CN109839451A - Rapid analysis method while perfluorinated compound, phenolic compound and estrogen in a kind of blood - Google Patents
Rapid analysis method while perfluorinated compound, phenolic compound and estrogen in a kind of blood Download PDFInfo
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Abstract
The invention belongs to technical field of analytical chemistry, and being related to one kind may be implemented rapid analysis method while perfluorinated compound in blood, phenolic compound and estrogen.The present invention, which is directed in present analysis, to be needed to detect the blood and urine of the same research object using three kinds of different analysis methods, analysis cost is expensive, analysis time is long, the defect of impact analysis efficiency, provide perfluorinated compound in a kind of blood, rapid analysis method while phenolic compound and estrogen, using GRD beta-glucuronidase, after acid or basic hydrolysis blood sample, solid phase extraction techniques purify blood sample, PFCs in blood is detected simultaneously using Liquid Chromatography-Tandem Mass Spectrometry, phenolic compound and estrogen, analysis is as the result is shown, a Sample pretreatment and chromatography can be passed through, PFCs in blood is analyzed simultaneously, phenolic compound and estrogen, the obvious deficiency for solving to analyze these three types of compounds simultaneously at present, improve analysis efficiency.
Description
Technical field
The invention belongs to technical field of analytical chemistry, and in particular to perfluorinated compound in blood, phenols may be implemented in one kind
Rapid analysis method while compound and estrogen.
Background technique
Prior art discloses perfluorinated compound (Perfluorinate chenimcals, PFCs) and phenolic compounds
It is all important plastic monomer, is widely used in industry and civil field.The former is typical Some Persistent Organic Pollutants, and
The latter is typical perishability environmental contaminants.More and more research discovery PFCs and phenolic compound have endocrine disruption
Effect can cause metabolic disease relevant to endocrine.People is had confirmed by detection blood or urine, epidemiological study
Group is extensively exposed to PFCs and phenolic compound, while also observing that PFCs and phenolic compound exposure change with estrogen stable state
It is related.Due in epidemiological study, in general research object usually hundreds of or even thousands large samples, in order to
Quickly and effectively interference of the research PFCs and phenolic compound to human body estrogen stable state, establishes and quick and precisely evaluates PFCs and phenol
The method of class compound exposure level and body estrogen stable state is particularly important.
It, can be in conjunction with albumin and the protein such as lipoprotein in blood plasma, in vivo after studies have shown that PFCs enters human body
Accumulation.It is similar to estrogen metabolism, after entering liver by blood circulation into the intracorporal phenolic compound of people, in liver microsomal
Body enzyme system effect under, with glucuronic acid ining conjunction with after, through urine exclude in vitro.Currently, most of research is all made of liquid phase color
PFCs in tandem mass spectrum detection blood is composed, using estrogen in immune reagent kit detection blood, is detected using Liquid Chromatography-Tandem Mass Spectrometry
Phenolic compound in urine.According to present analysis strategy, if studying PFCs and phenolic compound to the shadow of body estrogen level
It rings, needs to detect the blood and urine of the same research object using above-mentioned three kinds different analysis methods, not only analyze expense
With valuableness, analysis time is long, and a variety of methods and different samples increase potential analytical error, impact analysis efficiency.
Liquid Chromatography-Tandem Mass Spectrometry detection can be used based on phenolic compound and estrogen, while according to PFCs, phenols
Metabolic characteristic in compound and estrogen body, the present invention is quasi- to be provided perfluorinated compound in a kind of blood, phenolic compound and female swashs
Rapid analysis method while plain reaches while analyzing the deficiency of the three classes compound, improves analysis efficiency.
Summary of the invention
It is mesh of the invention for analysis cost valuableness of the existing technology, analysis time is long, and a variety of methods
Increase potential analytical error with different samples, the defect of impact analysis efficiency provides perfluorinated compound, phenols in a kind of blood
Rapid analysis method while compound and estrogen reaches while analyzing the deficiency of the three classes compound, improves analysis effect
Rate.
The present invention is based on phenolic compound and estrogen, and Liquid Chromatography-Tandem Mass Spectrometry detection, while basis can be used
Metabolic characteristic in PFCs, phenolic compound and estrogen body hydrolyzes blood sample, dissociation using GRD beta-glucuronidase, acid or buck
After the glucuronic acid that phenolic compound and estrogen combine, blood sample is purified using solid phase extraction techniques, uses liquid phase color
Spectrum tandem mass spectrum detects PFCs in blood, phenolic compound and estrogen simultaneously.The present invention can pass through a Sample pretreatment
And chromatography, while analyzing PFCs in blood, phenolic compound and estrogen, can preferably solve at present while analyzing these three types
The deficiency of compound improves analysis efficiency.
In the present invention, PFCs, phenolic compound and estrogen after solid phase extraction techniques are evolved, are adopted by hydrolyzing in blood
It is analyzed with reverse-phase chromatography mass spectrometric hyphenated technique single injected sampling while detecting PFCs in blood, phenolic compound and estrogen concentrations.
In the present invention, in blood before PFCs, phenolic compound and estrogen analysis, need by hydrolysis, hydrolysis method includes
GRD beta-glucuronidase, acid or basic hydrolysis.
In the present invention, in blood before PFCs, phenolic compound and estrogen analysis, need to purify by solid phase extraction techniques,
Solid phase extraction techniques include the reverse phase solid phase extraction technology based on C8, C18 or polymer material.
In the present invention, PFCs include perfluoro caprylic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonic acid (PFHxS),
Perfluorinated undecanoic acid (PFUdA), perfluoro-pelargonic acid (PFNA), perfluorobutyric acid (PFBA), perfluor valeric acid (PFPeA), perfluor caproic acid
(PFHxA), perfluoro-heptanoic acid (PFHpA), perfluoro decanoate (PFDA), perfluoro decane sulfonic acid (PFDS) and perfluor dodecanoic acid
(PFDoA)。
In the present invention, phenolic compound includes bisphenol-A (BPA), bisphenol b (BPB), Bisphenol F (BPF), bisphenol S (BPS), double
Phenol AF (BPAF), bisphenol-c (BPC) and bis-phenol E (BPE).
In the present invention, estrogen includes oestrone (E1), estriol (E3), beta estradiol (β-E2), 4- hydroxy-estrone (4-
OHE1), 2- hydroxy-estrone (2-OHE1), 16 Alpha-hydroxies-oestrone (16 α-OHE1), 2- methoxy-oestrone (2-MeOE1), 3- first
Oxygroup-oestrone (3-MeOE1), 16- ketone group-estradiol (16-ketoE2), 4- methoxyl group-estradiol (4-MeOE2), 2- methoxy
Base-estradiol (2-MeOE2) and 2- hydroxyl-estradiol (2-OHE2)。
The present invention is based on the concentration of PFCs, phenolic compound and estrogen in analysis blood, are this kind of compound crowds of evaluation
Exposed and health effect committed step.For being needed in present analysis using three kinds of different analysis methods to the same research pair
The blood and urine of elephant are detected, and analysis cost is expensive, and analysis time is long, and the defect of impact analysis efficiency provides a kind of blood
Rapid analysis method while middle perfluorinated compound, phenolic compound and estrogen, using GRD beta-glucuronidase, acid or alkali
After hydrolyzing blood sample, solid phase extraction techniques purify blood sample, detect PFCs in blood, phenol generalization simultaneously using Liquid Chromatography-Tandem Mass Spectrometry
Object and estrogen are closed, analysis is analyzed in blood the results show that can be by a Sample pretreatment and chromatography
PFCs, phenolic compound and estrogen, hence it is evident that solve the deficiency for analyzing these three types of compounds simultaneously at present, improve analysis efficiency.
Detailed description of the invention
Fig. 1 is analysis method flow chart of the invention.
Five kinds of perfluorinated compounds of Fig. 2 and Isotopic Internal Standard mixed standard solution chromatogram (50ng/ml).
Two kinds of phenolic compounds of Fig. 3 and Isotopic Internal Standard mixed standard solution chromatogram (50ng/ml).
Tri- kinds of estrogen of Fig. 4 and Isotopic Internal Standard mixed standard solution chromatogram (50ng/ml).
Specific embodiment
As shown in Figure 1, taking 20-500 μ l serum, corresponding Isotopic Internal Standard is added, mixes, with GRD beta-glucuronidase, acid
Or after alkali hydrolyzes under suitable conditions, blood sample is purified using solid phase extraction techniques, the impurity such as protein and lipid in blood is removed, makes
With reversed phase chromatography separation PFCs, phenolic compound and estrogen, Mass Spectrometer Method PFCs under ion mode, phenolic compound and female
Hormone molecule amount and signal strength, using internal standard curve standard measure.
Embodiment 1
Using common five kinds of PFCs, two kinds of phenolic compounds and three kinds of estrogen as target compound, the present invention point is shown
Detection limit, the range of linearity, precision and the accuracy of analysis method.
1 materials and methods
1.1 instruments and reagent
The ACQUITY Ultra Performance Liquid Chromatography instrument series connection SYNAPTG2 type high-resolution quadrupole rod of Waters, US/
The HLB solid phase extraction column of time of-flight mass spectrometer and 60mg/3ml specification.12 solid-phase extraction devices and OA-SYS-5085 type
Nitrogen evaporator is bought respectively in Supelco company, the U.S. and Organomation Associates company, the U.S..Five kinds of PFCs standards
Product (perfluoro caprylic acid (PFOA), perfluorooctane sulfonate (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorinated undecanoic acid (PFUdA) and
Perfluoro-pelargonic acid (PFNA)), two kinds of phenols (bisphenol-A (BPA) and bisphenol b (BPB)), three kinds of estrogen (beta estradiol (β-E2), it is female
Ketone (E1), estriol (E3)) and four kinds of Isotopic Internal Standard (BPA-d16、β-E2-d3、E3-d3And PFOA-13C8), Luo Man snail β-Portugal
Grape uronic acid enzyme aqueous solution (Type HP-2S) purchase in Cambridge Isotope Laboratories company, the U.S.,
Sigma company or Dr company, Germany；LC-MS grades of methanol, acetonitrile and pure water purchase are in Fisher company, the U.S..
1.2 method
1.2.1 chromatographic condition
Chromatographic column is ACQU ITY UPLC BEH C18 (1.7 μ m 3.0mm × 100mm)；Mobile phase is water and acetonitrile；
Flow velocity is 0.55mL/min；Use gradient elution: starting mobile phase linearly rises to 15% second in 0~2.0min for 5% acetonitrile
Nitrile linearly rises to 60% acetonitrile in 2.0~7.0min, linearly rises to 95% acetonitrile in 7.0~8.0min, in 8.0~9.0min
98% acetonitrile linearly is risen to, is linearly down to 5% acetonitrile in 9.0~9.5min, and keep 1.5min；Column temperature is 50 DEG C；Sample volume
For 10 μ l.
1.2.2 Mass Spectrometry Conditions
Using the electric spray ion source under ion mode；Capillary voltage is 2.2kV, orifice potential 35V, extracts cone
Voltage is 4V, and ion source temperature is 100 DEG C, and taper hole throughput is 40L/h, and desolvation temperature is 350 DEG C, desolventizing gas flow
For 800L/h, mass scan range is 100~1000Da, sweep time 0.2s.Each determinand quota ion is shown in Table 1.
1.2.4 sample analysis
100 μ l serum are taken, the 2 μ g/ml that 20 μ l are added contain β-grape of the methanol solution of 4 kinds of Isotopic Internal Standards, 20 μ l
After uronic acid enzyme aqueous solution and 50 μ l acetic acid ammonia buffers (pH=4.5), mixes, be placed in 37 DEG C of water enzyme digestion 7h or more, add
Enter the 8% phosphoric acid solution enzymolysis reaction of 200 μ l, 8000rpm is centrifuged after ten minutes, takes out whole supernatants.2mL is used in advance
Methanol and 2mL pure water activation balance HLB solid phase extraction column after, by it is above-mentioned by enzymatic hydrolysis after sample supernatant whole upper prop,
Impurity successively is eluted with 2mL pure water and 2mL30% methanol aqueous solution, elutes determinand with 3mL methanol.Under 45 DEG C of water-baths, use
Weak nitrogen stream will contain eluent respectively and be blown to close dry, and after being redissolved with 30% acetonitrile solution of 0.2mL, taking 10 μ l, sample introduction is analyzed.
PFCs, phenolic compound and estrogen extraction chromatography of ions figure are shown in Fig. 2-4 in chromatography column feed materials analysis.
1.2.5 the drafting of internal standard standard curve
Using mixed standard solution, be configured in 30% acetonitrile solution concentration be respectively 0.20ng/ml, 0.50ng/ml,
1.00ng/ml, 5.00ng/ml, 20.0ng/ml, 100.0ng/ml, 200.0ng/ml mixed standard solution series, addition with
The same amount of Isotopic Internal Standard of sample, taking 10 μ l, sample introduction is analyzed.Mass window is that 20mDa extracts determinand quota ion chromatography matter
Spectrogram, with the ratio between its chromatographic peak peak area and corresponding isotope quantitative ion chromatography peak peak area for ordinate, with determinand
Concentration is abscissa, draws internal standard standard curve.
2 results
2.1 linear and detection limit
As shown in table 1, each compound has good linear, and related coefficient is all larger than 0.992；With each of 3 times of baselines pair
Compound detection limit (LOD) is between 0.5-1.7ng/ml, with the corresponding each compound quantitative limit (LOQ) of 10 times of baselines in 1.7-
Between 5.6ng/ml.
1 method of table is linear and sensitivity
LOD: detection limit；LOQ: quantitative limit.
2.2 precision and the rate of recovery
As shown in table 2, it is respectively 10 and 50ng/ml target compound that concentration is added in blood, and each concentration repeats 5
When secondary, each compound rate of recovery is between 76-115%；Precision is indicated with relative standard deviation, between 5.3-11.2%.
The 2 method rate of recovery of table and precision (n=5)
3, conclusion
Using common five kinds of PFCs, two kinds of phenolic compounds and three kinds of estrogen as target compound, the present invention point is shown
Sensitivity, precision and the accuracy of analysis method, as the result is shown the present invention can be realized PFCs in blood, phenolic compound and it is female swash
Sample pre-treatments of element and a chromatography column feed materials analysis, solve the current defect for analyzing these three types of compounds simultaneously, improve
Analysis efficiency, the large scale analysis of these three types of compounds suitable for epidemiology.
Claims (7)
1. rapid analysis method, feature exist while perfluorinated compound PFCs, phenolic compound and estrogen in a kind of blood
In, using GRD beta-glucuronidase, acid or buck hydrolysis blood sample, the glucose that dissociation is combined with phenolic compound and estrogen
After aldehydic acid, blood sample is purified using solid phase extraction techniques, detects PFCs in blood, phenol generalization simultaneously using Liquid Chromatography-Tandem Mass Spectrometry
Close object and estrogen.
2. method according to claim 1, characterized in that PFCs, phenolic compound and estrogen are passing through water in the blood
Solution after solid phase extraction techniques are evolved, is analyzed using reverse-phase chromatography mass spectrometric hyphenated technique single injected sampling while detecting PFCs in blood, phenol
Class compound and estrogen concentrations.
3. method according to claim 1, characterized in that in the blood before PFCs, phenolic compound and estrogen analysis, need
Will be by hydrolysis, hydrolysis method includes GRD beta-glucuronidase, acid or basic hydrolysis.
4. method according to claim 1, characterized in that in the blood before PFCs, phenolic compound and estrogen analysis, need
It to be purified by solid phase extraction techniques, solid phase extraction techniques include the reverse phase solid phase extraction skill based on C8, C18 or polymer material
Art.
5. method according to claim 1, characterized in that the PFCs includes perfluoro caprylic acid (PFOA), perfluorooctane sulfonate
(PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorinated undecanoic acid (PFUdA), perfluoro-pelargonic acid (PFNA), perfluorobutyric acid (PFBA),
Perfluor valeric acid (PFPeA), perfluor caproic acid (PFHxA), perfluoro-heptanoic acid (PFHpA), perfluoro decanoate (PFDA), perfluoro decane sulfonic acid
(PFDS) and perfluor dodecanoic acid (PFDoA).
6. method according to claim 1, characterized in that the phenolic compound includes bisphenol-A (BPA), bisphenol b
(BPB), Bisphenol F (BPF), bisphenol S (BPS), bisphenol AF (BPAF), bisphenol-c (BPC) and bis-phenol E (BPE).
7. method according to claim 1, characterized in that the estrogen includes oestrone (E1), estriol (E3), β-it is female
Glycol (β-E2), 4- hydroxy-estrone (4-OHE1), 2- hydroxy-estrone (2-OHE1), 16 Alpha-hydroxies-oestrone (16 α-OHE1)、2-
Methoxy-oestrone (2-MeOE1), 3- methoxy-oestrone (3-MeOE1), 16- ketone group-estradiol (16-ketoE2), 4- methoxy
Base-estradiol (4-MeOE2), 2- methoxyl group-estradiol (2-MeOE2) and 2- hydroxyl-estradiol (2-OHE2)。
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| CN114720570A (en) * | 2020-12-22 | 2022-07-08 | 上海市环境科学研究院 | Method for detecting 8 estrogens in fish |
| CN114720570B (en) * | 2020-12-22 | 2023-08-29 | 上海市环境科学研究院 | Method for detecting 8 estrogens in fish meat |
| CN112611828A (en) * | 2020-12-29 | 2021-04-06 | 大连润生康泰医学检验实验室有限公司 | Method for purifying, enriching and detecting steroid hormone in blood |
| CN112611828B (en) * | 2020-12-29 | 2021-10-08 | 大连润生康泰医学检验实验室有限公司 | Method for purifying, enriching and detecting steroid hormone in blood |
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| WO2023005682A1 (en) * | 2021-07-27 | 2023-02-02 | 暨南大学 | Method for analyzing targeted exposure group of environmental pollutants in plasma, and use thereof |
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