CN107340340A - A kind of method for determining a variety of mycotoxin contents in fresh milk sample - Google Patents
A kind of method for determining a variety of mycotoxin contents in fresh milk sample Download PDFInfo
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- CN107340340A CN107340340A CN201710494560.0A CN201710494560A CN107340340A CN 107340340 A CN107340340 A CN 107340340A CN 201710494560 A CN201710494560 A CN 201710494560A CN 107340340 A CN107340340 A CN 107340340A
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- aflatoxin
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- 231100000678 Mycotoxin Toxicity 0.000 title claims abstract description 56
- 239000002636 mycotoxin Substances 0.000 title claims abstract description 56
- 235000013336 milk Nutrition 0.000 title claims abstract description 29
- 210000004080 milk Anatomy 0.000 title claims abstract description 29
- 239000008267 milk Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 20
- 239000000284 extract Substances 0.000 claims abstract description 17
- 239000007788 liquid Substances 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 12
- 239000003053 toxin Substances 0.000 claims description 12
- 231100000765 toxin Toxicity 0.000 claims description 12
- 108700012359 toxins Proteins 0.000 claims description 12
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 claims description 8
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 claims description 7
- DAEYIVCTQUFNTM-UHFFFAOYSA-N ochratoxin B Natural products OC1=C2C(=O)OC(C)CC2=CC=C1C(=O)NC(C(O)=O)CC1=CC=CC=C1 DAEYIVCTQUFNTM-UHFFFAOYSA-N 0.000 claims description 7
- KEOYKWIOAINZSQ-UHFFFAOYSA-N alpha-Zearalenol Natural products CC1CCCC(O)CCC=CCc2cc(O)cc(O)c2C(=O)O1 KEOYKWIOAINZSQ-UHFFFAOYSA-N 0.000 claims description 6
- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 claims description 5
- FPQFYIAXQDXNOR-QDKLYSGJSA-N alpha-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-QDKLYSGJSA-N 0.000 claims description 5
- DWTTZBARDOXEAM-GXTWGEPZSA-N alpha-Zearalanol Chemical compound O=C1O[C@@H](C)CCC[C@H](O)CCCCCC2=CC(O)=CC(O)=C21 DWTTZBARDOXEAM-GXTWGEPZSA-N 0.000 claims description 4
- 239000007864 aqueous solution Substances 0.000 claims description 4
- DAEYIVCTQUFNTM-ABAIWWIYSA-N ochratoxin B Chemical compound C([C@H](NC(=O)C1=CC=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 DAEYIVCTQUFNTM-ABAIWWIYSA-N 0.000 claims description 4
- FPQFYIAXQDXNOR-UHFFFAOYSA-N 7beta-trans-zearalenol Natural products O=C1OC(C)CCCC(O)CCCC=CC2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-UHFFFAOYSA-N 0.000 claims description 3
- 229930195730 Aflatoxin Natural products 0.000 claims description 3
- 241000228197 Aspergillus flavus Species 0.000 claims description 3
- VYLQGYLYRQKMFU-UHFFFAOYSA-N Ochratoxin A Natural products CC1Cc2c(Cl)cc(CNC(Cc3ccccc3)C(=O)O)cc2C(=O)O1 VYLQGYLYRQKMFU-UHFFFAOYSA-N 0.000 claims description 3
- 239000005409 aflatoxin Substances 0.000 claims description 3
- FPQFYIAXQDXNOR-PMRAARRBSA-N beta-Zearalenol Chemical compound O=C1O[C@@H](C)CCC[C@@H](O)CCC\C=C\C2=CC(O)=CC(O)=C21 FPQFYIAXQDXNOR-PMRAARRBSA-N 0.000 claims description 3
- 239000000706 filtrate Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- RWQKHEORZBHNRI-BMIGLBTASA-N ochratoxin A Chemical compound C([C@H](NC(=O)C1=CC(Cl)=C2C[C@H](OC(=O)C2=C1O)C)C(O)=O)C1=CC=CC=C1 RWQKHEORZBHNRI-BMIGLBTASA-N 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 150000002561 ketenes Chemical class 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000013642 negative control Substances 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 229960002300 zeranol Drugs 0.000 claims description 2
- 210000000481 breast Anatomy 0.000 claims 1
- 238000005374 membrane filtration Methods 0.000 claims 1
- 231100000614 poison Toxicity 0.000 claims 1
- 239000002574 poison Substances 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 abstract description 5
- 238000005259 measurement Methods 0.000 abstract description 3
- 239000012535 impurity Substances 0.000 abstract description 2
- 230000005686 electrostatic field Effects 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 15
- 239000011159 matrix material Substances 0.000 description 10
- 238000011084 recovery Methods 0.000 description 9
- 230000000694 effects Effects 0.000 description 7
- 238000000605 extraction Methods 0.000 description 5
- 239000012224 working solution Substances 0.000 description 5
- 238000001819 mass spectrum Methods 0.000 description 4
- 239000002115 aflatoxin B1 Substances 0.000 description 3
- 229930020125 aflatoxin-B1 Natural products 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- -1 therefore Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 150000002085 enols Chemical class 0.000 description 2
- 235000013350 formula milk Nutrition 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- MBMQEIFVQACCCH-UHFFFAOYSA-N trans-Zearalenon Natural products O=C1OC(C)CCCC(=O)CCCC=CC2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-UHFFFAOYSA-N 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 1
- 101710182532 Toxin a Proteins 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002108 aflatoxin M1 Substances 0.000 description 1
- 229930073161 aflatoxin M1 Natural products 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical class [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The present invention relates to a kind of method for determining a variety of mycotoxin contents in fresh milk sample, it comprises the following steps：S1：Mycotoxin is extracted from the fresh milk sample, obtains mycotoxin extract；S2：Liquid chromatogram separation is carried out to the mycotoxin extract, obtains elutriated fraction；S3：Mass Spectrometric Identification is carried out to the elutriated fraction using quadrupole rod electrostatic field orbit trap high-resolution mass spectrometer, determines the species and content of mycotoxin.The present invention can fully extract 14 kinds of mycotoxins, and using with high-resolution, the quadrupole rod electrostatic field orbit trap high resolution mass spectrometer of high-quality accuracy of measurement, can preferably isolating target compound and interference impurity；So as to provide reliable, accurate data.
Description
Technical field
The present invention relates to Food Inspection quarantine field, more specifically it relates to a variety of mould in a kind of measure fresh milk sample
The method of bacterium cellulose content.
Background technology
Milk has turned into the necessity of people's life, and especially infant's milk is the best substitute of breast milk, but mould is malicious
Element is as one of prevailing quality safety risk factor of milk, serious threat human health.Mycotoxin is processed in milk
Cheng Zhong, either pasteurize or high temperature sterilization can not degrade mycotoxin, and mycotoxin can completely remain to milk production
In product, therefore, mycotoxin increasingly gets more and more people's extensive concerning in milk.
At present, the method for monitoring mycotoxin contamination situation in milk is mainly enzyme linked immunosorbent assay, and this method is extensive
Analyze and detect applied to mycotoxin, but limit it due to false positive and the shortcomings of being unable to accurate quantification and further apply.Cause
This, the quantitative detecting method based on technologies such as thin-layered chromatography, gas-chromatography, high pressure liquid chromatographies is it has been established that but these sides
Method can only detect a kind of or a kind of mycotoxin, can not meet monitoring and the scientific research demand of mycotoxin in modern milk industry.
Therefore, it is necessary to a kind of method of a variety of mycotoxins of new measure fresh milk.
The content of the invention
To solve problem above, the invention provides a kind of side for determining a variety of mycotoxin contents in fresh milk sample
Method, it comprises the following steps：
S1：Mycotoxin extract is extracted from the fresh milk sample；
S2：Liquid chromatogram separation is carried out to the mycotoxin extract, obtains elutriated fraction；
S3：Mass Spectrometric Identification is carried out to the elutriated fraction using quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer, it is determined that
The species and content of mycotoxin.
The present invention is used with high-resolution, quadrupole rod-electrostatic field orbit trap high resolution mass spectrometer of high-quality accuracy of measurement,
Can preferably isolating target compound and interference impurity；Reliable, accurate data can be provided.This method passes through quadrupole rod-electrostatic
The selection ion scan of orbit trap high resolution mass spectrum extract each mycotoxin first mass spectrometric accurate mass number carry out it is qualitative with
It is quantitative, while automatic triggering second order mses collect the second order mses figure of each mycotoxin, the second order mses figure with standard items
Control, further confirms to compound.
Preferably, a variety of mycotoxins are a variety of in following mycotoxin：Aflatoxin M 2, aflatoxin
M1, aflatoxin G 2, aflatoxin G 1, aflatoxin B 2, aflatoxin B1, β-ZER, β-corn are red
Mould enol, α-zearalanol, α-zearalenol, zearelone, zearalenone, ochratoxin B and reddish brown song
Mould toxin A.
Preferably, described in the acetonitrile solution for using volume fraction to be 5-10% in S1 extracts from the fresh milk sample
Mycotoxin extract.Volume fraction is that 5-10% acetonitrile solution can effectively extract all 14 kinds of mycotoxins, recovery rate
More than pure water and the acetonitrile solution of volume fraction 20%.
Further, S1 comprises the following steps：
S11：Fresh milk sample is taken, Medium speed filter paper filtering is carried out after 4500rpm centrifugations；
S12：The filtrate being filtrated to get in S11 and the acetonitrile solution are mixed；
S13：Make the mixed solution that S13 is obtained by three-in-one more toxin antibody affinity columns, and eluted with methanol, dried up,
Mycotoxin residue is obtained, aspergillus flavus resisting toxin antibody, anti-Aspergillus ochraceus are fixed with three-in-one more toxin antibody affinity columns
Toxin antibody and the red enzyme ketenes toxin antibody of anti-corn；
S14：The mycotoxin residue is dissolved with the methanol aqueous solution of volume fraction 10%, with 0.22 μm of filter membrane mistake
Filter, obtains the mycotoxin extract.
Preferably, liquid chromatogram uses Kinetex C18 chromatographic columns in S2.
Preferably, the quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer takes SIM patterns to quantify.
Preferably, the fresh milk without mycotoxin is additionally provided with as negative control.
Brief description of the drawings
Fig. 1 is the rate of recovery statistical chart that 14 kinds of mycotoxins that mycotoxin obtains are extracted using different Extraction solvents；
Fig. 2 is the matrix effect statistical chart of 14 kinds of mycotoxins.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. the extraction of fresh milk mycotoxin
Fresh milks of the 100g added with 14 kinds of mycotoxins is taken, after 4500rpm centrifuges 10min, Medium speed filter paper filtering.Weigh
20.0g filtrates, 20mL10% acetonitrile solutions (volume ratio) are added, pass through the 3 more toxin immuno-affinity columns of unification after shaking up.Complete
Afterwards, affinity column is about cleaned with 20mL ultra-pure waters.Extract in affinity column after moisture, eluted using 3mL methanol, 40 DEG C are dried up with nitrogen
Eluent.Using 1.0mL10% methanol aqueous solutions (volume ratio) dissolved residue, 0.22 μm of PTFE filter membrane is crossed, treats that liquid chromatogram is surveyed
Examination.In described 14 mycotoxin be respectively aflatoxin M 2, Aflatoxins M1, aflatoxin G 2, aflatoxin G 1,
Aflatoxin B 2, aflatoxin B1, β-ZER, β-zearalenol, α-zearalanol, α-Gibberella zeae
Enol, zearelone, zearalenone, ochratoxin B and ochratoxin A.
Using without the fresh milk without mycotoxin as control.
2. liquid chromatogram
The sample that above-mentioned processing is obtained carries out liquid chromatogram, and condition is as follows：
1) chromatographic column：Kinetex C18,100*2.1mm (i.d.), 1.7 μm, or suitable person；
2) mobile phase：Mobile phase A：The aqueous solution (containing 0.01% formic acid/volume ratio, 0.1mM ammonium formates)；Mobile phase B：First
Alcohol.Gradient elution program is shown in Table 1；
Flow velocity：300 μ L/min (elution program 1)；
Column temperature：45℃；
Sample size：5μL.
The gradient elution program of the liquid chromatogram of table 1
3. mass spectrum
Detected using quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer, Mass Spectrometry Conditions and parameter are as follows：
Ionization mode：Electron spray ionisation negative ions pattern (ESI ±)
Voltage：4.0kV；
Scanning of the mass spectrum mode：SIM/dd-MS2 patterns；
Resolution ratio：M/z is 70 000 at 200；
Capillary temperature：250℃；
Sheath gas：45a.u.；
Aid in gas：10a.u；
S-lens level：50.0；
Automatic growth control：1.0E6；
Maximum injection length：250ms；
Normalize collision energy：40；
The UHPLC/ESI Q-Orbitrap parameters of 2 14 kinds of mycotoxins of table
4. measure
According to the content situation of test compound in sample liquid, the close standard working solution of selected concentration is analyzed.Press
The ascending order of concentration analyzes mixed-matrix standard working solution successively, obtain mixed-matrix standard working solution concentration with
The working curve of peak area.The response of testing compound all should be in the inspection of instrument in mixed-matrix standard working solution and sample liquid
In linear scope.To mixed-matrix standard working solution and the isometric sample introduction measure of sample liquid.
5. result calculates and statement
Using quantified by external standard method, residual quantity is calculated as follows.Result of calculation need to deduct blank value.
In formula：
Component residual quantity is tested in X-sample, unit is ng/kg (μ g/kg)；
The tested component solution concentration for c-obtained from standard working curve, unit is nanograms per milliliter (ng/mL)；
The final constant volume of V-sample solution, unit are milliliter (mL)；
Sample mass representated by m-final sample solution, unit are gram (g).
6. determine lower bound
Fresh milk aflatoxin M 2, Aflatoxins M1, ochratoxin B, ochratoxin A, measure lower bound are
0.0003μg/kg；Aflatoxin G 2, aflatoxin G 1, aflatoxin B 2, aflatoxin B1 measure lower bound are
0.001μg/kg；Zearelone, α-zearalenol, β-zearalenol measure lower bound are 0.003 μ g/kg；Corn
Zeranol, α-zearalanol, β-ZER measure lower bound is 0.008 μ g/kg.
7. the rate of recovery
The rate of recovery may indicate that the extraction efficiency of method.14 kinds of mycotoxins fresh milk addition concentration and the rate of recovery such as
Shown in table 3, as seen from the table, method of the invention is able to detect that this 14 kinds of mycotoxins, and is quantified.
Addition concentration and the rate of recovery of the 3 14 kinds of mycotoxins of table in fresh milk
8. influence of the different solvents to the rate of recovery
Following solvent extraction mycotoxin is used respectively to fresh milk：Do not extract, the acetonitrile water of pure water, volume fraction 5%
Solution, 10% acetonitrile solution and 20% acetonitrile solution.Obtained extract carries out liquid chromatogram and mass spectrum is detected, and obtains
To the rate of recovery as shown in figure 1, it can be seen that being carried using 5% and 10% acetonitrile solution to this 14 kinds of mycotoxins
Take efficiency all higher.
9. matrix effect
Extraction standard solution is quantified with reagent standard liquid, with the rate of recovery reflection matrix effect of extraction standard solution
Should, matrix effect measurement result is as shown in Fig. 2 the matrix effect of every mycotoxin to be measured illustrates three-in-one within 5%
More toxin antibody affinity column selectivity are very high, fine to the clean-up effect of sample, substantially remove matrix effect, can use
Neat solvent standard specimen is corrected.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.
Claims (7)
- A kind of 1. method for determining a variety of mycotoxin contents in fresh milk sample, it is characterised in that comprise the following steps：S1：Mycotoxin is extracted from the fresh milk sample, obtains mycotoxin extract；S2：Liquid chromatogram separation is carried out to the mycotoxin extract, obtains elutriated fraction；S3：Mass Spectrometric Identification is carried out to the elutriated fraction using quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer, determines mould The species and content of toxin.
- 2. according to the method for claim 1, it is characterised in that a variety of mycotoxins are more in following mycotoxin Kind：Aflatoxin M 2, Aflatoxins M1, aflatoxin G 2, aflatoxin G 1, aflatoxin B 2, aspergillus flavus poison Plain B1, β-ZER, β-zearalenol, α-zearalanol, α-zearalenol, zearelone, corn Zeranol, ochratoxin B and ochratoxin A.
- 3. according to the method for claim 1, it is characterised in that the acetonitrile solution that volume fraction is 5-10% is used in S1 The mycotoxin extract is extracted from the fresh milk sample.
- 4. according to the method for claim 3, it is characterised in that S1 comprises the following steps：S11：Fresh milk sample is taken, Medium speed filter paper filtering is carried out after 4500rpm centrifugations；S12：The filtrate being filtrated to get in S11 and the acetonitrile solution are mixed；S13：Make the mixed solution that S13 is obtained by three-in-one more toxin antibody affinity columns, and eluted with methanol, dry up, obtain Mycotoxin residue, aspergillus flavus resisting toxin antibody, anti-ochratoxin are fixed with three-in-one more toxin antibody affinity columns Antibody and the red enzyme ketenes toxin antibody of anti-corn；S14：The mycotoxin residue is dissolved with the methanol aqueous solution of volume fraction 10%, with 0.22 μm of membrane filtration, is obtained To the mycotoxin extract.
- 5. according to the method for claim 1, it is characterised in that liquid chromatogram uses KinetexC18 chromatographic columns in S2.
- 6. according to the method for claim 1, it is characterised in that the quadrupole rod-electrostatic field orbit trap high-resolution mass spectrometer SIM patterns are taken to quantify.
- 7. according to the method any one of claim 1-6, it is characterised in that be additionally provided with without the fresh of mycotoxin Breast is used as negative control.
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| CN201710494560.0A CN107340340B (en) | 2017-06-26 | 2017-06-26 | Method for determining contents of various mycotoxins in fresh milk sample |
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| CN201710494560.0A CN107340340B (en) | 2017-06-26 | 2017-06-26 | Method for determining contents of various mycotoxins in fresh milk sample |
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| CN107340340A true CN107340340A (en) | 2017-11-10 |
| CN107340340B CN107340340B (en) | 2020-04-24 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110749691A (en) * | 2019-12-23 | 2020-02-04 | 山东畜牧兽医职业学院 | HPLC-MS/MS method for determining aflatoxin and homologue thereof in infant auxiliary food |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110749691A (en) * | 2019-12-23 | 2020-02-04 | 山东畜牧兽医职业学院 | HPLC-MS/MS method for determining aflatoxin and homologue thereof in infant auxiliary food |
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