CN106932518B - Method for detecting endocrine disruptors in complex biological matrix - Google Patents
Method for detecting endocrine disruptors in complex biological matrix Download PDFInfo
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- CN106932518B CN106932518B CN201710318791.6A CN201710318791A CN106932518B CN 106932518 B CN106932518 B CN 106932518B CN 201710318791 A CN201710318791 A CN 201710318791A CN 106932518 B CN106932518 B CN 106932518B
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
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Abstract
The invention relates to a method for detecting endocrine disruptors in a complex biological matrix, which comprises the following steps of S1: accelerated solvent extraction and redissolution, S2: automatic gel permeation chromatography clarification and redissolution, S3: solid phase extraction and redissolution, S4: and (4) analyzing the dissolving solution III by using a liquid chromatography-mass spectrometry analyzer to finish the quantitative detection of the endocrine disruptors. The invention adopts accelerated solvent extraction-gel permeation chromatography purification and solid phase extraction technology to establish a simultaneous detection pretreatment technology for a plurality of EDCs in a living body, and adopts liquid chromatography-mass spectrometry technology to establish an instrument analysis method, so that the concentration of endocrine disruptors in a complex biological matrix can be effectively detected, and the quantitative analysis of the endocrine disruptors in the living body can be realized.
Description
Technical field
The present invention relates to a kind of detection technique of incretion interferent in complex biological matrix, especially a kind of complex biologicals
The detection method of incretion interferent in matrix.
Background technique
Incretion interferent (Endocrine Disrupting Chemicals, EDCs) is that one kind can interfere organism
Or the xenobiotics of human endocrinological's system.Existing research is it has been found that the generally existing surrounding medium of incretion interferent
In, including in each surrounding medium such as water, soil and air.Incretion interferent can move to each corner by food link network
And amplification is presented along food chain, grave danger is constituted to human and animal.In the body fluid of humans and animals, including blood,
The pollution for all detecting incretion interferent in urine, Cord blood or even neonatal body fluid extensively, such as bisphenol-A (BPA), nonyl
Base phenol (NP), octyl phenol (4-t-OP) and tert-butyl phenol (4-t-BP) etc..In addition, toxicological experiment and epidemiological survey result
Show that a large amount of existing EDCs may endanger endocrine, reproduction, nerve and immune system of the mankind and organism etc. in environment
Normal function, and likely result in developmental anomaly, endocrine disturbance, infant's deformity etc., seriously affect the existence of the mankind
With development.Therefore, world many countries government, related scholar and related international organization are caused the problem of incretion interferent
Pay much attention to, another the global environmental problem being considered as after depletion of the ozone layer, greenhouse effects.
Therefore, the concentration for detecting incretion interferent in organism, is clear incretion interferent to biological body pollution
Premise.But many biological samples, such as the organ of animal, bile, blood etc., the bio-matrix containing many complexity are difficult
Hydrolysis is complete.In addition, most of EDCs is more stronger than traditional pollutant polarity, and the low concentration in biology, so that detection point
Analysis method is more difficult and complicated, cannot achieve the quantitative analysis to EDCs in organism, especially Simultaneous Analysis for Multicomponent and meets with
Meet huge challenge.
For complicated bio-matrix, also it is rarely reported about the efficient rapid detection method of incretion interferent at present.
Therefore, need to propose it is a kind of easy to operate, can effectively in detection of complex bio-matrix incretion interferent concentration, realize pair
The rapid detection method of incretion interferent quantitative analysis in organism.
Summary of the invention
In order to overcome the drawbacks of the prior art, it is dry that the purpose of the present invention is to provide endocrines in a kind of complex biological matrix
The detection method of object is disturbed, the detection method is using accelerated solvent extraction-gel permeation chromatography purification, Solid Phase Extraction and liquid phase color
The technology that spectrum-mass spectral analysis combines, can effectively in detection of complex bio-matrix incretion interferent concentration, realize to life
The quantitative analysis of incretion interferent in object, the detection method is easy to operate, has consumption of organic solvent few, to determinand
The advantages that selectivity is high, rate of extraction is fast, sample recovery rate is high, it is interior low and complicated organism suitable for analysis content
Secrete chaff interferent.
The present invention is implemented as follows:
The detection method of incretion interferent, includes the following steps: in a kind of complex biological matrix
S1: accelerated solvent extraction simultaneously redissolves:
S11: accelerated solvent extraction: weighing sample to be tested, and sample to be tested is placed in freeze drier and is dried；Then
It is ground, grinding is placed in accelerated solvent extraction abstraction pool, and Isotopic Internal Standard mixture is added, in the temperature set
Under the conditions of degree, sample to be tested is extracted using extractant, collects and obtains the extract liquor dissolved with incretion interferent；
S12: it redissolves: evaporating the extractant in the extract liquor of step S11 acquisition, dissolve interior point using solvent I is redissolved
Chaff interferent is secreted, lysate I is obtained；
S2: automatic gel permeation chromatography is purified and is redissolved:
S21: automatic gel permeation chromatography purification: lysate I is subjected to automatic gel permeation chromatography purification analysis, collects stream
Liquid separation is organized out；
S22: it redissolves: evaporating the solvent in the outflow group liquid separation of S21 acquisition, the dissolution endocrine of solvent II is dry using redissolving
Object is disturbed, lysate II is obtained；
S3: Solid Phase Extraction is simultaneously redissolved:
S31: activation: activated solid extracts pillar, and lysate II is then passed through solid phase extraction column；
S32: collect eluent: vacuum drying solid phase extraction column, then elution stays in the sample on solid phase extraction column,
It is received with nitrogen blowpipe, is collected into eluent；
S33: nitrogen blowpipe is placed on nitrogen evaporator after nitrogen blows concentration, is dissolved incretion interferent using solvent III is redissolved, is obtained
To lysate III；
S4: lysate III is analyzed using liquid chromatograph-mass spectrometer, completes the quantitative detection of incretion interferent.
Preferably, the extractant in the step S11 is n-hexane and acetone, the dosage body of the n-hexane and acetone
Product is than being 1:1.
Preferably, it is ethyl acetate and hexamethylene, the ethyl acetate and hexamethylene that solvent I is redissolved in the step S12
Dosage volume ratio be 1:1.
Preferably, solvent II is n-hexane in the step S22.
Preferably, in the step S2, the Parameter Conditions of automatic gel permeation chromatography purification are as follows:
Filler is the neutral porous polystyrene-divinylbenzene microspheres body (Bio-Beads-X3) of 50g 200-400 mesh,
Column specification 40cm × 2cm i.d, UV detector Detection wavelength 240nm, mobile phase is ethyl acetate/ring that volume ratio is 1:1
Hexane, flow velocity 5mL/min, sample volume 5mL collect the outflow group liquid separation of 20~30min.
Preferably, solid phase extraction column activation method in the step S31 are as follows: use 5mL methanol, 5mL acetone and 5mL
Ethyl acetate passes sequentially through solid phase extraction column, then small by Solid Phase Extraction with the acetum of 5mL volume fraction 0.1%
Column.
Preferably, vacuum drying time is 10min in the step S32,
Nitrogen is added methanol after blowing concentration and is settled to 100 μ L in the step S33.
Preferably, the testing conditions in the step S4 are as follows:
Agilent EC-C18 column: 3.0mm × 100mm, 2.7 μm；
Using DMRM mode detection；
Ion injection electric is -4500V；
Dry temperature degree is 350 DEG C；
Dry gas stream speed is 10L/min；
Sampling volume is 5 μ L；
Mobile phase is the mixture of 0.4mL/min volume ratio 40% methanol and 60% water.
Compared with prior art, the invention has the following advantages:
(1) recovery of standard addition of the present invention is between 81.2%-98.3%, relative standard deviation 2.13%-4.89%；
(2) gel permeation chromatography combination solid phase extraction techniques have that rate of extraction is fast, determinand selectivity is high, to sample
The advantages that rate of recovery is high, it is low to be more suitable for reliable analytical concentration content, miscellaneous EDCs sample；
(3) biological sample is hydrolyzed compared to traditional chemical reagent, this method hydrolysis biological sample is more complete, is capable of handling
The increasingly complex biological sample of bio-matrix, at the same have the characteristics that consumption of organic solvent it is few, be more applicable for structure, ingredient
Complicated biological sample.
Detailed description of the invention
Fig. 1 is the rate of recovery of 5 kinds of EDCs in fish liver sample.
Fig. 2 is the LC/MS/MS analysis of spectra of 5 kinds of EDCs.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair
It is bright, rather than limit the scope of the invention.
The present invention provides a kind of detection method of incretion interferent in complex biological matrix, and this method is molten based on accelerating
Agent extraction-gel permeation chromatography purification, solid phase extraction techniques and liquid chromatography-mass spectrometry combines measurement complex biological matrix
In incretion interferent.
Below by taking the detection of 5 kinds of incretion interferents in fish liver as an example, detection method and detecting step are as follows:
S1: accelerated solvent extraction simultaneously redissolves
S11: accelerated solvent extraction: selecting the fish body sample of health to take 5g, (when fat content is about 1g, weighing sample size is
Liver 2-10g) is in sample bottle, and after freeze-dried machine is dry, grinding is placed in accelerated solvent extraction abstraction pool, uses
Diatomite filling, be added 100 μ L, 200 μ g/L Isotopic Internal Standard mixture (13C-BPA and13C-n-NP).It is 1 with volume ratio:
1 n-hexane and acetone is extracted at 80 DEG C of extraction temperature as extractant；
S12: it redissolves: evaporating the extractant in the extract liquor of step S11 acquisition, the acetic acid that volume ratio 1:1 is is added
The redissolution solvent I of ethyl ester and hexamethylene dissolves incretion interferent, and redissolves to 6mL, obtains lysate I；
S2: automatic gel permeation chromatography is purified and is redissolved:
S21: automatic GPC cleanup system: lysate I is purified through automatic gel permeation chromatography, automatic gel permeation chromatography
The Parameter Conditions of purification are as follows: filler is the neutral porous polystyrene-divinylbenzene microspheres body of 50g 200-400 mesh
(Bio-Beads-X3), column specification 40cm × 2cm i.d, UV detector Detection wavelength 240nm；Mobile phase be ethyl acetate/
Hexamethylene (1:1, V/V), flow velocity 5mL/min, sample volume 5mL collect 20~30min outflow component and are evaporated under reduced pressure to close and do,
S22: it redissolves: evaporating the solvent in the outflow group liquid separation of step S21 acquisition, 2mL n-hexane is added (to redissolve solvent
II) incretion interferent is dissolved, lysate II is obtained；
S3: Solid Phase Extraction is simultaneously redissolved:
S31: activation: OASIS HLB solid phase extraction column, using 5mL methanol, 5mL acetone and 5mL ethyl acetate successively
By solid phase extraction column, then with (attention is when replacing solvent after the acetum activation of 5mL volume fraction 0.1%
Solid phase extraction column can not be made dry, wet state must be kept), then lysate II is extracted slowly through solid phase dropwise
Take pillar；
S32: it collects eluent: after making the dry 10min of solid phase extraction column under vacuum conditions, being using 10mL volume ratio
The acetoneand ethyl acetate elution of 1:1 stays in the sample on solid phase extraction column, is received with nitrogen blowpipe, is collected into eluent；
S33: nitrogen blowpipe is placed on nitrogen evaporator after nitrogen blows concentration, and it is dry that methanol (redissolving solvent III) dissolution endocrine is added
It disturbs object and is settled to 100 μ L；
S4: analyzing lysate III using liquid chromatograph-mass spectrometer, complete the quantitative detection of incretion interferent,
Chromatographic column testing conditions are as follows:
Agilent EC-C18 column: 3.0mm × 100mm, 2.7 μm；
Using DMRM mode detection, there are cation and negative-ion mode detection；
Ion injection electric is -4500V；
Dry temperature degree is 350 DEG C；
Dry gas stream speed is 10L/min；
Sampling volume is 5 μ L；
Mobile phase is that the mixture of 40% methanol of 0.4mL/min volume ratio and 60% water is mobile phase.
The scanning feature ion of five kinds of substances is as shown in table 1:
Characteristic ion, retention time and the detection pattern of 1 five kinds of substances of table
Data analysis: the mixing mark of basic, normal, high 3 horizontal 5 kind EDCs is added in (methanol) respectively in blank assay
Quasi- solution, each level are done 3 parts, are measured in parallel by test method.
As shown in Figure 1, for the rate of recovery of 5 kinds of EDCs in fish liver sample；The LC/MS/MS analysis that Fig. 2 is 5 kinds of EDCs
Spectrogram is determined all kinds of for standard spectrogram by retention time (t) come 5 kinds of EDCs of qualitative determination by abundance (Abundance)
The response of determinand, and quantified using the method to compare with standard curve.
The testing result of Fig. 1 and Fig. 2 shows that for the rate of recovery of 5 kinds of EDCs between 81.2%-98.3%, relative standard is inclined
Difference is 2.13%-4.89%.2 kinds are added in fish liver specimens13C reagent (13C-BPA and13C-n-NP) make rate of recovery instruction
Agent, experiment measure13The rate of recovery of C reagent is respectively 82.5 ± 8.01% and 80.3 ± 9.55%, meets requirement of experiment.Experiment knot
The concentration range that fruit measures 5 kinds of EDCs in fish liver is 0.63-138 μ g/L, and it is good to show that the detection of target compound has
Reproducibility.Biological sample and traditional abstraction technique, this method few, water with consumption of organic solvent are hydrolyzed compared with chemical reagent
The advantages that the features such as solution biological sample is more complete, rate of extraction is fast, determinand selectivity is high, high to sample recovery rate, more
Suitable for structure, the biological sample of complicated component, similar to the detection and analysis of trace EDCs in this kind of biological sample of liver.
In this experimental method, detection limit concentration that the Determination Limit of EDCs is four times.This experimental method is also used to detect fish
The concentration level of 5 kinds of incretion interferents in body bile, each sample takes the bile of 20 μ L in centrifuge tube, through above-mentioned experiment side
Method carries out pre-treatment and instrument analysis detection, the results show that the rate of recovery of 5 kinds of EDCs is between 78.9%-113.7%, relatively
Standard deviation is 5.13%-7.89%.2 kinds added in bile sample13C reagent (13C-BPA and13C-n-NP) the rate of recovery point
Not Wei 87.9 ± 5.35% and 95.3 ± 6.31%, meet requirement of experiment.
Therefore, shown according to our result of study using accelerated solvent extraction-gel permeation chromatography purification, solid phase extraction
It takes and technology that LC-MS analysis combines, can effectively clear up Various Complex bio-matrix, specifically, efficiently
Detect incretion interferent.
Finally, it should be noted that above-described embodiments are merely to illustrate the technical scheme, rather than to it
Limitation；Although the present invention is described in detail referring to the foregoing embodiments, those skilled in the art should understand that:
It can still modify to technical solution documented by previous embodiment, or to part of or all technical features into
Row equivalent replacement；And these modifications or substitutions, it does not separate the essence of the corresponding technical solution various embodiments of the present invention technical side
The range of case.
Claims (3)
1. the detection method of incretion interferent in a kind of complex biological matrix, it is characterised in that: include the following steps:
S1: accelerated solvent extraction simultaneously redissolves:
S11: accelerated solvent extraction: weighing sample to be tested, and sample to be tested is placed in freeze drier and is dried；Then it carries out
Grinding, grinding is placed in accelerated solvent extraction abstraction pool, and Isotopic Internal Standard mixture is added, in the temperature strip set
Under part, sample to be tested is extracted using extractant, collects and obtains the extract liquor dissolved with incretion interferent；The wherein extraction
Solvent is n-hexane and acetone, and the dosage volume ratio of the n-hexane and acetone is 1:1；The sample to be tested be fish liver or
Fish body bile；
S12: it redissolves: evaporating the extractant in the extract liquor of step S11 acquisition, the dissolution endocrine of solvent I is dry using redissolving
Object is disturbed, lysate I is obtained；
S2: automatic gel permeation chromatography is purified and is redissolved:
S21: automatic gel permeation chromatography purification: lysate I is subjected to automatic gel permeation chromatography purification analysis, collects outflow group
Liquid separation；
The Parameter Conditions of automatic gel permeation chromatography purification are as follows: filler is the neutral porous polystyrene-two of 50g 200-400 mesh
Vinyl benzene microsphere (Bio-Beads-X3), column specification 40cm × 2cmi.d, UV detector Detection wavelength 240nm, flowing
It is mutually ethyl acetate/hexamethylene that volume ratio is 1:1, flow velocity 5mL/min, sample volume 5mL collect the outflow group of 20~30min
Liquid separation；
S22: it redissolves: evaporating the solvent in the outflow group liquid separation of step S21 acquisition, the dissolution endocrine of solvent II is dry using redissolving
Object is disturbed, lysate II is obtained；The redissolution solvent II is n-hexane；
S3: Solid Phase Extraction is simultaneously redissolved:
S31: activation: activated solid extracts pillar, and lysate II is then passed through solid phase extraction column；The solid phase extraction column
Activation method are as follows: pass sequentially through solid phase extraction column using the ethyl acetate of 5mL methanol, 5mL acetone and 5mL, then use 5mL
The acetum of volume fraction 0.1% passes through solid phase extraction column；
S32: collect eluent: vacuum drying solid phase extraction column, then elution stays in the sample on solid phase extraction column, uses nitrogen
Blowpipe receives, and is collected into eluent；
S33: nitrogen blowpipe is placed on nitrogen evaporator after nitrogen blows concentration, is dissolved incretion interferent using solvent III is redissolved, is obtained molten
Solve liquid III；
S4: lysate III is analyzed using liquid chromatograph-mass spectrometer, completes the quantitative detection of incretion interferent；Detector bar
Part is as follows:
Agilent EC-C18 column: 3.0mm × 100mm, 2.7 μm；
Using DMRM mode detection；
Ion injection electric is -4500V；
Dry temperature degree is 350 DEG C；
Dry gas stream speed is 10L/min；
Sampling volume is 5 μ L；
Mobile phase is 0.4mL/min, the mixture of volume ratio 40% methanol and 60% water；
According to above-mentioned testing conditions by fish liver or bile five kinds of incretion interferent BPA, NP, 4-t-OP, 4-t-BP,
NET is separated and be detected.
2. the detection method of incretion interferent in complex biological matrix according to claim 1, it is characterised in that: described
Solvent I is redissolved in step S12 for ethyl acetate and hexamethylene, the dosage volume ratio of the ethyl acetate and hexamethylene is 1:1.
3. the detection method of incretion interferent in complex biological matrix according to claim 1, it is characterised in that:
Vacuum drying time is 10min in the step S32,
Nitrogen is added methanol after blowing concentration and is settled to 100 μ L in the step S33.
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