CN106290694A - The Ultra Performance Liquid Chromatography level Four bar electrostatic field orbit ion trap mass spectrum screening method of preservative in milk and milk products - Google Patents
The Ultra Performance Liquid Chromatography level Four bar electrostatic field orbit ion trap mass spectrum screening method of preservative in milk and milk products Download PDFInfo
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- CN106290694A CN106290694A CN201610964638.6A CN201610964638A CN106290694A CN 106290694 A CN106290694 A CN 106290694A CN 201610964638 A CN201610964638 A CN 201610964638A CN 106290694 A CN106290694 A CN 106290694A
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Abstract
The invention discloses the Ultra Performance Liquid Chromatography level Four bar electrostatic field orbit ion trap mass spectrum screening method of preservative in a kind of milk and milk products, belong to technical field of food safety detection.It utilizes the QuEChERS method optimized that sample is carried out pre-treatment；Ultra Performance Liquid Chromatography level Four bar electrostatic field orbit ion trap mass spectrum is used to carry out examination, use full scan/data dependence collection, full scan/variable data non-dependent gathers, full scan/full ion fragmentation acquisition scans pattern, select cation or negative ion mode scanning, obtain complete one-level and second order spectrum；Use Exact Finder 2.5 software that the one-level obtained is processed with two grades of spectrograms, extract compound information.The information set up in spectrum storehouse is contrasted with measured object the selection result, the composition of preservative in confirmation milk and milk products.The main advantage of mass spectrometer used by the present invention is that resolution is high, qualitative and quantitative result is accurate, highly sensitive, Mass accuracy is high, can be applicable to the analysis of complex matrices.
Description
[technical field]
The invention belongs to technical field of food safety detection, particularly to the ultra high efficiency of preservative in a kind of milk and milk products
Liquid chromatograph-level Four bar electrostatic field orbit ion trap mass spectrum screening method.
[background technology]
Nowadays, during preservative has been widely applied to food production, milk and milk products adds preservative, it is possible to prolong
The shelf-life of long products.Along with growth in the living standard, people increasingly pay close attention to for the safety problem of milk and milk products, external source
Property specified risk material be affect milk and milk products safety an importance increasingly come into one's own.Preservative is in milk product one
Plant conventional food additive, such as benzoic acid, sorbic acid etc., play suppression growth of microorganism and breeding, the effect extended the shelf life.
Preservative function is that beta-unsaturated carbonyl structure can suppress microorganism containing α, beta-unsaturated carbonyl structure function mechanism, α
Repiration, thus cause energy matter adenosine triphosphate and reduced nicotinamide adenine dinucleotide to wane so that synthesis
Metabolism is obstructed, and Dynamic Membrane structure can not maintain activity, and metabolism direction trends towards hydrolysis, finally produces aqtocytolysis.But, use
Improper having certain seondary effect, the healthy of consumer can be caused certain infringement by long-term Excess free enthalpy.Therefore, it is necessary to it is right
The usage amount of preservative is controlled and monitors, and production and the use of preservative is controlled in the range of the most reasonably.
But, it is currently used for the high flux of milk and milk products exogenous specified risk material, method for quick less, front place
Reason operation complexity, material is high with equipment cost, is only limitted to several or quick detection of a compounds.External source in milk and milk products
The property high flux of specified risk material, rapid screening have become development trend.The present invention is directed to above-mentioned present situation, establish in milk and milk products
The combined gas chromatography mass spectrometry high flux of preservative, rapid screening analyze method.
[summary of the invention]
The present invention is directed to above-mentioned present situation, it is provided that the Ultra Performance Liquid Chromatography-level Four of preservative in a kind of milk and milk products
Bar electrostatic field orbit ion trap mass spectrum screening method, the main advantage of the method is that resolution is high, qualitative and quantitative result accurately,
Highly sensitive, Mass accuracy is high, can be applicable to the analysis of complex matrices.
The present invention is to be realized by following technical scheme:
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field orbit ion trap mass spectrum examination in a kind of milk and milk products
Method, comprises the following steps:
1) utilize QuEChERS method that the measured object sample of milk and milk products is extracted and purified, obtain measured object sample
The test solution of product；
2) use Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph that measured object sample is sieved
Look into, use 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 ms figure；
3) by software, the one-level obtained is processed with second order ms figure, extract preservative in measured object sample
Compound information；
4) the compound information of the preservative compounds information in preservative standard spectrum storehouse with measured object sample is compared
Right, carry out final decision structure and the group of preservative in quantitative milk and milk products by the chromatographic mass spectrometry information of analytical standard material
Become.
As a further improvement on the present invention, step 1) particularly as follows: weigh milk and milk products sample respectively in tool plug polyphenyl
In propylene centrifuge tube, adding volume ratio is 1:99 acetic acidacetonitrile mixed solution, adds anhydrous magnesium sulfate, pottery after vortex mixed
Homogenizing stone, anhydrous sodium acetate, after vortex, mechanical shaking extraction, then centrifuge tube is placed in centrifuge, after centrifugal treating, pipettes upper strata
Solution, in centrifuge tube added with C18, PSA and anhydrous magnesium sulfate in advance, is centrifuged with 10000r/min after vortex, upper solution warp
0.22 μm organic system micro-pore-film filtration, takes filtrate in sample introduction bottle, adds methanol and 8mmol/L ammonium formate aqueous solution.
As a further improvement on the present invention, step 2) particularly as follows: 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, extracts ion flow graph and carries out quantitatively, non-by variable data
Rely on collection vDIA Mode scans and obtain second order ms figure.
As a further improvement on the present invention, step 3) particularly as follows: by analyzing the fragmentation fragment that second order ms figure obtains
Data, tentatively judge this material belonging kinds, are then analyzed other sheet segment informations of this retention time chromatographic peak, including
Mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, thus infer this structure of matter and elementary composition.
As a further improvement on the present invention, step 4) in standard spectrum storehouse according to following steps set up:
1) standard solution of preservative standard substance is prepared；
2) Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph standard to preservative standard substance is used
Solution carries out examination, uses electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scans mould
Formula, obtains the complete one-level of standard solution and second order ms figure；
3) by software, the one-level obtained is processed with second order ms figure, extract the information of standard solution, set up
The standard spectrum storehouse of preservative.
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-column, 10mm × 2.1mm, 1.9 μm, Hypersil Gold aQ C-18 post,
100mm × 2.1mm, 1.9 μm；Column temperature: 35-40 DEG C；Flowing phase: A is percentage by volume 0.1% formic acid and 4mmol/L ammonium formate
Aqueous solution, Mobile phase B is the methanol solution of percentage by volume 0.1% formic acid and 4mmol/L ammonium formate；
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 velocity: 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 DEG C；Capillary temperature: 320-330 DEG C.
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 is:
Using Full MS/vDIA scan pattern, 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 ms 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, and isolation window ranges sets
For 25.0Da, Loop Count16；550.50011-850.63654 be second vDIA, dynamic background deduction 10.0s, isolation
Window ranges is set to 100.0Da, Loop Count16；Collision energy is respectively 17.5eV；35.0eV；52.5eV.
Relative to prior art, the invention have the advantages that
The present invention first passes through QuEChERS method and milk and milk products sample is extracted and purified, and then uses ultra high efficiency
Preservative in liquid chromatograph-level Four bar electrostatic field orbit ion trap mass spectrography detection milk and milk products, finally by standard spectrum storehouse
Compare, final decision structure and the composition of preservative in quantitative milk and milk products.The screening technique of the present invention, can be once
Property quantitatively, qualitative detection many kinds of substance (22 kinds of preservative), and have simple to operate, precision is high, resolution is high, qualitative and fixed
The amount advantages such as result is accurate, highly sensitive, Mass accuracy is high, can be applicable to the analysis of complex matrices.Especially, Q-Orbitrap
The resolution of (Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph) is high, is guaranteeing that trace compound is sensitive
Under degree, resolution is up to 70,000；Mass accuracy is high, it is possible to use external standard method, it is not necessary to internal standard, long Mass accuracy does not declines；
Highly sensitive, the frequency of detection ion cyclotron resonance, it is not required to extra detector；Wide dynamic range, reaches five orders of magnitude；
There is quick positive and negative switched scan pattern, be greatly saved analysis time, improve analysis throughput；Stability is high, only needs correction in a week
Once；Quantitation capabilities is high, can compare with triple quadrupole bar；Mass range is wide, can carry out polypeptide analysis；It addition, Q-
The instrumentation of Orbitrap is easy, and operating cost is low, can be applicable to the analysis of complex matrices.Result shows: 22 kinds of preservative
Having good linear relationship in the range of linear, correlation coefficient is all higher than 0.99, high (4 × CC β), in (2 × CC β), low by (1
× CC β) under three pitch-based sphere, the recovery of standard addition of substrate reaches 87%-108%, repeats to test 6 times, and its relative standard is inclined
Difference is 0.3%-6.4%.Determine that limit (CC α) and measurement capacity (CC β) are respectively 0.0012 μ g/kg-4.85 μ g/kg and 0.0017
μg/kg-8.32μg/kg。
Utilize the QuEChERS method after optimizing that milk and milk products sample is extracted and purified, simple and fast, efficiency
Height, can effectively get rid of the wherein impurity interference to subsequent detection.
[accompanying drawing explanation]
Fig. 1 is that the extraction of 2,4 dichlorophenoxyacetic acid is wandered about as a refugee subgraph.
[detailed description of the invention]
The invention provides the Ultra Performance Liquid Chromatography of preservative in a kind of milk and milk products-level Four bar electrostatic field track from
Sub-trap mass spectrum screening method, it is characterised in that: the 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, utilize the QuEChERS method optimized that milk and milk products sample is extracted and purified；
Secondly, Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum (Q-Orbitrap) is used to sieve
Look into, use electric spray ion source (ESI) and full scan/data dependence collection (Full MS/dd-MS2), the number of full scan/variable
According to non-dependent collection (Full MS/vDIA), full scan/complete three kinds of scan patterns of ion fragmentation collection (Full MS/AIF), obtain
To complete one-level and second order ms figure.
Again, use Exact Finder 2.5 software that the one-level obtained is processed with second order ms figure, extract
Compound information.
Finally, the information of the information in built vertical preservative spectrum storehouse with measured object is compared, including extracting flow chromatography
Retention time, molecular ion peak and its fragmentation of ions peak abundance ratio, the accurate mass number at molecular ion peak and fragmentation of ions peak,
Molecular ion peak and isotope ion peak abundance ratio, the composition of preservative in confirmation milk and milk products.
Below in conjunction with the accompanying drawings, the detailed description of the invention of the present invention is described in detail, but the invention is not restricted to this enforcement
Example.Understand thoroughly to make the public that the present invention to be had, present invention below preferred embodiment describes in detail concrete details.
1. chromatograph mass spectrum analysis condition
Key instrument: the Ultra Performance Liquid Chromatography-level Four bar electrostatic of U.S. Thermo Fisher Scientific company
Field orbit ion trap mass spectrum, is furnished with electric spray ion source.
(1) chromatographic condition
Chromatographic column: Accucore C-18aQ pre-column (10mm × 2.1mm, 1.9 μm, U.S. Thermo Fisher
Scientific company), Hypersil Gold aQ C-18 post (100mm × 2.1mm, 1.9 μm, U.S. Thermo Fisher
Scientific company)；Column temperature: 35 DEG C；Flowing phase: A is 0.1% (v/v) formic acid and 4mmol/L ammonium formate aqueous solution, flowing
Phase B is 0.1% (v/v) formic acid and 4mmol/L ammonium formate methanol solution.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
Measure 5 μ L；Collision gas: high-purity argon gas (purity >=99.999%).Electro-spray ionization (ESI) ion source: single holotype or negative
Pattern；Sheath gas velocity: 18L/min；Sheath atmospheric pressure: 275Kpa；Secondary air speed: 3L/min；Capillary voltage: negative ion mode
3000V, positive ion mode 3500V；Ion source temperature: 50 DEG C；Capillary temperature: 320 DEG C.
(2) Mass Spectrometry Conditions
Use Full MS/vDIA scan pattern.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 ms 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, and isolation window ranges sets
For 25.0Da, Loop Count16；550.50011-850.63654 be second vDIA, dynamic background deduction 10.0s, isolation
Window ranges is set to 100.0Da, Loop Count16.Collision energy is respectively 17.5eV；35.0eV；52.5eV.
Table 1 lists the information such as ionization pattern and the retention time of 22 kinds of preservative standard substances；Table 2 lists 22 kinds to be prevented
Rotten agent standard database partial content.
1 22 kinds of preservative standard substance information of table
2 22 kinds of preservative Ultra Performance Liquid Chromatographies of table/level Four bar-orbit ion trap mass spectrometry parameters
2. solution preparation
(1) preparation of standard solution
Weigh appropriate preservative standard substance 10mg (being accurate to 0.1mg) respectively, be placed in 10mL in volumetric flask, according to mark
Quasi-material dissolubility in different solvents (methanol, acetonitrile and toluene) needs with measuring, and selects suitable solvent to dissolve, is settled to
10mL, is configured to single compound standard reserving solution, keeps in Dark Place in-20 DEG C.Pipette single compound standard reserving solution in
In 100mL volumetric flask, diluting and be settled to scale with methanol-water solution (1:1, v/v), the standard substance of preparation respective concentration mixes
Close working solution.Mixed mark working solution keeps in Dark Place in-20 DEG C of the airtight bottle of brown.
(2) preparation of matrix matching solution
Pipette 12 parts of each 5mL milk and milk products blank samples (measuring before experiment) extracting solution, in 10mL volumetric flask, to use
Vacuum nitrogen dries up instrument and is blown near dry, be separately added into the preservative standard substance mixed solution 10 μ L 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, in volumetric flask, respectively add methanol
5mL, dilutes with 8mmol/L ammonium formate aqueous solution and is settled to scale, 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, 500 μ g/L corresponding matrix matching standard solution.
Matrix matching standard solution answers matching while using.
3. the pre-treatment of sample
Weigh mixing pasteurization milk, instantaneous ultrahigh-temperature sterilization breast, beverage containing milk and each 15g of fermentation milk sample (essence respectively
Really to 0.01g) in 50mL tool plug polystyrene centrifuge tube.Add 10mL acetic acidacetonitrile (1:99, v/v) mixed solution, vortex
Mixing 30s after add 6.0g anhydrous magnesium sulfate (through 500 DEG C of calcination 4h of Muffle furnace, be positioned in exsiccator), pottery homogenizing stone,
1.52g anhydrous sodium acetate, vortex 30s, mechanical shaking extraction 1min, centrifuge tube is placed in centrifuge, 4 DEG C, is centrifuged with 10000r/min
5min, pipettes upper solution 8mL in centrifuge tube added with 404mgC18,410mgPSA and 1.2g anhydrous magnesium sulfate in advance, high
Speed vortex 30s, is centrifuged 5min with 10000r/min, and centrifuging temperature is set to 4 DEG C, and upper solution is through 0.22 μm organic system microporous membrane
Filter, take 200 μ L filtrates in 2mL sample introduction bottle, add 300 μ L methanol and 500 μ L 8mmol/L ammonium formate aqueous solutions.For super
High performance liquid chromatography-level Four bar-orbit ion trap mass spectrum is measured.
4. analysis result
(1) foundation of method
Use Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrum to carry out examination, use electron spray ion
Source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scan pattern, by Ultra Performance Liquid Chromatography-level Four bar
Electrostatic field orbit ion trap mass spectrographic full scan pattern obtains one-level spectrogram, extracts ion flow graph and carries out quantitatively, by variable
Data non-dependent gathers vDIA Mode scans and obtains two grades of spectrograms.Two grades scanning obtained on the basis of full scan more for
The shatter segment data of property, can tentatively judge this material belonging kinds, then other fragments to this retention time chromatographic peak
Information is analyzed, and including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, thus infers this structure of matter and element group
Become.The fragment dissociation pathways inferred by this material and the preservative standard information set up compose other compounds belonging to class in storehouse
Dissociation pathways comparison, it is judged that the molecular formula of this compound and structural formula, is carried out by the chromatographic mass spectrometry information of analytical standard material
Final judgement is with quantitative.
(2) Method validation
The standard curve of a method, the range of linearity and correlation coefficient
Prepare 22 kinds of preservative series hybrid standard substance solutions, concentration is 0.1 μ g/kg, 0.5 μ g/kg, 1 μ g/kg, 2 μ
g/kg、4μg/kg、8μg/kg、10μg/kg、20μg/kg、40μg/kg、80μg/kg、100μg/kg、200μg/kg、400μg/
kg、500μg/kg.Result shows, in detection method, 22 kinds of preservative are in its corresponding concentration range, correlation coefficient (r2) all
More than 0.99, in good linear relationship.
The lower limit of quantitation of b method, recovery of standard addition and precision
The mensuration of CC α uses calibration curve, and CC α is equal to it is observed that the concentration value on axis of ordinates adds correspondence when responding
2.33 times of repeatability standard deviation.CC β is the value of CC α when adding CC α value 1.64 times of repeatability standard deviation.Determine limit
(CC α) and measurement capacity (CC β) are respectively 0.0012 μ g/kg-4.85 μ g/kg and 0.0017 μ g/kg-8.32 μ g/kg.
Precision is obtained by the relative standard deviation of 6 parallel laboratory test response rate results of statistics, is extracted by determinand clean
After change, use level Four bar-orbit ion trap Mass Spectrometer Method, calculate average recovery rate and the relative standard deviation of 22 kinds of preservative.
The response rate and relative standard deviation result are respectively 87%-108% and 0.3%-6.4%, the method accuracy set up and essence
Density is good.Table 3 below lists the methodology parameter of pasteurize Ruzhong preservative.
The range of linearity of table 3 pasteurize Ruzhong preservative, determine limit, measurement capacity, correlation coefficient, average recovery rate and
Precision (n=6)
(3) positive examination quantitative result
Table 4 positive examination quantitative result (n=6)
Note: No.1 is beverage containing milk sample；No.2,3 is instantaneous ultrahigh-temperature sterilization milk sample product.
Fig. 1 is with 2, and the extraction as a example by 4-dichlorphenoxyacetic acid is wandered about as a refugee subgraph, and chromatographic peak is Gauss distribution, without hangover with front
Stretch, thus the most accurate.The present invention use a pin input mode to complete examination in an experiment during screening, confirmation, quantitatively with
Meet high flux, the monitoring requirement quickly analyzed.In screening process, peak area threshold value is set as variable data non-dependent collection
The scan pattern response intensity 8.3 × 10 to molecular ion peak fragmentation threshold value4, signal-to-noise ratio settings is breast and breast system in national standard
In product, the quantitative limit setting 10 of relevant exogenous material mass spectrometric determination, chooses preservative mark in the milk and milk products set up
Quasi-data base, retention time is extracted window and is set as retention time ± 3 times shift of retention time standard deviation.Extraction chromatography peak matter
Lotus is 3ppm than allowing biggest quality deviation setting.Carry when using the retention time set and mass-to-charge ratio mass deviation to extract window
Get response intensity more than or equal to 8.3 × 104During chromatographic peak, sample is tentatively judged as positive, carries out next step confirmation work
Making, if the most not extracting chromatographic peak, preliminary judgement sample is negative.The sample being tentatively judged as feminine gender is entered
The extraction of row feature fracture fragment and analysis, carry out Screening analysis to unknown object material, and then reach a conclusion.Confirmation work master
Two aspects of isotope ion peak to be passed through information and two grades of fragmentation fragment abundance ratios are carried out, and solve isomers chromatograph
The confirmation difficult point flowed out altogether, will have M+1 feature13C, and has M+2 feature34S、81Br、37The isotope ion peak of Cl is rich
Degree ratio is applied to confirm step.
Current is still blank for the research of preservative high flux rapid screening technology in milk and milk products.For newborn and newborn
High flux, method for quick that in goods, preservative remains are less, and pre-treatment operation complexity, material is high with equipment cost, only
Be limited to the quick detection of a several or compounds, when many residuals are quickly analyzed often according to the construction features of compound by chemical combination
Detect after thing packet.In recent years, the application of QuEChERS wide spectrum extracting method and being made in milk and milk products by Instrument crosslinking
Exogenous specified risk material Screening analysis technology develops rapidly.Herein for above-mentioned present situation, use high resolution mass spectrum level Four bar electrostatic
Orbit ion trap mass spectrum, and combine Chemical Measurement to QuEChERS Pretreatment optimization and quantitative data analysis, for
Pasteurization milk, instantaneous ultrahigh-temperature sterilization butter newborn, dilute, fermentation milk and the baby formula breast living closely bound up with people
Powder, establishes exogenous specified risk material combined gas chromatography mass spectrometry high flux, rapid screening analysis method.
With milk and milk products complex matrices as model, establish exogenous specified risk material known target material Screening analysis side
Method.With methodology parameter plan of survey-EU criteria 2002/657/EC and SANCO/12571/2013 of system, super to using
High performance liquid chromatography-level Four bar electrostatic field orbit ion trap mass spectrum, the Screening analysis side of preservative in the milk and milk products of foundation
Method is examined, and in all storehouses, potential target compound has good linear relationship in the range of linear, and correlation coefficient is the biggest
In 0.99, under high, medium and low three pitch-based sphere, the response rate reaches 87%-108%, repeats to test 6 times, its relative standard deviation
For 0.3%-6.4%.Determine that limit (CC α) and measurement capacity (CC β) are respectively 0.0012 μ g/kg-4.85 μ g/kg and 0.0017 μ
G/kg-8.32 μ g/kg, is better than the limitation requirement that the national and relevant international organization such as China, Japan, USA and EU specifies, spirit
Sensitivity improves more than 5 times with degree of accuracy relatively national standard method.
These are only presently preferred embodiments of the present invention, be not limited only to the practical range of the present invention, all according to patent of the present invention
The equivalence that the content of scope is done changes and modifies, and all should be the technology category of the present invention.
Claims (7)
1. the Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field orbit ion trap mass spectrum examination side in a milk and milk products
Method, it is characterised in that: comprise the following steps:
1) utilize QuEChERS method that the measured object sample of milk and milk products is extracted and purified, obtain measured object sample
Test solution；
2) use Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph that measured object sample is carried out examination, adopt
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 ms figure；
3) by software, the one-level obtained is processed with second order ms figure, extract preservative compounds in measured object sample
Information；
4) the compound information of the preservative compounds information in preservative standard spectrum storehouse with measured object sample is compared, logical
The chromatographic mass spectrometry information crossing analysis standard substance carries out final decision structure and the composition of preservative in quantitative milk and milk products.
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field in a kind of milk and milk products the most according to claim 1
Orbit ion trap mass spectrum screening method, it is characterised in that: step 1) particularly as follows: weigh milk and milk products sample respectively and gather in tool plug
In propenyl benzene centrifuge tube, adding volume ratio is 1:99 acetic acidacetonitrile mixed solution, adds anhydrous magnesium sulfate, pottery after vortex mixed
Porcelain homogenizing stone, anhydrous sodium acetate, after vortex, mechanical shaking extraction, then centrifuge tube is placed in centrifuge, after centrifugal treating, pipettes
Layer solution, in centrifuge tube added with C18, PSA and anhydrous magnesium sulfate in advance, is centrifuged with 10000r/min after vortex, upper solution
Through 0.22 μm organic system micro-pore-film filtration, take filtrate in sample introduction bottle, add methanol and 8mmol/L ammonium formate aqueous solution.
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field in a kind of milk and milk products the most according to claim 1
Orbit ion trap mass spectrum screening method, it is characterised in that: step 2) particularly as follows: by Ultra Performance Liquid Chromatography-level Four bar electrostatic
Field orbit ion trap mass spectrographic full scan pattern obtains first mass spectrometric figure, extracts ion flow graph and carries out quantitatively, by variable number
Gather vDIA Mode scans according to non-dependent and obtain second order ms figure.
4. according to the Ultra Performance Liquid Chromatography of preservative in a kind of milk and milk products described in claim 1 or 3-level Four bar electrostatic
Field orbit ion trap mass spectrum screening method, it is characterised in that: step 3) particularly as follows: by analyzing the fragmentation that second order ms figure obtains
Fragment data, tentatively judges this material belonging kinds, is then analyzed other sheet segment informations of this retention time chromatographic peak,
Including mass-to-charge ratio, fragment abundance ratio and isotopic abundance ratio, thus infer this structure of matter and elementary composition.
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field in a kind of milk and milk products the most according to claim 1
Orbit ion trap mass spectrum screening method, it is characterised in that: step 4) in standard spectrum storehouse according to following steps set up:
1) standard solution of preservative standard substance is prepared；
2) Ultra Performance Liquid Chromatography-level Four bar electrostatic field orbit ion trap mass spectrograph standard solution to preservative standard substance is used
Carry out examination, use electric spray ion source and Full MS/dd-MS2, Full MS/vDIA, Full MS/AIF scan pattern,
The one-level complete to standard solution and second order ms figure；
3) by software, the one-level obtained is processed with second order ms figure, extract the information of standard solution, set up anticorrosion
The standard spectrum storehouse of agent.
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field in a kind of milk and milk products the most according to claim 1
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 spectrograph
Chromatographic condition be:
Chromatographic column: Accucore C-18aQ pre-column, 10mm × 2.1mm, 1.9 μm, Hypersil Gold aQ C-18 post,
100mm × 2.1mm, 1.9 μm；Column temperature: 35-40 DEG C；Flowing phase: A is percentage by volume 0.1% formic acid and 4mmol/L ammonium formate
Aqueous solution, Mobile phase B is the methanol solution of percentage by volume 0.1% formic acid and 4mmol/L ammonium formate；
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 velocity: 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 DEG C；Capillary temperature: 320-330 DEG C.
The Ultra Performance Liquid Chromatography of preservative-level Four bar electrostatic field in a kind of milk and milk products the most according to claim 1
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 spectrograph
Mass Spectrometry Conditions be:
Using Full MS/vDIA scan pattern, 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 deduction 10.0s；
Second order ms sweep parameter: sweep time: 0-15min；Resolution: 17500FWHM；The desired value of automatic growth control is fixed
It is 500,000；Maximum injection length 120ms；112.80116-487.97169 is first vDIA, and isolation window ranges is set to
25.0Da, Loop Count16；550.50011-850.63654 be 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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