CN110031561A - The dispersive solid-phase extraction gaschromatographic mass spectrometry detection method of organic tin environmental hormone in a kind of marine product - Google Patents

The dispersive solid-phase extraction gaschromatographic mass spectrometry detection method of organic tin environmental hormone in a kind of marine product Download PDF

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CN110031561A
CN110031561A CN201910336466.1A CN201910336466A CN110031561A CN 110031561 A CN110031561 A CN 110031561A CN 201910336466 A CN201910336466 A CN 201910336466A CN 110031561 A CN110031561 A CN 110031561A
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phase extraction
solution
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marine product
dispersive solid
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胡红美
王姮
孙秀梅
张博
应忠真
金衍健
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Zhejiang Marine Fisheries Research Institute
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Zhejiang Marine Fisheries Research Institute
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Abstract

The invention discloses a kind of dispersive solid-phase extraction gaschromatographic mass spectrometry detection methods of organic tin environmental hormone in marine product, using ultrasonic extraction, freeze degreasing, the purification of first time dispersive solid-phase extraction, derivative and back extraction, second of dispersive solid-phase extraction purification, gas chromatography mass spectrometry measures the organotin in marine product, this method is easy to operate, sample pre-treatments can be rapidly completed, wherein purified for the first time using dispersive solid-phase extraction twice, entire dispersive solid-phase extraction purification only needs 20~30min plus derivative and back extraction process, as a result accurate, nine kinds of organotin detections are limited to 0.4~1.0 μ g/kg, the rate of recovery is 80~117%, relative standard deviation (n=5) is 3.8~7.5%, sensitivity with higher and the satisfactory rate of recovery and reproducibility, it can be used for sea The assay of organotin in product.

Description

The dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product Spectrum detection method
Technical field
The present invention relates to a kind of marine product pollution detection technical fields, more particularly, to organic tin ring in a kind of marine product The dispersive solid-phase extraction gaschromatographic mass spectrometry detection method of border hormone.
Background technique
Organotin is widely distributed in atmosphere, water body, soil, bed mud, organism etc. respectively as a kind of important environmental hormone In kind medium, there is male sex hormone effect, disturbance endocrine system leads to reproduction, development and abnormal behavior, is often widely used as PVC stabilizer, antifouling of ship's hull material, timber preservative, insecticide, fungicide, wherein tributyl tin, triphenyltin and Its catabolite is classified as " priority pollutant blacklist " by multiple countries, the world.Although organotin pollution has caused whole world weight Depending on, but since half-life period is longer, can persistently exist.The organo-tin compound of solubilised state is easier to enter in seawater and migrates or pass through Biological concentration enters in food chain, and especially tributyl tin and triphenyltin are enriched with because fat-soluble compared with Gao Eryi in vivo. A large number of studies show that organotin, in fish, the organisms such as shellfish, gel-derived bioglass ceramic is up to 8700000, and has biology Amplification will finally threaten human health and life once being eaten by the mankind.There is report to point out that ocean lactation all over the world is dynamic There are the organo-tin compounds of high concentration in object, they can be such that immune system defence capability declines, make nerveous system System poisoning, mutagenesis, carcinogenic etc..Except marine food web, tributyl tin is had also discovered in human liver, this is likely due to The seafood of intake human body receives the pollution of organotin.It is reported that only tributyl tin, concentrations are not examine in the shellfish of the South Sea μ g/kg (in terms of Sn) out~249.9,67.6 μ g/kg of mean value (in terms of Sn)；Fishes In The South China Sea concentrations be not detected~ 1377.8 μ g/kg (in terms of Sn), 26.3 μ g/kg of mean value (in terms of Sn)；Bohai Sea Gulf mollusk concentrations be < 2.8~ 383.9 μ g/kg (in terms of Sn).
China is at present also not specified the limitation of organotin in food.European Food safety management bureau has announced two Butyl tin, tributyl tin, triphenyltin and dioctyl tin have immunotoxicity, and provide the maximum of these organotin summations in food Limit standard is 40 μ g/kg (in terms of Sn), and monomethyl tin and stannous methide maximum limit standard are 180 μ g/kg (in terms of Sn), one Tin octylate maximum limit standard is 1200 μ g/kg (in terms of Sn).However, the limit standard of other organo-tin compounds shows yet still Without final conclusion, this organo-tin compound type detected with method detection limit and a performance has direct relation.Existing report Most methods once detect 3~6 kinds of organo-tin compounds mostly.Therefore, organotin is particularly important in Analyzing marine products.
Currently, mainly using Soxhlet extraction, oscillation extraction, microwave radiation exaraction, accelerated solvent extraction, ultrasonic wave both at home and abroad The abstraction techniques such as extraction, in conjunction with chromatography column purification, Solid Phase Extraction, gel permeation chromatography purify etc. one or two kinds of purification sides Method, then using the technologies such as gas chromatography, high performance liquid chromatography, gas chromatography-mass spectrography, gas-chromatography tandem mass spectrometry into Organotin detects in row organism.
And marine product matrix is complicated, the key of pre-treatment and purifying step as organotin retention analysis in sample.To rouge The more marine product of fat, pigment content, it usually needs gel permeation chromatography and silicagel column or florisil silica column solid phase extraction Combined purifying, and gel permeation chromatography expensive equipment, need using a large amount of eluting solvent, and solid phase extraction is complicated for operation, It needs to carry out the processes such as extraction column activation, loading, elution, elution, take a long time, be not suitable for mass sample treatment.Dispersion Solid Phase Extraction has the characteristics that quick, simple, cheap, effective, reliable and safe, has been gradually applied to various agricultural and sideline products and ring Various detection of organic pollutants in the medium of border.Therefore, a kind of simple pre-treatment and purification method are developed, and is combined highly sensitive Gaschromatographic mass spectrometry detection technique is expected to solve prior art bottleneck.
Summary of the invention
Goal of the invention of the invention is to provide for a kind of dispersive solid-phase extraction of organic tin environmental hormone in marine product Gaschromatographic mass spectrometry detection method, the detection method is easy to operate, and sample pre-treatments, sensitivity with higher can be rapidly completed With the satisfactory rate of recovery and reproducibility, the assay of organotin in marine product can be used for.
To achieve the goals above, the invention adopts the following technical scheme:
The dispersive solid-phase extraction gaschromatographic mass spectrometry detection side of organic tin environmental hormone in a kind of marine product of the invention Method, comprising the following steps:
(1) acquisition and sample preparation of sample
By marine product sample smash to pieces it is homogeneous after, freeze-drying, in -18 DEG C refrigerate, it is to be measured.Marine product sample is fish: scale, Peeling, takes muscle along back；Marine product sample is shrimp, crab: decaptitating, removes appendage at decladding, takes muscle；Marine product sample is shellfish: Decladding, takes edible part, and sample is not more than 0.5cm × 0.5cm × 0.5cm；Marine product to be detected can be straight by fishing boat trawlnet Connect acquisition Yu Haiyang or purchase in aquatic products harbour, supermarket, the market of farm produce, aquatic products wholesale market, simultaneously using clean In Aluminium Foil Packing It is sealed in polyethylene bags and is transported.
(2) sampling and ultrasonic extraction
1.00g sample to be tested is weighed, acetic acid and methanol mixed solution that 5~10mL contains tropolone, vinegar is added For acid with methanol mixed solution, tropolone mass concentration is 0.03%, and the volume ratio of acetic acid and methanol is 1:9, is vortexed Ultrasonic extraction afterwards obtains extracting solution.When carrying out the selection of extraction mode, it is contemplated that traditional extraction such as oscillation extraction, Soxhlet extraction Method extraction process time-consuming, great work intensity are taken, and uses a large amount of toxic solvents, accelerated solvent extraction and microwave auxiliary extraction Equal instruments and technical costs are relatively high.And ultrasonic extraction can not only be effectively by the organic matter of stable structure from solid-like It is extracted in product, and has that high-efficient, instrument price is cheap, simple operation and other advantages, the present invention selects ultrasonic extraction Extract the organotin of marine product.
(3) degreasing is freezed
After extracting solution is placed in -85 DEG C of freezing degreasings, high speed centrifugation takes supernatant A.It is fairly simple to freeze defatting technology, Without using large-scale instrument and equipments such as additional agents and gel permeation chromatographies, and quite a few fat can be removed, be subsequent suitable Benefit carries out dispersive solid-phase extraction purification twice and provides strong guarantee.
(4) first time dispersive solid-phase extraction purifies
100~200mg N- propyl ethylenediamine solid-phase adsorbent is added in supernatant A, high speed centrifugation after vortex takes Clear liquid B.Carbohydrate, the fat for influencing target analyte detection can be effectively removed using N- propyl ethylenediamine solid-phase adsorbent The interference of acid, organic acid, phenols, carbohydrate and some polarity pigments, therefore before derivative and back extraction, first time dispersed solid phase extraction Take purification selection N- propyl ethylenediamine solid-phase adsorbent.
(5) derivative and back extraction
In supernatant B be added 30~40mL concentration be 1mol/L, pH be 4.5~5 NaAc_HAc buffer solution, 1g sodium chloride and 5mL n-hexane are vortexed and mix, add the tetrahydrofuran solution of the 200 μ L sodium of boronation containing tetraethyl, tetrahydrofuran The mass concentration of tetraethyl boronation sodium is 10% in solution, and high speed centrifugation after vortex oscillation takes upper layer n-hexane phase.
(6) second of dispersive solid-phase extraction purification
In n-hexane phase, the dehydration of 200mg anhydrous sodium sulfate and 50~200mg ketjenblack EC solid phase adsorption are sequentially added Agent takes supernatant after vortex, and membrane filtration takes filtrate.It is solid that inventor compares C18, N- propyl ethylenediamine, ketjenblack EC etc. The dispersive solid-phase extraction clean-up effect of phase adsorbent, the results showed that, using ketjenblack EC, extracting solution is substantially colorless, Suo Youhua It is relatively high (80~115%) to close the object rate of recovery, therefore second of dispersive solid-phase extraction purification final choice graphitization of the present invention Carbon black is as adsorbent.
(7) constant volume is concentrated
By filtrate at 35~40 DEG C of bath temperature, nitrogen flushing is evaporated to dryness, and with n-hexane dissolution, and is settled to 200 μ L, As upper machine solution.
(8) sample introduction needle extracts above-mentioned upper machine solution
Above-mentioned upper machine solution is extracted with sample introduction needle, carries out loading inspection by the gas chromatograph-mass spectrometer testing conditions set It surveys, obtains total ion chromatogram, select ion monitoring mode, qualitative and quota ion；GC conditions are as follows: injector temperature It is 260 DEG C；Carrier gas is high-purity helium, flow velocity 1mL/min；Sampling volume is 1 μ L；Input mode is Splitless injecting samples, It is purged after 0.75min with 15mL/min；260 DEG C of transmission line temperature；Gas chromatographic column temperature program are as follows: 60 DEG C of initial temperature, protect 1.0min is held, is warming up to 220 DEG C with 20 DEG C/min, 10min is kept, then be warming up to 280 DEG C with 15 DEG C/min, keeps 5.0min, Total run time 28min；It is the HP-35MS capillary gas phase color of 30m × 0.25mm × 0.25 μm that gas chromatographic column, which selects specification, Column is composed, stationary phase is the mixed liquor of diphenyl and dimethyl polysiloxane, and wherein the mass fraction of diphenyl is 35%, dimethyl The mass fraction of polysiloxanes is 65%；Mass Spectrometry Conditions are as follows: electron bombardment (EI) ion source, 230 DEG C of ion source temperature, ionization energy Measure 70eV；Solvent delay 3min；150 DEG C of level four bars temperature, 50~450amu of scanning range, retrieval spectrum library NIST.
(9) Specification Curve of Increasing
Salbutamol Selected Ion Monitoring mode carries out qualitative: taking concentration is Monobutyltin, dibutyl tin, the tributyl of 1000 μ g/L The organotin hybrid standard use of tin, a phenyltin, a tin octylate, tetrabutyltin, stannous phenide, dioctyl tin, triphenyltin 100 μ L of liquid, 200 μ L are settled to n-hexane respectively, and obtaining concentration is 500 μ g/L standard solution；By standard solution according to above-mentioned The requirement of step (8) is operated to obtain the total ion current figure chromatogram of standard solution, and the total ion current with single analyte Graph coloring spectrogram carries out qualitative ion, quota ion compares, and in combination with retention time, determines the type of nine kinds of organotins；This nine Nine kinds of organotin chromatographic peaks are identified when planting the qualitative ion, quota ion, retention time of organotin to as quantified by external standard method The foundation of identification；
Select quantified by external standard method: taking concentration is the organotin hybrid standard of 100 μ g/L using 20,50,200 μ L of liquid, simultaneously It separately takes the organotin hybrid standard of 1000 μ g/L of concentration using 50,100,200 μ L of liquid, is separately added into n-hexane and is settled to 200 μ L, Obtain the standard curve serial solution that six spiked levels ranges are 10~1000 μ g/L；By the mark of six kinds of different spiked levels Directrix curve serial solution establishes standard according to the corresponding relationship of organic tin concentration of addition and corresponding quota ion integrated peak areas Curve.
(10) sample measures
Organotin in total ion chromatogram obtained in step (8) is accordingly quantified into peak area, with above-mentioned steps (9) standard curve obtained in is compared, and the reality of nine kinds of organotins in sample marine product product can be obtained finally by conversion Content.
Preferably, be freeze-dried in step (1) using freeze drier, drying time 12~for 24 hours.
Preferably, 1~3g copper powder is added in acetic acid and methanol mixed solution in step (2), ultrasound is de- after vortex Sulphur, high speed centrifugation take centrifugal clear liquid, then 5~10mL of addition contains the acetic acid and methanol of tropolone in centrifugal clear liquid In mixed solution, acetic acid and methanol mixed solution, tropolone mass concentration is 0.03%, the volume ratio of acetic acid and methanol For 1:9, ultrasonic extraction after vortex obtains extracting solution.To prevent sulfur-containing compound in sample from interfering to result, add in extracting solution Enter copper powder and carries out desulfurization.
Preferably, the copper powder is first handled with dilute hydrochloric acid using preceding, then with distilled water flushing to pH be it is neutral, finally It dries up with acetone rinsing and under nitrogen flowing.It is first handled with dilute hydrochloric acid to remove its surface film oxide, then is removed with distilled water flushing The a small amount of acid for being attached to its surface is removed, finally drying is flowed down with acetone rinsing and in high pure nitrogen (v/v, 99.99%), to prevent copper Powder is again be oxidized, and is handled copper powder to guarantee desulfurization effect.
Preferably, high speed centrifugation speed is 3000~6000r/m, 3~5min of high speed centrifugation time.
Preferably, 1~3min of vortex time, 200~300W of ultrasonic power, 30~40 DEG C of ultrasonic temperature, ultrasonic time 10~20min.
Preferably, freezing degreasing 2h, high speed centrifugation speed 6000r/m, centrifugation time 5min in step (3).
Preferably, in step (4), 30~60s of vortex time, high speed centrifugation 3000~6000r/m of speed, high speed centrifugation 2~3min of time.
Preferably, in step (5), 10~15min of vortex oscillation time, 3000~6000 r/m of high speed centrifugation speed, 2~3min of high speed centrifugation time.
Preferably, 30~60s of vortex time, filter membrane micropore is 0.22 μm in step (6).
Therefore, the invention has the following beneficial effects: using ultrasonic extraction, freezing degreasing, first time dispersed solid phase extraction It takes purification, derivative and back extraction, second of dispersive solid-phase extraction to purify, is in gas chromatography mass spectrometry measurement marine product organic Tin, this method is easy to operate, and sample pre-treatments can be rapidly completed, wherein purified for the first time using dispersive solid-phase extraction twice, Entire dispersive solid-phase extraction purification only needs 20~30min plus derivative and back extraction process, as a result accurately, nine kinds of organotins Detection is limited to 0.4~1.0 μ g/kg, and the rate of recovery is 80~117%, and relative standard deviation (n=5) is 3.8~7.5%, have compared with High sensitivity and the satisfactory rate of recovery and reproducibility, can be used for the assay of organotin in marine product.
Detailed description of the invention
Fig. 1 is the total ion current figure chromatogram of nine kinds of organotin standard solution (500 μ g/L).
Wherein, 1. Monobutyltin；2. dibutyl tin；3. tributyl tin；4. a phenyltin；5. a tin octylate；6. the tetrabutyl Tin；7. stannous phenide；8. dioctyl tin；9. triphenyltin.
Specific embodiment
The present invention will be further described with reference to the accompanying drawings and detailed description.
Embodiment 1
Organotin detects in little yellow croaker
(1) acquisition and sample preparation of sample
It with clean In Aluminium Foil Packing and is sealed in polyethylene bags, the little yellow croaker sample of acquisition then with portable Refrigerating box transports laboratory back, scales, removes the peel, and takes muscle along back, and sample is not more than 0.5cm × 0.5cm × 0.5cm, with high speed Tissue mashing machine is homogeneous, and homogeneous to be placed in freeze drier dry 12 h, taking-up is placed in -18 DEG C of freezen protectives, to be measured.
(2) sampling and ultrasonic extraction
Weigh 1.00g sample to be tested, be added acetic acid and methanol mixed solution that 5mL contains tropolone, acetic acid with In methanol mixed solution, tropolone mass concentration is 0.03%, and the volume ratio of acetic acid and methanol is 1:9, is surpassed after vortex Sound extracts, and obtains extracting solution, vortex time 1min, ultrasonic power 200W, and 40 DEG C of ultrasonic temperature, ultrasonic time 20min.
(3) degreasing is freezed
After extracting solution is placed in -85 DEG C of freezing degreasing 2h, 6000r/m high speed centrifugation 5min takes supernatant A.
(4) first time dispersive solid-phase extraction purifies
100mg N- propyl ethylenediamine solid-phase adsorbent is added in supernatant A, high speed centrifugation after vortex takes supernatant B； Vortex time 30s, high speed centrifugation speed 3000r/m, high speed centrifugation time 3min.
(5) derivative and back extraction
In supernatant B be added 40mL concentration be 1mol/L, pH be 4.5~5 NaAc_HAc buffer solution, 1g chlorine Change sodium and 5mL n-hexane, is vortexed and mixes, add the tetrahydrofuran solution of the 200 μ L sodium of boronation containing tetraethyl, tetrahydrofuran solution The mass concentration of middle tetraethyl boronation sodium is 10%, and high speed centrifugation after vortex oscillation takes upper layer n-hexane phase；The vortex oscillation time 10min, high speed centrifugation speed 3000r/m, high speed centrifugation time 3min.
(6) second of dispersive solid-phase extraction purification
In n-hexane phase, the dehydration of 200mg anhydrous sodium sulfate and 50mg ketjenblack EC solid-phase adsorbent, whirlpool are sequentially added Supination takes supernatant, and membrane filtration takes filtrate；Vortex oscillation time 30s, filter membrane micropore are 0.22 μm.
(7) constant volume is concentrated
By filtrate at 35 DEG C of bath temperature, nitrogen flushing is evaporated to dryness, and with n-hexane dissolution, and is settled to 200 μ L, as Upper machine solution.
(8) sample introduction needle extracts above-mentioned upper machine solution
Above-mentioned upper machine solution is extracted with sample introduction needle, by gas chromatograph-mass spectrometer (the 7890B/5977A gas phase color set Compose mass spectrograph, Agilent Science and Technology Ltd., the U.S.) testing conditions carry out sample detection, obtain total ion chromatogram, select from Sub- monitoring pattern, qualitative and quota ion；GC conditions are as follows: injector temperature is 260 DEG C；Carrier gas is high-purity helium, stream Speed is 1mL/min；Sampling volume is 1 μ L；Input mode is Splitless injecting samples, is purged after 0.75min with 15mL/min；Transmission line 260 DEG C of temperature；Gas chromatographic column temperature program are as follows: 60 DEG C of initial temperature, 1.0min is kept, is warming up to 220 DEG C with 20 DEG C/min, 10min is kept, then is warming up to 280 DEG C with 15 DEG C/min, keeps 5.0min, total run time 28min；Gas chromatographic column selects rule Lattice are the HP-35MS capillary gas chromatographic column of 30m × 0.25mm × 0.25 μm, and stationary phase is diphenyl and the poly- silicon oxygen of dimethyl The mixed liquor of alkane, wherein the mass fraction of diphenyl is 35%, and the mass fraction of dimethyl polysiloxane is 65%；Mass Spectrometry Conditions Are as follows: electron bombardment (EI) ion source, 230 DEG C of ion source temperature, ionizing energy 70eV；Solvent delay 3min；Level four bars temperature 150 DEG C, 50~450amu of scanning range, retrieval spectrum library NIST.
(9) Specification Curve of Increasing
Salbutamol Selected Ion Monitoring mode carries out qualitative: taking concentration is Monobutyltin, dibutyl tin, the tributyl of 1000 μ g/L The organotin hybrid standard use of tin, a phenyltin, a tin octylate, tetrabutyltin, stannous phenide, dioctyl tin, triphenyltin 100 μ L of liquid, 200 μ L are settled to n-hexane respectively, and obtaining concentration is 500 μ g/L standard solution；By standard solution according to above-mentioned The requirement of step (8) is operated to obtain the total ion current figure chromatogram of standard solution, and the total ion current with single analyte Graph coloring spectrogram carries out qualitative ion, quota ion compares, and in combination with retention time, determines the type of nine kinds of organotins；This nine Nine kinds of organotin chromatographic peaks are identified when planting the qualitative ion, quota ion, retention time of organotin to as quantified by external standard method The foundation of identification；The qualitative ion of nine kinds of organotins, quota ion, retention time are as shown in table 1.
The retention time of 1 nine kinds of organotins of table, quota ion, qualitative ion
Select quantified by external standard method: taking concentration is the organotin hybrid standard of 100 μ g/L using 20,50,200 μ L of liquid, simultaneously It separately takes the organotin hybrid standard of 1000 μ g/L of concentration using 50,100,200 μ L of liquid, is separately added into n-hexane and is settled to 200 μ L, Obtain the standard curve serial solution that six spiked levels ranges are 10~1000 μ g/L；By the mark of six kinds of different spiked levels Directrix curve serial solution establishes standard according to the corresponding relationship of organic tin concentration of addition and corresponding quota ion integrated peak areas Curve, the equation of linear regression of standard curve, the range of linearity, related coefficient and detection limit are as shown in table 2.
The equation of linear regression of 2 nine kinds of organotins of table, the range of linearity, related coefficient and detection limit
Wherein, a:y and x respectively represents the reason of the corresponding quota ion integrated peak areas of analyte and analyte in n-hexane By concentration；B: the range of linearity represents concentration of the analyte in standard curve serial solution.
(10) sample measures
Organotin in total ion chromatogram obtained in step (8) is accordingly quantified into peak area, with above-mentioned steps (9) standard curve obtained in is compared, and the reality of nine kinds of organotins in little yellow croaker sample can be obtained finally by conversion Content.
1.00g is taken, 20 μ L, 200 μ are separately added into after the requirement processing of step (1) using above-mentioned little yellow croaker sample The organotin hybrid standard that L concentration is 100 μ g/L is made using the organotin hybrid standard that liquid and 200 μ L concentration are 1000 μ g/L With liquid, be configured to low (2 μ g/kg), in (20 μ g/kg) and high (200 μ g/kg) three kinds of addition concentration levels mark-on sample, whirlpool Rotation is uniformly mixed, and carries out five operation repetitives respectively according to above-mentioned steps (2)~(8) requirement, and obtain with above-mentioned steps (9) To standard curve compare, finally obtain the measurement concentration of nine kinds of organotins in mark-on little yellow croaker sample by converting；Under Formula carries out rate of recovery calculating:
In formula: R --- the rate of recovery, ﹪；
Cs--- the measurement concentration of nine kinds of organotins, μ g/kg in mark-on sample；
C0--- the concentration of nine kinds of organotins in actual sample, μ g/kg；
C --- the theoretical spiked levels of nine kinds of organotins, μ g/kg in mark-on sample.
Through detecting, nine kinds of organotins are not detected in little yellow croaker sample, different spiked levels water in mark-on little yellow croaker sample Flat recovery testu the results are shown in Table 3.
The precision (n=5) of the background value of nine kinds of organotins, recovery of standard addition and method in 3 little yellow croaker sample of table
Note: " ND " representative is not detected
As shown in Table 3, mark-on little yellow croaker sample recovery rate be 81~115%, relative standard deviation (n=5) be 4.1~ 7.5%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 2
Organotin detects in Penaeus Vannmei
(1) acquisition and sample preparation of sample
It by the Penaeus Vannmei sample of acquisition, with clean In Aluminium Foil Packing and is sealed in polyethylene bags, then with just The formula refrigerating box of taking transports laboratory back, and decaptitating, removes appendage at decladding, takes muscle, and sample is not more than 0.5cm × 0.5cm × 0.5cm, Homogeneous with high-speed tissue mashing machine, homogeneous be placed in freeze drier is dried for 24 hours, and taking-up is placed in -18 DEG C of freezen protectives, to It surveys；
(2) sampling and ultrasonic extraction
1.00g sample to be tested is weighed, acetic acid and methanol mixed solution that 10mL contains tropolone is added, in acetic acid With 3g copper powder is added in methanol mixed solution, ultrasound desulfurization, high speed centrifugation after vortex take centrifugal clear liquid, then in centrifugal clear liquid Acetic acid and methanol mixed solution that 10mL contains tropolone is added, ultrasonic extraction after vortex obtains extracting solution, acetic acid and first In mixed alkoxide solution, tropolone mass concentration is 0.03%, and the volume ratio of acetic acid and methanol is 1:9, vortex time 3min, ultrasonic power 300W, 30 DEG C of ultrasonic temperature, ultrasonic time 10min；Copper powder is first handled with dilute hydrochloric acid using preceding, then is used Distilled water flushing is neutrality to pH, is finally dried up with acetone rinsing and under nitrogen flowing.
(3) degreasing is freezed
After extracting solution is placed in -85 DEG C of freezing degreasing 2h, 6000r/m high speed centrifugation 5min takes supernatant A
(4) first time dispersive solid-phase extraction purifies
200mg N- propyl ethylenediamine solid-phase adsorbent, 6000r/m high speed centrifugation after vortex 60s are added in supernatant A 2min takes supernatant B.
(5) derivative and back extraction
In supernatant B be added 30mL concentration be 1mol/L, pH be 4.5~5 NaAc_HAc buffer solution, 1g chlorine Change sodium and 5mL n-hexane, is vortexed and mixes, add the tetrahydrofuran solution of the 200 μ L sodium of boronation containing tetraethyl, tetrahydrofuran solution The mass concentration of middle tetraethyl boronation sodium is 10%, and high speed centrifugation after vortex oscillation takes upper layer n-hexane phase, vortex oscillation time 15min, high speed centrifugation speed 6000r/m, high speed centrifugation time 2min.
(6) second of dispersive solid-phase extraction purification
In n-hexane phase, the dehydration of 200mg anhydrous sodium sulfate and 50~200mg ketjenblack EC solid phase adsorption are sequentially added Agent takes supernatant after vortex 60s, and 0.22 μm of membrane filtration takes filtrate；
(7) constant volume is concentrated
By filtrate at 40 DEG C of bath temperature, nitrogen flushing is evaporated to dryness, and with n-hexane dissolution, and is settled to 200 μ L, as Upper machine solution.
(8) sample introduction needle extracts above-mentioned upper machine solution
Above-mentioned upper machine solution is extracted with sample introduction needle, carries out loading inspection by the gas chromatograph-mass spectrometer testing conditions set It surveys, obtains total ion chromatogram, select ion monitoring mode, qualitative and quota ion；GC conditions are as follows: injector temperature It is 260 DEG C；Carrier gas is high-purity helium, flow velocity 1mL/min；Sampling volume is 1 μ L；Input mode is Splitless injecting samples, It is purged after 0.75min with 15mL/min；260 DEG C of transmission line temperature；Gas chromatographic column temperature program are as follows: 60 DEG C of initial temperature, protect 1.0min is held, is warming up to 220 DEG C with 20 DEG C/min, 10min is kept, then be warming up to 280 DEG C with 15 DEG C/min, keeps 5.0min, Total run time 28min；It is the HP-35MS capillary gas phase color of 30m × 0.25mm × 0.25 μm that gas chromatographic column, which selects specification, Column is composed, stationary phase is the mixed liquor of diphenyl and dimethyl polysiloxane, and wherein the mass fraction of diphenyl is 35%, dimethyl The mass fraction of polysiloxanes is 65%；Mass Spectrometry Conditions are as follows: electron bombardment (EI) ion source, 230 DEG C of ion source temperature, ionization energy Measure 70eV；Solvent delay 3min；150 DEG C of level four bars temperature, 50~450amu of scanning range, retrieval spectrum library NIST；
(9) Specification Curve of Increasing
Salbutamol Selected Ion Monitoring mode carries out qualitative: taking concentration is Monobutyltin, dibutyl tin, the tributyl of 1000 μ g/L The organotin hybrid standard use of tin, a phenyltin, a tin octylate, tetrabutyltin, stannous phenide, dioctyl tin, triphenyltin 100 μ L of liquid, 200 μ L are settled to n-hexane respectively, and obtaining concentration is 500 μ g/L standard solution；By standard solution according to above-mentioned The requirement of step (8) is operated to obtain the total ion current figure chromatogram (as shown in Figure 1) of standard solution, and and single analyte Total ion current figure chromatogram carry out qualitative ion, quota ion compares, in combination with retention time, determine nine kinds of organotins Type；Nine kinds of identification is organic when the qualitative ions of nine kinds of organotins, quota ion, retention time are to as quantified by external standard method The foundation of tin chromatographic peak identification, the qualitative ion of nine kinds of organotins, quota ion, retention time are as shown in table 1.
Select quantified by external standard method: taking concentration is the organotin hybrid standard of 100 μ g/L using 20,50,200 μ L of liquid, simultaneously It separately takes the organotin hybrid standard of 1000 μ g/L of concentration using 50,100,200 μ L of liquid, is separately added into n-hexane and is settled to 200 μ L, Obtain the standard curve serial solution that six spiked levels ranges are 10~1000 μ g/L；By the mark of six kinds of different spiked levels Directrix curve serial solution establishes standard according to the corresponding relationship of organic tin concentration of addition and corresponding quota ion integrated peak areas Curve, the equation of linear regression of standard curve, the range of linearity, related coefficient and detection limit are as shown in table 2.
(10) sample measures
Organotin in total ion chromatogram obtained in step (8) is accordingly quantified into peak area, with above-mentioned steps (9) standard curve obtained in is compared, and nine kinds of organotins can be obtained in Penaeus Vannmei sample finally by conversion Actual content.
Using above-mentioned Penaeus Vannmei sample, after the requirement processing of step (1), take 1.00g, be separately added into 20 μ L, The 100 μ g/L organotin hybrid standards of 200 μ L use liquid using the 1000 μ g/L organotin hybrid standards of liquid and 200 μ L, prepare At low (2 μ g/kg), in (20 μ g/kg) and high (200 μ g/kg) three kinds of addition concentration levels mark-on sample, vortex mixed is equal Standard song that is even, carrying out five operation repetitives respectively according to above-mentioned steps (2)~(8) requirement, and obtained with above-mentioned steps (9) Line compares, and the measurement concentration of nine kinds of organotins in mark-on Penaeus Vannmei sample is finally obtained by converting；It carries out according to the following formula The rate of recovery calculates:
In formula: R --- the rate of recovery, %；
Cs--- the measurement concentration of nine kinds of organotins, μ g/kg in mark-on sample；
C0--- the concentration of nine kinds of organotins in actual sample, μ g/kg；
C --- the theoretical spiked levels of nine kinds of organotins, μ g/kg in mark-on sample.
Through detecting, nine kinds of organotins are not detected in Penaeus Vannmei sample, different in mark-on Penaeus Vannmei sample to add The recovery testu of mark concentration level the results are shown in Table 4.
The precision (n=5) of the background value of nine kinds of organotins, recovery of standard addition and method in 4 Penaeus Vannmei sample of table
Note: " ND " representative is not detected
As shown in Table 4, mark-on Penaeus Vannmei sample recovery rate is 85~117%, and relative standard deviation (n=5) is 3.8~6.7%, meet analysis method to the rate of recovery and reproducibility requirement.
Embodiment 3
Organotin detects in oyster
(1) acquisition and sample preparation of sample
By the oyster sample of acquisition, with clean In Aluminium Foil Packing and it is sealed in polyethylene bags, then uses Portable type cold Hiding case transports laboratory back, and decladding takes edible part, and sample is not more than 0.5cm × 0.5cm × 0.5cm, uses high-speed tissue mashing machine Homogeneous, homogeneous to be placed in freeze drier dry 18h, taking-up is placed in -18 DEG C of freezen protectives, to be measured；
(2) sampling and ultrasonic extraction
1.00g sample to be tested is weighed, acetic acid and methanol mixed solution that 8mL contains tropolone is added, in acetic acid With 1g copper powder is added in methanol mixed solution, ultrasound desulfurization, high speed centrifugation after vortex take centrifugal clear liquid, then in centrifugal clear liquid Acetic acid and methanol mixed solution that 8mL contains tropolone is added, ultrasonic extraction after vortex obtains extracting solution, acetic acid and first In mixed alkoxide solution, tropolone mass concentration is 0.03%, and the volume ratio of acetic acid and methanol is 1:9, vortex time 2min, ultrasonic power 250W, 35 DEG C of ultrasonic temperature, ultrasonic time 15min；Copper powder is first handled with dilute hydrochloric acid using preceding, then is used Distilled water flushing is neutrality to pH, is finally dried up with acetone rinsing and under nitrogen flowing.
(3) degreasing is freezed
After extracting solution is placed in -85 DEG C of freezing degreasing 2h, 6000r/m high speed centrifugation 5min takes supernatant A
(4) first time dispersive solid-phase extraction purifies
200mg N- propyl ethylenediamine solid-phase adsorbent, 5000r/m high speed centrifugation after vortex 50s are added in supernatant A 2.5min takes supernatant B.
(5) derivative and back extraction
In supernatant B be added 30mL concentration be 1mol/L, pH be 4.5~5 NaAc_HAc buffer solution, 1g chlorine Change sodium and 5mL n-hexane, is vortexed and mixes, add the tetrahydrofuran solution of the 200 μ L sodium of boronation containing tetraethyl, tetrahydrofuran solution The mass concentration of middle tetraethyl boronation sodium is 10%, and high speed centrifugation after vortex oscillation takes upper layer n-hexane phase, vortex oscillation time 12min, high speed centrifugation speed 5000r/m, high speed centrifugation time 2.5min.
(6) second of dispersive solid-phase extraction purification
In n-hexane phase, the dehydration of 200mg anhydrous sodium sulfate and 50~200mg ketjenblack EC solid phase adsorption are sequentially added Agent takes supernatant after vortex 40s, and 0.22 μm of membrane filtration takes filtrate；
(7) constant volume is concentrated
By filtrate at 38 DEG C of bath temperature, nitrogen flushing is evaporated to dryness, and with n-hexane dissolution, and is settled to 200 μ L, as Upper machine solution.
(8) sample introduction needle extracts above-mentioned upper machine solution
Above-mentioned upper machine solution is extracted with sample introduction needle, carries out loading inspection by the gas chromatograph-mass spectrometer testing conditions set It surveys, obtains total ion chromatogram, select ion monitoring mode, qualitative and quota ion；GC conditions are as follows: injector temperature It is 260 DEG C；Carrier gas is high-purity helium, flow velocity 1mL/min；Sampling volume is 1 μ L；Input mode is Splitless injecting samples, It is purged after 0.75min with 15mL/min；260 DEG C of transmission line temperature；Gas chromatographic column temperature program are as follows: 60 DEG C of initial temperature, protect 1.0min is held, is warming up to 220 DEG C with 20 DEG C/min, 10min is kept, then be warming up to 280 DEG C with 15 DEG C/min, keeps 5.0min, Total run time 28min；It is the HP-35MS capillary gas phase color of 30m × 0.25mm × 0.25 μm that gas chromatographic column, which selects specification, Column is composed, stationary phase is the mixed liquor of diphenyl and dimethyl polysiloxane, and wherein the mass fraction of diphenyl is 35%, dimethyl The mass fraction of polysiloxanes is 65%；Mass Spectrometry Conditions are as follows: electron bombardment (EI) ion source, 230 DEG C of ion source temperature, ionization energy Measure 70eV；Solvent delay 3min；150 DEG C of level four bars temperature, 50~450amu of scanning range, retrieval spectrum library NIST；
(9) Specification Curve of Increasing
Salbutamol Selected Ion Monitoring mode carries out qualitative: taking concentration is Monobutyltin, dibutyl tin, the tributyl of 1000 μ g/L The organotin hybrid standard use of tin, a phenyltin, a tin octylate, tetrabutyltin, stannous phenide, dioctyl tin, triphenyltin 100 μ L of liquid, 200 μ L are settled to n-hexane respectively, and obtaining concentration is 500 μ g/L standard solution；By standard solution according to above-mentioned The requirement of step (8) is operated to obtain the total ion current figure chromatogram (as shown in Figure 1) of standard solution, and and single analyte Total ion current figure chromatogram carry out qualitative ion, quota ion compares, in combination with retention time, determine nine kinds of organotins Type；Nine kinds of identification is organic when the qualitative ions of nine kinds of organotins, quota ion, retention time are to as quantified by external standard method The foundation of tin chromatographic peak identification, the qualitative ion of nine kinds of organotins, quota ion, retention time are as shown in table 1.
Select quantified by external standard method: taking concentration is the organotin hybrid standard of 100 μ g/L using 20,50,200 μ L of liquid, simultaneously It separately takes the organotin hybrid standard of 1000 μ g/L of concentration using 50,100,200 μ L of liquid, is separately added into n-hexane and is settled to 200 μ L, Obtain the standard curve serial solution that six spiked levels ranges are 10~1000 μ g/L；By the mark of six kinds of different spiked levels Directrix curve serial solution establishes standard according to the corresponding relationship of organic tin concentration of addition and corresponding quota ion integrated peak areas Curve, the equation of linear regression of standard curve, the range of linearity, related coefficient and detection limit are as shown in table 2.
(10) sample measures
Organotin in total ion chromatogram obtained in step (8) is accordingly quantified into peak area, with above-mentioned steps (9) standard curve obtained in is compared, and is contained finally by the reality that nine kinds of organotins in oyster sample can be obtained in conversion Amount.
1.00g is taken, 20 μ L, 200 μ L are separately added into after the requirement processing of step (1) using above-mentioned oyster sample The organotin hybrid standard that the organotin hybrid standard that concentration is 100 μ g/L is 1000 μ g/L using liquid and 200 μ L concentration Using liquid, be configured to low (2 μ g/kg), in (20 μ g/kg) and high (200 μ g/kg) three kinds of addition concentration levels mark-on sample, Vortex mixed is uniform, carries out five operation repetitives respectively according to above-mentioned steps (2)~(8) requirement, and obtain with above-mentioned steps (9) To standard curve compare, finally obtain the measurement concentration of 9 kinds of organotins in mark-on oyster sample by converting；According to the following formula into The row rate of recovery calculates:
In formula: R --- the rate of recovery, %；
Cs--- the measurement concentration of 9 kinds of organotins, μ g/kg in mark-on sample；
C0--- the concentration of 9 kinds of organotins in actual sample, μ g/kg；
C --- the theoretical spiked levels of 9 kinds of organotins, μ g/kg in mark-on sample.
Through detecting, dibutyl tin, tributyl tin, a phenyltin have a detection in oyster samples, in mark-on oyster sample The recovery testu of different spiked levels levels the results are shown in Table 5.
The precision (n=5) of the background value of 9 kinds of organotins, recovery of standard addition and method in 5 oyster sample of table
Note: " ND " representative is not detected
As shown in Table 5, mark-on oyster sample recovery rate be 80~110%, relative standard deviation (n=5) be 3.9~ 7.1%, meet analysis method to the rate of recovery and reproducibility requirement.
Above-mentioned embodiment is only a preferred solution of the present invention, not the present invention is made in any form Limitation, there are also other variations and modifications on the premise of not exceeding the technical scheme recorded in the claims.

Claims (10)

1. the dispersive solid-phase extraction gaschromatographic mass spectrometry detection method of organic tin environmental hormone, feature exist in a kind of marine product In, comprising the following steps:
(1) acquisition and sample preparation of sample
By marine product sample smash to pieces it is homogeneous after, freeze-drying, in -18 DEG C refrigerate, it is to be measured；
(2) sampling and ultrasonic extraction
Weigh 1.00g sample to be tested, be added acetic acid and methanol mixed solution that 5~10mL contains tropolone, acetic acid with In methanol mixed solution, tropolone mass concentration is 0.03%, and the volume ratio of acetic acid and methanol is 1:9, is surpassed after vortex Sound extracts, and obtains extracting solution；
(3) degreasing is freezed
After extracting solution is placed in -85 DEG C of freezing degreasings, high speed centrifugation takes supernatant A；
(4) first time dispersive solid-phase extraction purifies
100~200mg N- propyl ethylenediamine solid-phase adsorbent is added in supernatant A, high speed centrifugation after vortex takes supernatant B；
(5) derivative and back extraction
In supernatant B be added 30~40mL concentration be 1mol/L, pH be 4.5~5 NaAc_HAc buffer solution, 1g chlorine Change sodium and 5mL n-hexane, is vortexed and mixes, add the tetrahydrofuran solution of the 200 μ L sodium of boronation containing tetraethyl, tetrahydrofuran solution The mass concentration of middle tetraethyl boronation sodium is 10%, and high speed centrifugation after vortex oscillation takes upper layer n-hexane phase；
(6) second of dispersive solid-phase extraction purification
In n-hexane phase, the dehydration of 200mg anhydrous sodium sulfate and 50~200mg ketjenblack EC solid-phase adsorbent are sequentially added, Supernatant is taken after vortex, membrane filtration takes filtrate；
(7) constant volume is concentrated
By filtrate at 35~40 DEG C of bath temperature, nitrogen flushing is evaporated to dryness, and with n-hexane dissolution, and 200 μ L is settled to, as upper Machine solution.
(8) sample introduction needle extracts above-mentioned upper machine solution
Above-mentioned upper machine solution is extracted with sample introduction needle, sample detection is carried out by the gas chromatograph-mass spectrometer testing conditions set, obtains Total ion chromatogram selects ion monitoring mode, qualitative and quota ion；GC conditions are as follows: injector temperature 260 ℃；Carrier gas is high-purity helium, flow velocity 1mL/min；Sampling volume is 1 μ L；Input mode is Splitless injecting samples, after 0.75min It is purged with 15mL/min；260 DEG C of transmission line temperature；Gas chromatographic column temperature program are as follows: 60 DEG C of initial temperature, 1.0min is kept, 220 DEG C are warming up to 20 DEG C/min, 10min is kept, then be warming up to 280 DEG C with 15 DEG C/min, keeps 5.0min, total run time 28min；It is the HP-35MS capillary gas chromatographic column of 30m × 0.25mm × 0.25 μm, stationary phase that gas chromatographic column, which selects specification, For the mixed liquor of diphenyl and dimethyl polysiloxane, wherein the mass fraction of diphenyl is 35%, dimethyl polysiloxane Mass fraction is 65%；Mass Spectrometry Conditions are as follows: electron bombardment (EI) ion source, 230 DEG C of ion source temperature, ionizing energy 70eV；It is molten Agent postpones 3min；150 DEG C of level four bars temperature, 50~450amu of scanning range, retrieval spectrum library NIST；
(9) Specification Curve of Increasing
Salbutamol Selected Ion Monitoring mode carries out qualitative: taking concentration is Monobutyltin, dibutyl tin, the tributyl tin, one of 1000 μ g/L Phenyltin, a tin octylate, tetrabutyltin, stannous phenide, dioctyl tin, triphenyltin organotin hybrid standard use 100 μ of liquid L is settled to 200 μ L with n-hexane respectively, and obtaining concentration is 500 μ g/L standard solution；By standard solution according to above-mentioned steps (8) Requirement operated to obtain the total ion current figure chromatogram of standard solution, and the total ion current figure chromatogram with single analyte Carry out qualitative ion, quota ion compares, in combination with retention time, determine the type of nine kinds of organotins；Nine kinds of organotins Qualitative ion, quota ion, retention time to as quantified by external standard method when nine kinds of organotin chromatographic peak identifications of identification according to According to；Select quantified by external standard method: taking concentration is the organotin hybrid standard of 100 μ g/L using 20,50,200 μ L of liquid, while separately being taken dense The organotin hybrid standard of 1000 μ g/L is spent using 50,100,200 μ L of liquid, is separately added into n-hexane and is settled to 200 μ L, obtains six Secondary spiked levels range is the standard curve serial solution of 10~1000 μ g/L；By the standard curve of six kinds of different spiked levels Serial solution establishes standard curve according to the corresponding relationship of organic tin concentration of addition and corresponding quota ion integrated peak areas；
(10) sample measures
By accordingly quantifying in peak area, with above-mentioned steps (9) for the organotin in total ion chromatogram obtained in step (8) Obtained standard curve is compared, and the actual content of nine kinds of organotins in sample marine product product can be obtained finally by conversion.
2. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 1 Spectrum detection method, which is characterized in that in step (1), it is freeze-dried using freeze drier, drying time 12~for 24 hours.
3. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 1 Spectrum detection method, which is characterized in that in step (2), 1~3g copper powder is added in acetic acid and methanol mixed solution, surpasses after vortex Sound desulfurization, high speed centrifugation take centrifugal clear liquid, then be added in centrifugal clear liquid 5~10mL contain the acetic acid of tropolone with In methanol mixed solution, acetic acid and methanol mixed solution, tropolone mass concentration is 0.03%, the body of acetic acid and methanol Product ratio is 1:9, and ultrasonic extraction after vortex obtains extracting solution.
4. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 3 Spectrum detection method, which is characterized in that the copper powder is first handled with dilute hydrochloric acid using preceding, then with distilled water flushing to pH is Property is finally dried up with acetone rinsing and under nitrogen flowing.
5. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 3 Spectrum detection method, which is characterized in that high speed centrifugation speed is 3000~6000r/m, 3~5min of high speed centrifugation time.
6. the dispersive solid-phase extraction gas phase color of organic tin environmental hormone in a kind of marine product according to claim 1 or 3 Compose Mass Spectrometry detection method, which is characterized in that in step (2), 1~3min of vortex time, 200~300W of ultrasonic power, ultrasound temperature 30~40 DEG C of degree, 10~20min of ultrasonic time.
7. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 1 Spectrum detection method, which is characterized in that in step (3), freeze degreasing 2h, high speed centrifugation speed 6000r/m, centrifugation time 5min.
8. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 1 Spectrum detection method, which is characterized in that in step (4), 30~60s of vortex time, high speed centrifugation 3000~6000r/m of speed are high Fast 2~3min of centrifugation time.
9. the dispersive solid-phase extraction gas-chromatography matter of organic tin environmental hormone in a kind of marine product according to claim 1 Spectrum detection method, which is characterized in that in step (5), 10~15min of vortex oscillation time, high speed centrifugation speed 3000~ 6000r/m, 2~3min of high speed centrifugation time.
10. the dispersive solid-phase extraction gas-chromatography of organic tin environmental hormone in a kind of marine product according to claim 1 Mass Spectrometry detection method, which is characterized in that in step (6), 30~60s of vortex time, filter membrane micropore is 0.22 μm.
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