CN111057055B - Multichannel mass spectrum derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon, and preparation method and application thereof - Google Patents

Multichannel mass spectrum derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon, and preparation method and application thereof Download PDF

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CN111057055B
CN111057055B CN201911257627.4A CN201911257627A CN111057055B CN 111057055 B CN111057055 B CN 111057055B CN 201911257627 A CN201911257627 A CN 201911257627A CN 111057055 B CN111057055 B CN 111057055B
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朱树芸
孙鲁平
赵先恩
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Qufu Normal University
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Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a multichannel mass spectrometry derivation reagent for detecting hydroxyl polycyclic aromatic hydrocarbons, and a preparation method and application thereof, in particular to a method for analyzing various free hydroxyl polycyclic aromatic hydrocarbons by using 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid as a derivation reagent and combining multichannel stable isotope labeling derivation with magnetic dispersion solid phase extraction and ultra-high performance liquid chromatography triple quadrupole mass spectrometry. The structural formula of the derivatization reagent is as follows:
Figure DEST_PATH_IMAGE002
. The invention can realize one-time sample introduction and simultaneous detection of multiple hydroxyl polycyclic aromatic hydrocarbons in 9 actual samples. The hydroxyl polycyclic aromatic hydrocarbon content in the actual sample can be obtained through quantification by an internal standard method. The method has the advantages of high flux, rapidness, high sensitivity, high accuracy, high selectivity and the like. The method provides an accurate and reliable technical means for evaluating the exposure level of the human polycyclic aromatic hydrocarbon.

Description

Multichannel mass spectrum derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon, and preparation method and application thereof
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a multichannel mass spectrometry derivation reagent for detecting hydroxyl polycyclic aromatic hydrocarbons, and a preparation method and application thereof, in particular to a method for analyzing various free hydroxyl polycyclic aromatic hydrocarbons by using 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid as a derivation reagent and combining multichannel stable isotope labeling derivation with magnetic dispersion solid phase extraction and ultra-high performance liquid chromatography triple quadrupole mass spectrometry.
Background
Polycyclic Aromatic Hydrocarbons (PAHs) exposure poses serious threats to human health, such as lung cancer, diabetes and cardiovascular and cerebrovascular diseases, and meanwhile, exposure of pregnant women to high-concentration polycyclic aromatic hydrocarbons to metabolism can cause miscarriage, premature birth, restricted intrauterine growth of fetuses and other adverse pregnancy fatalities and also influence intelligence development in childhood. Hydroxyl polycyclic aromatic hydrocarbons (OH-PAHs) are biomarkers for assessing human polycyclic aromatic hydrocarbon exposure levels. Simultaneous determination of a number of different hydroxyl polycyclic aromatic hydrocarbons is essential in exposure studies. In the human body, metabolites of polycyclic aromatic hydrocarbons mostly exist in the form of conjugates, and the matrix effect interference is serious, which is a great challenge for detecting trace amount of free hydroxyl polycyclic aromatic hydrocarbons. Therefore, it is important and necessary to establish a rapid, sensitive, accurate and high-flux analysis method for the free hydroxyl polycyclic aromatic hydrocarbon.
Chinese patent (CN103175920A) discloses a method for preparing 1-hydroxy phenanthrene, 2-hydroxy phenanthrene, 3-hydroxy phenanthrene, 4-hydroxy phenanthrene, 9-hydroxy phenanthrene, 1-hydroxy pyrene, 3-hydroxy benzo [ a ] alpha by using N, O-bis (trimethylsilyl) acetamide as a derivatization reagent through a gas chromatography-mass spectrometer]Pyrene and 7-hydroxybenzo [ a]Pyrene analysis and detection method using 2-hydroxy phenanthrene-d91-hydroxypyrene-d9And 3-hydroxybenzo [ a]Pyrene-d11The three deuterated compounds are used as internal standards, so that the derivatization time is long, the sensitivity is low, and the internal standard compounds are expensive and are not easy to purchase; chinese patent (CN106483230A) discloses that acetone and n-undecanol are used for carrying out dispersion liquid extraction-floating liquid drop solidification method enrichment on 2-hydroxynaphthalene, 2-hydroxyfluorene, 2-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene and 1-hydroxypyrene, and the analysis and detection are carried out by combining a high performance liquid chromatography-fluorescence detection method, so that the time consumption, the low sensitivity and the low analysis flux are realized. Therefore, the development of an analysis method for simultaneously detecting multiple components with high sensitivity and high flux has very important significance for evaluating and analyzing the exposure level of the polycyclic aromatic hydrocarbon in the human body.
Disclosure of Invention
The invention aims to solve the technical problem of how to sensitively, accurately and simultaneously detect various free hydroxyl polycyclic aromatic hydrocarbons with high flux, and provides a multichannel mass spectrum derivatization reagent for detecting the hydroxyl polycyclic aromatic hydrocarbons.
The invention also provides a preparation method of the multichannel mass spectrum derivatization reagent for detecting the hydroxyl polycyclic aromatic hydrocarbon.
The invention also provides a method for detecting hydroxyl polycyclic aromatic hydrocarbon by using the multichannel mass spectrometry derivation reagent, which designs and synthesizes a 10-channel stable isotope mass label (SIMT-332/338/346/349/351/354/360/363/374/377) for the first time, wherein, the derivative product of the SIMT-346 marked standard product is used as an internal standard, other 9 mass labels (SIMT-332/338/349/351/354/360/363/374/377) respectively mark 9 actual samples, 10 parts of solution are mixed in equal volume and then are subjected to the analysis method of magnetic dispersion solid phase extraction combined with ultra-high performance liquid chromatography triple quadrupole mass spectrometry detection, can realize one-time sample introduction and simultaneously analyze and determine various hydroxyl polycyclic aromatic hydrocarbons in 9 actual samples. The magnetic graphene oxide is used as an extraction material, and the method has the advantage of simple operation.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a multichannel mass spectrum derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon, which has the following structural formula:
Figure RE-GDA0002415695410000021
the R is1Is CH3，R2Is CH3、C2H5、C2D5、C3H7Or C4H9；
The R is1Is a CD3，R2Is a CD3、C2H5、C2D5、C3H7Or C4H9。
The invention also provides a preparation method of the multichannel mass spectrometry derivation reagent, which comprises the following steps:
a. 0.5g pipemidic acid was dissolved in 20mL acetonitrile and 0.59g K was added2CO3And adding 6.10mmol of iodomethane or deuterated iodomethane under stirring at room temperature, stirring the reaction mixture at room temperature for 8-9 h, carrying out vacuum filtration, and drying the obtained solid in an oven. Obtaining white solid powder, namely [ CH3]2/[CD3]2-PPA；
b. 0.5g pipemidic acid was dissolved in 20mL acetonitrile and 0.59g K was added2CO3Respectively adding 6.59mmol of bromoethane, deuterated bromoethane, 1-bromopropane or n-bromobutane under stirring at room temperature, stirring the reaction mixture at room temperature for 12-14 h, and respectively adding 6.10mmol of iodomethane or deuterated iodomethane and 0.59g K under stirring2CO3Continuously stirring for 22-25 h, carrying out vacuum filtration, drying the obtained solid in an oven to obtain white solid powder, namely [ CH3-C2H5]-PPA、[CD3-C2H5]- PPA、[CH3-C2D5]-PPA、[CD3-C2D5]-PPA、[CH3-C3H7]-PPA、[CD3-C3H7]-PPA、[CH3-C4H9]- PPA、[CD3-C4H9]-PPA；
c. 1.0g a and the product of b and 25mL of thionyl chloride were placed in a 50mL reaction flask connected to a reflux condenser with a NaOH fill tube. Stirring the mixture at 80 deg.C, refluxing for 3 hr, removing thionyl chloride by reduced pressure evaporation, and cooling to room temperature to obtain orange yellow solid, i.e. 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid (SIMT-332/338/346/349/351/354/360/363/374/377).
The invention also provides a method for detecting the hydroxyl polycyclic aromatic hydrocarbon by using the multichannel mass spectrometry derivation reagent, which comprises the following steps: carrying out derivatization reaction on a target substance by using a 10-channel stable isotope mass label 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid (SIMT-332/338/346/349/351/354/360/363/374/377): the method comprises the following steps of taking a derivative product of a SIMT-346 marked standard product as an internal standard, respectively marking 9 actual samples by other 9 mass labels (SIMT-332/338/349/351/354/360/363/374/377), carrying out magnetic dispersion solid-phase extraction by using magnetic graphene oxide after 10 parts of solutions are mixed in equal volume, and carrying out liquid-mass analysis to detect the hydroxyl polycyclic aromatic hydrocarbon after extract liquid is filtered by a filter membrane.
The detection method provided by the invention specifically comprises the following steps:
a. multi-channel stable isotope labeling derivatization process: respectively taking 9 parts of 10-50 μ L sample to be detected or 7 standard substance mixed solutions, 50-300 μ L NaHCO with pH of 8.0-103-Na2CO3Buffer solutions are respectively added into a centrifuge tube, then 100 mul of acetonitrile solution (SIMT-332/338/349/351/354/360/363/374/377) of 4 '- (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid and acetonitrile solution (SIMT-346) of 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid are respectively injected, the mixture is evenly shaken and sealed, and the mixture is subjected to oscillation reaction in a water bath at the temperature of 35-50 ℃ for 3-5 minutes; obtaining a derivatization product solution of a substance to be detected and an internal standard substance solution;
b. magnetic dispersion solid phase extraction process: taking 50-100 mu L of a mixed solution of a derivatization product solution of an object to be detected and an internal standard solution with the same volume, adding 6-10mg of an extraction material, dispersing the extraction material to form a suspension by ultrasonic assistance, then adjusting the pH of the solution to 7.0 by using a borate buffer solution, violently oscillating and adsorbing for 3-6 minutes, then separating the mixture by an external magnet, finally carrying out vortex operation for 1-5 minutes in 100-500 mu L of a desorbent solution, then drying under nitrogen flow, and re-dissolving the solid by acetonitrile to obtain a hydroxyl polycyclic aromatic hydrocarbon derivative extract solution;
c. and filtering the hydroxyl polycyclic aromatic hydrocarbon derivative extract solution by using a filter membrane, and analyzing and detecting by using an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system.
Further, the concentration of the standard substance mixed solution is 1.0X 10-5mol/L。
The extraction material used in the invention is magnetic graphene oxide, and is prepared by the following method: adding 0.1g of GO into 100mL of high-purity water, performing ultrasonic treatment for 2 hours to obtain 1g/mL of GO dispersed solution, and simultaneously purging 5.40g of FeCl by using nitrogen3·6H2O and 1.98g FeCl2·4H2O in 30mL water, then stirring the above solution and GO solution under nitrogen for 3h, adding 28% ammonia solution to adjust the pH of the solution, synthesizing magnetite material, stirring rapidly at 60 ℃ for 3h, cooling the solution to room temperature, then washing the dark black solution again with water/ethanol three times, and drying in vacuum at 65 ℃ to obtain Fe3O4a/GO composite material.
Further, the desorbent is methanol, ethanol, acetonitrile, acetone, methanol (0.1% formic acid), methanol (0.5% formic acid), methanol (1% formic acid), methanol (5% formic acid) or methanol (10% formic acid); preferably, the desorbent is methanol (1% formic acid); the resolution time was 2 minutes.
According to the detection target object, the free hydroxyl polycyclic aromatic hydrocarbon comprises one or more of 1-hydroxypyrene, 1-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene and 9-hydroxyphenanthrene.
The ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system used in the invention uses an Agilent SB C18 chromatographic column for chromatographic separation: 2.1mm × 50mm, 1.8 μm, 2 μ L of sample volume, 30 deg.C of column temperature, and gradient elution; the gradient elution method is characterized in that the time is 5.5min, the flow rate is 0.2mL/min, the mobile phase A is 10% methanol aqueous solution containing 0.1% formic acid, the mobile phase B is methanol containing 0.1% formic acid, the mobile phase composition at 0min is 35% A + 65% B, 15% A + 85% B at 1min, 2% A + 98% B at 3.5min, 2% A + 98% B at 4.5min, 0% A + 100% B at 5min, and 0% A + 100% B at 5.5 min.
The mass spectrum conditions of the ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system during analysis and detection are as follows: the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the air pressure of the sprayer is 40psi, the temperature of the sheath gas is 300 ℃, the flow rate is 11L/min, and the voltage of the capillary tube is 3.5 kV.
The invention provides an analysis method for simultaneously detecting multiple free hydroxyl polycyclic aromatic hydrocarbons in multiple biological samples in a high-throughput manner, wherein the hydroxyl polycyclic aromatic hydrocarbons are subjected to a derivatization reaction with a derivatization reagent 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid under a mild condition, an obtained derivatization product is subjected to magnetic dispersion solid-phase extraction through magnetic graphene oxide, an obtained derivatization extract solution is filtered through a filter membrane, and then an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system is used for analysis and detection.
Each fraction in the mobile phase of the present invention refers to a volume fraction.
The target analyte has the following structural formula:
Figure RE-GDA0002415695410000041
the method provided by the invention can bring a remarkable mass spectrum sensitization effect to the detection of the hydroxyl polycyclic aromatic hydrocarbon by combining a stable isotope derivatization technology with an ultra-high performance liquid chromatography triple quadrupole mass spectrometry. The derivatization reagent 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid containing one intramolecular permanent positive charge is used for the first time to derivatize the hydroxyl polycyclic aromatic hydrocarbon. Therefore, when the derivative obtained by the derivatization reaction of the hydroxyl polycyclic aromatic hydrocarbon is detected, the derivative product parent ion can generate specific reporter ions containing the pipemidic acid structure, and the remarkable mass spectrum sensitization detection effect is brought. By using the multi-channel quality label to mark the sample to be detected, the method can realize the simultaneous detection of multiple hydroxyl polycyclic aromatic hydrocarbons in 9 actual samples by single-needle sample injection, save a large amount of time and realize high-throughput analysis. The method has the advantages of good sensitivity, selectivity and accuracy and good recovery rate result through internal standard method quantification. The active phenolic hydroxyl contained in 7 kinds of hydroxyl polycyclic aromatic hydrocarbon can react with the active carbonyl chloride reactive group of the derivative reagent 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid quickly. The invention overcomes the problems of long reaction time, low sensitivity, serious matrix interference, low analysis flux and the like of the conventional method, and greatly improves the analysis flux, the sensitivity, the selectivity and the accuracy of liquid-mass detection. The invention provides an efficient and reliable technical means for simultaneously detecting various hydroxyl polycyclic aromatic hydrocarbons in human urine and blood plasma. Taking 1-hydroxypyrene as an example, the derivatization reaction process of 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid (SIMT-332) as a derivatization reagent is as follows:
Figure RE-GDA0002415695410000051
the invention has the advantages and beneficial effects that:
1. the invention designs and synthesizes a 10-channel stable isotope mass label 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid for the first time, and the label is used for stable isotope labeling derivatization of hydroxyl polycyclic aromatic hydrocarbon, wherein a standard substance labeled by SIMT-346 is used as an internal standard, and the rest 9 mass labels are respectively used for labeling 9 actual samples. High-throughput analysis for simultaneously detecting 9 actual samples by one-time sample introduction is realized. The derivatization reaction condition is mild and rapid, the derivatization product is stable, the analysis flux is high, and the technology obviously improves the analysis flux, the chromatographic resolution and the mass spectrum detection sensitivity of the analysis method.
2. The pretreatment technology of multichannel stable isotope labeling derivatization-magnetic dispersion solid phase extraction provided by the invention combines with an ultra-high performance liquid chromatography triple quadrupole mass spectrometry multiple reaction monitoring mode, and has the advantages of high flux, high sensitivity, high accuracy, simple and convenient operation and the like.
3. The detection and analysis method is successfully applied to the detection of various free hydroxyl polycyclic aromatic hydrocarbons in human urine and blood plasma, and the method has good applicability.
Drawings
FIG. 1 is a schematic diagram of the multi-pass stable isotope labeling derivatization combined with magnetic dispersion solid phase extraction and ultra-high phase liquid chromatography strategy.
FIG. 2 shows the mass spectrometric separation of 7 hydroxy polycyclic aromatic hydrocarbon derivatives from 10 stable isotope mass labels in example 1, (A): 2-hydroxy naphthalene derivative, (B): 1-hydroxy naphthalene derivative, (C): 2-hydroxy fluorene derivative, (D): 3-hydroxy phenanthrene derivative, (E): 9-hydroxy phenanthrene derivative, (F): 4-hydroxy phenanthrene derivative, and (G): 1-hydroxy pyrene derivative.
FIG. 3 is a schematic diagram showing the mass spectrometric cleavage mechanism of SIMT-332 and 1-hydroxypyrene derivatives in example 1.
FIG. 4 shows the mass spectrometric separation of hydroxy polycyclic aromatic hydrocarbon derivatives in urine in example 2, wherein (A) is 2-hydroxynaphthalene derivative, (B) is 1-hydroxynaphthalene derivative, (C) is 2-hydroxyfluorene derivative, (D) is 3-hydroxyphenanthrene derivative, (E) is 9-hydroxyphenanthrene derivative, (F) is 4-hydroxyphenanthrene derivative, and (G) is 1-hydroxypyrene derivative.
Detailed Description
The present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the invention.
The derivatization extract solution is filtered by a filter membrane of 0.22 mu m and then is analyzed and detected by an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometry system, and fig. 1 is an overview chart of the method.
The synthesis method of the derivatization reagent 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid used in the invention comprises the following steps:
a. 0.5g of pipemidic acid (1.65mmol), 20mL of acetonitrile was charged to a 50mL single-neck flask. Then 0.59g K was added2CO3(4.29mmol) and 6.10mmol of iodomethane or deuterated iodomethane are added at room temperature with stirring. And (3) reacting the reaction mixture at room temperature for 8-9 h, carrying out vacuum filtration, and drying the obtained solid in an oven. Obtaining white solid powder, namely [ CH3]2/[CD3]2PPA, yield 99.37%/99.42%.
b. 0.5g of pipemidic acid (1.65mmol), 20mL of acetonitrile was charged to a 50mL single-neck flask. Then 0.59g K was added2CO3(4.29mmol) and 6.59mmol of bromoethane, deuterated bromoethane, 1-bromopropane or n-bromobutane are added with stirring at room temperature. After 12h reaction at room temperature, 6.10mmol of iodomethane or deuterated iodomethane and 0.59g K were added under stirring2CO3(4.29 mmol). Stirring was continued for 22h, suction filtration under reduced pressure and the resulting solid dried in an oven. Obtaining white solid powder, namely [ CH3-C2H5]-PPA、[CD3-C2H5]-PPA、[CH3-C2D5]-PPA、[CD3-C2D5]-PPA、[CH3-C3H7]- PPA、[CD3-C3H7]-PPA、[CH3-C4H9]-PPA、[CD3-C4H9]PPA, all in yields greater than 92%.
c. About 1.0g a and the product of b, 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid, and 25mL of thionyl chloride were placed in a 50mL reaction flask connected to a reflux condenser with a NaOH fill tube. The mixture was stirred at 80 ℃ and refluxed for 3 hours. Removing thionyl chloride by reduced pressure evaporation, and cooling to room temperature to obtain orange solid, namely 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid (SIMT-332/338/346/349/351/354/360/363/374/377).
The extraction material magnetic graphene oxide used in the invention is prepared by the following method: adding 0.1g of GO into 100mL of high-purity water, and carrying out ultrasonic treatment for 2h to obtain 1g/mL of GO dispersion solution. While purging 5.40g FeCl with nitrogen3·6H2O and 1.98g FeCl2·4H2Solution of O in water (30 mL). And then stirring the solution and the GO solution for 3 hours under nitrogen, adding 28% ammonia solution to adjust the pH value of the solution, and synthesizing the magnetite material. After stirring rapidly at 60 ℃ for 3h, the solution was cooled to room temperature. The dark black solution was then washed three more times with water/ethanol and dried in vacuo at 65 ℃. To obtain Fe3O4a/GO composite material.
Example 1
Chromatographic separation and mass spectrum qualitative and quantitative analysis of 7 kinds of hydroxyl polycyclic aromatic hydrocarbon:
7 hydroxy polycyclic aromatic hydrocarbon standards (purchased from Sigma, J)&K & Aladdin reagent Co.) was made up with acetonitrile to a concentration of 1.0X 10-2And (3) a hydroxyl polycyclic aromatic hydrocarbon standard solution of mol/L. We centrifuged the analyte-free urine at high speed to remove impurities, added the analyte-free plasma to acetonitrile (1:3, v/v), centrifuged for 5 minutes (12000rpm), used the supernatant separated, and the treated analyte-free urine and plasma were mixed in the same ratio as a blank matrix. 0.2mL of the mixed standard stock solution of the hydroxyl polycyclic aromatic hydrocarbon is respectively taken from each single standard stock solution, and the volume is adjusted to 10mL by using a blank matrix to obtain the mixed standard stock solution of the hydroxyl polycyclic aromatic hydrocarbon. 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid is dissolved in acetonitrile to give 1.0X 10-3mol/L derivatizing reagentA solution; 9 parts of 20. mu.L standard substance solution are respectively put into a centrifuge tube of 1.5mL, and another 20. mu.L standard substance mixed solution (1.0X 10)-5mol/L) were placed in a 1.5mL centrifuge tube and 100. mu.L of NaHCO was added to each tube3-Na2CO3(pH 9.2) buffer solution, vortex mixer for 15s, add 200. mu.L each of the other 9 SIMTs (SIMT-332/338/349/351/354/360/363/374/377) solutions, SIMT-346 solution, seal, and react at 40 ℃ for 5 minutes. The SIMT-346 labeled standard derivative product solution (internal standard solution) and the other 9 derivatizing agent labeled standard derivative product solutions were then mixed in equal volumes in a 1.0mL tube (derivative product mixed solution) and subsequently used in the MDSPE procedure.
8mg of adsorbent (Fe)3O4/GO nanocomposite) was dispersed in the derivative product mix solution by ultrasound assisted dispersion to form a homogeneous suspension. The pH of the solution was then adjusted to 7.0 by using borate buffer. Adsorption equilibrium was reached by vigorous shaking for 5 minutes, and the mixture was then separated by an external magnet. The desorption was carried out by vortexing in 200 μ L of a 1% formic acid-containing methanol solution (as desorbent, where the desorbent was changed to methanol, ethanol, acetonitrile, acetone, methanol (0.1% formic acid), methanol (0.5% formic acid), methanol (5% formic acid) or methanol (10% formic acid), the analyte extraction efficiency was between 37-88% of that of methanol (1% formic acid) as desorbent) for 2 minutes. The eluate was collected and dried under a gentle stream of nitrogen and the solid material was redissolved and subjected to UHPLC-MS/MS analysis. The time is 5.5min, the flow rate is 0.2mL/min, the mobile phase A is 10% methanol aqueous solution containing 0.1% formic acid, the mobile phase B is methanol containing 0.1% formic acid, the mobile phase composition at 0min is 35% A + 65% B, 15% A + 85% B at 1min, 2% A + 98% B at 3.5min, 2% A + 98% B at 4.5min, 0% A + 100% B at 5min, and 0% A + 100% B at 5.5 min; the conditions of mass spectrum are: the temperature of the drying gas is 300 ℃, the flow rate is 10L/min, the air pressure of the sprayer is 40psi, the temperature of the sheath gas is 300 ℃, the flow rate is 11L/min, and the voltage of the capillary tube is 3.5 kV.
Better separation degree can be obtained according to the gradient elution procedure, and a graph 2 shows that the separation degree between 10 stable isotope mass labels and 7 hydroxyl polycyclic aromatic hydrocarbon derivatives is good, wherein the graph (A) is a mass spectrometric detection separation graph of 2-hydroxynaphthalene derivatives, (B) is 1-hydroxynaphthalene derivatives, (C) is 2-hydroxyfluorene derivatives, (D) is 3-hydroxyphenanthrene derivatives, (E) is 9-hydroxyphenanthrene derivatives, (F) is 4-hydroxyphenanthrene derivatives, (G) is 1-hydroxypyrene derivatives, and 70 hydroxyl polycyclic aromatic hydrocarbon derivatives are good. Mass spectrometry experimental results show that 70 hydroxyl polycyclic aromatic hydrocarbon derivatives produced the same major daughter ions m/z 332.2, m/z 338.2, m/z 346.2, m/z 349.2, m/z 351.2, m/z 353.2, m/z 360.2, m/z 363.2, m/z 374.2 and m/z 377.2 in the multiple reaction monitoring mode (MRM) for use as the quantitative daughter ions. Taking the derivative of SIMT-332 derived 1-hydroxypyrene as an example, FIG. 3 is a schematic diagram of mass spectrum cleavage mechanism of SIMT-332 and 1-hydroxypyrene derivatives, and a parent ion [ M ] + M/z 532.2 can generate a specific daughter ion M/z 332.2. Parameters of the MRM mode were optimized, quantitative ion pairs of 70 hydroxyl polycyclic aromatic hydrocarbon derivatives, optimized fragmentation voltage (V) and collision energy (ev), and correlation coefficient, detection limit, and quantitative limit of the analytical method are shown in table 1.
TABLE 1
Figure RE-GDA0002415695410000081
Figure RE-GDA0002415695410000091
Example 2
The detection and analysis of the free hydroxyl polycyclic aromatic hydrocarbon in the urine comprises the following operation steps:
centrifuging 500 μ L of urine of smoker or non-smoker at high speed to remove impurities, placing 9 50 μ L urine samples in 1.5mL centrifuge tube, and collecting 50 μ L standard substance mixed solution (1.0 × 10)-5mol/L) were placed in a 1.5mL centrifuge tube and 250. mu.L of NaHCO was added to each tube3-Na2CO3(pH 9.8) buffer solution, vortex mixer for 15s, add 550. mu.L each of the other 9 SIMTs (SIMT-332/338/349/351/354/360/363/374/377) solutions, SIMT-346 solution, seal, and react at 40 ℃ for 4.5 minutes. The SIMT-346 labeled standard derivative product solution (internal standard) was then addedSolution) and other 9 derivatization reagent-labeled urine sample derivatization product solutions were mixed in equal volumes in 1.0mL tubes (derivatization product mixture solution) and then used in the MDSPE procedure.
7mg of adsorbent (Fe)3O4/GO nanocomposite) was dispersed in the derivative product mix solution by ultrasound assisted dispersion to form a homogeneous suspension. The pH of the solution was then adjusted to 7.0 by using borate buffer. Adsorption equilibrium was reached by vigorous shaking for 5.5 minutes, and the mixture was then separated by an external magnet. The desorption was carried out by vortexing in 450. mu.L of a 1% formic acid-containing methanol solution for 3 minutes. The eluate was collected and dried under a gentle stream of nitrogen and the solid material was redissolved and subjected to UHPLC-MS/MS analysis. The content of free hydroxyl polycyclic aromatic hydrocarbon (n is 3) in the urine of smokers is respectively detected as follows: 0.0492ng/mL of 1-hydroxypyrene, 0.7644ng/mL of 1-hydroxynaphthalene, 1.3847ng/mL of 2-hydroxynaphthalene, 0.2486ng/mL of 2-hydroxyfluorene, 0.0273ng/mL of 3-hydroxyphenanthrene, 0.0135ng/mL of 4-hydroxyphenanthrene and 0.1419ng/mL of 9-hydroxyphenanthrene; the content of free hydroxyl polycyclic aromatic hydrocarbon (n is 3) in the urine of non-smokers is respectively as follows: 0.0042ng/mL of 1-hydroxypyrene, 0.2171ng/mL of 1-hydroxynaphthalene, 0.4069ng/mL of 2-hydroxynaphthalene, 0.0614ng/mL of 2-hydroxyfluorene, 0.0115ng/mL of 3-hydroxyphenanthrene, 0.0042ng/mL of 4-hydroxyphenanthrene and 0.0077ng/mL of 9-hydroxyphenanthrene, and the mass spectrometric detection chart of the sample is shown in FIG. 4.
Example 3
The detection of the free hydroxyl polycyclic aromatic hydrocarbon in the plasma comprises the following operation steps:
200 μ L of smoker or non-smoker plasma was taken, 100 μ L of acetonitrile was added, centrifuged at high speed for 5 minutes (12000rpm) and the supernatant was separated for use. 9 portions of 20. mu.L plasma samples were placed in a 1.5mL centrifuge tube, and another 20. mu.L standard mix solution (1.0X 10)-5mol/L) were placed in a 1.5mL centrifuge tube and 100. mu.L of NaHCO was added to each tube3-Na2CO3(pH 9.0) buffer solution, vortex mixer for 15s, add 150. mu.L each of the other 9 SIMTs (SIMT-332/338/349/351/354/360/363/374/377) solutions, SIMT-346 solution, seal, and react at 40 ℃ for 6 minutes. The SIMT-346 labeled standard derivatized product solution (internal standard solution) and the other 9 derivatization reagent labeled plasma samples were then derivatizedThe solution was mixed in equal volumes in 1.0mL tubes (derivatization product mix solution) and subsequently used in the MDSPE procedure.
8.5mg of adsorbent (Fe)3O4/GO nanocomposite) was dispersed in the derivative product mix solution by ultrasound assisted dispersion to form a homogeneous suspension. The pH of the solution was then adjusted to 6.8 by using borate buffer. Adsorption equilibrium was reached by vigorous shaking for 4.7 minutes, and the mixture was then separated by an external magnet. The desorption operation was performed by vortexing in 240. mu.L of a 1% formic acid-containing methanol solution for 1.7 minutes. The eluate was collected and dried under a gentle stream of nitrogen and the solid material was redissolved and subjected to UHPLC-MS/MS analysis. The content of 3 free hydroxyl polycyclic aromatic hydrocarbons (n ═ 3) detected in the plasma of smokers was: 0.0084ng/mL of 1-hydroxynaphthalene, 0.0151ng/mL of 2-hydroxynaphthalene and 0.0007ng/mL of 2-hydroxyfluorene; only 2 free hydroxyl polycyclic aromatic hydrocarbon contents (n ═ 3) were detected in the plasma of non-smokers: 0.0003ng/mL of 1-hydroxynaphthalene and 0.0008 ng/mL of 2-hydroxynaphthalene.
Example 4
The method for detecting the hydroxyl polycyclic aromatic hydrocarbon in the sediment comprises the following operation steps:
in reference (Zhongshan university journal (Nature science edition), 2015, 02:77-82), 9 river sediment samples (2.0 g) were taken, freeze-dried, ground through a 0.125mm metal sieve, subjected to accelerated solvent extraction with methanol, subjected to rotary evaporation concentration, redissolved with n-hexane and passed through a neutral alumina column; using dichloromethane containing 2% acetic acid as an elution solvent, and repeatedly eluting twice, wherein 5mL of the elution solvent is used each time; the eluted sample was dried with nitrogen, 20. mu.L acetonitrile was added for further use, and 20. mu.L of a mixed solution of standards (1.0X 10)-5mol/L) were placed in a 1.5mL centrifuge tube and 150. mu.L of NaHCO was added to each tube3- Na2CO3(pH 9.5) buffer solution, vortex mixer for 15s, add 200. mu.L each of the other 9 SIMTs (SIMT-332/338/349/351/354/360/363/374/377) solutions, SIMT-346 solution, seal, and react at 45 ℃ for 4.5 minutes. The SIMT-346 labeled standard derivatized product solution (internal standard solution) and the other 9 derivatization reagent labeled sediment sample derivatized product solutions were then mixed in equal volumes in a 1.0mL tube (derivatized)Production mix solution) and then used in the MDSPE program.
7.5mg of adsorbent (Fe)3O4/GO nanocomposite) was dispersed in the derivative product mix solution by ultrasound assisted dispersion to form a homogeneous suspension. The pH of the solution was then adjusted to 7.0 by using borate buffer. Adsorption equilibrium was reached by vigorous shaking for 5.5 minutes, and the mixture was then separated by an external magnet. The desorption operation was performed by vortexing in 200. mu.L of a 1% formic acid-containing methanol solution for 2.3 minutes. The eluate was collected and dried under a gentle stream of nitrogen and the solid material was redissolved and subjected to UHPLC-MS/MS analysis. 7 kinds of hydroxyl polycyclic aromatic hydrocarbons (n ═ 3) in a certain river bottom sediment are detected as follows: 0.0272ng/g of 1-hydroxypyrene, 0.7218ng/g of 1-hydroxynaphthalene, 0.7504ng/g of 2-hydroxynaphthalene, 0.7029ng/g of 2-hydroxyfluorene, 0.6953ng/g of 3-hydroxyphenanthrene, 0.5485ng/g of 4-hydroxyphenanthrene and 0.7739ng/g of 9-hydroxyphenanthrene.
Comparative example 1
The comparative example is the same as example 2, except that no magnetic dispersion solid phase extraction is performed in the derivatization reaction and sample pretreatment processes, and the derivatization reagent and derivatization conditions adopt N, O-bis tri silicyl acetamide disclosed in Chinese patent (CN103185762A) as the derivatization reagent.
Comparative example 2
The procedure of this comparative example is the same as that of example 2, except that the derivatization reaction and the sample pretreatment are carried out using dansyl chloride disclosed in the literature (Journal of Chromatography A, 2015, 1379: 51-55) as the derivatization reagent and liquid-liquid extraction.
Comparative example 3
The comparative example was conducted in the same manner as in example 2, except that no derivatization reagent was used and no magnetic dispersion solid phase extraction was performed as disclosed in Chinese patent No. CN106483230A during the pretreatment of the sample.
Comparative example 4
The comparative example is the same as example 2 except that during the sample pretreatment, derivatization was carried out without using a derivatization reagent and magnetic dispersion solid phase extraction was not carried out as disclosed in the literature (journal of analytical tests, 2019, 38: 270-276).
Table 2 below shows the results of the tests of example 2 in comparison with comparative examples 1 to 4.
TABLE 2
Figure RE-GDA0002415695410000121
As can be seen from Table 2, compared with the related reports, the method utilizes 4' - (N, N-dialkyl) -1-piperazinyl-6-carbonyl chloride-pipemidic acid to perform derivatization labeling on the hydroxyl polycyclic aromatic hydrocarbon, and has the advantages of mild and rapid derivatization conditions and high sensitivity. Compared with the comparative example 1, the method has the advantages of time saving, mild reaction conditions and higher sensitivity; compared to comparative example 2, the detection limit is similar, but the assay throughput of the present invention is 9 times it; compared with comparative example 3 and comparative example 4, the analysis flux is 9 times higher, and the detection limit is 2-6 orders of magnitude higher. In addition, the recovery rate result of the invention is good, and the accuracy of the analysis method is guaranteed.
To verify the applicability of the established assay, precision, accuracy (recovery) and matrix effect were examined in detail and the results are shown in table 3.
TABLE 3
Figure RE-GDA0002415695410000122
Figure RE-GDA0002415695410000131
As can be seen from tables 2 and 3, the linear range of 7 hydroxyl polycyclic aromatic hydrocarbon derivatives in 9 actual samples is 2-3000 pg/mL, the detection limit is distributed between 0.1-0.5pg/mL, and the quantification limit is distributed between 0.5-2.0 pg/mL. The result shows that the established analysis method has high analysis flux, high sensitivity and high accuracy, effectively reduces matrix interference and can be well applied to detecting the content of various hydroxyl polycyclic aromatic hydrocarbons in the three actual samples.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the embodiments, and any other changes, modifications, combinations, substitutions and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.

Claims (2)

1. A multichannel mass spectrometry derivatization reagent for detecting hydroxyl polycyclic aromatic hydrocarbon is characterized in that the derivatization reagent has the following structural formula:
Figure DEST_PATH_IMAGE001
the R is1Is CH3，R2Is CH3、C2H5、C2D5、C3H7Or C4H9；
The R is1Is a CD3，R2Is a CD3、C2H5、C2D5、C3H7Or C4H9。
2. A method for preparing the multichannel mass spectrometry derivation reagent for detecting hydroxyl polycyclic aromatic hydrocarbon of claim 1, comprising the following steps:
a. 0.5g pipemidic acid was dissolved in 20mL acetonitrile and 0.59g K was added2CO3Adding 6.10mmol of iodomethane or deuterated iodomethane under stirring at room temperature, stirring the reaction mixture at room temperature for 8-9 h, carrying out vacuum filtration, and drying the obtained solid in an oven;
obtaining white solid powder, namely [ CH3]2/[CD3]2-PPA；
b. 0.5g pipemidic acid was dissolved in 20mL acetonitrile and 0.59g K was added2CO3Respectively adding 6.59mmol of bromoethane, deuterated bromoethane, 1-bromopropane or n-bromobutane under stirring at room temperature, stirring the reaction mixture at room temperature for 12-14 h, and respectively adding 6.10mmol of iodomethane or deuterated iodomethane and 0.59g K under stirring2CO3Continuously stirring for 22-25 h, carrying out vacuum filtration, drying the obtained solid in an oven to obtain white solid powder, namely [ CH3-C2H5]-PPA、[CD3-C2H5]-PPA、[CH3-C2D5]-PPA、[CD3-C2D5]-PPA、[CH3-C3H7]-PPA、[CD3-C3H7]-PPA、[CH3-C4H9]-PPA、[CD3-C4H9]-PPA；
c. 1.0g a and the product of b neutralization and 25mL of thionyl chloride were placed in a 50mL reaction flask connected to a reflux condenser with a NaOH fill tube; stirring and refluxing the mixture for 3 hours at 80 ℃, evaporating under reduced pressure to remove thionyl chloride, and cooling to room temperature to obtain an orange solid, namely the multichannel mass spectrometry derivation reagent for detecting hydroxyl polycyclic aromatic hydrocarbon in claim 1;
the hydroxyl polycyclic aromatic hydrocarbon is selected from one or more of 1-hydroxypyrene, 1-hydroxynaphthalene, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene and 9-hydroxyphenanthrene.
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