CN109342633B

CN109342633B - Method for detecting metabolites of benzene, toluene and xylene in urine

Info

Publication number: CN109342633B
Application number: CN201811420940.0A
Authority: CN
Inventors: 乔荷; 刘晓东; 李珏; 孟祥娟; 牛东升; 丁晓文
Original assignee: Beijing Prevention And Treatment Hospital Of Occupational Disease Of Chemical Industry
Current assignee: Beijing Prevention And Treatment Hospital Of Occupational Disease Of Chemical Industry
Priority date: 2018-11-27
Filing date: 2018-11-27
Publication date: 2020-10-02
Anticipated expiration: 2038-11-27
Also published as: CN109342633A

Abstract

The invention discloses a method for detecting metabolites of benzene, toluene and xylene in urine, which comprises the following steps: (1) mixing urine with a first solvent, and centrifuging to obtain a supernatant; mixing the supernatant with a second solvent to obtain a first sample solution, and detecting a first metabolite in the first sample solution by adopting liquid chromatography-tandem mass spectrometry; the first metabolites include trimebuterol urate S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA; (2) mixing part of the first sample solution with a third solvent to obtain a second sample solution, and detecting a second metabolite in the second sample solution by adopting liquid chromatography-tandem mass spectrometry; the second metabolite comprises hippuric acid HA. The method of the invention can jointly detect more than six metabolites.

Description

Method for detecting metabolites of benzene, toluene and xylene in urine

Technical Field

The invention relates to a method for detecting metabolites in urine, in particular to a method for detecting metabolites of benzene, toluene and xylene in urine.

Background

Biological monitoring refers to monitoring the content of chemical substances and metabolites thereof in human biological materials (blood, urine, etc.) or the level of biological effects generated by the substances, and can reflect the total contact amount of chemical substances of different ways and sources of the organism. Thus, biological monitoring can provide an actual level of contact of the body and is therefore more advantageous than environmental monitoring.

The benzene compound is a chemical product with wide industrial application, and can be used as a chemical raw material, a solvent, a diluent, a fuel and the like. Benzene-based compounds are also a common class of environmental pollutants. Among the benzene-based compounds, benzene, toluene and xylene are most common. Benzene has hematological toxicity and carcinogenicity; toluene and xylene, while less toxic than benzene, can also have deleterious effects on the skin, endocrine, nervous system, etc., over prolonged contact. Therefore, biological monitoring of the exposure levels of benzene, toluene and xylene is highly desirable in occupational defense. However, there are few reports on the combined detection method of the metabolites of benzene compounds.

CN106680394A discloses a kit for determining the content of 14 environmental hormones in urine by liquid chromatography-tandem mass spectrometry, wherein the 14 environmental hormones are respectively: monomethyl phthalate, monoethyl phthalate, monobutyl phthalate, monobenzyl phthalate, monoethylhexyl phthalate, methyl paraben, ethyl paraben, propyl paraben, butyl paraben, nonylphenol, octylphenol, butylphenol, bisphenol a, triclosan. CN106680393A discloses a method for determining the content of 14 environmental hormones in urine by liquid chromatography-tandem mass spectrometry, which comprises the steps of quantitatively detecting 14 environmental hormones in a urine sample by adopting a high performance liquid chromatography-tandem mass spectrometry method, firstly carrying out primary separation on the urine sample subjected to pre-purification treatment by using the high performance liquid chromatography, scanning parent ion-daughter ion pairs of a target component by using a multi-ion reaction monitoring mode of tandem mass spectrometry negative ion electrospray ionization, accurately quantifying by using a deuterated isotope internal standard method, establishing a correction curve by using the ratio of standard substance concentration to internal standard substance concentration as an X axis and the ratio of standard substance response peak area to internal standard substance response peak area as a Y axis, and calculating the content of the 14 environmental hormones. CN103837624A discloses a liquid chromatography tandem mass spectrometry method for phenylglyoxylic acid and phenylglycolic acid in urine, which is characterized in that a sample is diluted and then directly introduced into a liquid chromatography-electrospray ionization-tandem mass spectrometer for measurement, and the content levels of phenylglyoxylic acid and phenylglycolic acid in the urine can be rapidly and accurately detected. CN102788852A discloses a method for detecting seven aromatic amine compounds in human urine by liquid chromatography-tandem mass spectrometry, namely detection of aniline, o-toluidine, m-toluidine, 1-aminonaphthalene, 2-aminonaphthalene, 3-aminobiphenyl and 4-aminobiphenyl. The method adopts liquid chromatography tandem mass spectrometry to detect some aromatic compounds and metabolites thereof, but none of the aromatic compounds are the metabolites of benzene, toluene and xylene.

CN103175921A discloses a method for analyzing four metabolites of benzene and toluene in urine by liquid chromatography-tandem mass spectrometry, which comprises preparing mixed standard sample working solutions with a series of concentrations, performing instrument analysis, and preparing a standard curve according to a linear relation existing between a response value and the concentration of the working standard sample; taking a urine sample to be detected, and adding a mixed working standard solution containing four characteristic metabolite isotope internal standard substances; adsorbing a target compound by using a solid phase extraction small column, and then carrying out instrumental analysis; obtaining response values of the four characteristic metabolites; the concentrations of the four characteristic metabolites in the urine samples were determined from the known coefficients f. The instrumental analysis adopts an ultrahigh pressure liquid chromatography-tandem mass spectrometry method, and a chromatographic column adopts a reversed phase C18 chromatographic column with the particle size less than 2 mu m. The method adopts a solid phase extraction method to separate metabolites, and can only detect four limited metabolites due to the limitation of extraction conditions, so that the metabolites of the benzene compounds cannot be detected comprehensively.

In recent years, many companies use mixtures of benzene-based compounds, and many benzene-based compounds are present on site. Therefore, there is still a need to develop a new detection method for comprehensively detecting metabolites of benzene-based compounds.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting metabolites of benzene, toluene and xylene in urine, which can jointly detect more than six biomarkers of benzene, toluene and xylene. It is a further object of the present invention to provide a detection method which can reduce the interference of a substrate and has high detection accuracy.

The invention provides a method for detecting metabolites of benzene, toluene and xylene in urine, which comprises the following steps:

(1) mixing urine with a first solvent, and centrifuging to obtain a supernatant; mixing the supernatant with a second solvent to obtain a first sample solution, and detecting a first metabolite in the first sample solution by adopting liquid chromatography-tandem mass spectrometry; wherein the first solvent is selected from one or two of methanol and acetonitrile, and the second solvent is selected from one or more of ultrapure water, methanol and acetonitrile; the first metabolites include trimebuterol urate S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA;

(2) mixing part of the first sample solution with a third solvent to obtain a second sample solution, and detecting a second metabolite in the second sample solution by adopting liquid chromatography-tandem mass spectrometry; wherein the third solvent is selected from one or more of ultrapure water, methanol and acetonitrile; the second metabolite comprises hippuric acid HA.

According to the detection method of the present invention, preferably, the first metabolite further comprises trans, trans-myfuroic acid t, t-MA.

According to the detection method of the present invention, preferably, the first metabolite further includes o-methylphenol.

According to the detection method provided by the invention, preferably, the volume ratio of the urine to the first solvent is 1: 1-20; the volume ratio of the supernatant to the second solvent is 1: 0.5-20; the volume ratio of the part of the first sample liquid to the third solvent is 1: 20-500.

According to the detection method of the present invention, preferably, in the step (1), the first solvent is methanol, and the second solvent is ultrapure water; in the step (2), the third solvent is a mixture of ultrapure water and methanol.

According to the detection method of the present invention, preferably, the volume percentage of methanol in the mixture of ultrapure water and methanol is 10 to 25 vol%.

According to the detection method of the present invention, preferably, the conditions of the liquid chromatography tandem mass spectrometry of the steps (1) and (2) are as follows:

liquid chromatography conditions: the liquid chromatographic column is a reversed phase chromatographic column, the grain diameter of a filler of the reversed phase chromatographic column is less than or equal to 5.0 mu m, and the column length is 30-150 mm; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 0.005-0.2 vol% of aqueous solution of formic acid or acetic acid; the mobile phase B is a methanol solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%, or an acetonitrile solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%; the flow rate of the mobile phase is 0.1-0.5 mL/min; the column temperature is 30-60 ℃; the elution mode is gradient elution; the sample injection amount is 1-10 mu L.

Mass spectrum conditions: adopting an electronic spray ionization source ESI, wherein the 8-hydroxy-2-deoxyguanosine adopts a positive ion mode ESI⁺Except for 8-hydroxy-2-deoxyguanosineHis metabolites adopt negative ion mode ESI^﹣(ii) a Adopting a positive and negative ion mode to collect in sections; monitoring by adopting a multi-reaction monitoring mode MRM; the capillary voltage is 0.5-5 kV; the ion source temperature is 120-180 ℃.

According to the detection method of the present invention, preferably, the gradient elution is as follows: keeping the volume percent of the mobile phase A unchanged for 0-1 min; 1-10 min, reducing the volume of the mobile phase A from 85 vol% to 70 vol%; reducing the volume of the mobile phase A from 70 vol% to 25 vol% for 10-13 min; 13-14 min, and the mobile phase A rises from 25 vol% to 85 vol%.

According to the detection method of the invention, preferably, in mass spectrum conditions, the collision energy is 5-25 eV, the cone hole voltage is 1-50V, and the quantitative ion pair is as follows:

compound (I)	Parent ion (m/z)	Ionic acid (m/z)
			8-OHdG	284	168
HA	178	134
			2-MHA	192	91
3-MHA	192	91
			4-MHA	192	91
S-PMA	238	109

According to the detection method, preferably, an external standard method is adopted for quantification, the concentration of a standard substance is taken as an X axis, the peak area of a quantitative ion response is taken as a Y axis, and a standard curve is established; quantifying the response peak area obtained by the liquid chromatogram tandem mass spectrum in the step (1) and the step (2) through a standard curve to obtain the measured concentration; converting the measured concentration to obtain the concentration c of the first metabolite and the second metabolite in the urine; the conversion formula is as follows:

c＝c₀×n

in the formula, c₀To measure concentration; and n is the dilution multiple.

The invention adopts methanol or acetonitrile to precipitate protein in urine, and then the protein is directly injected after being diluted by proper solvent. Compared with the traditional liquid-liquid extraction and solid-phase extraction, the method can jointly detect more than six biomarkers of benzene, toluene and xylene, thereby more comprehensively monitoring the exposure level of the benzene compounds. The invention adopts different dilution times aiming at different metabolites, thereby effectively reducing matrix interference and improving measurement accuracy.

Drawings

FIG. 1 is a graph of Multiple Reaction Monitoring (MRM) of example 2.

Detailed Description

The present invention will be further described with reference to the following specific examples, but the scope of the present invention is not limited thereto.

The method for detecting the metabolites of benzene, toluene and xylene in the urine is only used for evaluating the condition of the metabolites of the benzene compounds of a contacted person or a non-contacted person, and thus does not relate to the diagnosis and treatment of diseases. Furthermore, the metabolite status does not have a direct or indirect relationship to a disease and therefore cannot be used to diagnose a disease.

Benzene mercapto uric acid (S-PMA), trans-myfuroic acid (t, t-MA) and 8-hydroxy-2-deoxyguanosine (8-OHdG) are biomarkers for researching more benzene contacts and can better reflect the internal exposure level of benzene. Hippuric Acid (HA) and o-cresol (o-cresol) are biomarkers for toluene contact, and can better reflect the internal exposure level of toluene. 2-methylhippuric acid (2-MHA), 3-methylhippuric acid (3-MHA) and 4-methylhippuric acid (4-MHA) are the main metabolites of xylene (including three isomers of o-xylene, m-xylene and p-xylene), and can be used as biomarkers of xylene contact.

The method for detecting the metabolites of benzene, toluene and xylene in urine comprises the following steps: (1) a first metabolite detection step; (2) a second metabolite detection step. As described in detail below.

In the step (1), mixing urine with a first solvent, and then centrifuging to obtain a supernatant; and mixing the supernatant with a second solvent to obtain a first sample solution, and detecting a first metabolite in the first sample solution by adopting liquid chromatography-tandem mass spectrometry.

After mixing the urine with the first solvent, proteins in the urine can be precipitated, thereby reducing interference with the detection data. The protein was removed by centrifugation. The rotating speed of the centrifugation can be above 8000 rpm. The first solvent is selected from one or two of methanol and acetonitrile, preferably methanol or acetonitrile, more preferably methanol.

In the invention, the volume ratio of the urine to the first solvent can be 1: 1-20; preferably 1:1 to 10, and more preferably 1:2 to 5. The appropriate ratio of urine to first solvent can fully precipitate the protein, thereby improving detection accuracy and saving the amount of organic solvent. According to one embodiment of the invention, the volume ratio of the urine to the methanol can be 1: 1-20; preferably 1:1 to 10, and more preferably 1:2 to 5.

And mixing the supernatant obtained after centrifugation with a second solvent to obtain a first sample solution. Therefore, the urine sample can be diluted, and the interference of the urine matrix is reduced. The second solvent is selected from one or more of ultrapure water, methanol and acetonitrile; preferably ultrapure water or methanol, more preferably ultrapure water. The invention finds that the ultrapure water is more favorable for improving the accuracy of the detection result. Ultrapure water has the usual meaning in the art and is readily available and will not be described in further detail herein.

In the invention, the volume ratio of the supernatant to the second solvent can be 1: 0.5-20; preferably 1: 0.6-10, more preferably 1: 1-5. The appropriate dilution ratio can reduce matrix interference and improve detection accuracy. According to one embodiment of the invention, the volume ratio of the supernatant to the ultrapure water is 1: 0.5-20; preferably 1: 0.6-10, more preferably 1: 1-5.

Detecting the first metabolite in the first sample solution by liquid chromatography tandem mass spectrometry. The first metabolites include trimebuterol urate S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA. The method of the invention can simultaneously detect more than five metabolites of the benzene compounds. Preferably, the first metabolite may also comprise trans, trans-myfuroic acid t, t-MA. More preferably, the first metabolite may also include o-methylphenol. In certain embodiments, the first metabolite detected consists only of trimercaptouric acid S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA. In other embodiments, the first metabolite detected consists only of trimercaptouric acid S-PMA, trans-myfuroic acid t, t-MA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA.

And mixing part of the first sample solution with a third solvent to obtain a second sample solution. The third solvent may be selected from one or more of ultrapure water, methanol and acetonitrile; a ultrapure water-methanol mixture is preferred. In the ultrapure water-methanol mixture, the volume percentage of methanol is 10 to 25 vol%, preferably 15 to 20 vol%.

The invention finds that different dilution times are preferably used for different metabolites, which can effectively improve the accuracy of the detection result and can better maintain the instrument. The volume ratio of part of the first sample liquid to the third solvent may be 1:20 to 500, preferably 1:40 to 200, and more preferably 1:45 to 150. Appropriate dilution ratios can improve detection accuracy. According to an embodiment of the present invention, the volume ratio of the portion of the first sample liquid to the ultrapure water-methanol mixture may be 1:20 to 500, preferably 1:40 to 200, and more preferably 1:45 to 150.

Detecting a second metabolite in the second sample fluid using liquid chromatography tandem mass spectrometry. The second metabolite comprises hippuric acid HA, preferably, the only detected second metabolite is hippuric acid HA.

According to one embodiment of the present invention, the method for detecting metabolites of benzene, toluene and xylene in urine comprises the following steps:

(1) mixing urine and methanol according to a volume ratio of 1: 1-20, and centrifuging to obtain a supernatant; mixing the supernatant with ultrapure water according to the volume ratio of 1: 0.5-20 to obtain a first sample solution, and detecting a first metabolite in the first sample solution by adopting a liquid chromatography-tandem mass spectrometry; the first metabolite consists of benzene mercapto uric acid S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA and 4-methylhippuric acid 4-MHA;

(2) mixing part of the first sample liquid with a mixture of ultrapure water and methanol according to a volume ratio of 1: 20-500 to obtain a second sample liquid, and detecting a second metabolite in the second sample liquid by adopting a liquid chromatography-tandem mass spectrometry; the second metabolite is hippuric acid HA. The volume percentage of methanol in the ultrapure water-methanol mixture is 10-25 vol%.

The liquid chromatography tandem mass spectrum of the steps (1) and (2) can adopt ultra-high performance liquid chromatography-triple quadrupole mass spectrum; for example, ultra high performance liquid chromatography (Acquity UPLC I-Class, Waters corporation, usa) -triple quadrupole mass spectrometry (Xevo TQ-XS, Waters corporation, usa). The conditions of the liquid chromatography tandem mass spectrometry in steps (1) and (2) of the present invention may be the same or slightly different, and will be described in detail below.

First, the liquid chromatography conditions are described. The liquid chromatographic column is a reversed phase chromatographic column, the grain diameter of a filler of the reversed phase chromatographic column is less than or equal to 5.0 mu m, and the column length is 30-150 mm; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 0.005-0.2 vol% of aqueous solution of formic acid or acetic acid; the mobile phase B is a methanol solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%, or an acetonitrile solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%; the flow rate of the mobile phase is 0.1-0.5 mL/min; the column temperature is 30-60 ℃; the elution mode is gradient elution; the sample injection amount is 1-10 mu L.

In the present invention, the liquid chromatography column is a reverse phase chromatography column, and examples of the chromatography column include, but are not limited to, a HSS T3 type chromatography column, a BEH C18 type chromatography column. The particle size of the packing of the reverse phase chromatography column is 5.0 μm or less, preferably 2.0 μm or less. The length of the column is 30-150 mm, for example 100 mm.

The mobile phase of the liquid chromatography conditions comprises mobile phase a and mobile phase B. The mobile phase A is aqueous solution of formic acid or aqueous solution of acetic acid; preferably 0.005-0.2 vol% of formic acid or 0.005-0.2 vol% of acetic acid; preferably 0.01 to 0.1 vol% formic acid or 0.01 to 0.1 vol% acetic acid. According to one embodiment of the invention, the mobile phase A is 0.01 to 0.05 vol% formic acid solution or 0.01 to 0.05 vol% acetic acid solution; more preferably, the mobile phase A is 0.01-0.05 vol% formic acid aqueous solution.

The mobile phase B is methanol, acetonitrile, methanol solution of formic acid, methanol solution of acetic acid, acetonitrile solution of formic acid or acetonitrile solution of acetic acid; preferably, the mobile phase B is 0-0.2 vol% of methanol solution of formic acid, 0-0.2 vol% of methanol solution of acetic acid, 0-0.2 vol% of acetonitrile solution of formic acid or 0-0.2 vol% of acetonitrile solution of acetic acid; more preferably, the mobile phase B is 0-0.1 vol% methanol solution of formic acid, 0-0.1 vol% methanol solution of acetic acid, 0-0.1 vol% acetonitrile solution of formic acid or 0-0.1 vol% acetonitrile solution of acetic acid. According to one embodiment of the invention, the mobile phase B is 0-0.2 vol% formic acid in methanol or 0-0.2 vol% acetic acid in methanol; preferably 0 to 0.1 vol% of methanolic acid or 0 to 0.1 vol% of methanolic acetic acid; more preferably methanol.

The flow rate of the mobile phase is 0.1-0.5 mL/min; preferably 0.2-0.4 mL/min. The column temperature is 30-60 ℃; for example, 30 to 50 ℃. The sample amount is 1 to 10 μ L, preferably 3 to 6 μ L. The elution mode is gradient elution. Preferably, the elution procedure is as follows: keeping the volume percent of the mobile phase A unchanged for 0-1 min; 1-10 min, reducing the volume of the mobile phase A from 85 vol% to 70 vol%; reducing the volume of the mobile phase A from 70 vol% to 25 vol% for 10-13 min; 13-14 min, and the mobile phase A rises from 25 vol% to 85 vol%.

Mass spectrometry conditions are then introduced. Adopting an electronic spray ionization source ESI, wherein the 8-hydroxy-2-deoxyguanosine adopts a positive ion mode ESI⁺ESI in negative ion mode for metabolites other than 8-hydroxy-2-deoxyguanosine^﹣(ii) a Adopting a positive and negative ion mode to collect in sections; monitoring by adopting a multi-reaction monitoring mode MRM; the capillary voltage is 0.5-5 kV; the ion source temperature is 120-180 ℃.

The invention adopts positive and negative ion mode to collect in sections, and can detect a plurality of metabolites at one time, including but not limited to benzene mercapto uric acid S-PMA, trans-myxofuroic acid t, t-MA, 8-hydroxy-2-deoxyguanosine 8-OHdG, hippuric acid HA, o-methylphenol o-cresol, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA and 4-methylhippuric acid 4-MHA. The detection sensitivity and accuracy can be further improved by monitoring with the multi-reaction monitoring mode MRM. The capillary voltage is 0.5-5 kV; preferably 0.5-3.5 kV. According to one embodiment of the invention, the capillary voltage: 3.1kV (ESI)⁺)，1.0kV(ESI^﹣). The ion source temperature is 120-180 ℃, for example 150 ℃.

Under the mass spectrum condition, the collision energy is 5-25 eV, preferably 10-20 eV. The taper hole voltage is 1-50V, preferably 3-30V. During the period that the tested compound does not peak, the mass spectrum can be switched to waste liquid on line, so that the pollution of the sample to the mass spectrum is reduced.

Under mass spectrometry conditions, the ion pairs were quantified as follows:

compound (I)	Parent ion (m/z)	Ionic acid (m/z)
			8-OHdG	284	168；89
t,t-MA	141	53；45
			HA	178	134；77
o-cresol	107	92；76
			2-MHA	192	91；149
3-MHA	192	91；149
			4-MHA	192	91；149
S-PMA	238	109；192

Preferably, the quantitative ion pairs are as follows:

compound (I)	Parent ion (m/z)	Ionic acid (m/z)
			8-OHdG	284	168
t,t-MA	141	53
			HA	178	134
o-cresol	107	92
			2-MHA	192	91
3-MHA	192	91
			4-MHA	192	91
S-PMA	238	109

According to one embodiment of the invention, the quantitative ion pairs are as follows:

The invention adopts an external standard method for quantification. And establishing a standard curve by taking the concentration of the standard substance as an X axis and taking the peak area of the quantitative ion response as a Y axis. More than six of S-PMA, t-MA, 8-OHdG, HA, o-cresol, 2-MHA, 3-MHA and 4-MHA are selected to prepare a single standard stock solution and a mixed standard application solution, and then standard series solutions with different concentrations are prepared. The solvent used for preparing the stock solution, the application solution and the standard series solution is selected from one or more of ultrapure water, methanol and acetonitrile. The number of the standard series solutions is 4-10, preferably 5-8. The lowest concentration points of the standard solutions of the metabolites are the respective quantitative limits of the metabolites, and are concentrations with signal-to-noise ratios (S/N) of more than or equal to 10. The highest concentration points for the standard solutions of metabolites were:

S-PMA/8-OHdG：0.4～4ng/mL；

t,t-MA/o-cresol：20～200ng/mL；

HA/2-MHA/3-MHA/4-MHA：200～2000ng/mL。

and (3) quantifying the response peak area obtained by the liquid chromatogram tandem mass spectrum of the step (1) and the step (2) through a standard curve to obtain the measured concentration. And converting the measured concentration to obtain the concentration c of the first metabolite and the second metabolite in the urine.

c＝c₀×n

In the formula, c₀To measureConcentration; and n is the dilution multiple.

The following examples of instruments and reagents are described below:

ultra high performance liquid chromatography (Acquity UPLC I-Class, Waters corporation, usa), triple quadrupole mass spectrometry (Xevo TQ-XS, Waters corporation, usa); methanol and formic acid are LC-MS grade; the purity of the standard products of S-PMA, 8-OHdG, t-MA, HA, 2-MHA, 3-MHA and 4-MHA is more than 96 percent.

Example 1

Sample treatment:

placing 300 μ L urine in a centrifuge tube, adding 900 μ L methanol, vortex vibrating uniformly, and centrifuging at 15000rpm for 15min to obtain supernatant. And placing 500 mu L of the supernatant into a new centrifuge tube, adding 2mL of ultrapure water, and uniformly vortexing to obtain a first sample solution. 1mL of the first sample solution was placed in a 2mL sample injection bottle and injected to analyze the contents of S-PMA, 8-OHdG, 2-MHA, 3-MHA and 4-MHA.

mu.L of the first sample solution and 1980. mu.L of a 15 vol% methanol aqueous solution were placed in a new centrifuge tube and vortexed to homogenize the solution, thereby obtaining a second sample solution. 1mL of the second sample solution was placed in a 2mL sample introduction vial, and sample introduction was performed to analyze the HA content.

Preparing a standard solution:

mixed standard solutions with different concentrations are prepared by taking a 15 vol% methanol aqueous solution as a solvent. Concentration range of 8-OHdG: 0.002-2 ng/mL; concentration range of S-PMA: 0.001-2 ng/mL; concentration range of HA: 0.5-1000 ng/mL; concentration ranges of 2-MHA, 3-MHA and 4-MHA: 0.05-1000 ng/mL.

Liquid chromatography conditions:

the chromatographic column is HSS T3 type chromatographic column, and has filler particle diameter of 1.8 μm and column length of 100 mm. Mobile phase: the mobile phase A is 0.04 vol% of formic acid aqueous solution; the mobile phase B is methanol. Flow rate of mobile phase: 0.3 mL/min; column temperature: 30 ℃; sample introduction amount: 4 μ L. Gradient elution: keeping the volume percent of the mobile phase A unchanged for 0-1 min; 1-10 min, reducing the volume of the mobile phase A from 85 vol% to 70 vol%; reducing the volume of the mobile phase A from 70 vol% to 25 vol% for 10-13 min; 13-14 min, and the mobile phase A rises from 25 vol% to 85 vol%.

Mass spectrum conditions:

8-OHdG adopts positive ion mode ESI⁺S-PMA, HA, 2-MHA, 3-MHA and 4-MHA adopt negative ion mode ESI^﹣(ii) a Adopting a positive and negative ion mode to collect in sections; capillary voltage: 3.1kV (ESI)⁺)，1.0kV(ESI^﹣) (ii) a Multiple reaction monitoring mode (MRM); ion source temperature: at 150 ℃. The selection of the quantitative ion pairs, collision energy and cone hole voltage is shown in the following table:

TABLE 1

Compound (I)	Parent ion (m/z)	Ionic acid (m/z)	Collision energy (eV)	Taper hole voltage (V)
					8-OHdG	284	168	14	28
S-PMA	238	109	10	14
					HA	178	134	16	4
2-MHA	192	91	16	26
					3-MHA	192	91	16	26
4-MHA	192	91	16	26

And (3) verification of methodology:

quantifying by external standard method, establishing standard curve, and obtaining r of the standard curve²All above 0.995.

The concentration corresponding to a signal-to-noise ratio (S/N) ≥ 10 is determined as the limit of quantitation, and the limits of quantitation for 8-OHdG, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA are 2, 1, 500, 50 and 50pg/mL, respectively.

And (3) inspecting the accuracy and precision of the method by adopting a matrix labeling mode. Preparing low, medium and high concentration level standard urine respectively, treating 6 samples in each group, determining recovery rate and batch precision: the recovery rate of 8-OHdG, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA is within the range of 98-110%; the internal precision of the batch is within the range of 2.0-4.0%. Each concentration matrix standard sample is respectively processed for 6 times, and the precision among batches is measured: the batch precision of 8-OHdG, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA is in the range of 0.6-9.5%.

And (3) sample testing:

20 parts of urine of staff in a certain shoe factory is collected for testing, and the following results are obtained through urine specific gravity correction: the content of 8-OHdG, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA is 0.825-12.651 ng/mL, 0.024-0.186 ng/mL (6 cases are not detected), 8.619-811.166 mu g/mL, 12.952-5154.764 ng/mL, 18.650-10428.540 ng/mL and 11.077-4707.659 ng/mL respectively.

20 parts of urine of employees of a certain property company are collected for testing, and the following results are obtained after the urine specific gravity is corrected: the contents of 8-OHdG, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA are respectively 0.943-10.433 ng/mL, 0.020-0.034 ng/mL (14 cases are not detected), 6.118-452.130 mu g/mL, 5.760-40.313 ng/mL, 11.286-135.178 ng/mL and 6.270-67.055 ng/mL.

Example 2

Sample treatment:

placing 300 μ L urine in a centrifuge tube, adding 900 μ L methanol, vortex vibrating uniformly, and centrifuging at 15000rpm for 15min to obtain supernatant. And placing 500 mu L of the supernatant into a new centrifuge tube, adding 2mL of ultrapure water, and uniformly vortexing to obtain a first sample solution. 1mL of the first sample solution was placed in a 2mL sample injection bottle and sample injection was performed to analyze the contents of S-PMA, t-MA, 8-OHdG, 2-MHA, 3-MHA and 4-MHA.

Preparing a standard solution:

mixed standard solutions with different concentrations are prepared by taking a 15 vol% methanol aqueous solution as a solvent. Concentration range of 8-OHdG: 0.002-2 ng/mL; concentration range of S-PMA: 0.001-2 ng/mL; concentration range of t, t-MA: 0.04-100 ng/mL; concentration range of HA: 0.5-1000 ng/mL; concentration ranges of 2-MHA, 3-MHA and 4-MHA: 0.05-1000 ng/mL.

Liquid chromatography conditions:

Mass spectrum conditions:

8-OHdG adopts positive ion mode ESI⁺S-PMA, t-MA, HA, 2-MHA, 3-MHA and 4-MHA adopt anion ESI^﹣(ii) a Adopting a positive and negative ion mode to collect in sections; capillary voltage: 3.1kV (ESI)⁺)，1.0kV(ESI^﹣) (ii) a Multiple reaction monitoring mode (MRM); ion source temperature: at 150 ℃. The selection of the quantitative ion pairs, collision energy and cone hole voltage is shown in the following table:

TABLE 2

And (3) verification of methodology:

Concentrations corresponding to signal-to-noise ratios (S/N) ≥ 10 are defined as limits of quantitation, 8-OHdG, t-MA, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA limits of quantitation being 2, 40, 1, 500, 50 and 50pg/mL, respectively.

FIG. 1 is a graph of Multiple Reaction Monitoring (MRM) of example 2. And (3) inspecting the accuracy and precision of the method by adopting a matrix labeling mode. Preparing low, medium and high concentration level standard urine respectively, treating 6 samples in each group, determining recovery rate and batch precision: the recovery rate of 8-OHdG, t-MA, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA is within the range of 86-110%; the internal precision of the batch is within the range of 2.0-4.5%. Each concentration matrix standard sample is respectively processed for 6 times, and the precision among batches is measured: the batch precision of 8-OHdG, t-MA, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA is in the range of 0.6-9.5%.

And (3) sample testing:

20 parts of urine of staff in a certain shoe factory is collected for testing, and the following results are obtained through urine specific gravity correction: the contents of 8-OHdG, t-MA, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA are respectively 0.825-12.651 ng/mL, 6.708-131.461 ng/mL, 0.024-0.186 ng/mL (6 cases are not detected), 8.619-811.166 mu g/mL, 12.952-5154.764 ng/mL, 18.650-10428.540 ng/mL and 11.077-4707.659 ng/mL.

20 parts of urine of employees of a certain property company are collected for testing, and the following results are obtained after the urine specific gravity is corrected: the contents of 8-OHdG, t-MA, S-PMA, HA, 2-MHA, 3-MHA and 4-MHA are respectively 0.943-10.433 ng/mL, 4.173-48.504 ng/mL, 0.020-0.034 ng/mL (14 cases are not detected), 6.118-452.130 mu g/mL, 5.760-40.313 ng/mL, 11.286-135.178 ng/mL and 6.270-67.055 ng/mL.

The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.

Claims

1. A method for detecting metabolites of benzene, toluene and xylene in urine is characterized by comprising the following steps:

(1) mixing urine with a first solvent, and centrifuging to obtain a supernatant; mixing the supernatant with a second solvent to obtain a first sample solution, and detecting a first metabolite in the first sample solution by adopting liquid chromatography-tandem mass spectrometry; wherein the first solvent is methanol, and the second solvent is ultrapure water; the first metabolites include trimebuterol urate S-PMA, 8-hydroxy-2-deoxyguanosine 8-OHdG, 2-methylhippuric acid 2-MHA, 3-methylhippuric acid 3-MHA, and 4-methylhippuric acid 4-MHA;

(2) mixing part of the first sample solution with a third solvent to obtain a second sample solution, and detecting a second metabolite in the second sample solution by adopting liquid chromatography-tandem mass spectrometry; wherein the third solvent is a mixture of ultrapure water and methanol; the second metabolite comprises hippuric acid HA;

wherein, the conditions of the liquid chromatogram tandem mass spectrum of the steps (1) and (2) are as follows:

liquid chromatography conditions: the liquid chromatographic column is a reversed phase chromatographic column, the grain diameter of a filler of the reversed phase chromatographic column is less than or equal to 5.0 mu m, and the column length is 30-150 mm; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is 0.005-0.2 vol% of aqueous solution of formic acid or acetic acid; the mobile phase B is a methanol solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%, or an acetonitrile solution of formic acid or acetic acid with the volume percentage of 0-0.2 vol%; the flow rate of the mobile phase is 0.1-0.5 mL/min; the column temperature is 30-60 ℃; the elution mode is gradient elution; the sample injection amount is 1-10 mu L;

mass spectrum conditions: adopting an electronic spray ionization source ESI, wherein the 8-hydroxy-2-deoxyguanosine adopts a positive ion mode ESI⁺ESI in negative ion mode for metabolites other than 8-hydroxy-2-deoxyguanosine^﹣(ii) a Adopting a positive and negative ion mode to collect in sections; monitoring by adopting a multi-reaction monitoring mode MRM; the capillary voltage is 0.5-5 kV; the ion source temperature is 120-180 ℃.

2. The assay of claim 1 wherein the first metabolite further comprises trans, trans-myfuroic acid t, t-MA.

3. The method of claim 2, wherein the first metabolite further comprises o-methylphenol.

4. The detection method according to claim 1, wherein the volume ratio of the urine to the first solvent is 1: 1-20; the volume ratio of the supernatant to the second solvent is 1: 0.5-20; the volume ratio of the part of the first sample liquid to the third solvent is 1: 20-500.

5. The method according to claim 1, wherein the volume percentage of methanol in the mixture of ultrapure water and methanol is 10 to 25 vol%.

6. The detection method according to claim 1, wherein the gradient elution is as follows: keeping the volume percent of the mobile phase A unchanged for 0-1 min; 1-10 min, reducing the volume of the mobile phase A from 85 vol% to 70 vol%; reducing the volume of the mobile phase A from 70 vol% to 25 vol% for 10-13 min; 13-14 min, and the mobile phase A rises from 25 vol% to 85 vol%.

7. The detection method according to claim 1, wherein under the mass spectrometry conditions, the collision energy is 5-25 eV, the cone hole voltage is 1-50V, and the quantitative ion pair is as follows:

。

8. The detection method according to claim 1, wherein an external standard method is adopted for quantification, and a standard curve is established by taking the concentration of a standard substance as an X axis and taking the peak area of a quantitative ion response as a Y axis; quantifying the response peak area obtained by the liquid chromatogram tandem mass spectrum in the step (1) and the step (2) through a standard curve to obtain the measured concentration; converting the measured concentration to obtain the concentration c of the first metabolite and the second metabolite in the urine; the conversion formula is as follows:

c＝c₀×n

in the formula, c₀To measure concentration; and n is the dilution multiple.