CN112461979A - Purifying and filtering column and application thereof in determination of residual quantity of fipronil and metabolites thereof in eggs - Google Patents
Purifying and filtering column and application thereof in determination of residual quantity of fipronil and metabolites thereof in eggs Download PDFInfo
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- CN112461979A CN112461979A CN202011313270.XA CN202011313270A CN112461979A CN 112461979 A CN112461979 A CN 112461979A CN 202011313270 A CN202011313270 A CN 202011313270A CN 112461979 A CN112461979 A CN 112461979A
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- fipronil
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- 239000005899 Fipronil Substances 0.000 title claims abstract description 47
- 229940013764 fipronil Drugs 0.000 title claims abstract description 47
- 235000013601 eggs Nutrition 0.000 title claims abstract description 38
- ZOCSXAVNDGMNBV-UHFFFAOYSA-N 5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile Chemical compound NC1=C(S(=O)C(F)(F)F)C(C#N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl ZOCSXAVNDGMNBV-UHFFFAOYSA-N 0.000 title claims abstract description 37
- 239000002207 metabolite Substances 0.000 title claims abstract description 22
- 238000001914 filtration Methods 0.000 title abstract description 6
- 238000000746 purification Methods 0.000 claims abstract description 51
- 239000003463 adsorbent Substances 0.000 claims abstract description 28
- 239000012528 membrane Substances 0.000 claims abstract description 27
- -1 second sieve Substances 0.000 claims abstract description 26
- 239000000706 filtrate Substances 0.000 claims abstract description 16
- 238000001471 micro-filtration Methods 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 150000002500 ions Chemical class 0.000 claims description 69
- 239000000843 powder Substances 0.000 claims description 32
- 239000000243 solution Substances 0.000 claims description 23
- 239000011159 matrix material Substances 0.000 claims description 20
- 239000002245 particle Substances 0.000 claims description 18
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 14
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 11
- 239000007924 injection Substances 0.000 claims description 11
- 239000004743 Polypropylene Substances 0.000 claims description 10
- LGHZJDKSVUTELU-UHFFFAOYSA-N fipronil-sulfone Chemical compound NC1=C(S(=O)(=O)C(F)(F)F)C(C#N)=NN1C1=C(Cl)C=C(C(F)(F)F)C=C1Cl LGHZJDKSVUTELU-UHFFFAOYSA-N 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 229920001155 polypropylene Polymers 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 8
- 239000012086 standard solution Substances 0.000 claims description 7
- 239000000758 substrate Substances 0.000 claims description 7
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 5
- 238000005259 measurement Methods 0.000 claims description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000000605 extraction Methods 0.000 claims description 3
- 238000011049 filling Methods 0.000 claims description 3
- 235000019253 formic acid Nutrition 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000004949 mass spectrometry Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- 238000010812 external standard method Methods 0.000 claims description 2
- 230000010355 oscillation Effects 0.000 claims description 2
- 238000004451 qualitative analysis Methods 0.000 claims description 2
- 238000004445 quantitative analysis Methods 0.000 claims description 2
- 238000005185 salting out Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 21
- 238000001819 mass spectrum Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 235000013305 food Nutrition 0.000 abstract description 5
- 238000005119 centrifugation Methods 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 24
- 239000012071 phase Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 11
- 238000011084 recovery Methods 0.000 description 5
- 239000012488 sample solution Substances 0.000 description 5
- 239000013076 target substance Substances 0.000 description 4
- 239000012535 impurity Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000012629 purifying agent Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241000238876 Acari Species 0.000 description 1
- 241001674048 Phthiraptera Species 0.000 description 1
- 241000258242 Siphonaptera Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- CPPKAGUPTKIMNP-UHFFFAOYSA-N cyanogen fluoride Chemical compound FC#N CPPKAGUPTKIMNP-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 239000000273 veterinary drug Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/14—Preparation by elimination of some components
- G01N2030/146—Preparation by elimination of some components using membranes
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a purifying and filtering column, which comprises: syringe pipe, first sieve, purification adsorbent layer, microfiltration membrane, second sieve, filtrate export, first sieve is located the upper portion of syringe intraduct space, the purification adsorbent layer sets up between first sieve and microfiltration membrane, the microfiltration membrane sets up in the lower part of purification adsorbent layer, the second sieve sets up in the lower part of microfiltration membrane, the filtrate export sets up in the bottom of syringe pipe. The purification filter column is used for detecting the residual quantity of fipronil and metabolites thereof in the eggs, the operation is simpler, quicker and more sensitive than GB 23200.115-2018 liquid chromatogram-mass spectrum combined usage method for measuring the residual quantity of fipronil and metabolites thereof in eggs of national food safety standard, the purification effect is better, simultaneously, the steps of vortex and centrifugation in the national standard are saved in the purification process, and the one-step purification filtration is adopted, so the working efficiency is greatly improved.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of veterinary drug residue detection, and particularly relates to a purification filter column and application thereof in determination of fipronil and metabolite residues thereof in eggs.
[ background of the invention ]
Fipronil is an insecticide used for killing fleas, mites and lice, is used for preventing and controlling pests in a farm, can cause pollution to eggs when being used improperly, and has certain toxicity to human bodies after being eaten for a long time. The european union specifies: fipronil can not be used in the livestock and poultry breeding process of the human food industry chain. China GB2763-2019 defines the maximum residue of fipronil (the sum of fipronil, fipronil sulfone and fipronil thioether, expressed as fipronil) in eggs to be 0.02mg/kg, and a detection method GB 23200.115-2018 liquid chromatogram-mass spectrum combination method for determining the residue of fipronil and its metabolite in eggs, which is suitable for determining the residue of fipronil and its metabolite in eggs, is issued in 6.21.2018.6.2018. The dispersed solid phase extraction purification process needs the steps of vortex adsorption and then centrifugation to obtain supernatant, so that the operation steps are multiple, the time is consumed, and the purification effect is not ideal.
In addition, the purification process of the method adopts 50mgPSA powder, 50mgC18The powder and 150mg of anhydrous magnesium sulfate were subjected to vortex dispersion to adsorb interfering substances, and then centrifuged, and the supernatant was filtered. The purification process has multiple operation steps and low working efficiency.
[ summary of the invention ]
The invention aims to overcome the defects in the prior art and provide the application of the purification filter column which is simple and rapid and can achieve the same purification effect.
In order to solve the technical problems, the invention adopts the following technical scheme.
A purifying filter column comprising: syringe pipe, first sieve, purification adsorbent layer, microfiltration membrane, second sieve, filtrate export, first sieve is located the upper portion of syringe intraduct space, the purification adsorbent layer sets up between first sieve and microfiltration membrane, the particle diameter of adsorbent is greater than the aperture of first sieve and second sieve in the purification adsorbent layer, purification adsorbent layer constitution raw materials includes: PSA powder, PEP powder, C18The powder and the anhydrous magnesium sulfate are uniformly mixed to form the injection syringe, the microporous filter membrane is arranged at the lower part of the purification adsorbent layer, the second sieve plate is arranged at the lower part of the microporous filter membrane, and the filtrate outlet is arranged at the bottom of the injection syringe.
Further, the syringe tube was a polypropylene tube having an inner diameter of 12.9mm, a tube length of 67.6mm, and a volume of 6 ml.
Further, the first sieve plate and the second sieve plate are polypropylene sieve plates with the diameter of 13.0mm and the thickness of 1.2mm and the pore diameter of 20 mu m.
Further, the purification adsorbent layer comprises the following raw materials: 45-55mg of PSA powder, 45-55mg of PEP powder, 45-55mg of C18Powder, 135 and 165mg of anhydrous magnesium sulfate, and the raw materials are mixed uniformly.
Further, the particle size of the PSA powder is 40-60 μm, the particle size of the PEP powder is 40-60 μm, and C18The particle size of the powder is 40-60 μm.
Further, the microporous filter membrane is a polytetrafluoroethylene microporous filter membrane with the pore diameter of 0.2 mu m.
The invention also provides application of the purification filter column in determination of the residual quantity of fipronil and metabolites thereof in eggs, which comprises the following steps:
(1) sample preparation: taking an edible part of an egg sample, homogenizing, uniformly mixing, and freezing and storing;
(2) sample pretreatment: precisely weighing 5g of the uniformly homogenized and mixed egg sample obtained in the step (1), placing the sample in a 50mL polypropylene centrifuge tube, precisely adding 20mL acetonitrile, carrying out vortex mixing for 1min, carrying out oscillation extraction for 5min, adding 1.5g sodium acetate and 6g magnesium sulfate to carry out salting-out layering, carrying out vortex separation for 1min, centrifuging for 5min at 5000r/min, absorbing supernatant liquid, placing the supernatant liquid in a purification filter column, collecting filtrate, and directly filling the filtrate into a sample vial for determination by an ultra-high performance liquid-tandem mass spectrometer;
(3) preparation of matrix mixing standard working curve solution: preparing a blank substrate of the egg according to the steps (1) and (2) to obtain a blank substrate extracting solution; diluting the mixed standard solution with a blank matrix extracting solution to prepare a series of matrix mixed standard working curve solutions with gradient concentrations;
(4) and (3) determination: setting the determination conditions of the ultra-high performance liquid-tandem mass spectrometer, and injecting the matrix mixed standard working curve solution into the triple quadrupole ultra-high performance liquid-tandem mass spectrometer; the chromatographic measurement conditions were as follows: ACQUITY
BEH C182.1X 100mm, 1.7 μm; the mobile phase A is methanol, and the mobile phase B is 5mmol/L ammonium acetate-0.1% formic acid solution; column temperature: 35 ℃; flow rate: 0.3 mL/min; sample introduction amount: 2 mu L of the solution; mobile phase gradients and time were as follows: the gradient of the mobile phase is as follows: 10%, 90%, 10% of mobile phase A, and 10% of mobile phase B: 90%, 10%, 90%, mobile phase time: 1min, 3min, 6min and 10 min; the mass spectrometry conditions were as follows: an ion source: electrospray ion source (ESI), Multiple Reaction Monitoring (MRM); negative ion scanning mode: electrospray voltage (IS) -4500V; ion source Temperature (TEM)550 ℃; the flow rate of air curtain gas (CUR) is 35L/min; the flow rate of the atomizing gas (GS1) is 50L/min; the flow rate of the auxiliary heating gas (GS2) is 50L/min; injection voltage (EP) -10V; the ion pair, the declustering voltage, the collision energy and the collision chamber ejection voltage are specifically as follows:
firstly, fipronil: parent ions 434.9, quantitative ions 329.8, declustering voltage-101V, collision energy-21V and collision chamber ejection voltage-15V; parent ion 434.9, child ion 249.8, declustering voltage-101V, collision energy-34V and collision chamber ejection voltage-15V;
② fluorocarbonitrile: parent ions 386.9, quantitative ions 350.8, declustering voltage-66V, collision energy-22V and collision chamber ejection voltage-11V; parent ion 386.9, child ion 281.8, declustering voltage-66V, collision energy-46V and collision chamber ejection voltage-11V;
③ fipronil sulfone: parent ions 450.9, quantitative ions 281.8, declustering voltage-110V, collision energy-40V and collision chamber ejection voltage-15V; parent ions 450.9, child ions 243.8, declustering voltage-110V, collision energy-65V and collision chamber ejection voltage-11V;
fipronil sulfoxide: 418.9 parent ions, 382.8 quantitative ions, 80V cluster removing voltage, 17V collision energy and 15V collision chamber ejection voltage; 418.9 parent ions, 261.8 child ions, minus 78V cluster removing voltage, minus 36V collision energy and minus 10V collision chamber ejection voltage;
(5) qualitative and quantitative analysis: and comparing the retention time with the relative abundance of corresponding concentration to qualitatively analyze fipronil, fipronil sulfone and fipronil sulfoxide, and calculating quantitative results by using an external standard method.
Further, the temperature for cryopreservation of the sample in step (1) was-20 ℃.
Further, the centrifuge tube in step (2) is a 50mL polypropylene centrifuge tube.
Further, the concentrations of the series of gradient concentration matrix mixing standard working curve solutions in the step (3) are respectively: 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml and 20 ng/ml.
Compared with the standard method, the invention has the advantages that:
(1) the invention optimizes the components of the purifying agent by improving the prior purifying filter column, and adopts the following raw materials: 45-55mg of PSA powder with particle size of 40-60 μm, 45-55mg of PEP powder with particle size of 40-60 μm, and 45-55mg of C powder with particle size of 40-60 μm18The powder and 135-165mg of anhydrous magnesium sulfate are uniformly mixed to form the purifying agent, and the purifying and adsorbing effects are better.
(2) The purification filter column is used for detecting the residual quantity of fipronil and metabolites thereof in the eggs, the operation is simpler, quicker and more sensitive than GB 23200.115-2018 liquid chromatogram-mass spectrum combined usage method for measuring the residual quantity of fipronil and metabolites thereof in eggs of national food safety standard, the purification effect is better, simultaneously, the steps of vortex and centrifugation in the national standard are saved in the purification process, and the one-step purification filtration is adopted, so the working efficiency is greatly improved.
[ description of the drawings ]
FIG. 1 is a schematic diagram of the purification filter column of the present invention;
FIG. 2 is the ion mass spectra of 4 targets of fipronil, fipronil sulfone and fipronil sulfoxide in example 1.
[ detailed description ] embodiments
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. It should be emphasized that the following description is merely exemplary in nature and is not intended to limit the scope of the invention or its application.
As shown in fig. 1, a purification filter column includes: the injection device comprises an injection tube 1, a first sieve plate 2, a purification adsorbent layer 3, a microporous filter membrane 4, a second sieve plate 5 and a filtrate outlet 6, wherein the first sieve plate 2 is positioned at the upper part of the inner space of the injection tube 1, the purification adsorbent layer 3 is arranged between the first sieve plate 2 and the microporous filter membrane 4, the microporous filter membrane 4 is arranged at the lower part of the purification adsorbent layer 3, the second sieve plate 5 is arranged at the lower part of the microporous filter membrane 4, and the filtrate outlet 6 is arranged at the bottom of the injection tube 1.
The syringe tube 1 is a polypropylene tube with an inner diameter of 12.9mm, a tube length of 67.6mm and a volume of 6ml, and is used as a shell of a purifying filter column. The first sieve plate 2 and the second sieve plate 5 are polypropylene sieve plates with the diameter of 13.0mm and the thickness of 1.2mm and the aperture of 20 mu m, and mainly play roles in fixing the purifying adsorbent layer 3 and the microporous filter membrane 4 and controlling the flow rate of the column. The purifying adsorbent layer 3 comprises the following raw materials: 45-55mg of PSA powder with particle size of 40-60 μm, 45-55mg of PEP powder with particle size of 40-60 μm, and 45-55mg of C powder with particle size of 40-60 μm18The powder and 135-165mg anhydrous magnesium sulfate are uniformly mixed to form the extract, impurities in the extract can be selectively adsorbed without adsorbing the component to be detected, and the component to be detected is left in the purified filtrate to achieve the purpose of separating the impurities. The microporous filter membrane 4 is a Polytetrafluoroethylene (PTFE) microporous filter membrane with a pore diameter of 0.2 μm, and can filter purified liquid and prevent solution particles from blocking an instrument.
In the purification filter column, the purification adsorbent layer 3 is positioned at the upper part of the microporous filter membrane 4, and the first sieve plate 2 and the second sieve plate 5 are used for fixing the purification adsorbent layer 3 and the microporous filter membrane 4 tightly and firmly in order to ensure that the purification adsorbent is filled compactly and cannot be too loose.
The application of the purification filter column of the present invention is further described in detail below with reference to practical applications:
example 1
By adopting the application method, the residual quantity of fipronil, fipronil sulfone and fipronil sulfoxide in 10 batches of egg samples is respectively determined according to the following steps:
(1) sample preparation: respectively taking edible parts from 10 batches of egg samples, homogenizing and uniformly mixing, and freezing and storing at-20 ℃;
(2) sample pretreatment: precisely weighing 5g (to the accuracy of 0.01g) of the uniformly homogenized and mixed egg sample obtained in the step (1), and placing the sample in 50mL of polymerPrecisely adding 20mL of acetonitrile into a propylene centrifuge tube, uniformly mixing for 1min in a vortex manner, extracting for 5min in a vibration manner, adding 2g of sodium chloride and 6g of anhydrous sodium sulfate, carrying out 1min in a vortex manner, and centrifuging for 5min at the speed of 5000 r/min; taking the supernatant and placing the supernatant into the purification filter column of the invention, wherein the purification filter column is prepared as follows: the bottom of the injector tube 1 is arranged with a second sieve plate 5, and a filtrate outlet 6 is arranged under the second sieve plate 5, then the microporous filter membrane 4 is laid on the second sieve plate 5, the purifying adsorbent layer 3 is filled, and then the first sieve plate 2 is arranged to fix the purifying adsorbent layer 3 and the microporous filter membrane 4. The purifying adsorbent layer 3 comprises the following raw materials: 50mg of PSA powder with a particle size of 42-58 μm, 50mg of PEP powder with a particle size of 40-56 μm, and 50mg of C with a particle size of 41-55 μm18Powder and 160mg of anhydrous magnesium sulfate are uniformly mixed to form the magnesium sulfate powder;
collecting filtrate and directly filling the filtrate into a sample vial for determination by an ultra-high performance liquid-tandem mass spectrometer;
(3) preparation of matrix mixing standard working curve solution: taking the blank substrate of the egg to prepare the blank substrate extracting solution according to the steps (1) and (2). Diluting the mixed standard solution with blank matrix extract to prepare matrix mixed standard working curve solutions with series gradient concentrations of 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml and 20 ng/ml;
(4) and (3) determination: injecting the matrix mixed standard working curve solution and the sample solution into a triple quadrupole ultra-high performance liquid-tandem mass spectrometer for determination, wherein ion mass spectrograms of 4 target substances of fipronil, fluoroform nitrile, fipronil sulfone and fipronil sulfoxide are shown in figure 2, and the chromatographic determination conditions are as follows: ACQUITY
BEH C182.1X 100mm, 1.7 μm; the mobile phase A is methanol, and the mobile phase B is 5mmol/L ammonium acetate-0.1% formic acid solution; column temperature: 35 ℃; flow rate: 0.3 mL/min; sample introduction amount: 2 mu L of the solution; mobile phase gradients and time were as follows: the gradient of the mobile phase is as follows: 10%, 90%, 10% of mobile phase A, and 10% of mobile phase B: 90%, 10%, 90%, mobile phase time: 1min, 3min, 6min and 10 min; the mass spectrometry conditions were as follows: an ion source: electrospray ion source (ESI), Multiple Reaction Monitoring (MRM); negative ionSub-scanning mode: electrospray voltage (IS) -4500V; ion source Temperature (TEM)550 ℃; the flow rate of air curtain gas (CUR) is 35L/min; the flow rate of the atomizing gas (GS1) is 50L/min; the flow rate of the auxiliary heating gas (GS2) is 50L/min; injection voltage (EP) -10V; the ion pair, the declustering voltage, the collision energy and the collision chamber ejection voltage are specifically as follows:
firstly, fipronil: parent ions 434.9, quantitative ions 329.8, declustering voltage-101V, collision energy-21V and collision chamber ejection voltage-15V; parent ion 434.9, child ion 249.8, declustering voltage-101V, collision energy-34V and collision chamber ejection voltage-15V;
② fluorocarbonitrile: parent ions 386.9, quantitative ions 350.8, declustering voltage-66V, collision energy-22V and collision chamber ejection voltage-11V; parent ion 386.9, child ion 281.8, declustering voltage-66V, collision energy-46V and collision chamber ejection voltage-11V;
③ fipronil sulfone: parent ions 450.9, quantitative ions 281.8, declustering voltage-110V, collision energy-40V and collision chamber ejection voltage-15V; parent ions 450.9, child ions 243.8, declustering voltage-110V, collision energy-65V and collision chamber ejection voltage-11V;
fipronil sulfoxide: 418.9 parent ions, 382.8 quantitative ions, 80V cluster removing voltage, 17V collision energy and 15V collision chamber ejection voltage; 418.9 parent ions, 261.8 child ions, minus 78V cluster removing voltage, minus 36V collision energy and minus 10V collision chamber ejection voltage;
(5) and (3) characterization: when the sample is measured under the same experimental conditions, if the retention time of the detected chromatographic peak is consistent with that of the standard substance, and the mass spectrum quantitative ions and the mass spectrum qualitative ions of the target substance appear in the sample mass spectrogram after the background is deducted, and the same batch of tests are carried out, for the same compound, the relative abundance ratio of the qualitative ions and the quantitative ions of the target substance in the sample is compared with the matrix standard solution with the equivalent mass concentration, and the allowable deviation does not exceed the range specified in the table 1, the target substance in the sample can be judged to exist;
(6) quantification: quantifying by using an external standard curve method, in order to reduce the influence of matrix effect, adopting a working solution matched with a matrix to make a standard curve, determining the peak area of a quantitative ion, wherein the response value of a target object in a sample solution is in the linear range of quantitative determination detected by an instrument, detecting fipronil in 3 batches of egg samples according to the detection result, and the result is shown in table 4, and the calculation formula is as follows:
in the formula: omega-the residual amount of the target in milligrams per kilogram (mg/kg) in the sample; rho-mass concentration of target in nanograms per milliliter (ng/ml) in the matrix standard working solution; a-chromatographic peak area of the target in the sample solution; chromatographic peak area of the target in As-substrate standard working solution; v-final volumetric volume of sample solution in milliliters (ml); m-the mass of the sample represented by the sample solution in grams (g); 1000 is a scaling factor.
TABLE 4 detection results of fipronil and its metabolite residues in egg samples
Detection limit and quantification limit determination: the detection limit of the target object is determined by adding the standard solution into the blank egg matrix with the signal-to-noise ratio of 3 times, the quantitative limit of the target object is determined by using the signal-to-noise ratio of 10 times, the result is shown in table 5, and the result is far lower than the quantitative limit of 0.005mg/kg of GB 23200.115-2018 liquid chromatogram-mass spectrum combination method for determining the residual quantity of fipronil and metabolites thereof in eggs in national standard of food safety, which indicates that the method has high sensitivity.
TABLE 5 detection and quantification limits of fipronil and its metabolite residues in egg samples
| Target object | Detection limit (mg/kg) | Limit of quantitation (mg/kg) |
| Fipronil | 0.00001 | 0.00002 |
| Fluorocarbonitrile | 0.00001 | 0.00002 |
| Fipronil sulfone | 0.00002 | 0.00003 |
| Fipronil sulfoxide | 0.00002 | 0.00004 |
Determination of reproducibility and recovery: taking an egg blank matrix, adopting a method of adding a standard solution, measuring the repeatability and the recovery rate of three levels of three parts, wherein the results are shown in table 6, and the recovery rates are all in the range of 85-105%, which shows that the stability of the method is good.
TABLE 6 Repeatability (RSD) and recovery rate of detection method for residual quantity of fipronil and its metabolites in egg sample
The linear ranges, linear equations and related coefficients of the 4 targets are shown in table 7, the 4 targets of the method have good linear relation in the concentration range of 1-20 ng/ml, and the method can be used for quantitative determination of fipronil and metabolites thereof in eggs.
TABLE 7 Linear range, linear equation and correlation coefficient of detection method for residual quantity of fipronil and its metabolites in egg sample
Comparison of method accuracy: the standard solution is added into the negative egg sample according to the level of 0.01mg/kg to obtain a positive sample, GB 23200.115-2018 'liquid chromatography-mass spectrometry combined method for measuring the residual quantity of fipronil and metabolites thereof in eggs according to the national standard for food safety' and the method are respectively adopted for detection, the measurement results are shown in Table 8, the reproducibility of the measurement results of the two methods is less than 5.0, and the method is proved to have good accuracy.
TABLE 8 results of the two methods and reproducibility
And (4) conclusion: according to the method, acetonitrile is adopted for extraction, a purification filter column is used for purification and filtration, and the ultra-high performance liquid chromatography-tandem mass spectrometry is used for determining fipronil and metabolites thereof in the eggs, so that impurity interference can be effectively removed, and the method is simple and rapid to operate, high in sensitivity, stable in recovery rate and good in accuracy.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A purifying filter column, comprising: syringe pipe, first sieve, purification adsorbent layer, microfiltration membrane, second sieve, filtrate export, first sieve is located the upper portion of syringe intraduct space, the purification adsorbent layer sets up between first sieve and microfiltration membrane, the particle diameter of adsorbent is greater than the aperture of first sieve and second sieve in the purification adsorbent layer, purification adsorbent layer constitution raw materials includes: PSA powder, PEP powder, C18The powder and the anhydrous magnesium sulfate are uniformly mixed to form the injection syringe, the microporous filter membrane is arranged at the lower part of the purification adsorbent layer, the second sieve plate is arranged at the lower part of the microporous filter membrane, and the filtrate outlet is arranged at the bottom of the injection syringe.
2. The purifying filter column of claim 1, wherein the syringe tube is a polypropylene tube with an inner diameter of 12.9mm, a length of 67.6mm, and a volume of 6 ml.
3. The purification filter column of claim 1, wherein the first sieve plate and the second sieve plate are polypropylene sieve plates with a diameter of 13.0mm and a thickness of 1.2mm and a pore size of 20 μm.
4. The purifying filter column of claim 1, wherein the purifying adsorbent layer comprises the following raw materials: 45-55mg of PSA powder, 45-55mg of PEP powder, 45-55mg of C18Powder, 135 and 165mg of anhydrous magnesium sulfate, and the raw materials are mixed uniformly.
5. The purification filter column of claim 4, wherein the PSA powder has a particle size of 40-60 μm, the PEP powder has a particle size of 40-60 μm, C18The particle size of the powder is 40-60 μm.
6. The purification filter column of claim 1, wherein the microfiltration membrane is a 0.2 μm pore size polytetrafluoroethylene microfiltration membrane.
7. The use of a purification filter column according to any one of claims 1 to 6 for determining the residual amount of fipronil and its metabolites in eggs, comprising the steps of:
(1) sample preparation: taking an edible part of an egg sample, homogenizing, uniformly mixing, and freezing and storing;
(2) sample pretreatment: precisely weighing 5g of the uniformly homogenized and mixed egg sample obtained in the step (1), placing the sample in a 50mL polypropylene centrifuge tube, precisely adding 20mL acetonitrile, carrying out vortex mixing for 1min, carrying out oscillation extraction for 5min, adding 1.5g sodium acetate and 6g magnesium sulfate to carry out salting-out layering, carrying out vortex separation for 1min, centrifuging for 5min at 5000r/min, absorbing supernatant liquid, placing the supernatant liquid in a purification filter column, collecting filtrate, and directly filling the filtrate into a sample vial for determination by an ultra-high performance liquid-tandem mass spectrometer;
(3) preparation of matrix mixing standard working curve solution: preparing a blank substrate of the egg according to the steps (1) and (2) to obtain a blank substrate extracting solution; diluting the mixed standard solution with a blank matrix extracting solution to prepare a series of matrix mixed standard working curve solutions with gradient concentrations;
(4) and (3) determination: setting the determination conditions of the ultra-high performance liquid-tandem mass spectrometer, and injecting the matrix mixed standard working curve solution into the triple quadrupole ultra-high performance liquid-tandem mass spectrometer; the chromatographic measurement conditions were as follows:
BEH C182.1X 100mm, 1.7 μm; the mobile phase A is methanol, and the mobile phase B is 5mmol/L ammonium acetate-0.1% formic acid solution; column temperature: 35 ℃; flow rate: 0.3 mL/min; sample introduction amount: 2 mu L of the solution; mobile phase gradients and time were as follows: the gradient of the mobile phase is as follows: 10%, 90%, 10% of mobile phase A, and 10% of mobile phase B: 90%, 10%, 90%, mobile phase time: 1min, 3min, 6min and 10 min; the mass spectrometry conditions were as follows: an ion source: electrospray ion source (ESI), Multiple Reaction Monitoring (MRM); negative ion scanning mode: electrospray voltage (IS) -4500V; ion source Temperature (TEM)550 ℃; the flow rate of air curtain gas (CUR) is 35L/min; the flow rate of the atomizing gas (GS1) is 50L/min; the flow rate of the auxiliary heating gas (GS2) is 50L/min; injection voltage (EP) -10V; it is composed ofThe conditions of ion pairs, declustering voltage, collision energy and collision chamber ejection voltage are as follows:
firstly, fipronil: parent ions 434.9, quantitative ions 329.8, declustering voltage-101V, collision energy-21V and collision chamber ejection voltage-15V; parent ion 434.9, child ion 249.8, declustering voltage-101V, collision energy-34V and collision chamber ejection voltage-15V;
② fluorocarbonitrile: parent ions 386.9, quantitative ions 350.8, declustering voltage-66V, collision energy-22V and collision chamber ejection voltage-11V; parent ion 386.9, child ion 281.8, declustering voltage-66V, collision energy-46V and collision chamber ejection voltage-11V;
③ fipronil sulfone: parent ions 450.9, quantitative ions 281.8, declustering voltage-110V, collision energy-40V and collision chamber ejection voltage-15V; parent ions 450.9, child ions 243.8, declustering voltage-110V, collision energy-65V and collision chamber ejection voltage-11V;
fipronil sulfoxide: 418.9 parent ions, 382.8 quantitative ions, 80V cluster removing voltage, 17V collision energy and 15V collision chamber ejection voltage; 418.9 parent ions, 261.8 child ions, minus 78V cluster removing voltage, minus 36V collision energy and minus 10V collision chamber ejection voltage;
(5) qualitative and quantitative analysis: and comparing the retention time with the relative abundance of corresponding concentration to qualitatively analyze fipronil, fipronil sulfone and fipronil sulfoxide, and calculating quantitative results by using an external standard method.
8. The use of the purifying filter column according to claim 7 for determining the residual amount of fipronil and its metabolites in eggs, wherein the temperature of the sample frozen in step (1) is-20 ℃.
9. The application of the purification filter column in determination of residual quantity of fipronil and its metabolites in eggs according to claim 7, wherein the centrifuge tube in step (2) is a 50mL polypropylene centrifuge tube.
10. The use of the purifying filter column according to claim 7 for determining the residual amount of fipronil and its metabolites in eggs, wherein the concentrations of the series of gradient concentration matrix mixed standard working curve solutions in step (3) are respectively: 1ng/ml, 2ng/ml, 4ng/ml, 10ng/ml and 20 ng/ml.
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