridge in step b is an HLB cartridge.

9. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, Step b adopts elution procedure 2.

10. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The volume ratio of ethyl acetate to acetonitrile is 6:

1.

11. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The volume ratio of the sample to be tested, buffer solution, and enzyme is 1:1:0.01; the volume ratio of the sample to be tested to the water in the elution procedure is 5:2; the volume ratio of the sample to be tested to acetonitrile in the solvent for solid-phase extraction elution is 5:

1.

12. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, characterized in that, The volume ratio of the sample to be tested in step a to the diluent for reconstitution in step d is 5:

1.

13. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 12, characterized in that, It also includes passing the reconstituted sample to be tested through a 0.22 μm organic filter membrane and storing it in the dark and refrigerated for later use; steps a to d are carried out in the dark at a temperature below 20°C.

14. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 1, wherein The conditions of the mass spectrometry in step e are as follows: drying gas temperature 200 - 240°C, drying gas flow rate 5 L / min, nebulizer pressure 40 psi, capillary voltage 3000 - 5000 V, sheath gas temperature 250°C, sheath gas flow rate 12 L / min, nozzle voltage 0 - 1000 V.

15. The method for simultaneously detecting 16 kinds of hydroxy polycyclic aromatic hydrocarbons in urine according to claim 14, wherein The conditions of the mass spectrometry are as follows: drying gas temperature 240°C, drying gas flow rate 5 L / min, nebulizer pressure 40 psi, capillary voltage 3000 V, sheath gas temperature 250°C, sheath gas flow rate 12 L / min, nozzle voltage 1000 V.

16. The method for simultaneously detecting 16 hydroxy polycyclic aromatic hydrocarbons in urine according to claim 10, wherein The MRM information of the 16 kinds of hydroxy polycyclic aromatic hydrocarbons in step e is shown in Table 2: Table 2 MRM information of hydroxy polycyclic aromatic hydrocarbons 。

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