CN109001311A - The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products - Google Patents
The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products Download PDFInfo
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- CN109001311A CN109001311A CN201810699632.XA CN201810699632A CN109001311A CN 109001311 A CN109001311 A CN 109001311A CN 201810699632 A CN201810699632 A CN 201810699632A CN 109001311 A CN109001311 A CN 109001311A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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- G—PHYSICS
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- 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
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- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The present invention relates to a kind of Liquid Chromatography-Tandem Mass Spectrometry detection methods of interior exogenous female hormone in fish quality detection technique field more particularly to aquatic products, include the following steps (1) protein precipitation；(2) vortex shaking extracts；(3) gel permeation chromatography purifies；(4) liquid chromatography-tandem mass spectrometry detects.The present invention takes acetonitrile and ethyl acetate protein precipitation, the female hormone in vortex shaking extraction aquatic products is carried out using ethyl acetate as extractant, it will extract after supernatant liquid nitrogen blows concentration and be solved with cyclohexane-ethyl acetate redissolution, again through the chaff interferent in GPC cleanup system removal extract, scavenging solution is blown to close dry through rotary evaporation, multiple-reaction monitoring detection, inner mark method ration are carried out using Liquid Chromatography-Tandem Mass Spectrometry.This method is easy to operate, and reproducibility is preferable, and the qualitative accurate and rate of recovery is higher, can quickly analyze in aquatic products exogenous female hormone in 8 kinds.
Description
Technical field
The present invention relates to interior exogenous females in fish quality detection technique field more particularly to a kind of aquatic products
The Liquid Chromatography-Tandem Mass Spectrometry detection method of hormone.
Background technique
Estrogen is a kind of steroid hormone of ovarian secretion, and wherein estradiol, oestrone etc. belong to natural estrogen, hexene
Female phenol, Cycladiene etc. belong to synthetic estrogen.Female hormone drug, which has, to be promoted nutriment deposition in animal body and changes
The effect of kind production performance, can significantly improve the economic benefits of animal-breeding, be widely used in animal-breeding.Also there is illegal point
Son may will lead to it and remain in animal derived product in the breeding process using estrogen feed is contained.Measure aquatic products
Female hormone content in product is conducive to scientific research personnel and grasps and analyze the female hormone content in aquatic products, is conducive to country
The supervision of fish quality, to the secure effect of fish quality.
Estrogen level is not only influenced by breeding environment in aquatic products, and with its life habit, biological nature etc. because
Element is closely related.Therefore the female horizontal difference of different aquatic products is also very big, and aquatic products matrix is more complex.How effectively sample is passed through
Product pretreatment technology and instrument analysis technology are established female in the high aquatic products of a kind of rate of recovery height, favorable reproducibility, accuracy and are swashed
The detection method of element, becomes the key of research.
Related domestic and international estrogens detection method is concentrated mainly on environmental water sample, cosmetics, in dairy products at present, mainly
Detection technique have immunization, liquid chromatography, gas chromatography mass spectrometry, liquid chromatography-mass spectrometry etc., pretreatment technology is general
By Solid phase extraction, dispersive solid-phase extraction purification, molecular imprinting technology etc..Gel permeation chromatography is also known as molecular sieve gel color
Spectrum is a kind of purification separation technology of exploitation the 1960s.Since gel permeation chromatography is with big by solute molecule volume
Unique strong point of small separation, and the requirement to mobile phase is lower, and experiment condition is relatively mild, reproducible, the solute rate of recovery
The features such as high, therefore GPC possesses oneself significant advantage in terms of pigment, the sample purification rich in macromoleculars such as fat, is eating
Product secure context etc. is widely used, but it is less to be applied to estrogen retention analysis in aquatic products.Aquatic products sample substrate is complicated, if sample
In product pretreatment process impurity removal it is bad, can direct interference instrument measurement result, or even damage instrument.Therefore how efficiently
Ground extracts female hormone and is purified from aquatic products, the bottleneck analyzed at female hormone in aquatic products.
Summary of the invention
The present invention is high, dry with limitation, pre-treatment requirement in order to overcome female hormone analysis method in existing aquatic products
Disturb the problem more than factor, provide it is a kind of fast and accurately to the measuring method of exogenous female hormone interior in aquatic products, with reality
The quantitative and qualitative determination of interior exogenous female hormone in existing aquatic products.
To achieve the goals above, the invention adopts the following technical scheme:
The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products, comprising the following steps:
(1) protein precipitation: for the aquatic products after weighing about 2.0g homogenate in 50mL centrifuge tube, accurate addition 10 μ L internal standards mixing is molten
Acetonitrile-ethyl acetate mixtures of 6mL are added in liquid, avoid light place 2min；It covers oscillation and mixes 3min, protein precipitation；
(2) vortex shaking extracts: being added the ethyl acetate of 10mL, after vortex shaking 10min, 10000r/min is centrifuged at 4 DEG C
10min takes supernatant to be transferred in glass tube, then repeats to extract primary, merging upper layer organic liquor with the ethyl acetate of 5mL；?
It at 45 DEG C, is dried with nitrogen, residue 10.0mL cyclohexane-ethyl acetate mixed liquor dissolves, and crosses 0.45 μm of organic micro film, supplies
GPC cleanup system is used；
(3) gel permeation chromatography purifies: chromatographic column: 30mm × 210mm, filler Bio-BeadsSX3, neutral, porous polyphenyl second
Alkene-divinylbenzene microspheres body, mobile phase: cyclohexane-ethyl acetate mixed liquor, flow velocity: 5mL/min, sample volume: 5mL；It collects
The eluent of 15~26.7min is threaded to be dried with nitrogen under the conditions of 45 DEG C after closely doing at 45 DEG C, the methanol/water mixing of 1mL is added
Liquid redissolves solution, obtains redissolution liquid；
(4) liquid chromatography-tandem mass spectrometry detects: the redissolution liquid after taking constant volume, small to sample through 0.22 μm of organic membrane filtration
In bottle, liquid chromatography-tandem mass spectrometry detection is carried out, draws standard working curve, it is female according to calculated by peak area with inner mark method ration
The concentration of sex hormone.
Liquid Chromatography-Tandem Mass Spectrometry detection method of the invention can be used for detecting estriol, 17 alpha-estradiols, 17 β-female
Glycol, ethinyloestradiol, oestrone, diethylstilbestrol, hexestrol, dienestrol.
Preferably, in step (4),
Chromatographic condition are as follows: separated using ACQUITY UPLC BEH C18 column (2.1mm × 100mm, 1.7 μm)；Mobile phase: A phase:
0.1% ammonium hydroxide；B phase: acetonitrile；Sample temperature is 25 DEG C, and column temperature is 30 DEG C, and sampling volume is 5 μ L, flow velocity 0.2mL/min；Ladder
Degree elution: 0~1.0min, 30%B；1.0~1.5min, 30%~50%B；1.5~4.6min, 50%B；4.6~4.8min,
50%~100%B；4.8~5.3min, 100%B；5.3~5.5min, 100%~30%B；5.5~8.0min, 30%B；
Mass Spectrometry Conditions are as follows: mode is ionized using ESI-；Detection mode: multiple-reaction monitoring (MRM)；Capillary voltage: 2.5kV (-)；
Ion source temperature: 150 DEG C；Desolvation temperature: 500 DEG C；Desolventizing gas flow: 1000L/h；Collision cell pressure: 3.0 × 10- 3mbar.Liquid Chromatography-Tandem Mass Spectrometry parameter such as subordinate list 1 under MRM monitoring pattern；The lower 3 kinds of internal standard standards of ESI- ionization mode are molten
The MRM chromatogram of liquid as illustrated in the accompanying drawings from 1 to 3, the MRM chromatogram such as attached drawing 4-10 institute of the lower 8 kinds of female hormone of ESI- ionization mode
Show.
Preferably, in step (1), containing volume ratio is 1:1:1 estriol-d3 in the internal standard mixed solution, 17
Beta estradiol-d3 and diethylstilbestrol-d8.
Preferably, the volume ratio of acetonitrile and ethyl acetate is in the acetonitrile-ethyl acetate mixtures in step (1)
1:1。
Preferably, in step (2) and step (3), the cyclohexane-ethyl acetate mixed liquor cyclohexane and acetic acid second
The volume ratio of ester is 1:1.
Preferably, the volume ratio of first alcohol and water is 1:1 in the methanol/water mixed liquor in step (3).
Therefore, the invention has the following beneficial effects:
(1) acetonitrile and the effective protein precipitation of ethyl acetate are first used, then extracts the female hormone in aquatic products, extraction with vortex shaking
Effect is good, and equipment requirement is low, easy to operate；
(2) it is purified using gel permeation chromatography, good purification, significantly reduces in extract liquor impurity to liquid chromatogram-string
Join the interference of mass spectrometric procedure, high degree of automation；
(3) multiple-reaction monitoring, sensitivity with higher and accuracy are carried out using Liquid Chromatography-Tandem Mass Spectrometry；
(4) compared with existing measuring technology, method can carry out simultaneously it is qualitative and quantitative determination, operation is simple, consumption at
This is low, can quickly analyze the female hormone in aquatic products.
Detailed description of the invention
Fig. 1 is the MRM chromatogram of estriol-d3 under ESI- ionization mode.
Fig. 2 is the MRM chromatogram of 17 beta estradiol-d3 under ESI- ionization mode.
Fig. 3 is the MRM chromatogram of diethylstilbestrol-d8 under ESI- ionization mode.
Fig. 4 is the MRM chromatogram of estriol under ESI- ionization mode.
Fig. 5 is the MRM chromatogram of 17 alpha-estradiols and 17 beta estradiols under ESI- ionization mode.
Fig. 6 is the MRM chromatogram of ethinyloestradiol under ESI- ionization mode.
Fig. 7 is the MRM chromatogram of oestrone under ESI- ionization mode.
Fig. 8 is the MRM chromatogram of diethylstilbestrol under ESI- ionization mode.
Fig. 9 is the MRM chromatogram of hexestrol under ESI- ionization mode.
Figure 10 is the MRM chromatogram of dienestrol under ESI- ionization mode.
Specific embodiment
Below by specific embodiment, and in conjunction with attached drawing, the technical solutions of the present invention will be further described.
In the present invention, if not refering in particular to, all devices and raw material is commercially available or the industry is common are following
Method in embodiment is unless otherwise instructed conventional method in that art.
The present invention is surveyed using Waters of U.S. ACQUITYTM XEVO TQ-MS liquid chromatography-tandem mass instrument
It is fixed, Impact Vario full automatic gel purification system, using ACQUITY UPLC BEH C18 column (2.1mm × 100mm, 1.7
μm) realize component separation.
The Liquid Chromatography-Tandem Mass Spectrometry detection of interior exogenous female hormone in 1 mussel of embodiment
(1) protein precipitation: for the mussel after precise 2.0g homogenate in 50mL centrifuge tube, accurate addition 10 μ L internal standards mixing is molten
Liquid (estriol-d3,17 beta estradiol-d3, the mixing of diethylstilbestrol-d8 equal proportion), the acetonitrile of 6mL is added in avoid light place 2min
With ethyl acetate (1:1, v/v).It covers oscillation and mixes 3min, protein precipitation；
(2) vortex shaking extracts: being added the ethyl acetate of 10mL, after vortex shaking 10min, 10000r/min is centrifuged at 4 DEG C
10min takes supernatant to be transferred in glass tube.Repeat to extract primary, merging upper layer organic liquor with the ethyl acetate of 5mL again.?
It at 45 DEG C, is dried with nitrogen, residue 10.0mL hexamethylene-ethyl acetate (50:50, v/v) redissolves, and crosses 0.45 μm organic micro-
Pore membrane, for GPC cleanup system；
(3) gel permeation chromatography purifies: chromatographic column: 30mm × 210mm, filler Bio-BeadsSX3 (neutral, porous polyphenyl second
Alkene-divinylbenzene microspheres body), mobile phase: hexamethylene-ethyl acetate (50:50, v/v), flow velocity: 5mL/min, sample volume:
5mL.The eluent for collecting 15-26.7min is threaded to be dried with nitrogen under the conditions of 45 DEG C after closely doing at 45 DEG C, the methanol/water of 1mL
(1:1, v/v) dissolution；
(4) liquid chromatography-tandem mass spectrometry detects: the redissolution liquid after taking constant volume, small to sample through 0.22 μm of organic membrane filtration
In bottle, liquid chromatography-tandem mass spectrometry detection is carried out.Chromatographic condition are as follows: using ACQUITY UPLC BEH C18 column (2.1mm ×
100mm, 1.7 μm) separation.Mobile phase: A phase: 0.1% ammonium hydroxide；B phase: acetonitrile.Sample temperature is 25 DEG C, and column temperature is 30 DEG C, sample introduction
Volume is 5 μ L, flow velocity 0.2mL/min.Gradient elution: 0~1.0min, 30%B；1.0~1.5min, 30%~50%B；
1.5~4.6min, 50%B；4.6~4.8min, 50%~100%B；4.8~5.3min, 100%B；5.3~5.5min,
100%~30%B；5.5~8.0min, 30%B.Mass Spectrometry Conditions: mode, detection mode: multiple-reaction monitoring are ionized using ESI-
(MRM).Capillary voltage: 2.5kV (-)；Ion source temperature: 150 DEG C；Desolvation temperature: 500 DEG C；Desolventizing gas flow:
1000L/h；Collision cell pressure: 3.0 × 10-3mbar.Liquid Chromatography-Tandem Mass Spectrometry parameter such as subordinate list 1 under MRM monitoring pattern.It draws
Standard working curve calculates the concentration of female hormone with inner mark method ration；
(5) Specification Curve of Increasing
It is the mixing of 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL with bare substrate compound concentration
Standard solution is operated by the requirement of step (4), draws standard curve according to the corresponding relationship of concentration and peak area；
(6) measurement of the method rate of recovery
Respectively by 2.0 μ g/g, 5.0 μ g/g, 10.0 μ g/g concentration added in mussel sample female hormone hybrid standard it is molten
Liquid, each pitch-based sphere do six Duplicate Samples respectively；By step (1)-(4) carry out liquid chromatography-tandem mass spectrometry detection, and with it is upper
The standard curve (5) stated compares, and the concentration of female hormone in mussel sample to be measured is finally obtained by converting.Method recycling
Rate is in 78.9-108.7%, RSD≤15%.
Embodiment 2 is choked the Liquid Chromatography-Tandem Mass Spectrometry detection of interior exogenous female hormone in crab
(1) protein precipitation: choking crab in 50mL centrifuge tube after precise 2.0g homogenate, and accurate addition 10 μ L internal standards mixing is molten
Liquid (estriol-d3,17 beta estradiol-d3, the mixing of diethylstilbestrol-d8 equal proportion), the acetonitrile of 6mL is added in avoid light place 2min
With ethyl acetate (1:1, v/v).It covers oscillation and mixes 3min, protein precipitation；
(2) vortex shaking extracts: being added the ethyl acetate of 10mL, after vortex shaking 10min, 10000r/min is centrifuged at 4 DEG C
10min takes supernatant to be transferred in glass tube.Repeat to extract primary, merging upper layer organic liquor with the ethyl acetate of 5mL again.?
It at 45 DEG C, is dried with nitrogen, residue 10.0mL hexamethylene-ethyl acetate (50:50, v/v) redissolves, and crosses 0.45 μm organic micro-
Pore membrane, for GPC cleanup system；
(3) gel permeation chromatography purifies: chromatographic column: 30mm × 210mm, filler Bio-BeadsSX3 (neutral, porous polyphenyl second
Alkene-divinylbenzene microspheres body), mobile phase: hexamethylene-ethyl acetate (50:50, v/v), flow velocity: 5mL/min, sample volume:
5mL.The eluent for collecting 15-26.7min is threaded to be dried with nitrogen under the conditions of 45 DEG C after closely doing at 45 DEG C, the methanol/water of 1mL
(1:1, v/v) dissolution；
(4) liquid chromatography-tandem mass spectrometry detects: the redissolution liquid after taking constant volume, small to sample through 0.22 μm of organic membrane filtration
In bottle, liquid chromatography-tandem mass spectrometry detection is carried out.Chromatographic condition are as follows: using ACQUITY UPLC BEH C18 column (2.1mm ×
100mm, 1.7 μm) separation.Mobile phase: A phase: 0.1% ammonium hydroxide；B phase: acetonitrile.Sample temperature is 25 DEG C, and column temperature is 30 DEG C, sample introduction
Volume is 5 μ L, flow velocity 0.2mL/min.Gradient elution: 0~1.0min, 30%B；1.0~1.5min, 30%~50%B；
1.5~4.6min, 50%B；4.6~4.8min, 50%~100%B；4.8~5.3min, 100%B；5.3~5.5min,
100%~30%B；5.5~8.0min, 30%B.Mass Spectrometry Conditions: mode, detection mode: multiple-reaction monitoring are ionized using ESI-
(MRM).Capillary voltage: 2.5kV (-)；Ion source temperature: 150 DEG C；Desolvation temperature: 500 DEG C；Desolventizing gas flow:
1000L/h；Collision cell pressure: 3.0 × 10-3mbar.Liquid Chromatography-Tandem Mass Spectrometry parameter such as subordinate list 1 under MRM monitoring pattern.It draws
Standard working curve calculates the concentration of female hormone with inner mark method ration；
(5) Specification Curve of Increasing
It is the mixing of 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL with bare substrate compound concentration
Standard solution is operated by the requirement of (4) step, draws standard curve according to the corresponding relationship of concentration and peak area；
(6) measurement of the method rate of recovery
Respectively by 2.0 μ g/g, 5.0 μ g/g, 10.0 μ g/g concentration added in crab sample of choking female hormone hybrid standard it is molten
Liquid, each pitch-based sphere do six Duplicate Samples respectively；By step (1)-(4) carry out liquid chromatography-tandem mass spectrometry detection, and with it is upper
The standard curve (5) stated compares, and the concentration of female hormone in mussel sample to be measured is finally obtained by converting.Method recycling
Rate is in 90.4-112.6%, RSD≤15%.
The Liquid Chromatography-Tandem Mass Spectrometry detection of interior exogenous female hormone in 3 Larimichthys crocea of embodiment
(1) protein precipitation: the Larimichthys crocea after precise 2.0g homogenate is accurate that the mixing of 10 μ L internal standards is added in 50mL centrifuge tube
Solution (estriol-d3,17 beta estradiol-d3, the mixing of diethylstilbestrol-d8 equal proportion), the second of 6mL is added in avoid light place 2min
Nitrile and ethyl acetate (1:1, v/v).It covers oscillation and mixes 3min, protein precipitation；
(2) vortex shaking extracts: being added the ethyl acetate of 10mL, after vortex shaking 10min, 10000r/min is centrifuged at 4 DEG C
10min takes supernatant to be transferred in glass tube.Repeat to extract primary, merging upper layer organic liquor with the ethyl acetate of 5mL again.?
It at 45 DEG C, is dried with nitrogen, residue 10.0mL hexamethylene-ethyl acetate (50:50, v/v) redissolves, and crosses 0.45 μm organic micro-
Pore membrane, for GPC cleanup system；
(3) gel permeation chromatography purifies: chromatographic column: 30mm × 210mm, filler Bio-BeadsSX3 (neutral, porous polyphenyl second
Alkene-divinylbenzene microspheres body), mobile phase: hexamethylene-ethyl acetate (50:50, v/v), flow velocity: 5mL/min, sample volume:
5mL.The eluent for collecting 15-26.7min is threaded to be dried with nitrogen under the conditions of 45 DEG C after closely doing at 45 DEG C, the methanol/water of 1mL
(1:1, v/v) dissolution；
(4) liquid chromatography-tandem mass spectrometry detects: the redissolution liquid after taking constant volume, small to sample through 0.22 μm of organic membrane filtration
In bottle, liquid chromatography-tandem mass spectrometry detection is carried out.Chromatographic condition are as follows: using ACQUITY UPLC BEH C18 column (2.1mm ×
100mm, 1.7 μm) separation.Mobile phase: A phase: 0.1% ammonium hydroxide；B phase: acetonitrile.Sample temperature is 25 DEG C, and column temperature is 30 DEG C, sample introduction
Volume is 5 μ L, flow velocity 0.2mL/min.Gradient elution: 0~1.0min, 30%B；1.0~1.5min, 30%~50%B；
1.5~4.6min, 50%B；4.6~4.8min, 50%~100%B；4.8~5.3min, 100%B；5.3~5.5min,
100%~30%B；5.5~8.0min, 30%B.Mass Spectrometry Conditions: mode, detection mode: multiple-reaction monitoring are ionized using ESI-
(MRM).Capillary voltage: 2.5kV (-)；Ion source temperature: 150 DEG C；Desolvation temperature: 500 DEG C；Desolventizing gas flow:
1000L/h；Collision cell pressure: 3.0 × 10-3mbar.Liquid Chromatography-Tandem Mass Spectrometry parameter such as subordinate list 1 under MRM monitoring pattern.It draws
Standard working curve calculates the concentration of female hormone with inner mark method ration；
(5) Specification Curve of Increasing
It is the mixing of 1.0 μ g/mL, 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ g/mL, 50.0 μ g/mL with bare substrate compound concentration
Standard solution is operated by the requirement of (4) step, draws standard curve according to the corresponding relationship of concentration and peak area；
(6) measurement of the method rate of recovery
The hybrid standard of female hormone is added in rheum officinale fish sample by the concentration of 2.0 μ g/g, 5.0 μ g/g, 10.0 μ g/g respectively
Solution, each pitch-based sphere do six Duplicate Samples respectively；By step (1)-(4) progress liquid chromatography-tandem mass spectrometry detection, and with
Standard curve (5) obtained above is compared, and the concentration of female hormone in mussel sample to be measured is finally obtained by converting.Method is returned
Yield is in 85.4-108.4%, RSD≤15%.
Liquid Chromatography-Tandem Mass Spectrometry parameter under 1 MRM monitoring pattern of subordinate list
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, without departing from power
There are also other variations and modifications under the premise of technical solution documented by benefit requirement.
Claims (6)
1. the Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products, which is characterized in that including
Following steps:
(1) protein precipitation: for the aquatic products after weighing about 2.0g homogenate in 50mL centrifuge tube, accurate addition 10 μ L internal standards mixing is molten
Acetonitrile-ethyl acetate mixtures of 6mL are added in liquid, avoid light place 2min；It covers oscillation and mixes 3min, protein precipitation；
(2) vortex shaking extracts: being added the ethyl acetate of 10mL, after vortex shaking 10min, 10000r/min is centrifuged at 4 DEG C
10min takes supernatant to be transferred in glass tube, then repeats to extract primary, merging upper layer organic liquor with the ethyl acetate of 5mL；?
It at 45 DEG C, is dried with nitrogen, residue 10.0mL cyclohexane-ethyl acetate mixed liquor dissolves, and crosses 0.45 μm of organic micro film, supplies
GPC cleanup system is used；
(3) gel permeation chromatography purifies: chromatographic column: 30mm × 210mm, filler Bio-BeadsSX3, neutral, porous polyphenyl second
Alkene-divinylbenzene microspheres body, mobile phase: cyclohexane-ethyl acetate mixed liquor, flow velocity: 5mL/min, sample volume: 5mL；It collects
The eluent of 15~26.7min is threaded to be dried with nitrogen under the conditions of 45 DEG C after closely doing at 45 DEG C, the methanol/water mixing of 1mL is added
Liquid redissolves solution, obtains redissolution liquid；
(4) liquid chromatography-tandem mass spectrometry detects: the redissolution liquid after taking constant volume, small to sample through 0.22 μm of organic membrane filtration
In bottle, liquid chromatography-tandem mass spectrometry detection is carried out, draws standard working curve, it is female according to calculated by peak area with inner mark method ration
The concentration of sex hormone.
2. the Liquid Chromatography-Tandem Mass Spectrometry detection side of interior exogenous female hormone in a kind of aquatic products according to claim 1
Method, which is characterized in that in step (4),
Chromatographic condition are as follows: use ACQUITY UPLC BEH C18 post separation；Mobile phase: A phase: 0.1% ammonium hydroxide；B phase: acetonitrile；
Sample temperature is 25 DEG C, and column temperature is 30 DEG C, and sampling volume is 5 μ L, flow velocity 0.2mL/min；Gradient elution: 0~1.0min,
30%B；1.0~1.5min, 30%~50%B；1.5~4.6min, 50%B；4.6~4.8min, 50%~100%B；4.8
~5.3min, 100%B；5.3~5.5min, 100%~30%B；5.5~8.0min, 30%B；
Mass Spectrometry Conditions are as follows: mode is ionized using ESI-；Detection mode: multiple-reaction monitoring (MRM)；Capillary voltage: 2.5kV (-)；
Ion source temperature: 150 DEG C；Desolvation temperature: 500 DEG C；Desolventizing gas flow: 1000L/h；Collision cell pressure: 3.0 × 10- 3mbar。
3. the Liquid Chromatography-Tandem Mass Spectrometry inspection of interior exogenous female hormone in a kind of aquatic products according to claim 1 or 2
Survey method, which is characterized in that in step (1), the estriol-d3 for being 1:1:1 containing volume ratio in the internal standard mixed solution, 17
Beta estradiol-d3 and diethylstilbestrol-d8.
4. the Liquid Chromatography-Tandem Mass Spectrometry inspection of interior exogenous female hormone in a kind of aquatic products according to claim 1 or 2
Survey method, which is characterized in that in step (1), the volume ratio of acetonitrile and ethyl acetate is in the acetonitrile-ethyl acetate mixtures
1:1。
5. the Liquid Chromatography-Tandem Mass Spectrometry inspection of interior exogenous female hormone in a kind of aquatic products according to claim 1 or 2
Survey method, which is characterized in that in step (2) and step (3), the cyclohexane-ethyl acetate mixed liquor cyclohexane and acetic acid
The volume ratio of ethyl ester is 1:1.
6. the Liquid Chromatography-Tandem Mass Spectrometry inspection of interior exogenous female hormone in a kind of aquatic products according to claim 1 or 2
Survey method, which is characterized in that in step (3), the volume ratio of first alcohol and water is 1:1 in the methanol/water mixed liquor.
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| CN201810699632.XA CN109001311A (en) | 2018-06-29 | 2018-06-29 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products |
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| CN201810699632.XA CN109001311A (en) | 2018-06-29 | 2018-06-29 | The Liquid Chromatography-Tandem Mass Spectrometry detection method of interior exogenous female hormone in a kind of aquatic products |
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| CN112684030A (en) * | 2020-12-03 | 2021-04-20 | 大连理工大学 | Method for detecting perfluoroalkanoic acid compound in aquatic product by enrichment purification-liquid chromatography tandem mass spectrometry and application |
| 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 |
| CN115236221A (en) * | 2022-06-29 | 2022-10-25 | 江苏康达检测技术股份有限公司 | Method for detecting dihydric alcohol compound in environmental medium |
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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 |
| CN115236221A (en) * | 2022-06-29 | 2022-10-25 | 江苏康达检测技术股份有限公司 | Method for detecting dihydric alcohol compound in environmental medium |
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