CN106526020A - Ultra-high performance liquid chromatography-quadrupole static electric field orbitrap mass spectrometry screening method for mycotoxin in milk and dairy product - Google Patents
Ultra-high performance liquid chromatography-quadrupole static electric field orbitrap mass spectrometry screening method for mycotoxin in milk and dairy product Download PDFInfo
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- CN106526020A CN106526020A CN201610967234.2A CN201610967234A CN106526020A CN 106526020 A CN106526020 A CN 106526020A CN 201610967234 A CN201610967234 A CN 201610967234A CN 106526020 A CN106526020 A CN 106526020A
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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
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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
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
- G01N30/724—Nebulising, aerosol formation or ionisation
- G01N30/7266—Nebulising, aerosol formation or ionisation by electric field, e.g. electrospray
Abstract
The invention discloses an ultra-high performance liquid chromatography-quadrupole static electric field orbitrap mass spectrometry screening method for mycotoxin in milk and dairy products. The method is characterized in that a sample is pre-treated by an optimized QuEChERS method; a ultra-high performance liquid chromatography-quadrupole static electric field orbitrap mass spectrometry is used for screening, full-scanning/data-independent collection, full-scanning/variable data non-independent collection, and full-scanning/ion fragmentation collection scanning mode is employed, a positive ion or negative ion mode is selected for scanning to obtain complete first-order and second-order spectrums; Exact Finder 2.5 software is used for processing the obtained first-order and second-order spectrums, and the compound information is extracted. Compared the information in spectrum database and the screening result of the to-be-measured objects, composition of mycotoxin in the milk and dairy products can be confirmed. The mass spectrum apparatus has the advantages of high resolution, accurate qualitative and quantification result, high sensitivity, and high quality precision, and can be used for analyzing the complex matrix.
Description
【Technical field】
The invention belongs to technical field of food safety detection, the superelevation of mycotoxin in more particularly to a kind of milk and milk productses
Effect liquid phase chromatogram-level Four bar electrostatic field orbit ion trap mass spectrum screening method.
【Background technology】
Milk and milk productses are one of current food most popular to people, enjoy people due to its abundant nutritive value
Like.With growth in the living standard, people are increasingly paid close attention to for the safety problem of milk and milk productses, exogenous specified risk material
An importance for affecting milk and milk productses safety is increasingly taken seriously.Mycotoxin as a kind of exogenous material,
There are high risks to human and animal.Mycotoxin includes aflatoxins, ochracin class, zearalenones, volt horse
Endotoxin, deoxynivalenol class, Ergota bases.
Mycotoxin in milk and milk productses includes the higher metabolite of the parent toxin in feedstuff and its toxicity, wherein yellow
Aspertoxin M1 is two class carcinogens, and fumonisin B1 is a class carcinogen, can cause retardation of growth, acute poisoning, Acute Hepatic
Scorching and hepatic cell fattydegeneration etc..Therefore, it is necessary to the usage amount of mycotoxin is controlled and is monitored.
However, high flux, method for quick currently used for milk and milk productses exogenous specified risk material are less, front place
Reason complex operation, material are high with equipment cost, are only limitted to several or a class compound quick detection.Therefore, milk and milk productses
The high flux of exogenous specified risk material, rapid screening are into development trend.The present invention is directed to above-mentioned present situation, binding isotherm knowledge
With practical situation, mycotoxin combined gas chromatography mass spectrometry high flux, rapid screening analysis method is established.
【The content of the invention】
The present invention is directed to above-mentioned present situation, there is provided the Ultra Performance Liquid Chromatography-four of mycotoxin in a kind of milk and milk productses
Level bar electrostatic field orbit ion trap mass spectrum screening method, the main advantage of the method is high resolution, qualitative and quantitative result standard
Really, sensitivity height, Mass accuracy height etc., can be applicable to the analysis of complex matrices.
The present invention is realizing by following technical schemes：
The Ultra Performance Liquid Chromatography of mycotoxin-level Four bar electrostatic field orbit ion trap mass spectrum sieve in a kind of milk and milk productses
Checking method, comprises the following steps：
1) the measured object sample of milk and milk productses is extracted and is purified using QuEChERS methods, obtained measured object sample
The test solution of product；
2) measured object sample is sieved with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph
Look into, using electric spray ion source and Full MS/dd-MS2, tri- kinds of scan patterns of Full MS/vDIA, Full MS/AIF obtain
The complete one-level of measured object sample and second order mses figure；
3) one-level for obtaining is processed with second order mses figure by software, extracts mycotoxin in measured object sample
Compound information；
4) the compound information by the mycotoxin compound information in mycotoxin standard spectrum storehouse with measured object sample is entered
Row is compared, and carries out final decision structure with funguses poison in quantitative milk and milk productses by the chromatographic mass spectrometry information of analytical standard material
The composition of element.
As a further improvement on the present invention, step 1) it is specially：The milk and milk productses for having dissolved are weighed in tool plug polyphenyl
In propylene centrifuge tube, 45 DEG C ± 5 DEG C of water is added, and being vortexed fully to mix is completely dispersed sample, cools down in being put into circulating water；Plus
The volume ratio for entering the acetic acidacetonitrile/water mixed solution of percentage by volume 1%, acetonitrile and water is 84:16, after vortex mixed, add
Centrifuge tube, after vortex, mechanical shaking extraction process, is placed in centrifuge by anhydrous magnesium sulfate, ceramic homogenizing stone, anhydrous sodium acetate,
Filtrate is taken in sample introduction bottle, 8mmol/L formic acid aqueous ammonium and methanol is added.
As a further improvement on the present invention, step 2) it is specially：By Ultra Performance Liquid Chromatography-level Four bar electrostatic field rail
The full scan pattern of road ion trap mass spectrometry obtains first mass spectrometric figure, and extracting ion flow graph is carried out quantitatively, non-by variable data
Rely on collection vDIA Mode scans and obtain second order mses figure.
As a further improvement on the present invention, step 3) it is specially：By analyzing the fragmentation fragment that second order mses figure is obtained
Data, tentatively judge the material belonging kinds, and then other piece segment informations of the retention time chromatographic peak are analyzed, including
Mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, so that be inferred to the structure of matter and elementary composition.
As a further improvement on the present invention, step 4) in standard spectrum storehouse be according to following steps set up：
1) prepare the standard solution of mycotoxin standard substance；
2) use Ultra Performance Liquid Chromatography-mark of the level Four bar electrostatic field orbit ion trap mass spectrograph to mycotoxin standard substance
Quasi- solution carries out examination, using electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scanning
Pattern, obtains the complete one-level of standard solution and second order mses figure；
3) one-level for obtaining is processed with second order mses figure by software, extracts the information of standard solution, set up
The standard spectrum storehouse of mycotoxin.
As a further improvement on the present invention, Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap is mass spectrometric
Chromatographic condition is：
Chromatographic column：Accucore C-18aQ pre-columns, 10mm × 2.1mm, 1.9 μm, Hypersil Gold aQ C-18 posts,
100mm × 2.1mm, 1.9 μm；Column temperature：35-40℃；Mobile phase：A is 0.1% formic acid of percentage by volume and 4mmol/L ammonium formates
Aqueous solution, Mobile phase B is the methanol solution of 0.1% formic acid of percentage by volume and 4mmol/L ammonium formates；
Gradient elution program：0min, 100%A；1min, 100%A；7min, 0%A；12min, 0%A；13min, 100%
A；13-15min, 100%A；Flow velocity is 300 μ L/min；Sample size 5-10 μ L；Collision gas：Argon；
Electro-spray ionization ion source：Single holotype or negative mode；Sheath gas：18-19L/min；Sheath atmospheric pressure：
275Kpa；Secondary air speed：3-4L/min；Capillary voltage：Negative ion mode 3000V, positive ion mode 3500V；Ion source
Temperature：230-250℃；Capillary temperature：320-330℃.
As a further improvement on the present invention, Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap is mass spectrometric
Mass Spectrometry Conditions are：
Using Full MS/vDIA scan patterns, first mass spectrometric scanning resolution is 70000FWHM；Automatic growth control mesh
Scale value is set as 1,000,000；Maximum injection length 250ms；The quality error scope allowed is 5ppm；Dynamic background is deducted
10.0s；
Second order mses sweep parameter：Sweep time：0-15min；Resolution：17500FWHM；The target of automatic growth control
Value is set to 500,000；Maximum injection length 120ms；112.80116-487.97169 is first vDIA, isolates window ranges and sets
For 25.0Da, Loop Count16；550.50011-850.63654 being second vDIA, dynamic background deduction 10.0s, isolate
Window ranges are set to 100.0Da, Loop Count16；Collision energy is respectively 17.5eV；35.0eV；52.5eV.
Relative to prior art, the present invention has advantages below：
The present invention first passes through QuEChERS methods and milk and milk productses sample is extracted and purified, then using ultra high efficiency
Mycotoxin in liquid chromatograph-level Four bar electrostatic field orbit ion trap mass spectrography detection milk and milk productses, finally by standard spectrum
Storehouse is compared, the composition of final decision structure and mycotoxin in quantitative milk and milk productses.The screening technique of the present invention, can be with
Disposable quantitative, qualitative detection many kinds of substance (58 kinds of mycotoxins), and have it is simple to operate, it is high precision, high resolution, fixed
Property with the advantage such as quantitative result is accurate, sensitivity is high, Mass accuracy is high, can be applicable to the analysis of complex matrices.Especially, Q-
The high resolution of Orbitrap (Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph), is guaranteeing trace
Under compound sensitivity, resolution is up to 70,000；Mass accuracy is high, it is possible to use external standard method, without the need for internal standard, prolonged high in quality
Degree does not decline；Sensitivity is high, detects the frequency of ion cyclotron resonance, and does not need extra detector；Wide dynamic range, up to five
The individual order of magnitude；With quick positive and negative switched scan pattern, analysis time is greatly saved, improves analysis throughput；Stability is high, only needs
Correction in one week is once；Quantitation capabilities are high, can compare with triple quadrupole bar；Mass range is wide, can carry out polypeptide analysis；Separately
Outward, the instrumentation of Q-Orbitrap is easy, and operating cost is low, can be applicable to the analysis of complex matrices.As a result show：58 kinds true
Verticillium toxin has good linear relationship in the linear scope, and correlation coefficient is all higher than 0.99, high (4 × CC β), in (2 × CC
β), under low (1 × CC β) three pitch-based spheres, the recovery of standard addition of substrate reaches 81%-112%, repeats experiment 6 times, and which is relative
Standard deviation is 1%-6.4%.It is determined that limit (CC α) and measurement capacity (CC β) be respectively 0.0006 μ g/kg-0.55 μ g/kg and
0.001μg/kg-0.92μg/kg。
Milk and milk productses sample is extracted and purified using the QuEChERS methods after optimization, simple and fast, efficiency
Height, can effectively exclude wherein interference of the impurity to subsequent detection.
【Description of the drawings】
Fig. 1 be 3 kinds of QuEChERS sample pre-treatments Extraction solvent acetic acid/waters (MeOH/Water, 84:16, v/v), acetonitrile/
Water (MeCN/Water, 80:20, v/v) and acetonitrile/water (MeCN/Water, 84:16, v/v) to 58 kinds of target analytes response rate
(n=6, recovery of standard addition are 50 μ g kg to comparison diagram-1)；
Fig. 2 for milk, beverage containing milk and fermentation milk matrix effect scattergram (concentration be 10 μ g kg-1)；
Fig. 3 is Aflatoxins M1 [M+H]+329.06493 full scans of m/z and two grades of fragmentation fragment figures.
【Specific embodiment】
The invention provides in a kind of milk and milk productses mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic field track
Ion trap mass spectrometry screening method, it is characterised in that：Equipment used in the method includes Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrograph.Comprise the following steps：
First, matrix solution and standard solution are prepared；Using the QuEChERS methods for having optimized to milk and milk productses sample
Extracted and purified；
Secondly, sieved with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum (Q-Orbitrap)
Look into, using electric spray ion source (ESI) and full scan/data dependence collection (Full MS/dd-MS2), the number of full scan/variable
(Full MS/vDIA) is gathered according to non-dependent, full scan/full ion fragmentation collection (Full MS/AIF) three kinds of scan patterns is obtained
To complete one-level and second order mses figure.
Again, the one-level for obtaining is processed with second order mses figure with 2.5 softwares of Exact Finder, is extracted
Compound information.
Finally, the information of information and the measured object set up in mycotoxin spectrum storehouse is compared, and flows color including extracting
The accurate mass at the retention time of spectrum, molecular ion peak and its fragmentation of ions peak abundance ratio, molecular ion peak and fragmentation of ions peak
Number, molecular ion peak and isotope ion peak abundance ratio, confirm the composition of mycotoxin in milk and milk productses.
Below in conjunction with the accompanying drawings, the specific embodiment of the present invention is described in detail, but the invention is not restricted to the enforcement
Example.In order that the public has to the present invention thoroughly understanding, concrete details is described in detail in present invention below preferred embodiment.
1. chromatograph mass spectrum analysis condition
Key instrument：The Ultra Performance Liquid Chromatography of Thermo Fisher Scientific companies of the U.S.-level Four bar electrostatic
Field orbit ion trap mass spectrum, using electric spray ion source.
(1) chromatographic condition
Chromatographic column：Accucore C-18aQ pre-columns (10mm × 2.1mm, 1.9 μm, U.S. Thermo Fisher
Scientific companies), and Hypersil Gold aQ C-18 posts (100mm × 2.1mm, 1.9 μm, U.S. Thermo Fisher
Scientific companies)；Column temperature：35-40℃；Mobile phase：A is 0.1% (v/v) formic acid and 4mmol/L formic acid aqueous ammoniums, is flowed
Dynamic phase B is 0.1% (v/v) formic acid and 4mmol/L ammonium formate methanol solutions.Gradient elution program：0min, 100%A；1min,
100%A；7min, 0%A；12min, 0%A；13min, 100%A, 13-15min, 100%A；Flow velocity is 300 μ L/min；Sample introduction
Amount 5-10 μ L；Collision gas：High-purity argon gas (purity >=99.999%).Electro-spray ionization (ESI) ion source：Single holotype or
Negative mode；Sheath gas：18-19L/min；Sheath atmospheric pressure：275Kpa；Secondary air speed：3-4L/min；Capillary voltage：It is negative
Ion mode 3000V, positive ion mode 3500V；Ion source temperature：230-250℃；Capillary temperature：
320-330℃。
(2) Mass Spectrometry Conditions
Using Full MS/vDIA scan patterns.First mass spectrometric scanning resolution is 70000FWHM；Automatic growth control mesh
Scale value is set as 1,000,000；Maximum injection length 250ms；The quality error scope allowed is 5ppm；Dynamic background is deducted
10.0s。
Second order mses sweep parameter：Sweep time：0-15min；Resolution：17500FWHM；The target of automatic growth control
Value is set to 500,000；Maximum injection length 120ms；112.80116-487.97169 is first vDIA, isolates window ranges and sets
For 25.0Da, Loop Count16；550.50011-850.63654 being second vDIA, dynamic background deduction 10.0s, isolate
Window ranges are set to 100.0Da, Loop Count16.Collision energy is respectively 17.5eV；35.0eV；52.5eV.
Table 1 lists ionization pattern and retention time of 58 kinds of mycotoxin standard substances etc.；Table 2 lists 58 kinds of funguses
Toxin standard database partial content.
1 58 kinds of mycotoxin standard substance information of table
2 58 kinds of mycotoxin Ultra Performance Liquid Chromatographies of table/level Four bar-orbit ion trap mass spectrometry parameters
2. solution is prepared
(1) preparation of standard solution
Appropriate mycotoxin standard substance 10mg (being accurate to 0.1mg) is weighed respectively, is placed in 10mL in volumetric flask, foundation
Standard substance dissolubility in different solvents (methanol, acetonitrile and toluene) is needed with determining, and is dissolved from suitable solvent, constant volume
To 10mL, single compound standard reserving solution is configured to, is kept in dark place in -20 DEG C.Pipette single compound standard reserving solution in
In 100mL volumetric flasks, with methanol-water solution (1:1, v/v) scale is diluted and is settled to, the standard substance for preparing respective concentration is mixed
Close working solution.Mixed mark working solution keeps in dark place for -20 DEG C in brown airtight bottle.
(2) preparation of matrix matching solution
Pipette 12 parts of each 5mL milk and milk productses blank sample (determining before experiment) extracting solution, in 10mL volumetric flasks, to use
Vacuum nitrogen dries up instrument and is blown near dry, is separately added into the 10 μ L of mycotoxin standard substance mixed solution of 1000 μ g/L, 20 μ L, 40 μ
L, 80 μ L, 100 μ L, 200 μ L, 400 μ L, 800 μ L, 1000 μ L, 2000 μ L, 4000 μ L, 5000 μ L add first in volumetric flask, respectively
Alcohol 5mL, dilutes and is settled to scale with 8mmol/L formic acid aqueous ammoniums, is configured to 1 μ g/L, 2 μ g/L, 4 μ g/L, 8 μ g/L, 10 μ
G/L, 20 μ g/L, 40 μ g/L, 80 μ g/L, 100 μ g/L, 200 μ g/L, 400 μ g/L, the corresponding matrix matching standards of 500 μ g/L are molten
Liquid.Matrix matching standard solution answers matching while using.
3. the pre-treatment of sample
Milk power for infant and young children 3g (being accurate to 0.01g) is weighed in 50mL tool plug polystyrene centrifuge tubes, is added
20mL45 DEG C ± 5 DEG C of water, being vortexed fully to mix is completely dispersed sample, cools down in being put into circulating water.Mixing bar is weighed respectively
Family name's sterilization breast, instantaneous ultrahigh-temperature sterilization breast, beverage containing milk, fermentation milk and each 15g of the milk power for infant and young children sample (essences for dissolving
Really to 0.01g), in 50mL tool plug polystyrene centrifuge tubes.Add 10mL 1% (v/v) acetic acidacetonitrile/water (84:16, v/
V) mixed solution, adds 6.0g anhydrous magnesium sulfates, 1.45g anhydrous sodium acetates and ceramic homogenizing stone, is vortexed after vortex mixed 30s
30s, mechanical shaking extraction 1min.Centrifuge tube is placed in into centrifuge, 4 DEG C of temperature, 10000r/min centrifugation 5min pipette upper solution
8mL in advance added with the centrifuge tube of 405mg C18,108mg PSA and 1.2g anhydrous magnesium sulfates, high speed vortex 30s.With
10000r/min is centrifuged 5min, and centrifuging temperature is set to 4 DEG C, and 0.22 μm of organic system micro-pore-film filtration of upper solution Jing takes 200 μ L
Filtrate is into 2mL sample introduction bottles.Add 300 μ L methanol and 500 μ L 8mmol/L formic acid aqueous ammoniums.With ultra high efficiency liquid phase color
Spectrum-level Four bar-orbit ion trap mass spectrum is measured.
4. analysis result
(1) foundation of method
Examination is carried out with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum, using electron spray ion
Source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scan patterns, by Ultra Performance Liquid Chromatography-level Four bar
The mass spectrographic full scan pattern of electrostatic field orbit ion trap obtains one-level spectrogram, and extracting ion flow graph is carried out quantitatively, by variable
Data non-dependent collection vDIA Mode scans obtain two grades of spectrograms.Two grades of scannings have obtained more being directed on the basis of full scan
The fragmentation fragment data of property, you can tentatively judge the material belonging kinds, then other fragments to the retention time chromatographic peak
Information is analyzed, including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, so as to be inferred to the structure of matter and element group
Into.The fragment dissociation pathways that the material is inferred compose other chemical combination for belonging to class in storehouse with the mycotoxin standard information set up
Thing dissociation pathways are compared, and are judged molecular formula and the structural formula of the compound, are entered by the chromatographic mass spectrometry information of analytical standard material
Row is final judge with it is quantitative.
(2) Method validation
A matrix effects
The matrix effect (C%) of detection method is calculated as follows：
In formula：
C% matrix effects；
Ss target compounds make the slope of standard curve with neat solvent；
Sm target compounds add the slope of the matrix matching standard curve of bare substrate making.
As a result show that majority of compounds has matrix effect in 58 kinds of mycotoxins, wherein 5.17% belongs to medium strong
The matrix effect of degree, 89.66% is weaker matrix effect, and remaining minority mycotoxin belongs to strong basis mass effect.
The standard curve of b methods, the range of linearity and correlation coefficient
Prepare 58 kinds of mycotoxins series hybrid standard substance solutions, concentration be 0.1 μ g/kg, 0.5 μ g/kg, 1 μ g/kg, 2
μg/kg、4μg/kg、8μg/kg、10μg/kg、20μg/kg、40μg/kg、80μg/kg、100μg/kg、200μg/kg、400μg/
kg、500μg/kg.As a result show, in detection method 58 kinds of mycotoxins in its corresponding concentration range, correlation coefficient (r2)
0.99 is all higher than, in good linear relationship.
The lower limit of quantitation of c methods, recovery of standard addition and precision
The measure of CC α adopts calibration curve, and CC α are equal to it is observed that concentration value when responding on axis of ordinates adds correspondence
2.33 times of repeatability standard deviation.1.64 times of repeatability standard deviation when CC β add CC α values for the value of CC α.It is determined that limit
(CC α) and measurement capacity (CC β) are respectively 0.0006 μ g/kg-0.55 μ g/kg and 0.001 μ g/kg-0.92 μ g/kg.Precision
By count 6 parallel laboratory test response rate results relative standard deviation obtain, by determinand extraction and cleaning after, with level Four
Bar-orbit ion trap Mass Spectrometer Method, calculates average recovery rate and the relative standard deviation of 58 kinds of mycotoxins.The response rate with it is relative
Standard deviation result is respectively 81%-112% and 1%-6.4%, and the method accuracy set up and precision are good.Following tables
The 3 methodology parameters for listing mycotoxin in pasteurization milk.
The range of linearity of mycotoxin, determination limit, measurement capacity, correlation coefficient, average recovery rate in 3 pasteurization milk of table
And precision (n=6)
(3) positive examination quantitative result
4 positive examination quantitative result (n=6) of table
Note：No.2,3,4 pasteurization milk samples；No.1,5 is instantaneous ultrahigh-temperature sterilization milk sample product.
It will be seen from figure 1 that acetonitrile/water (MeCN/Water, 84:16, v/v) to 58 kinds of target analytes extraction effects most
It is good, effect be better than (MeOH/Water, 84:16, v/v) with acetonitrile/water (MeCN/Water, 80:20, v/v), thus select nitrile/
Water (MeCN/Water, 84:16, v/v) as Extraction solvent.Fig. 2 shows that majority of compounds is present in 58 kinds of mycotoxins
Matrix effect, the wherein 3 kinds matrix effects for belonging to moderate strength, 52 kinds of pigments are weaker matrix effect, remaining minority fungus
Toxin belongs to strong basis mass effect.Can be seen that by QuEChERS Pretreatments and chromatograph and matter from the distribution of matrix effect
The selection and optimization of spectral condition, the extraction and cleaning liquid-based mass effect for obtaining are relatively low.In order to reduce matrix effect interference as far as possible, building
During vertical calibration curve, standard substance mixed solution is prepared using matrix matching method.Blank sample substrate extracting solution is used respectively
Dilution mixture standard substance solution, has similar ionic environment to the extraction and cleaning liquid of quantitative analyses, so as to greatest extent
Matrix effect is eliminated to sensitivity and the impact of accuracy.Fig. 3 is the mass spectrum of aflatoxin in sample, and scanning of the mass spectrum point is in
Gauss distribution, scanning element are enough, quantitative accurate.
The present invention adopts a pin input mode to complete screening during examination, confirmation in an experiment, quantitatively to meet height
Flux, the monitoring requirement of quick analysis.In screening process, peak area threshold value is set as variable data non-dependent acquisition scans mould
Response intensity 8.3 × 10 of the formula to molecular ion peak fragmentation threshold value4, signal-to-noise ratio settings are phase in milk and milk productses in national standard
The quantitative limit setting 10 of exogenous material mass spectrometric determination is closed, mycotoxin criterion numeral in the milk and milk productses set up is chosen
According to storehouse, retention time is extracted window and is set as retention time ± 3 times shift of retention time standard deviation.Extraction chromatography peak mass-to-charge ratio
Biggest quality deviation setting is allowed to be 3ppm.Window is extracted when the retention time with setting and mass-to-charge ratio mass deviation to extract
Response intensity is more than or equal to 8.3 × 104During chromatographic peak, sample is tentatively judged as positive, carries out next step confirmation work, if
Chromatographic peak is not extracted under these conditions, preliminary judgement sample is feminine gender.Tentatively it is judged as that the sample of feminine gender carries out spy
The extraction and analysis of fracture fragment is levied, Screening analysis is carried out to unknown object material, and then is reached a conclusion.Confirmation work is mainly led to
Crossing two aspects of isotope ion peak information and two grades of fragmentation fragment abundance ratios is carried out, and is solved isomerss chromatograph and flowed altogether
The confirmation difficult point for going out, by with M+1 features13C, and with M+2 features34S、81Br、37The isotope ion peak abundance ratio of Cl
It is applied to confirm step.
At present for the research of mycotoxin high flux rapid screening technology in milk and milk productses is still blank.For breast and
In milk product, the high flux of mycotoxin residual, method for quick are less, pre-treatment complex operation, material and equipment cost
Height, is only limitted to several or a class compound quick detection, remains construction featuress when quickly analyzing often according to compound more
Detect after compound is grouped.In recent years, the application of QuEChERS wide spectrums extracting method and breast and breast are caused by Instrument crosslinking
Product exogenous specified risk material Screening analysis technology is developed rapidly.Herein for above-mentioned present situation, with high resolution mass spectrum level Four
Bar electrostatic field orbit ion trap mass spectrum, and Chemical Measurement is combined to the optimization of QuEChERS Pretreatments and quantitative data point
Analysis, for the pasteurization milk closely bound up with people's life, instantaneous ultrahigh-temperature sterilization breast, dilute cream, fermentation milk and infant
Prescription emulsifiable powder, sets up exogenous specified risk material combined gas chromatography mass spectrometry high flux, rapid screening analysis method.
With milk and milk productses complex matrices as model, exogenous specified risk material known target material Screening analysis side is established
Method.It is with the methodology parameter plan of survey of system-EU criteria 2002/657/EC and SANCO/12571/2013, super to using
High performance liquid chromatography-level Four bar electrostatic field orbit ion trap mass spectrum, the Screening analysis of mycotoxin in the milk and milk productses of foundation
Method is examined, and in all storehouses, potential target compound has good linear relationship in linear scope, and correlation coefficient is equal
More than 0.99, under high, medium and low three pitch-based spheres, the response rate reaches 81%-112%, repeats experiment 6 times, and its relative standard is inclined
Difference is 1%-6.4%.It is determined that limit (CC α) and measurement capacity (CC β) are respectively 0.0006 μ g/kg-0.55 μ g/kg and 0.001 μ g/
Kg-0.92 μ g/kg are better than the limitation requirement that the country such as China, Japan, USA and EU and relevant international organization specify, sensitive
Degree improves more than 5 times with degree of accuracy compared with national standard method.
Presently preferred embodiments of the present invention is these are only, the practical range of the present invention is not limited only to, it is all according to patent of the present invention
Equivalence changes and modification that the content of scope is done, all should be the technology category of the present invention.
Claims (7)
1. in a kind of milk and milk productses mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum examination
Method, it is characterised in that：Comprise the following steps：
1) the measured object sample of milk and milk productses is extracted and is purified using QuEChERS methods, obtained measured object sample
Test solution；
2) examination is carried out to measured object sample with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph, is adopted
With electric spray ion source and Full MS/dd-MS2, tri- kinds of scan patterns of Full MS/vDIA, Full MS/AIF obtain tested
The complete one-level of thing sample and second order mses figure；
3) one-level for obtaining is processed with second order mses figure by software, extracts mycotoxin chemical combination in measured object sample
Thing information；
4) the compound information by the mycotoxin compound information in mycotoxin standard spectrum storehouse with measured object sample is compared
It is right, final decision structure is carried out with mycotoxin in quantitative milk and milk productses by the chromatographic mass spectrometry information of analytical standard material
Composition.
2. in a kind of milk and milk productses according to claim 1 mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that：Step 1) it is specially：The milk and milk productses for having dissolved are weighed in tool plug
In polystyrene centrifuge tube, 45 DEG C ± 5 DEG C of water is added, being vortexed fully to mix is completely dispersed sample, be put into cold in circulating water
But；The volume ratio for adding the acetic acidacetonitrile/water mixed solution of percentage by volume 1%, acetonitrile and water is 84:16, vortex mixed
Afterwards, anhydrous magnesium sulfate, ceramic homogenizing stone, anhydrous sodium acetate is added, after vortex, mechanical shaking extraction process, centrifuge tube centrifugation is placed in into
Machine is centrifuged, and takes filtrate in sample introduction bottle, adds 8mmol/L formic acid aqueous ammonium and methanol.
3. in a kind of milk and milk productses according to claim 1 mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that：Step 2) it is specially：It is quiet by Ultra Performance Liquid Chromatography-level Four bar
The mass spectrographic full scan pattern of electric field orbit ion trap obtains first mass spectrometric figure, and extracting ion flow graph is carried out quantitatively, by variable
Data non-dependent collection vDIA Mode scans obtain second order mses figure.
4. in a kind of milk and milk productses according to claim 1 or 3, the Ultra Performance Liquid Chromatography-level Four bar of mycotoxin is quiet
Electric field orbit ion trap mass spectrum screening method, it is characterised in that：Step 3) it is specially：By analyze second order mses figure obtain it is broken
Sliver segment data, tentatively judges the material belonging kinds, then other piece segment informations of the retention time chromatographic peak is carried out point
Analysis, including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, so that be inferred to the structure of matter and elementary composition.
5. in a kind of milk and milk productses according to claim 1 mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that：Step 4) in standard spectrum storehouse be according to following steps set up：
1) prepare the standard solution of mycotoxin standard substance；
2) it is molten to the standard of mycotoxin standard substance with Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph
Liquid carries out examination, using electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scan patterns,
Obtain the complete one-level of standard solution and second order mses figure；
3) one-level for obtaining is processed with second order mses figure by software, extracts the information of standard solution, set up funguses
The standard spectrum storehouse of toxin.
6. in a kind of milk and milk productses according to claim 1 mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that：Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum
The chromatographic condition of instrument is：
Chromatographic column：Accucore C-18aQ pre-columns, 10mm × 2.1mm, 1.9 μm, Hypersil Gold aQ C-18 posts,
100mm × 2.1mm, 1.9 μm；Column temperature：35℃；Mobile phase：A is the water of 0.1% formic acid of percentage by volume and 4mmol/L ammonium formates
Solution, Mobile phase B are the methanol solution of 0.1% formic acid of percentage by volume and 4mmol/L ammonium formates；
Gradient elution program：0min, 100%A；1min, 100%A；7min, 0%A；12min, 0%A；13min, 100%A；
13-15min, 100%A；Flow velocity is 300 μ L/min；5 μ L of sample size；Collision gas：Argon；
Electro-spray ionization ion source：Single holotype or negative mode；Sheath gas：18L/min；Sheath atmospheric pressure：275Kpa；It is auxiliary
Help gas velocity：3L/min；Capillary voltage：Negative ion mode 3000V, positive ion mode 3500V；Ion source temperature：50℃；Hair
Capillary temperature：320℃.
7. in a kind of milk and milk productses according to claim 1 mycotoxin Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that：Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum
The Mass Spectrometry Conditions of instrument are：
Using Full MS/vDIA scan patterns, first mass spectrometric scanning resolution is 70000FWHM；Automatic growth control desired value
It is set as 1,000,000；Maximum injection length 250ms；The quality error scope allowed is 5ppm；Dynamic background deducts 10.0s；
Second order mses sweep parameter：Sweep time：0-15min；Resolution：17500FWHM；The desired value of automatic growth control is fixed
For 500,000；Maximum injection length 120ms；112.80116-487.97169 is first vDIA, isolates window ranges and is set to
25.0Da, Loop Count16；550.50011-850.63654 for second vDIA, dynamic background deduction 10.0s, isolation window
Mouth scope is set to 100.0Da, Loop Count16；Collision energy is respectively 17.5eV；35.0eV；52.5eV.
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